II 145 634 _THS_ A METHOD FOR 33mm mmm frH’PERSEE‘éSI'Tflilf‘Y m mo Thesis for the Degree of M. S. MSCHIGAN STATE UNIVERSETY S. K. QUADR! 1970 LIBRARY Michigan State U niversi‘. y "'1'? [HESIS 3 A METHOD FOR STUDYING DELAYED HYPERSENSITIVITY _I_[_1_\I_ VITRO by 1“" x“! . S. K;*Quadr1 A thesis submitted to Michigan State University in partial fulfillment of the requirements for the degree of Master of Science Department of Microbiology and Public Health 1970 ACKNOWLEDGMENTS The author expresses his sincere thanks and appreciation to Dr. V.H. Mallmann for her guidance throughout this study. Appreciation is also extended to Dr. W. L. Mallmann for his counsel. The author gratefully acknowledges the help and avice received from Dr. Philipp Gerhardt, Chairman, Department of Microbiology and Public Health, in the preparation of the manuscript. The author is obliged to Dr. Esther Smith, Chairman, Department of Medical Technology for her help in the preparation of photographs and to Dr. A. E. Lewis, professor, Department of Pathology, for his help in identification of peritoneal leukocytes. A special note of thanks goes to the Glass Blowing Shop, Department of Chemistry and to the Plastic Manufacturing and Supply Inc., Lansing, for the patience and generosity with which they manufactured innumerable designs that were tested in search of a newginlgitgg cytotoxic test. Last but not least, sincere appreciation is expressed to the Michigan Tuberculosis and Respiratory Disease Association and its. secretary, Mr. Irvin Nichols for financial support of this research project. TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEW MANUSCRIPT Introduction Materials and Methods Results Discussion Summary References APPENDIX Summary from Third Report Summary from Fourth Report Summary from Fifth Report REFERENCES 11 page 10 11 13 16 18 19 20 21 26 i 29 31 . LIST OF TABLES Table page 1. Effect of concentration of cells of peritoneal exudate 14 cells suspensions on the inhibition of migration of macrophages from tuberculin-sensitive guinea pigs in the presence of 25 ug PPD/ml. 2. Linear migration in microns of mononuclear cells from 24 guinea pigs infected with Mycobacterium bovis (BCG) in the presence and absence of PPD in vitro. 3. Linear migration in microns of mononuclear cells from 25 normal guinea pigs in the presence and absence of PPD in vitro. 4. Linear migration of peritoneal monocytes in vitro ' 28 from normal and tuberculin-sensitive guinea pigs after skin testing. 5. Linear migration of peritoneal monocytes in vitro from 30 normal and tuberculin-sensitive guinea pigs after skin testing. iii I'll-E..- ‘ll‘lllll.|f| l LIST OF FIGURES Figure page 1. Radial migration of macrophages. A migration spot before incubation (upper) and after incubation (lower) 15 iv INTRODUCTION There is a need for a simple and reliable‘ig'giggg test for delayed hypersensitivity. The first part of the present study was devoted to an investigation of the usefulness of the presently available jg_g;££g test which involves the migration of peritoneal exudate cells in capil- lary tubes. Results of this investigation are presented in the appendix. In the later part of this research project an alternate ig.yiggg test for delayed hypersensitivity was developed. The manuscript describing this method, along with a review of the pertinent literature are in- cluded in this thesis. LITERATURE REVIEW Delayed or tuberculin type sensitivity differs from immediate sensitivity in that there is a delay of 16-48 hours before the appearance of local reaction after contact with the specific antigen, whereas im- mediate type sensitivity reactions are visible within 5 minutes to 4 hours after contact with an antigen. Immediate sensitivity is anti- body associated and can be passively transferred in serum; delayed hypersensitivity has so far been passively transferred only by living lymphoid cells. ‘lg‘yiggg, mononuclear cells from animals with delayed hypersensitivity upon contact with specific antigen undergo morphologic and functional changes, viz. vacuolation of macrophages (1), a decrease in phagocytosis (2), differentiation of small mononuclear cells into macrophages (3), blast formation (4) and inhibition of macrophage migration (5). Several.ig'yiyg and in yitgg methods are used to detect or study delayed hypersensitivity. The former include an intradermal test or corneal injection. The intradermal test is most commonly used to detect the tuberculin type delayed hypersensitivity.' While it is still the best test available for this purpose, it nevertheless has many limitations. For example, in yiggg sensitivity has been demonstrated even after the loss of dermal sensitivity in animals dying of tuberculosis or suffering intercurrent infections (6). In another situation, infant guinea pigs which did not react to skin test showed specific in gigrg cytotoxic effects (7). Lurie et a1. (8) demonstrated in vitro cellular sensitivity in the absence of skin sensitivity in estrogen treated animals. Even in cases where animals develop slow dermal sensitivity or remain negative to skin test, in yitgg cytotoxicity can be demonstrated regularly (9). ‘As a matter of fact as early as 1936, Moen (10) stated that in gitgg macrophage reactivity was more reliable than intradermal test in de- tection of delayed hypersensitivity. DevelOpment of a suitable'in‘yigrg model for delayed hypersensi- tivity is also important from the point of techniques. Delayed hyper- sensitivity underlies similar if not identical reactions involved in several diseases, transplantation immunity, and autoimmune states. Not only the knowledge of this phenomenon at the molecular level is lack- ing, but the role of various types of cells and antibodies is very uncertain (11). The lack of a suitable‘in'giggg system has been one of ‘the main reasons for the slow progress in this area. In short, it is important from the points of view of both basic and applied research that a satisfactory in yiggg model for delayed hypersensitivity be developed. One of the earliest‘ig‘yi££g_studies on delayed hypersensitivity was done by Holst in 1922. He noted an inhibition of phagocytosis a- mond leukocytes_from tuberculous patients in the presence of tuberculin (2). Renewed interest in this area was initiated by the work of Rich and Lewis in 1928-1932 (5, 12). They cultured explants of buffy coat and bone marrow from guinea pigs in autologous plasma which coagulated Iand provided a semi-solid medium in which the cells migrated and grew. They demonstrated that addition of tuberculin to the explants from tuberculous guinea pigs caused increased destruction of cells and inhibited their growth and migration. Tuberculin had no effect on the explants from normal animals. These results were confirmed and extended by Aronson who reported that these cytotoxic effects were not noticeable in the case of immediate hypersensitivity (13). The explants from guinea pigs with immediate sensitivity to horse serum did not react to horse serum igflxiggg. In 1936 Moen and Swift (14) introduced the term cytotoxic index to express the extent of cytotoxic effect. The valueibr cytotoxic index was obtained by dividing the area of growth of the explants in the test cultures containing tuberculin by the area of growth of the explants in the control cultures. The Specificity of the ig‘yiggg cytotoxicity was confirmed by many workers (15-20). .lfl.!i££2 cytotoxic effects in guinea pigs with delayed hypersensitivity to tuberculin, ovalbumin, and diptheria toxoid were elicited only by the specific antigen (19). Cells from guinea pigs sensitized to dinitrophenylated proteins were affected only by the immunizing conjugate (20). Spleen as well as several tissue explants were explored for‘ig Igltgg cytotoxic effects. Carpenter (21) demonstrated in yiggg cyto- toxicity in the explants of lungs and lymph nodes. Several workers explored in great detail the lg‘yiggg reactivity of the epithelial cells to antigen. However, the results were not uniform. Packalen, et a1. (23) found specific inhibition of epithelial growth from kidney and liver. But Jacoby and Marcks (24) reported no cytotoxic effects in the same tissues. Negative results were also reported on skin (25) and corneal epithelium (26). ‘lg‘yitgg studies were also carried on in aggressor (target cell) cultures. Isoimmune mouse peritoneal macro- phages were found to cause cytolysis of target cells in monolayer cultures (27). In 1947 Favour (28) introduced an in giggg cytotoxic method based on the sharp decrease in the number of blood leukocytes which occurred on incubation with specific antigen. His initial observations were confirmed by many workers (29-32), but it was soon noted that similar results were obtained with various states of immediate hypersensitivity (33, 34). Although the iQIXiggg cytotoxicity of hypersensitive tissues‘ig 31333 cytotoxicity of hypersensitive tissues in the presence of specific antigen was firmly established, none of the several in yitgg systems studied was consistent and reliable enough to be adapted as a routine model. Kapral and Steinbring using cells from sensitive animals (1) noted several morphologic changes in the peritoneal monocytes due to tuberculin. The changes included vacuolation of macrophages, loss of phagocytic ability and cell detachment. Fabrizio (35) noted a decrease in the migration from plaques of peritoneal exudate cells. An imporved in gitgg test for delayed hypersensitivity was developed by George and Vaughan (36) in 1962. They used peritoneal exudate cells from guinea pigs with hypersensitivity to tuberculin. The washed cells were packed into capillary tubes which were placed in Mackaness chambers at 37°C. The cells migrated out of the capillary tubes in a fan-like fashion onto the surface of glass cover slips. In the presence of tuberculin their migration was markedly inhibited. The cells from normal animals migrated in the absence and presence of antigen. This procedure was further refined by David and coworkers (19, 20, 37, 38). They established that the inhibition of migration was the property of the cells and was not affected by circulating antibodies. They further found that the presence of a small number of sensitive cells (2.5%) in a population of normal cells would result in the inhibition of migration of the mixed population. A rough correlation was observed between the ‘ig.yi££g inhibition of migration and the size of the skin reaction. The capillary method was used to great advantage by Bloom and Bennet (39). .They used partially purified macrophages and lymphocytes from tuberculous anflnals and found that sensitive lymphocytes were necessary for inhibition of macrophage migration. They demonstrated that as few as 2% sensitive lymphocytes when mixed with normal exudate cells or purified macrophages, could induce inhibition of migration in the presence of antigen. .Furthermore, they obtained an "active" cell-free supernatant from sensitive lymphocytes which could sensitize normal macrophages. They suggested that lymphocytes are active cells in delayed hypersensi- tivity and upon contact with the antigen, they release a soluble factor-- the migration inhibition factor (MIF) which sensitized macrophages which are merely indicator cells. This hypothesis is Open to question, since in a recent report (40), both the serum and heat-eluates of macrophages from sensitized guinea pigs conferred sensitivity to migration-inhibition upon macrophages from normal guinea pigs. MANUSCRIPT A SIMPLE‘IN VITRO TEST FOR DELAYED HYPERSENSITIVITY BY SPECIFIC INHIBITION OF MACROPHAGE MIGRATION S. K. Quadri This research was supported in part by a Fellowship from the Michigan Tuberculosis and Respiratory Disease Association and Animal Disease and Research Division, USDA, Research Contract #12-14-100-8869 (45). Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article ACKNOWLEDGMENTS Sincere appreciation is expressed to Dr. Philipp Gerhardt, Chairman, Department of Microbiology and Public Health for his suggestions in the preparation of this manuscript. Introduction In 1932 Rich and Lewis (1) reported that the migration of cells from splenic and lymph node explants of tuberculous guinea pigs was inhibited in the presence of tuberculin. In 1962 George and Vaughan (2) used the relative distance of migration of peritoneal exudate cells out of capillary tubes onto a glass surface in the presence and absence of antigen. This method has been used, with slight modi- fications, by David et a1. (3) and others (4). Because the capillary technique requires a considerable amount of leucocytes, time and manipulations, we use a circular deposit of peritoneal exudate cells on a glass cover slip. The relative distances of radial migration, after attachment and incubation in the absence and presence of an anti- gen, are measured with a low-power microscope. lO Materials and Methods Cells: Three month old guinea pigs were sensitized by intraperitoneal (IP) injection of 5 mg (wet weight) of viable, attenuated Mg bggis (BCG). An additional 1 mg of BCG was injected 8-10 weeks later. After 2-8 months, peritoneal exudate was induced by an IP injection of 6-10 ml of sterile light mineral oil. Four to 6 days later, 100 ml of Hank's balanced salt solution (BSS) were injected IP. The peritoneal fluid was collected and centrifuged at 4C for 10 minutes at 160 xG. The sedimented cells were washed twice with B88 and centrifuged for 5 minutes at 90 xG. The final cell sediment was suspended in 4-5 ml of Medium 199 (M199) containing 15% normal guinea pig serum (Ml99-- 15% NGPS). The cell concentrations in the suspensions ranged from 9-16 x 103 cells/ml. Antigens: Tuberculin sensitivity was tested with purified protein derivative (PPD, Parke-Davis, Detroit, Michigan). Coccidiodin (Cutter Laboratories, Berkely 10, California), brucellergen (Merck Sharp and Dohme, West Point, Pennsylvania) and histoplasmin (Michigan.Department of Health) were used at the concentrations prescribed for skin tests. Migration Studies: Sykes-Moore chambers were autoclaved with the bottom cover slip and rubber ring in position. The cell suspension was drawn into a Pasteur pipette and five droplets were placed on the bottom cover slip, four around the periphery and one in the center. Each droplet was approximately 1.5 mm in diameter. The chambers were covered and incubated at 37C for 15-20 minutes. The cover slip was then floated, droplet side 11 Materials and Methods Cells: Three month old guinea pigs were sensitized by intraperitoneal (IP) injection of 5 mg (wet weight) of viable, attenuated M, bggis (BCG). An additional 1 mg of BCG was injected 8-10 weeks later. After 2-8 months, peritoneal exudate was induced by an IP injection of 6-10 m1 of sterile light mineral oil. Four to 6 days later, 100 m1 of Hank's balanced salt solution (BSS) were injected IP. The peritoneal fluid was collected and centrifuged at 4C for 10 minutes at 160 xG. The sedimented cells were washed twice with B88 and centrifuged for 5 minutes at 90 xG. The final cell sediment was suspended in 4-5 ml of Medium 199 (M199) containing 15% normal guinea pig serum (M199-- 15% NGPS). The cell concentrations in the suspensions ranged from 9-16 x 103 cells/ml. Antigens: Tuberculin sensitivity was tested with purified protein derivative (PPD, Parke-Davis, Detroit, Michigan). Coccidiodin (Cutter Laboratories, Berkely 10, California), brucellergen (Merck Sharp and Dohme, West Point, Pennsylvania) and histOplasmin (Michigan.Department of Health) were used at the concentrations prescribed for skin tests. Migration Studies: Sykes-Moore chambers were autoclaved with the bottom cover slip and rubber ring in position. The cell suspension was drawn into a Pasteur pipette and five droplets were placed on the bottom cover slip, four around the periphery and one in the center. Each droplet was approximately 1.5 mm in diameter. The chambers werecovered and incubated at 37C for 15-20 minutes. The cover slip was then floated, droplet side 11 Results The radial migration of macrophages from peritoneal exudates of tuberculin sensitive and normal guinea pigs was quantitatively measurable, Figure l. The migration of cells from tuberculin sensitive guinea pigs was markedly inhibited by 25 ug PPD/ml. The mean percentage inhibition of migration (PIM) from 10 animals was 61.2 :;lZ.5 (MEAN + Standard deviation). Cells from normal guinea pigs were not affected, with the PIM from 10 animals measured as 0.6 :_2.1. The inhibition was detectable at 1 hr and persisted beyond 48 hr. The mean PIM for 6 animals at 8 hr, 24 hr, and 48 hr was 47.9,: 7.1, 57.9,: 13.7 and 52.9,: 11.7, respectively. The inhibition was specific. A skin test dose of PPD (second strength) markedly inhibited the migration (mean PIM for 4 animals was 44.1 :;4.7), but skin test doses of histoplasmin, brucellergen and coccidiodin did not ( the mean PIM for 4 animals was 5.2,: 2.6, 0.8,: 1.4, and 3.4,: 1.9, respectively). Variation in the concentration of cell suspensions affected the percentage of migration but not the percentage of inhibition (Table 1). Differences in the percentage of migration for various cell concentra- tions in the absence of antigen corresponded to those in the presence of antigen, so that the percentage of inhibition was not affected appreciably. The extent of inhibition was influenced by the concentration of 'antigen. The mean PIM with 10 animals for 6.25 ug, 12.5 ug, 25 ug and 50 ug PPD/ml was 27.4 : 12.8, 45.6 : 16.2, 56.5,: 13.0 and 64.4,: 12.1 respectively. The lowest concentration of PPD tested and found to inhibit migration was 0.062 ug/ml. as so\maH x SH - moH x m.w mm3.aoamamamsm assuage as coaumuuamoaoo.aamo«, ZOHHHaHmme zHupommmeH on94mwwmimq_ mchmmemmw onHHmHmzH N sumo .He\mmm ms nu mo monomwum ecu a« mem moawnw u>wuwwaewucwasononsu scum mowmnmouome mo cowumuwwfi mo aoHanwscw ecu co wcowmaommnm HHoo oumwsxo HmoCOuHHmm mo mHHmu mo cowuwuucmucoo mo uoommm .H assay 14 Figure 1. Radial migration of macrophages. A migration spot before incubation (upper) and after incubation (lower). p—_ __ 15 Discussion These experiments have demonstrated that radial migration of macrophages from peritoneal exudates of tuberculin sensitive guinea pigs was specifically inhibited by PPD. This method for demonstrating inhibition of migration furnishes a simple means for studying factors associated with delayed hypersensitivity and has a number of economic and technical advantages over earlier methods. The inhibition was rapid and effectively accomplished by a range of tuberculin concentra- tions. Sensitivity was demonstrated by the inhibition of migration by as low as 0.062 ug of PPD/m1. Approximately 30-40 chambers can be made from 1 m1 of peritoneal cell suspensions containg l x 104 cellsfiml. Erythrocytes often constitute an undersirable contaminant in peritoneal exudate (3), but are easily eliminated in the present test. The test was not subject to error due to variations in the concentrations of cell suspensions. Lymphocytes are often considered to act as mediators of delayed hypersensitivity (5). The presence of l%-6% lymphocytes could have caused the inhibition of migration of macrophages through production of a presumed migration inhibition factor, MIF (4). However, the early inhibition (at 1 hr) would also favor the possibility of macrophages being sensitive cells after conversion from lymphocytes or from cyto- philic antibody (6). Recently both cytophilic antibody and MIF have been.imp1icated in the inhibition of migration of macrophages (7). Altogether, the present test is belieVed to be relatively simple to 16 17 use, permits quantitation of migration and cell types, has a high degree of accuracy, and should lend itself readily to various manipulations for further study of delayed hypersensitivity,:n vitro. Summary A rapid and reproducible test was developed for detection of delayed hypersensitivity,:n vitro. The basis of test was the inhi- bition of radial migration of sensitive peritoneal macrophages on a glass cover slip in the presence of sensitizing antigen. 18 (1) (2) (3) (4) (5) (6) (7) REFERENCES Rich, A.R. and Lewis, M.R. Bull. Johns Hopk. Hosp., 50:115, 1932. George, M. and Vaughan, J.H. Proc. Soc. Exp. Bio. Med., 111:514, 1962. David, J.R., Al-Askari, 8., Lawrence, H.S. and Thomas, L. J. Imm., 93:264, 1964. Bloom, B.R. and Bennet, B. Science, 153:80, 1966. Benacerraf, B. and Green, 1. Ann. Rev. Med. 20:141, 1969. Dumonde, D.C. Brit. Med. Bull., 23:9, 1967. Heise, E.L., Man, S. and Weiser, R.S. J. Imm., 101:1004, 1968. 19 ‘l‘i' APPENDIX APPENDIX The appendix consists of three reports submitted to the Animal. Health Division, ADE, USDA. The third report describes the successful use of capillary test and confirmation of the specific inhibition of migration of peritoneal exudate cells from tuberculin-sensitive guinea pigs in the presence of PPD. In the next stage attempts were made to determine the effects, if any, of the skin test on the migration of normal and sensitive peritoneal exudate cells. The results of this and a duplicate experhnent are contained in the fourth and fifth reports. The fourth report also contains procedures for isolation of migration inhibition factor and passive sensitization of normal cells. After the extensive use of capillary test in the above experiments, it was concluded that there was need to develop a more reliable and accurate,:n,y:£52 test for delayed hypersensitivity. The remainder of the research period was addressed to this problem and resulted in the development of the spot migration test described in the manuscript. 20 SUMMARY FROM THE THIRD REPORT Capillary method of miggation of peritoneal exudate cells: . Animals: Twenty four guinea pigs of mixed sexes were taken at 3 months of age from our colony. They were not tuberculin tested prior to use. The colony has been a closed colony for seven years and has remained tuberculin-negative. Sensitization of guinea pigs: Twelve of the guinea pigs were inoculated intraperitoneally with 5 mg wet weight of viable M5 bgyig (BCG). Collection and preparation of peritoneal exudate cells: Twenty ml of sterile light mineral ofl per guinea pig were injected intraperi- toneally. Three to five days later, 100 ml sterile Hanks Balanced Salt Solution without heparin or antimbtics (BSS) were injected intraperitoneally. The abdomen was kneaded gently and the fluid collected aseptically. The fluid was centrifuged for 10 minutes at 1200 rpm at 4 C and the supernatant fluid discarded. The cells were washed and centrifuged twice with BSS. The final sediment of cells was mixed with Medium 199-15% normal guinea pig werum to yield 10-20% cells by volume. The cell suspension was drawn into sterile capillary tubes. The tubes were sealed at one end and centrifuged for 5 min at 900 rpm. The capillary tubes were cut at 'the cell-fluid interface. Two or three tubes were placed in each Sykes-Moore chamber. 21 22 Migration-inhibition test: Four or more chambers containing two or more tubes of cells were prepared for each animal each time it was tested. One ml of M199-15% normal guinea pig serum was added to each of two chambers and one m1 of Ml99-15% normal guinea pig serum containing 16 ug protein of PPD-S added to the other two. After 20 hours incubation at 37 C, the tubes and cells in the chambers were observed under the microscope. The distance of the migration of the cells linearly from the tip of the capillary tube was measured by an ocular micrometer. The distance was recorded in microns. Results: The cells from infected guinea pigs migrated in the absence of PPD-S; their migration was inhibited by PPD-S (Table 2). The cells from non-infected guinea pigs migrated in the presence or absence of PPD (Table 3).. The distance of migration for each tube in each guinea pig is given. The average for each animal is given. While variations occur, the real difference of the cells from sensitive guinea pigs phs PPD-S is readily apparent. The range of linear migration for each group was as follows: Uninfected, no PPD: 510 to 1755 u Uninfected, with PPD: 435 to 2040 u Infected, no PPD: 510 to 2040 u Infected, with PPD: 0 to 255 u Effect of skin test on the in vitrofimigration of peritonealexudate cells: Following the above tests, six of the infected guinea pigs and six non-infected guinea pigs were tuberculin tested with 0.10 ug/0.l ml/guinea 23 pig intradermally. A delayed skin reaction from 10 mm to 16 mm deve10ped in the six infected guinea pigs. There was no reaction in the non- . infected guinea pigs. Two days after the tuberculin tests, migration-inhibition tests were begun to determine the effect of tuberculin tests on the results of migration-inhibition test. Four animals were tested daily: one infected, tuberculin tested; one infected, not tuberculin tested; one non-infected, tuberculin tested; one non-infected, not tuberculin tested. When the complete set of animals have been tested (3rd through the 10th day post-tuberculin), the tests were repeated (11th through 18th day post-tuberculin.) «oc.om . rmaoow emmwmnfio: m: amon03m 0m Benoncnuoow nouum «woe mcmnoo nmmm m: .BknoUonnowmc5.mc v.0m {.4Ioca was z_ax sec nymavow % . nymavow h N nyoacow a L nrmacaw % ~ nonm.emwx nmum~_ow< wamam_mw a L a N a _ a N >om a _ a N a _ a N >mm _ :8 So So 38 sum 8 o m. . o as N $0 :8 .80 Sm Sam :0 .5 mm S 2 w meo m_o mic mmo mom mm 0 ._ON 0 rm 1. :8 $0 N30 :8 :8. Nam so e. o :m m Nero .mmo mmo m_o ._rm .Nm m. mm mm m1 m .Nmo ~_mo mmo .mmo _ONo o 0 mm up No N mmo mmo .wNm Ammo .omr o o 0 mu “m m umNo ammo .wmm zNoo .No: 0 .N o as “N m .mwo mmo “.mo Ewmo .umo ”No w: w: wt .mm Lo mes Nam aNoo ammo __Nm 0 O we we N. .1 ammo mew zwmo _Nmo __NN _0N mm me No we LN ~_om mmo LNoo mm: LomN . 0 mm o 0 Ne reme>mm 1_NN mos _Nm1 ._sw .mzm Nu km :N mm mN mpzom .m_o - Nero 0 - Nmm 1!. O. 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INwm m mam __om _ONo _woo Home Now .LoNo .wmo _~mo Low: w mmo mam _1wo ._mo . _0Nm _uwm mmo ammo man .0N Lo ewmo LNmm No L_om LNNm Ammo _oNo mac LONO mum _e mmo zrpm .LONo mmo mmm .Nom “ONO m_o .0N0 mus 1N “mm _Lmo “.mo LONo __wo Ammo :Nm __oo Nose INmm yemwpom ~_om ~_NN _om~ .003. “.11 . ._Noo _ emu . 11x0 .os. _oww ‘1-.- u’,l.ul‘l 3|, 0 IIII'DI “'!I‘ii0'-‘t‘i‘!l-£""“"l‘-llll| SUMMARY FROM THE FOURTH REPORT Passive sensitization of mononuclear cells in:vitro: When lymphocytes from sensitive animals are treated with tuberculin, the supernatant fluid passively sensitizes monocytes from normal animals. Lymphocytes from normal animals or monocytes from normal or sensitive animals do not possess the passive sensitizing factor. We will attempt to isolate the factor. We hope from this study to not only gain an understanding of the mechanism of the phenomenon of delayed hypersensi- tivity but also to lead to the development of an,:n,y:££g test employing normal mononuclear cells or some primary or secondary cell cultures as index cells. In the passive sensitization,:n,y:££g study, peritoneal exudate cells were obtained from guinea pigs as described in the Third Semi- Annual Report. The cells were washed twice with Hank's balanced salt. solution and then suspended in Medium 199 containing 15% normal guinea pig serum. The cells were then incubated for 2 hours at 37°C in a glass petri dish. During this period the macrophages attached to the glass surface whereas the lymphocytes remained non-adherent and were removed with the fluid after gently shaking the plates. This cell suspension was carried through the above procedure for 3 more times. During this time, polymorphonuclear cells are generally lysed, monocytes remain attached to the glass and therefore, the cells remaining in the fluid were primarily lymphocytes. In addition, fresh medium was added to the plates to which '26 27 the monocytes attached and were incubated 2 hours. The fluid was removed after gently shaking the plates and discarded. The cells attached to the glass were released by adding ethylenediaminetetraacetate (1:5000) for 5 minutes. The released cells were monocytes. In the next step, the possibility of the elaboration by sensitized lymphocytes of a sensitizing factor was tested. The sensitized lympho- cytes obtained in the manner described above were cultured in the absence or presence of various quantitites of PPD. After 20-24 hours the cell suspension were centrifuged gently and the cells removed. The cell-free supernatants were further clarified by centrifugation at 250 r.p.m. for 15-20 minutes. The peritonea cells or macrophages obtained from normal donors were suspended and centrifuged in capillary tubes, placed in chambers which were filled with M199 with serum and various quantitites ‘ of the supernatants. Thus far, the results indicate that supernatants from sensitized lymphocytes cause inhibition of normal macrophages or normal peritoneal exudate cells in the presence of PPD whereas supernatants from normal lymphocytes fail to produce the same results. Data on the effect of skin test on the :3 313:9 migration of peri- toneal exudate cells is presented in Table 4. However, a duplicate experiment is also in progress. L. . .. . . . , _ . . _ . .1 _ . . . t. . . . . a . .,. . ‘illillL‘ll. III... I.ll...'ll.l1...u 1" 1" II! .1 Ell-ill! I t.li.t1[!tlll 1". l ‘10.... I: l .. -I! x |U Linea" 3' " ~’ _ k ., .. . . r: - H10~~uxca O; Pe“1ro““1‘ V - .. 1.“. 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Ci (n 0 *4 ' C: o o c» c') u: 0 O L” C‘ '0 V1 3 m o 0‘ m V’ F o u: m u. o c5 y; g N. U! U“ U' 0 . N c» e) C) s5 “ ’. J to C) \- f -I p. .l \r.) C) O 0 U1 680 0 1105 340 )/ ---- Died ---- 1020 1020 kl H V3 0 (D U1 0 Fl \J (D LO Is. r... “1 O \J C) U! bl O C) H N O V 00 U~ C) H. F“ 03 \9 U1 (1) O 1 a linear wie- h: a' a ‘ 1 oration PLIOL to tuberculin r'wL‘ . .ua in L" .i_.',“ h” - ‘ a L LL .-\aL—&\A’ :10 T3?) "1 \p_,' ‘-‘.(.‘. 'N .0. o. . .‘l; g ‘_ ‘ J . D. , t “ " “ ' ~ _ --—-.....__ ‘ 1‘ 'I ' - - ~ .- 'L C ‘ _y- L.‘ , 7‘ , . ..) b III I . . h _ v C, L1-4 PPD in v;‘ro. [a L'JD d o-T I 7" -- "- .— ' ' —~ -H ——.-‘.::_V“ . ‘ ’ "I \ 1"“ttrb, ‘0 REJ 1L V:“”q. 'JS ‘ ZvéU u ancc CE (I ‘7 “jI\-—_ ..——:..\.'.:_\_- ' 51“) - 4- A I A , 511.3 1'.” 1+, V:"'"" iU—rU u “#- fi-L.” " k, 0 0 . ' u 28 P4 C) to 03 C‘ U! 0" U‘ C) SUMMARY FROM THE FIFTH REPORT Effect of skin test on the in vitro migration: As reported in the Fourth Semi-Annual Report, an experiment was being performed on more animals to determine the effect of intradermal tuberculin tests on the in,gi£:2_migration-inhibition of macrophages from normal and sensitized animals prior to the collection of macrophages. Twelve BCG infected guinea pigs and 12 non-infected guinea pigs were tuberculin tested with 0.5 ug/.l m1/guinea pig intradermally. Delayed reactions from 9-15 mm developed at the site of all infected guinea pigs. The,:n,g:ggg migration tests were conducted on the tuberculin tested animals 2-6 days after the intradermal tuberculin injection. One animal from each group viz, sensitized non skin tested, sensitized skin tested, non-sensitized-nonskin tested, non-sensitized-skin tested, was tested on these days. The,:g,y:g£g tests were repeated on these animals in the same order from day 8 to day 13 so that animals tested on day 2 were again tested on day 8, and animals tested on day 3 were tested again on day 9 and so on. The results are presented in the following table. 29 30 1360 TABLE 5. Linear migration of peritoneal monocytes lg yitgg from normal and tuberculin-sensitive guinea pigs after skin testing. :gAg’ETREIEL BCG INFECTED GUINEA PIQ_S_ NON- INFECTED GUINEA PIGS HSRIHILIN Skin Tested No Skin Test §kin Tested No Skin Test TESXT no PPD with PPD no PPD with PPD no PPD with PPD no PPD with PPD Day 2 850 "221 850 68 1190 1020 1190 1190 ' 1105 ‘ _340 1190 51 1275 935 1190 1020 Dag; 3 1020 170 1190 136 1360 1190 1360 1190 1360 153 1020 153 1020 935 1530 1326 Day 4 680 o 1530 136 1020 680 1020 1105 935 170 -1495 170 850 935 1190 1360 Day: 5 1020 340 1530 255 1190 906 935 1105 ‘1105 255 1615 ‘170 1020 765 1020 1190 Day 6 765 221 1326 65 1020 595 1020 1360 850 289 1530 204 1156 850 1190 1445 Day 7 1870 170 1070 323 1190 1105 1020 680 1700 255 935 306 850 1190 1190 1020 Day 8 1190 340 1530 153 1190 1190 1360 680 1360 306 1360 136 1190 1020 1105 1020 Day 9 1870 510 850 255 1360 1190 1360 1190 1530 255 1360 170 1020 1020 1530 1020 Day 10 680 136 9 680 102 1530 1020 1020 1190 935 102 1020 ‘170 1020 765 850 850 Day 11 1700 255 1360 102 1360 1360 680 1020 1360 204 ' 1615 170 1020 1020 '265 850 Day 12 765 136 850 340 1190 1190 850 1020 1020 170 1360 510 1020' 1020 1360 1105 Day 13 1190 119 1360 136 1020 1020 1190 1360 1360 102 1020 85 1156 1020 1530 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) REFERENCES Kapral, F.A. and W.E. 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