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(Effixlfllk .in 83:1... 3 ifl’ftuv’él I“ v v i V first; gig .t 311%.?! 1 gal! , .fctfti: \ ‘ . gisttk {sf goixl-..‘ i--- .rnzé 5 its»: .Lirrtifvtzitatt.f3zt§r$:u, E31!¢t{t)rsllt§tétl l . ‘ . : $cf§.&l£7rltst¥¥t»¥;l::ritriizatEI:. , . . #2.} .2 :1 5?;5521Eirfgg‘ttrc}.. i?! fét...c.§c§lr4 12.5.5176: : . iv.XIHHHIIT.:I2‘.r)\¢l.|3..|a\(:rrl:?ek)? I , ‘ ‘ ..#.tt;5 .1» , “his LIBRARY ’ State x 0mm This is to certify that the thesis entitled THE ROLE OF HUMORAL AND CELLULAR FACTORS IN HEPATIC CLEARANCE AND KILLING OF SALMONELLA TYPHIMURIUM presented by Richard Lee Friedman has been accepted towards fulfillment of the requirements for Ph.D. . Microbiology degree in MajoxMofessor Date—31.221 1.9.7.9. .— 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to Book drop to remove this checkout from'your record. THE ROLE OF HUMORAL AND CELLULAR FACTORS IN HEPATIC CLEARANCE AND KILLING OF SALMONELLA TYPHIMURIUM By Richard Lee Friedman A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health l979 ABSTRACT THE ROLE OF HUMORAL AND CELLULAR FACTORS IN HEPATIC CLEARANCE AND KILLING 0F SALMONELLA TYPHIMURIUM By Richard Lee Friedman Clearance and killing of Salmonella typhimurium was studied in normal, immunized and Corynebacterium parvumytreated animals in the presence and absence of plasma. The perfused liver was the major experimental model used. The initial objective was to evaluate normal and experimentally altered plasma in bacterial trapping and killing. Addition of 5% normal rat plasma to perfusion media increased bacteri— cidal activity in the perfused rat liver. When complement activity was inhibited by heating at 57°C and 50°C, zymosan absorption or chelation with EDTA, bactericidal activity was significantly reduced. EGTA had no effect. Immune plasma heated to inhibit complement did not stimu— late bactericidal activity but significantly increased bacterial trap— ping by the liver. Absorption of normal and immune plasma with a specific anti-rat-C3—Sepharose 4B immunoabsorbant inhibited bacterici- dal activity. These results demonstrate that complement is required for bactericidal activity and is activated via the alternate in normal and via the classical pathway in immune plasma. A second objective was to examine experimentally altered livers. Livers from rats immunized with heat-killed §;_typhimurium were not Richard Lee Friedman statistically different from normal livers in bactericidal activity or distribution of recovered bacteria in the absence or presence of plasma. When the RES of mouse and rat livers was activated with §;_pgrvum vaccine 22% of the perfused bacteria were killed in the absence of plasma. Silica and phenylbutazone inhibited ifl_situ hepatic killing while EDTA had no effect suggesting that bactericidal activity reflected cellular killing. The addition of plasma did not significantly increase bactericidal activity over normal livers in the presence of plasma. Scanning electron microscopy of §;_parvum;treated livers revealed lar~ge rnumbers of white blood cells mostly macrophages and lymphocytes, adhering to portal veins and sinusoids. These macrophages were morpho- l ogically similar to Kupffer cells. A relative peripheral blood mono— c‘ytosis occurred simultaneously. Presumably the blood cell influx irito tlie liver plays an important role in the enhanced nonspecific antimi— cr‘obial resistance observed in §;_parvum—treated animals. I__vivo, EL; pairvum-treated mice were able to clear and kill I.V. injected S; txphimurium better and had a ten-fold increased resistance. Cumulatively the data shows that g; parvum vaccine enhances the ability of the liver to trap and kill bacteria in situ and increases clearance and resistance in_vivo. Livers from immune animals behaved silnilar to normal livers. To my wife, Ellen, for her love, perseverance and understanding during these past years. This degree is as much hers as it is mine. ii ACKNOWLEDGMENTS The author wishes to thank Dr. Robert J. Moon, my graduate advi- sor, for his time, patience, and understanding during the years I have been associated with him. I am forever grateful for his deep interest in my professional growth and development. I also thank Dr. Richard Sawyer for his collaborative work on the scanning electron microscopy in this thesis, for his thoughts and ideas on my research and above all for his friendship over the years. I wish to thank Dr. Elizabeth Werner, Jim Veselenak, Bob Leunk, Hassan Tavakoli, Marilyn Thelen and Laura Ruggeri for their help and friendship during my graduate studies. TABLE OF CONTENTS LIST OF TABLES .......................... LIST OF FIGURES ......................... INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . , . . . LITERATURE REVIEW ........................ Animals ........................... Bacteria ........................... Corynebacterium parvum . . . ................. Chemicals .......................... Rat Plasma .......................... Rat Plasma Treatments ................. . . . Ifl_vitro Liver Perfusion ................... Distribution and Survival of §;_typhimurium in Mice ..... §;_typhimurium Infection in Normal and §;_parvum-Treated Mice Total (WBCTCand Differential White Blood Cell Counts ..... Anti-CB—Sepharose 4B Immunoabsorbent ............. Assay for Hemolytic Complement ................ Immunodiffusion ....................... Scanning Electron Microscopy .............. . . . Statistics . . . . ...................... RESULTS .......................... Trapping and Killing of §;_typhimurium by Perfused Rat Livers in the Absence or Presence of Normal Rat Plasma ...... Effect of Heat, Zymosan and Chelators on Bactericidal Activity of Plasma in the Perfused Liver .............. Effect of Immunoabsorption of C3 on the Ability of Normal Rat Plasma to Stimulate Bactericidal Activity in the Perfused Liver ........................... Bactericidal Activity of Perfused Livers in the Presence of Immune Plasma ....................... Trapping and Killing of §;_typhimurium by Livers from Humor- ally Immunized Rats in the Absence and Presence of Normal Plasma ........................... Trapping and Killing of §;_typhimurium by Perfused Livers from Normal and C;_parvum-Treated Mice ............. iv Page vi 22 22 24 25 30 31 mg wwfi-u" .7 . . LL .. Page Effect of EDTA, Silica and Phenylbutazone on Bacterial Killing by Perfused Livers from C. parvum-Treated Mice ....... 3l Trapping and Killing of L. typhimurium by Perfused C. parvum- Treated Rat Livers in the Absence or Presence of Plasma . . 33 Scanning Electron MiCroscopy of Livers from Normal and C; parvum-Treated Animals ................... 35 Scanning Electron Microscopy of C. parvum-Treated Mouse Livers Perfused with S. typhimurium ................ 47 White Blood Cell Kinetics in Normal and C. parvum-Treated Rats and Mice .......................... 54 In vivo Distribution and Survival of S. typhimurium in Normal —and C. parvum- -Treated Mice ................. 54 Survival of Normal and C. parvum-Treated Mice Infected with L. gyphimurium . . . . . ............ . . 56 DISCUSSION ................... . . . . . . . . . 59 LITERATURE CITED . . . . . . . .................. 7l LIST OF TABLES Page 20 23 23 25 28 29 29 3l 32 32 34 55 Table 1 Bacterial trapping and killing by the perfused rat liver in the absence or presence of rat plasma .......... . . . . . 2 Percentage of depression of total hemolytic complement levels in treated normal rat plasma .......... 3 Role of complement in bacterial killing by perfused rat livers 4 Differentiation of classical and alternate complement pathway activity in mediating plasma killing in the perfused liver 5 Immunoabsorption of C3 from rat plasma and its effect on plasma's bacterial killing in the perfused liver ....... 6 Effect of immune plasma treatment on bactericidal activity in vitro and CH titers . . . .................. —————- 50 7 Bacterial killing in the perfused rat liver in the presence of immune rat plasma either untreated or treated by heat, zymosan, EGTA or C3-absorption . . . . . . ........ . . . . . 8 Trapping and killing of L. typhimurium by livers of immunized rats in the absence and presence of normal rat plasma . . 9 Clearance of g; typhimurium by perfused livers from normal and §;_parvum-treated mice . . . . . . 10 Effect of EGTA, silica and phenylbutazone on bacterial killing by perfused livers from L. parvum-treated mice . . ll Bacterial trapping and killing by §;_parvum-treated and normal rat liver in the absence or presence of plasma ........ l2 Total and differential white blood cell counts in normal and_g; parvum-treated rats and mice ................. l3 Recovery of S. typhimurium 15 and 60 minutes after I. V. injec- tion into normal and L. parvum-treated mice . . vi 55 LIST OF FIGURES Figure l Immunodiffusion slides of C3 titers from normal and immune plasma before and after anti-rat-C3 immunoabsorption . . SEM of normal and 9; parvum-treated liver SEM of various white blood cell types observed in L. parvum- treated livers . . . . . . . . . . . . . . SEM of Sinusoidal areas of normal andg.~ parvum—treated livers SEM of macrophage- macrophage interaction in L. parvum- treated liver . . . . . . .. . . . SEM of macrophage— lymphocyte interaction in L. parvum- treated liver . . . . . . . . SEM of a sinusoidal macrophage from a §;_parvum-treated liver SEM of macrophage phagocytizing L. typhimurium in Q; parvum- treated liver . . . . . . . . . . . . . . . . SEM of macrophage phagocytizing L. .typhimurium in_g; parvum— treated liver . . . . . . . . . . . . . . . . Susceptibility of normal and L. oparvum-treated mice to infec- tion by L. typhimurium . . . . . . . . . . . . . . . . Page 27 37 39 42 44 46 49 5l 53 58 INTRODUCTION The relative role of humoral and cellular factors in the antimi- crobial properties of the liver is not well defined. The liver Kupffer cells make up a major percentage of the reticuloendothelial system in the body and are a key initial cellular defense barrier against micro- bial invasion of the blood (44, 92). Studies by Moon et al. (6T) suggest that the removal of bacteria by hepatic tissue involves both phagocytic and non-phagocytic parameters of the liver and that plasma is obligatory for bactericidal activity. The initial observation that non-phagocytic cells play an important role in microbial filtration by hepatic tissue was extended by Friedman and Moon (35) who demonstrated that while treatment with the macrophage toxin, silica (4, 5T), decreases bacterial trapping by 50% it did not abolish it. The initial objective of this thesis is to confirm and extend earlier studies suggesting a role for both complement and antibody in the antimicrobial activities of the liver (13, 43, 59, 60, ll7). Research progress, particularly in the complement system, warrants a more critical evaluation of the role of humoral elements in bacterial killing at this time. A second objective is to describe the relative roles of antibody and complement in trapping of bacteria by both normal and immune livers. A third objective involves a study of livers from thpagyumy treated animals. §;_parygm_is a macrophage activator (4l, 98, ll8) which in the course of our experiments, was fbund to induce bacterici- dal activities of liver in the absence of plasma. This observation led to additional investigation to determine more of the nature and biolo- gical significance of §;_pgrygm_induced changes jfl_yiyg, particularly with respect to bacterial infection. LITERATURE REVIEW The reticuloendothelial system (RES) is the major cellular defense barrier against microbes which enter the bloodstream (44, 92). It consists of fixed phagocytic cells including liver Kupffer cells, splenic macrophages, microglial cells of the brain, pulmonary alveolar macrophages and lymph node macrophages. In addition to clearance of microbes, the RES also functions in hemoglobin uptake and breakdown (39), protein transport (48), uptake and metabolism of steroids and lipids (9, 25), degradation of red blood cells (7), clearance of auto- logous tissue debris (ll6), endotoxin uptakeauuidetoxification (32), phagocytosis of foreign colloidal and particulate material from the blood (ll, lll), antigen processing (34) and immunity and destruction of tumor cells (8, 50). As early as l886 Wyssokowitsch (l25) demonstrated that bacteria and fungal spores injected intravenously into rabbits were cleared by Specific endothelial cells in the liver and spleen. Bull (l5) in l9l5 studied the fate of intravenously injected Salmonella typhi into rabbits. Fifteen minutes after injection the number of bacteria per ml of blood dropped from l07 to 40 bacteria per ml. Bull found that 2l minutes after injection, the majority of the §;_3yphi could be recovered from the spleen, liver and lung. He concluded that bacilli accumulated mainly in the liver and spleen and were taken up by assem- bled PMN (RES cells) and destroyed. Rogers categorized bloodstream clearance of bacteria (92) into three phases. Initially, when large numbers of viable bacteria were injected I.V. into rabbits, 99.9% were cleared in the first ten minutes to 5 h, the rate depending on the organism being studied. This rapid clearance phase was followed by a period when microbes persisted in the circulation due to slower removal rates. A final phase occurred in which bacteria either disappeared or multiplied to high levels leading to death. Rogers states that the fixed phagocytic cells sequestered the majority of the bacteria during the initial rapid clearance phase. Kupffer cells and splenic macrophages were most active in this process. Sixty to 95% of the bacteria were sequestered in these organs. This thesis primarily evaluates Kupffer cell function during the early clearance phase. Kupffer cells reside in the lumen of hepatic sinusoids adhering to the fenestrated endothelium by fine cytoplasmic filopodia (T4, 62- 64, l23). They occupy a large area in the lumen of the sinusoid and are usually found at sinusoidal junctions. The body of the Kupffer cell is covered by numerous folds, ruffles, invaginations and micro- villi (60). Thirty-eight percent of the total number of liver cells are Kupffer cells (45). The liver perfusion model offers a unique opportunity to study the interaction of cellular and humoral factors in bacterial trapping and killing by the liver. In l9l6 Manwaring and Coe (59) did rabbit liVer perfusions with pneumococci. Bacteria suspended in Ringer solu- tion or Ringer plus l-lO% normal sera were not cleared by the liver. When 1% immune sera was added to the perfusion media, the liver retained all bacteria that were perfused. The serum component involved was heat-stable at 60°C for 30 minutes (antibody) and was called an endothelial opsonin. Manwaring and Fritschen (60) did further perfused liver experiments using Staphylococcus aureus, §;_coli and Bacillus anthracis with similar results. Howard and Wardlaw (43) found that human, rat and mouse sera increased clearance of §;_ggli_by the perfused rat liver when added to the perfusion media. The opsonic activity of sera was reduced by heating at 56°C for 30 minutes, absorp- tion with homologous strain of bacteria or with an antigen-antibody complex. Opsonic activity was abolished by homologous absorption followed by heating at 56°C or absorption with zymosan. They concluded that the serum factors involved in clearance of bacteria by the liver were specific antibody, complement and probably properdin. In the experiments, the actual number of viable bacteria in the perfused livers was not determined, so what effect these serum treatments had on actual bacterial killing is not known. Experiments were also done using other strains of gram negative and gram positive bacteria (ll7). The presence of serum enhanced bacterial clearance of all the gram negative organisms studied and heating serum reduced this activity. The gram positive bacteria (§;_aureus, Strep, pyogenes, B; cereus and §;_murium) were cleared in the absence of serum and its presence reduced their clearance. Bonventre and Oxman (l3) studied the role of humoral and cellular factors on clearance and killing of Staphylococcus aureus and Salmo? nella enteritidis by the perfused rat liver. Their results indicate the immunological status of the liver or of the serum had no effect on clearance or killing of_§; aureus. Experiments with §;_enteritidis showed that the presence of immune humoral and/or cellular factors profoundly affected the removal of bacteria by the liver. When normal livers were perfused with immune serum the rate of clearance of g; enteritidis was greatly increased. In the non-immune liver perfusion system, 200% of the §;_enteritidis perfused were recovered in the liver as determined by bacterial plate counts. In the presence of immune serum bacterial multiplication did not occur and with both immune liver and serum only 5% of the infused bacteria were recovered from the liver. The above mentioned studies suggest that antibody and comple- ment play a role in bacterial trapping and killing in the liver. More specific treatments of normal and immune serum or plasma must be done to critically determine the role of these humoral factors in the liver's interaction with bacteria. Complement is critically involved in immune processes and also interacts with the kinin, clotting and fibrinolytic systems (88). Complement is also involved in phagocytosis of foreign matter, neutrali- zation of viruses by antibody, bactericidal reactions, immune adherence, chemotaxis and histamine-regulated vascular permeability (T6, 38, 93). The proteins that make up the complement system account for 5-l0% of the weight of all plasma proteins (20). Complement is synthesized by RES cells in the liver, intestine, bone marrow, spleen, lung, lymph nodes and macrophages (20). The critical step in generation of the biological activities of the complement system is the production of the major cleavage fragment of C3, C3b. Both the classical and alternate complement pathways can cleave C3 to yield C3b but both do it by different mechanisms (3l). In the classical pathway, activation of complement (C) is initiated by an antigen-antibody complex that binds and converts Cl to its activated state CT (56) which then cleaves C4 (65) and C2 to form the classical C3 convertase, CT42 (66) which cleaves C3 to produce C3a and C3b. The alternate pathway of complement activation was discovered when it was observed that zymosan, an insoluble polysaccharide-containing deriva- tive of yeast cell walls, inactivated serum C3 without depletion of Cl, C4 or C2 (84). Alternate pathway activity requires low levels of C3b which is produced by interaction of C3 and B in the presence of'D or P to form the amplification C3 convertase, C3bBb (22, 30, 31) which is stabilized by properdin (P) (30). C3b acts as a receptor for B in a Mg++-dependent reaction that exposes a site on B to cleavage by‘D (3T). D causes release of Ba fragment from B to uncover the C3-cleaving site in the Bb fragment that remains bound to C3b (3T). Once C3b is produced by either pathway it can be involved in a number of biological activities. Foremost of these is C3b interaction with C5 to generate the C5b6789 cytolytic complex (54) which can lyse red blood cells or bacteria. C3b is also involved in immune adherence (7l), enhances phagocytosis of C3b coated particles (l0), induces secretion of lysosomal enzymes by cultured peritoneal macrophages (lOl), secretion of a chemotactic factor by B lymphocytes (96) and is involved in lymphocyte differentiation (82). Data reported in this thesis point to a critical role of C3 in the bactericidal properties of perfused livers in the presence of plasma. Little is known about rat C3, while human (73), guinea pig (l05) and murine C3 (83) have been isolated and characterized. Recently Daha et al. (23) isolated and characterized C3 from rat plasma by precipitation with polyethyleneglycol in the presence of benzamidine which inhibited proteolytic enzymes and kept C3 functionally active during chromatographic purification. The percipitate was chromato- graphed on CM-cellulose, hydroxyapatite and QAEASO—Sephadex, and gel filtered on Sephadex G-200 superfine. Recovery of C3 was 18-26% and the material was homogeneous on SDS-PAGE analysis. Rat C3 has a mole- cular weight of l87,000, which is similar to human, mouse and guinea pig C3. It is composed of 2 disulphide linked polypeptide chains of l23,000 and 76,000 daltons. The isolated protein was proven to be rat C3 by its ability to react with EACl42 and cause subsequent blood cell lysis. In the assay rat and human C3 functional activities were simi- lar. The plasma concentration of rat C3 in Wistar rats was calculated to be 58li49 ug/ml. Rat C3 was able to react with human components of the alternate pathway to form the C3 convertase, showing compatibility between rat and human C3. lg_yi£§9_studies on isolated Kupffer cells by Munthe-Kaas et al. (67, 68) demonstrated that they have C3b receptors. C3b-coated SRBC attached to Kupffer cells but less than 20% were phagocytosed in the absence of serum. When cultures were rinsed and incubated in media containing 30% newborn calf serum, the majority of the attached red blood cells were phagocytosed. Mouse peritoneal exudate cells did not phagocytose C3b-coated SRBC when incubated with newborn calf serum. The C3b receptor was sensitive to treatment with trypsin and pronase as was the C3b receptor of peritoneal cells. The ability of Kupffer cell C3b receptors to induce phagocytosis is unique and does not occur in other mononuclear cells (58). ljlgjy9_studies of clearance and destruction of erythrocytes by Schreiber et al. (75) suggest that Kupffer cell C3b receptors are involved. Kupffer cells also have Fc receptors for 196 (47, 67-69). Huber et al. (47) using red blood cells sensitized with 196, demonstrated attachment and phagocytosis of the RBC in more than 90% of the isolated human Kupffer cells. Munthe-Kaas et al. (67-69) demonstrated Fc receptors on rat Kupffer cells jfl_yi§rg, Seventy-five percent of the cultured cells by two days and 96% by Six days had ingested IgG-coated SRBC. At 4°C, IgG-coated SRBC attached but were not internalized and upon transfer to 37°C, phagocytosis occurred. Peak phagocytosis of IgG-coated SRBC occurred within 30 minutes after their addition to Kupffer cell cultures. Kupffer cell Fc receptors were sensitive to trypsin and mercaptoethanol. The role of Kupffer cells in trapping and killing of bacteria in the liver has not been well characterized. Early work by Manwaring et al. (59, 60) and Howard and Wardlaw (43, ll7), using the perfused liver, studied clearance of bacteria from perfusion media. Any bac- teria not recovered from the media after perfusion through the liver were considered phagocytosed and killed by Kupffer cells, but no direct quantitation of viable bacteria in the liver was done. Moon et al. (6T) recently showed that clearance of §;_typhimurium by the perfused mouse and rat liver was not synonymous with phagocyto- sis and killing by Kupffer cells. No killing of_§L typhimurium occurred in the perfused liver when the perfusion media contained only M-l99 but greater than 70% of the bacteria were cleared in a single pass. By phase, electron and scanning electron microsc0py (35, 61), bacteria appeared trapped in the liver sinusoids giving a "log-jam" appearance. Fifty-percent of the trapped bacteria were killed when blood or plasma was added to the perfusion media. No killing occurred when bacteria l0 were incubated with blood or plasma alone. These experiments demon- strate that bacterial clearance in the perfused liver may not reflect phagocytosis but actually trapping of bacteria in liver sinusoids. These results suggest a number of research questions regarding the mechanism of bacterial trapping by Kupffer cells. In an earlier study Friedman and Moon (35) evaluated the effects crystalline silica had on bacterial trapping by the perfused liver. The working hypothesis was that destruction of Kupffer cells should Significantly decrease trapping. Crystalline silica is a specific macrophage toxin (4, 51). When taken up by macrophages it interacts with lysosomal membranes making them permeable. Lysosomal enzymes leak into the cytoplasm and cause death of the cell (4, 42, 70). Normal mouse livers trapped 63.5% of an infused dose of §;_typhimurium with 42.3% recovered in the effluent. Silica-treated livers trapped only 3l.l% with 65.9% recovered in the effluent (35). Scanning elec- tron microscopy revealed that silica caused damage and destruction of Kupffer cells but had no other histotoxic effects on the liver (35). Destruction of Kupffer cells significantly decreased bacterial trapping by the liver, but still liver bacterial trapping occurred. These experiments indicate that maximal bacterial trapping by the liver devoid of most Kupffer cells can still trap bacteria in sinusoids, which demonstrates that bacterial trapping also involves non—Kupffer cell components of the liver. Another tool used to study the role of Kupffer cells in bacterial- liver interactions is phenylbutazone (PB), an anti-inflammatory drug that inhibits phagocytosis (55, l08, llO) and intracellular killing 4 (77, l08, ll0). Whitehouse (l2l) observed that 2.5xl0- M PB inhibited ll phosphorylation coupled to succinate oxidation in rat liver mitochon— dria. PB uncouples oxidative phosphorylation (production of ATP) with- out inhibiting oxidative metabolism and cellular respiration. Strauss et al. (ll0) studied the effect of PB on phagocytosis and intracellular killing by guinea pig PMN. PB inhibited intracellular killing and uptake of §;_ggli_by PMN. When 5 u moles/ml of PB were added to PMN homogenates bactericidal activity was inhibited. When PB was added to PMN in the process of killing bacteria, killing was immediately stopped. These results demonstrate that PB inhibits killing by an effect on intracellular activities and shows that inhibition of phago- cytosis and bactericidal activity are independent events. Metabolic 14C-formate studies showed that the drug inhibited glucose-l—14C and oxidation of both resting and phagocytizing PMN which suggest an effect on hexose monophosphate shunt and H202 formation. The study also showed that PB inhibits glucose-6-phosphate and 6-phosphogluconate dehydrogenase activity. Similar effects of PB on human PMN (l08) and monocytes (77) have been observed. Work by Leijh et al. (55) demonstrated that l mM PB inhibited the intracellular killing of Q; albicans by human PMN and blood monocytes and had no effect on phagocytosis. Kjosen et al. (53) verified this by showing that low levels of PB inhibit intracellular killing of bacteria by PMN by blocking the hexose monophosphate shunt (required for bacterial killing) and not the Embden Myerhoff glycolytic pathway (required for phagocytosis). Another method to evaluate the role of Kupffer cells in bacterial trapping and killing is to stimulate RES cell activity. Halpern et al. (4l) while examining the genus Corynebacterium for Species related to 12 mycobacteria in their ability to stimulate the RES, observered that g;_ parvum was a powerful macrophage activator. §;_parvum causes a vast array of biological responses L vivo and _i_n_ vitro. Injection of L pagygm I.V. causes enlargement of liver, spleen, and lung and increases carbon clearance (2, 17, 57), transient anaemia (57), adjuvant effect on antibody response (46, 79, 106) and stimulates RES activity (98, 118). §;_pa§ygm_depresses T-cell—mediated immune phenomena which include delayed hypersensitivity (19, 99), phytohemagglutinins, mixed lympho— cyte, graft-v.s.-host (100) and homograft responses (l7). £;_E§:lwfl vaccine also has anti—tumor activity (98, 113, 114, 124). Treatment of experimental animals with §;_pa§ypm_enhances their resistance to Listeria monocytogenes (6, 95, 112), Brucella abortus (1), Salmonella enteritidis (19), Staphylococcus aureus (103), Herpes Sim— plex virus (52), Plasmodium berghei (76), I;_gondii (112) and Q; albicans infection (103). §;_parvum treatment reduces resistance of mice to Aspergillus nidulans infection and leads to fatal murine asper- gillosis (87). IQ; parvum exerted an immunosuppressive effect on Trichinella §piralis infections in rats and prolonged the infection (94). Treatment of rabbits with Q; parvum did not enhance resistance to infection with Treponema pallidum (6). lg_gi§:g,and_in.xiyg_studies show that §;_pa§ygm stimulates immu- nological activities via direct interaction with lymphocytes and RES cells (12, 18). lg_yi§:g, Christe and Bonford (18) showed that oil- induced CBA mouse peritoneal macrophages could be activated by contact with §;_pg§ypm_alone, while normal macrophages required simultaneous exposure to Q;_pa:ygm and spleen cells from mice immunized with_QL parvum. Treatment of immune spleen cells with anti-theta serum and l3 complement inhibited activation. They observed in ligg that T-cell depleted mice were able to respond to §;_pg§!gm, in demonstrating macro- phage activation and splenomegaly, but the ability of spleen cells to activate normal macrophages ig_xitrg_was reduced (12). From these experimental results Christe and Bonford concluded that §;_pg:ygm_acti- vated macrophages by both direct and immunological mechanisms, i.e. via T-cells. The immunopotentiating activity of §;_pa§ygm seems to reside in the phospholipid of the bacteria (27). Fauve and Hevin found that bacterial phospholipid extracts of §;_pa31gm injected into mice enhanced blood clearance of §;_typhimurium (27). The extract also increased resistance to L;_monocytogenes infection and inhibited multiplication of the organism in the liver and spleen (27). MATERIALS AND METHODS Animals Sprague-Dawley male rats, 300 to 350 g were obtained from Harlan Industries (Indianapolis, Ind.). Female Carworth CF-l mice weighing 18 to 25 g were obtained from Charles River, Wilmington, Delaware. All animals were maintained under standard laboratory conditions with Purina Laboratory Chow and water available ad libitum. Bacteria Eighteen to 24 h cultures of Salmonella typhimurium, strain SR-ll, were grown in brain heart infusion broth and centrifuged at 8,000 x g for 15 minutes. The bacteria were resuspended in either M-l99 (Gibco, Grand Island, N.Y.), Ca++ and Mg++ free Hanks balanced salt solution or sterile saline. Corynebacterium parvum Corynebacterium parvum vaccine was supplied as a gift by Dr. Richard Tuttle, Burroughs Wellcome Co., Research Triangle Park, N.C. The two lots of vaccine (CA528A and CA580A) used throughout this study were formalin killed suspensions supplied at a concentration of 7 mg dry weight/m1 containing thiomersal. Both lots were washed three times in sterile saline to remove the preservative and stored at 4C. No variation in response between vaccine lots was observed. 14 15 Rats were injected with 350 pg of C;_parvum intravenously two days before examination. Mice were injected with 700 ug §;_parvum nine days before examination. Chemicals Dorenturp crystalline silica (0012), particle size 5 pm or less, was supplied by Dr. Ing M. Reisner, Steinkohlenbergbauereiw, 43 Essen- Kray, Frillendolfer Strabe 351, W. Germany. Before injection it was autoclaved in powder form, suspended in sterile saline at a concentra- tion of 20 mg/ml, and sonicated in a Bronsonic ultrasonic cleansor (no. 8220, Branson Instruments Co., Sketon, CT). Silica was given to mice six days after §;_pa§!gm_injection. A total of 10 mg was given intravenously over a three day period, i.e., 3 mg on days six and seven and 4 mg on day eight. Mice were studied on the ninth day after_g; pargpm_injection. Phenylbutazone (Lot #127C-0083, Sigma Chemical Co., Columbus, OH) was dissolved in 95% ethanol at a concentration of 10 mM and added to M-l99 to yield a final concentration of 1 mM (pH adjusted to 7.3 by 1N NaOH). The solution wasfiltersterilized. Experimentally, 1 ml of 1 mM phenylbutazone was infused into §;_pa§ygmytreated mouse livers and the livers washed for 20 minutes with M-199 prior to infusion of bacteria. EDTA (disodium ethylenediaminetetraacetate) (Matheson Coleman and Bell, Cincinnati, OH) was added to Ca++ and Mg++ free Hanks balanced salt solution (HBSS) at a concentration of 0.01 M and the pH readjusted to 7.3 by lN NaOH. l6 RatLplasma Blood was obtained from heparinized rats by cardiac puncture and centrifuged to obtain plasma. Plasma was pooled and stored at -70°C until use. Immune rat plasma was obtained from rats immunized with heat-killed §;_typhimurium vaccine. The vaccine was made by heating BHI cultures of §;_typhimurium at 65°C for 1.5 h, centrifuged and washed three times in sterile saline and resuspended in saline at 1 x 1010 bacteria/m1. Rats were immunized via IP injections of 1 ml of a 1/100 dilution of vaccine on day one and then injected with 1 ml of vaccine 4, 8, 12, and 16 days later. Immune plasma was pooled and used~ in experiments. The tube agglutination titer of pooled immune plasma to §;_typhimurium was 6,400. Rat plasma treatments Zymosan Type A (Sigma Chemical Co., Cleveland, OH) was prepared by the method of Fine et al. (33). Zymosan was boiled in normal saline, centrifuged and washed and stored in saline at -20°C at a concentration of 2 mg/ml. Zymosan was added to plasma at 2 mg/ml and incubated for 1 h, 37°C and removed by centrifugation at 3000 x g for 15 minutes. EGTA (ethyleneglycol-bis-(8-aminoethyl ether) N, N'-tetraacetic acid) (Sigma Chemical Co., Cleveland, OH) and EDTA were used as chelating agents. EDTA and EGTA with MgCl2 (Mg EGTA) were added to rat plasma at a concentration of 0.01 M and pH was adjusted to 7.3 with NaOH. EDTA inhibits the classical and alternate pathways of complement (107) while EGTA inhibits only the classical pathway (24, 33, 107). Rat plasma was heated to 57°C for 1 h to destroy complement activity (104) or to 50°C for 30 minutes to inhibit alternate pathway activity (40). l7 The effect of these treatments on hemolytic complement activity are presented in Tables 2 and 6. In vitro liver perfusion Procedures for animal surgery and liver perfusion have been described in detail (61, 97). Briefly, in both rats and mice, the por- tal system was exposed by a midline abdominal incision. The portal vein was cannulated efferently and the cannula secured by ligatures. The thorax was exposed and the inferior vena cava was cannulated above the hepatic vein. The inferior vena cava was closed by a ligature placed above the right renal vein and livers were perfused with sterile M-l99 or HBSS to remove gross blood. After washing, 1 ml of S;_ typhimurium was slowly infused and followed immediately by perfusion media. The effluent was collected from the efferent cannula into a sterile graduated cylinder. Perfusions lasted 30 minutes and were done at room temperature. The liver was disconnected from the perfusion apparatus and homogenized in sterile distilled water and the collected effluent was also blended. Quantitative tryptose agar pour plates were made from the homo- genate to determine the number of trapped bacteria which remained via- ble in the liver and on the effluent to determine the number not trapped in the organ. The percentage of viable bacteria trapped in the liver plus the percentage of recovery in the effluent could then be subtracted from the percentage of bacteria infused (100%) to give the percentage of killing. l8 Distribution and survival of S. typhimurium in mice Mice were killed 15 or 60 minutes after intravenous injection of 9 1.0 x 10 §;_typhimurium. The liver, spleen and lungs were removed and homogenized with a Teflon and glass homogenizer in 9 ml of sterile dis- tilled water. The carcass, excluding the stomach, intestinal tract, skin, paws and tail was homogenized in 99 ml of distilled water in a Waring blender for four minutes. Quantitative pour plates of all homogenates were prepared. S. typhimurium infection in normal and C.¥parvum-treated mice Normal and §;_parvum-treated mice were infected by intravenous 5 7 injection of 4.1 x 10 to 4.1 x 10 .S; typhimurium. Survival was observed for two weeks. The LD50 for normal and §;_parvum—treated mice was determined by the method of Reed and Muench (89). Total (NBC) and differential white blood cell counts Leukocyte NBC from rats and mice were performed using Becton, Dickinson Unopettes for manual white blood cell enumeration. Cells were counted in an improved Neubauer chamber. Differential white cell counts were determined on air dried peripheral blood smears. Blood was obtained by cardiac puncture in rats or from the retroorbital plexus of mice. Slides were stained with Wright stain. Monocytes, polymor- phonuclear leukocytes and lymphocytes were expressed as a relative percentage of 100 total cells counted. Anti-C3-Sepharose 4B immunoabsorbent Goat anti-rat C3 sera was provided by Dr. Jeffrey Williams. Its specificity for rat C3 was verified by immunoelectrophoresis against l9 normal rat sera in which only a precipitin line to C3 was obtained. The anti-rat-C3 antibody fraction of the sera was isolated by precipi- tation with 50% (NH4)2504 two times. The precipitate was resuspended in 0.1 M PBS and dialyzed until no (NH4)2504 remained. The Sepharose 4B immunoabsorbent was prepared by the method of Cuatrecasas and Anfinsen (21) with modification. The antibody fraction from the anti-C3 sera was added to CNBr-activated Sepharose 48 at a concentration of 10 mg protein/ml of gel in coupling buffer (NaHCO3 buffer, 0.1 M, pH 8.3; containing 0.5 M NaCL) and mixed for 2 h at room temperature. The gel was washed with coupling buffer and excess active groups on the gel were blocked with 1 M ethanolamine (pH 9.0) and then the gel was washed with coupling buffer and acetate buffer (0.1 M, pH 4.0 with 0.5 M NaCl) three times. The prepared immunoabsor- bant was stored at 4°C in 0.1 M PBS with 0.01% sodium azide. A control Sepharose 4B immunoabsorbant was also made with anti-Salmonella H antigen antibody in the same manner. Normal and immune plasma was absorbed two times with the anti- C3-4B absorbant by adding 20 ml of plasma to 10 ml of centrifuged absorbant in a capped plastic centrifuge tube and mixed at room temper- ature for 1 h. The absorbed plasma was recovered by pelleting of the immunoabsorbant at 11,000 x g for ten minutes. The used anti-C3-4B was re-activated by washing with glycine-HCl (0.2 M, pH 2.8 and 0.2 M, pH 2.2) and used again. Assay for hemolytic complement Hemolytic C titers were measured in CH50 units following a modification of Kabat and Mayer (49) on untreated and treated rat 20 plasmas (Table 1). Sheep erythrocytes (Colorado Serum Co., Denver, CO) were sensitized with rabbit anti-SRBC hemolysin (Microbiological Assoc., Walkersville, MD). Sensitized calls (EA) in 0.5 m1 of Veronal- -4 -4 M Mg++’ buffered saline containing 1.5 x 10 M Ca++ and 5 x 10 pH 7.4 were added to 0.5 m1 of rat plasma dilutions. The tubes were incubated at 37°C for 1 h and then 2 m1 of buffer were added to each and centrifuged at 1000 x g for ten minutes and the absorbance read at 541 nm. TABLE 1. Bacterial trapping and killing by the perfused rat liver in the absence or presence of rat plasma.a Recovery (%) Experimental M-199 Plasma Liver 61.1 i.9 27.815b Effluent 40.4 :_8 36.1 :_10 Total 101.5 : 12 62.1 :14b Killing o 37.7b aAverage + standard deviations from at least six separate experimental determinations. bp wF _mELo: .m> _o.o n a u .mcowmseema Lm>WP _mELo: .m> _oo.o v a n .mcowumcwEmemv _mpcmswgmaxm mpmcmamm xwm pmmmp pm Eoee mcovumw>mc vemvcmpm H.m=_m> mmmxm>< m m.Ne o.N_ 2.25 m.me ee.m_ ee__e¥ w.fl N.Nm _P H.o.wm m.fl m.mm N H.N.Om em.fl.e.om Feeee m_H.N.mN m H.m.m m H.N.NN em.fl.m.m_ em.fl _.e_ eee=Peem eflmém :fl_de eflfiz “Hm;m N_fl@8 e9: mEmwFa .Q.U + mEmmPa mczEEH< + wEmmE .a.u + wEmmpa FMELOZ + mmpiz + ee>eu _eeeez ee>eu .e.o ee>_u .e.u ee>es .e.o ee>wu .e.u _eeeeeeeeexm 11. m.msmm_a mo mocmm .898 so mucwmnm wcp cw Lw>w_ we; _mseo: ucm vmpmmepiss>emm .0 >2 ace—_wx use mewaqmgu _mwgmpumm .FF MAmnon-specifically activate hepatic RES by C:_pg:ypm_vaccine. Immunized rats developed humoral titers of 6,500 to the bacteria, but livers from these animals behaved as normal in their ability to trap and kill bacteria in the absence or presence of plasma (Tables 1 and 8). This demonstrates that Kupffer cells cannot be activated by a heat-killed vaccine and strongly suggests that macro- phage cytophilic antibody does not play a role in the bactericidal and trapping activity of hepatic tissue. The hepatic RES was non-specifically activated with C:_pggypm_ vaccine, a macrophage stimulator (41, 98, 118) and the resultant effects on trapping and killing of §:_typhimurium ip_situ in mice and rats and ig_yiyg_in mice were studied. Data presented in previous papers (35, 61, 92) and in this thesis demonstrate that both the mouse and rat models yield similar data in correlative experiments. The initial C:_pg§ypm studies were in the absence of plasma. In normal perfused mouse livers, over 10 % of the bacteria were recovered (Table 9) while in C:_pp§ypm7treated livers only 77.4% were recovered indicating 22.6% of the bacteria were killed. The bactericidal acti- vity correlates with the decrease in percent viable bacteria recovered from the liver, i.e. 45.8% in C: pggvpmytreated livers versus 67.6% in normal. In both normal and §:_pa§ypmytreated livers the effluent contained the same percent bacteria (Table 9). These data represent the first successful demonstration of bactericidal activity in perfused liver in the absence of plasma and suggest that activated RES cells 64 populating §:_p3§ypmytreated mice are functionally different from nor- mal Kupffer cells. To be certain that cellular and not residual humoral factors were the active component in bactericidal activity, perfusion experiments using EDTA, silica and phenylbutazone were performed. For EDTA studies, Ca++ and Mg++ free HBSS containing 0.01 M EDTA was used as perfusion medium instead of M-l99. EDTA at this concentration inhibits both the classical and alternate complement pathways (107). The data show (Table 10) that EDTA did not alter the distribution or bactericidal activity of C:_pg§ypm7treated perfused livers indicating that the classical and/or alternate pathways were not participating in the bac- tericidal activity. Silica treatment blocked the ability of C:_pg§ypm_to stimulate bactericidal activity in livers due to destruction of RES cells. Sili- ca treatment also decreased the ability of the perfused livers to trap bacteria. These results are similar to those previously reported (35) and reaffirm that viable RES cells are required for Optimal bacterial trapping in the perfused liver even though they alone are not the only factor involved. Phenylbutazone (PB) is an anti-inflammatory drug that inhibits phagocytosis (55, 108, 110) and intracellular killing (77, 108, 110). Infusion of PB into C:_pp§ypm7treated livers both inhibited bacterici- dal activity (Table 10), and decreased bacterial trapping. Since both silica (a specific RES toxin) and PB inhibited killing to the same extent, it can be concluded that the bactericidal activity of C:_ parvum-treated livers resides in activated liver RES cells. 65 C:_pg§ypm¢treated rat livers were used to study the effect of plasma on bacterial trapping and killing in the activated organ. 9: pgrgpmytreated rat livers had similar bactericidal activity as C:_ pgrypmftreated mouse livers (Tables 11 and 9). §;_pgpypm_treatment also enhanced rat livers ability to trap bacteria as demonstrated by the decreased number of bacteria recovered in the effluent (Tables 11 and 1). This enhanced trapping ability was not observed in C;_p§rgp£y treated mouse livers (Table 9). C:_pg§ypmgtreated rat liver in the presence of plasma did not have greater bactericidal activity over normal liver, but significantly enhanced bacterial trapping did occur in the presence of normal plasma (Tables 11 and l). C;_pg:ypm_treatment had no effect on plasma factors involved in liver bacterial trapping and killing. This is shown by results of normal liver perfusion experiments using §;_pg:ypm7treated liver and plasma (Tables 11 and 1). These results suggest that bac- terial killing by liver Kupffer cells, whether they are normal or activated, in the presence of plasma can kill only a certain percentage of bacteria that are cleared by the organ in the time period investi- gated (30 minutes). SEM was used to morphologically study C:_pgrypm;treated livers to determine why these livers were able to kill bacteria in the absence of plasma. Observations made by SEM revealed that C: pgpvpm_treatment in both rats and mice caused an influx of white blood cells into the liver. White blood cells consisting mainly of macrophage and lympho- cytes were observed adhering to portal veins, portal venules and sinu- soids (Figures 2-4). This white cell influx into the liver occurred concomitant with a relative peripheral blood monocytosis (Table 12). 66 This study corroborates the observation by Warr and Sljivic (118) that §:_parvum treatment results in the accumulation of extra-hepatic mononuclear cells in the liver. Similar results have been obtained by North (75) and Volkman (115) using Listeria monocytpgenes which showed that cells emigrating into the liver were peripheral blood monocytes. This response is not surprising since the major portion of an intrave- nous C:_pggxpm_injection is cleared by the liver (26). The morphological features of T and B lymphocytes, blood mono- cytes and polymorphonuclear leukocytes (PMN) have been studied in detail by SEM (3, 5, 74, 85, 86, 120). While there is general agree- ment that macrophages and lymphocytes may be differentiated, Polliack et a1. (85, 86) showed that jp_yitrp_lymphocytes of known T and B cell identity display a considerable amount of surface variation. Barber and Burkholder (5) and Albrecht et al. (3) found that macrophage mor- phology is extremely variable and may reflect the functional state of these cells. By SEM alone this study did not reveal the histological type of white blood cell adhering to portal vasculature in anything other than general terms. It is unlikely that cells identified as macrophages adhering to portal veins represent Kupffer cells emigrating from liver sinusoids though their morphological features were strikingly similar to those of Kupffer cells and macr0phages of extra-hepatic origin were morphologi- cally very similar (Figure 4). A difference in attachment to the sinu- soidal wall in Figure 4B can be observed between the two macrophages. One macrophage has no observable filopodia attached to the sinusoidal endothelium while the other macr0phage is attached to the endothelium by numerous cyt0plasmic filopodia. This morphological difference may 67 suggest that the highly attached macrophage is a Kupffer cell while the other cell is a blood derived macrophage. No direct evidence sup- porting this contention is presently available. North (75) could only make this distinction by the use of autoradiographic studies of charac- teristic labeling patterns of Kupffer cells and blood monocytes. It was found that macrophages characteristically adhere in a ”head-tail" orientation in portal veins (Figure 3A and 3B). This head- tail orientation was not observed in smaller portal venules or in sinusoids. The dynamics of fluid movement in portal veins of larger diameter may influence such cell orientation. This orientation might also be due to macr0phage motility (5). A considerable amount of cell-cell contact or interaction between adhering white blood cells was observed in C:_pg§ypmytreated liver. These interactions occurred in both portal veins and sinusoids. Macro- phages were observed interacting with macrophages (Figure 5) and lym- phocytes (Figure 6) by direct cell surface contact and by the extension of cytoplasmic appendages between cells. Frost and Lance (36) showed that C: pgrgpm treatment results in the sequestering of lymphocytes in lympoid tissue, a process termed lymphocyte trapping. This study demonstrates that lymphocyte trapping also occurs in §;_p§§ypmytreated rat and mouse livers. Neilsen et al. (72) and Werdelin et al. (119) used SEM to study macrophage-lymphocyte cluster formation during the jg_yit§9_induction of the immune response to soluble protein antigens. Roelants (91) reviews the significance of such a model in some detail. It is unknown if similar macr0phage-1ymphocyte interactions observed in this study have functional significance pertinent to these models, 68 although, the immunopotentiating ability of C:_p3§1gm is a function of its direct stimulation of both lymphoid and RES cells (12, 18, 41, 98, 118). SEM of C:_pg§ypmgtreated mouse livers perfused with §:_typhimu- .pimm revealed numerous macrophage-bacterial interactions. The host cells were stellate in appearance with surface invaginations, ruffles and microvilli (Figures 7, 8 and 9). In C;_pg§ypmytreated livers it was difficult to differentiate morphologically between blood-derived macrophages and liver Kupffer cells. The macrophage in Figure 7 is attached to the sinusoidal endothe- lium by numerous cytoplasmic filopodia while the macrophages in Figures 8 and 9 have no observable filopodia attached to the endothelium. This difference in attachment to the sinusoidal wall may be significant, possibly suggesting that the macrophage in Figure 7 is a Kupffer cell while those in Figures 8 and 9 are blood-derived macrophages. The macrophages actively phagocytizing bacteria in Figures 8 and 9 show specific surface areas demarcated into regions involved or not involved in phagocytosis. Regions of the macrophage directly involved in bacterial phagocytosis are highly blebbed and invaginated (Figures 8B and 9B). In these areas no surface microvilli are present. Parakkal et al. reported similar loss in surface microvilli on macro- phages during phagocytosis jp_yjt:p_(80). Regions of the macrophage not directly involved in bacterial phagocytosis retain the surface microvilli (Figures 88 and 9B). While SEM fail to distinguish Kupffer cells from extra-hepatic macrophages in C:_pgrypm7treated liver it demonstrates the accumulation of white blood cells in the liver. This study also suggests that 69 several immunologically significant phenomena such as macrophage- macrophage and macrophage-lymphocyte interactions occur in §:_pg§ypm7 treated liver. The anatomical alterations in liver most likely are an integral part of the increased microbial resistance observed in the C:_ pggypmftreated host (1, 6, 19, 52, 76, 103, 112) and is the reason for the perfused liver's ability to kill bacteria in the absence of plasma. The effect of C:_pg:ypm on the ability of mice to clear and kill microbes jp_vivo were investigated to determine if the perfused liver was a true indicator of RES activity in the whole animal. Recovery of §:_typhimurium 15 and 60 minutes after intravenous injection varied significantly between normal and §;_pg:ypm¢treated mice. In normal mice approximately 42% of the bacteria were killed after 15 minutes. No additional killing occurred after 60 minutes (Table 13). There was a slight rise in the percent recovery in liver after 60 minutes, possi- bly due to some bacterial multiplication. In C: pggypmytreated mice 74.2% of the bacteria were killed by 15 minutes and 93.5% by 60 minutes. The majority of viable bacteria were recovered in the liver and carcass in normal mice while the majority were recovered in the liver and spleen in C:_pg:ypmytreated mice. The increased splenic trapping in C:_p§:ypm7treated mice may be due to its increased size. Cumulatively these data demonstrate that activation of the RES by C:_pggypm_signifi- cantly increases the ability of liver and Spleen to clear and kill S; typhimurium ip_vivo and that the perfused liver is an accurate indica- tor of RES function as previously shown (35). §:_parvum treatment 1ncreased the LD50 of§L typh1mur1um 1n m1ce 5 6 ( from 6.4 x 10 to 4.1 x 10 Figure 10). 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