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Inn..- .‘ 1’: r.» . \mefciéfiw'l ‘I A - a 11' :»)‘N.w> 5719?;- ““535 K .". auggrm <2a1ized injection of C. parvum results in a regional lfifflflph node response, primarily a proliferative response of small lymphocytes (O'Neill et al., 1973) . Ogmundsdottir and Weir (1976) showed that in vitro C. parvum adheres to 'tllea surface of PEC. A HCl-lipid extract, when preincubated vvjxth PEC, inhibits the binding of whole C. parvum to PEC. Trypsinization of PEC has no effect on adherence obviating a possible role for macrophage associated antibody in k>iaiding C. parvum. Addition of the major sugars found in C. parvum cell walls to the incubation medium and removal (bf? divalent ions from the medium inhibited binding. Christie and Bomford studied the in vitro (Christie ade Bomford, 1975) and in vivo (Bomford and Christie, 1975) “mechanism of macrophage activation by C. parvum. Their Studies show that in vitro and in vivo C. parvum stimulates “macrophage both directly and through immunological nRadiation by T lymphocytes. Studies by Tuttle and North (1976a, 1976b) demonstrated that C. parvum treatment ‘generates a subpopulation of tumor specific, short lived, replicating T lymphocytes which passively transfer Specific resistance to syngeneic fibrosarcoma in mice. These studies suggest that in vivo C. parvum derives immunopotentiating abilities from its interaction with lymphocytes and cells of the MPS. Subsequent interactions 44 of these activated or sensitized lymphocytes and macro— phages result in the variety of in vitro and in vivo biological activities seen in C. parvum stimulated experi- mental animal models (Howard et al., 1973) . This is only a working hypothesis based on a few experimental models. The sequence of events in vivo following C. parvum treatment which results in the expression of acquired CMI to infection is essentially unknown. In addition to a search for potentiators of both Specific and nonspecific antimicrobial host defense systems a considerable amount of study has been devoted to the evaluation of inhibitors of these systems. Three such inhibitors employed in this study are crystalline silica, iodoacetic acid (IAA) , and phenylbutazone (PB). Crystalline silica is a specific cytotoxin for cells Of the MPS (Allison, 1970, 1971; Allison et al., 1966; Comvili and Perin, 1963; Friedman and Moon, 1977; Kessel et al., 1963; Marks and nagelschmidt, 1959; Van Loveren et al., 1977) . Kassel et a1. (1963) clearly demonstrated that PMN are not susceptible to the cytotoxic action of Silica. Silica enhances susceptibility of experimental ‘hosts to Friend virus (Larson et al., 1972), Coxsackie B-3 virus (Roger-Zisman and Allison, 1973), Herpes virus (Zisman et al., 1970) , Mycobacterium tuberculosis (Vorwald and Delahunt, 1938; Vorwald et al., 1954) both in vivo and in vitro (Allison and Hart, 1968), Trypanosoma cruzi 45 (Kierszenbaum et al., 1974), and S. typhimurium (Friedman and Moon, 1977). Nadler and Goldfischer (1970) demonstrated that ingestion of silica by macrophages results in release of lysosomal enzymes into the macrophages' cytoplasm. The macrophage essentially commits suicide. Rios and Simons (1972) observed that poly—2-vinylpyridine n-oxide (PVO) reverses the ability of silica to induce macrOphage intracellular degranulation by combining with the lysosome membrane and stabilizing it. Allison (1967) reviewed the mode of toxic action of silica on macrophage lysosomes. It is thought that silica is converted rapidly into silicic acid. The hydroxyl groups of silicic acid form hydrogen bonds with lysosome cell membrane phospholipids and proteins disrupting the integrity of the membrane. This would explain the stabilizing effect of PVO, its' oxygen atoms form hydrogen bonds with the hydroxyl groups of silicic acid to block hydrogen bonding between silicic acid and lysosome membrane components. Levy and Wheelock (1975) observed that intravenous silica injection rapidly depresses the ability of the MP8 to clear colloidal carbon from the bloodstream of mice. It reduces the in vitro phagocytic ability of PEC harvested three days after silica injection. Friedman and Moon (1977) found a slight depression in the carbon clearance ability of silica treated mice. In this study profound 9'" 46 effects were observed in the ability of silica compromised mice to clear intravenously injected S. typhimurium and to effectively kill the bacteria in vivo and in vitro. In vitro evaluation of the perfused mouse liver showed that silica treatment kills Kupffer cells without altering other anatomical features of the liver. The ability of the liver to trap perfused bacteria is reduced in these livers as is the rate of bacterial killing. Iodoacetic acid (IAA), C2H3IOZ, prevents glycolysistnr inhibiting phosphoglyceraldehyde dehydrogenase. It alters function of this enzyme by alkylation of cysteine 149 at the enzymes' active site. IAA also splits ribonuclease into two inactive fragments at pH = 5.5 (White et al., 1973). Assidao et al. (1964) found that 0.1 M IAA inhibited carbon clearance from perfusion medium in per— fused rat liver. IAA was found to block oxidative Kupffer cell metabolism. Pisano et a1. (1968) found that 3 mM IAA blocks phagocytosis of RE test lipid emulsion by Kupffer cells and 0.1 M IAA inhibited Kupffer cell hexose monophosphate shunt utilization of glucose (Pisano et al., 1970). Leijh et a1. (1977) found that 1 mM IAA abrogated the ability of both PMN and BM to ingest C. albicans. The use of IAA to inhibit phagocytosis has been reviewed by Karnovsky et a1. (1970). Taken together these studies indicate that IAA has an inhibitory effect on macrophages in vitro and in vivo. 47 Phenylbutazone (PB), C O 4-Butyl—1,2—diphenyl— 19H20N2 2' 3,5-pyrazolidinedione, was found by Whitehouse (1964) to be an uncoupler of oxidative phosphorylation selectively inhibiting the biogenesis of ATP without blocking oxidative metabolism and cellular respiration. This metabolic inhibitor is used to effectively block the ability of phagocytic cells to kill intracellularly phagocytized microorganisms. Weissman (1966) found that high concentrations of PB prevented lysosomal enzymes from being released in vivo but it had no effect on isolated lysosomes in vitro. This observation appears to be incidental to the more profound effect of PB as a metabolic inhibitor in phagocytic cells. Steigbigel et a1. (1974) found that inclusion of 2 mg/ml PB in the incubation medium of either PMN or BM completely inhibited the ability of these cells to kill phagocytized E. coli, L. monocytogenes, S. typhimurium, and S. aureus. Solberg and his colleagues have studied the inhibitory effects of PB on PMN function. Killing of phagocytized S. aureus was inhibited by 2 mg/ml PE in vitro (Solberg, 1972). It was found that high concentra— tions of PB caused reduced phagocytic activity by PMN in addition to blocking intracellular killing of S. aureus (Solberg, 1974). At lower doses PB inhibited PMN CO2 production by blocking the utilization of glucose in the hexose monophosphate shunt pathway inhibiting intracellular 48 killing. Strauss et a1. (1968) studied the effects of varying concentrations of PB on the ability of guinea pig PMN to phagocytize and kill E. coli. Bactericidal PMN homogenates are also inhibited by PB in vitro. Metabolic evaluation of the effects of PB and PMN showed that the drug inhibits l4C—glucose and 14C-formate oxidation indicating an inhibitory effect on the hexose monophosphate shunt preventing H202 generation. This study also showed that PB inhibits glucose 6—phosphate dehydrogenase and 6—phosphogluconate dehydrogenase activity. Leijh et al. (1977) found that 1 mM PB inhibited the intracellular killing of C. albicans by PMN and BM. This study implied that 1 mM PB inhibits only intracellular killing and not phagocytosis. This observation is substantiated by the work of Kjosen et al. (1976). They found that low concentrations of PB inhibit intracellular killing of S. aureus by human PMN by selectively blocking the hexose monophosphate shunt and not the Embden Myerhoff glycolytic pathway. Lehrer (1975) also found that PB inhibited the ability of BM to kill ingested C. albicans. Taken together these studies show that silica, IAA, and PB, inhibitors of phagocytic function in vivo and in vitro, may be used as tools to manipulate the macrophage-microbe interaction. MATERIALS AND METHODS Animals Sprague—Dawley—derived male rats weighing 300 to 400 g were purchased from Spartan Research Animals, Inc., Haslett, MI. Animals were maintained under standard laboratory conditions with Purina Laboratory Chow and water available ad libitum. Microorganisms The strain of Candida albicans used in this study was isolated from a case of candidal vaginitis at the Olin Health Center of Michigan State University. Identity was confirmed by fermentation of glucose and maltose but not sucrose or lactose, chlamydospore production on 1% Tween 80 corn meal agar, and germ tube formation in 2 h at 37 C. Stock cultures were maintained on Sabouraud dextrose agar (SDA) slants at room temperature. The inoculum was prepared from a fresh transfer incubated under continuous agitation at 37 C overnight in 100 ml of Trypticase soy broth (Difco), pH = 7.4, supplemented with 4% d-glucose (Baine et al., 1974). Cells were harvested and washed three times in sterile saline. Standardization of the 49 50 inoculum was by hemocytometer counts and pour plates of lO-fold dilutions in Sabouraud dextrose agar. Chemicals Sodium barbital injection, 65 mg/ml (W. A. Butler, Co., Columbus, Ohio) was stored by refrigeration and rats were anesthetized by intraperitoneal injection of 9.5 mg/ml of barbital in sterile saline. Heparin, sodium salt (Sigma Chemical Co., St. Louis, MO), Grade II was filter sterilized in sterile saline using Falcon 0.22 micron filters, No. 7103 (Falcon Div. of Becton Dickinson & Co., Oxnard, CA), to a concentration of 20,000 USP JA units/ml and stored as stock at 4 C. A 1:10 dilution of stock was made in sterile saline and rats were given 2,000 units of heparin intravenously (i.v.) in the dorsal vein of the penis. Phenylbutazone (Lot No. 127C—0083) and iodoacetic acid (as free acid, Lot No. 37C—0375) were both obtained from Sigma Chemical Co., Columbus, Ohio. Both drugs were used in 1 mM concentrations prepared in fresh Medium 199 (M 199, Gibco). Upon suspension of each drug the pH of the solution was adjusted to 7.3 with l N NaOH and the solution resterilized by filtration in Falcon 0.22 micron filters. Corynebacterium parvum vaccine was obtained as a gift from Dr. Richard L. Tuttle, Burroughs Wellcome Co., 51 Research Triangle Park, N.C. Two lots of vaccine were used throughout this study. Lots CA 528A and CA 580A were formalin killed suspensions supplied at a concentration of 7 mg dry weight of bacteria/m1 with 0.01% thiomersal. Control studies using stock vaccine indicated that 350 ug of C. parvum killed 106 C. albicans within 30 min at 37 C. This was due to the preservative. Despite the fact that various blood fractions from C. parvum—treated rats were not toxic for C. albicans, all experiments were conducted with C. parvum vaccine washed free of toxic perservative. No variation in response between lots of vaccine was observed. Dorentrup silica (DO 12), particle size approximately 5 microns, was kindly supplied by Dr. Robert J. Moon, Department of Microbiology and Public Health, Michigan State University. All suspensions were autoclaved in powder form and suspended in sterile saline at a concentration of 100 mg/ml. Prior to i.v. injection, DO 12 silica was exposed to ultrasonic vibration by a Sonifier Cell Desruptor Model W 1400 (Heat Systems~ Ultrasonics Inc., Plainview L.I., N.Y.) at 75 watts for 1 min to suspend the silica. Two 5 mg injections were given i.b. 48 h apart. The last silica injection was given 24 h prior to experimentation. 52 Corynebacterium parvum treatment Rats were given either 350 ug or 1.4 mg washed C. parvum i.v. Each dose was administered under light barbital anesthesia either two days or ten days prior to experimentation. Complete blood counts (CBC) and differential white blood counts Leukocyte CBC were performed using Becton Dickinson Unopettes for manual white blood cell enumeration. Cells were counted in an improbed Neubauer chamber. Differential white blood cell counts were determined at the appropriate times from air dried, peripheral blood smears, obtained by cardiac puncture, stained with Wright stain. Monocytes, polymorphonuclear leukocytes (PMN), and lymphocytes were expressed as a relative percentage of the 100 total cells counted. In vivo clearance and tissue distribution of C. albicans in rats To measure clearance of C. albicans from the blood- stream of normal rats approximately 106 or 108 yeast cells were injected i.v. into the dorsal vein of the penis of barbital anesthetized, heparinized rats. A needle was inserted into the heart, and 1 ml blood samples were withdrawn at 0, 2, 5, 10, 15, 20, and 30 min. Quantita— tive plate counts determined viability. The distribution 53 of yeast cells among liver, lungs, spleen, kidnesy, and peripheral blood was determined 30 min and 60 min after injection in normal and C. parvum—treated rats. Viability was determined by standard tissue homogenization and quantative plate count methods (Moon et al., 1974). Rat surgery and in vitro liver perfusion Rats were anesthetized with barbital, heparinized and bled by cardiac puncture. When appropriate, plasma was immediately separated from peripheral blood cells by a single centrifugation at 2,000 X g for 10 min in a Phillips Drucker L—780 combination centrifuge. In one set of experiments blood was collected in Becton Dickinson Vacutainers containing 10.5 mg disodium Edetate (EDTA, ethylene diamine tetraacetic acid). For all other experiments blood was separated in Becton Dickinson red top Vacutainers. Procedures for liver perfusion have been described in detail by Moon et a1. (1975) and Exton (1975). A full length midline incision was made, the abdominal walls retracted and the viscera displaced to one side exposing the portal vein. A ligature was placed under the inferior vena cava above the right renal vein. Two ligatures were placed under the portal vein, one above the splenic vein and one below it. The portal vein was held taut with a forceps and a small cut made in the wall of the vein below 54 the two ligatures. A sterile polyethylene cannula (Becton Dickinson Co., Rutherford, NJ, ID, 0.046 inch; OD, 0.066 inch or Becton Dickinson Intramedic Tubing, ID, 0.047 inch; OD, 0.067 inch) filled with slowly flowing Gibco M 199 was inserted into the lumen of the portal vein and tied into place by the two ligatures. The cannula led to a three way valve (Becton Dickinson Co., Model No. MS 3033) one port of which contained a 1 ml syringe containing sterile M 199 and the second port of which was attached to two 50 ml glass syringes filled with prewarmed sterile M 199. Since the valve was open to prevent introduction of air bubbles into the portal vein, the inferior vena cava was cut below the renal vein allowing a slow flow of M 199 through the liver and preventing swelling. The tip of the cannula did not extend into the portal hepatis. The thorax was reflected and a ligature placed under the inferior vena cava distal to the hepatic vein. The right atrium was held taut with a forceps, cut, and the efferent polyethylene cannula (ID, 0.046 inch; OD, 0.066 inch) was inserted, pushed into the inferior vena cava, and secured with the ligature. The ligature on the inferior vena cava above the right renal vein was tied and the perfusion medium collected from the efferent cannula. In most cases the time elapsed from the initial incision to tying the last ligature on the inferior vena cava was between 5 and 10 min. Theliver 55 was kept moist by bathing it with warm sterile M 199 and covering it with a sterile plastic petri dish. The liver was washed free of blood cells with a sterile M 199 (ca. 50 ml). The flow rate was carefully adjusted to establish a constant flow through the liverand did not exceed suggested rates of flow for isolated per— fused livers (Miller, 1973). Prior to perfusion with yeasts the effluent was treated for sterility and any liver having more than ten colony forming units (CFU) per m1 of effluent was excluded from the experiment. At the end of each experiment sterility of the effluent was checked in a similar manner. After washing the liver, the 1 m1 syringe containing .M 199 on the three way valve was exchanged for a 1 m1 syringe containing 1 m1 of sterile saline and either 106 or 108 CFU of C. albicans. A sterile 125 m1 Erlenmyer flask with a foil cover was placed at the efferent cannula which was inserted through the foil. The yeast cells were slowly and steadily infused through the three way valve and followed immediately by perfusion of 100 ml of M 199 collected in 30 min. In some experiments perfusion medium was supplemented with either 10% whole rat blood or 5% rat plasma. For 60 min and 3 h perfusions the Erlenmyer flasks were changed at 30 min intervals and 100 ml of perfusion medium was collected in 30 min. The previous 100 ml was held on ice until ready for dilution plating. 56 For quantitative plate counts, the liver was dis~ connected from the perfusion apparatus, excised,and placed in a 100 m1 graduated cylinder. The volume was adjusted to 100 ml with sterile saline, and the livers were homogenized in a Waring blender for 2 min. Quantitative Sabouraud dextrose agar pour plates were made using 10 fold serial dilutions of the control, C. albicans in saline, liver homogenate, and effluent. Plates were incubated 48 h at 37 C and the number of CFU was counted manually. The percentage of untrapped yeasts was calculated by the formula: N = number of CFU recovered in effluent number of CFU infused X 100 VThe difference between the percentage of CFU recovered in ‘the effluent and the total infused (100%) suggests the 19ercentage remaining in the liver. The percentage of ‘viable yeasts remaining in the liver plus the percentage .recovered in the effluent when subtracted from the jpercentage infused (100%) indicated the percentage killed. Control experiments characterizing the isolated jperfused rat liver using 106 and 108 CFU indicated that liver flow rates remained normal after the yeasts were infused. After perfusion for either 30 min, 60 min, or 3 h with sterile M 199, an additional 1 ml portion was 57 collected and tested for sterility. This sample consis— tently yielded less than 10 CFU indicating that the yeast cells trapped in the liver could not be removed by continuous perfusion. Macrophage inhibition studies Both phenylbutazone and iodoacetic acid were infused directly into isolated perfused livers of rats receiving 350 ug C. parvum two days prior to use. Both drugs were used in 1 mM concentrations prepared in sterile M 199. These concentrations are non-toxic to C. albicans (Leijh et al., 1977). Once infused with either drug, livers were washed for 15 min with sterile M 199 prior to infusion of C. albicans. Rats were given 10 mg DO 12 silica i.v. as described above. Rats were injected i.b. with 350 ug C. parvum 24 h after the first 5 mg silica injection and two days prior to experimentation. Results from the silica injected C. parvum-treated rats were compared to control rats receiving only C. parvum. Scanning electron microscopy (SEM) Isolated perfused livers from normal and C. parvum— ‘treated rats, with or without infused C. albicans were Iprepared for SEM by the methods of O'Donnell and Hooper (1977), with the following modifications. After perfusion tuith 108 yeast cells, the upper reservoir of the perfusion 58 apparatus was filled with freshly prepared warm 2% glutaraldehyde (Eastman Kodak, Rochester, NY) in sterile 0.2 M sodium phosphate buffer (2% GA—PB) at pH = 7.4, and 100 ml was perfused in 30 min. Fixed livers were excised, cut into small blocks, and allowed to stand in 100 ml of fresh cold 2% GA—PB overnight. Blocks were dehydrated in sequential 30 min steps with 10, 20, 40, 70, 90, 95, and 100% ethanol. The tissue was held overnight at 4 C in a fresh change of 100% ethanol. The dehydrated blocks were cyrofractured in liquid nitrogen. Fractured tissue was placed in metal baskets under liquid nitrogen and dried in an Omar SPC 900/EX critical point dryer using CO2 as the carrier gas. The specimens were coated with gold (200—300 A) using the EMS—41 Minicoater (Film Vac. Inc., Englewood, NJ) and viewed in either an AMR—900 SEM or an ISI Super Mini II SEM. Statistics Statistical analysis was performed by the White Rank order test (Wilcoxon and Wilcox, 1949) and all data was reported with its standard deviation. RESULTS (Slearance and tissue distribution of intravenously injected C7. albicans in normal rats In vivo clearance of 106 and 108 CFU of C. albicans :Erom the bloodstream of normal rats is shown in Figure l. 13y 5 min over 90% of the intravenously injected yeasts Vvere cleared. Between 5 and 30 min the percentage of \riable yeasts remaining in circulation fell below 1%. One Ilundred percent of the injected cells was recovered from aceticuloendothelial (RES) organs as viable yeasts, 30 min aafter injection, at both dose levels. Over 90% were accounted for in the liver and lungs (Table 1) . Less tllan 10% of the yeasts were in the kidneys. Sixty minutes after intravenous injection of 106 CFU irlto normal rats only 56% of the total number of yeast <:e:11s were recovered from the same RES organs, 44% being 1 Hzmomma HJNHNUTES TUNE 62 Table 1. Survival of C. albicans 30 min and 60 min after intravenous injection into ratsa. EXPERIMENTAL ORGAN 30 min after 106 30 min after 108 60 min after 106 CFU injected i.v. CFU injected i.v. CFU injected i.v. Liver 63 3: 10b 79 i 4 23 _+_ 4 Lungs 27 i 5 19 i 2 27 i 7 Spleen 4 i l 4 i <1 2 i <1 Kidneys 10 i 2 2 i <1 3 i <1 Peripheral Blood 1 i <1 1 i <1 1 i <1 TOTAL 105 i 11 104 i 5 56 i 2 KILLING o 0 44 i 2 a I I Each value represents at least 31x separate experimental determinations. bPercentage : standard deviation. 63 same dose recovered 60 min after injection (P = 0.001). Hepatic clearance of C. albicans by perfused livers from normal rats The perfused rat liver cleared over 85% of 106 or 10 yeasts on a single pass (Table 2). In all but one instance (line 2, Table 2) all yeasts remained viable. The 15% killing found in this experimental group was not statistically significant. A similar experiment in the presence of blood did not significantly change trapping or killing (Table 3), even in perfusions lasting as long as three hours (Table 4), the total recovery of C. albicans being essentially 100%. There was no decrease in yeast cell viability when 106 CFU of C. albicans were incubated at 37 C, under gental agitation for 60 min, in the presence of normal rat plasma or normal rat blood (data not shown) . §E§4 of normal rat liver and in vitro clearance of £;_£1lbicans from the perfusion medium Figure 2 shows micrographs of normal rat liver Siruasoidal tissue. Figure 2A is a low magnification of crYofractured liver showing a portal vein with bifurcation. Small portal venules branch away from the portal vein toWard central veins. Material in the portal vein is ar"tifactual. Radiating from the portal venules are Silnasoids which extend into a central vein. Higher 64 Table 2. Trapping of a single pass of viable C. albicans by the perfused rat liver after 30 min in the absence of blooda. % Rmxnmmy No.of yeasts % perfused . Killing Effluent Liver Total Homogenate Recovery 1.38 x lo6 18 2‘. 12b 84 i 13 102 i 13 o 2.39x1o6 15:3 70:1 85:2 15 1.49 x 108 14 i 2 87 i 13 101 i 14 o a . . Each value represents at least six separate exper1mental determin— ations . Percentage : standard deviation. 65 Tablee3. Trapping and killing of 106 C. albicans by perfused rat livers after 30 min in the presence of whole blooda. % RECOVERY Experimental Kilslkin Liver Total 9 Effluent Homogenate Recovery No additives Dkadium b 199 only 18 4_- 2 84 i 13 102 i 13 0 Medium 199 + 10% whole rat blood 15 i 7 104 _-+_-_ 17 119 i 17 O a . Each value represents at least six separate experimental determinations . b Percentage : standard deviation. 66 Tablja 4. Trapping and killing of 106 C. albicans by perfused rat liver after 30 min, 60 min or 3 h continuous perfusion in the absence and presence of whole blood plasmaé % RECOVERY % Experimental . . Liver Total Kllllng Effluent Homogenate Recovery 30 MIN PERFUSION M1990n1y 4:2b 90:7 94:8 6:5C M 199 + 5% plasma 6 : 4 96 : 3 102 : 7 O 60 MIN PERFUSION Ml99only 4:1 91:3 95:4 5_2 M 199 + 5% plasma 9 : 6 90 : 10 99 : 5 0 3 H PERFUSION M 199 only 7 : 3d 103 : 10 110 : 14 0 M 199 + 10% whole rat 20 : 7 85 : 20 105 : 17 0 blood a Each value represents at least five separate experimental determinations . Percentage : standard deviation. c P = 0.50 Average of four 100 m1 fractions collected over 3 h, the last three fractions contained less than 10 CFU/fraction. Figure 2. 67 SEMS of normal rat liver A. Cryofractured rat liver with branching portal vein (PV), portal venules (Pv), sinusoids (S), and central vein (CV). Material in the portal vein is arti— fractual (arrow). X 90. Central vein (CV) with sinusoids (8). Areas indicated by arrows are magnified in C. and D. X 210. Sinusoidal area (S) with fenestrated endothelial lining (E), parenchymal cells (PC), and Kupffer cells (KC). X 2,100. Sinusoidal area (S) with Kupffer cell (KC) and attached erythrocyte (arrow). X 2,100. \ o 3 OD. , \ -/.' . . Vi 69 magnification of this area is shown in Figure 28. Figures 2C and 2D are higher magnifications of the sinusoidalareas indicated by arrows in Figure 2B. Figure 2C shows the liver sinusoid, the lumen of which is lined by a double layer of fenestrated endothelium. Beneath the endothelial net, parenchymal cell microvilli project into the Space of Disse (not shown). Two Kupffer cells are attached to the endothelial cell cytoplasmic net by fine cytoplasmic dendritic processes. The surface of Kupferr cells had numerous folds and small villous projections. Figure 2D shows a Kupffer cell with an attached erythrocyte. Figure 3 shows low magnification micrographs (380- 760 X) of yeasts trapped in the liver following perfusion. Figure 3A is a micrograph of sinusoids packed with numerous yeast cells. Figure BB shows a venule of the portal system also filled with yeasts. Figure 3C is a micrograph of a branch of the portal vein filled with yeasts. Figures 4A and 4B are higher magnifications of the sinusoidal areas containing C. albicans. Both figures show that yeast cells fill a considerable portion of the sinusoidal lumen. These figures also show that trapped yeasts were most often not associated with Kupffer cells. Frequently pseudohyphae were observed. In Figure 5, venous sinusoids in the lower right of the micrograph contain C. albicans. The branches of the portal vein are also filled with yeasts. The inset in Figure 5 is a IPigure 3. 70 Low-magnification SEMS of C. albicans trapped by the perfused rat liver. A. Liver sinusoids (S) with numerous trapped C. albicans (arrows). X 380. B. Portal venule (PV) and sinusoids (8) containing trapped C. albicans. )(760. C. Arteriole (A) and branch of the portal vein (PV) containing trapped C. albicans. X 760. 71 Figure 4. 72 High-magnification SEMs of C. albicans trapped in liver sinusoids. A. C. aZbficans trapped inside liver sinusoids (S) partially filling the lumen of the sinusoid. X 2,150. B. Kupffer cell (KC) and C. albicans trapped inside a liver sinusoid. x 2,200. 73 Figure 5. 74 SEM of trapped C. albicans showing pseudohyphae (arrow), liver sinusoids (S), and branching portal vein (PV) with trapped C. albicans. X 380. (Inset) Higher magnification of C. albicans with pseudohyphae (area indicated by arrow). X 3,800. #1.. _L‘ ' u. “no. ‘ - 1 .I 76 higher magnification of the area indicated by the arrow showing pseudohyphae. The pseudohyphae observed in these sections do not represent new elements formed during the short perfusion time since they were also observed in the inoculum. Clearance and tissue distribution of intravenouslyinjected C. albicans in C. parvum-treated rats. Thirty minutes after intravenous inje-tion of 106 CFU of C. albicans into C. parvum—treated rats approximately 1% of the total inoculum was recovered from the peripheral blood (Table 5). Sixty minutes after injection, peripheral blood contained less than 1% of the total number of injected CFU. A total of 65% of the inoculum was recovered from various RES organs, with 26% in the liver 30 min after injection. The total recovery 60 min after injection was 56% with 27% in the liver. Sixty minutes after injection approximately 44% of the yeast cells were killed by C. parvum-treated rats. Hepatic clearance of C. albicans by perfused rat livers from C. parvum—treated rats. When C. albicans was infused into C. parvum—treated rat livers 95% of the yeasts were recovered after 30 min in the absence of plasma. Extending the perfusion time in C. parvum-treated rat livers to 60 min decreased the percentage of recovery to 78%, with 5% in the effluent 77 'Table 5. Survival of 106 C. albécans 30 min and 60 min after injection into C. parvum-treated rats. EXPERIMENTAL., ORGAN Distribution 30 min Distribution 60 min after l.v. injection after i.v. injection Liver 26 : 8b 27 : 2 Lungs 32 : 8 23 : 5 Spleen 2 : 0 3 : l Kidneys‘ 4 : l 3 : 1 Peripheral l 0 Blood TTOTAL“ 65 : 8 56 : 4 KILLING 35 : 6 44 : 4 aEach value represents at least five separate experimental determinations. bPercentage : standard deviation. 78 and 73% in the liver. Addition of 5% plasma to the perfusion medium decreased the total percent of recovery to 66% with approximately 1% in the effluent and 64% in the liver (Table 6). There was no decrease in yeast cell 6 CFU of C. albicans was incubated at viability when 10 37 C, under gentle agitation for 60 min, in the presence of plasma or blood from C. parvum—treated rats (data not shown). C. albicans by perfused livers from C.;parvum-treated‘rats. When rats receiving 350 ug C. parvum were perfused ten days after injection there was no increase in the killing of infused C. albicans in the absence or presence of plasma (Table 7). Treating rats with 1.5 mg C. parvum either two days or ten days prior to perfusion experiments in the absence or presence of plasma, did not significantly increase the ability of hepatic tissue to trap and kill 0. albficans (Table 7). ‘White blood cell kinetics in normal! C. parvum and silica injected C. parvum—treated rats. The CBC for normal rats was approximately 10,092 cells/mm3, 5% of which were monocytes, 14% PMN, and 81% lymphocytes (Table 8). Forty eight hours after injection of 350 pg C. parvum the CBC increased to ca. 16,819 cells/ mm3 with the monocyte count elevated to 19% of the 79 Trapping and killing of 106 {Fable 6. C. albicans by perfused rat livers from C. parvum-treated rats in the absence and presence of rat plasmaa. % Rmxwmmy % Experimental Killing Effluent Liver Total Homogenate Recovery 30 MIN PERFUSIONb M 199 only 3 : 1C 92 : 95 : 5 : 2 6 0 MIN PERFUSION M 199 only 5 : 2 73 : 78 : 22 : 5d M 199 + 5% 1 + <1 64 + 66 + 34 + 5 plasma — — — — a . . Each value represents at least five separate experimental determinations. 1DInjection of 350 119 C. parvum i.v. two days prior to experimentation. c: . . Percentage :_standard dev1ation. d]? = 0.001 .r_—__ ,. 1 4 80 (Fable 7. Trapping and killing of 106 C. albicans by perfused rat livers from C. parvum—treated rats in the absence and presence of rat plasmaa. Experimental % RECOVERY % (C. parvum '. Killing dose/time of Effluent Liver Total exposure) Homogenate Recovery 350 ug/ten days M 199 only 4 : lb 87 : 5 91 : 5 9 : 5 M 199 + 5% plasma 1 : 0 85 : 2 86 : 2 l4 : l .1.4 mg/two days M 199 only 2 : l 84 : 6 86 : 6 14 + 6 M 199 + 5% 4 + 1 68 + 5 73 + 6 27 + 6 plasma — — — — 114 mg/ten days M 199 only 5 : 2 82 : 5 87 :’4 l3 : 3 M 199 + 5% 2 + 1 81 : 5 83 : 6 17 + 6 plasma — k aEach value represents at least five separate experimental determinations . Percentage : standard deviation. P" A.“_' ““1”“ ‘1 l. j 4 . 81 (iifferential count. Ten days after injection, the CBC and.percentage of monocytes remained elevated. The CBC of rats treated with 1.4 mg C. parvum decreas- ed to ca. 6,000 cells/mm3 two days after treatment but was statistically elevated by ten days after treatment. At 'both times the monocytes were elevated relative to the CBC. In both normal and C. parvum-treated rats the relative percentage of lymphocytes remained unchanged. The relative percentage of PMN decreased slightly in rats 'treated with 350 ug C. parvum. In C. parvum—treated rats given silica the CBC was <:a. 8,688 cells/mm3 and the relative differential cell (:ount was unchanged compared to normal rats (Table 8). ggnhibition of phagocytosis and phagocytic killing of _92 albicans by perfused livers from C. parvum—treated rats. In the presence of 5% plasma perfused livers from C. parvum-treated rats killed approximately 40% of 106 infused C. albicans after 60 min (Table 9). Corynebacteri— um parvum-treated rat livers exposed to either 1 mM phenylbutazone (PB) or 1 mM iodoacetic acid (IAA) were unable to kill hepatically trapped C. albicans in the presence of 5% plasma (Table 9). Trapping ability was :not effected by treatment of livers with either drug since 'the percent recovery in the effluent was not significantly different from controls. Collection of whole rat blood 82 Table 8. White blood cell kinetics in normal, C. parvum and C. parvum-treated rats injected with silicaa. Experimental (C.gxumum REUHEVESE dose/time of 3 exposure) CBC cells/mm Monocytes ‘ PMN Lymphocytes Normal rats 10,092 : 320b 5 : 1 14 : 2 81 _+_ 2 350 ug/two days 16,819 : 6945 19 +_3 6 : 3 75 : 3 350 ug/ten days 11,913 : 1593C 18 _+_ : 75 : 11,013 : 3371 19 : : 76 : 1.4 mg/two days 6,644 : 276 15 : : 76 : 5,088 : 3028 14 : : 79 : 6 7,783 : 3322 9 : 11 : 80 : 6 1.4 mg/ten days 16,472 : 3000 ll : 9 : 80 : 12,793 : 2601 13 : 11 : 76 : 350 ug/two days d 10 m9 DO 12 silicaB'688 i 3600 5 2 18 : 4 77 + 2 H- if aEach value represents at least six separate experimental determinations. bPercentage : standard deviation. cSeparate experimental groups of at least six animals per group. dExperimental group of ten animals. 83 (table 9. Inhibition of phagocytosis and phagocytic killing of 106 C. albicans by perfused rat livers from C. parvum—treated ratsi Experimental ‘( C. parvum % RECOVERY % dose/60 min K' 11. perfusion : Effluent Liver Total 1 lng 5% plasma) Homogenate Recovery 350 pg C. parvum M 199 + 5% 2 : lb 59 : 8 61 : 8 39 : 8 plasma 350 ug C. parvumc Dd 199 + 5% plasma + 1 mM PB 7 : 3 93 : 10 100 : 7 0 b4 199 + 5% plasma + 1 mM IAA 2 : 1 111 : 12 113 : 12 0 D4 199 + 5% plasma + 10.5 mg EDTA 33:1 99 : 9 102 : 2 0 350 ug C. parvum 10 mg DQ 12 silica 1% 199 only 17 : 10 88 : 7 105 : 4 0 I“ 199 + 5% 19 + 10 83 + 2 102 + 5 0 plasma — g a. . Each value represents at least five determinations. Percentage :_standard deviation separate experimental C . . . . .Addltlves to perfus1on medium were PB (phenylbutazone), IAA (iodoacetic acid), and EDTA (ethylene diamine tetraacetic acid). 84 .in.the presence of 10.5 mg EDTA abrogated the ability (pf plasma to enhance in vitro killing of C. albicans by 19erfused rat livers from C. parvum—treated rats (Table 9). ZFive percent of the infused yeasts were recovered in the eeffluent and 99% were recovered in the liver homogenate after 60 min. Silica treated C. parvum rat livers, in the absence of plasma, were not as efficient in trapping or killing [W' ‘7‘“ —-l yeasts as were livers from normal rats (Table 4) or rats ‘ treated with C. parvum alone (line 1, Table 9). Seventeen percent of the infused yeasts were recovered in the effluent and 88% in the liver homogenate. Addition of jplasma to the perfusion medium did not significantly alter this distribution. In the absence or presence of plasma silica treatment decreased the trapping ability, and abolished killing of C. albicans by C. parvum-treated Ihepatic tissue. SEM of C. parvum-treated rat liver. SEM studies of C. parvum—treated rat livers revealed :morphologically dissimilarity compared to normal livers. ZForty eight hours after treatment with C. parvum, a loranch of the portal vein had numerous blood cells Eidhering to the wall (Figure 6A and 6B). Several cells Inorphologically similar to lymphocytes with round cell loodies and numerous villi can be seen. In addition two m..- a ... Figure 6. 85 SEMS of rat livers two days after injection of 350 pg C. parvum. A. Portal vein (PV) with adhering white blood cells (arrow). X 700. White blood cells which adhere to portal vein walls (PV) in A, showing lymphocytes (L) and macrophages (M). X 3,000. Portal vein with branches (PV) and adhering macrophage (M) with tail (arrow). X 200. Higher magnification of a macrophage (M) in a sinusoid (S). The macrophage surface has numerous folds and projections and is attached to the endothelium by cytoplasmic processes. X 3,000. 86 87 cells believed to be phagocytic cells of unknown identity can be observed. Figure 6C shows another portal vein with bifurcation. Macrophages observed adhering to the vein wall displayed typical "tails" which extended downstream or away from the "head" of the macrophage which was upstream or against the flow of blood. Figure 6D shows a sinusoid almost completely occluded by a macrophage. The cytoplasmic dendritic processes extend away from the . .*"“‘“~'”‘:1 macrophage body anchoring it to the endothelial lining of the sinusoid. I Figure 7A is a higher magnification of a portal vein with white cells adhering to the vein wall. There was a close association between lymphocytes and macrophages adhering to vein walls. Many fine cytoplasmic filaments extend between adjacent cells. Figure 7B and 7C show high magnifications of liver sinusoids. Figure 7B shows a macrophage and several attached lymphocytes. Numerous cytoplasmic filaments extend from the macrophage surface trapping the lymphocytes. Figure 7C shows a macrophage adhering to the endothelium of a sinusoid and attached to, or interacting with, a lymphocyte. Fine cytoplasmic appendages extending away from the folded surface of the macrophage contact the surface of the lymphocyte. Figure 7. 88 High magnification SEM of cellular interactions in C. parvum-treated rat livers. A. Portal vein (PV) with clusters of macrophages (M), lymphocytes (L) and cytoplasmic filaments (arrow) attached to the portal vein wall. X 700. Macrophage (M) in a sinusoid (S) with attached lymphocytes (L) which are surrounded by cytoplasmic filaments (arrow). X 5,000. Macrophage (M) in a sinusoid (S) and a lymphocyte (L) attached by several cytoplasmic appendages (arrow). X 4,000. 89 90 SEM of C. albicans trapped in perfused liVers of C. parvum-treated rats. Figures 8 through 10 show micrographs of livers from C. parvum-treated rats following perfusion with C. albicans Figure 8 shows trapping of yeast cells along the walls of portal veins of decreasing diameter. Figure 8A shows :massive accumulation of white cells and yeast cells in 3“ clusters in a large portal vein. Figure 8B shows a smaller portal vein, its‘ branches, and adjacent sinusoids, all of which were filled with adhering white cells and yeast clusters. Figure 88 shows a portal venule which was clogged with white cells and yeast clusters. A higher magnification of yeast clustering is shown in Figure 9. Yeast clusters consist of numerous tightly adhering yeast cells, blastospores, and occasionally pseudohyphae. The yeast clusters adhered to the walls of ‘portal veins. Many fine cytoplasmic filaments entangled the clusters. Pseudohyphae were considerably larger than the cytoplasmic filaments (Figure 9A). Yeast clusters were often associated with, or surrounded by, several macrophages. The macrophages were either attached to the portal vein wall or in the process of phagocytizing the yeasts. Cytoplasmic filaments interconnected macrophages and yeast clusters (Figure 9B). Yeasts, with and without attached filaments, were log jammed in portal venules prior to entering sinusoidal areas Figure 8. 91 SEM of C. albicans clearance in portal veins of perfused livers from C. parvum- treated rats. A. Portal vein (PV), and sinusoids (S), filled with white blood cells and yeast clusters. X 100. Portal vein branches (PV), and sinusoids (S), filled with white blood cells and yeasts. X 100. Portal venule (Pv) with adjacent sinusoids (S) both containing clusters of white blood cells and yeasts. X 200. 92 Figure 9. 93 SEM of C. albicans trapping by perfused livers from C. parvum-treated rats. A. Yeast cluster with pseudohyphae (arrow) and attached cytoplasmic filaments (F arrow). X 1,000. Yeast cluster with attached macro— phages (M) and cytoplasmic filaments (arrow). X 1,000. Portal venule (PV) with log jammed C. albicans which fills the adjacent sinusoids (S). Cytoplasmic filaments are attached to several yeasts (arrow). X 700. C. albicans (Ca) in a sinusoid (S) attached to and in proximity to a macrophage (M). X 2,800. 94 Figure 10. 95 SEM of macrophage phagocytosis of C. albicans in perfused rat liver from C. parvum-treated rats showing a parenchymal cell (PC) and a sinusoid (S) with a macrophage (M) attached by cytoplasmic processes (arrow) to the endothelium. The macrophage has extended a cytoplasmic filament around a yeast cell (F arrow) pinning it to the endothelial lining of the sinusoid. X 6,000. 96 97 (Figure 9C). Candida albicans was also trapped inside liver sinusoids near several macrophages occluding the sinusoid. Figure 10 is a high magnification of a macrophage, located in a sinusoid, in the process of engulfing several C. albicans. The macrophage was attached to the endothelium by its cytoplasmic processes. The macrOphage extended a cytoplasmic filament around one of the yeast cells, pinning it against the endothelial lining of the sinusoid, engulfing the yeast. DISCUSSION The ability of the liver to clear and kill C. albicans is evident in clinical literature. Eras et Fa 4"“.- '~ 8. A J ‘ a a1. (0972) found that in human candidiasis the route of infection is frequently penetration of the gastrointestinal epithelium with subsequent hematogenous dissemination. Myerowitz et a1. (1977) observed that gastrointestinal infection preceeds systemic invasion in cases of disseminated candidiasis. They found that 75% of their patients with proven disseminated candidiasis had hepatic in addition to kidney involvement. Louria et a1. (1962) found primarily kidney involvement in their study. The mechanism by which C. albicans is eliminated from the liver, and the mechanism of its initial clearance in this organ, is poorly understood. It might be postulated that during human and experimental candidiasis the liver serves as a site where host defense mechanisms would restrict further hematogenous dissemination from the gastrointestinal tract. Useful information might be obtained from an in vitro model which could be experimentally manipulated and which 98 99 approximates the initial bloodstream clearance of C. albicans. Moon et a1. (1975) demonstrated, with respect to vascular clearance of Salmonella typhimurium, that the isolated perfused liver is an experimental model which approximates in vitro the in viva clearance and killing of bacteria. The perfused rat liver not only reflects the ability of the host to kill hepatically trapped S. typhimurium, but the model clearly defines a ET‘_‘—"-‘1 fl I new functional role for the liver, that of microbial trapping in liver sinusoids. Phase contrast and transmission electron microscopy of sinusoidal areas showed bacteria "log jammed" in sinusoids and outside liver Kupffer cells. Trapped bacteria are killed by Kupffer cells when humoral factors are added to perfusion medium In contrast to the bacterial model, when a single pass of C. albicans was infused into normal rat livers there was no significant killing of the yeasts in the absence or presence of humoral factors (Table 2 and 3). At both doses studied yeast cells were cleared with equal efficiency (Figure 1, Table 2 and 3). When the perfusion medium consisted of M 199 plus 10% rat blood there was no change in the ability of the perfused rat liver to clear and kill C. albicans (Table 3). In fact total recovery in the absence or presence of whole blood or plasma exceeded 1000%, even after three hours (Table 4). lOO Baine et al. (1974) showed that C. albicans was rapidly removed from the bloodstream of rabbits. Distribution of yeast cells among various RES organs was dependent on the route of intravenous injection. Iannini et a1. (1977) also studied the vascular clearance of C'. albicans in rabbits comparing clearance of yeast cells to pseudohyphae clearance and found that the liver is extremely efficient to the clearance of both, In vivo clearance (Figure 1) and tissue distribution data (Table l) confirm that in rats the majority of injected yeast cells were removed by the liver within 30 min. After 60 min the distribution of yeast cells had not significantly changed With respect to the lungs, spleen, kidneys, and peripheral blood. The percent recovery in the liver decreased 40%. Hence, there is general agreement with the literature that the liver and lungs are the major organs involved in bloodstream clearance of C. albicans (Baine et al., 1974; Hurley, 1966; Hurley and Fauci, 1975; Iannini et al., 1977; Kemp and Solotorovsky, 1962) , and that subsequent to hepatic clearance yeast cells are rapidly killed. In the study by Baine et al. (1974) it was found that in vitro, 90% of the yeasts were cleared from Krebs— Hanseleit buffered perfusion medium over a 60 min perfusion time. Addition of 5% heat inactivated normal rabbit serum to-the perfusion medium increased clearance to 96%. In buffer with fresh 5% normal rabbit serum lOl clearance increased to 98%. The increase was found to be statistically significant. After examining the hepatically cleared yeasts by transmission electron microscopy it was concluded that clearance was promoted by heat stable and heat labile serum factors and that Kupffer cells accounted for the uptake of C. albicans by rabbit liver. On closer analysis certain discrpancies between the rabbit and rat models become apparent. For example, in the rat model, addition of whole blood (Table 3 and 4) to the perfusion medium did not enhance trapping or killing, even after extended perfusion times. In the rabbit, the liver clears C. albicans more efficiently in the presence than in the absence of 5% rabbit serum. Viability alone was measured in our study while Baine et al. (1974) measured the percentage of recovery by a quantitative Comparison of radioactively labeled yeasts with viability. In the rat no problem with aggregation of yeasts was encountered using M 199. Baine et a1. (1974) employed a buffered perfusion medium containing bovine serum albumin (BSA) to reduce aggregation of yeasts. Aggregation is probably due to low levels of anti—Candida agglutinating antibody (Mathews and Inman, 1968) which may clump C. albicans in rabbit serum (Smith and Louria, 1972) . Unpublished observations in the Medical Mycology laboratory of Dr. Everett S. Beneke, Michigan ‘ ETu—l-m-W" , 102 State University, indicate that normal rat serum does not clump C. albicans when incubated at 37 C for up to 60 min. Jeunet and Good (1969) studied the effects of soluble and heat aggregated BSA on the clearance ability of the perfused rat liver. Aggregated BSA produced reticulo— endothelial blockage due to exhaustion of plasma opsonin and stauration of Kupffer cells. A possible explanation of why rabbit liver clear 98% of infused yeasts in the presence of humoral factors while rat livers clear only 80% may be that a C. albicans—BSA interaction enhances trapping of yeasts. Whether differences between the rat and rabbit models reflect animal, experimental procedure, assay, or observational variations is not presently known. When hepatic clearance of C. albicans is compared to hepatic clearance of bacteria, insights into the interaction of hepatic tissue with bacteria and fungi may be obtained. Howard and Wardlaw (1958), using the perfused rat liver, found that normal human, rat, and mouse sera were opsonic. These sera enhanced phagocytosis of Escherichia coli by Kupffer cells. The cpsonic component of human serum was heat labile, suggesting a complement origin, and absorbable, implying the presence of E. coli specific opsonic antibodies. They suggest that opsonins were also generated from preperdin activation of 103 complement. Bonventre and Oxman (1965) used the perfused rat liver to evaluate the phagocytosis and killing of a gram positive and gram negative bacteria. They found that the immunological status of the rat had no effect on clearance and killing of Staphylococcus aureus by the perfused liver. Immune serum increased the rate and degree of phagocytosis of S. enteritidcs resulting in their complete destruction by the perfused rat liver. Moon et a1. (1975) employed normal rat serum in their perfusion system. Addition of whole blood or plasma to the perfusion medium reduced the percent recovery in the effluent and in the liver resulting in approximately 55% of the infused bacteria being killed by the liver. This study made no attempt to characterize the plasma components in normal rat plasma which had such a profound effect on the fate of S. typhimurium. These studies suggest that humoral components from normal and immune rats enhance the ability of liver Kupffer cells to kill hepatically trapped bacteria. Solomkin et a1. (1978) showed that PMN phagocytic killing of C. albicans occurs optimally when yeasts are opsonized by specific opsonic antibody and complement opsonins, derived from either the classical or alternate pathways. Data presented in this study (Table 3 and 4) suggests that plasma factors do not enhance the ability of normal hepatic tissue to trap and kill C. albicans. 104 The data, when taken together with data obtained using bacterial systems, confirms Manwaring and Fritschen‘s (1923) "second law" of microbic—tissue affinity, namely "The microbic-tissue affinity varies with the micro— organism tested". A correlary to this "Law" might be postulated to state that tissue affinity varies also with respect to trapping and killing of the particular microbe being tested. Moon et a1. (1975) showed that S. typhimurium is initially log jammed in liver sinusoids extracellularly. Scanning electron microscopy (SEM) studies (Friedman and Moon, 1977) showed bacteria adhering to the sinusoidal endothelium. SEM studies were made to find out whether hepatic clearance of C. albicans in normal rats is a function of Kupffer cell or sinusoidal trapping. Previous studies on the SEM of C. albicans in vitro (Joshi et al., 1973) and on the SEM of rat liver sinusoids (Motta and Porter, 1974) are consistent with the anatomical relationships demonstrated in this study. Light microscopic studies have indicated similar relationships to the SEM studies presented here (Hurley, 1966; Kemp and Solotorovsky, 1962; Louria et al., 1960, 1963). Extensive studies evaluating various tissue preparation methodologies show that perfusion—rapid fixation techniques including cryofracturing, and critical point drying are least likely to introduce artifacts in ‘tm——.—_' *~ 105 host tissues (O'Donnell and Hooper, 1977; Weiss, 1972). Transmission electron microsc09ic studies (Weiss, 1972) and SEM studies (Motta and Porter, 1973) are not particularly clear as to the nature of Kupffer cell orientation within the sinusoid. Kupffer cells have large bodies covered with many folds and microvilli which cover the body of the cell (Figure 2 and 4). The cell occupies a considerable portion of the lumen of the sinusoid, a? I usually at the junction of anastomosing sinusoidal branches. Bloodstream borne microbes must come into intimate contact with the surface of Kupffer cells as they pass through the sinusoid. Extending away from the body of the cell fine, cytoplasmic dendritic processes anchor the cell to the fenestrated endothelial lining of the sinusoid. CytOplasmic processes vary in length, shape and size and may be branched. They terminate, in almost all cases observed in this study, on the surface of the fenestrated endothelial cells. Termination of these processes on parenchymal cell microvilli was not seen in this study. Both transmission electron microsc0py (Weiss, 1970, 1972) and SEM (Motta and Porter, 1973) suggest that they occasionally do. When normal livers were infused with C. albicans small numbers of yeast cells became log jammed in sinusoid- al spaces (Figure 3, 4, and 5). Yeasts appear to adhere to the endothelium of the sinusoid. It is envisaged that 106 txrapping within the sinusoid restricts passage of more yeasts through the lumen. As yeasts filled the sinusoids ‘tl1ey bagan to back up into portal venules and eventually loeacame trapped in branches of the portal system. IEridividual yeast cells occupy a portion of the sinusoidal llimen making passage through the lumen restricted. It is 11(3t known whether trapping is a physical phenomenon or iJTVOlVeS chemical interaction of yeast cell walls with sinusoidal endothelial cell membranes. King et a1. (1977) found that C. albicans readily arlhere to vaginal epithelium although the mechanism of adherence was not examined. If physical trapping were the cuily trapping mechanism it might suggest that a smaller ornganism'would pass through the liver and not be trapped. Trlis is not the case. Salmonella typhimurium also afilheres to sinusoidal endothelium (Friedman and Moon, 31977). Sterility controls taken from perfusion medium 30 ruin after infusion of yeasts contained fewer than 10 CFU/ml indicating that the yeasts were stuck in the liver. In normal rat liver C. albicans trapping occurs ‘without Kupffer cell involvement (Figure 4). Baine et a1. (1974) show one transmission electron micrograph in which a yeast is being phagocytized. Our SEM study suggests that this may be an isolated event. There is no anatomical basis for their statement that the cell phagocytizing the yeast is, in fact, a Kupffer cell. The 107 majority of yeast cells seen in the SEM characterization were not associated with Kupffer cells. The inability to resolve the identity of Kupffer cells by either transmission electron or light microscopy shed light on the observation by Kemp and Solotorvsky (1962) that C. albicans was inside "endothelial phagocytes". It also points out the superior value of SEM to clearly identify Kupffer cells in sinusoidal areas of the liver. These observations taken together characterize the initial trapping of C. albicans by normal rat hepatic tissue. It does not explain how the trapped yeasts are eliminated. If tissue distribution was examined 60 min after intravenous injection it was found that there was a significant decrease in the number of viable yeasts trapped in the liver (Table 1). How are these cells eliminated? This is an expecially acute question since extended perfusion times (Table 4), in the absence or presence of plasma, did not increase killing of C. albicans. Control studies indicated that whole rat blood is not candidacidal in vitro. There is ample evidence that once C. albicans is trapped inside hepatic tissue a rapid PMN leukocytosis occurs (Baine et al., 1974). This response may be initiated either by the release of chemotactic factors from.the yeast itself (Denning and Davies, 1973; Cutler, r——-.—-‘r ‘11 108 1978) or by complement activation (Solomkin et al., 1978). Numerous in vitro studies have clearly demonstrated the candidacidal activity of the PMN (Arai et al., 1977; Davies and Denning, 1972; Denning and Davies, 1973; Glasser et al., 1977; Lehrer, 1970, 1972; Lehrer and Cline, 1969; Leijh et al., 1977; Schmid and Brune, 1974; Solomkin et al., 1978; Venkatraman et al., 1973; Yamamura et al., 1976; Zeya and Spitznagel, 1966). The sequence of ‘1"! . 1 events by which hepatically trapped C. albicans is eliminated in vivo in normal rats involves an initial trapping of yeasts in liver sinusoids followed by PMN inflammation (Hurley, 1966; Hurley et al., 1975; Louria et al., 1960, 1963; Kemp and Solotorvsky, 1962; Rogers and Balish, 1977). Influx of the highly candidacidal PMN into the liver (Louria et al., 1960) immediately after trapping and the subsequent killing of the yeasts accounts for the loss in viability seen in normal rat liver after 60 min (Table 1). This would explain observations by <3thers indicating that liver populations of C. albicans (decline rapidly after injection. Failure to recognize 'the activity of PMNs in the liver may also explain many .assumptions that hepatic candidacidal activity is due to IKupffer cells (Meister et al., 1977a, 1977b). Blood :monocytes may also emigrate into the liver, as shown by North.(l970) using Listeria monocytogenes, develop into macrophage and contribute to the elimination of yeasts. 109 It is unknown if lymphocytes play a role in this response in normal hepatic tissue. Clearly, the normal rat liver does not interact with C. albicans in a manner analogous to its interaction with bacteria. The second objective of this study is to ask whether hepatic tissue can be manipulated so that it would kill trapped yeast cells. Ideally the manipulatory process would be nonspecific enough to insure an optimal response by hepatic tissue. Characterization of this enhanced nonspecific response would of necessity have to differentiate between activity due to PMN only, as opposed to activity due to Kupffer cells. The immuno— potentiator Carynebacterium parvum was chosen as it is known to be an activator of the MPS. It was theorized that stimulation of rats in viva would be manifested by enhanced in vitro Kupffer cell activity. In viva bloodstream clearance data showed that when C. albicans was injected into C. parvum-treated rats yeast cells were rapidly cleared fromthe peripheral blood by 30 min (Table 7). Organ distribution data showed that the percentage of viable cells distributed among lungs, spleen, kidneys, and blood were not statistically distinguishable from the distribution in normal rats the total in viva loss in viability is reflected primarily by the loss of viable yeast cells in the liver alone. To obviate the possibility that killing in vitro 110 by C. parvum-treated hepatic tissue could be due to peripheral blood PMN only plasma was used to supplement perfusion medium in all of the remaining experiments. Moon et al. (1975) found that extended perfusion times in the absence of humoral factors increased the ability of the perfused rat liver to kill S. typhimurium. Killing in vitro was the same as that observed in viva when perfusion medium contained whole blood or plasma but not PM! waif-VT: V . blood cells. Both antibody opsonins and complement derived serum factors participate in Kupffer cell killing (Ruggiero et al., 1977). Leijh et a1. (1977), in a comparison of the kinetics of phagocytosis and killing of C. albicans by PMS and blood monocytes (BM), found that both cells phagocytize yeasts at equal rates for the first 30 min. Yeasts are killed during the next 30 min after ingestion. Extended perfusion times in C. parvumétreated rat livers might increase the models' sensitivity in detecting C. albicans killing. Perfusion of C. albicans into livers from C. parvum-treated rats was candidacidal both in the absence and presence of plasma. Killing was enhanced by humoral factors. Plasma did not alter the ability of the liver to trap yeasts but enhanced killing. The optimal response was detected two days after injection of 350 pg C. parvum. The 44% loss of viable yeasts in viva (Table 5) was approximated by a 34% loss in viable yeast cells 111 in vitro (Table 6). Intravenous injection of C. parvum results in high numbers of bacteria in the liver (Demitrov et al., 1976). Inflammation following hepatic clearance of C. parvum could result in the accumulation of large numbers of PMNs and BMs in the liver. These cells would in all probability adhere to liver sinusoids and not be washed out of the liver prior to infusion of C. albicans. They are washed out of normal rat liver prior to infusion of yeasts. Killing could be due to the PMN and/or BM derived macrophages. The maximum response of C. parvum-treated hepatic tissue occurs at two days. At this time the meximum PMN and BM response to C. parvum in the liver would be expected to occur. Perfusion studies indicated that in vitro killing only approximates in viva killing further strengthening the possibility that phagocytic cells other than Kupffer cells might be in the liver. In addition to stimulating macrophage directly C. parvum also stimulates T and B lymphocytes directly (Bomford and Christie, 1975; Christie and Bomford, 1975; Tuttle and North, 1975a, 1975b). Both events could generate activated lymphocytes and macrophages which release nonspecific lymphotoxins (Cerottini and Brunner, 1977; Pearsall et al.,l974) or macrophage cytotoxins (Ghaffar and Cullen, 1976; Lohman—Matthes, 1973, 1976; Melson et al., 1974). The mechanism of C. albicans 112 killing by C. parvum-treated hepatic tissue might not be d11€2 to phagocytic killing. To further investigate this possibility perfusion studies were performed in C. parvum- ‘trweated rats given metabolic inhibitors of phagocytic activity. Plasma chelated with 10.5 mg EDTA lost the ability to enhance hepatic killing. Plasma ions are essential for both phagocytic engulfment of microbes (Stossel, 1974a) arud for complement activation. Both activities are in— hiJoited by chelation of essential plasma divalent ions, snlch.as Mg++ and Ca+fi from the perfusion medium. Our dirta suggests that ions are necessary for the enhanced (effect of plasma on hepatic killing. Whether complement alone is involved was not clarified by the use of EDTA. Further, use of EDTA does not indicate what type of cell kills C. albicans, the PMN or macrophage. To clarify this point, perfusion studies with 1 mM PB and 1 mM IAA were performed to determine whether killing of yeasts was due to phogocytosis. Both PB and IAA completely abrogated the killing of trapped yeasts in C. parvum-treated rat liver. Leijh et al. (1977) showed similar results in vitro with C. albicans. Phenylbutazone specifically inhibited intracellular killing of yeasts and IAA specifically blocks phagocytosis of C. albicans. When coupled with the EDTA data (Table 9) the results indicate that killing of trapped yeasts was ‘ F ”on. Carl, 4‘ 113 nrot; due to extracellular white cell toxins. Friedman and Moon (1977) showed that crystalline ssifiLica is a Kupffer cell or macrophage-specific cytotoxin i!) 'the perfused liber. Silica treatment of rats in viva lcifills cells of the MP8. The PMN population remains flnrlctional. Injection of silica abolished the ability of hepatic tissue from C. parvum—treated rats to kill C7. albicans. Cumulatively the studies with inhibitors suggest that C. parvum stimulated liver macrophage, and not PMN, Efliagocytized and killed hepatically trapped C. albicans. Ii consistent feature of C. parvum treatment was a :relative monocytosis. Blood monocytes were elevated four fold over normal values in C. parvum—treated rats. Injection of silica into C. parvum—treated rats depressed this monocytosis. Silica treatment either depresses the release or kills the monocytes when they are released from the bone marrow. North (1970) and Volkman (1976) observed that when L. manacytagenes is cleared by the liver a rapid influx of peripheral blood monocytes into the liver occurs. These cells are indistinguishable from Kupffer cells when they take up residence in liver sinusoids as examined by light microscopy. Killing of C. albicans in C. parvum- treated rats could be due to newly recruited blood monocytes. They would have enhanced nonspecific phagocytic killing activity. To further clarify this possibility, P . .4... .5). 9m- :1». ‘ - A . 114 extensive SEM investigation of hepatic tissue from 6?. parvum-treated rats and from C. parvum-treated rats perfused with C. albicans was performed. SEM of liver from rats treated with C. parvum :reyvealed striking morphological differences when compared ‘tc> the anatomical features of normal rat liver (Figure 2). I?cxrty eight hours after injection of the bacteria there ‘flEiS a massive influx of white blood cells into the liver. 'Fliis influx coincided with a relative monocytosis (IFable 8). White cells adhere to the walls of portal Vwains including large and small branches of the portal ‘Jein, portal venules, and sinusoids. White cells adhering 'to portal veins appeared to be macrophages and lymphocytes (Figure 6 and 7). The morphological features of T and B lymphocytes, blood monocytes, and PMN have been studied in detail by SEM (Barber and Burkholder, 1974; Noonan and Riddle, 1977; Polliack et al., 1973; Wetzel et al., 1973). Nielsen et a1. (1974) and Werdelin et a1. (1974) employed SEM to study macrophage—lymphocyte cluster formation during the in vitro immune response to soluble protein antigen. Roelants (1977) discusses this model and the significance of macrophage—lymphocyte clustering in detail. All of these SEM studies indicate that it is not all clear, based on SEM alone, that white cells can be distinguished. It is agreed that cells with characteristic surface morphologies may be generally categorized as 115 either macrophages or lymphocytes. For purposes of discussion cells morphologically similar to those cells described by these studies will be referred to simply as macrophages and lymphocytes. The cells with a satellite appearance and numerous surface folds adhering to portal vein walls appearing to be macrophage are probably of blood monocyte origin. Observations were made that the macrophages characteristi- cally adhere to the wall with the largest portion of the cell body or "head", upstream or facing the flow of blood. A long "tail" extends downstream from the head of the macrophage. This head-tail orientation was seen most often in larger portal veins and not in smaller veins. The reasons for such an orientation are not readily apparent from the literature. The greater volume of fluid in the larger veins may influence such an orientation. Such orientation of macrophages might also be due to motility (Barber and Burkholder, 1974) . Macrophages in the sinusoids of C. parvum—treated rat livers have morphological features similar to those described for macrophages adhering to portal veins and to normal rat liver Kupffer cells. They occupy a significant Portion of the sinusoidal lumen, have folded surfaces, Stellate appearance and fine cytoplasmic processes anchoring them to the endothelium. Are they the original, resident population of Kupffer cells or are they blood 116 monocyte derived macrophage? North (1970) could only make this distinction by the use of autoradiographic studies of characteristic labeling patterns. Volkman 1976) showed that Kupffer cells, contrary to previous data (van Furth et al., 1975), are not derived from blood monocytes. They are a resident, self-sustaining population of cells. For purposes of discussion cells morphologically identifiable as macrOphage bound either in the sinusoid or in the portal veins will be referred to simply as macrophage. There is a considerable amount of contact or inter— action between adhering white cells in C. parvum—treated liver. For example, the macrophages in Figure 7 have trapped lymphocytes. Macrophage—lymphocyte contact was envisaged to occur in two ways represented. Lymphocytes appear to sit in a cup-like depression on the surface of the macrophage with numerous fine cytoplasmic processes surrounding them. In the second type of interaction cytoplasmic processes extend between the surface of the macrophage contacting the surface of the lymphocyte. It is unknown if these relationships have any functional significance or if they contribute to the enhanced killing ability of C. parvum—treated hepatic tissue. When compared to the ultrastructural morphology of normal rat hepatic tissue (Figure 2) it becomes apparent that when C. albicans enters hepatic tissue from 117 C. parvum-treated rats (Figure 6, 7, and 8) it encounters a liver strikingly dissimilar, functionally and morpholog— ically, from normal liver. Microsc0pic examination of the inoculum prior to perfusion, and of the effluent after perfusion revealed only yeasts, blastospores and rarely a germ tube. Yeast cells observed in the perfusion medium were not aggregated, either before or after perfusion, and there was no change in the number of CFU recovered from F""""’J ‘ the effluent if the effluent was homogenized or held on ice prior to plating. Yeast cells formed large aggregates or "yeast clusters" only in perfused C. parvum—treated rat livers. Yeast clusters adhere to the walls of portal veins and venules. Portal vein walls contain large numbers of yeast clusters. Yeast clusters consist of numerous tightly adhering yeast cells, blastospores, and an occasional germ tube. They were entangled by many fine cytoplasmic filaments. SEM showed yeast clusters with cytoplasmic filaments and associated macrophages, yeast clusters alone adhering to portal vein walls, and yeast clusters with or without cytoplasmic filaments but with numerous macrophages. The macrophages either adhered to the clusters surrounding them or phagocytized yeasts. Observations made by comparing SEM of clearance in normal and C. parvum-treated rat liver indicated that the trapping mechanism in C. parvum-treated hepatic tissue 118 was different from normal liver trapping. In normal rat liver, in the presence of plasma, trapping occurred primarily in sinusoids and not in portal veins. Yeasts back up into portal veins but they occluded the lumen of the vessel and did not adhere to the walls in any great numbers. Yeast clustering in which cytOplasmic filamentation occurred was in the C. parvum—treated rat. Friedman (1978, personal communication) has observed similar cytoplasmic filaments entangling S. typhimurium in C. parvum-treated mice. In the C. parvum—treated rat liver a significant portion of trapping appears to occur by yeast cluster formation on the walls of the portal vein. The origin of the cytoplasmic filaments attached to C. albicans is unknown. They were not present in the inoculum. Candida albicans forms filaments in vitro which are 0.8-1.3 microns in diameter, but, only after 18 h of culture (Yamaguchi et al., 1974). There are no reports of in viva filamentation. When coupled with the observation that these filaments are seen with S. typhimurium trapping and C. albicans trapping it may be postulated that they are of host origin. This conclusion does not conflict with reports of macrophage filamentation in vitro (Barber and Burkholder, 1974). Log jam trapping of yeasts in sinusoids and portal venules, similar to log jamming in normal livers, also 119 occurs in C. parvum—treated liver. Unlike yeast cells trapped in normal liver sinusoids, yeasts in C. parvum— treated liver sinusoids usually were attached to cytoplasmic filaments or were being phagocytized. It may be that as yeast cells contact macrophages adhering to the portal vein walls they temporarily stick to the macrophage surface. If they were pulled away, by the flow of perfusion medium or by the impact of other yeast cells, they might draw out a thin cytoplasmic filament from the macrophage which would break off. The yeast—filament would be carried into the liver eventually ending up in a yeast cluster or in a sinusoid. Whatever their origin, filaments do not increase trapping above that seen in normal liver. Another explanation for their origin might be that they are formed by some interaction of C. albicans with a factor unique to C. parvum-treated rat plasma. Yeast clustering could artifactually alter viability counts when plasma is added to the perfusion medium. The loss in viability in C. parvum—treated rat liver would then be an artifact of aggregated yeast clusters. In all of the experiments using inhibitors of phagocytosis 100% of the inoculum was consistently recovered suggesting that the loss in viability was not an artifact. SEM studies did not resolve the question of which cell, the resident Kupffer cell or the blood monocyte derived macrophage, was responsible for killing 120 C. albicans. Both cells were obviously present and indistinguishable in C. parvum—treated livers while only Kupffer cells were present in normal rat liver. Since normal hepatic tissue does not kill C. albicans while C. parvum-treated macrophages kill the yeast one conclusion is that Kupffer cells do not participate in the hepatic clearance of C. albicans. Several studies (Bomford and Christie, 1975; Christie and Bomford, 1975; Scott, 1972; Tuttle and North, 1975a, 1975b) have investigated the in viva and in vitro mechanism of action of C. parvum. No studies to date have evaluated the initial response of the host to C. parvum treatment. Data obtained in this study indicate that early in the hosts' exposure to C. parvum the MP8 is nonspecifically stimulated, in the absence of lymphocyte— mediated stimulation. This stimulation results in increased hepatic killing ability which may play a significant role in antimicrobial resistance. This study characterized the initial hepatic clearance of C. albicans by normal rat hepatic tissue. The mechanism of clearance was different from that of bacterial hepatic clearance. Clearance of yeasts occurs primarily by nonphagocytic trapping in liver sinusoids. In viva and in vitro pathophysiological experimentssmggest that sinusoidal trapping was the major way in which yeasts were cleared. SEM confirmed this. Treatment of rats with 121 C. parvum resulted in an influx of white blood cells into the liver. Pathophysiological experiments showed that the macrophages present in these adhering white cells killed the yeasts. Killing occurred optimally in the presence of humoral factors. 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