Secretion of the C3 Component of Complement by Peritoneal Cells Cultured with Encapsulated Cryptococcus neoformans

Transcription

1 INFECTION AND IMMUNITY, Oct. 1997, p Vol. 65, No /97/$ Copyright 1997, American Society for Microbiology Secretion of the C3 Component of Complement by Peritoneal Cells Cultured with Encapsulated Cryptococcus neoformans REBECCA BLACKSTOCK* AND JUNEANN W. MURPHY Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma Received 7 April 1997/Returned for modification 26 May 1997/Accepted 9 July 1997 Two isolates of Cryptococcus neoformans were identified as being widely divergent in pathogenic potential after intratracheal infection of mice. These isolates differed in their ability to upregulate capsule synthesis when grown under tissue culture conditions, and this property correlated with virulence. We postulated that differential capsule synthesis may cause differential stimulation of macrophages to produce products such as complement components. To test this hypothesis, heat-killed yeast cells were incubated with normal mouse peritoneal cells (PC) before the level of C3 secreted was determined. Cryptococcal stimulants were grown on mycological agar, which does not promote capsule synthesis, or in RPMI 1640 at 37 C in an atmosphere of 5% CO 2, which stimulates capsule synthesis, to determine the role that the capsule plays in the induction of C3 secretion. C3 levels were elevated in cultures containing cryptococci grown in RPMI 1640 at 37 C in an atmosphere of 5% CO 2, and the level of C3 detected was correlated with the amount of capsule expressed by the yeast cell stimulant. Nonencapsulated mutants of C. neoformans did not stimulate C3 secretion. Purified capsular polysaccharide (glucuronoxylomannan [GXM]) also stimulated the PC to secrete C3. Two signals were required before GXM stimulated C3 secretion. The second signal was identified as endotoxin present in small amounts (0.06 ng per ml) in tissue medium. Endotoxin may provide a priming stimulus for PC to express receptors or other cytokines needed for effective stimulation of C3. These experiments show that enhancement of C3 secretion by C. neoformans is due to GXM and is correlated with the virulence of the cryptococcal isolate. Cryptococcus neoformans is a yeast-like organism which is found in nature in highly nitrogenous soil such as in areas rich in pigeon droppings (25). When examined in tissue sections, the organisms are surrounded by a large gelatinous capsule. However, in the soil, the capsule collapses to form a thin cellophane-like barrier (10). Under conditions found in the soil, capsule synthesis is repressed and the organism becomes small enough to be inhaled into the lungs (37). After inhalation of infective particles, the residual capsule rehydrates and capsule synthesis is upregulated. The capsule has long been recognized as a virulence factor of cryptococci because of its antiphagocytic activity (6, 40) and its ability to induce host T-cell responses which downregulate protective immunity (3 5). More recently, the capsule has been shown to have more diverse effects such as induction of cytokines from macrophages and neutrophils (32, 44), inhibition of neutrophil migration by loss of L-selectin and tumor necrosis factor alpha (TNF- ) receptors from the surface of the polymorphonuclear leukocyte (13), and potentiation of human immunodeficiency virus infection (29). The capsule is not the only virulence factor of C. neoformans, and others such as the ability to synthesize melanin (34, 39) on medium containing polyphenolic compounds and the ability to synthesize mannitol (46) have been shown to help the yeast survive killing mechanisms provided by macrophages and polymorphonuclear leukocytes (9, 45). While cryptococci are known to possess several virulence factors, very little is known about the expression of these factors as the organism moves from its primary site of infection in the lung to other tissues of the body and ultimately to the brain, where it causes death due to meningoencephalitis. Some * Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK Phone: (405) Fax: (405) becky-blackstock@uokhsc.edu. cryptococcal isolates are known to be highly pathogenic for mice, while others are relatively avirulent. A recent histologic comparison of two such strains of C. neoformans revealed striking differences in host inflammatory responses when the two isolates were inoculated into the lungs of mice (12). We recently initiated investigations of differential host responses that occur in response to cryptococci which vary widely in virulence. For these studies, we used two C. neoformans isolates which are serotype A. One is highly virulent for mice, while the other is classified as weakly virulent. Initial experiments were undertaken to examine expression of capsule synthesis by these two isolates under conditions which might be expected in vivo. Only the highly virulent isolate had the ability to significantly increase its capsule size under these conditions. The alternative complement pathway is known to be activated by the capsule of cryptococci (23, 24) and provides opsonization which allows organisms to be engulfed by phagocytes (40). In addition, complement split products contribute to the influx of inflammatory cells (35) and regulation of the development of cell-mediated immunity (15). For these reasons, the influence of our highly virulent and weakly virulent isolates on complement secretion was of interest. In tissues, local synthesis of complement components may participate in the generation of inflammation and immune responses (35, 36). Therefore, the ability of highly encapsulated and weakly encapsulated organisms to stimulate secretion of complement components and generate complement split products may be a major contributor to the regulation of the inflammatory response. In this investigation, we examined the ability of our two cryptococcal isolates to induce C3 secretion from normal mouse peritoneal cells (PC) cultured in vitro. Our results showed that C3 secretion did occur in response to cryptococci and that increases in C3 secretion were associated with the virulence of the cryptococcal isolate. The capsule of the organism was found to be responsible for inducing C3 secretion. 4114

2 VOL. 65, 1997 STIMULATION OF C3 SYNTHESIS BY CRYPTOCOCCAL GXM 4115 However, a second signal was required by the PC population before C3 was secreted in vitro under tissue culture conditions. MATERIALS AND METHODS Animals. CBA/J female mice were purchased from Jackson Laboratories, Bar Harbor, Maine. The mice arrived at 7 weeks of age and were used in experiments from 8 to 12 weeks of age. The mice were housed in the University of Oklahoma Health Sciences Center Animal Facility, which is approved by the American Association for the Accreditation of Laboratory Animal Care. Maintenance of low-level endotoxin conditions. Experiments were done to maintain endotoxin levels at the lowest levels achievable. This included the use of sterile plastic culture dishes and pipettes, glassware baked at 180 C for 3 h, and reagents which were certified to contain less than 8 pg of endotoxin per ml (minimal detectable level) by the Limulus assay. All tissue culture medium and buffers prepared locally were tested by the Limulus assay to ensure that detectable endotoxin was not present. Fetal bovine serum was certified to contain low concentrations of endotoxin at endotoxin unit per ml. Reagents. RPMI 1640 and penicillin-streptomycin were purchased from Gibco BRL, (Grand Island, N.Y.). Dulbecco s phosphate-buffered saline (PBS) and Limulus assay kits were obtained from BioWhittaker (Walkersville, Md.). Hy- Clone (Ogden, Utah) was the supplier of fetal calf serum. O-phenylenediamine (OPD) substrate tablets were purchased from Sigma Chemical Co. (St. Louis, Mo.). Mycological agar was purchased from Difco Laboratories (Detroit, Mich.). Kirkegaard & Perry (Gaithersburg, Md.) was the supplier of bovine serum albumin (BSA) blocking solution and PBS-Tween solutions used in the enzymelinked immunosorbent assay (ELISA). Endotoxin (lipopolysaccharide [LPS]) was from Escherichia coli serotype O127:B8 and was purchased from Sigma. Fungal strains. Encapsulated C. neoformans strains used in this investigation included strain NU-2, which was originally obtained from the University of Nebraska Medical School, and strain 184A, which was originally obtained from L. Friedman, Tulane University Medical School. Both of the encapsulated cryptococcal isolates are serotype A. Nonencapsulated mutants of C. neoformans included strains M7 (7) and 602 (22). All isolates were maintained in the laboratory by culture on mycological agar (Difco). In some instances, organisms were grown in RPMI 1640 containing bicarbonate at 37 C in an atmosphere of 5% CO 2 to enhance capsule production. Organisms grown for low-level capsule production were grown on low-ph Sabouraud agar (14), on mycological agar, or in RPMI 1640 containing bicarbonate at room temperature and without CO 2. Determination of the relative virulence of the two C. neoformans isolates. The comparative virulence of cryptococcal strains 184A and NU-2 was assessed by infection of mice with 10 4 yeast cells via the intratracheal route. Mice were anesthetized with ketamine (5 mg/kg of body weight) and xylazine (5 mg/kg). After surgical exposure of the trachea, a 22-gauge angiocatheter was inserted into it. Twenty-five microliters of a solution of cryptococci (10 4 organisms) was injected into the angiocatheter tube, followed by 50 l of air to expel all of the inoculum into the lungs. The incision was closed with wound clips. Animals were observed daily for deaths to determine the mean survival time for each isolate of C. neoformans. Quantitation of capsule synthesis. To examine the ability of cryptococcal isolates to upregulate their capsule synthesis under tissue culture conditions, strains 184A and NU-2 were cultured on low-ph modified Sabouraud agar (14) or in RPMI 1640 containing bicarbonate at 37 C in an atmosphere of 5% CO 2. The diameter of the entire organism and the diameter of the yeast cell were measured (in micrometers) to determine the mean capsule width (MCW), which was calculated by subtraction of the diameter of the yeast cell by the following formula: (cell diameter yeast diameter)/2. The MCW was derived from the average of 100 individual yeast cells. Preparation of heat-killed cryptococci. Cryptococci were grown for low-level capsule synthesis on low-ph modified Sabouraud agar (16% glucose, ph 5.0), on mycological agar, or in RPMI 1640 at ambient conditions for 3 days. These media, under these conditions, have been found to support equivalent levels of capsule synthesis by a given encapsulated strain of C. neoformans. Cells grown for high-level capsule production were grown for 3 days in RPMI 1640 containing bicarbonate at 37 C in an atmosphere of 5% CO 2. At the end of the culture period, the cells were harvested and washed three times in RPMI 1640 and resuspended to a concentration of 10 7 per ml. The cryptococcal suspensions were placed in a water bath at 60 C for 2 h. After heating, subcultures were made from the suspensions onto mycological agar to ensure that all of the cryptococci were killed. These heat-killed cells were refrigerated as a stock solution until used. At the time of use, aliquots of the heat-killed cryptococci were removed from the stock solution, washed with RPMI 1640, and resuspended in RPMI 1640 containing 10% fetal calf serum at a concentration of 10 6 yeast cells per ml. Preparation of cryptococcal GXM. Capsular polysaccharide (glucuronoxylomannan [GXM]) of C. neoformans NU-2 was prepared as described previously (2). Because the method of preparation of GXM includes dialysis, it is impossible to maintain conditions which are completely endotoxin free. However, precautions such as those described above were maintained as much as possible. Because of the chemical structure of the capsular polysaccharide, testing for endotoxin is not possible. The polysaccharide contains glucuronic acid, which causes positive reactions in the Limulus assay (28). PC monolayers. Normal (unstimulated) PC were harvested from mice by lavage of peritoneal cavities with cold Hanks balanced salt solution containing 10 U of heparin per ml. Because of the number of cells required for each experiment, it was necessary to pool PC collected from four to five individual mice. The PC were centrifuged at 200 g and resuspended in RPMI 1640 containing 10% fetal calf serum, 50 U of penicillin per ml, and 50 g of streptomycin per ml. The suspensions were adjusted to contain 10 6 cells per ml, and 0.1 ml of cells was distributed into the wells of flat-bottom 96-well microtest plates. The cells were incubated at 37 C in an atmosphere of 5% CO 2 for1h prior to the addition of cryptococci or cryptococcal GXM. As determined by nonspecific esterase staining, the PC populations contain 50 to 56% macrophages. Because nonadherent cells were not removed from the cell cultures and due to the fact that a variety of cell types can secrete C3, the cultures studied for their C3 secretion are referred to as PC cultures. Stimulation of C3 synthesis. For stimulation of C3 synthesis by PC, the PC cultures were treated with 0.1 ml of heat-killed cryptococci containing 10 5 yeast cells or PC and yeast cells at a 1:1 ratio. Preliminary experiments showed that this was the best ratio for stimulation of C3 synthesis and that increased amounts of cryptococci did not enhance C3 secretion above the level provided by the 1:1 ratio. Heat-killed cryptococci were used because viable organisms would overgrow the PC under tissue culture conditions. Each experimental condition was set up as three to four replicate cultures. Supernatants were harvested 1, 3, or 5 days after C. neoformans cells were added to the PC and analyzed for the amount of murine C3. Results are expressed as the mean concentration of C3 the standard error of the mean (SEM) of three to four replicate cultures of the pooled PC per experiment. ELISA for C3. An ELISA for murine C3 was developed as described by Goodrum (16). Wells of 96-well, flat-bottom microtiter plates (Immulon IV) were coated with 0.1 ml of a 1:750 dilution of goat anti-mouse C3 (Cooper Biomedical) diluted in coating buffer (0.1 M NaHCO 3 ; ph 8.2). The plates were incubated overnight at 4 C. The next day, the antibody solution was removed from the wells and the wells were blocked with 0.2 ml of 1% BSA in PBS solution. After incubation for 1 h atroom temperature, the blocking solution was removed and the wells were dried by inversion of the plate on a paper towel. Complement standards or dilutions of unknown samples were added to the wells of the plates in duplicate in a volume of 0.1 ml. The plates were incubated for 4 h at room temperature. After removal of the samples, the wells of the plates were washed three times with PBS-Tween, and 50 l of detecting antibody (peroxidase-labeled anti-mouse C3; Cooper Biomedical) diluted 1:4,000 in 1% BSA PBS was added to each well. The plates were incubated for an additional 2 h before removal of the detecting antibody followed by three washes of the wells with PBS-Tween. Sigma Fast OPD tablets were used as described in the manufacturer s instructions to prepare OPD substrate containing urea hydrogen peroxide. One-tenth milliliter of substrate solution was added to each well, and after incubation for 5 to 15 min, the reaction was stopped by the addition of 0.1 ml of 3 N HCl. The optical density was read at 490 nm and compared to the standard curve to determine the units of C3 secreted into the tissue culture medium. For the standard curve, pooled normal mouse plasma (stored in aliquots at 70 C) was used. A unit of C3 was defined as the amount of C3 in 1 ml of mouse plasma diluted 1:10 7. The standard curve was linear between 1,000 and 15.6 U per ml. Because there is no source of purified mouse C3, data cannot be expressed as nanograms per milliliter. Statistical analysis. Student s t test was used to analyze C3 data and the MCW of cryptococcal isolates. Survival data were analyzed with Kaplan-Meier survival plots followed by log rank tests (JMP Software; SAS Institute, Cary, N.C.). Data having a P value of 0.05 were considered significant. Experiments were performed at least two times with equivalent results. RESULTS Comparison of the levels of virulence of cryptococcal strains 184A and NU-2. The levels of virulence of strains 184A and NU-2 were directly compared after intratracheal infection of mice with 10 4 cryptococci. The animals were monitored for deaths daily. Survival curves for the first 95 days are shown in Fig. 1. Strain NU-2 was highly virulent for mice and killed 100% of the animals by day 67. In contrast, only 40% of animals infected with strain 184A were dead by day 67 and the remaining animals survived until day 95, when the experiment was terminated. The mean survival time for NU-2-infected mice was days, and 184A-infected mice had a mean survival of days (P 0.039). This experiment identified these two isolates as being divergent in their capacity to cause lethal infection and suggested that differences in host responses to the two isolates may be utilized to identify factors which contribute to the pathogenesis of C. neoformans.

3 4116 BLACKSTOCK AND MURPHY INFECT. IMMUN. FIG. 1. Survival of mice infected by the intratracheal route with cryptococcal isolates NU-2 and 184A. Mice were infected intratracheally with 10 4 C. neoformans cells and examined daily for deaths. FIG. 2. Capsule synthesis by cryptococcal isolates 184A and NU-2 after culture on low-ph Sabouraud agar or in RPMI 1640 at 37 C in an atmosphere of 5% CO 2. Data are expressed as the MCW (in micrometers) SEM of 100 yeast cells., P , compared to 184A grown in RPMI Comparison of the levels of capsule synthesis by cryptococcal strains 184A and NU-2. It has been known for some time that capsule synthesis by cryptococci grown in vitro depends upon the culture conditions used. Routine culture media used for the cultivation of fungi usually results in growth of yeast cells expressing a small amount of capsule, and some formulations have been designed to limit capsule formation (42). On the other hand, growth of cryptococci under tissue culture conditions (i.e., RPMI 1640 containing bicarbonate and in an atmosphere of 5% CO 2 ) enhances the ability of the organism to synthesize capsule (17). The important factor for capsule formation in this medium is the growth of the organism at 37 C in 5% CO 2 since incubation of these same cultures at room temperature without CO 2 produces yeast cells with limited amounts of capsule (17). We directly compared the MCWs of strains 184A and NU-2 when grown on low-ph modified Sabouraud agar and when grown in RPMI 1640 incubated under tissue culture conditions to determine if the two isolates differed in their ability to upregulate synthesis of their capsules. The results are shown in Fig. 2. When grown on the low-ph Sabouraud agar for 18 h, strain 184A had an MCW of 0.8 m. In RPMI 1640 under 5% CO 2 at 37 C, the capsule of 184A was slightly larger (MCW, 1.07 m) after 18 h of growth. The capsule of strain NU-2 was only slightly larger than that of strain 184A when grown on the low-ph agar (MCW, 0.9 m). However, after growth in RPMI 1640 in CO 2 at 37 C, NU-2 significantly upregulated capsule synthesis, with an MCW of 2.54 m. This experiment showed that under tissue culture conditions which are similar to the temperature and CO 2 concentration in tissues of the body, strain NU-2 produced much more capsule and that this characteristic may contribute to the pathogenic potential of this highly virulent isolate of C. neoformans. Examination of PC C3 secretion in response to encapsulated and nonencapsulated C. neoformans strains. Macrophages are natural effector cells which infiltrate tissues infected with C. neoformans. Although macrophages in tissues may directly kill C. neoformans, they may also exhibit other functions which might have an impact on the pathogenesis of C. neoformans. For this reason, we began our examination of the differential host response to C. neoformans NU-2 and 184A by examination of macrophage responses to each isolate. We hypothesized that clearance from the lung may depend upon opsonization of cryptococci by the complement component C3, which is known to enhance phagocytosis of the organism (40). However, in the lung environment, complement is not constitutively present at very high levels and must be synthesized by macrophages and other cells in the lung (36). Because we needed a large number of macrophages for our experiments, we first examined C3 secretion by PC which are comprised of approximately 55% macrophages. The PC were stimulated with heat-killed NU-2 or 184A grown under conditions which resulted in the production of small capsules (i.e., mycological agar) and compared to PC stimulated by the same cryptococci grown under conditions which favored capsule production (tissue culture conditions). Supernatants of these cultures were examined for C3 secretion into tissue culture medium. Initially, C3 levels were monitored on days 1, 3, and 5 after the initiation of the culture. In these experiments, C3 was not detected in the C. neoformans-stimulated cultures above the background level of PC (cultured without stimulus) until day 3 of culture. C3 secretion by PC stimulated with cryptococci was significantly elevated at days 3 and 5 of culture, and the highest concentration of C3 was detected at day 5. For this reason, day 5 was chosen as the best time to assay C3 levels in the cultures. Stimulation of PC with cryptococcal strain NU-2 enhanced C3 secretion from the PC slightly when the organism was grown on ordinary mycological culture medium, which does not favor capsule production (Fig. 3A). However, after growth under tissue culture conditions, NU-2 stimulated a dramatic increase in the amount of C3 secreted by the PC. On the other hand, stimulation with strain 184A grown under conditions which do not favor capsule synthesis did not increase C3 synthesis above background levels. After growth in RPMI 1640 in CO 2, strain 184A did increase the amount of C3 secreted by the PC; however, this increase was significantly less than that observed when PC were stimulated with the highly encapsulated strain NU-2. Therefore, the amount of C3 secreted by the PC was directly related to the amount of capsular polysaccharide expressed on the yeast cell and to the relative virulence of the two isolates. The amount of C3 secreted by the PC was related to the

4 VOL. 65, 1997 STIMULATION OF C3 SYNTHESIS BY CRYPTOCOCCAL GXM 4117 FIG. 3. Stimulation of C3 synthesis by PC cultured with encapsulated cryptococcal isolates NU-2 and 184A (A) or nonencapsulated mutants 602 and M7 (B) grown on mycological agar (M) or in RPMI 1640 (R) at 37 C in 5% CO 2. Data are expressed as mean units of C3 (per milliliter) SEM., significant enhancement of C3 secretion (P 0.05), compared to 184A grown in RPMI 1640;, significant enhancement of C3 secretion (P ), compared to NU-2 grown on mycological agar. amount of capsule expressed on the yeast cells. Therefore, nonencapsulated mutants were used to induce C3 to determine the role of the capsular polysaccharide in eliciting the C3 response. Encapsulated and nonencapsulated strains of C. neoformans were used to stimulate C3 from PC cultures (Fig. 3). Cryptococci were grown on mycological agar to obtain cells expressing low amounts of capsular polysaccharide and in RPMI 1640 in an atmosphere of 5% CO 2 to induce upregulation of capsule synthesis. Nonencapsulated mutants were grown under both conditions to control for possible changes in cryptococci when grown under tissue culture conditions which may occur in addition to increases in capsule synthesis. As observed in other experiments, strain NU-2 significantly upregulated C3 secretion by PC when the yeast expressed large amounts of capsular polysaccharide. Nonencapsulated mutant strains M7 and 602 did not induce C3 secretion from PC above background levels. Since no differences were seen between nonencapsulated mutants grown under the two conditions which change expression of capsular polysaccharide, it was concluded that expression of other, noncapsular factors by cryptococci grown under these conditions was not responsible for the increased stimulation of C3 detected when the highly encapsulated isolate was used to stimulate PC cultures. Examination of the ability of purified cryptococcal capsular polysaccharide to induce C3 from PC. To further identify cryptococcal capsular polysaccharide as the inducer of C3 in this system, PC were cocultured with the purified GXM fraction of C. neoformans. GXM was added to the cultures in doses of 100 to 3.13 g per ml. The greatest amount of C3 was detected when the PC were exposed to 50 to 100 g of purified GXM per ml (Fig. 4). However, stimulation greater than background levels occurred when GXM was added at doses as low as 3.13 g per ml. Contribution of endotoxin to the simulation of C3 synthesis by PC. Having observed that GXM concentrations of 6.25 and FIG. 4. Stimulation of C3 synthesis by PC cultured in the presence of highly encapsulated cryptococci (NU-2 RPMI) or purified cryptococcal capsular polysaccharide (GXM) at the concentrations indicated (micrograms per milliliter). Data are expressed as mean units of C3 (per milliliter) SEM. Significant enhancement above background (BG) levels (P ) was found with yeast cells and all doses of GXM g per ml stimulated about the same levels of C3 synthesis and that the level was significantly above the background level of C3, we decided to assess the possibility that endotoxin may be contaminating some of the solutions used and thereby augmenting C3 production. To analyze the contribution of endotoxin to our system, we blocked endotoxin activity with polymyxin B in doses ranging from 25 g per ml to 2.5 ng per ml. C3 secretion was inhibited by the addition of polymyxin B in doses as low as 0.25 g per ml. Polymyxin B inhibited both the background levels of C3 secreted by the PC and secretion of C3 stimulated with GXM (Table 1). These observations left open the question as to whether trace amounts of endotoxin found in the tissue culture medium acted synergistically with GXM or whether our GXM preparation contained enough endotoxin to initiate C3 secretion alone. Polymyxin B was tested for its ability to inhibit the induction of C3 secretion when highly encapsulated, heat-killed cryptococci were used as the stimulant. It is far less likely that endotoxin contaminated our heatkilled organisms because the cryptococci were grown in RPMI 1640 in 5% CO 2. This tissue culture medium did not contain Stimulant TABLE 1. Inhibition of GXM-stimulated C3 secretion by polymyxin B a Polymyxin dose ( g) C3 secreted (U/ml) ( SEM) None None 1,700 ( 275) ( 15) ,310 ( 181) GXM None 8,390 ( 242) ,430 ( 112) ,635 ( 35) a PC were cultured with or without 25 g of cryptococcal GXM in the presence or absence of various doses of polymyxin B. C3 levels in 5-day culture supernatants were measured by ELISA.

5 4118 BLACKSTOCK AND MURPHY INFECT. IMMUN. DISCUSSION FIG. 5. Stimulation of C3 synthesis by PC cultured in the presence of NU-2 or 184A grown in RPMI 1640 under ambient conditions for low-level (L) capsule synthesis or in RPMI 1640 grown at 37 C in 5% CO 2 for high-level (H) capsule synthesis in the presence or absence of 25 g of polymyxin B per ml. Data are expressed as mean units of C3 (per milliliter) SEM. BG, background. fetal calf serum, which is the major contributor of endotoxin in this system. In these experiments, polymyxin B inhibited C3 secretion induced by heat-killed cryptococcal cells (Fig. 5). After examination of several lots of tissue culture media, we found one lot in which the background levels of C3 secretion by the PC was very low and which did not support stimulation of C3 secretion by cryptococcal GXM. Using this lot of tissue culture medium, we studied the effect of stimulating PC cells with endotoxin alone, GXM alone, or various combinations of endotoxin and GXM (Fig. 6). GXM was added to the cultures at a dose of 25 g per ml, and endotoxin was added in various doses ranging from 0.25 to 0.03 ng per ml. In this lot of tissue culture medium, cryptococcal GXM (25 g per ml) did not stimulate PC to secrete C3. Combinations of lower doses of endotoxin, known to be incapable of stimulating C3 synthesis alone, with 25 g of GXM per ml resulted in synergistic stimulation of C3 secretion (Fig. 6). Doses of endotoxin required for this synergistic effect could be as low as 0.06 ng per ml. These data indicate that GXM is not able to stimulate C3 secretion alone but depends upon the combined effects of other signals, which were supplied in this study by extremely low doses of endotoxin. FIG. 6. Stimulation of C3 synthesis by PC cultured with GXM (25 g per ml) in the presence or absence of low doses of endotoxin (LPS). Data are expressed as mean units of C3 (per milliliter) SEM., significant enhancement of C3 secretion (P 0.001), compared to PC stimulated with 25 g of GXM alone. While a large body of information has been obtained concerning virulence factors and host responses to C. neoformans over the past 20 years, very few papers have compared relative virulence with differences in host responses to different cryptococcal isolates. In the current investigation, we have initiated such a comparison of two isolates of C. neoformans which vary widely in virulence. A direct comparison of the survival of CBA/J mice infected intratracheally with cryptococcal isolate NU-2 or 184A revealed that these two cryptococcal isolates are widely divergent in their pathogenic potential. Strain NU-2 killed 100% of mice by day 67 after intratracheal inoculation of 10 4 yeast cells, whereas only 40% of 184A-infected mice died over the same time period. Of those 184A-infected mice that survived beyond day 67, all survived until day 95 after infection, when the experiment was terminated. Since capsule size is a recognized virulence factor, we examined these two isolates for expression of capsule synthesis under conditions which are known to diminish capsule synthesis and under conditions known to enhance capsule production. The highly virulent isolate NU-2 was able to significantly upregulate capsule synthesis when grown under tissue culture conditions. Considering that tissue culture conditions are more like growth conditions in the body, we propose that the upregulation of capsule synthesis by NU-2 in vivo could contribute to the more aggressive infection caused by this isolate. Our findings in the mouse model are in accord with the observations of Granger et al. (17), who reported that cryptococcal variants that lost their ability to upregulate capsule synthesis under tissue culture conditions are less virulent in steroidtreated rabbits. It has been known for many years that the size of the cryptococcal capsule influences the ability of macrophages to engulf cryptococci (26), and, therefore, inhibition of clearance of cryptococci after the organism enters the body represents one mechanism whereby upregulation of capsular polysaccharide could contribute to the pathogenic potential of individual isolates. However, additional factors which contribute to the inflammatory response may also impact host resistance to cryptococcal isolates which vary in virulence. For instance, it has been previously shown that inflammatory reactions in the lungs of mice differ after infection with two C. neoformans isolates which differ in virulence (12). In that investigation, a weakly virulent isolate (strain 52D) induced a strong inflammatory response characterized by the presence of more neutrophils than macrophages on day 7 of infection and with macrophages predominating by day 14 of infection. In contrast, scant infiltrates were observed when mice were infected with the highly virulent cryptococcal isolate. Infiltrates detected with the virulent isolate were described as lymphoid in nature, and no neutrophils were found (12). The differences in inflammatory infiltrates to the two different isolates in the previous study showed that mechanisms responsible for eliciting an early neutrophil influx into the lung were quite different between these two isolates. One mediator which may be responsible for influx of neutrophils into infected tissues is C5a, a split product of the complement cascade known for its chemotactic activity for neutrophils. Complement components are synthesized by many different cell types in lungs and other tissues (36). The requirement for local synthesis of complement components could explain the delay of 7 days before neutrophils appeared in lungs of mice infected with a weakly virulent isolate of C. neoformans. To examine the relative abilities of the two cryptococcal isolates which we were studying to initiate C3 secretion, we initially chose to use an in vitro culture system. Normal

6 VOL. 65, 1997 STIMULATION OF C3 SYNTHESIS BY CRYPTOCOCCAL GXM 4119 unstimulated PC were cultured in the presence of cryptococcal strain 184A or NU-2. The two isolates were grown under conditions which favored capsule synthesis or under conditions in which capsule expression was low. Heat-killed cryptococci were cocultured with PC, and C3 secretion was determined by an ELISA. We initially predicted that expression of small amounts of capsule would favor C3 secretion. To our surprise, the opposite was true. C3 secretion in response to the highly encapsulated isolate, strain NU-2, was greater than that secreted in response to strain 184A. Growth under conditions which enhanced capsule expression enhanced the ability of NU-2 and 184A to elicit C3 secretion from PC cultures. However, the magnitude of the response was related to capsule size and the virulence of the isolate, with strain NU-2 consistently eliciting the greatest amount of C3 from the PC cultures. These results suggested that the cryptococcal capsular polysaccharide was responsible for eliciting C3 from the PC. Evaluation of the ability of nonencapsulated mutants of C. neoformans revealed that these isolates did not induce C3 synthesis by PC. Comparison of C3 secretion by PC stimulated with nonencapsulated cryptococcal mutants grown in tissue culture medium with and without CO 2 indicated that other cryptococcal components, potentially upregulated in CO 2, were not responsible for the stimulation of C3 secretion in the PC cultures. Purified cryptococcal GXM also induced C3 from PC, confirming the role of the capsule in eliciting the response. Because the GXM preparation was not heated, as were the whole-cell preparations, these experiments ruled out effects of heat on the cryptococcal capsule which might have contributed to the results obtained when yeast cells were used as the stimulant. During the 5-day culture period, no phagocytosis occurred when encapsulated cryptococci were used as stimulants, while the nonencapsulated mutants were phagocytized during the first few hours of culture. Although C3 is synthesized in culture, the amount produced is apparently insufficient to provide adequate opsonization of the encapsulated cryptococcal isolates. Phagocytosis of the nonencapsulated isolates might explain why they did not induce C3 secretion; however, C3 was secreted in response to phagocytized Candida albicans (data not shown). Goodrum (16) reported that stimulation of C3 from macrophages by group B streptococci could be inhibited by cytochalasin B but that this treatment did not inhibit induction of C3 by endotoxin. The phagocytic process per se was not sufficient to induce C3 from macrophages, since ingestion of latex beads did not induce C3. These observations reveal that macrophages utilize a variety of signals for the secretion of C3 and that the signals used may vary depending upon the nature of the stimulant provided. The fact that soluble GXM also induced C3 secretion by PC suggests that receptor-mediated mechanisms are responsible for the C3 secretion induced by C. neoformans. We examined the ability of polymyxin B to inhibit the induction of C3 secretion by cryptococcal GXM to control for possible endotoxin contamination of our GXM preparations. In these experiments, polymyxin B was able to completely block C3 secretion induced by GXM. In addition, polymyxin B inhibited background C3 levels detected in cultures of PC which were not stimulated with GXM. These results suggest that low levels of endotoxin, known to be in tissue culture media containing fetal calf serum, could be responsible for the background levels of C3 detected. Based on these findings, we assessed the possibility of synergistic interactions between endotoxin and cryptococcal GXM in the induction of C3 by PC. It is also important to mention that experiments which utilize polymyxin B as an inhibitor of endotoxin activity must be viewed with caution. First, polymyxin B has been reported to stimulate cytokines and C3 (8, 19) from macrophages. Although we did not see secretion of C3 by PC cells exposed to 25 g to 2.5 pg of polymyxin B, we have no information regarding induction of cytokines in our experiments. Second, polymyxin B is a polycationic molecule, and it is possible that it can interact directly with GXM, which is polyanionic. Our GXM preparations were made under conditions to minimize endotoxin contamination to the greatest extent possible. However, it is virtually impossible to obtain GXM which does not have the potential for endotoxin contamination. Because the glucuronic acid present in GXM causes positive results in the Limulus assay (28), it is not possible to confirm that GXM preparations are free of endotoxin. To examine the potential role of endotoxin in our system in another way, we first studied the ability of polymyxin B to inhibit stimulation of C3 secretion induced by encapsulated cryptococci grown under low-level endotoxin conditions. In these experiments, cryptococci were grown in RPMI 1640 which did not contain fetal calf serum. For low-level capsule expression, the cultures were grown at room temperature in ambient air, and for high-level capsule expression, the cultures were incubated in 5% CO 2 at 37 C. Since our tissue culture medium contained undetectable amounts of endotoxin, these cryptococcal yeast cells were free of significant endotoxin contamination. When polymyxin B was used to block C3 secretion in cocultures of PC and encapsulated yeast, the polymyxin was able to completely inhibit the secretion of C3. Although this experiment further implicated endotoxin as a costimulant of C3 secretion in this system, the data were not conclusive. We examined several lots of tissue culture medium for their ability to support GXM-induced C3 secretion. One lot was identified in which GXM did not induce PC to secrete C3. Using this lot of tissue culture medium, we directly demonstrated that the addition of small amounts of endotoxin in the presence of 25 g of cryptococcal GXM per ml stimulated C3 from murine PC synergistically. In these experiments, doses of endotoxin as low as 0.06 ng per ml acted as an effective costimulant. When tested alone, this dose of endotoxin did not provide sufficient stimulus for the secretion of C3. The identification of a lot of tissue culture medium which does not support secretion of C3 from PC after stimulation with GXM is rare, and these results show that the additive accumulation of endotoxin in tissue culture medium after the addition of all components is sufficient to provide the amount of endotoxin needed for costimulation of C3 secretion in the presence of cryptococcal GXM. In this system, endotoxin may provide a priming stimulus which allows the PC to express receptors needed for GXM stimulation or it may induce other cytokines from the PC which are needed as costimulatory molecules. The results of this investigation reveal that C3 secretion can be stimulated by cryptococci from PC cultures. In vivo endotoxin is not available as a costimulant for the induction of C3 secretion. However, the regulation of C3 synthesis by cytokines is complex, and in vivo costimulation could be provided by one or more of these molecules. In particular, interleukin 6 (IL-6) is a potent inducer of C3 (31), and low levels of IL-6 along with GXM could provide the necessary signals responsible for increasing C3 synthesis in tissues. Other cytokines have also been reported to upregulate C3 secretion, and these include IL-1, macrophage colony-stimulating factor (M-CSF), and gamma interferon (1, 31, 38). Gamma interferon has also been reported to downregulate the C3 mrna response (11). Granulocyte-macrophage CSF (GM-CSF) downregulates C3 secretion elicited by endotoxin and by Candida species (18), and GM-CSF mrna expression is inhibited in NK cells exposed to cryptococci (27a). Because the capsular polysaccharide can induce expression of IL-1 and IL-6 (32) and potentially down-

7 4120 BLACKSTOCK AND MURPHY INFECT. IMMUN. regulate GM-CSF expression in vivo, the combination of these factors could allow cryptococci with large capsules to significantly upregulate synthesis of C3 and possibly other complement components. The correlation between increased C3 secretion and virulence of the organism is not one that we expected. However, it is of interest to note that a similar correlation has been made with human peripheral blood mononuclear cells stimulated with Candida species (20). One would expect that increases in C3 secretion would enhance the inflammatory responses generated upon infection with C. neoformans. The cryptococcal capsule is known to activate the alternative complement pathway and release complement split products which are known to be chemotactic for neutrophils. While C5a is known to enhance chemotaxis (35) and participate in the elicitation phase of delayed-type hypersensitivity responses (43), C3a can exert a negative influence on the development of immunity, including inhibition of NK activity, inhibition of antibody responses (27), and downregulation of TNF- and IL-1 expression by macrophages (41). Once encapsulated cryptococci induce sufficient expression of complement components, then activation of the alternative pathway with the generation of C3a could significantly alter the inflammatory response elicited to these highly encapsulated isolates, as compared to cryptococcal isolates which express a relatively small amount of capsule. The importance of the expression of TNF- early in lung infections with cryptococci (21) suggests that the effects of C3a on TNF- expression could play a pivotal role in the ability of the host to elicit protective anticryptococcal immune responses. Alternatively, activation of C3 by large amounts of GXM may deplete C5 in the local tissue environment. It has been known for many years that mice which are deficient in C5 are more susceptible to cryptococcal infection than those which are not (33). Our current investigations are directed at examining the ability of these two cryptococcal isolates to upregulate C3 synthesis in lung tissues where basal complement levels are low and to determine the influence that C3 and C3 split products play in the generation of the early inflammatory response which develops as a result of infection with C. neoformans. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. We are indebted to Ronald Greenfield for the statistical analysis of our survival experiments. REFERENCES 1. Andoh, A., Y. Fujiyama, K. Kitoh, M. Niwakawa, K. Hodohara, T. Bamba, and S. 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