MSU LIBRARIES n V RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. FUNCTICNAL CAPACITY OF THE RESIIXJAL IEUKCXIYTES m 211‘!) DEFICIEIT MICE By Joan Marie Cook—Mills A DISSMATIQI Suhnitted to Michigan State University in partial fulfillment of the requiranents for the degree of WOF Flam Department of Biochemistry 1987 ABSTRACT FUNCTIONAL CAPACITY OF THE -IINAL LEUKGZYTES m ZINC DEFICIENT MICE By Joan Marie Cook-Mills Zinc deficiency drastically compromises cell and antibody medi- ated immune responses in both htmns and animls. This is due, in part, to a significant rethiction in nunbers of leukocytes. Since numerous zinc dependent enzymes are necessary for cell function, it was important to determine the functional capacity of the residual leukocytes from zinc deficient mice _i_n _v_i_t_z_‘g while minimizing zinc repletion. In response to T—cell stimulants, residual splenic lymphocytes from zinc deficient mice could proliferate, produce interleukin-2, and produce interleukin-2 receptors as well as splenocytes from zinc adequate or restricted mice in y_i_t_rg. In response to antigenic stimulation, antibody production per antibody secreting cell was the same for splenocytes from zinc deficient and zinc adequate mice. Thus, the residual splenic lymphocytes of zinc deficient mice were able to carry out many fundamental inmme responses. In contrast, resident peritoneal macroflmages from zinc deficient mice have been reported to associate with and destroy fewer m; m 912; (L M) parasites than Macrophages from adequately fed mice. Modifications of previous assays designated herein made it possible for the first time to directly quantitate 1-120: production by resident macroflaages using L c_ru_gi as the stimulant. It was found that production of I130; by macrophages stimlated with L cruzi trypomastigotes or opsoni zed trypomstigotes but not amstigotes correlated with L c__ru_z_i-mcrophage association. Furthermore, it was shown that arachidonate metabolites my be intermediates in trypomastigote stimulation of 1120: production by resident macrophages. Upon deprivation of dietary zinc, the total amount of L 9_r_g_z_;_ trypouastigote-stimulated 11:0 produced/ an; macrofllage protein was reduced but HaOz production/ parasite associated with the morophages was unaltered. Also, the reduced L c_r\£i_-mcrophage association in the zinc deficient group we. not due to a decrease in long-chain unsaturated fatty acids, which is known to decrease phagocytosis, or to a decrease in production of leukotrienes, which may decrease trypounstigote-mcrophage association. Therefore, some other pro- cess(es) critical to association and destruction of L QLuz_i must be aberrant in morophages from zinc deficient mice. Dedicated to mr husband, James Mills and m parents, Norman and Barbara Cook for their patience, love, understanding, and support iv Acknowledgements I would like to thank Dr. Pamela Fraker for her guidance, sup- port, and frienship. I am grateful to the members of m comitee - Dr. Steven Aust, Dr. William Smith, Dr. Shelagh Ferguson-Miller, Dr. Estelle McGroarty, and Dr. Ronald Patterson - for their contents and suggestions. Also, thank you to Dr. Richard Luecke for his good advice throughout my undergraduate and graduate studies . Thank you Dr. Julie Wirth for your advice and frienship and for batch after batch of L _c_r\_iz_i. I would especially like to acknowledge the lab members of the present - Joseph Gibbons, Gerry Morford, Andra Cress, Dr. Lewis King, Lorri Teper - and of the past - Dr. Carmen Medina, Dr. Li Gang, Paul Keller, and Roger Morford - for their help, encourage- ment, and in particular all the good times. Also, thank you to Mildred Rivera for her friendship and help. Most importantly, thanks to w parents and grandparents for instilling in me a love of learning and being a constant source of support. Special thanks to my husband for his love, emotional sup— port, and teaching me to relax. TABLE OF (DNTENTS Page LISTOFTABIEOOOOOO0..OOOOOOOOOOOOOOOOOOOOOOOOO00.00.000.00x LISI‘OFFIMOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Xi LIST. OF WATIwSOOOOOOOOOO0.0..00...OOOOOOOOOOOOOOOOOOO xv IWIwOOOOOOOOOOOO0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOO0.. 1 CHAPTERI: lem We...ooooooo00000000000000.0000... I. Zim mfifimyOOO...00.0.00...OOOOOOOOOOOOOOOOOOOOO. A.) Causes of Zinc Deficiency...................... B.) Alterations in the Imme System by Zim mprimtimOOOOOOOOOOOOOO0.000000000000 CO) maismtoInfmtionOO0.00000000IOOOOOOOOOOO II. Possible Roles for Zinc in the Destruction of mmozens by mmmeSIOOOOOOOOOOOOOOOOOOOOOO00.0. A.) Production of Toxic Oxygen Metabolites by Mononuclear Phagocytes: Possible R01“ forZimOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOO B.) Possible Alterations in the "Oxygen Burst" of Phcrofllages from Zinc Deficient Mice. . . . . . . . C. ) Possible Alterations in Membranes of Macrophages from Zinc Deficient Mice........... WOOOOOOO0.0.0.000000000000000000000000000000... CHAPTERZ: HJNCTICNAL CAPACITY OF THE RESIIXJAL LW IN ZINC DHICIM mmOOOO0.000000000000000000000 -vi 11 12 12 14 19 21 21 37 43 48 58 MACr0000..00000000000 OOOOOOOOOOOO 000 ..... 0 ........ 000 59 IWIw0000000000000000.00000.....000000000000000.. 61 MA'I'ERIAISANDWoooooO 0000000000000 0 ..... 0.00000... 63 Animals............................................ 63 Diets.............................................. 63 Collection of autologous serum..................... 64 Zinc analysis...................................... 64 Cell cultmoooooo0.0000000000000000000000000000... 64 Mitogenic stimulation.................. ..... ....... 65 Mixed lymphocyte culture 65 Assay for ILPZ activity............................ 66 mintenance of cytolytic T—cell line............... 67 ILPZ receptors..................................... 67 Antibody production................................ 68 Radioimmunoassay................................... 69 Statistics.............. ..... ...................... 7O MISC-000.000.000000000000000000.00000000000000.0000. 71 DIWIW.000000000.0000000000..0.0000000000....000.000106 W0000000000.000000.0000.0.0000.00000000000000.0111 CHAPTER 3: WED (XJNDITIONS I'm MEASURING H20: WION BY RESIDENT MACROPHAGES: MODIFICATIONS FOR USE mmPm1m0000000000000000.00000000000000.000114 W000.000.000000.0000......00000000.......0000000.115 1W1m000000000000000....000...00000000.00000....0117 MAMIAIS AND W0000000000000000000 0000.00.00.00...119 Animals............................................119 Collection of mouse serum..........................119 Isolation of Iznggpsoma cruzi.....................119 Preparation of opsonized.zymosan and opsonized I; cruzi.........................................120 Collection, isolation, and identification of peritoneal macrophages...........................120 Phenol red assay...................................121 Homovanillic acid assay for mo...u...............122 Association of T; cruzi with macrophages...........123 ‘Macrophage viability...............................123 Statistics.........................................124 mm000000 ....... 0000.0...0.0..000.00000.0.000000000.125 vii DIm1m000.0000.000000000 0000000 0 ...... 000000....0000156 W0.000000 00000 0 0000000000 0000000 000000000000000 160 CHAPTER 4: I;_cruzi STIMULATED H30! PRODUCTION: QUANTITATION AND A.POSSIBLE MECHANISM. ........ ...163 W000000000000000000000000.00.000.0.00.00000000000164 W1m0.0000..0.000000.00.000 000000 0 000000 000.0000166 mmmsmm000000000 ........ 0.00.0000....00000169 Animals............................. ........... ....169 Isolation of Irnggpsoma cruzi.....................169 Preparation of opsonized I; cruzi..................169 Collection, isolation, and indentification of peritoneal macrophages........................170 Homovanillic acid assay for 11203...................170 Association of T; cruzi with macrophages...........172 Synthesis of margaric phosphatidylcholine..........172 Fatty acid.composition of macrophage phospholipids.174 Arachidonic acid release by'macrophages............175 3H—arachidonic acid labelled macrophage phospholipids....................................177 Macrophage viability....................... ...... ..177 mm000000000000.0000.000.000.00.0.0000 00000 . 00000 0.0178 DISCIJSSImoooooooooooooooo 0000000 0.0.0.00 00000 0.00.000.0200 REFERENCES.......................... .......... ..........205 CHAPTER 5: FUNCTIONAL CAPACITY OF RESIDENT PERITONEAL MACHDPHAGES m ZINC DEFICIENI‘ MICE.............209 W0000000000.0000000000000...00.0000000000000000.0210 mm.0.000.0000.000....0000..00.....000.00.00000213 MAMIALS Am Woeooooooooooooooo00.000000000000000217 ms00000000.0.0.00..00000...0.00000000000000000217 Diets000000.0000000.00.00.0000.00.00.00.000...00.00217 Zinc mnis. 0 0 0 0 0 0 0 . 0 0 . 0 0 0 0 0 . 0 . 0 0 . . 0 0 0 . 0 0 . 0 0 0 0 0 0 0217 Isolation of Irnggpsoma cruzi.....................218 Preparation of opsonized.zymosan...................218 viii Collection, isolation, and identification of peritoneal macrophages...........................219 Macrophage viability...............................219 Association of I; cruzi with macrophages...........219 Phenol red assay...................................220 HOmovanillic acid assay for H302...................221 Synthesis of margaric phosphatidylcholine..........222 Fatty acid composition of macrophage phospholipids.223 Arachidonic acid release by macrophages............225 3H-arachidonic acid labelled macrophage wOSWOIj-pids00000000000000...0000....00000000000226 StatistiCSooooooooooooooooooooooooooooo 0000000 0.00.227 Mime... 00000 0.000.00.00000.00000.00000.000.00.000000228 DISQ-BSIW...000 0000000 000.00.00.0000..000000000 00000 00.256 mm0000000000.00.00.00.0000000000000 00000 .0.00000262 WWWIWS..0.0 ....... 0.0.0 0000000 0.00.00.0000..268 Amm000000.0.00.0...00.000.000......000.0.0 ..... 00000....276 TABLE LIST OF TABLES PAGE CHAPTER 2 Body Weight, Diet Consunption, Degree of Parakeratosis, Thymus Weights, and Serun Zinc Levels of Mice After 30 Days on Zinc Deficient or Zinc Adequate Diet. . . . .72 Immoglobulin Production/P1313104 CHAPTER3 I130; Production by Resident Peritoneal Phcrophages. . . . . . . 126 CHAPTER4 “lease Of 3H‘81‘80hidonate mmliteSOooooooooooooooooo0195 CHAPTER 5 Incorporation of 3H—arachidonate into Phospholipids. . . . . . 254 Release of 3H—arachidonate metabolites...................255 LIST OF FIGURES Figure Page CHM 1 1 The "oxygen burst" - Mechanisms for production of oxygen metabolites by morophageszz 2 The "oxygen burst" - Mechanisms for activation of mm OXidaseoooooooooooooooooooooooooooooooooooooocoo000026 3 Synthesis of arachidonate from cis-linoleic acid. . . . . . . . . . 30 CHAPTERZ 1 Dose curve and kinetics in a serun free system of the proliferative response to Con A by splenocytes prepared from mice consuning norml laboratory chow................74 2 Dose curve for the proliferative response in a serun free system to Con A by splenocytes prepared from severely zinc deficient, moderately zinc deficient, restricted, and control mice.76 3 IL—2 activity in serun free culture median harvested at 24 hours from triplicate cultures with 2 ug/ml Con A- stimulated splenocytes from severely zinc deficient, moderately zinc deficient, restricted, or control mice. . . .78 4 Kinetics of the MIC proliferative response in 5% FCS Sllpplmtw. Mim00.000.00000.000.00.00.00.000..0.000..081 5 Dose curve for the MIC proliferative response in 5% PCS S‘Jpplmted Mim0000000000000000.00.000000000000000000083 6 Kinetics of the MLC proliferative response in 0.5% zinc deficient autologous serun supplemented medium. . . . . . . . . . . .85 7 Kinetics of the MIC proliferative response in 0. 5% ‘zinc adequate autologous serun supplemented median. . . . . . . . . . . . .87 xi 10 ll 12 13 Dose curve of the MLC proliferative response in 0.5% zinc deficient autologous serum supplemented median. . . . . . . 89 Dose curve of the MLC proliferative response in 0.5% zinc adequate autologous seran supplemented median. . . ..... 91 IL—2 activity in 5% FCS supplemented median harvested at 48 hours from quintriplet cultures of a MLC. . . . . . . . . . . .94 Proliferation of mitomvcin C-treated splenocytes from mice ffinOMI lawmmwChWoooooooooooooooooooo000000097 IL-2 activity in median harvested from mitomycin C- treated splenocytes from mice fed normal laboratory chow. . 99 Percentage of splenocytes with IL—2 receptors. . . . . . . . . . . . 102 CHAPI‘ER3 Standard curve for H30; production in the modified 11181101 m ”say0000000000000000.0000.000000000.00000000.128 Modified phenol red assay: Atmospheric conditions for optiml I-IzOz production by adherent uncrophages from 2.1 x 10‘ resident peritoneal cells.................130 Modified phenol red assay: Requirements for 0.5 nM 03.012 and 2 m NaNa for optimal 1130. production by adherent mcrophages from 2. 1 x 10' resident peritoneal cells.0000000.00.000.0000.0000000...0000.0000000000000000132 Modified phenol red assay: Measurement of total, extracellular, and intracellular H201 produced by adherent macrophages from 2 . 1 x 10‘ resident peritoneal Cell-80000000000000.0.0.0000.0.0.00.0.0.000000000000000000135 Viability of L cruzi after 2 hours in solutions containing phenol red or homovanillic acid, substrates for we”saysmmmm0000.0.00000000000000.000000138 Modified HVA assay: Atmospheric conditions for optiml Hz 02 production by adherent macrophages from 2 x 10‘ resident peritoneal cells...................141 Modified HVA assay: Requirements for 0.09 m 08.01: and 0.05 n1"! MgClz for optimal I130; production by adherent macrophages from 2 x 10" resident peritoneal cells....................................................143 Modified HVA assay: Requirements for 0.09 m CaClz - xii 10 11 12 and 0.05 IIM MgClz for optimal HzOz production by adherent morophages from 2 x 10° resident peritoneal “11800000000000.00000000000....00.0000.00000000000000000145 Modified HVA assay: Requirements for 2 HM NaNc for optimal 1120: production by adherent macrophages from 2 x 10° resident peritoneal cells................... ..... 147 Standard curve for 1130; production in the modified HVA ”say000000000000000.000.000.000000000000000000 000000 149 Modified HVA assay: Measurement of total, extra- cellular, and intracellular H30; produced by adherent mcrophages from 2 x 10‘ resident peritoneal cells. . . . . . .152 Modified HVA assay: I-IzO: production by resident macrophages incubated with L cruzi or opsonized L cruzi at a parasitezmacrophage raio of 5:1, 10:1, or 201154 CHAPI‘ER4 Correlation between I-IaO: production and proportion of morophages associated with L cru21179 Correlation between 1120: production and nunber of L cruzi per 100 macrophages at low levels of L cruzi- mmme”swiation00.000000000000000000000000000000.0181 Correlation between 11.0. production and killing of tryponnstigotes oramastigotes..................... ...... 185 Corrrelation between 1130; production and killing of opsonized tryponnstigotes................................187 Correlation between 1130; production and mmber of I. . cruzi per 100 macrophages at high levels of L cruzi- mmme ”axiation000000000000000000000000000000000.0189 Fatty acid quantities in phospholipids from resident mmmes0000.000.00.00.000.000.00000000.00.00.00...00.193 H202 production by resident morophages stimulated with exogeneous fatty wid800000000000000000000000000000000000198 xiii 10 CHAPTERS Opsonized zymosan-stimulated HzOz production by resident peritoneal macrophages preapred from severely zinc deficient, moderately zinc deficient, restricted, or control mice as measured by the phenol red assay. . . . ..... 229 Arachidonate—stimulated 1130; production by resident peritoneal macrophages prepared from severely zinc deficient, moderately zinc deficient, restricted, or controlmiceasmeasuredbytheHVAassay........... ..... 232 INA-stimulated H30; production by resident peritoneal macrophages prepared from severely zinc deficient, moderately zinc deficient, restricted, or control mice as determined by the phenol red assay234 Experiment 1: L cruzi-stimulated 1120: production by resident peritoneal necrophages prepared from severely zinc deficient, moderately zinc deficient, restricted, or control mice as measured by the HVA assay.............237 Experiment 2: At high levels of L cruzi-merophage association, L cruzi-stimulated 1120: production by resident peritoneal mcrophages prepared from severely zinc deficient, moderately zinc deficient, or control mice as measured by the HVA assay239 Nanber of L cruzi associated with resident peritoneal macrophages prepared from severely zinc deficient, moderately zinc deficient, or control mice. . . . . . . . . . . . . . .241 Proportion of macrophages from severely zinc deficient, moderately zinc deficient, or control mice that were ”axiaMWj-tlhlgcmzi000000000000000000.000.00.000...0244 Amount of H30; produced per L cruzi associated with resident peritoneal macrophages prepared from severely zinc deficient, moderately zinc deficient, or control mice as calculated from figures 6 and 7246 Mole percent of fatty acids in phospholipids from resident peritoneal mcrophages prepared from severely zinc deficient, moderately zinc deficient, restricted, or control mice249 Nanomoles fatty acid per milligram uncrophage protein in phospholipids from resident peritoneal macromages prepared from severely zinc deficient, moderately zinc deficient, restricted, or control mice. . . . . . . . . . . . . . . . . . .251 xiv (8) Con A 0m EDTA (8) HVA IflIh 13th NADPH PGEz LIST OF ABBREVIATIONS aqueous bovine serum albumin concanavalin A counts per minute cytotoxic T—lymphocyte line dihomo-gamma—linolenic acid Dulbecco’s modified minimal essential median delayed type hypersensitivity disodium ethylene-diaminotetracetate fetal calf serum gaseous hydroxyeicosatetraenoic acid homovanillic acid interleukin-2 lipopolysaccharide leukotriene B. leukotriene C4 mixed.lymphocyte culture nicotinamide adenine dinucleotide phosphate phosphate buffered saline plaque foaming cell prostaglandin.E2 phorbol 12-myristate 13-acetate protein purified.derivative radioimmunoassay standard error of the mean superoxide dismutase sheep red.blood cells Irypggpsoma 922g; T—cell dependent antigens T-helper cell T—cell independent - class 1 T-cell independent - class 2 trinitrophenyl conjugate tetraphenylporphyrin INTIDDUCTION Nutitional deficiencies compromise the inane systan (1). Defi- ciencies in vitamins (2), protein or amino acids (3), essential fatty acids (4) or trace elements (5) rapidly alter inlnune function causing an increase in smceptibility to infections (7, 8). A deficiency of the trace element zinc is a prevalent halan nutritional problem throughout the world, including the USA (7 , 9, 10, 11). Severe cases of zinc deficiency most often occur in de- veloping nations where poor diets, diseases, atd omortunities for infection are prevalent. In both zinc deficient halnns atd animls, rapid std severe depressions occur in antibocw atd cell mediated imme responses (12-19). These reductions in inlune function may be due to a reduction in cell nanbers std/or functional capacity of the cells or subsets of lymphoid cells. There is a drastic reduction (50%) in absolute nanbers of lymthocytes in the blood, thymus, std spleen of the mouse, yet, the relative percentage of T- atd B—cells (types of lymphocytes) remains antranged at least in the spleen (12). The total mnber of morophages in the blood atd spleen is also reduced (50%) (20). 'lhese reductions in total cell nanbers, at least in part, explain the reduced inlnune responses of the zinc deficient animl. Maven the reduction in imme function my also be due to a decrease in functional capacity of the residual lymphocytes. It is possible that cellular functions are compromised since many enzymes inclaiing the RNA polymerases are zinc dependent 2 (7). In the past, the functional capacity of residual T—cells, B- cells, atd macrophages has not been carefully characterized while regulating the presence of the deficient nutrient i_n m in either zinc deficient or other nutritionally deficient animl models. To evaluate the functional capacity of T-cells, we emined proliferation atd production of interleukin-2 (IL-2, a hormone-like protein secreted by T—cells) by T—cells from zinc deficient mice in response to Con- canavalin A (a mitogen) or allogeneic cells. We also examined the acquisition of IL-2 receptors by these T-cells upon stimulation with allogeneic cells. 'Ihe functional capacity of residual B—cells was determined by measuring the average smart of IgM ard IgG secreted per antibody producing cell during a primary response to sheep red blood cells. In previous i_n vitro studies, the availability of large amounts of zinc present in fetal calf seran (350 ug Zn/dl) had not been cmsidered as a source of zinc that might facilitate repair or improvement in function of lymphocytes from zinc deficient animals or patients. To greatly reduce exogenous zinc in our studies, we utilized autologous seran frun zinc deficient mice (50 ug Zn/dl) or a seran free. systan. 'Ihe data in chapter 2 shows that T-cells std B-cells fran zinc deficient mice have norml or elevated functional capacities regardless of whether zinc was readily available or limited in the culture system. In contrast to norml functioning of residual T-cells atd B- cells, resident peritoneal mcrophages from zinc deficient mice have a reduced functional capacity (21, 22). Phcrophages, which are impor- tant as one of the first lines of host defense against infections, 3 are not able to associate with or destroy the parasite W gguz__i_ (L guz_i) i_n Mpg. 'Ihus, susceptibility to some pathogens is increased in zinc deficient mice. A low zinc diet resulted in increased infections among normally resistant mice to Catdida albicatns (23) , and zinc deficient mice have a reduced ability to combat L as; infections (21, 22, 23). L M infected zinc deficient mice had a 50 fold increase in blood parasitemias atd an increased mortality rate as compared to infected zinc adequate mice or nonin- fected zinc deficient mice (24). The increased smceptibility to L gr_'u_z_i infections is probably, in part, due to the inability of uncro- phages to associate with or destroy L gm__z_i_. 'lhis is especially interesting since these defects of the macrophage function were reversed by a short period of incubation with zinc at five times physiological concentrations (21, 22) . Several other metals at similar concentrations did not restore the ability of zinc deficient macrophages to combat L M (21, 22). Since destruction of L mi has been correlated with 11:02 prochlction by moroflnages (25, 26), we examined the ability of zinc deficient resident morofinages to produce 11:0: . Usually, IhOn produc- tion is measntred using artificial stimlants like phorbol wristate acetate (PMA, a chemical) or opsonized zyunosan (an antibody coated yeast cell extract) with. activated rather than resident macrophages. Activated macrophages are normally used to assay for 1130; because the levels of 1120: produced by resident moroflnages were previously too low to detect. Since it was necessary to use resident macrophages from zinc deficient mice to measure 1120: production, the assay for 4 1130: production was improved. Modifications to accomplish this goal are presented in Chapter 3. Using this assay, 1130; production by resident moroflnages from zinc deficient and zitnc adequate mice was measured after stimulation with PMA, opsonized zymosan, or the secord messenger in opsonized zymosan-stimulated 1130: production, arachi- donate (Chapter 5). To minimize the possibility of restoration of macrophage functions i_t_n m, all studies on resident macrophages from zinc deficient mice were done using bovine seran albunin (0.4 I13 211/ g albanin) or a seran free system. Further, since PMA and opsonized zymosan were known to activate 1130; production by different mechanism, it is possible that L M used yet another route for stinmlation of 1130: production. 'Iherefore, it was also necessary to use L gn__n_z_i as the stimulant when measuring 11:02 production by zinc deficient atnd zinc adequate resident macrophages. However, stimulation of Hao. production by resident nacrofinages with L gr_'n_.nz_i_ posed yet another series of problms. L M were destroyed by the substrate (phenol red) connonly used to assess 3.0. production. 'lhe corncentration of this substrate could not be reduced sitnce it would then become limiting in the reactions. 'lherefore, an alternative substrate which did not destroy L cruzi was fourd, and the assay for 11:03 was again altered atd improved for use with resi- dent morophages with L M as the stimulant (Chapter 3). 'lhis assay represents a valuable new procedure that will enable inves- tigators to evaluate I-IcOn production by resident morophages using natural pathogens such as L cruzi (Chapter 4); heretofore, this was 5 not possible. L M stimulated 3:0: production by zinc deficient std zinc adequate resident macrophages was measured (Chapter 5) . L m; enters the macrophage by active penetration of the macrophage membrane or by phagocytosis. 'Ihe mechanism(s) for L _C_131__Zi penetration or phagocytosis is unknown. Hmaever, it has been shown that fluidity of the macrophage plasma membrane atd phagocytosis by macrophages is depressed by an increase in the plasnn membrane in the proportion of fatty acids with shorter chain length atd more saturation than arachidonate (27, 28) . 'Ihe percentage of these shorter-chain , more-saturated fatty acids have been shown to be elevated in livers of zinc deficient mice (29-35). Itdeed, defi- ciencies in essential fatty acids std zinc share some similar charac- teristics such as immune disorders, parakeratosis, and diarrhea (36, 37) . Perhaps macrophages from zinc deficient mice have an altered fatty acid composition which would reduce membrane fluidity and phagocytosis std therefore decrease the association of L 9_ru_z_i. Fatty acids such as arachidonic, linolenic, linoleic, and stearic acid added exogenously have also been shown to stimulate production of oxygen metabolites by morophages, and as chain length decreased or saturation irncreased these fatty acids were less stimulatory for production of the oxygen metabolites (38, 39). 'lhe mechanism for L g_m_zi_ stinllated mo. production is unknown. Honeever, L. Q_1f_|_lzj._ does stimulate the release of leukotrienes (40) which are lipoxygemse metabolites of the fatty acid, arachidonate. In addition, fatty acids are intermediates in stimulation of HaOn production by opson- ized zymosan (41) std my also be involved in L cruzi stimulated 6 Bio. production. Alterations in fatty acid composition of zirnc deficient macrophages could canme a reduction in both the nanber of L 9M associated with the macrophage std the degree of stimulation of H30; production. 'lherefore, the fatty acid composition of phospho- lipids from zinc deficient std zirnc adequate resident macrophages was determined (Chapter 5) . In addition, there is the possibility that release of fatty acids upon stimulation of the mcrophage by L c_ruzi_ my be a zinc dependent process. (he process for liberation of fatty acids from phospholipids is known to be zinc dependent. Phospholipase C, which in conjunction with diacylglycerol lipase liberates fatty acids from rhospholipids , requires zinc for activity (42). Perhaps, in the mcrophages from zinc deficient mice, rhospholipase C lacks adequate amounts of zinc std thus has reduced activity. To examine this possibility, L 9&2; stimulated release of rsdiolabelled arachidonic acid std it’s metabo- lites from zinc deficient std zinc adequate uncrophages was mneasured while carefully regulating zinc levels i_n vi__tro (Chapter 5). In samry, staiies were performed to detemine the ability of T-cells from zinc deficient mice to proliferate and produce inter- leukin-2 atnd interleukin-2 receptors, the ability of B-cells from deficient mice to produce antibodies, std the ability of zinc defi- cient moropinages to produce mo. std release fatty acids. Phorophage fatty acid composition was also examined. In contrast to previous studies, the level of zinc introduced i_n thg was carefully regu- lated. Thus, real std not artifactail functional capacities of leukocytes from zinc deficient and zinc adeqmte mice were studied. 3. 8. 10. 11. 12. 13. References Chandra, R. (1972) J. Pediatr. 81, 1184. Hodges, R., Bean, W., anlson, M., std Bieler, R. (1962) Am. J. Clin. Nutr. _1_l, 187. Gershoff, s., Gill, 'r., Simonisr, s., and Steinberg, A. (1968) J. Nutr. g, 184. Schaedler, R. std Dubos, O. (1956) J. Exp. Med. Log, 64. DeWille, J., Fraker, P., and Roneos, D. (1979) J. Nutr. 1Q, 1018. Prasad, A. S., bebani, P., Abbssii, A., Bowersox, E., std Fox, M. (1979) Ann. Int. Med. §_9_, 483. Prasad, A. S. (1979) Ann. Rev. Phamcol. Toxicol. 10, 393. Gordon, J., Jansen, A., std Asoli, W. (1965) F. mdiat. 66, 679. ' Hambridge, K. N., Walrsvens, 9., Brown, R., Webster, 3., White, M., Anthony, M., ed Roth, M. (1976) Amer. J. Clin. Nutr. Q, 734. Ssndstead, H. (1973) Amer. J. Clin. Nutr. 26, 1251. Satdstead, H., Henriksen, L., Gregor, J., Prasad, A., and Good, R. (1982) Amer. J. Clin. Nutr. 36, 1046. Fraker, P. J., DePasqnele-Jardieu, P., std Cook, J. (1988) Arch. Derm. (in press). Fraker, P. J., Haas, S. M., and Luecke, R. W. (1977) J. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Nutr. _1_0_Z, 1889. Luecke, R. W., Simonel, C., std Fraker, P. J. (1978) J. Nutr. 198, 881. Fraker, P. J. (1983) Survey Imlnutnol. Res. _2_, 155. Zwickl, C. M., and Fraker, P. J. (1980) Immmol. Commrn. _9_, 611. Fraker, P. J., Hildebrsndt, E., and Luecke, R. W. (1984) J. Nutr. ;1_4_, 170. Fraker, P. J., Zwickl, C. M., and Luecke, R. W. (1982) J. Nutr. L2, 309. Fernatdes, C., Nair, M., Once, E., Tanks, '1‘., Floyd, R., std Good, R. A. (1979) Proc. Natl. Acad. Sci. U.S.A. E, 457. Wirth, J. J., Fraker, P. J., stdKierszenbanln, F. (1984) J. Nutr. 1_1_4, 1826. Wirth, J. J., Fraker, P. J., std Kierszenbaal, F. (mnuscript in preparation). Fraker, P. J., Jardieu, P., and Wirth, J. (1986) In "Nutritional Diseases: Research Directions in Comparative Pathobiology" pp 197-213. Alan R. Liss, Inc. Salvin, S. B., and Ihbin, D. S. (1984) Cell. Imnel. 81, 546. Fraker, P. J., Caruso, R., std Kierszenbaan, F. (1982) J. Nutr. 13, 1224. Villalta, F., std Kierszenbaan, F. (1983) J. Imml.‘_1_3l, 1504. Villalta, F., and Kieszenbaan, F. (1984) J. Immol. L33, 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 3338. Phhoney, E. M., Hamill, A. L., Scott, W. A., std Cohn, Z. A. (1977) Proc. Natl. Acad. Sci. U.S.A. fl, 4895. l‘hhoney, E. M., Scott, W. A., Lstndsberger, F. R., Hamill, A. L., std Cohn, Z. A. (1980) J. Biol. Chem. 2_5_5_, 4910. Huang, Y. 8., Game, 8. C., Horrobin, D. F., std Davignon, J. (1982) Atherosclerosis 1L1, 193. Cutntnstne, S. C., Horrobin, D. F., std Phnku, M. S. (1984) Proc. Soc. Exp. Biol. Med. _lfl, 441. Curntnstne, S. C., std Horrobin, D. F. (1985) J. Nutr. _1_;§, 500. Bettger, W. J., Reeves, P. C., Moscatelli, E. A., Reynolds, 0., std O'Dell, B. L. (1979) J. Nutr. _198, 480. Clern, 8., Castro-We, M., Collipp, P. J., Jonas, E., std Phddaish, V. T. (1982) Lipids 11, 129. Field, H. P., std Kelleher, J. (1983) Proc. Nutr. Soc. g, 54A. Tsai, S. L., Craig-Schnidt, M. C., Weete, J. D., std Keith, R. E. (1983) Fed. Proc. _4_2, 823 (abs. 3110). Horrobin, D. F., and Cunnsne, S. C. (1980) Med. Hypothesis 8, 277. Cunnsne, S. C., Huang, Y. S., Horrobin, D. F., std Davignon, J. (1981) Prog. Lipid Res. 89, 157. Bromberg, Y., std Pick, E. (1984) Cell. Immol. 88, 213. Kskinnme, K. (1974) Biochim. Biophys. Acta m, 76. Wirth J. J., and Kierszenbann, F. (1985) J. Immol. 134, 1989 . 41. 42. 10 Wirth, J. J., Kierszenbaan, F., (1985) Mol. Biochem. Parasitol. _8_7, 546. Phridonneau-Parini, I., and Tsuber, A. I. (1986) Clinical Research fl, 661A. Chapter 1 LITEATUREREVIEW 11 12 The purpose of this research was to analyze the functional capa- city of leukocytes from zinc deficient mice. T-cell proliferation was analyzed as well as two processes important in stimulating the proliferation, IL—2 production and IL-2 receptor proulction by T cells. The ability of B-oells to produce antibodies was measured on a per cell basis. Phorophsges, which are important as one of the first lines of defense against pathogens, was also analyzed. Mea- surements of macrophage prochlction of toxic oxygen metabolites, release of fatty acids, and macrophage fatty acid composition were performed. Therefore, this review will provide the reader with information concerning zinc deficiency std it’ 8 effects on the immune system. Also, the possible roles for zinc in the phagocytosis std destruction of pathogens by mecrofinsges will be discussed. 1. Zinc Deficiency A.) Causes of Zinc Deficiency. A suboptimal intake of zinc in the hnmsn diet is not uncommon (1, 2, 3, 4). Severezinc deficiencies are most prevalent in uderdeveloped nations since people from these countries consane large quantities of unleavened grain products containing phytate, which chelates zinc making it unavailable for absorption from the intestine (1). Deficiencies in zinc are not confined to the uderdeveloped nations; in fact, it has also been observed in middle std upper income families of the United States (2, 3). 'lhese deficiencies were probably caused by imnproper eating l3 habits resulting in imbalanced diets. In addition, a study of chil- dren from low income families in the Denver area revealed that several children were zinc deficient; they had poor taste acuity, slow growth std low hair zirnc levels (2). Upon supplementation with zinc, these conditions were alleviated (5). It should be noted that children and pregnant women (6) who my have an increased nutrient require- ment for zinc are at a greater risk of becoming deficient. A genetic disorder called Acrodermetitis enteromthica also results in a deficiency in zinc (1). Acrodemtitis enteropathica is an autosomel recessively inherited trait which redress the in- dividuals capacity to absorb zinc from the diet. 'lhese patients have a severe dermtitis, reduced plane zinc levels, reduced cellular immunity, std irncreased susceptibility to infections (7, 8), comon characteristics accomanitng zinc deficiency. Upon zinc therapy, these symptome are alleviated (7, 8). A similar disorder occurs in Freisisn cattle (9). It has been suggested that the reduced absorp- tion of zinc is due to lack of a zinc birding protein in the intestine of patients with Acrodemtitis (10). In addition to improper diets or melsbsorbtion of zinc, several disease states my result in zinc deficiency. With catabolism of bodymess, therewillbeloss of zitncsswellssshigh zinc require- ment for repair. Illnesses resulting in zitnc deficiency itnclude chronic renal disease, burns or psoriasis, malignancies, parasitic infections, diabetes, cirrhosis of the liver, alcoholism, std collagen disease (11). Drug treatments can also cause depletion of zinc; these include sntimetabolites, antisnabolic agents, penacillsmine 14 therapy, prolonged intraveneous therapy with nutrient solutions containing insufficient amounts of zinc, std oral contraceptives (11). B.) Alterations in the immune sy_s_tem Q: zinc demivstion. Hnmn studies show that zirnc deficiency alters T-cell std B-cell responses. A female child with Acrodermtitis enterogthica ms foud to have mnstny alterations in her immune system (12). Analysis of her peripheral lymphocytes showed that there was a decrease in the nanber of B- std T—cells. In addition, the lymphocytic response per 10‘ cells to mitogens specific for T—cells or B—cells (Con A, PHA, PM!) was reduced. Delayed-type-hypersensitivity responses were also reduced. She also had hypogsnleglobulemia, a characteristic of Acrodermatitis. Upon zinc therapy, all responses returned to normal levels. Animal studies have also shown that cellular std antibab medi- ated responses are affected by zinc deprivation i__n_ 35112. The T.- cell depedent antibody mediated response per spleen to sheep red blood cells (SR3?) is 10% of controls (13), but upon reconstitution of the zinc deficient mice with thymocytes, the response increases to 61% of controls (13). Therefore, the response may be due to reduced nanbers std/or impairment of B-cells std/or T-cells. The reduced antibody mediated response to SR3: is partly the result of reduced nanbers of splenocytes since the response per 10' splenocytes is normal (14). However, the ability of the residnnl B—cells to produce antibodies was unknown. Chapter 2 examines the average 15 amount of antibody production per antibody producing cell from zinc deficient and zinc sufficient mice. There is also loss of some '1‘- cell functions including cytotoxic T-cell responses against allogeneic tunor cells and decreased delayed-type-hypersmsitivity (DI'H) respon- ses (inflammatory responses mediated by Tp-cells and nacrofliages) to dinitrofluorobenzene (15, 16). The altered T—cell mediated responses seen with zinc deprivation may be due to impairment of usual developent of T-cells in the thyuus or to impairment of the functions of more mtme T-cells. The functional capacity of the residial T—cells had not been examined. Chapter 2 discusses the ability of T-cells to proliferate and produce interleukin-2 and interletkin-Z receptors . T-cell develoment seam to be altered. Nash gt. al_. (17) found an increase in nanber of imture T-cells in spleens of zinc deficient mice as determined by autologous rosette formtion. Impaired developnent of T—cells my be a result of alterations in the thynn. In the zinc deficimt animal the thy” is severely atrofiiied with preferential loss of the thymic cortex, the location of inature thymocytes (18). The thymic atrophy is caused, in part, by the high corticosterone levels found in the zinc deficient animls (19). Adrenalectanized zinc deficient mice are protected fra- thymic atromy (20); yet, there is only marginal (20%) improvement in antibody mediated responses per spleen to Slim, a T-cell dependent antigen. In addition, with in- creased severity of the zinc deficient condition, in both the adrenal- ectomized and nonadrenalectomized mice, the T—cell dependent response to SR3) continues to decrease on a per spleen basis (21). Therefore, 16 thymic atrophy (me to high corticosterone levels is not the only factor contributing to impairment of the response to 81280 by zinc deficiency. Not only is the thymus atrophied but several thymic hormones are altered with zinc deficiency. These hormones may play a role in development of T-cells in the thymus (22, 23). Iwata (22) ard Chardra (23) observed that zinc deficient mice had redmed ac- tivity of thymulin, a zinc dependent hormone (24). Another thymic hormone, thymopoietin was fomd to be present in reduced levels in the serun of zinc deficient patients (25). A reduction in these hormones may cause improper differentiation std mturation of T- cells in the thymus ard peripheral lymphoid organs of zinc deficient irdividuals . Improper development of the T—cell could at least in part explain altered T-cell responses upon zinc deprivation. B—cell development may also by halted at an intermediate stage; that is, the developnent of the immature B-cell in the bone narrow to the nature peripheral resting B-cell may be impaired. Ability to respond to certain polyclonal B-cell mitogens and/or antigens arise at different stages during ontogeny. Mitogens which stimulate B-cells include dextran sulfate, lipopolyssccharide (LPS) ard protein purified derivative (PPD) . Dextran sulfate stimulates immature B-cells (26- 28) . LPS stimlates B-cells in intermediate stages of development, while PPD stimrlates more mature resting B—cells (26-29) . It has been demonstrated that when equal numbers of lymphocytes from! zinc deficient mice are stimulated with dextran sulfate or LPS, the re- sponse is twice that of controls (14, 30). However, when cells are stimulated with PPD there is not a significant difference from the Hifl . ..M. M... «M 17 controls (14) . T-cell itdeperdent antigenic responses by immature B—cells were also elevated. Responsiveness to T—cell itdependent (TI) antigens develops at different stages of B-cell ontogeny. Trinitro— phenyl conjmated LPS ('I'NP-LPS) , a "TI-1" antigen, stimulates immature B—cells; whereas, TNP-Ficoll, a "TI—2" antigen, stimulates B—cells in intermediate stages of development (31). Again, the _it_1 m mitogenic response of splenocytes of zinc deficient mice to either of these TI-sntigens is elevated (14, 30). The increased B—cell responses to antigens or mitogens which stimulate relatively imm- ture B—cells suggests that resting B-cells from zinc deficient mice might be halted in some intermediate stage of development. Although B—cell std T-cell subsets appear to be differentially affected by zinc deprivation (14), the relative percentage of B- std T—cells in the spleen does not change in zinc deficient versus zinc adequate mice (32). However, the total number of lymrhocytes in the spleens of zinc deficient mice is reduced (14) (50% of controls). 'lhe total umber of mononuclear phagocytes, magocytic cells of the immune system, is also reduced (50%) in the spleens of zinc deficient mice (33). Men together, the reduced cell atdsntibody mediated responsesareinpartduetoreducedcellnunber. Anadditional explanation could be reduction in the ability of the residual leuko- cytes to carry out specific functions especially since about 200 zinc metalloenzymes have been described (1) . The zinc depetdenoy of these enzymes has ustally been identified by loss of enzyme activity due to renoval of zinc by chelation (34). Amoung the zinc dependent enzymes are thymidine kinase, DNA deperdait INA polymerase, std .5. x We. BCI‘. sf, 18 termiral deoxynucleotidal transferase. Loss of enzyme activity due to loss of zinc could explain several alterations in cellular func- tions of zinc deficient animals. However, in a zinc deficient state, loss of enzyme activity due to loss of zinc has not been demonstrated. In fact, Luecke g. _a_l_. has shown that in the zinc deficient weanl- ing pig the activity per cell of several zinc depetdent enzymes remains normal; yet, the total amount of the enzyme in the tissue is usually reduced due to atrophy of the organ (35). If there is altered enzyme activity, one would expect to fitd altered functional capaci- ties. Therefore, to determine if zinc regulates the functional capacity of B-cells and T-cells, proliferation std cytokine production by residual lymphocytes frcn zinc deficient mice was measured in Chapter 2. It has been shown that some functional capacities of peritoneal macrofiiages from zinc deficient mice are aberrant. This will be discussed in detail in section I C. Although the total number of peritoneal macrophages from deficient mice is normal (33) std they are able to magocytose norml numbers of latex beads (33), they have a redieed capacity to ingest std destroy pathogens (36, 37). The inability of the zinc deficient macrophages to destroy L c_ng; was further studied in Chapter 5. In salary, zinc deficiency alters lymfliocytic responses in both animals std hmns. B-cells my be arrested in intermediate stages of maturation. T—cell mediated responses are decreased although thymic atrophy was of only minor importance in T-cell impairment. However, the reduced levels of thymic hormones in zinc deficient LL 19 itdividuals my also arrest development of T-cells . Macrophages are unable to associate std destroy the pathogen '_l_‘_._ c_ryz_i; however, they are able to associate with inert particles. The reduced immune responsesareinpartduetoreducedcellnumberandmyalsobedue to reduced functional capacity of the residual leukocytes. C.) Resistance to infection. Zinc deficient mice are more suscep- tible to pathogens than their zinc adeqnate counterparts . Salvin and Robin (38) demonstrated that a low zinc diet increased the infec- tion of normally resistant mice to Catdida albicans. Also, Fraker gt. $1,. (39) showed that zinc deficient mice have a reduced ability to combat Mom 9:22;, an obligate intracellular parasite which causes Chagas’ disease. This was demonstrated by a 50 fold increase in blood parasitenias in zinc deficient mice compared to controls (39) . In addition, zinc deficiency and infection with T‘_. gang had a synergistic effect on the death rate (39) . Twenty-two days after infection, 80% of the infected zinc deficient mice died, whereas there were no deaths among the infected zinc adeqnate mice or the uninfected zinc deficient mice (39) . The inability of the zitnc deficient mice to protect against infection by I. c_ru_zi is mnost likely due, in part, to the inability of the resident moroflnages from the zinc deficient mice to phagocy- tose std destroy the parasite. I_n_ vitro the percentage of mcrophsges associated (a term including membrane boutd std phagocytosed para- sites) with I. M std the number of _T‘_. c_nru_zi_ per 100 morophsges 20 was substantially reduced in the zinc deficient groups at time zero after infection (36, 37) . After 24 hours, mcrofinages from the zinc deficient groups were not able to destroy as mny parasites as mero- phages from the zinc adequate std the pair-fed zinc adequate groups (36, 37). That is, at 24 hours as compared to zero hours, there was a reduction (20-50%) in the percentage of mcrophages associated with I. M std the number of I. 91112; per 100 macrophages in the zinc adequate groups but not the zinc deficient groups. This defect was particularly interesting because it was readily reversed by i_n vi_t_ro addition of zinc. Before infection, a 1 hour treatment of the moroflnsges with long ZnCh /ml (5 times physiologi- cal senm concentrations of zinc) repaired the ability of the zinc deficient groups to associate with std destroy the Z. M (36, 37 ) . The pretreatment with zinc did not affect the ability of the macrophages from zinc adeqnate mice to associate with std destroy the I. c_rnfl (36 , 37 ). This ability of ZnCla to reverse the defects of zinc deficient morophsges was specific in that pretreatment _in $313 with several other trace metals (Cnfila std NiSO.) at five times physiological concentrations did not significantly reverse the defect of the zinc deficient moroflnages to associate with and destroy the Iarasite (36, 37). Pretreatment with MnCla had a slight restora- tive affect on the ability of the zinc deficient morophages to associate°with the z. cruzi (36, 37). In emery, zinc my play some role in the association with and destruction of 1‘. cruzi by resident macrophages. Thus, zinc deficient macrophages provided an excellent 21 opportunity to investigate possible zinc dependent mechanisms in the association with std destruction of L cruzi (see chapter 5). II. Possible Roles for Zinc in the Destruction of Pathogens by Macrophages . Since macrophages from zitnc deficient mice are utnable to destroy the pathogen L cruzi (36, 37 ) std L cruzi are destroyed by toxic oxygen metabolites produced by macrophages (40) , perhaps zinc par- ticipates in the "oxygen burst", the consumption of oxygen and produc- tion of toxic oxygen metabolites by leukocytes. Other metals such as iron and copper (41) are known to participate in the "oxygen burst" . However, heretofore, the importatnce of zinc in the "oxygen burst" has gone unrecognized. In this section, possible roles for zinc in the production of toxic oxygen metabolites and the impaired destruction of pathogens by mcroflnagas from zinc deficient snimls will be discussed. Also, since mcrophsges from zinc deficient mice have reduced ability to associate with L 3%, there will be a discussion on possible roles for zinc in macrophage-L c_rliz__i associa- tion including zinc-membrane interactions std zinc in fatty acid production for membrane phospholipids. A.) Production of Toxic 0_xygen Metabolites by Mononuclear Mo- cyte_sr: Pogible Roles for Zinc. The "oxygen burst" (Figure 1) can be activated by fincrbol myristate acetate (PMA) (42, 43) , a slall molecule which can diffuse into the cell. The "htrst" can also be 22 Figure 1. The "oxygen burst" - Mechanism for production of oxygen metabolites by morofinsges. 23 cytoplasm H202 2H' M ZNADPH Glucose-64’ ., . . a 2 (Home monophoophoto shunt) 202 5 @H . ' 2NADP" Glucooo / oupotorido dismutoso 2(Cu, Zn) / o2 phogoc tosis H (cs3) ' y / 202 Zalutoflnlono ZNADP+ hoxooo monoph H odolooo j shunt | 2° os-so emopu Glucose-G-phoophoto 6, ”2° Hobot Wolu Roocflon acid hydrolosos phogolysosome 24 activated by stimulants which are phagocytosed via specific receptors such as opsonized zymosan (42) , an antibody coated yeast cell extract, or L Eng; (44, chapter 3 std 4), a pathogen. These agents eventual- ly promote activation of NADPH oxidase, an enzyme located in the plasla membrane, which, in turn, catalyzes the production of super- oxide (03') from molecular oxygen in the extracellular environment or phagosome/phagolysosome. This reaction requires two NADPH mole- cules which are provided by the hexose monophosphate shunt in the cytoplasm. The Or , then, either nonenmatically diemutates to HmOm or superoxide dismutase in the cytoplasm catalyzes the dismutstion. AsOr ammo. areproduced, theycsndiffneethroughtheplssm membrane. Extracellular/phagosome/phagolysosome Or and 3.0. are used to destroy pathogens while cytoplasmic On‘ std 11303 are scaven- ged by superoxide dismutsse std catalase , respectively, to protect the mcrorinage. Glutathione peroxidase also scavenges 8.0. while oxidizing glutathione. Within the thagosome/phagolysosome, HxOn reactswithOn‘ toproduceOn, Gl- atd-(li. TheOn‘, 3:02, andoai are toxic to various pathogens. Phagolysosomes also contain lysosoml enzymes . Myeloperoxidsse which catalzyes the production of highly reactive hypohalous acids from Hzon is present in finsgolysosomes of mnonocytes, immature mono- nuclear finagocytes of the peripheral blood, std polymorphonuclear leukocytes but not moroflnages (45) . Therefore , the mture macro- Iinage is unable to produce hypohalous acids. Thus, we will not be concerned with the possible effects of zinc deficiency on these 25 processes since all experiments reported herein were done with mture macrophages. Mechanism of the "oxygen burst" will now be described while focussing on possible roles for zinc in these processes. The mecha- nisms of stimulation of NADPH oxidase by MA std opsonized zymosan are known (Figure 2). PMA stimulates NADPH oxidase by birding to protein kinase C (46, 47) which, in turn, linosflnorylates std thus activates NADPH oxidase (48-53). On the other hand, opsonized zymnosan stimu- lates NADPH oxidase via arachidonic acid released from phosflnolipids (20:4) (48, 49, 54, 55, 56, 57). The following is the sequence of events most likely to occur upon stimulation of macrophages by op- sonized zymnosan. The opsonized zymnossn birds to receptors on moro- phages for the Fc portion of the antibody. Suzuki et. a1. (58) showed that a FoliZb binding protein of mcrophsge membranes has phosrinolipase 1h activity sirnce it specifically cleaves fatty acids from the C2 position of the glycerol backbone of phosphatidylcholine. The activity of this protein irncreased four fold upon birding of IgGZb (58) . Therefore, upon birding of opsonized zymosan to FcXZb receptors, the rhospholipsse A. portion of the receptor could liberate 20:4 fral phospholipids in the morophage plasma membrane. A con- siderable amount of the fatty acid (25%) in morophsge phosrinolipids is 20:4 (59). Once 20:4 is liberated from the plasma membrane phos- pholipid, it my then stimulate NADPH oxidase. It has been shown that 20:4 std lipoxygenase metabolites of 20:4 such as leukotrienes, 15-HETE or 15- , stimulate NADPH oxidase std that this stimula- tion is not inhibited by cyclo-oxygenase or liPOXYIenase inhibitors Figure 2. 26 The "oxygen burst" - Mechani- for activation of NADPH oxidase . arm-c mm? mm 27 phorbol Stimulation myristato acetate 202 Stimulation 20; - aotivit ZNADP" : ' PMA- Protein Kinaso c Protein Kinase C cyclo- oxygenaso ZNADPH P5‘52 2mm?“ phoopnolipaoo A2 I phosphoiipid phosphoiipaso C Zn L diacylglycerol 20,4(J lipase Liipoxygonases Iou kottionos NADPH oxidaso7 .1 active NADPH oxidase 202 20:; 28 (48, 54, 55, 56, 57, 60). In contrast, 20:4 metabolites of the cyclo-oxygenase pathway, the prostaglandins, are not stimulatory for NADPH oxidase (48, 54). Thus, upon binding of opsonized.zymossn to Fc&2b receptors, phospholipase A; activity of the receptor liberates 20:4 from membrane phospholipids std 20:4 or a lipoxygensse (but not a cyclo-oxygenase) metabolite of 20:4 stimulates NADPH oxidase for the production of superoxide. Resident peritoneal macrophages have three phospholipase ac- tivities (61-65): a phospholipase A; active at pH 4.5, a Gait-depen- dent phospholipase A; active at pH 8.5, and a phosphatidylinositol- specific phospholipase C. The particular phospholipases involved in the degredation of phospholipids differs with different stimulants. Zymosan stimulates degradation via both phospholipase A and phos- ' pholipsse C whereas INA stimulates deacylation of phosphstidylino- sitol via phospholipase A (64) . The FHA stimlsted release of 20:4 does not activate NADHH oxidase (Figure 2) since, with this stimu- lant, the 20:4 is converted to prostsglsndins (66, 67) which are not stimulatory for NADPH oxidase (48, 54). 20:4 released.by phospholi- pase A.snd.the lipoxygenase metabolites are stimulatory for the "oxygen.burst” (48, 54, 55, 56, 57). FUrther evidence for the in- volvement of phospholipase C in the stimulation of the "oxygen burst" includes an increase in.On uptake snd.hexose monophosphste shunt activity and a 41% decrease in phospholipid content as determined by lipidébound phosphate content upon addition of phospholipase C to polymorphonuclear leukocytes (68) . Phospholipase C stimulation of 29 the "oxygen burst" prdbably involves diacylglycerol lipase catalyzed release of 20:4. There are several possible roles for zinc in the stimulation of NADPH oxidase by 20:4. Zinc may be important for the release of 20:4 from phospholipids, since phospholipase C, a zinc dependent enzyme (69, 70), in conjunction with diacylglycerol lipase can liber- ate 20:4 from phospholipids (Figure 2) (70). Phospholipase c has two zinc atoms in the active site (70). Both of which are required for activity (70). Removal of even one zinc atom reduces the en- zymatic cleavage of phosphatidylcholine to 3-11%iof control and removal of the second zinc atom reduces the activity to less than 1% (70). These zinc atoms are tightly bound to the active site (70). In fact, EDTA.does not seem to remove zinc from.the enzyme since addition of EDTA has no effect on enzyme activity (70). Zinc has also been implicated in the regulation of phospholipase A; activity (71-73). Phospholipase A3 liberates fatty acids from.the CZ posi- tion of the phospholipid.glycerol backbone. Manku et. al. (72) have suggested.that in the presence of physiological levels of zinc, dihomno-gsmIIa-linolenic acid (DHGL) is mobilized from plasma menbrsne phospholipids of the rat superior mesenteric vascular bed. DHCL is the immediate precursor to either 20:4 (Figure 3) or prostsglsndins of the 1 series. If DHGL is converted to 20:4, mdbilization of DHGL could.play a significant role in stimulating the oxygen burst via 20:4. Zinc mObilizstion of DHGL for prostaglandin synthesis would not be stimulatory for NADPH oxidase since prostaglandins do not stimulate NADPH oxidase. waever, inhibition of prostsglsndin 30 Figure 3. Synthesis of arachidonate fret cis-linoleic acid. 31 cis - linoleic acid' NA DP” Zn delta - 6 - desaturase NADPH gamma - linoleic acid dihomogommo linoleic acid delta -5 - desaiurase arachidonic acid (20=4) 32 synthesis my allow Dim. or its metabolite, 20:4, to stimulate NADPH oxidase. This is a possibility since addition of 2m zinc i_n v_it_ro has been shown to inhibit prostaglandin synthesis by polymorphonuclear leukocytes (73). In addition, perhaps DHGL itself could stimulate NADPH oxidase since shorter chain fatty acids can, albeit to a lesser extent, stimulate NADPH oxidase (48, 54). In contrast to the phos- pholipase A: activity in the presence of zinc reported by Phnku et. al. (72), Wells (74) has reported that zirnc inhibits the calcium depedent activity of phosflnolipsse As from Crotalus adsmnteus (snake) venom by birding to the active site std itducing confomtion- a1 changes in the enzyme. Inhibition of phospholipase A; activity by addition of zinc has also been reported by others (75, 76). In sumsry, zirnc my be important in the release of 20:4 from phospho- lipids via rinospholipsse C - diacylglycerol activity or some phos- finolipase A: like activity. Zinc my also be important in the stabilization of 20:4 against oxidation std enhatnce the probability of stimulation of NADPH oxidase by 20:4. It is known that zinc or iron can complex with 20:4 std oxygen (77, 78). Iron catalyzes the oxidation of 20:4 whereas zinc does not (78). 20:4 + o. + Fe“ ) [20:4, 02, Fez’kolplor > lipid peroxides -l- 03' -l- Fe“ 20:4 + 0: + Zn“ -—> [20:4, 0:, antkupnu Zinc also competes with iron for formation of this complex (78). Perhaps, zinc stabilizes the 20:4 so that 20:4 is not available for 33 oxidation in the iron complex std therefore can stimulate the NADPH oxidase. Zinc my also play a role in the catalysis of 03‘ production by NADPH oxidase since nucleotides, such as NADPH, are able to comnplex with zirnc as determined by elution patterns of NADPH from Sephadex G—10 columns equilibrated with zinc ions (79, 80). Zinc birds to NADPH in a 2:1 mnolsr ratio (80). The first zinc atom birds between the monophosphate on the C2 of the sdenirne-ribosyl portion std the diflnosrinate group (80). The accord zinc atom birds to the remaining oxygen of the motnophosphate std the diphosphate, the forming a ten member ring (80). When zirnc complexes with NADPH, it interferes with oxidation of NADPH by making it unavailable as an enzyme sub- strate (80). The activity of the enzymes, NADPH oxidase derived from liver microsomes and mixed function oxidsses from smooth en- doplasmic reticulum, are inhibited in the presence of zinc (Kn =7.22 ”M Zn“) (80). In the presence of Iinysiological or deficient levels of zinc, the formation of the Zn; -NADPH complex probably plays only a minor, if arm, regulatory role in the oxygen burst by morophsges since, with zitnc deficiency, n.0, production by macrophages is de- creased (Chapter 5); thus, it is doubtful that NADPH oxidase activity would be increased. However, supraphysiological levels of zinc my inhibit NADPH oxidase via complex formtion with NADPH. Perhaps this explains the inhibition of oxygen consumption std hexose mono- phosphate shunt activity in polymorflnonuclear leukocytes and perito- neal mcrophsges by high concentrations of zinc i_t_n vitro (81-84). 34 Superoxide, produced by NADPH oxidase, either nonenzymatically dismutates to 11.0. , or superoxide dismutase (SOD) catalyzes the dismutstion (Figure 1). The enzymatic dismutation of 0.- is metal depedent. There is a cytoplasmic Cu/Zn SOD std a mitochondrial Mn- SOD. SOD is a dimer of two identical subunits (85). Each subunit of the cytoplasmic SCI) contains one molecule of copper std one mole- cule of zirnc located far apart (about 34 angstroms) (85). The two copper ions are catalytic cofactors whereas the two zirnc ions are structusl cofactors (86). Upon rmoval of zinc from Cu/Zn SOD, the enzyme activity at pH6 is still 90% of the intact Cu/Zn SOD (86). At pH>7, the copper ion migrates to the zinc free site, (86). So, at pH8.5, the zinc free so) has only 50% activity (86). The rate con- stants for interaction of Or with zinc free St!) at pH6 is similar to the intact Cu/Zn SOD (114). From this, the authors suggested that the catalytic mechanism (a ping-pong mechanism) for zirnc free 301313 thessmessthe intactCu/Znsw. Zinc is thoughttoplsys structural role since it has been shown that zirnc stabilizes SOD against inactivation by SCN' (86) . The mjor biological role for Cu/Zn St!) has not been elucidated. It may be important in the produc- tion of H30: for destruction of pathogens or it’s mjor role my be scavenging On' to protect the morophage from Or that has diffused through the membrane. Whatever the biological role for SOD in the morophage, zinc is not crucial for activity of the Cu/Zn 8(1). The nonenzymatic dismutstion of 03' my also be metal depen- dent. Iron has been shown to react with 03'. Furthermore irdirect evidence suggests that zirnc my also be involved in some reactions 35 with oxygen species. The reaction with iron is called the Haber- Weiss reaction (Fig. 2) (41). Fe“ +Or ——> Fe“ + 0; Fe“ +1130. —> Fe“ +0}!- +-OH -G~I+H:Oa —)H:O+I'Dn' ID;- +H.o. -—-->o. +mo+~an -on+ Fe“ —> Fe“ +0}!- The resulting oxygen metabolites are (H- and the toxic -a-I. These reactions occur at acidic pH’s found in the phagosome/phagolysosome. The phagosome/phagolysosome contains the engulfed pathogen/ stimulant (Figure 1). These reactions do not occur at higher pH’s found in the cytoplasm. This is important since production of highly reac- tive oxygen metabolites in areas other than the phagolysosome could destroy the leukocyte . Zinc may also be involved in the nonenzymtic disnmtation of Or . There is evidence, albiet inconclusive, that zinc catalyzes reactions with oxygen. In the following reactions, reduction of oxygen in the presence of zinc and bipyridine (bipy) yields highly reactive radicals and zinc peroxides (87) . Zn“ (biPY)a'* + 0n + 9- i’L.» [Znu (biPYhOO- 1* {mz(b1py).oo 1+ + e- --—> Zn” (bipy)a(02) These chemical reactions were determined electrochemically under aprotic conditions (dimethylformmide) . Aprotic conditions were used to stabilize the superoxide ion so that it would not dismutate to 1120) . Since an aprotic and not an aqueous envirenent was used, the significance of these reactions for biological systens remins 36 to be determined. In addition, it has also been observed that oxygen reacts with metal surfaces of zinc at extremely low temperatures (77K) (88) . The molecular steps determined for the dissociative chenisorption of oxygen at the metal surface is as follows: 0; (g) —> 03(a) —> 0'(a) —> Ol-(a) (88). Again, since these reactions were done at extremely low temperatures, the biological relevance is unknown. Several other metals (iron, copper, manganese, and cobalt) are known to react with oxygen (89). These metals are more reactive with oxygen than zinc since they have unfilled d-shell orbitals. Previously, zinc was thought to be unreactive with oxygen because zinc has a filled d-shell orbital (du). However, a zinc metal- loporphyrin is reactive with superoxide and forms a superoxo complex (89) . Superoxide has a relatively high affinity for Zn—meso—tetra- phenylporphyrin [Zn(T‘PP)] at 25°C, since it competes with Me3S0 for birding even when MezSO is the solvent (89). Zn(T'PP) birds only one axial ligand electrostatically since zinc is a d” metal. The reac- tions of 03" with Zn(T‘PP) are as follows: 2h(T‘PP) —> [Zn(TPP)0:]' —?—> [Zn(T'PP)Gl]‘ (117). Perhaps, sale form of zinc or complex containing zinc will, in the future, prove to have biological reac- tivity with oxygen species that participate in the oxygen burst. In sumry, zinc may participate at mny steps in production of toxic oxygen nnetabolites by uncrophages. It my be important in the stimulation of NADPH oxidase via phosrinolipase A: or phopholipase C - diacylglycerol lipase catalyzed release of 20:4 from phospholipids. Zinc is an important cofactor for phospholipase C. Zinc my also stabilize 20:4 against oxidation in a Fe—20:4-0; complex and thus 37 allow 20:4 to stimulate NADPH oxidase. Perhaps, zinc or some yet unknown complex containing zinc will prove to have biological reac- tivity with oxygen or its metabolites. Zinc deficient macrophages which have a reduced ability to destroy L ”A would hopefully provide an opportunity to pursue identification of the important zinc dependent processes in the "oxygen burst". B.) Possible Alterations in the "mmt" of MacroMes from Zinc Deficient Mice. The toxic oxygen metabolites of the "oxygen burst" are important in the destruction of certain pathogens inclLd- ing L c_n_ru_gi_. If zinc is important for normal production of these metabolites, one would expect ahnormel functioning of the oxygen burst with zinc deficiencies. This is the case. As discussed in section I C above, zinc deficient mice are unable to combat L 9&2; infections (39). I}; xi_t_r9_ the zinc deficient macrophage is unable to destroy the (L cruzi (36, 37). Also, addition of zinc but not other metals restored the ability of macrophages from zinc deficient mice to destroy L M (36, 37). H30: produced by macrophages is thought to be important in the destruction of L m (40, 90). It has been shown that L mi are killed by 11:03 (40) ard the ability of macrophages to kill L M correlates with the level of 1130; produced by macrophages stimulated with phorbol 12—myristate 13-acetate (PMA) (91). Catalase, a scavenger of 830; , abrogates the ability of the macrophages to kill L cruzi (90, 91). In addition, scavengers of other oxygen metabolites such as the superoxide scavenger, superoxide dismutase, 38 the hydroxyl radical scavengers, nennitol and sodium benzoate, and the possible scavenger of singlet oxygen (10: ), histidine, do not affect the ability of mecroflnages to kill L M (90, 91) . In fact, L M itself contains superoxide dismutase (92) but not catalase (92-94). Thus it is unable to scavenge the mo. . Further evidence for the role of I-IaOn in killing L (£112.! includes cytochemi- cal indentification of 11.0. in polymorphonuclear leukocyte vacuoles containing L cruzi (40). ' Despite this evidence for the importance of 1130: in the destruc- tion ong_r_1_nzi, theamount of 11:0: producedbngLuz_i infected macrophages has never been directly measured. It is also important to use L M as the stimulant when investigating the ability of zinc deficient resident mecrofinages to produce 3.0. since other agents such as BIA probably use different receptors and/or mechanism of stimulation. Further with regards to association and phagocytosis, it is also important to use L 9_ru_zi rather than other stimulants since resident macrophages from zinc deficient mice are able to phagocytose norml numbers of latex beads (33) but not L 99421 (36, 37) . Also in the past, it has not been possible with the available assays to detect 1130: produced by resident mecroflnages (95-99) . Usually, merophages that have already been activated i_n 2319 are used for measuring 1130; production in order to obtain detectable levels of H10; . A modification of the Pick and Mizel assay that can be used to detect 1120; production by unactivated resident macrophages will be described in Chapter 3. As a result of the modification 39 mde, the amnount of 1120: produced by resident mcrophages stimulated with L 9_ru_zi_ can nowbe measured (Chapter 3, 4, 5). A reduction in the "oxygen burst" would in part explain the reduced ability of zinc deficient snimls to combat infections. Sinnce HaOn production is important for destruction of L (mi. HaOn production by zinnc deficient and zinnc adequate mcrophages was ex- aminned in Chapter 5. The mechanism of stimulation of the "omen burst" by L Q_ru_£i_ is unknown. However, if it is through release of 20:4 and 20:4 stimulation of NADPH oxidase (Figure 2), zinnc could be crucial at several steps in the mechanism. As already discussed in the previous section II A, 20:4 levels in phospholipids, the release of 20:4, or the ability of 20:4 to stimulate NADPH oxidase my be altered in zinnc deficient morophages. If there is a reduction in level of 20:4 in deficient macrophages, one would expect reduced stimulation of NADPH oxidase and ultimately a reduced level of HaOn production by the deficient mcrophages. 20:4 levels in other tissues from zinnc deficient snimls are altered. The level of 20:4 in skin is influenced by the level of zinnc (79). Also, in the liver of zinc deficient rats, there is a reduced level of 20:4 (100-106). This reduction in 20:4 is accompanied by an increase in cis-linoleic acid, a 20:4 precursor, as a result of a reduction in delta-6-desa- turase activity (Figure 3) (100, 107). Delta-6-desaturase is the enzyme which catalyzes the conversion of cis-linoleic acid to game linolenic acid. Game-linolenic acid is the inediate precursor to dihomogamm linolenate which is then converted to 20:4 by delta-5- desaturase. Clejan et. al. (104) and Field et. al (105) reported 40 that delta-5-desaturase activity of the liver is also reduced (50- 70% of control) in zinc deficient rats. Horrobin and Cunnane (107) have also suggested that zinc my play some role in the mobilization of 20:4 and in delta-S-desaturase activity (Figure 3). Perhaps, zinc deficient mcrophages also contain less 20:4 and more of the fatty acid precursors for 20:4. If so, the deficient morophages would contain fatty acids of shorter chain length and higher degrees of saturation than zinc adeqnete macrophages. The fatty acid chain length and degree of saturation have been shown to determine the level of superoxide production by NADPH oxidase (48, 108); longer chain fatty acids with higher degrees of unsaturation stimulate production of larger amounts of superoxide. Therefore, upon stimula- tion of zinc deficient morophages with L (£12; , there would be release of shorter-chain more-saturated fatty acids and consequently less stimlation of NADPH oxidase as compared to zinc adequate moro- phages. Thus, there would mnost likely be a reaction in 11.0. produc- tion and in turn reduced killinng of L gr_uz__i. The results of fatty acid analysis of phosflnolipids from zinc deficient and zinc adequate macrophages is presented in Chapter 5. Besides a possible reduction in synthesis and incorporation of 20:4 into moaninolipids of zinc deficient mcroflnages, there my also be a reduction in release of 20:4 from the phospholipids. As discussed earlier in section II A , zinc my be important in the release of 20:4 from phcsmonipids since phospholipase c is a zinc dependent enzyme (Figure 2) (69, 70). Depletion of zinc fram the enzyme would reduce- ginosfinolipase C activity which, in turn, would 41 reduce the amount of 20:4 released for stimulation of NADPH oxidase resulting ultimtely in less 1130. production. Also, a rednction in the stimulation of the release of 20:4 my in part be due to the rednced number of L gru_zi_ associated with the zinc deficient mcro- phages. So, zinc my play a role in the events of bindinng and/or stimulation of morophage I-hOn production. If there is a rednction in 20:4 in or released from plasm membranes of zinc deficient macrofinages, there would probably also be a reduction in production of 20:4 metabolites of the cycle-oxyge- nase and/or lipoxygenase pathways. It has been reported that lipoxy— -oxygenase but not cyclo-oygennase products stimulate NADPH oxidase (54) . However, lipoxygenase products are not absolutely required for stimulation of NADH-I oxidase since lipoxygenase inhibitors do not affect the "oxygen burst" (54) . Most likely, with inhibition of lipoxygenase, the lipoxygenase substrate 20:4 stimulates NADPH oxi- dase. To further demcnstrate the importance of lipoxygenase products in the "oxygen burst" and the killing of L M, Wirth e_t_. 9;. (109, 110) showed that addition of leukotriene B. or leukotriene C4 , lipoxygenase products, increased killing of L 9312; and that this effect of the leukotrienes is abrogated by the 11.0; scavenger catalase (109. 110). Since leukotrienes can stimulate the "oxygen burst" (60), perhaps, the increase in killing of L gru__;i_ was the result of increased stimulation of 1130: production by leukotrienes. If leuko- triene production is reduced in the zinc deficient macrophages, this could also explain the rednction in the association with and H30: mediated destruction of L cruzi by zinc deficient macrophages. 42 Once 20:4 is liberated from the macophage phospholipid, zinc may compete with iron for complex formation with 20:4 and oxygen (78). Since the complex containing iron but not zinc oxidizes 20:4, zinc may stabilize 20:4 against oxidation. In the case of the zinc defi- cient macrophage, perhaps, a larger portion of the 20:4 is oxidized by the iron complex and, as a consequence, less 20:4 is available for stimulation of NADPH oxidase. Thus, there would be less produc- tion of 03' and consequently Hzon by deficient mcrophages. The analysis of the release of 20:4 and its metabolites upon stimulation of zinc deficient macrophages with I; gggzi is presented in Chapter 5. In summary, if levels of 20:4 released by zinc deficient macro- phages is reduced, it may be due to reduced synthesis of 20:4 for incorporation into phospholipids, reduced stimulation for release of 20:4 from.phospholipids, or increased oxidation of the released.20:4. Still, there exists other possibilities for a reduction in the "oxygen burst" of zinc deficient macrophages. Unfortunately these reactions are not well defined. For example, a reduction in HhCh production could be due to reduced.availability of zinc for catalysis of some as yet unknown chemical reactions with oxygen. Or, zinc my be unavailable for the formation of some zinc nucleotide complexes other than Zna-NADPH that perhaps participate in the "oxygen burst". Also, it remains to be determined whether or not any of the known mechanisms for stimulation of the "oxygen burst" by macrophages hold true when I; 932;; is the stimulant. Even though not much is known about 2; Qgggi-macrophage interactions or the importance of—zinc in the "oxygen burst", the literature discussed in this chapter 43 implicates mnny speculative roles for zinc in the "oxygen burst". Much work must be done to elucidate the role(s) for zinc in the production of toxic oxygen metabolites by macrophages as well as the defect of zinc deficient macrophages in associating with and destroy- ing the pathogen, L cruzi. C.) Posgble Alteraiiogs in Membranes of Mgcronflges from _LZinc Defi- cient Mice. Zinc my also be important in the association of L gn;uz_i with the macrophage since, as discussed in section I C, associa- tion with zinc deficient mcrophages is reduced (36, 37). Additon of zinc repairs this defect (36, 37). Zinc has been shown to play a role in stabilization of membranes against perturbing agents or oxidations. Zinc is also necessary for synthesis of some important components of the membrane snch as 20:4. Whether these or other interactions between zinc and the plasm membrane are altered in the zinc deficient mcrophage remins to be determined. Since zinc has been shown to stabilize membranes, perhaps, this trace element is also important for association of the parasite with the mcrophage membrane. The evidence for stabilization of membranes by zinc is far from complete. So far, it has been shown that plasm membrannes contain substantial amcunnts of zinc (79) , that supra-physio- logical levels of zinc stabilizes membranes against perturbing agents (79) , and that sub-physiological levels of zinc leads to loss of plasma membrane zinc and destabilization of the membrane (79). The level of zinc (64-222 ug/g protein) in the plasma membrane (111) my have considerable physiological relevence since the plasm membrane 44 has rather diverse functions including trannsport of nutrients , min- tenance of osmolarity, and stimuli transduction through receptors. Although the role(s) for zinc in the membranne has not been delin- eated, zinc has been found to be associated with several membranes (plant membranes, plasm and lysosomnl membranes of lung alveolar mcrophages, lysosoml membranes of liver cells, neurotubules, micro- tubules, and plasm membranes of erythrocytes (79). A substantial level of zinc is in the membranes of erythrocytes (115 i 9.7 ug/g phospholipid) (111). A similar amounnt of another metal, copper, is associated with the erythrocyte membrane (128 ug/g phospholipid) (111). The zinc in erythrocyte plasma membranes is minly associated with the lipid phase (69% of the zinc), in particular the phospho- lipids, with some zinc bound to membranne proteins (111). As stated earlier, zinc my stabilize 20:4 from oxidation in the iron-20:4- oxygen complex (78) . Zinc has been shown to prevent lipid peroxida— tion of 20:4 in liver microsomes i_n vi_tro (79). anapil et. al. (111) speculate that this is due to an interaction of zinc with polyunsaturated fatty acids of phospholipids or with proteins of mem- branes. Also, high dietary zinc inhibits i_n_ v_itr_o induction of lipid peroxidation of liver microsomes (79). Thus, addition of zinc ionns seem to inhibit lipid peroxidation of biological membranes (79) . In fact, high levels of zinc are used to stabilize various membranes during membrane purification procedures (79) . Membrane integrity is altered by a deficiecy in zinc. Histol- ogy of membranes from several zinc deficient snimls, including rat pancreas and intestinal cells, tnmnor cells, and Elena ilis, 45 showed alterations in membrane mcrphology (73) . In the zinc deficient state, the chick embryo is unable to mintain norml ionic balance or cell volume (79). Erythrocytes from zinc deficient rats are more prone to hemolysis and this correlates with the level of reduced serum zinc (79). In addition, when zinc was added to norml eryth- rocytes, they acquired an increased stability against osmotic shock (79) . Zinc deficiecies also have adverse effects on leukocyte per- meability to sodium (73). Thus, it is possible that pretreatment of zinc deficient mcrophsges with zinc my stabilize the mcrophage membranes making possible norml levels of association of the para- sites. In turn, there would be norml stimulation of the "oxygen burst" since the level of L c_ru__zi_-mcrophage association correlates with L <_:_rn_i_z_i_ stimulated 11:0; production by resident mcrophages (Chapter 4). Another explanation for the reduced association of L c_ruzi_ with zinc deficient mcrofinages my be alterations in the fatty acid composition of the mcrofinage membrane. If zinc deficient mcro- phages have a reduction in the more unsaturated fatty acid, 20:4, andanincreaseinmore saturatedprecursors, thismylesdtoa reduction in phagocytosis and possibly the decrease in association of L 9_rg_z_i_ seen with zinc deficient mcrophages. It has been re- ported that a rednction in unsaturated fatty acids and a concomitant increase in more saturated fatty acids leads to reduced fluidity of the membrane bilayer, rednced phagocytosis, and increased activation energy for phagocytosis in mcrophages (112, 113). As discussed in section II B, deficiecies in zinc reduce the level of 20:4 in the 46 rat liver (100-106). This rednction was accompanied by an increase in cis-linoleic acid, a precursor to 20:4, due to reduced activity of delta-5- and delta-6-desaturase (100, 104, 105, 106) (Figure 3). These alterations my also occur in the deficient mcrophages. If the fatty acid composition is altered in zinc deficient mcrophages, one would expect that a 30 minute incubation with zinc would restore the plasma membrane fatty acid cmposition to norml since L 9_ru_Li- mcrophage association is restored. The possible restoration of fatty acid composition in the presece of zinc my occur during recycling of the plasma membrane of zinc deficient mcrophages. The resident mcrophage has an extremely high rate of plasma membrane pinocytosis; the entire membrane is edocytosed approximtely every 33 minutes (114). The mjority of this edocytosed membranne is recycled to the cell surface (115, 116). Unfortunately, the mechan— ism for membrane recycling are not well understood (115). However, it is thought that the golgi apparatus (115-118) and the endoplasmic reticulum (118) my be involved in this recyclinng process. Exposure of the internnalized membrane to these organelles during recyclying my be important for repair of membrane components especially after exposure to the degredative processes occuring in the phagolysosome. Since fatty acid elongation and desatuation normlly occur in the endoplasmic reticulum and these processes my be decreased in defi- cient macrophages, perhaps, the fatty acid composition of the mem- branes of zinc deficient macrophages can be significantly restored by the membrane recycling processes (hiring the consecutive incubations of a half hour with zinc and one hour with L cruzi. To repair 47 deficient mcrophages, modifications of existing fatty acids is more probable than synthesis of new fatty acids since synthesis requires several hours (118) yet repair occurs within 1 1/2 hours (36, 37). One process of phosflnolipid synthesis/repair has been shown to occur rather rapidly during phagocytosis; finosphatidyl inositol turnover as measured by P“ incorporation can be detected in 10 minutes with a mximl rate between 1 and 6 hours (119). In sumery, the redtced association of L M with zinc deficient mcrophages my be due to an altered fatty acid composition of the plasm membrane and these alterations my be repaired by addition of zinc. The fatty acid composition of the fincsfinolipids from zinc deficient and zinc sufficient mcrophages was determined and will be discussed in Chapter 5. 2. 10. 11. 12. 13. References Prasad, A. S. (1979) Anm. Rev. Pharmcol. Toxicol. 20, 393. Hambridge, K. N., Walravens, P., Brown, R., Webster, 8., White, M., Anthony, M., and Roth, M. (1976) Amer. J. Clin. Nutr. 2_9, 734. Sandstead, H. (1973) Amer. J. Clin. Nutr. 26, 1251. Sandstead, 11., Henriksen, L., Gregor, J., Prasad, A., and Good, R. (1982) Amer. J. Clin. Nutr. g, 1046. I-Iambridge, R., Hambridge, C., Jacobs, M., and Banmn, J. (1972) Pediatr. Res. 6, 868. Henkin, R., nan-chain, J., and Meret, s. (1971) Am. J. Obstet. Gynecol. 119, 131. Portnoy, B., and mlokhia, M. (1974) Lancet g, 663. anandra, R. R., and Dayton, D. H. (1982) Nutr. Res. 2, 721. Moynahan, E., and Barnes, P. (1973) Lancet 1, 676. Lombeck, I., Schnippering, M., Ritzl, F., Feinedegin, L., and Brenner, H. (1975) Lancet, _2_, 855. Schloen, L., Fernanades, C., Gsrofalo, J., and Good, R. (1979) Clin. Bul. _9_, 63. Oleske, J. M., Westphal, M. L., Shore, J., Gordon, D., Gogden, J., Nshmias, A. (1979) Am. J. Dis. Child. m, 915. Fraker, P. J., Mass, 8. M., and Luecke, R. W. (1977) J. Nutr. _1_(_)1, 1889. 48 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 49 Fraker, P. J., DePasquale-Jardieu, P., and Cook, J. (1988) Arch. Derm. (in press) Fernandes, C., Nair, M., Omoe, K., Tannaka, T., Floyd, R., and Good, R. A. (1979) Proc. Natl. Acad. Sci. U.S.A. E, 457. Fraker, P. J., Zwickl, C. M., and Luecke, R. W. (1982) J. Nutr. Q2, 309. Nash, L., Iwata, T., Fernandes, 6., Good, R. A., and Incefy, G. (1979) Cell. Immnol. _48, 238. DePssqnele-Jardieu, P., and Fraker, P. J. (1979) J. Nutr. mg, 847. DePasquale-Jardieu, P., and Fraker, P. J. (1979) J. Nutr. 199, 1847. Fraker, P. J., DePasquale-Jardieu, P., Zwickl, C. M., and Luecke, W. (1978) Proc. Natl. Acad. Sci. U.S.A. Z_5_, 5660. DePasquale-Jardieu, P., and Fraker, P. J. (1980) J. Immunol. $4, 2650. Iwata, T., Incefy, C., Tanska, T., Ferannanndes, C., Menedez, C. J., Pih, K., and Good, R. A. (1979) Cell. Immunol. fl, 100. Chandra, R., Heesi, C., and Au, B. (1980) Clin. Exp. Immunol. 43, 332. Dardenne, M., Pleau, J., Lefrancier, P., and Bach, J. (1981) CR Acad. Sci. Paris gig, 793. Cunningham-Bundles, C., Cunningham-Rundles, S., and Garafolo, J. (1979) Fed. Proc. §_8_, 1222. Gronowicz, E., Coutinho, A., and Moller, G. (1974) Scand. J. Immunol. _3_, 413. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 50 Bona, C., Yano, A., Dimitrio, A., and Miller, R. (1978) J. Exp. Med. 118, 136. Gronowicz, E., and Coutinho, A. (1975) Scand. J. Immunol. _4, 429. Goodmn, M., and Weigle, W. (1980) Clin. Immunnol. Immunopath. 15, 375. Fraker, P. J. (1983) Survey Immunol. Res. _2_, 155. McKearn, J., and Qnintains, J. (1979) Cell. Immunol. 14, 367. DePasqunle-Jardieu, P. (1982) Doctoral Thesis, Michigan State University, East lensing, Michigan. Wirth, J. J., Fraker, P. J., and Kierszenbaum, F. (1984) J. Nutr. 1_1_4_, 1826. Vallee, B., and Koch, F. (1957) J. Biol. Chem. 2_25, 185. Miller, E., and Luecke, R. (1969) J. Nutr. 95, 278. Wirth, J. J., Fraker, P. J., and Kierszenbaum, F. (mnuscript in preparation). Fraker, P. J., Jardieu, P., and Wirth, J. (1986) In "Nutritional Diseases: Research Directions in Comparative Pathobiology" pp 197-213. Alan R. Liss, Inc. Salvin, S. B., and Rabin, D. S. (1984) Cell. Immunol. 81, 546. Fraker, P. J., Caruso, R., and Kierszenbaum, F. (1982) J. Nutr. 112, 1224. Villalta, F., and Kierszenbaum, F. (1983) J. Immunol. 1_1, 1504. 1 Aust, S. D., Morehouse, L. A., and Thomas, C. E. (1985) J. Free Rad. Biol. Med. 1, 3. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 51 Johnston, P. A., Adams, D. 0., and Hamilton, '1‘. A. (1986) Cell. Immunol. 1%, 400. Johnston, R. B. (1981) In "Lymphokines" (Pick, E., ed.) _3, pp 33-56. Academic Press, Inc., New York. Docampo, R., Cssellas, A. M., Madeira, E. D., Cardoni, R. L., Moreno, S. N. J., and mson, R. P. (1983) FEBS Lett. m, 25. Simona, S. R., and Karnovsky, M. L. (1973) J. Exp. Med. 138, 44. Castagnna, M., Takai, Y., Kaibuchi, K., Sanno, K., Kikkawa, U., and Nishizuka (1982) J. Biol. Chem. 251, 7847. Nishizuka, Y. (1984) Nature 1_0_8, 693. Bromberg, Y., and Pick, E. (1984) Cell. Immunol. g, 213. Maridonneau-Parini, I., and Tauber, A. I. (1986) Clinical Research 31, 661A. Tauber, A. I., Cox, J. A., Jeng, A. Y., and Blumberg, P. M. (1986) Clinical Research 21: 664A. McPhail, L., Clayton, C. C., and Snydermn, R. (1984) Science 2_21, 622. Fujita, I. Irita, K., Takeshige, K., and Minnakami, S. (1984) Biochem. Biophys. Res. Commun. 12_Q, 318. Robinson, J. M., Badwey, J. A., Karnovsky, M. L., and Karnovsky, M. J. (1984) Biochem. Biophys. Res. Comm. 122, 734. Bromberg, Y., and Pick, E. (1983) Cell. Immol. 19, 240. McPhail, L. C., Shirley, P. 8., Clayton, C. C., and 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 52 Snnydermn, R. (1985) J. Clin. Invest. E, 1735. Curnnette, J. T‘. (1985) J. Clin. Invest. fl, 1740. Vercauteren, R. E., and Heynemen, R. A. (1984) J. Leuk. Biol. g, 751. Suzuki, T., Saito-Taki, T., Sadasivan, R., and Nitta, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 72, 591. Scott, w. A., Zrike, J. M., Hamill, A. L., Kempe, J., and Cohn, Z. A. (1980) J. Exp. Med. 15;, 324. Flohé, L. Beckmnannn, R. Giertz, H., and Goschen, G. (1985) In "Oxidative Stress" (Seis, B., ed.) pp 403-435. Academic Press, Inc., New York. Wightmn, P. D., Dahlgren, M. E., Davies, P., and Bonney, R. J. (1981) Biochem. J. _2_99, 441. Wightmn, P. D., Dahlgren, M. E., Hall, J. C., Davies, P., and Bonney, R. J. (1981) Biochem. J. 19_7, 523. aninsson, A., and Sundler, R. (1985) Biochim. Biophys. Acta 8_1§, 265. Fmilsson, A., and Sundler, R. (1986) Biochim. Biophys. Acta fl, 533. Moscat, J., Aracil, M., Diez, E., Balsinde, J., Barreno, P. C., and Municio, A. M. (1986) Biochem. Biophys. Res. Commun. 1%, 367. Brune, K., Aehrinnghaus, U., and Peskar, B. A. (1984) Agents Actions 1_4, 729. Bonney, R. J., Wightmn, P. D., Dahlgren, M. E., Davies, P., Kuehl, F. A., Jr., and Rules, J. L. (1980) Biochim. Biophys. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 53 Acts 6;), 410. Patriarca, P., Zatti, M., Cramer, R., and Rossi, F. (1970) Life Sci. g, 841. Ottolenghi, A. C. (1965) Biochim. Biophys. Acta 1_0_6_, 510. Dennis, E. A. (1983) In "The Enzymes" (Boyer, P., ed.) _1fi, 307. Academic Press, Inc., New York. Horrobin, D. F., Phnku, M. 8., Cunnane, S., Karmzyn, M., Morgan, R. 0., Ally, A. I., and Karmall, R. A. (1978) Can. J. Neur. Sci. 5, 93. Manku, M. S., Horrobin, D. F., Karmzyn, M., and Cunnnnane, S. C. (1979) Endocrinclogy 10_4_, 774. Bettger, W. J., and O’Dell, B. L. (1981) Life Sci. 2_8_, 1425. Wells, M. A. (1973) Biochemistry g, 1080. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G., and Field, J. B. (1969) J. Biol. Chem. 2_44, 5189. Stossel, T. P., Murad, F., mson, R. J., and Vaughan, M. (1970) J. Biol. Chem. 2_45, 6228. Peterson, D. A., Gerrard, J. M., Benton, and M. A. (1981) Med. Hypothesis _7_, 1389. Peterson, D. A., Gerrard, J. M., Peller, J., Res, G. H. R., and White, J. G. (1981) Prostaglandins Med. 6, 91. Chvapil, M. (1973) Life Sci. _1_3_, 1041. Slater, T. F. (1974) In "Molecular Mechanisms of Oxygen Activation" (Hayaishi, Q, ed.) pp 143- 176. Academic Press, Inc., New York. Stankova, L., Drach, G. W., Hicks, T., Zukoski, C. F., and 82. 83. 84. 85. 86. 87. 89. 90. 91. 54 Chvapil, M. (1976) J. Lab. Clin. Med. 88, 640. Chvapil, M., Stankova, L., Zukoski, C., IV, and Zukoski, C. III (1977) J. Lab. Clin. Med. 89, 135. Chvapil, M., Stannkova, L., Berhard, D. S., Weldy, P. L., Carlson, E. C., and Campbell, J. B. (1977) Infect. 1mm. 18, 367. Chvapil, M., Stannkova, L., Bernhard, D. 8., Zukoski, C. F., and Drach, G. W. (1977) Invest. Urol. 15_, 173. Ricchelli, F., Rossi, E., Salvato, B., Jori, G., Bannister, J. Y., and Bannister, W. H. (1983) In "Oxy Radicals and Their Scavenger Systems" (Cohen, G., and Greenwald, R. A., eds.) 1, 320-323. Elsevier Science Publishing Co., Inc., New York. O’Neill, P., Fielden, E. M., Cocco, D., Calabrese, L., and Rotillo, 'G. (1983) In "any Radicals and Their Scavenger Systems" (Cohen, G., and Greenwal , R. A., eds.) 1, 316-319. Elsevier Science Publishing Co. , Inc. , New York. Sawyer, D. T., Roberts, J. L., Jr., T‘snchiya, T., and Srivatsa, G. S. (1984) In "Oxygen radicals in Chemistry and Biology" (Bors, W., Saran, M., Tait, D., eds.) pp 25-34. Walter de erter & Co., New York. Au, C. T., and Roberts, M. W. (1986) Nature 3_19_, 206. Kasai, P. B., Pbleod, D., Jr., andWatanabe, T. (1977) J. Am. Chem. Soc. _9_8, 3522. Villalta, F., and Kieszenbaum, F. (1984) J. Immunol. 1__3, 3338. Nathan, C. F., Silverstein, S. C., Brukner, L. 11., and Cohn, 55 Z. A. (1979) J. Exp. Med. _lfi, 100. 92. Boveris, A., Sies, H., Martino, E. E., Docsmpo, R., Torrens, J. F., and Stoppani, A. O. M. (1980) Biochem. J. 188, 643. 93. Docsmpo, R., De Boiso, J. F., Boveris, A., and Stoppani, A. O. M. (1976) Experientia 12, 972. 94. Cardoni, R. L., Docsmpo, R., and Cssellas, A. M. (1982) J. Parasitol. 88, 547. 95. Badwey, J. A., Robinson, J. M., Lazdins, J. K., Briggs, R. T., Karnovsky, M. J., and Karnovsky, M. L. (1983) J. Cell. Physiol. 118, 208. 96. Tsunawski, 8., and Nathan, C. F. (1984) J. Biol. Chem. 858, 4305. 97. Dean, J. H., Lauer, L. D., House, R. V., MurrayM. J., Stillmn, W. S., Irons, R. D., Steinhagen, W. H., Phelps, M. C., and Adam, D. O. (1984) Tox. Appl. Phanm. 7_2, 519. 98. Murray, H. W., Nathan, C. F., and Cohnn, Z. A. (1980) J. Exp. Med. L52.» 1610. 99. Phrioka, A., and Kobayashi, A. (1985) J. Protozool. 82, 153. 100. Huang, Y. 8., Cunnnnane, S. C., Horrobin, D. F., and Davignnon, J. (1982) Atherosclerosis 11, 193. 101. (humane, S. C., Horrobin, D. F., and mnku, M. S. (1984) Proc. Soc. Exp. Biol. Med. 171, 441. 102. Cunnnane, S. C., and Horrobin, D. F. (1985) J. Nutr. 115, 500. 103. Bettger, w. J., Reeves, P. G., Moscatelli, E. A., Reynolds, G., and O’Dell, B. L. (1979) J. Nutr. m, 480. 104. Clejsn, S., Castro-Magma, M., Collipp, P. J., Jones, E., 105. 106 . 107 . 108 . 109 . 110. 111. 112. 113. 114. 115. 116. 56 and Maddaiah, V. T. (1982) Lipids u, 129. Field, H. P., and Kelleher, J. (1983) Proc. Nutr. Soc. 48, 54A. Tsai, S. L., Craig-Schmidt, M. C., Weete, J. D., and Keith, R. E. (1983) Fed. Proc. g, 823 (abs. 3110). Horrobin, D. F., and Cunnnane, S. C. (1980) Med. Hypothesis 8, 277. Kakinun, K. (1974) Biochim. Biophys. Acta 848, 76. Wirth, J. J., and Kierszenbann, F. (1985) J. Immunnol. 13_4, 1989. Wirth, J. J., and Kierszenbaum, F. (1985) Mol. Biochem. Parasitol. 8, 97. Chvapil, M., Montgomery, D., Ludndg, J. C., and Zukoski, C. F. (1979) Proc. Soc. Exp. Biol. Med. 168, 480. Mahoney, E. M., Hamill, A. L., Scott, W. A., and Cohn, Z. A. (1977) Proc. Natl. Acad. Sci. U.S.A. fl, 4895. Phhoney, E. M., Scott, W. A., Lsndsberger, F. R., Hamill, A. L., and Cohn, Z. A. (1980) J. Biol. Chem. _2_85, 4910. Steinmn, R. M., Brodie, S. E., and Cohn, Z. A. (1976) J. Cell Biol. Q, 665. Tulkens, P. Schneider, Y. J., and Trouet, A. (1980) In "Pionnonuclear Phagocytes. Functional Aspects." (van Furth, R., ed.) 1, 613-647. Pertinum Nijhoff Publishers, Boston, MA. Farquhar, M. G. (1982) In "Membrane Recycling" (Ciba Foundation Symposium 92) (Everard, D., and Collinns, G. M., $80) We 157-1830 mm BOOkB, Ltdo, 10m. 57 117. Cohn, Z. A., and Steinmen, R. M. (1982) In "Membrane Recycling" (Ciba Foundation Symposium 92) (Everard, D., and Collins, G. M., eds.) pp. 15-34. Pitmn Books, Ltd., London. 118. Scott, W. A., Money, E. M., and Cohn, Z. A. (1980) In "Mononnclear Phagocytes. Functional Aspects." (van Furth, R. , ed.) 1, 685-701. Martinum Nijhoff Publishers, Boston, MA. 119. Ogmmmdsdbtter, H. M., and Weir, D. M. (1979) Immunology 81, 689. Chapter 2 FUNCTICNAL CAPACITY OF THE FESIDUAL LYMPHCXNTES IN ZINC DEFICIENT MICE 58 ABSTRACI‘ Zinc deficiency, a condition not infrequently encountered in humans, drastically rednces cell and antibody mediated responses. Since there are numerous zinc depedent metalloenzymes necessary for lymphocyte and mcrophage function, it was of interest to determine the capacity of the residual lymphocytes from zinc deficient mice to proliferate and produce lymginokines in response to stimulation. In these studies, miniml levels of zinc were introduced i_n_n vi_tro by using autologonm sernmn from zinc deficient mice or a serum free system. Splenocytes from mcderstely or severely zinc deficient adult A/J mice gave norml proliferative responses and generated adequate interleukin 2 (IL-2) activity when stimulated with Con- cannavalin A in a serum free system. When stimulated with allogeneic target cells, splenocytes from deficient mice exhibited enhanced proliferation and IL-2 production (180%) compared to the splenocytes from adequately fed mice regardless of whether the amount of zinc in the culture medium was ample or limiting. The increased prolif- eration was probably not due to a lower threshold for stimulation since the number of IL-2 receptors per cell from the deficient mice was the same as controls. However, more cells were available for stimulation in the deficient group since a greater proportion of cells bore IL-2 receptors. Although B—cells from zinc deficient mice stimulated i_n mg with sheep red blood cells produced fewer total numbers of plaque forming cells (PFC) per spleen, the propor- tion of PFC per 100 viable splenocytes and the amounnts of IgM and 59 60 IgG produced per plaque were norml. Based on the tests performed thus far, ample or limiting amounts of zinc in the culture medium do not affect lymphocyte responses and it would appear that the residual splenic lymphocytes of zinc deficient mice are able to carry out mny fundamental immune processes. INTKIXKZTION Zinc deficiency is a prevalent human nutritional problem through- out the world, including the USA (1-4). In both man and animals, zinc deficiency causes rapid and severe depressions in immune function (5). In.the mouse, zinc deficiency causes drastic reductions (40- 50%) in the absolute numbers of lymphocytes and.macrophages in the blood, thymus, and spleen (5, 6). Yet, the relative percentage of TL and.B-cells and.mononuclear cells remained unchanged (5, 6). Antibody mediated responses to both thymus independent and thymus dependent antigens in mice show marked.depression (50-70%) depending on the degree of the deficiecy (5, 7, 8, 9, 10, 11). Other T-cell dependent responses such as delayedrtype hypersensitivity and cyto- lysis of tumor cells are also substantially reduced by the deficiency (12, 13). Because of the greatly reduced.capacity of zinc deficient mice to mount immune responses the question arose as to whether the depres- sion in immune function was due only to decreased.numbers of lymc phocytes and/or to a decrease in functional capacity of the residual lymphocytes. There are over 100 enzymes that are dependent upon zinc for function.including many enzymes associated.with RNA and.DNA synthesis (2). SUboptimal dietary zinc might, therefore, affect the activity of these enzymes snd.other zinc dependent processes thereby altering proliferation and production of lymphokines by lymphocytes. For this reason, the ability of T and.B-cells from zinc deficient mice to respond to various stimulants was assessed along with their 61 62 capacity to proliferate and produce interleukins and antibodies. Care was also taken to regulate the level of exogenous zinc in the culture medium. To this end, lymphocytes were cultured in medium supplemented with sera from zinc deficient mice or in a serum free system containinng concentrations of zinc that were below physiological levels to reduce the possibility of 1p 11t_r9 rejuvenation of zinc depedent processes . These results were compared to data obtained with cultures supplemented with fetal calf serum which contains significant levels of bioavailable zinc. The results will show that lymphocytes from zinc deficient mice are able to perform mnanny key immune functions even in those culture systems where the availability of zinc was limited. MATERIAIS ANDME'IHODS Animls. NJ and CS7Bl/6 fenele mice were purchased from the Jackson laboratory, Bar Harbor, Maine. Female Lewis rats were pur- chased from Charles River Breeding Laboratories, Portage, Michigan. Egg. Six week old A/J female mice were placed in stainless steel cages with mesh bottom to rednce recycling of zinc. They were fed g libitum a biotin fortified egg white diet containing either deficient (0.8ug Zn/g) or adequate (27ug.Zn/g) levels of zinc. The composition of the diet is described in detail in Appen- dix. Since inanition accompanies zinc deficiency, a third group, the restricted mice, were fed zinc adequate diet equivalent to the average amount of food consumed the previous day by zinc deficient mice. All mice had free access to deionized distilled water ((0.2ug Zn/K) . Feed jars and water bottles were washed with 4N HCl and rinsed with deionized water to remove zinc. The mice were weighed at least once a week. At the end of the dietary period, those mice that received zinc deficient diet and weighed 65-68% of the average body weight of the control mice were designnated as severely zinc deficient mice. Moderately deficient mice were defined as weighing 70-74% of the average control mouse body weight. Previous studies indicated the latter group is only modestly effected by inanition (8). The total degree of parakeratosis per mouse was determined by summing the degree of parakeratosis assigned on a scale of 0 to 4 to the eye, tail, ears, anus, and coat. Thymuses were also weighed for each dietary group. 63 64 Collection of atholggous serum. Serum was collected by sever- ing the subclavian artery of severely zinc deficient or control mice. The blood was incubated for 15 minutes at 37°C followed by several hours at 4°C. Mouse serum (0. 5% v/v) supplemented culture medium was filter sterilized using a 0.2 micron Nalgenne filter. Zinc analgis. The diets and sera were annalyzed for zinc content by atomic absorption spectrophotometry (Varian Techron AA-17 5 , Spring- vale, California) as described in earlier publications (7,- 14). Cell Culture. The culture medinmn consisted of Rafi-1640 (M.A. Bioproducts, Walkersville, MD) buffered with 0.01M Hepes pH 7.4 (Gibco Laboratories, Grand Island, N.Y.), 0.06%w/v NaHOO: (Gibco), and supplemented with 0.1m nonessential amino acids (M.A. Biopro- ducts), ZmM glutamine, 1M Na pyruvate (M.A. Bioprodncts), 100 unnits penicillin, 100ug/m1 streptomycin (M.A. Bioproducts), 50 ug/ml gen- tamwoin (M.A. Bioprodncts), 5 x 10'5M 2-mercaptoethanol, and 0.1x BME vitamin mix (M.A. Bioproducts) (15). The medium was supplemented with 0.5%v/v serum collected from deficient or control mice and 0.8%w/v bovine serum albumin (tissue culture grade BSA, Sigma, St. Louis, no), or with 5%v/v fetal calf serum (FCS, M.A. Bioproducts). Spleens were remcved asceptically, minced, and pressed through a sterile stainless steel mesh (100 gauge). The single cell suspension was washed and cell viability was determined using the trypsn blue dye exclusion method. Cultures were incubated at 37°C uder a humid- ified atmcsphere of 10% 00:. N02, and 83% N: or 7% 00: and ambient air. 65 Mit__ogenic stimulation. Splenocytes (2.5 x 105) were cultured in 96 well flat bottom microtiter plates (Falcon Plastics, Oxnnand, CA) in serum free medium containing Concanavalin A, (Con A, Sigma), at concentrationns rannginng from 0.5 to 5 ug/ml. After 24 hours, luCi of methyl-[3HJ-thymidine (2 Ci/mfl, Armersham) was added to each well. Eighteen hours later, the cultures were harvested using a multiple sample harvester (Otto Hiller Co., Madison, WI), and the DNA was precipitated with cold trichloroacetic acid onto glass fiber filters. The amount of radioactivity incorporated into the DNA was measured by a Delta 300 scintillation counter (Tracor Analytic). Unstimulated A/J splenocytes incorporated less than 10,000 cpm of methyl-[3H]- thymidine. At the time of harvest, cell viability, as determined using the trypan blue exclusion method, was 70% 1 6%. Also, 24 hour supernatants from Con A stimulated A/J splenocytes were collected for annalysis of interleukin-2 (IL-2) activity. Mixed lmflnocyte culture (LL01. The culture meditn described above was supplemented with either 0.5% autologone serum from zinc deficient mice (50 mg Zn/dl) or control mice (100 ug Zn/dl) and 0.8% BSA (tissue culture grade) or with 5% PCS. In order to reder the target cells unresponsive, splenocytes from 057Bl/6 (H-2") mice were incubated with mitomroin C (25 ug/ml) for 30 minutes. 2.5 x 105 A/J splennocytes (H-Z') were incubated with 1 x 10‘, 2.5 x 105, or 7.5 x 105 mitomycin C treated C57Bl/6 splenocytes (H-2°). Methyl-[3H]- thymidinewasaddedforthefinal 18hoursofa3, 4, 5, or6day incubation period. The level of radioactivity incorporated into the DNA was determined as described for the Con A assay. Mitomycin C 66 treated 057Bl/6 splenocytes incorporated less than 1000 cpm of methyl- [3H]-thymidine. At the time of harvest, A/J splenocyte viability as determined by trypan blue exclusion was 91 1_4%. In addition, super- natants were collected and pooled from identically stimulated cultures (triplicates) at 48 hours for analysis of IL-2 activity. The ability of mitomycin C treated cells to absorb, produce, or proliferate in response to IL-2 was also analyzed. Absorbtion of IL-2 by mitomycin C treated cells was determined.by adding IL-2 to mitomycin C treated cells and measuring IL-2 activity in 24 hour supernatants. Production of IL-2 was determined.by measuring IL-2 activity in 24 hour super- natants from mitomycin C treated A/J and.mitomyoin C treated C57Bl/6 cells. Proliferation in response to IL-2 was determined from.methyl- [3H]-thymidine incorporation by mitomycin C treated cells supplemented with IL-2. Assay for IL-8_ggtivity. CTLL-Z cells (1 x 10°), an ILPZ depen- dent cytolytic T-cell line (Scripps Clinic, La Jolla, CA), were supplemented with various dilutions of standard or test supernatants from either Con A or allogeneic cell-stimulated splenocytes so that the final culture volume was 100 pl. Supernatants from the zinc adequate splenocytes served as the standard. The cultures were pulsed with 1 “Ci methyl-[3H]-thymidine for the final 18 hours of a 42 hour incubation followed.by determination of the amount of methyl- [3H]-thymidine incorporated into the DNA. One unit/ml of ILPZ ac- tivity was assigned to that dilution of the supernatant from.zinc adequate splenocytes which induced 50% of the maximum 67 methyl-[3H]-thymidine incorporation. IL-2 activity was quantified by prdbit analysis (16). Maintenance of cytolytic T-cell line. CTLL-2 cells (5 x 103/ml) were cultured in IL-2 supplemented culture medium (50%/50% v/v) and subcultured.every 2-3 days when saturation density (approximately 5 x 10° to 10° CTLL-2 cells/ml) was reached. ILPZ supernatants to maintain this cell line were obtained fromlCon A.(2 pg/ml) stimulated female Lewis rat splenocytes (1 x 10° cells/ml) after 48 hours of culture. IL-81receptorg. IL-2 receptors on splenocytes were labeled by indirect immunofluorescense using 7D4, a rat IgMCmonoclonal antibody which is specific for the ILr2 receptor (17, 18). Cells from each group were collected on day 3, 4, 5, or 6 of the MLC, pooled and placed over a lympholyte M gradient (Accurate Chemical and Scientific Corp.). The lymphocytes in the interface of the gradient were col- lected and washed three times with 0.1M phosphate buffered saline (PBS, pH 7.4) - 0.15%.BSA - 0.15%.NaNa. Viable lymphocytes (1 x 10°) were placed on ice and resuspended in 200 pl of 7D4 antibody with 0.15% NaNa for 30 minutes at 4°C. The cells were washed three times and resuspended in 100 pl of PBS-BSA-NaNa followed by the addition of 70 pl of a 1/2 dilution of fluorescein isothiocyanste (FITC) conjugated affinity purified mouse anti-rat kappa (FITC-Mar 18.5, Becton Dickinson). After a 30 minute incubation at 4°C, the cells were washed three times in the cold.with PBS-BSA-NaNs and resuspended to 2 ml. The relative number of IL-2 receptors per cell and the percentage of cells with receptors was analyzed using an CL a, I‘.‘ 68 Orthocytofluorograph 50-H connected to a 2150 computer (Ortho Diagnos- tic Systems Inc., Raitan, N.J.) The percentage of fluorescently labelled cells and cellular debris was Obtained.from a cytogram of forward.scatter versus 90 degree angle scatter of the fluorescently labelled material. Less than 15%.fluorescence was cellular debris. A background of less than 3%.fluorescence was Observed when FITC- MAR18.5 antibody was added alone. The data is shown as the percentage of cells, excluding cellular debris, that were fluorescently labelled. Antibggy production. A/J mice in each dietary group were im- munized intraperitoneally with 1 x 10° SRBC in sterile PBS. Five days later, spleens were removed for the determination of the number of immunogIObulin secreting cells and the average amount of im- mnunnoglobulin secreted per cell. The number of direct (IgM) and indirect (IgG) anti-SEE: plaque forming cells (PFC) from each spleen was determined.using a modification of the Jerne plaque assay as described.in detail elsewhere (19). A.nonimmunized.mouse, included as a control, produced less than one PFC per million spenocytes. For the determination of the average amount of immunogldbulin.produced per PFC, 10 x 10° splenocytes were suspended.in 1 m1 of culture medium supplemented.with 1% gamma gldbulin.free BSA (Calbiochem) and placed.in.a 12 x 75mm culture tube (Falcon) for two hours at 37°C and 7%.003. Supernatants were collected and the amount of IgG and IgM produced.was determined.by radioimmunosssay as described.below. Radioiodination. Mouse gsmma-gldbulin (Calbiochem), and mouse IgM (MOPC 104E, Bionetics) were radioiodinated in the presence of Iodogen, as previously described (20). Specific activity of the 69 iodinated immunoglobulin was 1.3 x 10° cpm/pg for IgM and 2.1 x 105 cpm/pg for IgG. Radioimmunoasgy. The amount of IgM or IgG produced in 2 hours by cultured splenocytes from mice immunized with SRBC was determined by radioimmunoassay. Supernatants to be tested or unlabeled im- munoglobulinns to be used as standards (mouse IgM, I‘DPC 104E, Bionetics or mouse IgG, Calbiochem) and 5 x 10’ pg of radioiodinnated IgM or IgG were added to 10 pl of norml rabbit serum, along with either affinity pm'ified rabbit anti-muse p or rabbit anti—mcuse gamma (Zymed) . The antibody complexes were allowed to form for 1 hour at 37°C. Then, the complexes were precipitated with 90 pl of goat anti-rabbit serum, which had been adsorbed with mouse imlmrnoglobulins. The precipitate was washed twice with borate buffered saline (pH 8.0) - 1%w/v BSA, dissolved in 0.1N W, and counted in a game counter. There was no cross reactivity as detected by this assay for mouse IgM with rabbit anti-mome game or for mcuse IgG with rabbit anti-mouse p. The amcunnt of immunoglobulin in the test super- natant was determined from a standard curve usingknown amcunts of IgM or IgG. The average amount of IgG or IgM produced per IgG or IgM plaque forming cell (PFC) was calculated as follows: Amount of immunoglobulin prodnced by 10° cells divided by PFC/10° cells = average amount of immunoglobulin produced/PFC For splenocytes from nonimlmunized mice, antibody production per PFC was zero as determined by this assay. 70 Statistics. The mean and standard error of the mean were calcu- lated for each treatment group. Probability values for the comparison of the zinc deficient and restricted groups to the control group were determined by a completely random ANOVA followed by Dunnett’s t Test . RESULTS The effects of a zinc deficient diet on growth, diet consnmnp- tion, thymus weight, degree of parakeratosis, serum zinc and splen- ocyte numbers are shown in Table 1. After a 30 day dietary period, the zinc deficient mice had consnmned 12% less diet than the control mice. The average severely zinc deficient mouse selected for study weighed 66% of that of control mice, mile the mcderstely zinc defi- cient mice weighed 72% of that of control mice. Past data indicates that inanition will have a greater effect on the lower than the higher weight group with regard to T-cell dependent antibody mediated responses and thymus weights (8) . The restricted mice, which consumed the same amount of diet as the deficient mice, weighed 82% of that of control mice. The degree of parakeratosis for the severely zinc deficient mice was about twice that of moderates (Table 1); restricted and control mice showed no signs of parakeratosis. The thymus weights of the severe mice were significantly smller than the moderates which were significantly lower (approximtely 50% of controls) than the restricted or control mice (Table 1) . There was no differece between the thynmns weights of the restricted and. control mice. In addition, the average number of cells per spleen from the severely and moderately zinc deficient mice was reduced by 43% and 47x, respec- tively, compared to zinc adequate mice. However, the number of splenocytes in the restricted group was rednced by only 17% compared to controls. Sera collected from the severely and moderately defi- cient mice to test zinc status and for use in culture contained 50 71 72 TABLE 1 Body Weight, Diet Consumption, Degree of Parakeratosis, Thymus Weights, and Serum Zinc Levels of Mice After 30 Days on Zinc Deficient or Zinc Adquste Diet Dietary Severely Moderately Zinc Group Zinc Zinc Restricted Adequnte Deficient Deficient Initial Body 16.9 1 0.07' 16.9 1 0.07 17.0 1 0.25 17.1 1 0.15 Wt. (a) Finnal Body 14.3 1 0.52° 15.8 1 0.15° 17.7 1 0.05° 21.8 1 0.20 Wt. (8) Food Consunption 73.4 1 4,453.: 73.4 1 4.45° 92.7 1 4.82 (3) Degree of 8.3 1 1.2» 3.1 1 0.4» 0.0 1 0.0 0.0 1 0.0 Parakeratosis‘I Thymus Wt. 6.5 1 0.8° 17.6 1 1.35 30.4 1 1.3 33.7 1 1.0 (8) Cells per Spleen 1.9 1 0.06° 1.8 1 0.10° 2.7 1 0.20 3.3 1 0.25 (x10'7) Serum Zinc levels 52.4 1 LB" 48.5 1 2.2° 101.9 1 2.7° 98.5 1 2.1 (nil/d1) Mean1SEdof5or8mice °p<0.01ascomparedtozincadequatemice 0 Average diet consumption by zinc deficient mice prior to separation into severely and mcderstely deficient groups 4 Sum of degree of parakeratosis assigned to eye, tail, ears, coat,ardanusonascaleof1to4 fr: fol flifi. 73 pg Zn/dl compared to 100 pg Zn/dl for control mice (Table 1). This was similar to zinc levels reported.in previous studies (15). In the first study, the capacity of residual splenic T-cells of deficient mice to proliferate and produce ILPZ in response to stimula- tion with Con.A was assessed. In this case, the splenocytes were cultured in serum free medium since FCS contains high levels of zinc (350 118 Zn/dl) which, in turn, my initiate i_n 11119 repair by cells from zinc deficient mice. Serum supplementation was not necessary fOr good.cell viability (70 1_6%) or proliferation in response to Con A. In these experiments the level of zinc in the medium was 8 pg Zn/dl. Optimal splenocyte proliferation by the zinc adequate group was Obtained on day 1 (24 hour incubation with Con A plus an l8 hour pulse with 3H Thymidine) (Figure 1). When pulsed on day 1, optimal proliferation for all dietary groups was Obtained.using 2 pg/ml of Con A (Figure 2). As can be seen from Figure 2, there was no significant difference in the proliferation of splenocytes among the four dietary groups regardless of the concentration of Con A. Optimal ILPZ production was Observed at 24 hours after stimulation with 2 pg/ml of Con A (data not shown). From Figure 3, it is apparent that production of IL-2 by the moderately zinc deficient and re- stricted.dietary groups was similar to that by the zinc adequate group. The production of IL-2 by cells from the severely zinc defi- cient mice though slightly lower than the control group was not sig- nificantly different . Figure 1. 74 Dose curve and kinetics in a serum free system of the proliferative response to Con A by splenocytes prepared from mice consuming nnorml laboratory chow. On day 1, 2, 3, or 4, 3H-thymidine was incubated with the splenocytes for an additional 18 hours. Incorporation of °H-thymidine into the DNA was determined. Each point represents themean1SEVIof 3 mice. 75 F70. x :33 3:28.68. 8632..-...» ConA(p.g/mi) Figure 2 . 76 Dose curve for the proliferative response in a serum free system to Con A by splenocytes prepared from severly zinc deficient, moderately zinc deficient, restricted, and control mice. an day 1, 3H-thymidine was incubated with the splenocytes for an additional 18 hours. Then, incorporation of 3H-thymidine into the DNA was determined. Each point respresents the mean 1 SE1 of five or six mice. 3H—Thymidine Incorporation (cpm x IO'3) 77 $3 3 1‘3 I O—-o Severe Zn Deficient o—-o Moderate Zn Deficient o---o Restricted a...“ Control _ Con A( [1.9/ml) Figure 3 . 78 IL-2 activity in serum free culture medium harvested at 24 hours from triplicate cultures with 2 pg/ml Con A- stimulated splenocytes prepared from severely zinc deficient, mcderstely zinc deficient, restricted, or control mice. IL-2 activity was measured by the assay using the IL-2 dependent CTTL-Z cell line. Each bar represents the mean 1 SE! of five or six mice. -._2‘- l O _n‘l--l‘-- I00 — q 0' ii IL - 2 activity (units/ml) N 0| I //////////]—+ Moderate Restricted Control 80 The ability of residual Tm -cells to respond to allogeneic cells during a MLC was also studied by measuring splenocyte proliferation and IL-2 prodtction. The MIC was supplemented with serum in order to obtain good cell viability and proliferation. In 5% FE supple- mented medium, optimal splenocyte proliferation was achieved for all dietary groups on day 4 (Figure 4) using 7.5 x 10° mitomycin C treated CS7Bl/6 stimulator cells. In a subsequent study using 5% PCS, optiml splenocyte proliferation on day 5 was obtained using 2.5 x 10° mito- mycin C treated cells (Figure 5). Surprisinngly, the zinc deficient groups had significantly higher proliferative responses than the control group; the proliferation by splenocytes from severely and mcderstely zinc deficient A/J mice were 185% and 142%, respectively, of controls (Figure 5). Since FCS conntains high levels of zinc (351 pg Zn/dl) , there was concern that the higher response by the deficient groups my have been due in part to the availability of zinc resulting in 1n_n v_it_ro repair by the deficient groups. To test this, we mini- mized the addition of zinc by usinng 0.8% EA (0.4 pg Zn/dl) and 0.5% autologous serum from zinc deficient (40 pg andl) to supplement the culture medium. It should be noted that the RENT-1640 and the various medium supplements contain only 8 pg Zn/dl. Figures 6 and 7 show the kinetics, and figures 8 and 9 show the dose response in zinc deficient or zinc adequate autologous serum plus EA. Using EA and autologous deficient sera (40 pg Zn/dl) or autologous adequate sera (107 pg Zn/dl), the optimal proliferation was to day 5 (Figures 6, 7) usinng 1 x 10° mitomycin C treated C57Bl/6 target cells (Figures Figure 4. 81 Kinetics of the MIC proliferative response in 5% FCS supplemented medium. A/J responder splenocytes (2.5 x 10°) from severely zinc deficient, moderately zinc deficient, restricted, or control mice were cultured with 7 .5 x 10'- mitomycin C-treated CS7Bl/6 splenocytes and pulsed for 18 hours with 3H-thymidine on day 3, 4, or 5. Each bar represents the mean 1 SD! for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses. 3H-Tinymidine Incorporation (x i0'3) 82 MIXED LYMPHOCYTE CULTURE (5% FCS) maven Zn Deficicni DModeroic Zn Deficient Restricted - Conlrol 3 4 5 Day Pulsed Figure 5 . 83 Dose curve for the MLC proliferative response in 5% FCS supplemented medium. A/J responder splenocytes (2.5 x 10°) from severely zinc deficient, moderately zinc zinnc deficient, restricted, or control mice were cultured with mitomycin C-treated C57Bl/6 splenocytes and pulsed for 18 hours with °H-thymnidine on day 5. Each point represents the mean 1 SE“! for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses . 3H-Thymidine Incorporation (x IO'3) 84 5% FCS l O—c Severe Zn Deficioni O-0 Moderate Zn Deficlonn O--0 Restricted but Control a l b ‘\ \ 00 a t \ I 25 . _ 7.5 C578l/6 cells (x IO 5) Figure 6. 85 Kinetics of the MIC proliferative response in 0.5% zinc deficient autologous serum supplemented medium. A/J responder splenocytes (2.5 x 10°) were cultured with 1 x 10° mitomycin C-treated C57Bl/6 spleccytes and pulsed for 18 hours with °H-thymidinne on day 4, 5, or 6. Each bar represents the mean 1 SIM for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses. Malacca: Scrum) 86 MIXED LYMPHOCYTE CULTURE (Zn Deficient 1m Day Pulsed / // TI r] cm mm am an an.“ / / ammm «dam amen I .mua- . a l25 - so). _ m 7 :7. .3 56.098... 36.82.75 Figure 7. 87 Kinetics of the MLC proliferative response in 0.5% zinc adeqnete autologous serum supplemented meditn. A/J responder splenocytes (2.5 x 10°) were cultured with 1 x 10° mitomycin C-treated C57Bl/6 spleccytes and pulsed for 18 hours with °H-thymidine on day 4, 5, or 6. Each bar represents the mean 1 SE! for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses. 3H-Tinyrrnidirno Incorporation (cpm 11 Id") 88 MIXED LYMPHOCYTE CULTURE (Zn Adequate Auroioqous Serum) m 5mm Zn Deficient D Moderate Zn Deficient '25 Ruiniciod - Control ‘1- ‘1 Oi 0| 0 4 5 Day Pulsed Figure 8. 89 Dose curve of the MU: proliferative response in 0.5% zinc deficient autologous serum supplemented median. A/J responder splenocytes (2.5 x 105) from severely zinc deficient, moderately zinc deficient , restricted, or control mice were cultured with mitomycin C-treated C57B1/6 splenocytes and pulsed for 18 hours with 3H—thymidine on day 5. Each point represents the mean 1 SIM for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses . 90 Molagous Serum Zn Deficient l l l H Severe Zn Deficient '2 e--e Moderate Zn Deficient 5 - °---0 Restricted .. h-uO Control I. ,. I . r. \ \ IOO , \ \ - . \ r \ \ . \ m... \ I so. . \ \\ 5 \ 75— , I \\ \ \ '- e‘ \ e“ \ t \ H-Thymidine Incorporation (com 3: l0'3) 3 25 l 2.5 _J‘J. CS7Bl/6 cells (x :6“) 7'5 91 Figure 9. Dose curve of the MLC proliferative response in 0.5% zinc adequate autologous senn supplemented median. A/J responder splenocytes (2.5 x 105) from severely zinc deficient, moderately zinc deficient, restricted, or control mice were cultured with mitanycin C-treeted C57Bl/6 splenocytes and pulsed for 18' hours with 3H--thymidine on day 5 . Each point represents the mean 1 SD! for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to control responses . 3H—Tliymidine Incorporation (cpm x IO'BI .24 mi 25 92 Zn Adequate Autoloaaue Serum ' o——e Severe Zn Deficient r --e Moderate Zn Deficient 0""0 Reetrlcted 5"“ Control ‘ 2.5 7.5 C57Bl/6 cells(x I65) 93 8, 9) . Regardless of whether the serum supplement contained adequate or limiting levels of zinc, proliferation by splenocytes from zinc deficient A/J mice was increased compared to proliferation by splen- ocytes from zinc adequate mice (Figures 8, 9). When zinc deficient autologous serun was used (40 pg Zn/dl) , the severely and moderately deficient groups gave significantly increased proliferative respon- ses of 141%and 127%, respectively (Figure 8), of the zinc adeqmte group. Using zinc adequate autologous serul (107 pg Zn/dl) (Figure 9), the severe and moderate groups again exhibited enhanced prolifera— tive responses (137% and 122%, respectively, of controls). However, statistical analysis indicates that only the severe group was sig- nificantly different from control. In zinc adequate and deficient autologous serun, the proliferation by the restricted group was not significantly different from the control group at the optiml stimu- latory condition of 1 x 105 C57Bl/6 target cells but was significantly different from controls at 2.5 x 105 057Bl/6 target cells. Even though FCS supported greater overall proliferation than autologous serun, there was increased proliferation by the deficient groups regardless of the source of sera. Therefore, the level of zinc present in the sera does not appear to account for the enhamed proliferative response of splenocytes from the zinc deficient dietary groups. Since proliferation by cells from zinc deficient mice was in- creased both in FCS and autologous serun, IL-2 activity was measured in supernatants from a Mm supplemented with FCS. Figure 10 shows that IL-2 activity was increased in the 48 hour supernatants from Figure 10. 94 IL-2 activity in 5% FCS supplemented medim harvested at 48 hours from quintriplet cultures of a MLC. A/J responder splenocytes (2.5 x 105) from severely zinc deficient, moderately zinc deficient, restricted, or control mice were cultured with 2.5 x 10' C57Bl/6 splenocytes. IL-2 activity was measured by the assay using the IL-2 dependent GILL-2 cell line. Each bar represents the mean 5; SE“! for five or six mice. Asterisk indicates significance of p < 0.05 or better as compared to the respective control for each concentration of CS7Bl/6 target cells. C to...» M 2 m a man u p u s (é T%//////////M/+///////////////////// ei%/////////////////#//////////////////////////////// in; at at Le 2.5223 23.85.... 96 the severe, moderate, ard restricted groups (185%, 131%, ard 155% of controls, respectively) at optiml conditions with 2.5 x 103 CS7Bl/6 target cells The mitoumcin C-treated cells serving as stimulator cells were unresponsive in the MLC. 'lhe mitouwcin C treated CS7Bl/6 cells did not proliferate in the presence of IL-2 nor did they respond to mitomycin C treated A/J splenocytes (Figure 11). Birthermore, IL-Z added to the culture containing only target cells was not absorbed nor was IL-2 activity produced by mitomycin C treated cells (Figure 12). Therefore, the proliferation ard the production of IL-2 in the MID was due only to the AM splenocytes. The observations that both IL-2 activity and cellular proli- feration of splenocytes from the zinc deficient groups were elevated suggested that there might be a concomitant modulation of IL—2 receptor nanbers. Therefore, it was of interest to examine the acquisition of IL-2 receptors during the MIC. The relative nunber of receptors was qmntitated by indirect inmnuncfluorescense of pooled cells‘fromeachdietarygroupondays3, 4, 5, ard60faMleupple- mentedwith 5%1'133. Qaday 1 ard20f theMlCthe percentage of fluorescence was very close to backgrourd (data not shown). Cyto- fluorographic analysis of labelled cells from the four dietary groups showed that the nanber of receptors per cell was not altered by zinc deficiency since, for all groups, the mean channel nunber of fluores- cence was approximately 555. However, the percentage of cells with IL-2 receptors was considerably increased for the severely zinc deficient group from day 3 (120% of controls) to day 6 (180% of Figure 11. 9? Proliferation of mitomycin C-treated splenocytes from mice fed norml laboratory chow. In the presence or absense of IL-2, mitonwcin C-treated 057Bl/6 splenocytes pooled from six mice were incubated in 5% FCS supplemented medium with untreated or mitomycin C- treated A/J splenocytes from 3 mice for 5 days and pulsed for 18 hours with 3H-thymidine. Each bar represents an individual sample. [inhumane (25200“ I M m carat/6' 'I-I-Thynnidine Incorporatianoth") No. mu -Cnnito.-C lL-Z 20 40 so so I00 + - + .. ' ' + ... _ .— I __ + + _ - + + + + - ‘+ - .+ _ _ _ . 2 - + + — - + + + + - + - + — _ .— 3 .. + + ._ — T + -I- + - - + + .. .. - + ...y Figure 12. 99 IL—2 activity in mediun harvested from mitomcin C- treated splenocytes from mice fed norml laboratory chow. In the presence or absense of IL-2, mitouwcin C treated C57Bl/6 splennccytes pooled from six mice were incubated in 5% PCS supplemented median with untreated or mitomycin C-treated A/J splenocytes from 3 mice. IL-2 activity was measured by the assay using the Ila-2 dependent GILL-2 cell line. Each bar represents an individual sample . 100 WRSXIO‘I 2501'! AN AN Bra “:2 lL-Zactlvlty (Unite/nil) "°"" u c l 2 a I U I + - + — I - + + — — + + + + — + - 2 — + + _ u l + +++ l 101 controls) of the MIC (Figure 13) . The percentage of fluorescently labelled splenocytes from the moderately zinc def icent group was the same as that of the zinc adequate group. There was a slight increase compared to controls in the nanber of splenocytes in the restricted group with IL-2 receptors; however, this increase occurred in the final days of the MIC whereas the percentage of splenocytes with IL- 2 receptors from the severely zinc deficient group was increased in the early stages of the MIC. In summary, allogeneic cell-stimulated proliferation and IL-2 production by the residml splenocytes from zinc deficient mice were increased compared to that for splenocytes from control mice. There was also an increase in the nanber of splenocytes with IL-2 receptors in the zinc deficient group; however, the number of IL-2 receptors per cell was the same for all groups. Next, the functional capacity of the residual B-cells of the deficient mice was assessed by examining the ability of antigen- activated B-cells to produce immoglobulins in response to SR3}. Splenocytes were removed from mice injected 5 days earlier with SRBC and analyzed for the nanbers of cells producing antibody (plaque forming cells, PFC) using the Jerne plaque assay and the amount of antibody produced per PFC using a radioinlnunoassay. 'lhe level of zinc intronhnced in £11.22 was minimized sinnce the cells were inncubated in median supplemented with 1% BSA (0.5 pg Zn/dl). As in several previous studies (5, 7, 8, 14, 19, 21), the nunber of PFC per spleen was reduced to 50% in the deficient groups whereas the nunber of PFC per million splenocytes reunined unaltered in the zinc deficient groups (data not shown). Table 2 shows that the average amount of Figure 13. 102 Percentage of splenocytes with IL-2 receptors. A/J responder splenocytes (2.5 x 109) from severely zinc deficient, moderately zinc deficient, restricted, or control mice were cultured with 7.5 x 105 057Bl/6 splenncytes in 5% FCS. On day 3, 4, 5, and 6, the IL-2 receptors on these splenocytes were labelled by indirect immofluorescense. Each bar represents the percent of cells, excluding cellular debris, that were fluorescently labelled for pooled cells of five or six mice. 103 Restricted a; ...new.nouen.nonewneuenomeuovwnovu noueneuewwwwwwnensue...” Q - severly zinc deficient 1:] - moderately zinc mm 7) - - Controlled no”Renown.“ewonononfiowfiorfio”Rowenowflononou.H $2.83.». _ _ m m 2.8 eozono. 328230.“. e\o IO- 5 7 A4 Days 104 TABLEZ Immoglobulin Production/PFC Dietary GNUPB P8 WI!“ PFC P8 180/ 13G PFC Severely Zinnc Deficiennt 22.1 1 4.7' 15.5 1 3.1 Moderately Zinc Deficient 29.6 i 5.2 6.9 i 2.8 Restricted 18.3 1 3.5 17.0 ~_f; 5.8 Zinc Adequate 25.4 1; 2.2 12.9 1 3.4 Unprimed Mouse N.D.b N.D. 'Meanis‘nEdoflltonice 1’ Not detectable 105 IgM produced per IgM PFC and the average amount of IgG production per IgG PFC for the zinc deficient and restricted groups is not signnificantly different than those values for the control group. Thus, the proportion of residual B—cells from zinc deficient mice which respond to sheep red blood cells and the amount of antibody produced by antibody secreting cells was normal. DISCUSSICN Although a variety of inmune responses are decreased in the zinc deficient animal (5, 7, 8, 9, 10, 11, 12, 13), the data pre- sented in this chapter indicates that the residual lymflnocytes from zinc deficient mice fuction as well as or better than lymphocytes from control mice. In response to Con A, splenocytes from either severely or moderately zinc deficient mice have the same response kinetics, proliferative capacity and ability to produce IL-2 as splenocytes from adequately fed mice uder serun free conditions. ’lhis was so even though serun free conditions were used to alleviate the possibility of repair due to the addition of serum zinc. Ex- emplifing the importance for careful regulation of the addition of the deficient nutrient in i_n_n_ vi_t_rg studies, are other studies which have shown that microbicidal defects of mscroplcges from zinc defi- cient mice were rapidly reversed by the addition of exogenous zinnc 11.1 m (22. 23) . Contradictory results are reported in the literature for stimula- tion of zinc deficient splenocytes by Con A (24, 25, 26, 28). Dif- ferences in experimental approaches account for mat of the differen- ces in results. Flynn (24) and Zanzonico e_t_.. a_._l. (25) who utilized i_n_n £121.? depletion of zinc by chelation found decreased proliferative splenocyte responses to Con A by splennocytes frm deficient mice. Flynnnn (24) used chelex 100 for depletionn of zinc from media to be used in his assays whereas annzonico e_t. _a_l. (25) added EDTA directly to the stimulated splennocytes. 'lhe EDTA would chelate a lot of 106 107 other metals since it has a low affinity for zinc making comparisons between studies difficult. Indeed in those studies, there was no evidence that zinc was, in fact, inaccessible to the cells. With regard to Flynn’s studies, it my be that short term i_n vi_tro deple- tion of zinc simply does not have the same effects as 30 days of zinc deprivation in gigg, especially, since in zigg zinc depeletion leads to activation of the stress axis (27) that adversely effects lymphocyte function. Studies by Gross gt. al. (26) using zinc defi- cient rats showed that the Con A response was reduced for splenocytes but normal for thymocytes. Hawever, there has been some controversy over this data since proliferation.was presented.as a stimulation index instead of counts per minute of total radioactivity. Data presented as stimulation indices can be misleading unless the counts per minute of the unstimulated controls are the same. Kramer gt. al. (28) found.that Con A.responses by splenocytes from zinc deficient rats were increased in 15% heat-inactivated fetal calf serun but normal in 2% autologom serum from zinc deficient, restricted, or zinc adequate rats. The latter agrees with our results for zinc deficient mice. In contrast to the normal mitogenic responses to Con A, proli- feration and.IL~2 production.by'Ti-cells in.response to allogeneic cells was increased in both the severely and.mnderately zinc defi- cient groups; values for the severely zinc deficient group were most elevated, The increased response was not the result of repair by the deficient cells through utilization of zinc present in FCS since there was also an increased response in the presence of zinc deficient 108 autologous serun. The increased response to allogeneic cells may have been due to an increase in the proportion of cells with IL-2 receptors and/or to an increase in nanber of IL-2 receptors per cell during the onset of the MIC response. An increase in nanber of receptors per, cell my lower the threshold for stimulation needed to begin cell-cycle progression (29) . The data showed that there was no measurable increase in nanber of IL-2 receptors per cell in the zinc deficient groups during the MIC repsonse since the mean channnel nunber of fluorescence was the same for all dietary groups. In contrast, there was a modest but measurable increase in the nanbers of cells with IL-2 receptors in the severely zinc deficient group by day 3 of the MIC. There are several possible explanations for the enhanced MLC responses . There may be acre immature T—cells with the surface mrkers Lyt 2,3 and high levels of Lyt 1 (this cell type is now identified by Lyt 2,3 and L3T4) in the zinc deficient mice, since this cell type is thought to be the mjor responders in the primary MLC (30). Indeed, Nash g. al. (31) ncted an increase in the propor- tion of inmture T—cells in the spleens of zinc deficient mice. Therefore, in the deficient group, more cells would be available to respond in the MIC with a concomitant increase in IL-2 production and IL-2 receptors as was observed. Thus, in contrast to Con A, which stimulates manny T-cell subsets (32) , the MIC stimulates a more narrow subset of T-cells. Therefore, with proportional channges in cell nanbers amoung T-cell subsets, one would not expect alterations in polyclonal proliferation in response to Con A but would expect 109 increased proliferative levels in a MLC with an increase in number of cells in the responding T-cell subset. An alternative explanar tion for the enhanced.MLC may be that there is loss of ILPZ inhibitor activity in the zinc deficient mice. It has been suggested that there is an inhibitor of ILPZ driven T—cell proliferation (33). Tadmori, Kant and Kamoun (34) also propose that an inhibitor protein controls ILrZ mRNA production. Perhaps zinc plays some role in the production or function of this inhibitor so that with loss of zinc there is a loss of inhibition and consequently overproduction of IL~ 2 followed by increased cellular proliferation. Conversely, the degree of production of the inhibitor may be altered.by the presence of corticosterone produced.during the course of the zinc deficiency. Perhaps, the Thcell subset responding in the MLC has lost ILPZ in- hibitor activity. Another possibility is that some other mechanism of suppression, such as loss of suppressor cell activity towards the MLC response but not the Con A response, was also impaired. Al- though the Tl-cell responses to allogeneic cells were increased, the residual Ti-cells were able to function at least as well as the zinc adequate controls. Therefore, the reduced T-cell dependent responses in giyg (5, 7, 8, 9, 10, 11, 12, 13) may be due to the reduced numbers of cells (5, 6) since those residual T-cells responding to Con A or allogeneic cells retain normal functional capacity. The residual B~cells from zinc deficient mice also retain normal capacity to produce Ig. With initiation of the immune response to SRBC in the host, normal quantities of antibodies were produced by individual activated Bncells from zinc deficient mice compared to 110 B—cells from control mice. In addition, the number of antibody secreting cells was normal per million splenocytes, but the number of antibody secreting cells per spleen was reduced (5, 7, 8, 14, 19, 21). Previous reports also show that proliferation by residual B- cells from zinc deficient mice is nonmal or increased in response to several mitogens (5). The mitogenic response to lipopolysaccharide and dextran sulfate is increased for zinc deficient mice whereas the mitogenic response to purified protein derivative is the same as the zinc adequate controls. It was suggested that the elevated responses were due to an increase in nanber of inmture B—cells in zinc defi- cient mice since the relatively immature subset of B—cells responds to lipopolysaccharide and dextran sulfate (35, 36). The above cited results and the present findings indicate that the reduced B—cell responses by the zinc deficient mouse are most likely due to reduced numbers of cells and not reduced functional capacity of the residual B-cells. In conclusion, the functional capacities of the residual T- cells and.B~cells were normal for zinc deficient mice with regard to stimulation,with Con A or allogeneic cells, and SRBC, respectively. This is the first diverse study of the function of residual lym- phocytes in a nutritionally'depleted.animal where the in xitgg ex- posure to the deficient nutrient was regulated” The data indicated that the surviving lymphocytes in zinc deficient mice are normal for many immune functions and do not show evidence of significant altera- tion in cell processes dependent on zinc. 10. 11. 12. Hambridge , White, M. , References K. N., Walravens, P., Brown, R., Webster, 8., Anthony, M., and Roth, M. (1976) Amer. J. Clin. Nutr. _2_9, 734. Prasad, A. Sandstead, Sandstead, (1979) Ann. Rev. Phsrnnacol. Toxical. 2_9, 393. H. (1973) Amer. J. Clin. Nutr. _2_6_, 1251. H., Henriksen, L., Gregor, J., Prasad, A., and Good, R. (1982) Amer. J. Clin. Nutr. 3Q, 1046. Fraker, P. J., DePasquale-Jsrdieu, P., and Cook, J. (1988) Arch. Derm. (in press) Wirth, J. J., Fraker, P. J., and Kierszenbaun, F. (1984) J. Nutr. _1_1_4_, Fraker, P. Nutr. L”, Luecke, R. Nutr. _1_08, Fraker, P. Zwickl, C. 611. Fraker, P. Nutr. 13, Fraker, P. Nutr. 1_1_2_, 1826. J., Haas, S. M., and Luecke, R. W. (1977) J. 1889. ' W., Simonel, C., and Fraker, P. J. (1978) J. 881. J. (1983) Survey Imnnnol. Res. -_2_, 155. M., and Fraker, P. J. (1980) Imnnol. Comm. Q, J., Hildebrandt, K., and Luecke, R. W. (1984) J. 170. Je’ ZWiCkl, Ce Me, Elli we, Re We (1982) Je 309 . 111 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 112 Fernandes, G. Nair, M., Chloe, K., Tannaka, T., Floyd, R., and Good, R. A. (1979) Proc. Natl. Acad. Sci. U.S.A. Z_6_, 457. DePasqnnle-Jardieu, P., and Fraker, P. J. (1980) J. Inmunol. _1_24, 2650. Mishell, R. I., and Dutton, R. W. (1967) J. Exp. Med. 13_6, 423. Gillis, S., Ferm, M. M., Ou, W., and Snnith, K. S. (1978) J. Imnnol. 1_29, 2027. Malek, T. R., Robb, R. J., and Shevach, E. M. (1983) Proc. Natl. Acad. Sci. U.S.A. Q, 5694. Ortega, R. G., Robb, R. J., Shevach, E. M., sndi‘hlek, T. R. (1984) J. Imunol. 1513, 1970. Fraker, P. J., DePasquale-Jardieu, P., Zwickl, C. M., and Luecke, R. W. (1978) Proc. Natl. Acad. Sci. U.S.A. 15, 5660. Fraker, P. J., Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Coumnun. 89, 849. Luecke, R., and Fraker, P. (1979) J. Nutr. 109, 1373. Wirth, J. J., Fraker, P. J., and Kierszenbann, F. (manuscript in preparation). Fraker, P. J., Jardieu, P., and Wirth, J. (1986) In "Nutritional Diseases: Research Directionns in Comparative Pathobiology" pp. 197-213. Alan R. Liss, Inc, New York. Flynn, A. (1984) J. Nutr. M: 2034. Zanzonico, P., Fernandes, G., and Good, R. A. (1981) Cell. Inmmnnnol. g, 203. Gross, R. L., Osdin, N., Fang, L., and Newbernne, P. M. (1979) 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 113 Am. J. Clin. Nutr. 3_2, 1260. DePasquale-Jardieu, P., ard Fraker, P. J. (1979) J. Nutr. _1_9_9_, 1847. Kramer, T. R. (1984) J. Nutr. 1L4, 953. Smith, K. A. (1984) Ann. Rev. Inlmnnol. g, 319. Simon, M. M., and Abenhsrdt, B. (1980) Eur. J. Immol. 19, 334. Nash, L., Iwata, T., Fernandes, G., Good, R. A., and Incefy, G. (1979) Cell. Imnnol. $8, 238. Jandinski, J., Cantor, H., Tadakune, T., Peavy, D. L., Pierce, C. W. (1976) J. Exp. Med. _14_3, 1382. Harowitz, J. B., Kaye, J., Conrad, P. J., Katz, M. E., and Janeway, C. A., Jr. (1986) Proc. Natl. Acad. Sci. U.S.A. Q, 1886. Tadmori, W., Kant, J. A., andKamoun, M. (1986) J. Inmuncl. 136, 1155. Gronowicz, F., and Coutinho, A. (1975) Scand. J. Imnnol. _4_, 429. Gronowicz, F., Coutinho, A., and Moller, G. (1974) Scand. J. Immol. _3_, 413. Chapter 3 WED ”EDITIONS PCB MEASURING Heoe WION BY RESIDENT W: mDIFICATImS FOR USE WITH PARASITES 114 The assay conditions described by Pick and Mizel which utilizes phenol red to measure HaOn production by neutrophils or elicited macrophages has been improved so that the amount of mo, produced by resident macrophages can now be measured. As opposed to the negli- gible amounts of 1130; production (0—5 M) previously obtained, phorbol 12-myristate 13-acetate (INA) or opsonized zymosan but not zymosan alone readily stimlated i120: production (30 (M) by resident peri- toneal nnacrophages using the modified conditions for the Pick assay. The modifications made for optiml Heoe production included an in- crease in the concentration of resident peritoneal macrophages, addition of 0.5 m CaCla to the reaction solution and an incubation at 37°C uder ambient air. Addition of 2 I“ W: to inhibit scaveng- ing of H20; by catalase did not affect H20; production by resident nnacrophages . When attempting to stimulate resident macrophages with the parasite Mosaic c_rngi (L M), it was foud that the phenol red employed as the substrate in the Pick assay rapidly killed the parasites. This problem was rectified by using an alternnative substrate, homovanillic acid (HVA), and eliminating the M: from the reaction solution. With HVA as substrate, the optimal conditions for H30; prodnction included the presence of 0.09 an M12 and 0.05 M MCI: and incubation at 37°C with 7% co. and ambient air. In the past it had not been possible to directly measure P130: production by mcrophages stimulated with _T_. c_rgz_i. With the modifications sug- gested herein, consistent and good production of Knox (3-10 am 115 116 could be obtained for the first time. Further, it was noted that opsonized L c_ru_z_i elicited at least twice as nnch H20] production as nonopsonized parasites. In addition, as the number or ratio of parasites to mcrophages increased, there was a corresponding increase in H20: production. In smllery, now it is possible to measure i120; production by resident necrophsges stimulated with nonliving agents such as PMA or opsonized zyncsann or with a living pathogen such as L cruzi . Futher- more, the HVA assay will now make it possible to better assess the significance of 1120; in the killing of L cruzi by infected mcro- phages. Introdnction Ann assay described by Pick e_t. a_.‘l_. (1) has beern conlnonly used to measure 3202 production by neutrophils and elicited necrophages (1-4) . This assay utilizes the horseradish peroxidase catalyzed oxidation of phenol red to an unknown prodnct that absorbs at 610 run. It mnct be ennphssized that in the past HeOn PPOd'UCtion by resi- dent necrophages has not been measurable since the sensitivity of this assay was far too low for quantitation of resident necrophage 1130. production (2, 5, 6, 7, 8, 9). Conditions were described herein that made it possible to assess 11:0: prodnction by resident necro- rinsges. However, for our studies, it was necessary to measure HzOn prodnction from resident nnacrophages using T_‘_. c_ru_z_i since we were I interested in examining the fuctionnal capacity of resident mcro— phages from zinc deficient mice. These macrophages are deficient in their ability to associate with and kill the unicellular parasite, Mosaic £112.; (L M) (10, 11). Since destruction of L c_ru_z_ihasbeenassunedtoberelatedto the ancuntlofI-hon produced (12, 13), we wanted to compare the ability of macrophages from zinc deficiennt mice and those from zinc adequate mice to produce H30: . Defective 11.0. production was presnmned to be one explanation for the reduced ability of macrophages from deficiency to kill parasites. For this part of the studies, it became necessary to modify the prevailinng assay conditions so that the amount of 1430. produced by resident macrophages could be measured. This was done. However, it 117 118 was subsequently foud that L M was killed by phenol red which is the major substrate of the Pick assay (1) . This would explain why in the past other investigators were unable to use the parasite as the stinnnlant for i130; production. Instead, the ability of macro- phages to produce H30. upon stimulation with the chemical PMA had been mathenctically correlated with killinng of L cruzi by the mcro- phages (14). In addition, 14.0. production by infected cells had ' been demonstrated qualitatively by a cytochemical stain for H20; (12). Thus, it was presuned that HaOn was essential to killing of L Qua; since the ability of phagocytic cells to kill L c_ru_z_i had been shown to be inhibited by the 8.0. scavennger, catalase, but not by inhibitors of other oxygen metabolites (13, 14). The above experiments were the sole evidence that L gru__§i_ stimulated H30: production and that the HeOn produced was essential to the killing of L cruzi. Yet, it had never been shown directly that L 9M; stimulated HeOn production by macrophages. Therefore, additional modifications were mde in the 11:02 assay using HVA as a substrate so that L mi could be used as a stimulant of resident peritoneal necrofinages. This ncdified assay will be important in determining the significance of 11303 to the killing of L cruzi. Likewise, it will also be possible to more clearly assess the role of Han in the killing of other living pathogenns and parasites such as Leshmania and Plasmodiun falcim. MATERIALS ANDME'ITDDS Aninnals. A/J female mice were purchased from Jackson labora- tory, Bar Harbor Maine. Fennale Crl-CD—l (IRC)BR Swiss mice were purchased from Charles Rivers, Portage, Michigan. Collection of mouse seran. Seran was collected from mice by severing the subclavian artery. The blood was incubated for 15 minutes at 3700 followed by several hours at 4°C. Mouse seran was complement inactivated by incubating at 56°C for 30 minutes. Isolation of T_rmosona cru_zi (T. cruii). Four week old Crl- CD-l (IRC)BR Swiss fencle mice were infected intraperitoneally with 2 x 105 blood forms (tryponcstigotes) of the Tulahuén strain of L M. Twelve to 14 days later, blood was collected from the axillary artery of mice anesthetized with ether. Blood was collected in tubes containing heparin or disodian ethylene-diaminotetrscetate (EUl‘A) powder so that the final concentrationns were 25 U/ml or 2.5 mg/ml, respectively. Parasites from the blood were separated on a lympholite gradient (isolymph, Canard-Schlesinger, Carle Place, N.Y.) (15) and passed through a diethylaminoethylcellulose column (16). The parasites were centrifuged (800 x G, 15 minutes, 4°C) and resuspended in Dulbecco’s modified miniml essential median (mm, Gibco, Grand Island, NY) supplemented with 100 IU penicillin and 100 pg streptomycin / ml. (he hudred percent of the parasites were viable and in the tryponnastigote form as determined by morphology and nctility. Before use in the assays for I120: . the parasites were 119 120 mshed two times in phosphate buffered saline (PBS) with 1% glucose and resuspended in the reaction solution for the HaOn assay. Mich of opsonized zmsan and opsonized T. cruz' . (me mg of zymosan, a yeast cell extract (Sigma, St. Louis, m), was incubated with one milliliter of complement inactivated norml ncuse seran from NJ mice for 1 hour at 37°C. The opsonized zymosan was washed 2 times and resuspended in 10 nfl PBS (pH 7.4). L _c_ru_zi_ trypomastigotes were opsonized by incubation for 1 hour at 37°C in 10 m PBS (pH 7.4) with 1% glucose and 20% complement innactivated seran from chronic L c_ru_Li infected CD1 mice. The opsonized L wiweremshedZtincsandresuspendedinIOnfims (1117.4). The seran was obtained during the chronic phase of experimental ' Chagas’ disease which occurs 5-6 weeks after the initial infection. Chronic mice have normal inlnune responsiveness but continue to harbor the parasite and produce L carpi-specific antibodies (17, 18) . Collection, isolation, and identification of Eritoneal necro- Mgg. Cells were harvested from the unelicited ncuse peritonean by lavage with 5 ml of cold (4°C) mm and 0.5% w/v gamma. globulin free bovine seran albanin (BSA, Calbiochem, La Jolla, CA) (19). Cells were adhered to tissue culture plates for 1 1/2 hours at 37°C and 7% (Dz-ambient air. Nonadherent cells were removed by washing with 37° C THEM. The percentage of mcrophages/ncnccytes adhered to the culture plate was determined by staining for nonspecific ester- ase activity as detailed in the modification (20) of the technique described by Yam et. al. (21). Always, 94-100% of the adhered cells 121 were identified.as mcnocytes/macrophages by staining for nonspecific esterase and.by morphology. PhenglfRed.A§§§y. The assay conditions described by Pick and Mizel (1) were ncdified for use with resident macrophages. (he hundred.microliters of 7 x 10° resident peritoneal exudate cells/ml were added.per well to a 96 well flat bottom plate (Corning Glass Works, Corning, NY) and allowed to adhere for 1 1/2 hours. .After removing nonadherent cells, the adherent macrophages were washed with 10 M phosphate buffered saline (PBS) - 1% glucose (37°C). Forty microliters of the assay solution consisting of 140 nM NaCl, 0.5 nfi C8013, 10 nM potassian phosphate buffer (pH 7.4), 2 all We, 5.5 a“ glucose were supplennented with 0.56 nM phenol red (United States Biochemical Corporation, Cleveland, Ohio), and 19 U/ml horse- radish peroxidase (Sigma, St. Louis, MO) and.added to each well. NaNa was included since it inhibits catalase, the cytoplasmic scaven- ger of H30; . Phorbol 12-nwristate 13-acetate (PMA, Consolidated Chemicals) was diluted in ethanol and.added to the cells in a volume of 2 pl per well so that the ethanol concentration was less than 1%. Opsonized zymosan or PMA stimulated cells were incubated for 90 minutes at 37°C uder a hanidified atmosphere of 7% (Dz-ambient air, ambient air, or 10% (I): , 7% On and 83% N: . Standards consisted of wells containing reaction solution and 0-60 “M 14:03 but no cells. Nonspecific production of 1120: was determined by including wells containing cells but no stimulant or stimulant but no cells. The reactions were stopped and protein dissolved by the addition of 2 pl of 10 N NaOH. The absorbance at 610 nm of pooled triplicates was 122 determined using a microcuvette and a spectrophotonneter (Gilford, Oberlin, Ohio) . bbcrophage protein was determined by the Lowry (22) . The data are presented as nmoles Hem/mg macrophage protein from the following calculation: (11M HzOz derived from the standard curve) X (1 liter/1000 ml) X (0.132 ml final volanne of sample) X (1000 nmoles/1 pmole) / (mg macroninage protein determined by the Lowry) = nmoles of HeOn/m macrophage protein. Homovanillic acid assay for Hag; . The assay conditions described by Rush e_t_. a_]_.. (23) were ncdified for use with resident nnacrophages with L 931an trypomstigotes as the stimulant. (he milliliter of 2 X 10° resident peritoneal cells/m1 was added per well to a 24 well flat bottom plate (Coming) and allowed to adhere for 1 1/2 hours. After rencval of the nonadherent cells, the adherent macrophages were washed with PBS - 1% glucose (37°C). Two hudred microliters of reaction solution which consisted of PBS, 1% glucose, 0.09 nM CaCla , 0.05 n1“! MgClz , 200 pM homovanillic acid, and 2 U/ml horseradish peroxidase were added per well. The opsonized zymosan or EMA was added in a smll volane (2-10 pl). When parasites were the stimulant, they were resuspended in the 200 ul of reaction solution inmediately prior to addition to the nnacrophsges. Standards consisted of wells containing reaction solution and 0-20 pM HzOn but no cells. Non- specific production of PhD; was determined by including wells contain- ing cells but no stimulant or stimulant but no cells. The plates were centrifuged at 50 x G for 3-4 minutes to increase parasite- nccrophage interactions and incubated for 90 minutes in a hanidified atmosphere at 37°C and 7%VCXh-smbient air. The reaction was stopped 123 by the addition of 25 pl of 25 uni EDTA and 0.1 M glycine at pH 12. The relative fluorescence of the homovanillic acid dimer was deter— mined using a‘spectrofluorometer (Perken-Elmer 650-40) equipped with a microcuvette. The excitation and emission wavelengths were 312 nm and 420 nm, respectively. The slit widths for excitation and emis- sion were 2 nm and 5 nm, respectively. The ancunt of 1120: in the samples was analyzed by linear regression of the standard curve. ' The amount of mcrophage protein was determined by the Lowry (22) . The data are presented as nncles Hzoz / mg macrophage protein from the following calculation: (HM 1420; derived from the standard curve) X (1 liter/1000 ml) X (0.225 ml final volane of sample) X (1000 nncles/1 pmole) / (mg nnacrophage protein as determined by the Lowry) = nmoles H202 / mg mcrophsge protein. Association of T. orgzci with macroMes. After incubating the necrophages with parasites for 90 minutes in THEM at 37°C and 7% (Dz-ambient air, the nonassociated parasites were rencved by washing three times with PBS - 1% glucose. The cultures were incubated in DIEM for another 18 hours and/or fixed with methanol for 5 minutes, allowed to dry, and stained for 1 hour with Giemsa in 10 nM phos- phate buffer (pH 6.8). The cells were washed one time with 10 nM phosphate buffer (pi-I 6.8). Nanbers of parasites per 100 macrophages and percent of macroflnages associated with parasites were determined by connnnting at least 200 cells per replicate. MacroQage viability. Viability of the adherent nnacrophsges was determined usinng the trypsn blue exclusion method. Macrophage viability was always >95%.. 124 Statistics. The mean and standard error of the mean were calcu- lated for each group. Probability values were determined by a com- pletely random Anova followed by Thmnett’s t Test. RESULTS The standard assay for 1130; described by Pick and Mizel (1) utilizes the horseradish peroxidase catalyzed reduction of phenol red to an unknown derivative absorbing at 610 nnm. In the past, resident macrophages (i.e. macrophages not activated or elicited i_n_n y_i_vg ) have been shown to prodnce very low or nonmneasurable ancunts of H302 even though a variety of stimulants were tested (2, 5, 6, 7, 8, 9) . Therefore, to improve the assay conditions for use with resident macrophages , the concentration of mcrophages was increased from the usual rsnnge of 3 x 105 peritonneal cells plated/ml reaction solution to 1 x 10" peritoneal cells plated/m1 of reaction solution (1) to 1.75 x 10" peritoneal cells plated/ml reaction solution. Thus, the concentration of macrophages herein was 2 to 58 times that described in Pick and Mizel (1) . Using these concentrations of cells, it became possible to readily measure peroxide production by resident nccrophages using a variety of probes (Table 1). With the standard assay, 20 pg of PMA elicited the production of 10 pmoles of mo. per mg mcrophage protein which was the optiman that could be obtained uder these conditions. However, as little as 12.5 pg of EMA elicited four times as much peroxide production using the suggested cell concentrations (Table 1) . More striking differences were obtained with opsonized zymosan which must be phagocytosed by the mcrophage. Using the ncdified conditions, a rsnnge of concentrations of opsonized zymposan failed to produce detectable quantities of 1120; (approximately 1.0 nmoles 125 126 Table 1 Hz 03 Production by Resident Peritoneal Macrophages Assay Stimulant Ancunt of nncles 1130; /mg Conditions Stinmlant nnacrophage protein --- 0 3e5 i 1e7 Pick & PMA 2 ng 3.4 1 0.6 Mizel Phenol 20 ng 10.0 1 2.4 Red Assay‘ 80 ng 8.8 1 1.3 opsonized 20 pg 1.1 1 7.2 zymosan 50 pg 0.0 1 4.8 100 pg 1.4 1 5.7 --- 0 7.0 1 1.3 Modified Phenol PMA 0.0125 ng 8.8 Red Assay" 12.5 ng 40.5 1 2.9 250 ng 40.4 1 1.0 500 ng 4.3.6 1 3.9 opsonized 12 pg 42.6 1 4.0 zymosan 1' Assay described in Pick, E., and Mizel, D. (1981) J. Inlnunnol. Methods 46, 211-226. Conditions for comparisonn: 0.3 x 105 to 10 x 105 peritoneal cells in 100 pl reaction solution, no CaCla , no NaN; , incubation in 5% (1);-ambient air at 37°C. 1’ Modified Phenol Red Assay described herein. Conditions for comparison: 21 x 105 peritoneal cells in 120 pl reaction solution, 0.5 nfl CaClz , 2 n1“! NaNa ,. incubation in ambiennt air at 37°C. 127 1130; /mg nnscrophage protein) (Table 1). However, 40 pmoles Pinon/IDS necrophage protein were readily obtained with the modified assay system. It should be noted that the qnmntities of 1130; produced by both PMA and zyncsann were readily and reliably quantitated since they coincided with the middle of the standard curve (Figure 1) . Since previous investigators had used a variety of atncspheric conditions with mononuclear cells (1-14) , various incubation condi- tions were also evaluated for the modified phenol red assay. The conditions tested were ambient air, 7% (1);-ambient air, and the conditions described by Mishell and Dutton for leukocyte cultures that uses (24) 10% (D3, 7% 01 and 83% Na. Under ambient air (about 20% On and (0.1% 003) or 7% (Dr-ambient air, there was twice as much 1130; production (Figure 2) by resident nnscrophages whether opsonized zymosan orPMAwereusedthanwhen 10%CDn, 7%02, and83%Nz was used. Thus, an increase in atmospheric 0: caused a significant increase in HzOn production (Figure 2) when utilizing the phenol red assay. This is probably due to the presence of more 0: as a substrate for NADPH oxidase which catalizes the production of On‘ which in turn dismutates to H.203 . Therefore, the remaining studies using the phenol red assay for H202 were carried out uder ambient air. In an attempt to optimize the amount of H:On produced by the resident macrophages, CaCla (0.5 M) which is required for phagocy- tosis and NaNa (2 nfl) which inhibits the Kroc-scavenger catalase were added to the reaction solution (Figure 3). When present, calcian increased the ancunt of 1130; production (525%) when nacrophages were stimulated with opsonized zymom, a particle which is phagocytosed Figure 1. 128 Standared curve for 1130. production in the modified phenol red assay. Various concentrations of reagent 11:02 was incubated with the reaction solution for one hour since the reaction goes to canpletion by 1 hour and the product is stable for several hours. 129 0.8 - _ 6 4. 0. O A E: 0.3 3:35.361 0.2 - 40 50 60 30 20 H202 (FM) 130 Figure 2. Modified phenol red assay: Atncsrineric conditions for optimal n.0, production by adherent mcroflnages from 2. 1 x 10° resident peritoneal cells. mcrophages were incubated with 12 pg opsonized zymosan or 3 pg PMA for 2hoursinthepresence of 0.5MCaCla andZnMNaNa. nmoles H202/ mg macrophage protein 80 7O 60 50 4o. 30 20 131 now. 00,. 7% oz, 83% N2 D 7% cat, ambient air . ambient air 4 '27“; opsonized zymosan , no stimulant Figure 3. 132 Modified phenol red assay: Requirements for 0.5 nfl CaClz and 2 nfl NsN; for optiml HaOn production by adherent mcrophsges from 2.1 x 10° resident peritoneal cells. Macrophages were incubated with 6 or 12 pg opsonized zymosan or 3 pg INA for 2 hours uder ambient air. 133 3% PMA ..a/////////.///////////////////// 4 mr. mm mum sax/zzzzzznr/z/z/z/zé m m 0 M d c. n. .m. m. ozm ... Mm WWW ZZZ/Ac w an. - mm 7%? m _ _ — _ _ _ _ F O O O 8 7 6 w w w m m 5295 massacres... uE \NONI $.05: 134 upon binding to receptors on macrophages (Figure 3) . Addition of calcian did not significantly increase 1120: Production (7%) when the stimulant was the nclecule PMA. This is probably due to the fact that EMA diffuses through the nccrophage membrane; thus , receptor mediated phagocytosis does not occur (Figure 3). The concentration of NaNa usually used to inhibit cellular catalase (25-32) did not affect the ancunt of 1130. produced by the macrophages stimulated with PMA or opsonized zymosan. This suggests that cytoplasmic cata- lase is not scavenging significant amounts of 11302 . In summary, uder the optinnal conditions described above, H30; production by resident macrophages can be measured since the ancunt of 1130; produced is now near the middle of the linear portion of the standard curve. To determine the distribution of HaO: production, extracel- lular, intracellular, and total amounts of 1120: were measured. The total ancunnt of 1130: produced by the macrophages can be assessed by adding New directly to the cells and reaction solution. Extracel- lular 1130; may be measured by adding the NaOH to the reaction solution after rencval from the cell monolayer. Fresh reaction solution and A NaOH was added to the rennaining cell monolayer to determine the ancunnt of intracellular H30; . As can be seen from Figure 4, the majority of detectable 1130; was in the extracellular environment with negligible amcunts remaining in the intracellular environment. Although this modified phenol red assay can be used with nonliv— ing stimulants, whenn the stimulant is a living parasite such as the obligate intracellular parasite L cruzi, no mo. was produced. It was noted that the parasites died rapidly in the reaction solution. Figure 4 . 135 Modified phenol red assay: Measurement of total, extracellular, and intracellular 1130. produced by adherent macrophages from 2.1 x 10‘ residennt peritoneal cells. Phcrophages were incubated with 14 pg opsonized zymosan in 0.5 mid 0801:, and 2 m We uder ambient air. Total H202 wasmeasuredbyaddingNaDHtostop the reactions to the cells and reactionn solution. Fbctracellular 1130; was measured by sddinng the Nam! to the reaction solution after removal from the cells. Intracellular 11:03 was measured by adding fresh reaction solution to the remaining adherent macrophages and adding Neal. 80 20 nmoles H202/mg macrophage protein \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ A intracellular extracellular total (i4pg opsonized zymosan) 137 Annalysis of the components of the reaction solution indicated that phenol red, the substrate for the assay, was toxic to L m (Figure 5) . The toxicity of phenol red was tested by incubating the para- sites at 7% (X); -ambient air for 2 hours in several solutions (without macrophages) . Parasites were 100% viable in HEM. Upon addition of 0.56 m phenol red to either IMF)! or the assay solution, no viable parasites remained after 2 hours. Interestingly, if horseradish peroxidase was added to the normal reaction solution at the same time as phenol red, it provided some protection to the parasites (approximately 40% viability). The concentration of phenol red can not be reduced in the assay since the substrate would becoming limit- ing in the reaction (1). An alternative substrate for the assay, homcvannillic acid (HVA), has been described by Rush gt. a_l. (23). A HVA has been rarely used as a substrate when assaying for cellular production of H20: . However, HVA proved very useful in our system since the HVA concentration (200pM) necessary for measuring H30: production did not destroy L $32; either in the presence or absence of horseradish peroxidase in the reaction solution (Figure 5) . Asinthecaseforphennol red, itwasnecessarytomodifythe conditions of the HVA assay described by Rush e_t. e_l. (23) for me with resident peritoneal cells. The concentration of cells was increased from 2.5 x 105 cells plated/ml reaction solution (23) to 1 x 10" resident peritoneal cells plated/ml reaction volumne. Thus, the macrophage concentration was 40 times that described by Ruch 91. g. (23) To further mcdify Ruch’s assay, the reaction volane was reduced from 2 ml to 0.2m to increase the concentration of 1120: . 138 Figure 5. Viability of L cruzi after 2 hours in solutions containing phenol red or homovanillic acid, substrates for the assays measuring HaOn . Viability was determined by counting the remaining parasites. 139 3H :3 .03.... 22.32... 63.328 EEO. .8830 2:66 3.000 SEcd ..002 260! do 332.00 8.3.3 .336s o 0 ill Om or Cd .32. N 3 Eco F 2......' .\. 03033 or. 2.0 3.... 1.: 0::0n neat-5 20.33%. 2E0. c. 300123 528.020.. .EBN 2.0 0.00 2:30.669. zaoow 030303. ecu 3!... ma. 2...: “380:2. 30:30.... EEO. c. 0.00 0......0359. Zaoow 5.3.0... 030 of c. 2.03.8.3 5.00.3.0... .555. 2.0 no. .23.... EEwnd 83.33.. 030 e. no. .05.... 2:500 2min c. to. .oeoca .28de AZUEOV 52002 no_oow 00.2002 u.oouon_:o ON —. 30.3.3 30... 140 Again, as with the ncdified phenol red assay, the optimal atmospheric conditions for the HVA assay was uder either ambient air or 7% (X):- ambient air (Figure 6) . Since calcium and magnesium were included in Ruch’s assay, the requirement for these metals in stimulation of optimal P1202 production by EMA or opsonized zymcsan was determined. When opsonized zymcsan was the stimulant, addition of calcium or magnesium doubled the level of H30: produced. However, simultaneous additions of calcium and magnesium resulted in a 3.75 fold increase compared to the amouunnt of 1130; in the absence of calcium and mag- nesium. Thus, both calcium and magnesium were required for optimal mo. production (Figure 7). However, when EMA was the stimulant, either calcium or magnesium but not both were required for optimal 1120: production (Figure 8). Addition of calcium or magnesium in- creased HzOn production about 50%. Simultaneous addition of calcium and magnesium did not increase 1130: production. This is in contrast to the results obtained with the phenol red assay where calcium was not required for optimal PMA stimulated 1130; production. In addition the ability of NaNa to increase H30; measurements by inhibiting macrofinage cytoplasnnic catalase was again determined. Figure 9 shows that upon addition of 2 mi“! NaN; , a concentration normally used to inhibit cellular catalase (25-32), Heoz production was not in- creased suggesting that as before cytoplasmic catalase was not sca- venging detectable amounts of Hz 0; . The standardcurve for theHVAassaywss linear fromOto 10pM HzOz (Figure 10). In this assay, H30: produced by resident macro- phages was between the concentrations of 3 and 10 M 1130; , or about Figure 6. 141 Modified HVA assay: Atmcspheric conditions for optimal HzOz production by adherent macrophages from 2 x 10' resident peritoneal cells. Macrophages were incubated with 6 or 12 pg opsonized zymosan or 1 or 10 pg EMA for 2 hours in 0.09 m CaCla and 0.05 m MCI: . 142 ._ //V////¢////////// I50 } 582.“ 829.82: Sex «on... 3.9:: m. 3///////////////§ m m 0.. n. .m u. m m w m m a. D I Figure 7 . 143 Modified HVA assay: Requirements for 0.09 m 09.01. and 0.05 m MgClz for optimal 1120; production by adherent macrophages from 2 x 10‘ resident peritoneal cells. Macrophages were ircubated with 6 or 12 pg opsonized zymosan for 2 hours under 7% (Dz-ambient air. 144 E T7?//////////////////////// .. We. a: m”. a a. W MWWW £555532222293 /W mm w m 0%. a D E I 5203 oooeaoboe 95.04.. 8.9:: 145 Figure 8. Modified HVA assay: Requirements for 0.09 m 08.01: and 0.05 m MgClg for optiml P130: production by adherent macrophages from 2 x 10° resident peritoneal cells. Macrophages were ircubated with 1 or 10 pg PMA for 2 hours under 7% (Dz-ambient air. 146 6 0.09m Coo, . 0.05m M901, D 0.09...» Cock. no men, 5 no Coat, 0.05m ma, - 006061,, no ma, ' wwfiowwwu u ”on.“ “an." Nona.” a. “assess.“ o ;/////////////////339. v a guesses” seweeewsefi$3 §//////////////////////// b 200 - O 5 £29... 82382: 95 ~o u... 3.06: I50 - IOO L Figure 9. 147 Modified HVA assay: Requirements for 2 m MN: for optimal P1203 production by adherent macrophages from 2 . x 10° resident peritoneal cells. Macrophages were incubated with 6 or 12 pg opsonized zymosan or 1 pg EMA for 2 hours under 7% (1);-ambient air. 148 8 no NON, El am Mon, 17 a?///////////////////////// '21“: T7///////////V///// ,fi 6M no stimlan: m 200 *- l50 *- 0 5 £92.. 393236 95 ~o“... 3.9:: IOO r- 'M opsonized zymosan PMA Figure 10 . 149 Starriared curve for 1130: production in the ncdified HVA assay. Various concentrations of reagent {-1303 was ircubated with the reaction solution for one hour since the reaction goes to completion by one hour and the product is stable for several hours. 150 H202 (FM) I 0|23456789|0 20— . _ w m 5 85820:: 3:28 151 40 to 150 nncles HzOz/ m8 macrophage protein (Figures 7 and 8). These [130; concentrations are within the linear portion of the stan- dard curve . The total and extracellular concentrations of P120: (Figure 11) were also assessed for the modified HVA assay. As for the phenol red assay, the majority of 11:0: produced by either zymosan or EMA was found in the extracellular environment (Figure 11). As can be clearly seen in Figure 12, the modified HVA assay made it possible to demonstrate for the first time that L gru_zi_ could indeed initiate I-hO; production. In the first experiment where the parasites endnibited a high degree of infectivity, macrophages stimulated with nonopsonized L c_ru_z_i generated about 30 nmoles of 11:0: per mm of macrophage protein. Resident macrophage infected with opsonized L c_ruii produced twice as much 14:0: . Variation in infectivity of the parasites from experiment to experiment is common being a troublesome variable not yet under the control of parasitologists. At lower but nevertheless ample levels of infection (30-40%) , a second experiment yielded a similar pattern of results. At a 10:1 ratio of L mi to macrophages, the nonopsonized parasites caused production of half the ancunt of 11.0. as opsonized L 9M. Further, at the 10:1 ratio, there was definite correlations between the degree of infectivity of the L cruzi and amount of 1130; produced. In this, the parasites of experiment one which were 2.5 to 3.0 times more virulent, caused about 2.5 times more peroxide to be produced. Figure 11. 152 Modified HVA assay: Measurement of total, extra- cellular, and intracellular I-hOn produced by adherent macrophages from 2 x 10' resident peritoneal cells. Macrophages were ircubated with 6 or 12 ug opsonized zymosan or 1 or 10 ug PMA in 0.09 MCaCla, and 0.05 m MgCl; umnder 7% (Dz-ambient air. Total 11:02 was measured by adding glycine-EDTA to stop the reactions to the cells and reaction solution. Extracellular 11:02 was measured by adding glycine-EDTA to the reaction solution after rencval from the cells. Intracellular P120: was measured by adding fresh reaction solution to the renining adherernt macrophages and adding glycine-RUM. 153 200? D intracellular H‘O2 ISO _. extracellular H20: I total H10: 5 O l nmoles 14,0g lmq macrophage protein on o I 1'" A no 6 l2 stinulant opsonized zymosan PMAlpql (ll-9) Figure 12. 154 Modified HVA assay: 11:0: prochction by resident macrophages incubated with L c_ru_ii or opsonized L M at a parasitemacrofinage ratio of 5:1, 10:1, or 20:1. 1120: was measured after two hours incubation of parasites with adherent macrophages in the reaction solution with 0.09 mM CaCl; and 0.05 m MgCla under 7% (Dz-ambient air. L QM and nacrophages were incubated in W for 2 hours and then the proportion of macrophages associated with parasites which is indicated as percent infection was determined. Protein content was detemined for mcrophages not incubated with parasites . nmoles Hzozl mg macrophage protein 80 70 155 P I opsonized I cruzi _ experiment I T. cruzi experiment 2 90% infection 30-40‘lo infection 5 I / / / / I / / / 5 4 \\\\\\\\\\\\\‘ 7 a 6 § l0=l o 5:: l0=l 20:1 T. cruzi = macrophage DISCUSSION Modifications of the assay conditions for measurement of I-hO; production as described in this chapter have now made it possible to measure H30: production by resident macrophages . The concentration of resident peritoneal macrophages was increased in order to increase the amount of H301 produced in a given volume of assay solution. Thus, the concentration of the H30; and therefore the concentration of the phenol red product was increased. Also, the addition of 0.5 mM CaCla increased the ancunt of H30: production when opsonized zymosan was the stimulant. This was probably due to the calcium requirement for cell phagocytosis since opsonized zymosan binnds to receptors and is then phagocytosed. Most likely, other stimulants which involve receptor mediated phagocytosis will also require calcium for optimal H30; production. In contrast, calcium did not improve an stimulated 11.0. production. This is probably because an diffuses through the cell menbrane thus not requiring calcium for receptor mediated phagocytosis. Also, in an attempt to increase HzOn measure- ments , NaNa was added to inhibit macroplage cytoplasmic catalase which scavenges HaO; . However, addition of NaNo had no effect on the ancunt of H10: produced. This was predictable since murine nacrophages contain relatively low to negligible levels of catalase activity as compared to macrophages from humans or guinea pigs (31) . When studing mcrophages from other species, it is important to add NaNa to inhibit scavenging of 1120; by cytoplasmic catalase. Also, 156 157 optimal P120: production was obtained uunder ambient air. This is probably a result of the presence of a greater supply of oxygen which is the substrate for NADPH oxidase. In some previous studies where the oxygen supply was low due to use of other atmospheric conditions (5-9) , there was probably an underestimate in the amount of 11:02 that the cells were capable of producing. Thus, many previous reports suggest that very small to negligable amoumnts of H30: are produced by resident macrofinages (2, 5, 6, 7, 8, 9). In our hands, similar results were obtained uaing the published assay conditions (0-7 nmoles I-IzOz/ mu macrofinage protein). In contrast, using the ncdified phenol red assay described in this chapter, much higher amounts of H20; were produced by resident macrophages (40-60 nmoles H201/ mg macrophage protein). So, with an increase in cell concentra- tion, addition of calcium, and incubation under ambient air, the amount of 11:0; produced by resident macrophages is increased to the middle of the standard curve. Thus, these modifications now make it possible to measure H30; production by resident macrophages. Additional modifications of the assay for I120: production were made for use with the parasitic stimulant, L c_ru_z_i. Since the substrate, phenol red, was toxic to the parasite, another nontoxic substrate, HVA, was used. Ruch e_t. §_1. (23) described the rarely used HVA assay. The conditions for this assay were also improved by increasing the cell concentration when using resident macrofinages. In addition, since Ruch’s assay included calcium and magnesium in the reaction solution, the requirements for these in optimal stimula- tion of mo. production were determined. Calcium and magnesium were 158 required for optimal opsonized zymosan stimulated 1120: production. However, only calcium or nagnesium but not both were required for optimal INA stimulation. The calcium requirenent for opsonized zymosan stimulation was probably due to the need for calcium for phagocytosis of the opsonized zymosan. This modified.HVA.acid.assay was more sensitive than the phenol red assay since lower concentra— tions of H302 could.be measured. Using the modified HVA assay for 11.0. . L c_n_lz_i trypomastigotes produced from 10-30 nmoles 830: depending on ratio of L 9Lu2_i_ to mononuclear cells and degree of infectivity of the parasite. Those which had been opsonized.with chronic serum stimulated twice as much Hz 02 production as nonopsonized trypomastigotes . This would make sense since once the immune systemumounts an antibody response to the parasite, trypomastigotes (the blood form of I; ggggi) may actual— ly be opsonized ig xigg. Generally speaking, the second phase of any immune response is more intense. The HVA.assay'can.now be used to answer questions about the role of opsonization in parasitic defense. For the most part, the amount of I; 939;; stimulated.HaOI production.depended.upon the amount of I; ggggi_present and degree of infection. This is the first instance where L guz_i stimulated ago; production has been directly and quantitatively measured, since as pointed out in the introduction, previous assessments had.been done indirectly or nonquantitatively (12-14). The HVA assay will be a vital addition to immunoparasitology by making it possible to answer many key questions regarding the killing of L cruzi by mononuclear cells. The question of whether or not 159 HzOz is a key element in the killing of L gluz_i can now Mgin to be addressed. It will also be important to knnow what the relationship is between the amounnt of H30; produced and the number of parasites that can associate with a particular phagocytic cell. In other words, as the burden of potentially infective L c_ru_z_i increases, can the macrophage respond with incremented increases in 112°: produc- tion. If this is so, is the amounnt of H30: produced directly propor- tional to the degree of associated parasites or are there limitations and thresholds that might explain why Cnagas’ disease is never com- pletely eliminated by the immune system. Assessment of the ability of the various life-cycle forms of L m to initiate I-IzOn produc- tion might shed additional light on the ability of the various forms to escape the host defense system. Finally, the ability of the various form of the mcnnonuclear cells to produce 1130: upon stimula- tion by L M can also be evaluated. Thus comparisons in intensity of HaOn production between resident and peritoneal elicited macro- phages and perifineral or tissue-localized phagocytes can be made. Finally, this assay made it possible to do the studies in chapter 5 of this thesis. There, it was essential that L gr_u_u_zi_ stimulate measurable quantities of 1130. production by resident peritoneal macrophages so that the deficit in killing L _c_ru_zi by zinc deficient resident macrophages could be investigated. Also, if mo. production was reduced in L Qg_nz_i_ stimulated zinc deficient macrophages, it may help identify the zinc depedent step(s) in the mechanism(s) of parasite destruction. 10. 11. 12. References Pick, E., and Mizel, D. (1981) J. Immunol. Methods g, 211. Marioka, A., and Kobayashi, A. (1985) J. Protozool. 3, 153. Pick, E., and Keisari, Y. (1981) Cell. Immunol. 53, 301. Freund, M., and Pick, E. (1985) Immunology fl, 35. Badwey, J. A., Robinson, J. M., Lazdins, J. R., Briggs, R. T., Karnovsky, M. J., andKannovsky, M. L. (1983) J. Cell. Physiol. 1_15, 208. T‘sum'awaki, S., and Nathan, C. F. (1984) J. Biol. Chem. g, 4305. Dean, J. H., Lauer, L. D., House, R. V., Murray, M. J., Stillman, W. S., Irons, R. D., Steinhagen, W. H., Phelps, M. C., and Adams, D. 0. (1984) Tax. Appl. Pharm. 12, 519. Murray, H. W., Nathan, C. F., and Cohn, Z. A. (1980) J. Exp. Med. $2, 1610. Ito, M., Karmali, R., and Erin, M. (1985) Immunology _5_§, 533. Wirth, J. J., Fraker, P. J., and Kierszenbaum, F. (manuscript in preparation). Fraker, P. J., Jardieu, P., and Wirth, J. (1986) In "Nutri tional Diseases: Research Directions in Comparative Pathobiology" pp. 197-213. Alan R. Liss, Inc., New York. Villalta, F., and Kierszenbaum, F. (1983) J. Immunol. m, 160 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 161 1504. Villalta, F., and Kierszenbaum, F. (1984) J. Immunol. _1_113, 3338. Nathan, C. F., Silverstein, S. C., Brukner, L. H., and Cohn, Z. A. (1979) J. Exp. Med. L49, 100. Budzko, D. B. (1974) J. Parasitol. Q, 1037. Mercado, T. I., Katusha, K. (1979) Prep. Biochem. _9_, 97. Hayes, M. M., and Kierszenbaum, F. (1981) Infect. Immun. 31, 1117. Kierszenbaum, F. (1981) Immunclogy g, 641. Conrad, R. E. (1981) In "Phnual of Macrophage Methodology V13" (Herscowitz, H. B., Holden, H. T., Bellanti, J. A., Ghaffar, A., eds.) pp. 5-12. Parcel Dekker, Inc., New York. Bozdeck, M. J., and Bainton, D. F. (1981) J. Ecp. Med. 1_5_§, 182. Yam, L. T., Li, c. Y., and Crosby, w. H. (1971)'Am. J. Clin. Pathol. _55, 283. Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. (1951) J. Biol. Chem. 193, 265. Ruch, W., Cooper, P. H., and Baggiolini, M. (1983) J. Immunnol. Methods _6_3, 347. Mishell, R. I., and Dutton, R. W. (1967) J. Encp. Med. lag, 423. R008, D., van Schaik, M. L. J., Weening, R. S., Weever, R. (1977) In "Surperoxide and Superoxide Dismutases" (Michelson, A. Ma, mm, Jo Mag-hidOVich, I, as.) pp. 307-3160 26. 27. 28'. 29. 30. 31. 32. 162 Academic Press, Inc., New York. Babior, B. M. (1977) In "Surperoxide and Superoxide Dismutases" (Michelson, A. M., biz-Cord, J. M., Fridcvich, I, eds.) pp. 272-281. Academic Press, Inc., New York. Rossi, F., Bellavite, P., and Berton, G. (1982) In "Phagocytosis - Past and Furture" (Kamovsky, M. L., and Bolis, L., eds.) pp. 167-192. Academic Press, Inc., New York. Ohno, Y., and Gallin, J. I. (1985) J. Biol. Chem. _2_6_0, 8438. Nicholls, P. (1964) Biochem. J. 99, 331. Theorell, H., and Ehrenberg, A. (1952) Arch. Biochem. Biophys. 4_1, 462. Simona, S. R.,and Karnovsky, M. L. (1973) J. Exp. Med. 1_38, 44. ' Daminani, G., Kiyotaki, C., Soeller, W., Sasada, M., Peisach, J., and Bloom, B. R. (1980) J. Exp. Med. 152, 808. Chapter 4 L cruzi-STIMILATED 11:0: WON: WANT‘ITAT‘ICN AND A PCBSIBLE MECHANISM 163 For the first time, erpgnosoma c_ruii (L c_ru_z_i_)-stimulated HaOu production by leukocytes was directly quantitated. Assessment of the ability of leukocytes to produce Ego. is very important since H30; is thought to be crucial in the destruction of L clung; para- sites. Blood fornms of L cruzi (trypomastigotes) that were opsonized with heat inactivated serum from chronically infected mice stimulated four times as much 1120) production by resident macrophages as nonop- sonized trypomastigotes. If the intracellular form of L (_3_ru_;z_i, the amastigote, was the stimulant, half as much HxOn was produced by resident macrophages as compared to stimulation by the trypomastigo- te. Further, it was demonstrated that 1130; production stimulated by trypomastigotes and opsonized trypomastigotes bunt not amastigotes correlated with the level of parasite-macrophage association. More- over, an increase in 1120: production coincided with an increase in killing of trypounstigotes, amastigotes, or opsonized trypomastigotes. The amouunnt of Heoa Produced when 50% of the trypomastigotes or amas- tigotes were destroyed was the same. When 50% of the opsonized trypomastigotes were killed about 3.5 times as much HaOu was produced. Hal); production was characterized further by investigating a possible mechanism or route for intiation of L 9.9121. stimulated 1130; production. The following preliminary results suggest that arachidonate metabolites are second messengers in the activation of H20: production by L c__ruAi_ . The macrophage phospholipids contained a considerable ancunnt of arachidonate as a source for production of 164 165 these arachidonate metabolites; 21% of the fatty acids were arachi- donate. Also, addition of I; Qgpgi to resident macrophages prein- cubated.with 3H-arachidonate stimulated the release of PGE2, HETE’s and what may be leukotrienes at a ratio of 2:1:4. It is known that HETE's and leukotrienes but not PGEu stimulate resident macrophages to produce H502. If I; QEQg; also stimulates release of other fatty acids that have shorter chain length or less saturation than arachi- donate (20:4) , they would not activate nearly as much 11:02 Produc— tion as 20:4. Furthermore, exogenous 20:4 was shown to stimulate 2 to 3 times as much H30. production as the exogenous addition 18:3, 18:2, or 18:0. In summary, since I; Qgggi stimulates release of HE'I‘E’s, what may be leukotrienes, and 11:0; and since HE'I'E’s and leukotrienes activate 1130: production, HE'I‘E’s and leukotrienes may be second messengers for L c_ru_z_i stimulated 1130: production by resident macrophages . INTRODIL'I‘ION Previous studies have suggested that W c_ru_z_i (L gru__z_i_) stimulates phagocytic cells of the immune system to produce H30: and that P130: is critical for the destruction of L c_ru_z_i. n.0, production by infected cells has been demonstrated qualitative- ly by a cytochemical stain for P130: (1); the stain was present in macrophage vacuoles containing L 9&2; . However, it had never been demonstrated in a direct, quantitative way that L c_rlzi could indeed initiate 1130; production. Instead, the chemical phorbol myristate acetate (INA) was used to stimulate 1420; production by activated macrophages and then the 1130; production was matheuati- cally correlated with destruction of L c_ruin by activated macro- phages as determined in another assay (2) . In a further attempt to demonstrate that 1130; was important to the destruction of L c_ru_zi, killing of L as; by inflammatory cells has been inhibited by the H; O; scavenger , catalase , buut not by inhibitors of other oxygen metabolites (2, 3). Qapter 3 demonstrated that L gguz__i does in fact stimulate quantitifiable amoumnts of 11:0: (IO-30 nmmoles [1103/ m8 macrofinage protein). Whether or not the ancunnt of 1130: produced correlated with the number of L M associated with the macrophages relained unknnown. Therefore, to further characterize H20: produc- tion by L cruzi infected macrophages, studies were done herein to assess the amcuurnt of H302 produced by resident macrophages as related to the levels of infection of macrophages by several forms of the Tulahuén strain of L cruzi. Also, correlations were done herein 166 167 between L c_m_zi_-stimulated 1120: production and destruction of L c_ru_g_i_ in an attempt to determine whether or not 1130; was indeed critical to the killinng of L gr'_uz_i by macrophages. The mechanism by which L c_ruzi, might initiate i130: production by macrophages is also umknnown. Mechanisms of stimulation by agents other than L M such as PMA and opsonized zymosan have been identified. PMA diffuses into the cell mnembrane and activates protein kinase C to phosphorylate NADPH oxidase which in turn catalyzes production of 02'; Or dismutates to 14:02 (4-11). (In the other hand, opsonized zymosan enters the macrophage through receptor(Fc)- mediated phagocytosis and stimulates the release of arachidonate which then activates NADPH oxidase (6, 7, 12, 13, 14, 15, 16). The mechanism(s) for L c_ru_gi stimulated 1130; production has not been identified. Identification of the mechanism(s) whereby L gnluz__i_ activates 1130; production was difficult since the mechanism for L gu_z_i_ invasion of macrophages and the receptors on macrophages for phagocytosis of L c_ruii are umnknown. In this chapter, information that begins to elucidate a possible mechanism for L c__ru_z_i_ stimu- lated H30; production by macrophages is presented. Since exogeneous addition of MC. or LTBo has been shown to increase killing of L g_ru_gi by macrophages (17, 18) and since L 9&2; is phagocytosed as is opsonized zymosan, it seemns probable that 20:4 or 20:4 metabolites might also be second messengers in L grlugi-stimulated P120: produc- tion. Therefore, release of 20:4 and its metabolites by 3H-20:4 labelled resident macrophages incubated with L c_ru_zi was measured. The endogenous 20: 4 content of the phosphol ipids of resident 168 macrophages from A/J mice was analyzed. Also, since other fatty acids may be released from macrophage phospholipids upon infection with L SEW—ii the ability of several exogenously added fatty acids to activate i120: production by resident macrophages from A/J mice was compared. MATERIALSANDMEI‘HODS Animals. A/J female mice were puurchased from Jackson Labora- tory, Bar Harbor Maine. Female Crl-CD-l (IRC)BR Swiss mice were purchased from Charles Rivers, Portage, Michigan. Isolation of W cruii 1T. cruzi . Four week old Crl- CD—l (IRC)BR Swiss feuale mice were infected intraperitoneally with 2 x 105 blood forms (trypomstigotes) of the T‘ulahuén strain of L _c_r_u_z_i_. Nelve to 14 days later, blood was collected from the axillary artery of mice anesthetized with ether. Blood was collected in tubes containing heparin or disodium ethylene-diaminotetracetate (EUPA) powder so that the final concentrations were 25 U/ml or 2.5 mg/ml, respectively. Parasites from the blood were separated on a lympholite gradient (isolymnph, Gallard-Schlesinger, Carle, N .Y.) (19) and passed through a diethylaminoethylcellulose column (20). The parasites were centrifuged (800 x G, 15 minutes, 4°C) and re- suspended in mlbecco’s ncdified minimal essential medium (wan, Gibco, Grand Island, NY) supplemented with 100 IU penicillin and 100 pg streptomycin / ml. One hundred percent of the cells were viable parasites in the trypomastigote form as determined by morphology and motility. Before use in the assays for 11:02 , the parasites were washed two times in phosphate buffered saline (PBS) - 1% glucose and resuspended in the reaction solutionn for the HaOz assay. Amastigotes were collected from the supernatants of L $12—1- infected rat heart moblast cultuures as previouusly described (21) . Pr__e_p_a_r_ation of mized T. cruzi. L cruzi trypomastigotes 169 170 were opsonized by incubation for 1 hour at 37°C in 10 mt! PBS (pH 7 .4) with 1% glucose and 20% complement inactivated serum from chronic L gig; infected CD1 mice. The opsonized L g_ru_u_z_i_ were washed 2 times and resuspended in 10 a“ PBS (pH 7.4). The serum was obtained during the chronic phase of experimental Chagas’ disease which occurs 5-6 weeks after the initial infection. Chronic mice have normal immuune responsiveness buut continue to harbor the parasite and produce L M-specific antibodies (22, 23). CollectionL isflationLaLd idgntificglgn of mfltoneal macro- m. Cells were harvested from the uunelicited mouse peritoneum by lavage with 5 ml of cold (4°C) [HEM and 0.5% w/v gamma globulin free bovine serum albumin (BSA, Calbiochem, La Jolla, CA) (24). Cells were adhered to tissue cuulture plates for 1 1/2 houurs at 37°C and 7% (Dz-ambient air. Nonadherent cells were remnoved by washing with 37°C DIEM. The percentage of macrophages/monocytes adhered to the culture plate was determined by staininng for nonspecific ester- ase activity as detailed in the modification (25) of the technique described by Yam et. al. (26). Always, 94-100% of the adherent cells were identified as mcnocytes/macrophages by staining for non- specific esterase and by morphology. Willie acid sag: for Hag; . The assay conditions described by Ruch gt. a_l. (27) were modified for use with resident macrophages and the stimulant, L M trypomastigotes. One milliliter of 2 x 10‘ resident peritoneal cells/ ml was added to a 24 well flat bottom plate (Corning) and allowed to adhere for 2 hours . Nonadherent cells were removed from the adherent macrophage mnonolayer by washing 171 three times with PBS - 1% glucose (37°C). Two hundred microliters of reaction solution which consisted of PBS, 1% glucose, 0.09 mM CaClz , 0.05 mM MgCl: , 200 pM homovanillic acid, and 2 U/ml horse— radish peroxidase were added per well. The fatty acid stimulants 20:4, 18:3, 18:2, or 18:0 were diluted in ethanol and 2 u]. added per well so that less than 1% of the reaction solution was ethanol and the final concentration was 10 “M or 50 “M. When parasites were the stimulant, they were resuspended in the 200 pl of reaction solution to be added to the macrophages. Standards consisted of wells contain- ing reaction solution and 0-20 “M mo. but no cells. Nonspecific production of HaOu was determined by including wells containing cells bunt no stimulant or stimulant but no cells. The plates were (centrifuged at 50 x G for 3-4 minutes to increase parasite-macro- phage interactions and incubated for 90 minutes in a humidified atmosphere at 37°C and 7% (Dz-ambient air. The reaction was stopped by the addition of 25 p1 of 25 mt! 1mm and 0.1 M glycine at pH 12. The relative fluorescece of the homovanillic acid dimer was deter- mined using a spectrofluorometer (Perken-Elmer 650-40) equipped with a microcuvette. The excitation and emission wavelengths were 312 nm and 420 nm, respectively. The slit widths for excitation and emis- sion were 2 nm and 5 mm, respectively. The amouunt of i120: in the samples was analyzed by linear regression of the standard curve. The amouunnt of macrophage protein was determined by the method de- scribed by Lowry (28). The data are presented as nmoles I-Iaon/ mi macrophage protein. Nmoles of P130: / mg macrophage protein was calcu- lated as follows: (HM H20: calculated from the standard cuurve) x (l 172 liter/ 1000 ml) x (0.225 ml final volume of reaction solution) x (1000 nmoles/ lumole)/ (mg macrophage protein determined from the Lowry) = nmoles H202/ m8 macrophage protein. _As_sroci§tion of T. 0&in with grow. After incubating the macrophages with parasites for 90 minutes in mm at 37°C and 7% (Dz-ambient air, the nonassociated parasites were removed by washing three times with PBS - 1% glucose. The cultures were incubated in DIEM for another 18 hours and/or fixed with methanol for 5 minutes, allowed to dry, and stained for 1 hour with Giemsa in 10 mM phos- phate buffer (pH 6.8). The cells were washed one time with 10 mM phosphate buffer (pi-I 6.8). Numbers of parasites per 100 macrophages and percent of macrophages associated with parasites were determined by couunnting at least 200 cells per replicate. The percent decrease in number of L gru__z_i associated with macrophages from the time of removal of nonassociated parasites to 19 to 24 houurs later represented killing of L _c_ru_zi_ by macrophages. At 19 to 24 hours, the number of nonassociated parasites was less than 3% of the number of parasites associated with the macrophages. Thuus, a reduction in number of parasites associated with the macrophages was due to killing and not simply escape of the parasites from the macrophages. Smthesis of mic mosQQtigylcholine (29). Phrgaric (17:0) phosphaticb'lcholine was chosen as an internal standard for the analy- sis of fatty acid composition of macrophage phospholipids since methylated margaric acid has a different retention time during gas liquid chromatography than fatty acids normally found in good quan- tities in mammalian phospholipids. Phrgaric acid (200 mg) was placed 173 in a small teflon capped tube with 300 pl thionyl chloride, puulsed with nitrogen, and placed on a steam bath for 1 hour. The products were evaporated to dryness under nitogen in a 80°C water bath, washed successively with water, 10% sodium carbonate, and water, and then dried uunder nitrogen. The fatty acyl chloride (50 mg) was reacted with the CdClr complex of L-alpha-glycerophosphorylcholine (10.45 mg) while stirrinng for 2 hours in 150 pl chlorform and 9 pl pyridine under an atmosphere of nitrogen. The stirrer was rinsed with chloro- form and the solvents were evaporated under nitrogen. The reaction products were dissolved in 166 pl ether (dry), cleared by centri- fugation, and the solvents removed under nitrogen. The lipids were dissolved in 500 ul acetone (dry), placed on a acetone-(X): bath for 1 hour and isolated by centrifugation in the cold. They were preci- pitated again with 250 pl acetone. The lipids were dried under nitrogen, dissolved in chlorcform:methanol:water (5:4:1, v:v:v) and passed over a 21ml column of amberlites IRSO:IK350 (50: 50). The column was washed with the solvent mixture. The effluent was dried under nitrogen, dissolved in chloroform and placed over a 1 ml silicic acid column. The silicic acid column was washed with 30 column volumes of chloroform and the phospholipid was eluted with 15 column volumes of chloroform:methanol (1:1) followed by 10 column volumes of methanol. The eluuent was evaporated uunnder nitrogen and the phos- pholipid was stored dry uunnder nitrogen at -20°C. The last 5ml frac- tion of the silicic acid column effluuent did not contain margaric acid as measured by gas liquid chromatography of the fatty acid methyl ester. Esterification is described below. The phospholipid 174 fraction from the silicic acid column contained margaric acid as determined by gas liquid chromatography of the methyl ester. Fatty acid mitiop of upgcrophgge gospholipids. Macro- phages were adhered for 2 hours to 24 well tissue culture plates in DIEM-0 . 5% BSA. Nonadherent macrophages were remcved by washing three times in DIEM-0.5% BSA. The adherent macroplages were col— lected by scraping into 0.15M NaCl. Since one twelfth of the amouunnt of the synthesized internal standard, margaric phosphatidlycholine, gave a peak height durinng gas liquid chromatography that was 80% of the chart range, this amount of internal standard was added to each sample to control for sample loss during the purification of the macrophage phospholipids. The lipids were extracted by the method described by Bligh and Dyer (30). Briefly, methanol:chloroform:wa— ter (4:2:8, v:v:v) was added to the macrophages. Then one volume of chloroform followed by one volume of water was added to the extraction mixture. The mixture was centrifuged and the chloroform layer was collected and evaporated under nitrogen. The lipids were resuspended in chloroform and placed over a 0.5 ml heat activated silicic acid column in a pasteur pipette. The neutral lipids which do not bind to the column were washed through the column with chloroform. The phospholipids bound to the column were then eluted with chloroform: - methanol (1:1) followed by methannol. The organic solvents were evaporated from the neutral and phospholipids under nitrogen . The fatty acids were converted to methyl esters (31) by incubating over- night at 75°C in 6% P101 in methanol under nitrogen. The fatty acid methyl esters were extracted with three equal volumes of hexane and 175 washed once with water. Water was removed from the hexane by the addition of anhydrous sodium sulfate. The hexane was collected and evaporated uunnder nitrogen. The fatty acid methyl esters were dis- solved in hexane and analyzed (32) using a Hewlett-Packard gas chroma- tograph equipped with a 6 foot x 1/8 inch i.d. glass column packed with 10% SP-2330 Chromasorb W/AW. Analysis was performed isothermally at 180°C for 7 minutes followed by an increase of 4°C /minute to a limit of 200°C. The carrier gas-flow rate was 30 ml/minute. Peak identifications were based on relative retentionn times of standard methyl esters (Nu Chek Prep, Elysian, Minnesota). Relative peak areas were measured by a Hewlett-Packard 3380A electronic integrator. The quantity of the various fatty acids was obtained using linear regression from a standard curve of peak area versus amount of fatty acid methyl ester. Total macrophage fatty acid was determined by comparing peak area of the internal standard in the samples of macro— phage phospholipida to peak area of a sample of internal standard alone that had not been submitted to the phospholipid isolation procedure. Also, samples of macrophage phospholipids from each severely zinc deficient, moderately zinc deficient, restricted, and zinc adequate mice, which did not contain the internal standard, did not have a peak with the same retention time as the internal standard. Thus , since the macrophage phospholipids did not contain margaric acid, margaric acid was a valid internal standard. Arachidonic gcid release by macroflnages (33, 34). Macrophages adhered to 24 well plates were incubated with 1.5 pCi (5, 6, 8, 9, - 11, 12, 14, 15-3H)arachidonic acid (Amersham) in 2 ml IMF}! overnight 176 and washed three times with serum free DIEM at 37°C . The macro- phages were stimulated by nonopsonized trypomastigotes of L gru_z_j._ for 2 hours. in serum free mm. Nonstimulated macrophages were included to control for spontaneous release of radioactivity by the macrophages . The culture medium was removed from the adherent macro- phage ncnolayer, centrifuged, and acidified to 0.03M with citric acid. The acidified media was extracted three times with 3 ml of chloroform:methanol (2:1, v:v) . The chloroform layers were combined, washed two times with 2 ml of methanolmater (2:1, v:v) and evapor- ated to dryness under nitrogen. The residue was dissolved in 40 u]. of ethylacetatezmethanol (3:1, v:v) and applied to precoated plates (Silica Gel 60, Merck) for thin-layer chromatography. The internal standards PCB: and arachidonate were added to each sample. Other standards 6-keto-PGFun pin , LTBa , LTD. , LTD. , LTEc , and ricinoleic acid (an oxidized product of arachidonate) were run separately along with PCB; and arachidonate. Arachidonmte and its metabolites were separated by developnent twice in the solvent system chloroform: ethyl- acetatezmethanolzacetic acidzwater (140:60:16:2:1, v:v:vzvzv). The internal standards were visualized by exposure to I; . Half centimeter semnents for each ascending sample of the silicic acid coated plates were scraped and radioactivity was measured by a Delta 300 scintilla— tion counter (Tracor Analytic) . Radioactivity for each segment of a sample was plotted versus distance (cm) on the plate. The data for each radioactive peak are presented as 3H—cpn of stimulated release minus 3H-cpn spontaneous release from nonstimulated samples. 177 3H-a;ac;fihidonic acid labelled ymrflge mosmolipids. The methods for identification of those macrophage phospholipids that contained 3 H-arachidonate used herein was described by Enilsson et . al. (35, 36). Briefly, radiolabelled resident mcrophages were collected by scraping into 1 ml of ice-cold 10 II“ m1. Lipids were extracted with 6 ml of chloroform:methanol (1:1, v:v). Phase separa- tion was obtained by addition of 2 ml of 10 I!“ 1101 followed by centri- fugation. The chloroform layer was removed, evaporated to dryness, and dissolved in 40 pl ethylacetatemethanol (3:1, v:v). The lipids were separated by thin layer chromtography on precoated plates (Silica Gel 60, Merck) with the solvent system chloroform:methanol- :acetic acidzwater (25:20:3:0.3, v:v:v:v). Lipids were visualized by exposure to I; . The plates were scraped in 0.5 cm sements for each ascending sample and radioactivity was measured by a Delta 300 scintillation counter (Tracor Analytic) . Radioactivity for each segment of a sample was plotted versus distance on the plate. The total radioactivity for each peak was'calculated. The data for each radioactive peak are presented as radioactivity released by L 9%; stimulated mcrorhages minus spontaneous release of radioactivity by nonstimulated macrophages . mcroQQge viability. Viability of the adherent macrophages was determined using the trypan blue exclusion method. Always >95% of the macrorhages were viable. RESULTS For the first time, it was possible to demonstrate that measur- able amounts of H20: could be detected in cultures of macrophages stimulated with L gru_rzi_ trypomastigotes using the modified homo- vanillic acid assay for Hsz (Figure 1) . The increased sensitivity of this assay nude it possible to use resident macrophages instead of the activated macrophages as was normlly used in the past. Thus, L c_ruii trypomastigotes were capable of stimulating the genera— tion of 11:02 by mcrophages. This is important since P120: is thought to destroy L c_rugi. Previously, indirect evidence suggested that mcrophages produced P120; in response to L c_ru_zi_ since it has been shown that L gru__zi stimulates the uptake of oxygen (37), that a cytochemical stain for I120: is present in vacuoles containing L gru_zi (1), and that catalase, the scavenger for I120: abrogates killing of L p_r_'uz_i_ by mcrophages. Finally, herein, L c_ru_zi_ has. been directly shown to stimulate H20: production in measurable quantities. To determine if the amount of H20; produced correlated with macrophage infection, the percentage of macrophages associated with trypomstigotes and the number of parasites per 100 mcrophages from several experiments were compared with the macrophage production of 1130; (Figures 1 and 2) . As the percentage of mcrophages associated with the trypomastigotes increased, there was a linear increase in 1130; production (correlation coefficient = 0.957, Figure 1). When trypomstigotes were opsonized with chronic serum, a similar cor- relation was found between parasite association with macrophages and 178 Figure 1 . 179 Correlation between I130: production and proportion of mcrophages associated with L gr_uz_i. For measurement of P120: production, L c_ru_z_i amstigotes, L M trypomastigotes , or opsonized L QM trypanstigotes were incubated for 2 hours with adherent mcrophages in in the reaction solution. Parasites were incubated for 2 hours with macrophages in THEM and then the proportion of macrophages associated with L cruzi was determined. The points for each form of parasite were frm experiments performed on several days with freshly prepared macrophages, parasites, and reaction solution. O) 0 0| 0 A O 01 0 /mg macrophage protein N O 5 nmoles H202 180 O trypomastigotes I opsonized trypomastigotes ‘ amastigotes 4&1! l 0 25 50 75 mo °/o macrophages associated with T.cruzi Figure 2. 181 Correlation between HaOn production and nanber of L m per 100 macrophages at low levels of L c_ru_ii- macrophage association. For measurement of H30: production, L m trypomstigotes were incubated for 2 hours with adherent macrophages in the reaction solution. Parasites were incubated for 2 hours with macrophages in IMF)! and then the number of L c_ruii associated with macrophages was determined. The points were from experiments performed on several days with freshly prepared mcrophages, parasites, and reaction solution . nmoles H202/ mg macrophage protein IO 182 l L l 50 IOO l50 number of Icruzi/ IOO macrophages 183 production of 8202 (correlation coefficient = 0.950, Figure 1). However, opsonized trypomstigotes stimulated considerably ncre I120: production than did nonopsonized trypomastigotes (Figure 1) . For example, when 50% of the mcrophages were associated with parasites, opsonized trypomastigotes stimulated over four times as much HzOz as did nonopsonized L QM. Interestingly, when amastigotes, the noninvasive intracellular form of L c_ru_rz_i_ was tested, 1130: production did not correlate well with the percentage of mcrophages associated with parasites (correlation coefficient = 0.535, Figure 1). Also, amastigotes stimulated lower ancunts of 1130: production at a given level of association than trypomastigotes (Figure 1). This was true even though high numbers of amstigotes had associated with the macrophages. Thus, the ability of the parasite to activate production of 11.0. by resident mcrophages depended upon the form of the para- site. Furthermore , a good correlation was also observed between the number of trypomastigotes per 100 macrophages and the amount of 1110; production (Figure 2). The relationship was linear from 0 to ap- proximtely 80 parasites per 100 mcrophages, at which point the amount of I110; produced reached a plateau (Figure 2). Thus, uncro- phages were capable of producing acre 11:0; to presumably destroy the greater number of trypomastigotes with it. (he would expect that with an increase in Hsz production more parasites would be destroyed if 11:03 was truly critical for killing. Indeed, using data from several experiments, it was found that, generally, there was a direct relationship between the amount of 184 killing of trypomastigotes, amatigotes, and opsonized trypomastigotes after 19-24 hours and the amount of 1130: production (Figures 3 and 4). For trypomastigotes and amastigotes, approximately the same ancunt of 11:0: (15 nncles/ m macrophage protein) caused a 50% de- crease in number of parasites per 100 macrophages (Figure 3). Ap- proximately 3.5 times that ancunt of HaOn coincided with a 50% de- crease in number of opsonized trypomastigotes associated.per 100 macrophages (Figure 4). For destruction of 25% of the parasites, 5 nncles Haozlmz protein were produced by trypomstigotes and amas- tigote-stimulated.macrophages whereas opsonized trypomastigotes stimulated production of 50 nmoles 11:0: lung mcrophage protein. An increase in killing of L _c_rl2_i with an increase in i120: production by macrophages agrees with previous literature that suggests that HzOn production is important in destruction of L c_ru_z_i (1, 2, 3, 37) since killing of I; 929;; was abrogated.by scavengers of H30: (2, 3). waever, this is the first instance where I; cruzi-stimulated P130. has been quantitated, correlated with degree of L cruzi-macro- phage association, or correlated.wdth.degree of killing of I; cruzi by'macrophages. The characteristics and.degree of infectivity of the parasite are highly variable. This may be related to the progression of the parasites through each form in its life cycle. In a sabsequent study which had.much higher levels of association (260-900 I; 923;; trypomstigotes/ 100 mcrophages) , P1202 production again correlated with the number of I; cruzi/100 macrophages (correlation coeffecient = 0.961, Figure 5). At these high levels of association, 85-100% of Figure 3 . 185 Correlation between H202 production and killing of trypomastigotes or ‘amastigotes. For measurement of P120; production, T; cruzi trypomastigotes were incubated for 2 hours with adherent macrophages in the reaction solution. Parasites were incubated for 2 hours with macrophages in DMEM and then the number of L c_ruii associated with macrophages were determined. The number of parasites associated with the macrophage at 2 hours was compared to the number still associated 19-24 hours later to determine killing of L $112; by mcrophages. The points were from experiments performed on several days with freshly prepared mcrophages, parasites, and reaction solution. 8 N O nmoles Hp: lmg macrophage protein 5 186 O trypomastigotes C] amastigotes a a a DO 8 fi 0 D D a l l l l 25 50 75 I00 56 decrease in number of T.cruzi IIOO macrophages Figure 4 . 187 Correlation between 1120: production and killing of opsonized trypomastigotes. For measurement of H30; production, L M trypomstigotes were incubated for 2 hours with adherent macrophages in the reaction solution. Parasites were incubated for 2 hours with macrophages in DIEM and then the number of L gr_'u_rz._i associated with mcrofluages was determined. The number of parasites associated with the mcrophage at 2 hours was compared to the number still associated 19-24 hours later to determine killing of L 9&2; by macrophages. The points were frcm experiments performed on several days with freshly prepared mcrophages, parasites, and reaction solution. 188 .5 60!- O 8 g . . C i 50" O o C 5 on a I E O e 40 = E . . 1 25 5O 75 96 decrease in number of T.cruzi IIOO macrophages Figure 5 . 189 Correlation between H20: production and number of L g_r_uz__i per 100 mcrophages at high levels of L c_ru_z_i- macrophage association. For measurement of 1130. production, trypamstigotes were incubated for 2 hours with adherent mcrophages in reaction solution. For the determination of the number of L M associated with the macrophages, parasites were incubated with macrophages in 11134. Points are from a single assay. nmoles H202 / mg macrophage protein 190 l l l l 250 500 750 IOOO number of parasites per IOO macrophages 191 the macrophages had associated with T. mi (data not shown). Therefore, for this set of data, the percentage of mcrophages as- sociated with parasites had reached a maximum degree of infectivity; thus, correlations between percentage of macrophages associated with L M and 8303 production could not be done. At these high levels of association where significant ancunts of 11202 are produced, macro- phage membrane integrity is compromised and the cells died within 24 hours (data not shown). Thus, correlations between H302 production and killing of the parasite could not be done for this set of data. Although, in Figure 2 and 5, the ancunt of H30: production, limits for maximal 1130; production, and degree of L _cr_'u_z_i_-nncrophage as— sociation were quite different, the amount of HzOz consistantly correlated with L c_ruz_i_-macrofiuage association. The observed varia- tions in degree of association of L c_ru_z_i with the macrophages are probably due to variablities in the ability of the parasite to infect and activate the macrophage. . The mechanism(s) for stimulation of P120: production by L gru_zi_ were unknown. However, one pathway by which L c_ruz_i_ could stimu- late n.0, production was analyzed. It was hypothesized that 20:4 or its metabolites may be involved in L m-stimulated H202 production since 20:4 metabolites are known to be stimulatory for the "oxygen burst" (12, 39) and, most importantly, exogeneous leukotrienes, an 20:4 metabolite, have been shown to increase L c_ru_zi association and killing (39, 40). The following preliminary data suggests that 20:4 metabolites my also be second messengers in L gig-stimulated HzOz production by resident mcrophages. First, the amount of 192 endogenous 20:4 naturally present in macrophage phospholipids as a source for release of 20:4 was determined. Figure 6 shows that macrophage phospholipids contain a considerable amount of 20:4 (71 nmoles/mg macrophage protein); 21% of the fatty acid was 20:4 which is very close to the reported.25% (41). Next, release of 20:4 and its metabolites by macrophages stimulated with T; cruzi was inves- tigated. Table 1 shows that incubation of T; cruzi trypomastigotes with resident macrophages stimulated the release of the arachidonic acid (20:4) metabolites, PGEz, HETE’s, and.what may be leukotrienes, from macrophages in a ratio of 2:1:4. It has been shown by Fels gt. al. (34) that the opsonized.zymosan-stimulated.release of radioac- tivity from 3H-20:4 labeled.macrophages is 20:4 and its metabolites. Further analysis by high pressure liquid chromatography of the radio- active peak comigratory with the leukotrienes would.be necessary to confirm identification of the radioactive peak as leukotrienes. If these are identified as leukotrienes, one would.want to try to corre- late leukotriene production with infection and.killing of T; cruzi. Furthermore, the amount of nonmetabolized 20:4 that was released upon stimulation of macrophages with I; ggg§i_was not greater than the spontaneous release of nonmetabolized 20:4 by nonstimulated macrophages (Table 1). Thus, T; cruzi trypomastigotes did.stimulate the release of 20:4 but all of it was metabolized to PGE, HETE’s and perhaps leukotrienes . Assuming that leukotrienes were produced, most (71%) of the 20:4 was metabolized via the lipoxygenase pathway and 29% went via the cyclo—oxygenase pathway. One may assume that the 20:4 released was derived from the phospholipids since others have Figure 6. 193 Fatty acid quantites in phospholipids from resident macrophages . Adherent macrophages were scraped into 0.15 M NaCl. Phrgaric phosriuotidlycholine was added as an internal standard. Lipids were extracted by the Bligh and Dyer method. Phospholipids were separated from neutral lipids using a silicic acid colum. Then, the fatty acids were transmethylated and quantitated by gas liquid chromatography techniques. The nanomoles of each fatty acid were calculated using the peak area of the internal standard. Protein content of the scraped cells was determined by the Lowry method. 194 Tm r 0 5 150 - IOO - £306 moccaoeooe oExEoo >23 3.2:: l852 20=4 |8=I fatty acid l8=0 |650 195 TABLE 1 Release of 3H-arachidonate Metabolites' Rfb Comigratory Stimulated Spontaneous Standards Release Release aH (era-II)c ’11 (cpm)° 0.02 L'I‘Cu LTD“ ME‘ 302 1 48 66 1 12 0.35 PCB: 150 1 14 67 1 7 0.67 HE'I‘E’s 84 1 48 205 1 27 0.88 arachidonate -11 1 9 90 1 14 3H—arachidonate metabolites in supernatants frm resident mcrofluages stimulated with L cruzi at a L cruzi:mcrophage ratio of 25:1 Rf of standards: 0.02 = (LTCu, LTDa, LT'Eu), 0.28 = Tng, 0.32 = 6-keto PGF; , 0.35 = PGEg, 0.52 =LT‘Bu, 0.67 = I-lETE’s, 0.79 = ricinoleic acid, 0.88 = arachidonate mean 1 SEM for 3H on released by stimulated mcrophages minus 311 cpn of spontaneous release by nonstimulated mcrofiuages for each radioactive peak. 311 cpn released by nonstimuulated macrophages in the area coinciding with a stimulated radioactive peak 196 reported that themjority of the 3H-20:4 is incorporated into the macrophage phospholipids (34) . In this study, the mcrophage phospho- lipids at an Rf of 0.94 on thin layer chromatography contained 49256 1 9912 cm of '11. Thus, the ancunt of radioactivity released (about 600 cm 3H) was smll (1.2%) compared to the amount of 3H incorpo- rated into the phospholipids . Other stimulants such as opsoni zed zymosan have been shown to stimulate release of 6% to 15% of the 311 that was incorporated into phospholipids of resident macrophages (36) . Thus, L 0M may stimulate the release of snaller ancunts of 20:4 than opsonized zymosan or phorbol. Also, L M may be less stimulatory than opsonized zymosan or phorbol since in Chapter 3 it was shown that L c_ru_zi was less stimulatory for Hzo, production than either opsonized zymosan or fluorbol. Another possibility is that the assay conditions my need to be optimized. Also, since the data suggested that trypomastigotes my stimulate release of 20:4 from phospholipids to stimulate the "oxygen burst", the quuestion comes to mind, "Are other fatty acids also involved in the stimulation of the oxygen burst by L M?" This was doubtful since fatty acids of shorter chain length and higher degrees of saturation than 20:4 have been reported to stimulate production of much smller ancunts of 1130: than 20:4 (42 , 43). To confirm that this relationship among the. fatty acids for stimulation of 1130: is truue in our system with resident peritoneal macrophages from NJ mice, several fatty acids fouund in nacrophage membranes were used to stimulate Haoz producti on . 197 Figuure 7 shows that several exogeneously added fatty acids did stimulate H302 production by resident peritoneal macrophages from A/J mice. Also, as reported previously (42, 43), the amouunt of 1120: produced was dependent upon chain length and degree of saturation; 20:4 stimulated twice as much 1130: production as 18:3, 18:2, or 18:0. Therefore, if L c_ruz_i_ stimulates release of fatty acids of shorter chain length or more saturation than 20:4, these shorter chain more-saturated fatty acids would probably not be as important in the oxygen burst stimulated by L g_r_uz__i, since they were less stimulatory for 1120: production than 20:4 and perhaps 20:4 metabo- lites. In summary, these preliminary results suggested that 20:4 metabolites my be second messengers in H30: production by macrophages stimulated with L M. 1 Another conceivable approach to showing that 20:4 metabolites are intermediates in L c_ru_zi-stimulated 1130: production would be to inhibit lipoxygenase and/or cyclo-oxygenase activity of the mcro- phages. If this reduced 20:4 netabolite release as well as 1130: production, it would suggest that 20:4 metabolites are involved in 1120: production. However, although the inhibitors of lipoxygenase and cyclo-oxygenase are supposedly specific for lipoxygenase versus cyclo-oxygenase, these inhibitors alter mny other cellular activities such as glucose metabolism (44, 45). Thus, this approach is dependent upon the development of a specific inhibitor of lipoxygenase or cyclo-oxygenase which does not affect other cellular processes . Figure 7 . 198 1130: production by resident mcrophages stimulated with exogeneous fatty acids. For determination of 1130. production , exogeneous fatty acids were incubated with adherent mcrophages for 2 hours in the reaction solution. Protein content was determined for mcrophages incubated in mm without fatty acids . nmoles H202/mg macrophage protein 199 IO— a no fatty acid IOpM fatty acid [3 50pm fatty acid J4 _ g / é / / LE / r _ / o' l8=0 l8=2 use 20:4 fatty acid DISCUSSION Although a considerable number of studies have been done to link H.202 production by necroriuages to destruction of L my 1120: production by mcrophages stimulated with L QLuz_i_ has not been quantitated. Using the modified HVA assay described in chapter 3, one may now measure L c_ruii stimulated 1120; production by resident mcrophages. In this chapter, the modified HVA assay was used to characterize L g_r_uz_i stimulated 1120: production. L EDA—21. trypo- mastigotes that had been opsonized with chronic serum stimulated four times the ancunt of 1130; production as nonopsonized trypouas- tigotes. Indeed, once the inmuune system mounts an antibody response to the parasite, trypomastigotes, the blood form of L c_ru_z_i, may actually be opsonized i_n yi_vg. In comparison to the trypomastigotes, amastigotes, the noninvasive intracellular form of L gru__z_i, stimu- lated half as much 1120: production at a given level of association with the macrophages. Therefore, some forms of L grluz_i_ are more stimulatory than others for Heou production by resident macrophages. The mechanisms for association of the nonopsonized forms of L c_ru_z_i are unknown. However, the opsonized trypomastigotes associate with the Fc receptors on the macrophages and are then phagocytosed (6, 7, 12, 13, 14, 15, 16). It has been shown that opsonization of L 9;in facilitates uptake and destruction of the parasites by necro- phages (46 , 47). The enhanced destruction of opsonized parasites is probably related to the enhanced production of HzOu by the macro- phage. Still, until mechanisms for association of trypouastigotes 200 201 and amastigotes with macrophages are better defined, it is difficult to surmise a reason for why some L (£22.; forms are more stimulatory for 11202 production than others at a given level of association. It was also shown that 1130: production by trypomastigotes and ap- sonized trypomastigotes but not amstigotes correlated well with the level of L c_ruz_i-mcrophage association. Thus, there was a linear relationship between trypomstigote stimulation and H20; production. In addition, the maximum amouunt of 11:0: production by the mcrophages varied with the batch of L gag; Since from experiment to experi- nent the sane number of L M incubated with macrophages will have varied degrees of association due to variations in infectivity of the parasites, correlations between H202 production and association are very important. The variations in infectivity by the parasite are probably related to the progression of the parasite through each of the forms in its life cycle. Moreover, it was shown that H30; production is related to de- struction of L c_ru_z_i as suggested by others (1, 2, 3, 37), since as the amouunt of L c_ruzi_-stimulated 1120: production by necrophages in- creased, the amount of killing of L c_rug; by mcrophages also in- creased. The ancunt of HzOz produced at 50% killing of trypoma— tigotes and amastigotes was the same. However, 3.5 times as much 1130; was produced at 50% killing of opsonized trypomastigotes. This suggests that more HzOr production my be required to kill opsonized trypomstigotes than trypomastigotes or amastigotes. However, since other factors besides HzO: my be involved in the killing of L cruzi, relative susceptibility of these forms of L cruzi to {-1303 or 202 some HaOz metabolite is still unknown. Still, there is one major problem with correlating H202 DI‘Oduction to killing of L c_ruii. For detection of good quantities of H103 , relatively high numbers of parasites must be associated with the macrophages and, at high levels of association, the parasites destroyed the macrophages at the 6 to 24 hours when killing of L c_ryz_i_ by macrophages is determined. Therefore, herein, at high levels of 11 Qgggi-macrophage association correlations could not be made between 1130; production and killing of T_-. c_ruzi_- Also, in this chapter, the following possible mechanism.for 11 w; trypomstigote-stimulated 11:0: production was suggested. L cruzi trypomastigotes stimulate release of the 20:4 metabolites, HETE’s and.what may be leukotrienes, which, in turn, stimulate NADPH oxidase located in the plasma membrane of the macrophage. NADPH oxidase catalyzes production of Or which then dismutates to H302 . In support of this series of events, preliminary'data showed that trypomastigotes stimulated the release of ncstly lipoxygenase pro- ducts of 20:4; 71% of 'H—cpn of stimulated release was HE'I'E’s and what may be leukotrienes. These results need to be verified by high pressure liquid.Chromatography before any sound conclusions can be made. Farther support for the hypothesized.mechanism is the fact that leukotrienes and HE'I'E’s are known to stimulate HaO: production (12, 39). Moreover, L M did stimulate 1130: production. The possibility that other fatty acids are also second.messengers is doubtful since exogeneous addition of the fatty acids 18:0, 18:2, or 18:3 was mch less stimulatory than 20:4. In fact, these results 203 agree with the literature which shows that, as the fatty acid chain length is shortened and the degree of fatty acid saturation is in- creased, there is less stimulation of 1130; production by the fatty acid (42, 43) . A considerable amouunt of endogeneous 20:4 was present as a possible source for release of 20:4 since 21% of the fatty acids in macrophage phospholipids was 20:4. This 21% is similar to the 25% reported in the literature for 20:4 in resident mcrophages (41). 20:4 is also an intermediate in stimulation of NADPH oxidase by opsonized zymosan (6, 7, 12, 13, 14, 15, 16). However, whether opsonized zymosan and L 9% use the same receptors or mechanism for stimulation of the release of 20:4 from phospholipids remains to be determined. Opsonized zymosan enters the cell through receptor mediated phagocytosis. Unfortunately, the mechanism for L cruzi invasion of macrophages and phagocytosis of L c_ruz_i_ by macrophages is unknown. In summary, for the first tine, L c_ru_z_i has been used as the stimulant in the quantitation of 11:0: production. Also, this quan- titation was done with resident mcrophages instead of the activated macrophages. L c_ru_z; (trypomstigote and opsonized trypouastigote but not amstigote)-stimulated 1130; production correlated with the level of parasite-mcrophage association. Opsonization of trypoma- tigotes increased the amount of i130; produced. Amastigotes stimulated production of very low levels of 1130: at a given level of association. Also, increased killing of trypomastigotes, opsonized trypomstigotes, and amastigotes seemed to be directly related to increased H30: production. Finally, thenechanism for L cruzi trypomastigote 204 stimulated H: 0; production by macrophages probably involves 20: 4 and its metabolites as second messengers. Future research my include verification of leukotriene production by L gnu—ml-stimulated macro- phages and, then perhaps, correlations between leukotriene production and L c_ru__z_i_-macrophage association or destruction of L g_ru_zi_ by mcrophages. Also, in order to better understand L gru__z_i_ infections, future research must include studies on variability in L M infectivity, mechanisms for association of L m with the macro- phage, and the influence of factors mde by other cell types on these processes as well as 11:0: production. 6. 10. 11. 12. References Villalta, F., and Kierszenbaum, F. (1983) J. Immunol. 1 1, 1504. Villalta, F., and Kierszenbaum, F. (1984) J. Immunol. 1 3, 3338. Nathan, C. F., Silverstein, S. C., Brukner, L. H., and Cohn, Z. A. (1979) J. Exp. Med. mg, 100. Castagna, M., Takai, Y., Kaibuchi, H., Sana, K., Kikkawa, U., and Nishizuuka (1982) J. Biol. Chem. 251, 7847. Nishizuka, Y. (1984) Nature 3_08, 693. Bromberg, Y., and Pick, E. (1984) Cell. Immunol. eg, 213. Maridonneau-Parini, I., and Tauber, A. I. (1986) Clinical Research 3_4, 661A. Tauber, A. 1., Cox, J. A., Jeng, A. Y., and Blumberg, P. M. (1986) Clinical Research 31, 664A. McPhail, L., Clayton, C. C., and Snydermn, R. (1984) Science 2%, 622. Fujita, I. Irita, H., Takeshige, K., and Minakami, S. (1984) Biochem. Biophys. Res. Comm. 129, 318. Robinson, J. M., Badwey, J. A., Kamovsky, M. L., and Kamovsky, M. J. (1984) Biochem. Biophys. Res. Canmuun. _1_2, 734. Bromberg, Y., and Pick, E. (1983) Cell. Immunol. Z_9_, 240. 205 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 206 MoPhail, L. C., Shirley, P. 8., Clayton, C. C., and Snydermn, R. (1985) J. Clin. Invest. fl, 1735. Curnuette, J. T. (1985) J. Clin. Invest. 7_5_, 1740. Vercauteren, R. E., and Heynenan, R. A. (1984) J. Leuk. Biol. 36, 751. Suzuki, T., Saito-Taki, T., Sadasivan, R., and Nitta, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 19, 591. Villalta, F., and Kierszenbaum, F. (1983) J. Immunol. 1_1, 1504. Aust, S. D., Morehouse, L. A., and Thoma, C. E. (1985) J. Free Rad. Biol. Med. 1, 3. Budzko, D. B. (1974) J. Parasitol. 69, 1037. Mercado, T. I., Katusha, K. (1979) Prep. Biochem. _9, 97. Villalta, F., and Kierszenbaum, F. (1982) J. Protozool. 2_9, 570. Hayes, M. M., and Kierszenbaum, F. (1981) Infect. Inmuun. 3_1, 1117. Kierszenbaum, F. (1981) Immunology fi, 641. Conrad, R. E. (1981) In "Phnual of Macrophage Methodology V13" (Herscowitz, H. B., Holden, H. T., Bellanti, J. A., Ghaffar, A., eds.) pp. 5-12. Marcel Dekker, Inc., New York. Bozdeck, M. J., and Bainton, D. F. (1981) J. Earp. Med. 153, 182. Yam, L. T., Li, C. Y., and Crosby, W. H. (1971) Am. J. Clin. Pathol. fi, 283. Ruch, W., Cooper, P. H., and Baggiolini, M. (1983) J. Immunol. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 207 Methods 63, 347. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. _1_9_3, 265. Baer, E., and Buchnea, D. (1959) Can. J. Boichem. Physiol. g, 953. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 61, 911. Mahoney, E. M., Hamill, A. L., Scott, W. A., and Cohn, Z. A. (1977) Proc. Natl. Acad. Sci. U.S.A. 11, 4895. Scott, W. A., Pawlowski, N. A., Murray, H. W., Andreach, M., Zrike, J., and Cohn, Z. A. (1982) J. Exp. Med. _1_56, 1148. Humes, J. L. (1981) In "Methods for Studying Mononuclear Phagocytes" (Adams, D. 0., Edelson, P. J., and Karen, H. S., eds.) pp. 641-654. Fels, A. O. S., Pawlowski, N. A., Abraham, E. L., and Cohn, Z. A. (1986) J. Exp. Med. 1Q, 752. Enilsson, A., and Suundler, R. (1985) Biochim. Biophys. Acta 616, 265. Enilsson, A., and Suundler, R. (1986) Biochim. Biophys. Acta 616, 533. Docsmpo, R., Cssellas, A. M., Ithdeira, E. D., Cardoni, R. L., Moreno, S. N. J., and Mason, R. P. (1983) FEBS Lett. 1_55, 25. Flohé, L. Becknenn, R. Giertz, H., and Goschen, G. (1985) In "Oxidative Stress" (Seis, H., ed.) pp 403-435. Academic Press, Inc., New York. 39. 40. 41. 42. 43. 44. 45. 46. 47. 208 Wirth, J. J., and Kierszenbaum, F. (1985) J. Immunol. 13_4_, 1989. Wirth, J. J., and Kierszenbaum, F. (1985) Mol. Biochenn. Parasitol. 6, 97. Scott, W. A., Zrike, J. M., Hamill, A. L., Kempe, J., and Cohn, Z. A. (1980) J. Exp. Med. 1_52, 324. Bromberg, Y, and Pick, E. (1984) Cell. Immunol. 66, 213. Kakinum, K. (1974) Biochim. Biophys. Acta 316, 76. Anderson, B. R., Amirault, H. J., LeBreton, G. C. (1981) Prostaglandins Q, 469. Tsunawaki, S., and Nathan, C. (1986) J. Biol. Chem. _2_6_1, 11563. Zingales, B., and Colli, W. (1985) Current Top. Micro. Immunol. m, 129. Nogueira, N., Chaplan, S., and Cohn, Z. (1980) J. Exp. Med. _1_53, 447. Chapter 5 FUNCTIONAL CAPACITY OF RESIDENT PERITONEAL MACWPHAGES Find ZINC DEFICIENT MICE 209 ABSTRACT Zinc deficiency greatly increases the mortality rate of A/J mice infected with the parasite macaw c_ru_u_z_i_ (L c_rluz_i_.). This is in part due to the inability of resident macrophages from zinc deficient mice to phagocytose and destroy the parasite. Since de- struction of L M is thought to be dependent on H20: production by the macrophage, we investigated the ability of resident peritoneal macrophages from zinc deficient mice to produce 11:0; . Upon stimula- tion with the coumonly used agents, phorbol lZ-myristate 13-acetate (PMA, a chemical), or opsonized zyncsan (a yeast cell extract), or arachidonate (the second messenger in opsonized zymosan stimulated 1130; production), zinc deficient macrophages produced normal ancunts of 11202 as compared to zinc adequate mcrophages. However, when L <_:ru_z_i was the stimulant, macrophages from severely and moderately zinc deficient mice produced lower amounts of H30: than the zinc adequate controls (66% and 83%, respectively, of controls; both of which are significantly different than control). However, when H30; production was expressed as amount of 1130: per L mug; associated with the macrophages, similar quantities of 11202 were produced by nuacrophages from all dietary groups. Thus, necrophages from zinc deficient mice could produce norml ancunuts of 1120: upon stimulation with :1; c_ruii when the degree 0. number of associated parasites was compared to the amouunt of H30; production. This was not due to repair 210 211 i_g 3113.22 since the amount of zinc in culture medium was always limit- ing (less than 1.5 ug/ d1). The latter observations indicated that the failure of mcro- phages from zinc deficient mice to kill L guz_i might be more related to reduced rate of association of the parasite with deficient mcro- phages. Association of L gru_z; trypomstigotes with macrophages involves both direct invasion by the parasite as well as macrophage phagocytosis. The receptors and mechanisms for association are unknown. However, the ability of mcrophages to carry out phagocy- tosis has previously been found to be reduced if the content of long-chain saturated fatty acids in the phospholipids of the plasma membrane is increased. Also, previous studies have shown that livers from zinc deficient snimls have an increased amount of long-chain saturated fatty acids. Thus, zinc deficient mcrophages my also contain these fatty acids. However, when analyzed the ancunt of 16:0, 18:0, 18:1, 18:2, and 20:4 in the phosfiuolipids, fatty acids were found to be the sane for resident macrophages from zinc defi- cient and zinc sufficient mice. Since exogeneouus addition of leukotrienes has been shown to increase L c_ru_ii association and destruction, it remained to be determined which 20:4 metabolites were released upon stimulation with L qu_z_i_ and if mcrophages from deficient mice were capable of producing these metabolites. Before analyzing release of radio- labelled 20:4 metabolites, it was important to determine if mcro- phages from the deficient mice incorporated the same amount of radio- activity as controls. Indeed, mcrophages from the zinc deficient 212 mice incorporated 3H—arachidonic acid into the same types of phos- pholipids at equal quantities as mcrophages from zinc adequate mice. Preliminary data indicated that, When stimulated with L M, macrophages from zinc deficient mice released three times as much of an arachidonic acid metabolite, comigratory with leukotrienes, as macrophages from zinc adequate mice. However, the release of the arachidonate metabolites PGE; and HE'I'E by L 9122.1 stimulated mcro- phages from deficient mice was norml as compared to macrophages from zinc adequate mice. Therefore, the increased amcumt of arachi- donate released by mcrophages from deficient mice compared to norml mcrophages was metabolized to what my be leukotrienes. Before sound conclusions can be made concerning the radioactive peak com- igratory with leukotrienes, the identity of this peak must be veri- fied. However, it can be concluded that the decrease in association with and/or decreased destruction of L gru__z_i by macrofluages from deficient mice ms not due to altered fatty acid campositionof the mcrophage phospholipids or reduced production of H30: , reapectively. IN'I'RDDUCI‘ION Zinc deficiency increases the susceptibility of both humans and animals to disease and infections (1-6). Zinc deficient mice are more susceptible to infection with Egypggpsoma gggg; (I1 Qgggi) (3), an obligate intracellular parasite which causes Chagas’ disease in humans. Fraker et. al. (3) showed that there is a synergy between zinc deficiency and infection with I; Qgggi. Upon infection, the mortality rate and the level of blood parasitemias were drastically increased in the zinc deficient mice as compared to infected.zinc adequate controls or noninfected zinc deficient mice. The inability of the mice to combat the :1 916g; infection was shown to be due, at least in part, to a reduced capacity of resident peritoneal macrophages to phagocytose and.destroy the parasite (7, 8). lg ELISE: the percentage of macrophages which had.I1 926g; bound to their surface and the number of T1 916;; per 100 macrophages were substantially reduced in the zinc deficient groups (7, 8). In addition, after 24 hours, macrophages from.the zinc deficient groups were not able to destroy as many parasites as the zinc adequate group (7, 8). These defects were reversed by a half hour preincuba- tion i_n m of the mcrophages from zinc deficient mice with zinc at five times the physiological concentration (7, 8). Other metals such as copper and nickel were unable to restore parasite uptake and killing (7). Manganese had.a slight restorative effect (7). 213 214 Therefore, the defect(s) in the zinc deficient macrophage seemed to be either directly or indirectly dependent on zinc. Hydrogen peroxide produced by macrophages has been shown to correlate with destruction of L gruii (9-11). Also, addition of physiological levels of 11302 has been shown to destroy L c_ruz_i_ (9, 10). Other oxygen metabolites such as OH- and 02' are probably not involved in the destruction of L c_ru_z_i since scavengers of these oxygen species do not affect the ability of the macrophages to kill L gu_z_i (10). FXurthermcre, in chapter 4 of this thesis, it was shown directly that L 9_n_'u_z; could indeed stimulate the production of quantifiable amouunts of mo. . However, taken together, this does not indicate that 11202 is the afferent molecule that destroys L c_ru_zi. In fact, L c_ruii itself contains superoxide dismutase, the O;- scavenger, (12) but not catalase, the 11202 scavenger, (12-14). Still, ago, is involved in the destruction of L gru__z_i by mcro- phages. Since zinc deficient mcrophages have a reduced capacity to destroy L mug, and H¢O; is important in the destruction of this parasite, it was presumed that, in the absense of zinc, macrophages might have a reduced capacity to produce n.0, sinceit was demon- strated in chapter one that zinc was important for mny reactions associated with the production of Hsz . In this chapter, the ability of zinc deficient mcrophages to produce Han was analyzed. In all studies on zinc deficient mcrophages, the ancunt of zinc available was less than 1.5ug/dl. Thus, the amcuunt of zinc available for 215 restoration of function by mcrophages from zinc deficient was mini- mal. Because of the reduced ability of L M to associate with mcrophages from zinc deficient mice, it was also possible that alterations in the composition of the membrane or secondary mes- senger system were created by the deficiency. A reduced amount of 20:4 has been fouund in phospholipids of zinc deficient rat livers (15-22) . Further, decreases in unsaturated long chain fatty acids such as 20:4 have been shown to result in reduced phagocytosis (23, 24). Therefore, since zinc is required for synthesis of long chain uunsaturated fatty acids such as arachidonic acid (20:4) and altera- tions in fatty acid composition affects phagocytosis, it was thought that the mcrophages from zinc deficient mice my have an altered fatty acid composition within their phospholipids. To test the possibility of a reduction in long chain unsaturated fatty acids such as 20:4 in phospholipids of mcrophages from zinc deficient mice, the fatty acid composition of macrophage phospholipids from zinc deficient and zinc sufficient mice were compared. Fuurthermore, exogeneous addition of leukotrienes, lipoxygenase products of 20:4, has been shown to increase association with and killing of L .C_I'U_£i_ by mcrophages (25, 26). Therefore, production of 20:4 metabolites my be altered in mcrophages from zinc deficient mice resulting in reduced association with and killing of L c__ru_z_i_ by deficient mcro- phages. Preliminary data in Chapter 4 indicated that L gr;_uz_i_ stimu- lated the release of HE'I'E’ s, PGE’s and another 20:4 metabolite com- igratory with leukotrienes by mcrophages from normal mice. 216 Therefore, the relative amounts of these 20:4 metabolites released by the macrophages from zinc deficient and zinc adequate mice were compared. Also, to determine if any alterations among the dietary groups was due to altered release or incorporation of the 3H-20:4, the amount of radioactivity incorporated into the macrophage phos- pholipids from the dietary groups was compared. In summary, 1130; production by mcrophages and possible altera- tions in the membranes of macrophages from zinc deficient and zinc adequuate mice was the subject of these studies. MATERIALS ANDMEI'I-DIB Animals. Femle Crl—CD-l (ICR)BR Swiss and femle A/J mice were purchased from Jackson laboratories, Bar Harbor, Maine. CD1 female mice were puurchased from Charles Rivers, Portage, Michigan. mpg. Six week old A/J female mice were placed in stainless steel cages with mesh bottom to reduce recycling of zinc. They were fed 66 libitum a biotin fortified egg white diet containing either deficient (0.8 pg Zn/g) or adequate (27 pg Zn/g) levels of zinc. The composition of the diet is described in Appendix. Since inanition accompanies zinc deficiency, a third group, the restricted mice, were fed zinc adequate diet equivalent to the average ancunt consumed the previous day by zinc deficient mice. All mice had free access to deionized distilled water ((0.2 pg Zn/g). Feed jars and water bottles were washed with 4N HCl and rinsed with deionized water to remOve zinc. The mice were weighed at least once a week. At the end of the dietary period, those mice that received the zinc deficient diet and weighed 65-68% of the average body weight of the control mice were designated as severely zinc deficient mice. Mod- erately deficient mice were defined as weighing 70-74% of the average control mouse body weight. Previous studies indicated the latter group is only modestly effected by inanition (27, chapter 2). Zinc analgis. The diets, culture media and sera were analyzed for zinc content by atomic absorption spectrophotometry (Varian 217 218 Techron AA—175, Springvale, Australia) as described in earlier publi- cations (28, 29). Isolation of Trypgnosom§,cruzi (T. cruzi . Four week old Crl- CD—1(ICR)BR Swiss mice were infected.intraperitoneally with 2 x 105 blood forms (trypomastigotes) of I; Qgggi. TWelve to 14 days later, blood.was collected.from the axillary artery of mice anesthetized with ether. Blood was collected in tubes containing heparin or disodium ethylene-diaminotetracetate (EDTA) powder so that the final concentrations were 25 U/ml or 2.5 mg/ml, respectively. Parasites from the blood were separated on a lympholite gradient (30) and ' passed through a diethylaminoethylcellulose column (31). The para- sites were centrifuged (800 x G, 15 mdnutes, 4°C) and resuspended.in Dulbecco’s modified miniml essential medium (and, Gibco, Grand Island, NY) supplemented with 100 IU penicillin and 100 pg strep— tomycin / ml. Greater than 99% of the cells were viable parasites in the trypomastigote form. Before use in the assays for 11:02 , the parasites were washed.two times in phosphate buffered.saline (PBS, pH 7.4) containing 1%,glucose and resuspended in the reaction solution for the H30: assay. Eggpgrgtion of opsonized gyggggp. One mg of zymosan, a yeast cell extract (Sigma, St. Louis, m), was incubated with one mil- liliter of complement inactivated.mouse serum from.A/J mice for 1 hour at 37°C. The opsonized zymosan was washed 2 times and resuspend- ed in 10 mM PBS (pH 7.4). The zinc content of opsonized zymcsan was 55 pg Zn/ g zymosan. 219 Collection, isolation, apd identification of peritonealgmgcro— meg. Cells were harvested from the unelicited ncuse peritoneum by lavage with 5 ml of cold (4°C) DMEM and 0.5% w/v gamma globulin free bovine serum albumin (BSA, 0.4 ui Zn/ 8 BSA, Calbiochem, La Jolla, CA) (32). Cells were adhered to tissue culture plates for 1 1/2 hours at 37°C and 7% (Du-ambient air. Nonadherent cells were removed by washing with 37°C m. The percentage of mcrophages- /monocytes adhered to the culture plate was determined by staining for nonspecific esterase activity as detailed.in.the modification (33) of the technique described.by'Yam.et. al. (34). Always, 94- 100% of the adherent cells were identified as monocytes/macrophages by staining for nonspecific esterase and by morphology. Macrophage vigbility. Viability of the adherent macrophages was determined using the trypan blue exclusion method.at the end of each assay. Always >95% of the macrophages were viable. Association of T. crugi with macrophages. After incubating the mcrophages with parasites for 90 minutes in m ((1 pg Zn/ dl) at 37°C and 7% (Du-ambient air, the nonassociated parasites were remcved by washing three times with PBS - 1%.glucose. The cultures were incubated.in DMEM for another 18 hours and/or fixed with methanol for 5 minutes, allowed to dry, and stained for 1 hour with Giemsa in 10 mM phosphate buffer (pH 6.8). The cells were washed one time with 10 mM phosphate buffer (pH 6.8). NUmbers of parasites per 100 macrophages and percent of macrophages associated with parasites were determined by counting at least 200 cells per replicate. 220 Phenol Red Assay. The assay conditions described by Pick and Mizel (35) were ncdified for use with resident macrophages. Resident peritoneal exudate cells (7 x 105) in 100 pl were allowed to adhere to a 96 well flat bottom plate (Corning Glass Works, Corning, NY) for 1 1/2 hours. After removing nonadherent cells, the adherent mcrophages were washed with 10 m phosphate buuffered saline (PBS) - 1% glucose (37°C) . Forty microliters of the reaction solution con- sisting of 140 mM NaCl, 0.5 mi“! CaClz , 10 I!“ potassium phosphate buffer (pH 7.4), 2 mm NaNa, 5.5 mM glucose, 0.56 mM phenol red (United States Biochemical Corporation, Cleveland, Ohio), and 19 U/ml horse- radish peroxidase (Sigma, St. Louis, in) were added to each well. The reaction solution contained 1.4 pg Zn/ dl. NaNa was included since it inhibits catalase, the cytoplasmic scavenger of H20; . Phorbol 12—myristate 13-acetate (PMA, Consolidated Chemicals, Midland, MI) was diluted in ethanol and added to the cells in a volume of 2 pl per well so that the ethanol concentration was less than 1%. Opsonized zymcsan or EMA stimulated cells were incubated for 90 minutes at 37°C umder a humidified atmcsphere of ambient air. Stan- dards consisted of wells containing reaction solution and 0-60 pM H302 but no cells. Nonspecific production of 11:02 was determined by including wells containing cells but no stimulant or stimulant but no cells. The reactions were stopped and protein dissolved by the addition of 2 pl of 10 N Naa-I. The absorbance at 610 nm of pooled triplicates was determined using a microcuvette and a spectrophoto- meter (Gilford, Oberlin, Ohio). Macrophage protein was determined by the Lowry (36) . The data were presented as nmoles I-hOg/mg 221 macrophage protein from the following calculation: (pM H202 derived from the standard curve) X (1 liter/1000 ml) X (0.132 ml final volume of sample) X (1000 nmoles/1 pmole)/(mg mcrophage protein determined by the Lowry) = nmoles of 1120: /mg macrophage protein. Homovanillic acid assay for Hag; . The assay conditions described by Ruch e_t_.. al. (37 ) were modified for use with resident macrophages and the stimulant, L gr_uz_i_ trypomastigotes. Resident peritoneal cells (2 x 10') in 1 ml DMEM - 0.5% BSA (1.5 pg Zn/ dl) were allowed to adhere to a 24 well flat bottom plate (Corning). Nonadherent cells were removed from the adherent macrophage monolayer by washing three times with PBS - 1% glucose (37°C). Two hundred microliters of reaction solution which consisted of PBS, 1% glucose, 0.09 nM CaClz , 0.05 n“ MgClz , 200 pM homvanillic acid, and 2 U/ml horseradish peroxidase (1.4 pg Zn/ dl) were added per well. Arachidonate was diluted in ethanol and 2 pl added per well so that less than 1% of the reaction solution was ethanol and the final arachidonate con- centration was 50 pM. However, when parasites were the stimulant, they were resusperded in the 200 p1 of reaction solution to be added to the mcrophages. Standards consisted of wells containing reaction solution ard 0-20 pM 11302 but no cells. Nonspecific production of P120: was determined by ircluding wells containing cells but no stimu- lant or stimulant but no cells. The plates were centrifuged at 50 x G for 3-4 minutes to increase parasite-macrophage interactions and incubated for 90 minutes in a humidified atmosfiuere at 37°C ad 7% (Dz-ambient air. The reaction was stopped by the addition of 25 pl of 25 mM ED'I'A and 0.1 M glycine at pH 12. The relative fluorescence 222 of the homovanillic acid dimer was determined using a spectrofluoro- meter (Perken-Elmer 650-40) equipped with a microcuvette. The excita- tion ard emission wavelengths were 312 nm and 420 nm, respectively. The slit widths for excitation and emsision were 2 nm and 5 nm, respectively. The amount of H20; in the samples was analyzed by linear regression of the standard curve. The amount of macrophage protein was determined by the method described by Lowry (36) . The data are presented as nmoles Hzozl m macrophage protein from the following calculation: (pM H202 derived from the standard curve) X (1 liter/1000 ml) x (0.225 ml final volume of sample) x (1000 uncles/1 pmole) / (mg mcrophage protein as determined by the Lowry) = nncles HzOz / ms mcrophage protein. Synthesis of margaric mosguétidylcholine (38). Margaric (17:0) phosphatidylchol ine was chosen as an internal standard for the anal- ysis of fatty acid composition of mcrophage phospholipids sirce methylated margaric acid has a different retention time during gas liquid chromtography than fatty acids normlly found in good quan- tities in mamlian phospholipids. Margaric acid (200 mg) was placed in a sunll teflon capped tube with 300 pl thionyl chloride, pulsed with nitrogen, and placed on a steam bath for 1 hour. The products were evaporated to dryness under nitogen in a 80° C water bath , washed successively with water, 10% sodium carbonate, ard water, and then dried under nitrogen. The fatty acyl chloride (50 mg) was reacted with the CdClz complex of L—alpha-glycerophosphorylcholine (10.45 113) while stirring for 2 hours in 150 pl chlorform and 9 pl pyridine under an atmosphere of nitrogen. The stirrer was rinsed with 223 chloroform ard the solvents were evaporated under nitrogen. The reaction products were dissolved in 166 pl ether (dry), cleared by centrifugation, ard the solvents removed under nitrogen. The lipids were dissolved in 500 pl acetone (dry), placed on a acetone-(X); bath for 1 hour and isolated by centrifugation in the cold. They were precipitated again with 250 pl acetone. The lipids were dried under nitrogen, dissolved in chloroform:methanol:water (5:4:1, v:v:v) and passed over a 2ml column of amberlites IR50:IH350 (50: 50). The column was washed with the solvent mixture. The effluent was dried under nitrogen, dissolved in chloroform ard placed over a 1 ml silicic acid column. The silicic acid column was washed with 30 column volumes of chloroform ard the phospholipid was eluted with 15 column volumes of chloroformzmethanol (1:1) followed by 10 column volumes of methanol . The eluent was evaporated under nitrogen ard the [nos- pholipid was stored dry under nitrogen at -20°C. The last 5ml frac- tion of the silicic acid column effluent did not contain nnrgaric acid as measured by gas liquid chrountography of the fatty acid methyl ester. Esterification is described below. The phospholipid fraction from the silicic acid column contained mrgaric acid as determined by gas liquid chromtography of the methyl ester. Fatty acid maition of mcroflge mosgolipids. Macro- phages were adhered for 2 hours to 24 well tissue culture plates in DIEM-0.5% BSA (1.5 pg Zn/ dl). Nonadherent macrophages were removed by washing three times in RTFM-0.5% BSA. The adherent mcrophages were collected by scraping into 0.15M NaCl. Sirce one twelfth of the amount of the above synthesized internal standard, mrgaric 224 phosphatidlycholine, gave a peak height during gas liquid chromtog- raphy that was 80% of the chart range, this amount of internal stan- dard was added to each sample to control for sample loss during the purification of the macrophage phospholipids. The lipids were ex- tracted by the method described by Bligh ard Dyer (39) . Briefly, methanol:chloroform:water (4:2:8, v:v:v) was added to the macrophages. Then one volume of chloroform followed by one volume of water was added to the extraction mixture. The mixture was centrifuged ard the chloroform layer was collected ard evaporated under nitrogen. The lipids were resuspended in chloroform and placed over a 0.5 ml heat activated silicic acid column in a pasteur pipette. The neutral lipids which do not bind to the column were washed through the column with chloroform. The phoslnolipids bound to the column were then eluted with chloroformzmethanol (1:1) followed by methanol. The organic solvents were evaporated from the neutral atd phospholipids under nitrogen . The fatty acids were converted to methyl esters (40) by incubating overnight at 7 5°C in 6% I-ICl in methanol under nitrogen. The fatty! acid methyl esters were extracted with three equal volumes of hexane and washed orce with water. Water was removed from the hexane by the addition of anhydrous sodium sulfate. The hexane was collected ard evaporated under nitrogen. The fatty acid methyl esters were dissolved in hexane and analyzed (41) using a Hewlett-Packard gas chromatograflu equipped with a 6 foot x 1/8 itch i.d. glass column packed with 10x sp-2330 Chromasorb W/AW. Analysis was performed isothermally at 180°C for 7 minutes followed by an increase of 4°C /minute to a limit of 200°C. The carrier gas-flow 225 rate was 30 ml/minute. Peak identifications were based on relative retention times of standard methyl esters (Nu Chek Prep, Elysian, Minnesota). Relative peak areas were measured by a Hewlett-Packard 3380A electronic integrator. Fatty acid quantities were obtained by linear regression from a standard curve of peak area versus amount of fatty acid methyl ester. Total mcrophage fatty acid was determined by comparing peak area of the internal standard in the samples of macrophage phospholipids to peak area of a sample of internal standard alone that had not been sutmitted to the phospholipid isolation procedure. Also, samples of macrophage ficspholipids from severely zinc deficient, moderately zirc deficient, restricted, and zirc adequate mice, which did not contain the internal standard, did not have a peak with the same retention time as the internal standard. Thus, since the macrophage phospholipids did not contain margaric acid, margaric acid was a valid internal standard. Arachidonic _aLcid release by macroQgges (42, 43). Macrophages adhered to 24 well plates were ircubated with 1.5 pCi (5, 6, 8, 9, 11, 12, 14, 15-3H)arachidonic acid (Amersham) in 2 m1 of W over- night ard washed three times with serum free man (<1 pg Zn/ d1) at 37°C. The mcrophages were stinulated by trypomastigotes of L c_ruzi_ for 2 hours in serum free IMEM. Nonstimulated mcrophages were ircluded to control for spontaneous release of radioactivity by the macrophages. The culture medium was removed frcm the adherent macrophage monolayer, centrifuged, ard acidified to 0.03M with citric acid. The acidified media was extracted three times with 3 ml of chloroform:methanol (2:1, v:v). The chloroform layers were combined, 226 washed two times with 2 ml of methanol:water (2:1, v:v) ard evaporated to dryness under nitrogen. The residue was dissolved in 40 pl of ethylacetatezmethanol (3: 1, v:v) ard applied to precoated plates (Silica Gel 60, Merck) for thin—layer chromatography. The internal standards PGE: ard arachidonate were adied to each sample. Other standards 6-keto-PGFuu no , LT34 , UPC. , LTD. , LTE4 , and ricinoleic acid (an oxidized product of arachidonate) were run separately along with PGE: ard arachidonate. Arachidonate ard its metabolites were separated by developnent twice in the solvent system chloroform:ethyl- acetate:methanol:acetic acid:water (140:60:16:2:1, v:v:v:vzv). The internal standards were visualized by exposure to I: . Half centimeter segments for each ascerding sample of the silicic acid coated plates were scraped and radioactivity was measured by a Delta 300 scintilla- tion counter (Tracor Analytic). Radioactivity for each segment of a sample was plotted versus distarce (cm) on the plate. The data for each radioactive peak are presented as ’11an of stimulated release minus 3H—cpn spontaneous release from nonstimulated samples . 3H—a_r_achidonic acid labelled macroflge gosgolipids. The identification of those macrophage phospholipids- that contained 3 H- arachidonate was described by Emilsson g. _a_l. (44, 45) . Briefly, radiolabelled resident macrophages were collected by scraping into 1 ml of ice-cold 10 m HCl. Lipids were extracted with 6 ml of chloro- form:methanol (1:1, v:v). Phase separation was obtained by addition of 2 ml of 10 I!“ 1101 followed by centrifugation. The chloroform layer was removed, evaporated to dryness, ard dissolved in 40 pl ethylacetatezmethanol (3:1, v:v) . The lipids were separated by thin 227 layer chromatography on precoated plates (Silica Gel 60 , Merck) with the solvent system chloroform:methanol:acetic acid:water (25:20:3:O.3, v:v:v:v) . Lipids were visualized by exposure to I: . The plates were scraped in 0.5 cm segments for each ascending sample ard radioac- tivity was measured by a Delta 300 scintillation counter (Tracor Analytic). Radioactivity for each segment of a sample ms plotted versus distarce on the plate. The total radioactivity for each peak was calculated. Statistics. The mean ard standard error of the mean were calcu- lated for each treatment group. Probability values for the comparison of the zirc deficient ard restricted groups to the control group were determined by a completely random Anova followed by Dunnett’s Test . RESULTS We analyzed the ability of resident peritoneal mcrophages from zinc deficient mice to produce 11an upon stimulation with several agents. The ancunt of zirc present during these studies was minimal since the medium for isolation of the macrophages contained <1 pg~ Zn/ dl and the reaction solutions for measuring Th0; production contained less than <1 pg Zn/ d1. First, opsonized zymosan, a yeast cell extract to which autologous serum antibodies have been bound, or the secord messenger in opsonized zymosan stimulation of [hot production, arachidonate (20:4) , were exogeneously added to the macrophage monolayer to stimulate 14an production. If opsonized zymosan-stimulated 1120: production by macrophages from zirc deficient mice was reduced, stimulation of the deficient mcrophages with exogeneous 20 : 4 would help delineate whether a defect was in the release of the secord messenger 20:4 or in the stimulation of the "oxygen burst" by 20:4. However, at several corcentrations of op- sonized zymosan, resident peritoneal macrophages from zirc deficient mice were capable of producing norml amounts of I130: over a period of 90 minutes when compared to macrolnages from zirc adequate mice (Figure 1). At the highest dose of 12 pg of opsonized zymosan, 140 i 10 nmoles P120; were produced per mg protein by macrornages from zinc deficient or zirc sufficient mice. Also, exogeneous addition of 20:4 (50 pM) stimulated prochction of the same quantities of mo: 228 229 Figure 1 . Opsonized zymosan-stimulated 1120: production by resident peritoneal macrophages prepared from severly zirc deficient, moderately zirc deficient, restricted, or control mice as measured by the phenol red assay. Adherent macrophages from 2.1 x 10‘ peritoneal cells were incubated for 2 hours with 4, 8, or 12 pg opsonized zymosan in the reaction solution and H20: content determined. Protein content of macrophages ircubated in IMF}! for 2 hours was the same per well for all dietary groups. Each point represents the mean 1 SEN for triplicates for pooled peritoneal cells from 10 to 15 mice. 230 o—osevcneuv zmc amcueuu Hmoaumsuv zmc oer-mam o—o asmucueo I50 ~Hcormuou 3r. N. IOO 50 nmoles H202, mg macrophage protein 3:3 ' l l I 4 8 l2 pg OPSONIZED ZYMOSAN 231 (about 9 nmoles H202/ m8 macrophage protein) by macrophages from severe, moderate, restricted, and control mice (Figure 2). For these and all subsequent experiments, the amount of macrophage protein as determined by the Lowry was the same for all dietary groups (data not shown). Thus, the same number of macrophages had adhered to the plate for each dietary group and the same number of cells in each dietary group were responding in these experiment. Also, greater than 95% of the adherent cells for all dietary groups were viable and were identified as macrophages by staining for nonspecific ester- ase and by morphology. Therefore, the mechanism for opsonized zymc- san—stimulated 1130; production was unaltered in the macrophages from zinc deficient compared to zinc adequate mdce. Although opsonized zymosan is a more natural stimulant, phorbol 12-myristate 13-acetate (PMA), a chemical, seems to be the agent most often used to stimulate H302 production. Upon stimulation with PMA, macrophages from zinc deficient mice again produced normal amounts of P130; in 90 minutes as compared to 1120: production by macrophages from zirc adequate mice (Figure 3). Therefore, P120; production and the mechanism for stimlation of H202 production by PMA was unaltered by the deficiency. Since I; ggugi may have a different mechanism of entry into the macrophages than opsonized zymcsan or PMA, it was important to measure 1130; production by L gm__z_i-stinmlated macrophages from zirc deficient and zinc adequate mice. Using the improved assay conditions for measuring H302 production described in Chapter 3 and!L cruzi as the stimulant, the macrophages from severely zinc deficient mice produced 232 Figure 2. Arachidonate-stimulated 1130: production by resident peritoneal mcrophages prepared from severly zirc deficient, moderately zirc deficient, restricted, or control mice as measured by the HVA assay. Adherent macrophages from 2 x 10' peritoneal cells were incubated for 2 hours in the reaction solution with 50 pM arachidonic acid and.HaOh content determdned. Protein content of mcrophages ircubated in 11491 for 2 hours was the same per well for all dietary groups. Each bar represents the mean 1 SD! of triplicates of pooled peritoneal cells from 10 to 15 mice. 233 % I5? 0 5 5205 00939.02: oEx NON... 8.06: MODERATE RESTRICTED CONTROL SEVERE Figure 3 . 234 PMA-stimulated HaOz production by resident peritoneal macrophages prepared frtm severely zinc deficient, moderately zirc deficient, restricted, or control mice as determined by the phenol red assay. Adherent mcrophages from 2.1 x 10‘ peritoneal cells were ircubated for 2 hours with 3 pg INA in the reaction solution ard H30: content determined. Protein content of macrophages incubated in 111E! for 2 hours was the same per well for all dietary groups. Each bar represents the mean 1 SE! of triplictes of pooled peritoneal cells from 10 to 15 mice. 235 W- _7//////////// c T .wuase«saga m w m m 7 £0.05 32380:. 9: \NONI $.06: Ja' SEVERE MODERATE RESTRICTED CONTROL 236 63 to 65% as much H302 as zinc adequate controls for the various I; ggug;:macrophage ratios tested (Figure 4). The H202 production by macrophages from.mcderately zinc deficient mice was also significantly lower than controls (65-73% of controls). However, the level of I; ggugizmacrophage association was too low for accurate quantitation of association since there was only one parasite per macrophage and only 10 i 5% of the macrophages had.parasites associated.with them. Unfortunately, the degree of infectivity of this parasite is highly variable and unpredictable since not enough is known about the mechan- ism(s) of parasite-macrophage association. Quantitation of I; qugi- macrophage association is very important for interpretation of the results, since in chapter 4 it was shown that H202 production corre- lates with I; ggugi-macrophage association. An interpretation of the reduced H202 production by I; ggugI-stimulated.macrophages from zinc deficient mice can not be made without knowing the I; ggugi- macrophage association. In a subsequent experiment, the parasites were much more infec- tive ard, again, 1120: production by the macrophages from the deficient mice was significantly less than controls (Figure 5). In this case, at the optimal I; ggugi:macrophage ratio of 30:1, the severely and moderately zinc deficient groups were 66% and.83%, respectively, of controls. However, the number of I; 923;; associated with severely and moderately zinc deficient macrophages was also significantly reduced (71% and.75%, respectively, of controls at a I; qugi:macro- phage ratio of 30:1) (Figure 6). This reduction in the number of Figure 4. 237 Experiment 1: L gr_u_zI-stimulated P1302 production by resident peritoneal macrophages prepared from severly zinc deficient, moderately zinc deficient, or control mice as measured by the HVA assay. Adherent macrophages from 2 x 10° peritoneal cells were incubated for 2 hours in the reaction solution with parasites at a L c_ru_gmacrophage ratio of 20:1 or 35:1 and HaOz content was determined. Protein content of mcrophages ircubated in DMEM for 2 hours was the same per well for all dietary groups. Each bar represents the mean 1 SEM of triplicates from pooled peritoneal cells of 10 to 15 mice. Asterisk indicates significance of p < 0.05 or better as compared to the control group for the same L cruzi :macrophage ratio . 238 L - , Tv/é/Z/Zé/éé??? . s11 ié/éé/é/é/é/Q Ts was .é/é/é/é/é/é/w 5305 002328.: oE\~o~I 8.9:: MODERATE CONTROL SEVERE Figure 5 . 239 Experiment 2: At high levels of L m-macrornage association, L _c_ru__z_i_-stimlated 1'1302 production by resident peritoneal mcrophages prepared from severly zinc deficient, moderately zinc deficient, or control mice as measured by the HVA assay. Adherent mcrophages from 2 x 10‘ peritoneal cells were incubated for 2 hours in the reaction solution with parasites at a L c_rpz__i:macrophage ratio of 5:1, 10:1, 20:1 or 30:1, and H202 content was determined. Protein content of macrophages incubated in DIEM for 2 hours was the same per well for all dietary groups. Each bar represents the mean _4_- SFM of triplicates from pooled peritoneal cells of 10 to 15 mice. Asterisk irdicates significarce of p < 0.05 or better as'compared to the control group for the same L cruziznncrornage ratio. 240 ”an.”ovuaaaaaaaov”Oaxaca.” m. . a m w. ._ mwmmm macs- e t * m usea.€%...a§£$55a a VII/III!!! m * 34%.«owovwowaouaouou H. * riff/I’d are m 301' O O 2 £82.. 32328:. oExaou: 3.9:: Figure 6. 241 Number of L c_rin associated with resident peritoneal macrophages prepared from severely zirc deficient, moderately zirc deficient, or control mice. Macrophages ard parasites are the same as those used in figure 6. Trypomstigotes were incubated for 2 hours with macrophages from each dietary group at a L c_ruLih macrophage ratio of 5:1, 10:1, 20:1, or 30:1 in DIEM. Nonassociated parasites were removed by washing. The remaining parasites ard macrophages were stained with Giemsa. The number of parasites and number of macrophages with parasites were counted. Each bar represents mean 1 SE“! of triplicates of pooled peritoneal cells from 10 to 15 mice. Asterisk indicates significance of p < 0.05 or better as compared to the control group for the same L cruzimncrofiuage ratio. 242 Control . an e .. - s m M... m m m *1 ..m_ D a E I * .. .a.3.......................a 551...”. _W .. . * L m m m a «82832. 8. \ g as .352. 243 parasites associated with mcrophages from zinc deficient mice is consistent with results in several previous studies (8, 9) . The percentage of macrophages from zinc deficient versus zinc adequate mice, which had one or more L M associated with it, however, was not significantly different (Figure 7). In contrast, previous studies have shown that, at lower levels of association, the per- centage of zinc deficient macrophages associated with L gru_£_i was also significantly lower than that with zinc adequate macrophages (8, 9) . This inconsistency is most likely due to much higher levels of parasites that had to be used in these studies in order to detect P120; in a quantitative manner. At the high levels of association required for production of good quantities of Han , the macrophages were always viable for the few hours during the assay for mo. . Furthermore, when the amount of 1120: produced was calculated per number of parasites associated with 100 macrophages in these par- ticular experiments (Figure 8 derived from Figures 5 & 6), there appeared to be no differerce in the ancunnt of P120: produced amounng macrophages from the various dietary groups. For all groups, there was about 3 _4_- 1 nmoles HaOz/ mg mcrophage protein/ L _c_ru_zi_ as- sociated with macrophages produced. This suggested that deficient macrophages can produce normal amounts of H20: upon stimulation with I; gag; but there was simply less stimulant ard therefore less peroxide production due to the decrease in number of L 9_ru__z_i as- sociated with the deficient macrophages. The data suggested that the mjor defect in P1202 production by macrophages from zinc deficient mice was a defect in ability of L Figure 7 . 244 Proportion of macrophages from severely zirc deficient, moderately zirc deficient, or control mice that were associated with L m. Pbcrophages and parasites are the same as those used in figure 6. T‘rypomastigotes were incubated for 2 hours with nncrofinages from each dietary group at a L m: macrophage ratio of 5:1, 10:1, 20:1, or 30:1 in THEM. Nonassociated parasites were removed by washing. The remaining parasites and macrophages were stained with Giemsa. The number of mcrophages with parasites was counted. Each bar represents the mean 5; sea of triplicates of pooled peritoneal cells from 10 to 15 mice. 245 IOO ' 3 75- 0| 0 1 °/. macrophages associated with T..cr N 0| I \\\\\h Moderate ‘licruzi; macrophage - D 5:l Q IO=I B 20=I I 30=I Control Figure 8. 246 Amount of HzOz produced per L c_ru_gi associated with resident peritoneal mcrophages prepared from severely zinc deficient, moderately zinc deficient, or control mice as calculated from figures 6 and 7. (nanomoles Ham/mg macrophage protein)/(number of T.cruzi/100 macrophages). 247 PM” m .... u m ..... m ..m 5 m 2 w m We s _. we ,2? c ..a.. a a m a .. wassewage...was.usage... . T J7 I A m r m. 9,. m. . _ $033 80232005 55 3.3380 qua Fxéofih 03.1286 oExaoa: 8.2:: 248 933;; to associate with the macrophages. The mechanisms for I; ggggi penetration of macrophages and phagocytosis of I; 922g; by macrophages is unknown. In an attempt to explain the deficit in association of deficient macrophages with I; ggggi, we analyzed fatty acid composition of phospholipids from zinc deficient macro- phages and I; Qgggi stimulated 20:4 release by zinc deficient macro- phages. The relative proportions of the fatty acids in the macrophage phospholipids were the same in the zinc deficient, restricted, and zinc adequate groups (Figure 9). The relative ratios for 16:0, 18:0, 18:1, 18:2 to 20:4 were 4:3:1:1:3. Thus about 25% of the fatty acid of the phospholipids was 20:4. The total amount of each fatty acid in the phospholipids was also the same for macrophages from zinc deficient and zinc adequate mice (Figure 10). In con- clusion, the reduced association of I; ggg§I_to macrophages from zinc deficient mice was not due to an altered composition of fatty acids in the macrophage phospholipids. Although the fatty acid composition of the macrophage phospho- lipid was normal, the possibility remained.that I; ggggi-stimulated release of fatty acid.metabolites was reduced in.macrophages from zinc deficient mice. To analyze the release of 20:4 metabolites, adherent resident macrophages from mice in each dietary group were incubated overnight with 3H+20:4. Then, I; ggugi-stimulated release and nonstimulated spontaneous release of 20:4 and its metabolites into the medium were analyzed. Since the fatty acid composition of the macrophage phospholipids was normal for zinc deficient mdce, it was likely that deficient macrophages would.incorporate 3H-20:4 in Figure 9 . 249 Mole percent of fatty acids in phospholipids from resident peritoneal mcrophages prepared from severely zinc deficient, moderately zirc deficient, restricted, or control mice . Adherent macrophages were scraped into 0.15 M NaCl. Margaric phosphotidlycholine was added as an internal standard. Lipids were extracted by the Bligh and Dyer method. Phospholipids were separated from neutral lipids using a silicic acid column. Then, the fatty acids were transmethylated and quantitated by gas liquid chromatography techniques. Protein content of the scraped cells was determined by the Lowry method. Each bar represents the mean _t SE! of triplicates of pooled peritoneal cells from 10 to 15 mice. 250 Severe a {919233191026202926233633939331’3231’ , "k\\\\\\\\\\\\\\\\\\\\\\\\ *4 S!’ O N N .53 i- Q .3 13%;: w 20:0 UB- l8= I fatty' acid |8*O >10110193333333333Ioloiolotoioioiozototom«or.» 4o- 30" 0 IO” *7. Now moo Km: Figure 10 . 251 Nanomoles fatty acid per milligram mcrophage protein in phospholipids from resident peritoneal macrophages prepared from severely zinc deficient, moderately zirc deficient, restricted, or control mice. The nanomoles of each fatty acid was calculated for figure 10 using the peak area of the internal standard. Each bar represents the mean 1 SIM of triplicates of pooled peritoneal cells from 10 to 15 mice. 252 Dietary groups 0363014763333333“ItYeYeYeYoYe'e‘Zfl N \\\\\\\\\X\\\\\\\‘] 2 E 9,: 5, ii 9 E! C] U I mm 5 L\\\V 'IKOX Ithhfthbidth!626382626?" l8=O t-i\\\\\\\\\\\\\\\\ III32692936292020!'20320326203323!$363 20X I-—-|X\\\\\\\\\\\\\\\\\\\\\X\ \ I l |6=O ISO - 8 8 ugonond abandoning: buu/ pm 4:10; se|ouuu fatty acid 253 the same manner as the controls. Table 1 shows that indeed the zinc deficient macrophages incorporated 3H—arachidonic acid into the same phospholipids and to the same extent as control mcrophages. However, L c_ru_gI stimulated release of 20:4 and it’s metabolites from zinc deficient macrophages was altered (Table 2) . Although the same amount of PGE; and HE'I‘E’s were released by mcrophages from zinc deficient and zinc adequate mice (Table 2) , there was a 319% and 249% increase in radioactivity for the severely and moderately defi- cient groups as compared to the zinc adequate control at a Rf of 0.02 on the thin layer chromtogram (Table 2). This peak of radioac- tivity my be leukotrienes since leukotrienes were comigratory. However, the total release of 3H was small (about 2%) compared to the amount incorporated into the mcrophage phospholipids . 254 TABLE 1 Incorporation of 3H—arachidonate into Phospholipidsa Dietary Group 3H (cpnP Severely Zinc Deficient 44680 1 4727 Moderately Zinc Deficient 50934 1 8191 Restricted 34972 1 2763 Zinc Adequate 49256 1 9912 ' Adherent mcrophages from 2 x 10' peritoneal cells prepared from severely zinc deficient, moderately zinc deficient, restricted, or zinc adequate mice were incubated with 1 pCi of (5, 6, 8, 9, 11, 12, 14, 15-3 H) arachidonic acid overnight. Excess 3H—arachidonic acid was removed by washing. mcrophages were collected by scraping, and the lipids extracted. Lipids were separated by thin layer chromato- graphy in a solvent system of chloroform:methanol:acetic acid:water (25:20:3:O.3, v:v:v:v). Plates were scraped in 0.5 cm ascending segments for each sample and 311 measured by scintillation counting. The amount of macrophage protein per sample was the same for the severely zinc deficiennt, moderately zinc deficient, restricted, and zinc adequate groups. 5 Mean 1 SEM of the radioactivity in the one radioactive peak at Rf = 0.94 for triplicates of pooled peritoneal cells from 12 to 20 mice from each dietary group. 255 TABLE 2 Release of 3H-arachidonate metabolites' ’11 (cpm) be Comigratory Severec Moderate Restricted Control Standards 0.02 L'I‘Ce. LTD“ 964 1 16‘” 751 1 22' 345 1 60 302 1' 48 LTE. 0.35 PCB; 179 1 13 153 1 24 142 1 14 150 1 14 0.67 HE'I‘E’s 166 1 54 121 1 20 123 1 5 84 1 48 a. Release of 3H-arachidonate metabolites form adherent resident macrophages stimulated with L cruzi at a L cruzizmncrophages ratio of 25:1. Briefly, medium removed from the adherent macrophage monolayer was centrifuged. Supernatants were acidified with 0.03 M citric acid and extracted three times with chloroform: methanol (2:1, v:v). The chloroform layer was washed with methanol:water (2:1, v:v), evaporated to dryness under N: and applied to silicic acid coated plates. The plates were developed twice in chloroform:ethylacetate:methanolzacetic acid:water (140:60:16:2:1, V:V:V:V:V). Plates were scraped in 0.5 cm ascending segments for each sample and 3H measured by scintillation counting. Rf of standards: 0.02 = (LTCe, LTD), LTEe), 0.28 = 15:33, 0.32 = 6- ketO-sz 9 035 RE), 0052 =Lm, 0067 = HETE’B, 0079 = riCin" oleic acid, 0.88 arachidonate Dietary groups Mean of (cm of 3H of peak - cpm of spontaneous release) 1 SE! for triplicates of pooled peritoneal cells from 12 to 20 mice from each dietary group. Spontaneous release of 86 1 27 cm or leukotrienes, 74 1 14 cpm for PGEa, and 160 1 32 cpm for HE'I‘E’s was the same for macrophages from mice in all dietary groups. Significance of p < 0.05 or better as compared to control group DISCUSSION Since zinc deficient macrophages are unable to destroy the L cruzi associated with them (7, 8) and HeOz Production is deemed important in the destruction of L cruzi, it was hypothesized that macrophages from zinc deficient mice produced less P120: per amount of stimulant than macrophages from zinc adequate controls. However, it was shown that macrophages from deficient mice produced normal amcunts of H202 . Upon stimulation with the commonly used agent, INA, zinc deficient macrophages produced as much 1130: as zinc adequate macrophages . We also exogeneously added a more natural agent, op— sonized zymosan, or the second messenger in opsonized zymosan stimula- tion of NADPH oxidase, 20:4, to stimulate HzOz production. Again, zinc deficient macrophages produced normal amounts of 11:02 as compared to zinc adequate macrophages when stimulated with opsonized zymosan or 20:4. Since PMA and opsonized zymosan stimulate NADPH oxidase through different mechanisms (46-58), it was possible that L _c_rug; stimulates NADH! oxidase through yet another mechanism. Therefore, it was important to use L c_r_ug;_ as the stimulant for HaOn production. It was shown that macrophages from severely and moderately zinc deficient mice produced significantly less 11202 per macrophage (63% to 73% of control) than zinc adequate macrophages. However, this was determined to be due to less overall activation of the deficient macrophages since fewer parasites had associated with these cells. When considered from this point of view, the amount of 1120: produced per parasite was the same for macrophages from each dietary group of 256 257 mice. This relationship between association and 1130: production is in agreement with the results in chapter 4 where the amount of mo. produced correlated.with the level of I; QgggI-macrophage associa- tion. Furthermore, one would expect that destruction of a lower burden of parasites such as that associated with the deficient macro- phages would require less HaOa . With this assumption, the conclusion can be made that the mechanism for production of P130: is unaltered in the macrophages from zinc deficient mice. The macrophages had prObab- ly not undergone repair ig 21129 since, in all studies herein, the amount of zinc in the cultures was less than 1.5 pg Zn/ dl and similar amnounnts were present in previous studies where microbicidal activity of deficient macrophages was aberrant (7, 8). As a comparison, 1000 pg ZnClaldl which is about 500 pg zinc/d1 was used in other studies to restore microbicidal activity by macrophages from zinc deficient mice to control levels (7, 8). Therefore, the absense of dietary zinc must alter some other micrObicidal process of the macrophages since previous studies showed that macrophages from.zinc deficient mice had a reduced.abi1ity to destroy those parasites that had.as- sociated with it (7, 8). Unfortunately, the amount of killing of the parasite by the macrophages from deficient and control mice could not be measured since in the first experiment the level of I; qugI-macrophages association was too low for accurate measurement. When repeated at the higher level of infection required for stimulation of good levels of H30; production, the macrophages were destroyed by the parasites after the 6 to 18 hours, when degree of killing of L cruzi was 258 measured. As mentioned before, the degree of infectivity of the parasite is highly variable and unpredictable. waever, herein, the macrophages were prepared.under the same zinc free conditions as in previous studies where it was shown that there was a reduction in as- sociation of I; 922;; with and killing of I; qugi by the macro- phages from.zinc deficient mice. Also in this study, the association of I; 922;; with the zinc deficient macrophages was reduced. So, one may assume that in this study, the microbicidal activity of the macrophages fromnzinc deficient mice was also aberrant. A high percentage of macrophages (85-100%) associated with parasites were used when it was shown that 1130; production per para- site was normal for deficient macrophages. In contrast, in previous studies, lower levels of association (18—45%) were used to show that killing of I; 933;; by zinc deficient macrophages was reduced compared to controls. Thus, it is important to show that, also, at lower levels of association, 1130; production per parasite was the same for the deficient and control groups. The two studies herein, where the I; ggggI-macrophage association was very low (10 1 5%rof the macro- phages were associated with parasites), 1130; production by macrophages fromuzinc deficient mice was 63% to 73% of controls and, in previous studies, association with I; ggggi by deficient macrophages was in the range of 50% to 75%.of controls. Therefore, even at low levels of association, the ratio between deficient and control groups far HzOz production and for association was the same. Thus, the ancunnt of 1130; per parasite was probably the same among the dietary groups at both low and high levels of infection. 259 Still, previous evidence (9-11) and the data in Chapter 4 sug- gest that 1120; is important in destruction of L 911$. Perhaps some process in the killing following the production of 1130; re- quires zinc. However, the actual process of destruction of L 9_r_uz_i_ and whether or not some agent in addition to H202 is important remains to be determined. The reduced association of parasites with macrophages from zinc deficient mice compared to controls was also hypothesized to be due to a reduction in long-chain unnsaturated fatty acids such as 20:4 since this has been shown to reduce membrane fluidity and phagocytosis (23, 24). If fatty acid composition is altered in deficient macro- phages and it is important in L c_ru_z_i-macrophage association, the macrophages from zinc deficient mice must also be able to restore ‘ the fatty acid composition to normal in a half hour incubation with zinc followed by a one hour incubation with L c_r_u_rz_; since, in this time span with zinc, the association of L M with macrophages from zinc deficient mice is restored to control levels (7, 8). Synthesis and incorporation of new fatty acids requires too much time (59). However, the entire macrophage membrane is recycled via pinocytosis every 33 minutes (60). Perhaps, during the recycling of the membrane which has been suggested to involve the Golgi apparatus and the endoplasmic reticulum (61-63) , the existing fatty acids can be modified to restore the fatty acid composition to normal. However, the hypothesis that fatty acids were altered was incorrect. The fatty acid composition and total amount of each of the fatty acids of the phospholipids from zinc deficient macrophages was the same as 260 controls. The presence of normal amounts of 20:4 in phospholipids of deficient macrophages is in agreement with the finding that op— sonized zymosan stimulated production of normal amcunts of 11.0. by zinc deficient macrophages since 20:4 is a second messenger in op- sonized zymosan stimnulation of 1130: production (48, 49, 54, , 46, 47). Although the membrane fatty acid composition is normal for zinc deficient macrophages, the L gryz_i stimulated release of 20:4 meta- bolites, which may be involved in association and killing of L £11411. (7, 8) , may by altered. Zinc deficient macrophages and zinc adequate macrophages released normal amounts of PCB; and HETE’s, 3H- arachidonate metabolites, into the culture medium upon stimulation with L gull. Spontaneous release of PGE; and HETE’s was very low for each dietary group. However, these preliminary results did indicate that there was an increase in what may be leukotrienes. This was not due to altered incorporation of 3H—arachidonic acid since the same amount of radioactivity was incorporated into phospho- lipids of mononuclear cells from zinc deficient and zinc adequate mice. Also, the release of this 20:4 metabolite was several times the spontaneous release by nonstimulated macrophages from all dietary groups. Since this radioactive peak comigratory with leukotrienes was very close to the origin, it could be phospholipids instead of leukotrienes. However, one would not expect viable macrophages to release large amounts of entire phospholipids upon stimulation with L 9&2}; This data is very preliminary and identification of the increased production of this 20:4 metabolite that was comigratory with leukotrienes must be verified by high pressure liquid 261 chromatography. Also, the total release of 3H was small (about 2%) compared to that incorporated into the macrophage phospholipids. Thus, L g_r;_uz__i may be less stimulatory than other agents such as zymosan reported to cause release of 6% to 15% of the tritium labelled 20:4 metabolites from macrophage phospholipids. Another possibility is that optimal conditions for L gru__g__i_ stimulation of macrophages were not used. Further, one would expect leukotriene production to be reduced in deficient macrophages since exogenous addition of LTD. or LT'B; have been shown to increase not decrease L c_ru_z_i-macrophage association (25, 26), killing of L gru__z_i by macrophages (25, 26), and stimulation of the "oxygen burst" (54, 64). Although from the data collected to date, it is also not known if the supposed leuko- trienes were active. Leukotrienes can be oxidized by the oxygen metabolites of the burst (65) . In the interim, no sound conclusions can be made about leukotrienes until further studies are done to identify the 20:4 metabolites produced. In summary, zinc deficient macrophages produced normal amounts of HzOz per amount of stimulant. The reduced association of L 9%; with macrophages from zinc deficient versus zinc adequate mice was not the consequence of altered fatty acid composition of the deficient macrophages. Also, preliminary data suggests that L g_ru__z_i_-stimulated release of an 20:4 metabolite was three times greater by macrophages from zinc deficient mice compared to controls. The potential importance and role of this metabolite in association and destruction of L gru__zi_ by macrophages should be the subject of future studies . 10. 11. References Prasad, A. S. (1979) Ann. Rev. Pharmacol. Toxicol. 29, 393. Gordon, J., Jansen, A., and Asoli, W. (1965) J. Pediat. 66, 679. Fraker, P. J., Caruso, R., and Kierszenbaum, F. (1982) J. Nutr. 112, 1224. McClain, C. J., Soutor, C., Steele, N., Levine, A. S., Silvis, S. E. (1980) J. Clin. Gastroenterol. g, 125. Prasad, A. (1963) Arch. Intern. Med. 1_1_1, 407. Weston, W. L., Huff, J. C., Humbert, J. R., Hambridge, K. M., Nelder, K. H., and.Walravens, P. A. (1977) Arch. Dermatol. 113, 422. Wirth, J. J., Fraker, P. J., and Kierszenbaum, F. (manuscript in preparation). Fraker, P. J., Jardieu, P., and.Wirth, J. (1986) In "Nutritional Diseases: Research Directions in Comparative Pathobiology " pp. 197-213. Alan R. Liss, Inc, New Yark. Villalta, F., and Kierszenbaum, F. (1983) J. Immunol. 131, 1504. Villalta, F., and Kierszenbaum, F. (1984) J. Immunol. _1_3_, 3338. Nathan, C. F., Silverstein, S. C., Brukner, L. H., and Cohn, z. A. (1979) J. EXp. Med. _1_43, 100. 262 263 12. Boveris, A., Sies, H., Martino, E. E., Docampo, R., Turrens, J. F., and Stoppani, A. O. M. (1980) Biochem. J. 1_8_§, 643. 13. Docampo, R., DeBoiso, J. F., Boveris, A., and Stoppani, A. O. M. (1976) Experientia 12, 972. 14. Cardoni, R. L., Docampo, R., and Casellas, A. M. (1982) J. Parasitol. 6_8, 547. 15. Huang, Y. S., Cunnnane, S. C., Horrobin, D. F., and Davignon, J. (1982) Atherosclerosis 11, 193. 16. Cunnnane, S. C., Horrobin, D. F., and Manku, M. S. (1984) Proc. Soc. Exp. Biol. Med. m, 441. 17. Cunnnane, S. C., and Horrobin, D. F. (1985) J. Nutr. 15_5, 500. 18. Bettger, W. J., Reeves, P. G., Moscatelli, E. A., Reynolds, G., and O’Dell, B. L. (1979) J. Nutr. ICE, 480. 19. Clejan, S., Castro-Maganna, M., Collipp, P. J., Jones, E., and Maddaiah, V. T. (1982) Lipids 11, 129. 20. Field, H. P., and Kelleher, J. (1983) Proc. Nutr. Soc. 4_5_, 54A. 21. Tsai, S. L., Craig-Schmidt, M. C., Week, J. D., and Keith, R. E. (1983) Fed. Soc. 11g, 823. (abs. 3110). 22. Horrobin, D. F., and Cunnnane, S. C. (1980) Med. Hypothesis _6, 277. 23. Mahoney, E. M., Hamill, A. L., Scott, W. A., and Cohn, Z. A. (1977) Proc. Natl. Acad. Sci. U.S.A. fl, 4895. 24. Mahoney, E. M., Scott, W. A., Iandsberger, F. R., Hamill, A. L., and Cohn, z. A. (1980) J. Biol. Chem. g, 4910. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 264 Villalta, F., and.Kierszenbaum, F. (1983) J. Immunol. 131, 1504. Aust, S. D., Morehouse, L. A., and Thomas, C. E. (1985) J. Free Rad. Biol. Med. 1, 3. Luecke, R. W., Simonel, C., and Fraker, P. J. (1978) J. Nutr. 198, 881. Fraker, P. J., Haas, S. M., and Luecke, R. W. (1977) J. Nutr. 191, 1889. DePasquale-Jardieu, P., and Fraker, P. J. (1980) J. Immunol. 121, 2650. Budzko, D. B. (1974) J. Parasitol. 69, 1037. Mercado, T. I., Katusha, K. (1979) Prep. Biochem. 9, 97. Conrad, R. E. (1981) In "Manual of Macrophage Methodology V13" (Herscowitz, H. B., Holden, H. T., Bellanti, J. A., Ghaffar, A., eds.) pp 5-12. Marcel Dekker, Inc., New Yark. Bozdeck, M. J., and Bainton, D. F. (1981) J. Exp. Med. 153, 182. Yam, L. T., Li, C. Y., and Grosby, W. H. (1971) Am. J. Clin. Pathol. 55, 283. Pick, E., and.Mizel, D. (1981) J. Immunol. Methods 16, 211. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. Ruch, W., Cooper, P. H., and.Baggiolini, M. (1983) J. Immunol. Methods 63, 347. Baer, E., and Buchnea, D. (1959) Can. J. Boichem. Physiol. 11, 953. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 265 Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 31, 911. Mahoney‘, E. M., Hamill, A. L., Scott, W. A., and Cohn, Z. A. (1977) Proc. Natl. Acad. Sci. U.S.A. Z_4_, 4895. Scott, W. A., Pawlowski, N. A., Murray, H. W., Andreach, M., Zrike, J., and Cohn, Z. A. (1982) J. Exp. Med. _1___5_§_, 1148. Humes, J. L. (1981) In "Methods for Studying Mononuclear Phagocytes" (Adams, D. O., Edelson, P. J., and Koren, H. S., eds.) pp. 641-654. Fels, A. O. S., Pawlowski, N. A., Abraham, E. L., and Cohn, Z. A. (1986) J. Ebcp. Med. 16g, 752. Enilsson, A., and Sundler, R. (1985) Biochim. Biophys. Acta 8_1_6_, 265. Emilsson, A., and Sundler, R. (1986) Biochim. Biophys. Acta m, 533. Castagna, M., Takai, Y., Kaibuchi, K., Sano, H., Kikkawa, U., and Nishizuka (1982) J. Biol. Chem. fl, 7847. Nishizuka, Y. (1984) Nature £8, 693. Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213. Maridonneau—Parini, I., and Tauber, A. I. (1986) Clinical Research fl, 661A. Tauber, A. 1., Cox, J. A., Jeng, A. Y., and Blumberg, P. M. (1986) Clinical Research 31, 664A. McPhail, L., Clayton, C. C., and Snyderman, R. (1984) Science Q, 622. Fujita, I. Irita,‘ K., Takeshige, K., and Minakami, S. (1984) 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 266 Biochem. Biophys. Res. Commun. IZ_0, 318. Robinson, J. M., Badwey, J. A., Kamovsky, M. L., and Karnovsky, M. J. (1984) Biochem. Biophys. Res. Commun. _1_2_2_, 734. Bromberg, Y., and Pick, E. (1983) Cell. Immunol. 19, 240. McPhail, L. C., Shirley, P. S., Clayton, C. C., and I Snyderman, R. (1985) J. Clin. Invest. E, 1735. Curnette, J. T. (1985) J. Clin. Invest. fl, 1740. * Vercauteren, R. E., and Heynelan, R. A. (1984) J. Leuk. Biol. 31, 751. SW1, To, saito-Taki, To, SfidflSiVan, Re, 8111 Nitta, To (1982) Proc. Natl. Acad. Sci. U.S.A. E, 591. Scott, W. A., Mahoney, E. M., and Cohn, Z. A. (1980) In "Mononuclear Phagocytes. Flnctional Aspects." (van Furth, R. , ed.) _1_, 685-701. Phrtinum Nijhoff Publishers, Boston, MA. Steinmnan, R. M., Brodie, S. E., and Cohn, Z. A. (1976) J. Cell Biol. 6_8, 665. T‘ulkens, P. Schneider, Y. J., and Trouet, A. (1980) In "Mononuclear Phagocytes. Functional Aspects." (van Furth, R. , ed.) 1, 613-647. Martinum Nijhoff Publishers, Boston, MA. Farquhar, M. G. (1982) In "Membrane Recycling" (Ciba Foundation Symposium 92) (Everard, D., and Collins, G. M., eds.) pp. 157-183. Pitman Books, Ltd., London. Cohn, Z. A., and Steinman, R. M. (1982) In "Membrane Recycling" (Ciba Foundation Symposium 92) (EVerand, D., and Collins, G. M., eds.) pp. 15-34. Pitman Books, Ltd., London. 64. 267 Wirth, J. J., and Kierszenbaum, F. (1985) J. Immunol. 1_4, 1989. Flohé, L., Beckmnannn, R., Giertz, H., and Loschen, G. (1985) In "Oxidative Stress" (Sies, H., ed.) pp. 403-435. Academic Press, Inc. , New York. SUP’MARY AND CDNCLUSIONS Zinc deficiency, not infrequently encountered in humans, drasti- cally compromizes cell and antibody mediated immune responses in both humans and snimls. The reduced immune capacity is at least in part due to a 50% decrease in number of leukocytes in animals. The functional capacity of the residual cells may also be altered upon suboptimal intake of zinc since over 100 enzymes are dependent upon zinc for activity. Therefore, the purpose of this research was to examine the functional capacity of the residual leukocytes in an attempt to identify an aberrant zinc dependent process. In all these studies, the possibility for _iLn 113410 repair due to addition of serum zinc was minimized by using autologous serum from zinc deficient mice (50 pg Zn/dl) or a serum free system rather than fetal calf serum which contains relatively high levels of zinc (350 pg Zn/dl) and is commonly used for culturing leukocytes. The results of these experiments showed that residual splenic lymphocytes from zinc deficient mice could function at least as well as splenocytes from zinc adequate mice. Proliferation and production of interleukin 2 (IL-2) in repsonse to the T-cell mitogen concanavalin A was the same for splenocytes from severely and moderately zinc deficient mice as for splenocytes from zinc adequate and resticted mice. These results are in agreement with a previous report on Con A proliferation by splenocytes from zinc deficient mice where the i_n; vitro ancunt of zinc was limited. However, other reports shorting 268 269 reduced proliferation in response to Con A for the deficient group contradict the results herein. This is probably due to their deplet- ing zinc in m instead of dietary depletion of zinc. With i_n m depletion, there is simply removal of zinc from the existing cells whereas depletion of zinc i_n _v_i_v_c_> may also affect cell develop- ment as well as cause production of factors such as cortisone from other tissues which may play a part in the deficiency. During a T- cell response to allogeneic cells in a one-way mixed lymphocyte culture (MLC) , splenocyte proliferation, IL-2 production by splen- ocytes, ard the number of splenocytes with IL-2 receptors for the zinc deficient groups was twice that of the zinc adequate groups. This increased response to allogeneic cells may be due to altera- tions in the proportions of T-cell subsets since relatively immature T-cells respord in a primary MLC whereas concanavalin A, a poly- clonal activator, stimulates many T-cell subsets. This possibility could be analyzed in the future by fluorescently labelling cell surface markers. Another possible explanation for the increased response in the MLL‘ may be that some inhibitory activity is impaired in splenocytes from zinc deficient mice. Residual B—cells from zinc deficient mice also seemed to fume- tion normlly. Splenocytes of zinc deficient mice activated in _vi_v_g with sheep red blood cells produced the same amount of IgM ard IgG per IgM ard IgG plaque forming cell (PFC, antibody secreting cell), respectively, as splenocytes from zinc adequate mice. Also, in agreement with many previous reports, the number of PFC per million splenocytes was the same among the experimental dietary groups 270 although the number of PFC per spleen was reduced (about 50% of control) for the deficient groups. In conclusion, residual splenic lymphocytes from zinc deficient mice retained normal functional capacity at least for those responses tested. In contrast, resident peritoneal macrophages from zinc def i- cient mice have a reduced capacity to associate with ard destroy the obligate intracellular parasite L 9&1. which causes Chagas’ disease in humans. Since H30: is thought to play a critical role in the destruction of L M, it was important to compare 1130: production by resident peritoneal macrophages from zinc deficient mice with H202 production by macrophages from zinc adequate mice. However, in order to measure H20: production by resident macrophages instead of the commonly used i_n yi_vg activated macrophages, the assay for measur- ing P120: production needed to be improved. Previously, the amount of H20: produced by resident macrophages was reported to be low to negligable. Also, although a considerable number of studies have been done to link H302 production to destruction of L M, L gag-stimulated 1130; has not been directly quantitated. Modifications that were made in the corditions for the commonly used phenol red assay to optimize P120: production by resident macro- phages stimulated with morbol or opsonized zymosan included an increase in cell concentration, addition of calcium ard an incuba- tion in ambient air at 37°C. However, when the stimulant was the living pathogen L gru_lz_i_, H20: production could not be measured since the high concentrations of phenol red required for the assay were toxic to L cruzi. The assay was further modified by using 271 another substrate homovanillic acid (HVA) . To improve the HVA assay for use with resident macrophages , the concentration of macrophages was again increased. Also, CaCl; ard MgClz , which were included in the previously described assay with HVA, were determined to be re- quired for optimal HaOz production by resident peritoneal macro- phages stimulated with phorbol or opsonized zymosan. Using this modified assay, the amount of H20; produced by resident macrophages could now be quantitated. In addition, with the HVA assay, it was now possible for the first time to quantitate I130; production by macrophages stimulated with L mi. This assay will be essential for future studies on L c_ru_lz_i destruction ard may be important when other living pathogens such as Leshmania or Plasmodium falcim are used to activate 1120: production by macrophages. L c_ru_zi_-stimulated H30: production by macrophages from mice consuming normal laboratory chow was studied since there is no avail- able literature on the quantitation or mechanism of L gguz__i_—stimu- lated I-hO. production. Resident peritoneal macrophages incubated with amastigotes, the intracellular forum of L c_ruz_i, produced half as much rho. as macroplages incubated with trypomastigotes , the blood form of L gru_z_i_. T‘rypomastigotes that were opsonized with heat inactivated serum from chronically infected mice stimulated four times as mmch H302 production as nonopsonized trypomastigotes. Also, upon stimulation with trypomastigotes or opsonized trypomas- tigotes but not amastigotes, the amount of mo. produced correlated with the proportion of macrophages associated with parasites ard with the number of parasites per macrophage. The maximum amount of 272 H302 produced by the macrophages stimulated with trypomastigotes varied. Perhaps the maximal amount of H202 produced by macrophages depends upon the "stage" of the parasite in its progression through each of the forms of its lifecycle. Furthermore, a pathway by which I; cruzi trypomastigotes may stimulate H202 production was analyzed. Preliminary data indicated that I; eggs; trypomastigotes stimulated the release of the arachi- donate (20:4) metabolites PGEz, HETE’s and.what may be leukotrienes in a ratio of 2:1:4. HETE’s and leukotrienes are known to stimulate the production of oxygen metabolites such as tho; . Therefore, 20:4 and its metabolites may be second messengers in the production of H302 by resident macrophages. In addition, as a source for production of 20:4 metabolites, resident peritoneal macrophages from A/J mice contained a considerable amount of endogeneous 20:4 (21% of the fatty acid) in the phospholipids. If trypomastigotes also stimulate the release of shorter—chain, more-saturated fatty acids than 20:4, these fatty acids would not activate nearly as much 1120; production as 20:4 and perhaps its metabolites since 20:4 stimulated 2 to 3 times as much H30; prouhction as 18:0, 18:2, or 18:3. Taken together, the data suggested that 20:4 and its metabolites may be intermediates in one pathway for trypomastigote-stimulation of P130; production by resident macrophages. Now that the ancunt of 1120: produced by L M-stimulated macrophages could.be measured.and preliminary'data indicated one possible mechanism for L gag-stimulation of I120; production, possible alterations in the ability of resident macrophages from 273 dietary zinc deficient mice to produce P1202 could be studied. It was shown that, upon incubation with I; cruzi trypomastigotes, the total amount of H302 produced per macrophage from severely and.moder- ately zinc deficient mice was 66% and 83%, respectively, of that by macrophages from zinc adequate mice. However, this reduction in H302 production was due to less stimulation of the macrophages from zinc deficient mice since fewer I; cruzi associated.with the defi- cient macrophages. Thus, the amount of 1120: produced per L _c_ru_Li associated.with the macrophages was the same for all experimental dietary groups. One would expect that a reduced number of parasites would also require proportionally less H30; for destruction. The H302 production by macrophages stimulated with the commonly used nonliving agents phorbol, opsonized zymosan, or 20:4 was also the same for all dietary groups. Therefore, at a given amount of stimu- lant, H303 production by macrophages from NJ mice was not altered by deprivation of dietary zinc. Since, macrophages from zinc deficient mice destroyed.a smaller proportion of the parasites associated with them than macrophages from zinc adequate or restricted.mice, some process in the destruction of E; cruzi besides the production of H202 must directly or irdirectly require zinc. Perhaps zinc is required in some process in killing following production of neon. The mechanisms fer'1;_gguzi invasion of macrophages and.macro- phage phagocytosis of I; cruzi is unknown. However, since a de- crease in long chain unsaturated fatty acids is known to decrease phagocytosis and since exogeneous addition of leukotrienes, 20:4 metabolites, are known to increase the association of trypomastigotes 274 with macrophages, it was hypothesized that the reduction in number of trypomastigotes associated.with deficient macrophages may be due to an altered fatty acid.composition. It was shown that the ratios among and total amount of 16:0, 18:0, 18:1, 18:2, and 20:4 were unal- tered in the phospholipids of macrophages from.zinc deficient mice as compared to macrophages from zinc adequate or restricted mice. Thus, the reduced association of T; cruzi with macrophages from zinc deficient mice was not due to an altered composition of fatty acids in the phospholipids. Preliminary data suggested.that the amount of release of the 20:4 metabolites, HETE's and PGE2, was the same for all experimental dietary groups. Hewever, macrophages from zinc deficient mice released.three times as much of an 20:4 metabolite, that may be some leukotriene, than macrophages from zinc adequate mice. Iden¥ tification of this metabolite and its role in the association of T; cruzi with macrophages requires futher study. In summary, dietary zinc deprivation did.not reduce several functions of residual lymphocytes from.zinc deficient mice but did reduce some functions of residual peritoneal macrophages from zinc deficient mice. The reduced ability of deficient macrophages to destroy L _c__ru_z_i was not the result of reduced production of H30; per given amount of stimulant. Also, the reduced.1; cruzi-macro- phage association for the experimental deficient group was not a consequence of an altered fatty acid.composition within the macro- phage phospholipids or the result of reduced production of leuko- trienes. In fact, compared to controls, macrophages from zinc defi- cient mice produced three times as much of an 20:4 metabolite. In 275 conclusion, some other processes must be aberrant in the association with ard destruction of L c_ru_z_i by macrophages from zinc deficient mice. Also, an important contribution was made to the study of L w; destruction by leukocytes since the improved assay conditions for P120: production now makes it possible to directly quantitate the amount of P120: produced by leukocytes stimulated with L cruzi. APPENDIX Composition of the Zinc Deficient ard Zinc Adequate Diet 276 277 The zinc deficient diet (Table 1) was designed and extensively examined by Luecke, R. W. and Fraker, P. J. ((1979)J. Nutr. 109, 1373-1376). Briefly, it was shown that A/J female mice fed zinc deficient diet supplemented with 5.9 to 31.4 pg Zn/g attained maximum growth and had.normal antibody-mediated responses to sheep red blood cells. However, drastic reductions were observed for the antibody- mediated responses by splenocytes from A/J mice fed.the zinc defi- cient diet containing less than 1 pg Zn/g. Therefore, in the studies described in this thesis, zinc deficient diet contained less than 1 pg Zn/g and the zinc adequate diet contained 27 pg Zn/g. Table 1 Composition of the zinc deficient diet z/kg Glucose monohydrate 590 Egg white solids (spray-dried)1 220 Corn oil 100 Fiber1 30 Salt mix2 40 Vitamin mix3 10 Ethoxyquin! 10 1Cellulose-type fiber, Teklad.Test Diets, Madison, Wisconsin, 2Bernhart, F. W. &.Tommarelli, R. M. (1966) J. Nutr. 89, 495, except that a U.S.P. grade of CaHPOh was used instead of reagent grade, and also zinc carbonate was omitted, 3Composition similar to the AIN—76 mixture (1977) J. Nutr. 107, 1340, except that the biotin level was increased to provide an additional 4 mg/kg diet. ‘Santoquin, Monsanto Chemical Co., St. Louis, Missouri.