INFECTION AND IMMUNITY, Mar. 2001, p. 1808–1815 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.3.1808–1815.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
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Cryptococcus neoformans Induces Macrophage Inflammatory Protein 1␣ (MIP-1␣) and MIP-1 in Human Microglia: Role of Specific Antibody and Soluble Capsular Polysaccharide DAVID GOLDMAN,1,2* XIANYUAN SONG,3 RYUHEI KITAI,3 ARTURO CASADEVALL,2,4 MENG-LIANG ZHAO,3 AND SUNHEE C. LEE3 Departments of Pediatrics,1 Pathology,3 Medicine,4 and Microbiology and Immunology,2 Albert Einstein College of Medicine, Bronx, New York Received 25 October 2000/Returned for modification 7 December 2000/Accepted 12 December 2000
We characterized the expression of the -chemokines macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1, and RANTES by primary human microglia after exposure to Cryptococcus neoformans. In the absence of specific antibody, C. neoformans failed to elicit a chemokine response, while in the presence of specific antibody, microglia produced MIP-1␣ and MIP-1 in amounts comparable to those induced by lipopolysaccharide. RANTES was also induced but at much lower levels. In addition to MIP-1␣ and MIP-1 mRNA, we observed a robust induction of monocyte chemoattractant protein 1 and interleukin-8 mRNA following incubation of microglia with opsonized C. neoformans. In contrast, cryptococcal polysaccharide did not induce a chemokine response even when specific antibody was present and inhibited the MIP-1␣ induction associated with antibody-mediated phagocytosis of C. neoformans. The role of the Fc receptor in the observed chemokine induction was explored in several experiments. Treatment of microglia with cytochalasin D inhibited internalization of C. neoformans but did not affect MIP-1␣ induction. In contrast, treatment with herbimycin A, a tyrosine kinase inhibitor, inhibited MIP-1␣ induction. Microglia stimulated with immobilized murine immunoglobulin also produced MIP-1␣ and RANTES (MIP-1␣ > RANTES). Our results show that microglia produce several chemokines when stimulated by C. neoformans in the presence of specific antibody and that this process is likely to be mediated by Fc receptor activation. This response can be down-regulated by cryptococcal capsular polysaccharide. These findings suggest a mechanism by which C. neoformans infections fail to induce strong inflammatory responses in patients with cryptococcal meningoencephalitis and have important implications for antibody therapy. Cryptococcus neoformans is a fungal pathogen that is remarkable for its ability to cause central nervous system (CNS) infections (6, 7, 10, 14). C. neoformans elicits a wide range of tissue responses (30). There is evidence that the variability in tissue inflammatory response is due to both host immune status (30) and attributes of fungal cells including the polysaccharide capsule and phenotypic switching (20). Granulomatous inflammation is the tissue response associated with control of infection (30). In most patients with AIDS, however, cryptococcal meningoencephalitis is associated with minimal inflammation (2, 39). There is increasing evidence that microglia play a central role in the host response in cryptococcal meningoencephalitis. Microglia can ingest and limit the growth of C. neoformans (4, 31). Histopathological studies of AIDS patients with cryptococcal meningoencephalitis have shown that perivascular microglia act as important phagocytes for C. neoformans (30). Glucuronoxylomannan (GXM), the main constituent of the C. neoformans polysaccharide capsule, is found in proximity to and inside microglia during cryptococcal meningoencephalitis (29). GXM has powerful immunoregulatory effects that in-
clude cytokine dysregulation, shedding of selectin, and inhibition of leukocyte migration (3, 15, 28, 38, 42). The role of microglia in regulating the inflammatory response in cryptococcal meningoencephalitis is poorly understood. Among the factors which are necessary to generate an appropriate inflammatory response are the production of proinflammatory cytokines and chemokines (34, 43). The ␣-chemokine interleukin-8 (IL-8) is an important chemoattractant for neutrophils, and the -chemokines macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1 are involved in chemoattraction of T cells and monocytes. Monocyte chemoattractant protein 1 (MCP-1) has also been shown previously to be important in recruitment of monocytes to the brain (46). Many of these chemokines are produced by activated microglia and astrocytes after stimulation with endotoxin and proinflammatory cytokines (22, 35), but little is known about their production following interaction with microorganisms. In addition, some chemokines also function as modulators of human immunodeficiency virus type 1 (HIV-1) infection in the brain (1, 21, 27). These include -chemokines that bind to the chemokine receptor CCR5, namely, RANTES, MIP-1␣, and MIP-1. Since cryptococcal meningoencephalitis is often associated with AIDS, C. neoformans-induced chemokines could also modulate HIV-1 infection in microglia. CCR5 may also have a crucial role in defense against C. neoformans in the CNS. Studies of mice with targeted deletions of the CCR5 gene demonstrate that these mice cannot mount appropriate
* Corresponding author. Mailing address: Department of Pediatrics, Golding 703, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-4259. Fax: (718) 430-8701. E-mail:
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inflammatory responses in the brain against C. neoformans infection, although normal inflammatory responses are observed in the lung (24). In this report, we examine the chemokine expression of human microglia in response to C. neoformans exposure in the presence and absence of capsule-specific antibody and determine how this response is modified by capsular polysaccharide. MATERIALS AND METHODS Microglia. This study is part of an ongoing research protocol that has been approved by the Albert Einstein College of Medicine Committee on Clinical Investigations. Informed consent was obtained from participants. Fetal brains were obtained from elective terminations of pregnancy from healthy women with no risk factors for HIV-1 infection. Fetal microglia were cultivated from secondtrimester abortuses as described previously (31, 32). Briefly, the brain tissues were mechanically and enzymatically dissociated and passed through nylon meshes with 130- and 230-m pores to generate a suspension of mixed brain cell populations. Cells were seeded at 108 cells per 75-cm2 tissue culture plate in medium (Dulbecco modified Eagle medium with 4.5 g of glucose/liter, 4 mM L-glutamine, and 25 mM HEPES buffer) supplemented with 5% fetal calf serum, penicillin (100 U/ml), streptomycin (100 g/ml), and amphotericin B (Fungizone; 0.25 g/ml; Life Technologies, Bethesda, Md.). After 2 weeks of culture, microglia were harvested by aspiration of culture medium, pelleted, and seeded in 96-well culture plates at a density of 4 ⫻ 104 cells per well. Microglia medium was the same as mixed culture medium but without amphotericin B. Organism. C. neoformans American Type Culture Collection strain 24067, a serotype D strain, was used in this study. Serotype D strains are responsible for most cases of cryptococcal meningoencephalitis in certain parts of the world, especially northern Europe. In New York City, N.Y., serotype D strains make up 15% of clinical cryptococcal isolates (44). Cells were grown in Sabouraud dextrose broth in a rotary shaker at 30°C until stationary phase. Cells were then washed three times in sterile phosphate-buffered saline and counted with a hemocytometer. Cryptococcal polysaccharide. Polysaccharide was prepared from culture supernatant of strain 24067 by alcohol precipitation followed by repeated extractions with 1:1 chloroform-butanol solution, as described previously (16). Polysaccharide consists of more than 90% GXM and for this study is referred to as GXM (9). Antibodies. Protein G-purified, murine monoclonal immunoglobulin G1 (IgG1) antibodies from two different hybridomas (2H1 and 18B7) that bind GXM (monoclonal antibody [MAb]) were used in this study (8, 37). These antibodies are similar in both characteristics of binding to GXM and biological activity in vivo and were used interchangeably. The Limulus lysate assay (BioWhittaker, Walkersville, Md.) revealed endotoxin levels of ⬍0.1 endotoxin unit in the antibody preparations. Inoculation of microglia with C. neoformans or soluble polysaccharide. C. neoformans cells were added to microglia cultures at 4 ⫻ 105 cells per well to yield a C. neoformans cell/microglia ratio of 10:1 in the presence or absence of MAb. After 90 min of incubation at 37°C, nonphagocytosed C. neoformans cells were removed by washing the cells twice. Cultures were fed again with fresh medium. Soluble GXM at 0.1 to 50 g/ml was added to microglial cultures in the presence and absence of MAb and incubated for 90 min, and unbound GXM was removed by washing the cells twice. Some cultures were inoculated with both C. neoformans cells and GXM. For these experiments, fungal cells and GXM were added simultaneously and cultures were washed after 90 min. Induction of chemokines by immobilized immunoglobulin. Ninety-six-well Nunc-Immuno plates with a Maxisorp surface (Nunc, Naperville, Ill.) were precoated with various concentrations of normal mouse IgG2a or IgG1 (PharMingen, San Diego, Calif.) in phosphate-buffered saline at 37°C for 1 h. The plates were washed twice with phosphate-buffered saline, and then microglia were seeded at 4 ⫻ 104 cells per well. Chemokine levels were determined by enzymelinked immunosorbent assay (ELISA) after 16 h. Microglia seeded on wells coated with vehicle alone served as controls. Effects of cytochalasin D. Cytochalasin D (Sigma) in dimethyl sulfoxide (DMSO) was added to microglial cultures at 2 to 40 M, and cultures were challenged with C. neoformans in the presence of MAb. Cultures were washed after 90 min to remove nonadherent organisms and suspended in medium containing cytochalasin D. Levels of MIP-1␣ were determined by ELISA after 16 h. DMSO alone (0.02%) did not alter MIP-1␣ production. Effects of herbimycin A. Herbimycin A (Calbiochem) was diluted in DMSO and then added to microglial cultures at a final concentrations of 10 ng/ml to 1
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g/ml. One hour later, cultures were challenged with C. neoformans and MAb. Levels of MIP-1␣ were determined by ELISA after 16 h. Lactate dehydrogenase efflux (Sigma) was measured in parallel to determine the effect of herbimycin A on cell death. Chemokine ELISAs. The concentrations of chemokines (MIP-1␣, MIP-1, and RANTES) in microglial culture supernatants were determined by ELISA using ELISA kits from R&D Systems (Minneapolis, Minn.). For some experiments, ELISA was performed using capture and detection antibody pairs from R&D Systems. The sensitivities of detection were similar in the two ELISA systems. Microglial culture supernatants were diluted 1:5 to 1:10 before ELISA. RNase protection assay. RNA was extracted from 6 ⫻ 105 microglial cells using TRIZOL (Molecular Research Center, Cincinnati, Ohio), according to the manufacturer’s instructions. RNA was analyzed using the PharMingen human chemokine RNase protection assay kit according to the manufacturer’s instructions. Images were developed by autoradiography, and densitometry was performed using Ambis ImageQuant software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used as a loading control. GXM immunohistochemistry. The immunohistochemistry assay for GXM was performed as described previously (29). Cells were fixed with methanol and incubated with another MAb against GXM, 3E5, at a concentration of 2 g/ml. The 3E5 MAb used here is a murine IgG2a antibody. Secondary antibody was horseradish peroxidase-labeled goat anti-mouse IgG2a (Southern Biotechnology, Birmingham, Ala.). Color was developed with diaminobenzidine. Statistics. Chemokine levels and phagocytic indices were compared using the Student t test. P values of ⬍0.05 were considered significant.
RESULTS C. neoformans induces MIP-1␣, MIP-1, and RANTES production in microglia. Control microglia treated with medium alone produced low levels of chemokines, while treatment of microglia with lipopolysaccharide (LPS) resulted in a marked induction of all three chemokines as described previously (27, 35) (Fig. 1). Treatment with C. neoformans in the absence of antibody to GXM (MAb) induced little or no MIP-1␣, MIP1, or RANTES (Fig. 1). In contrast, the combination of C. neoformans and MAb consistently resulted in the induction of large amounts of MIP-1␣ and MIP-1 with levels comparable to those produced by LPS-treated microglia (Fig. 1A and B). RANTES production was also increased in cultures treated with both C. neoformans and MAb relative to controls, but the concentration was much lower than that induced by LPS (Fig. 1C). Occasionally, microglial cultures treated with MAb alone released detectable amounts of MIP-1␣ and MIP-1 (for example, see Fig. 3B; also see Discussion). Immobilized immunoglobulin induces chemokine production in microglia. Because C. neoformans induced chemokines only in the presence of MAb, and because MAb alone also sometimes induced chemokines, we tested whether microglial Fc receptor (FcR) cross-linking could result in chemokine production. To determine this, microglia were plated on tissue culture wells precoated with known concentrations of normal mouse immunoglobulins (IgG2a and IgG1). Levels of MIP-1␣ and RANTES were then determined by ELISA after 16 h. Cells treated with LPS or medium alone without immunoglobulin coating served as controls. Figure 2 shows that immobilized IgG2a induced both MIP-1␣ and RANTES in microglia in a dose-dependent manner (0.6 to 100 g/ml) and that IgG2a was more potent than IgG1 when used at 25 g/ml (P ⬍ 0.05). Immobilized immunoglobulins had different potencies for the induction of -chemokines, with the IgG2a concentration that induced 50% of the LPS effect being approximately 1 g/ml for MIP-1␣ and 25 g/ml for RANTES. Soluble C. neoformans capsular polysaccharide does not induce MIP-1␣ in microglia. Next, we determined if soluble
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FIG. 1. Production of MIP-1␣, MIP-1, and RANTES by human microglia exposed to C. neoformans and MAb. Microglia at 4 ⫻ 104 cells per well were stimulated with 4 ⫻ 105 C. neoformans cells (CN), 20 g of anti-GXM MAb per ml (MAb), or a combination of both (CN ⫹ MAb) for 1.5 h. Cultures were then washed to remove extracellular C. neoformans and MAb. After addition of fresh medium, cells were incubated for another 16 h; then chemokine levels were determined in the supernatants by ELISA. Positive and negative controls consisted of microglia treated with 10 ng of LPS/ml and with medium alone, respectively. The results are means ⫾ standard deviations from triplicate wells. (A) MIP-1␣. Induction of MIP-1␣ was detected in all cases in cultures treated with C. neoformans plus MAb (ⴱ, P ⬍ 0.05 compared with controls [medium or C. neoformans]), in amounts comparable to those induced by LPS. The results of an experiment representative of 11 separate experiments are shown. (B) MIP-1 . Induction of MIP-1 was seen for microglia following inoculation with C. neoformans and MAb in all cases (ⴱ, P ⬍ 0.05 compared with medium- or C. neoformans-treated controls). The results of an experiment representative of four separate experiments are shown. (C) RANTES. Small but significant (ⴱ, P ⬍ 0.05) amounts of RANTES were induced in cultures treated with C. neoformans plus MAb, at levels much lower than those induced by LPS. The results of an experiment representative of three separate experiments are shown.
cryptococcal capsular polysaccharide (GXM) could induce MIP-1␣ in the presence or absence of MAb. Microglial cultures were incubated with 20 g of MAb/ml in the presence of various concentrations of GXM (0.1, 1, and 10 g/ml), and
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FIG. 2. Immobilized immunoglobulin induces chemokine production in microglia. Microglia were seeded on plates coated with known concentrations of normal mouse IgG1 (25 g/ml) or IgG2a (0.4 to 100 g/ml, as indicated), and then chemokine production was examined by ELISA after 16 h. Controls were microglia seeded on uncoated plates and treated with medium alone (media) or 10 ng of LPS/ml. Concentration-dependent induction of both MIP-1␣ (A) and RANTES (B) was observed with immobilized IgGs, with IgG2a being more potent than IgG1 (ⴱ, P ⬍ 0.05). Values are means ⫾ standard deviations from triplicate wells. Results are representative of two separate experiments with similar results.
levels of MIP-1␣ were determined (Fig. 3A). GXM alone or in combination with antibody did not induce MIP-1␣ in microglia, while C. neoformans plus antibody or LPS readily induced this chemokine. Increasing the concentration of GXM to 200 g/ml failed to elicit a chemokine response (data not shown). To determine if MAb failed to induce internalization of soluble polysaccharide in microglia, cultures were examined for GXM by immunohistochemistry. In cultures incubated with GXM plus MAb, immunostaining demonstrated internalized polysaccharide in microglia (Fig. 4B), while almost no staining was detected in cultures treated with GXM alone (Fig. 4A). Thus, in the presence of MAb, soluble polysaccharide was internalized in microglia presumably via FcRs, and yet, unlike phagocytosis of C. neoformans fungal cells, opsonized polysaccharide failed to induce MIP-1␣. Addition of capsular polysaccharide inhibits MIP-1␣ induction in microglia by antibody-opsonized C. neoformans. Since large amounts of shed GXM coexist with the fungal organisms in the CNS of patients with cryptococcal meningoencephalitis (29), we determined the effect of GXM on the production of MIP-1␣ in microglia triggered by antibody-opsonized C. neo-
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FIG. 3. Production of MIP-1␣ by microglia exposed to GXM. MIP-1␣ levels were determined in microglial cultures exposed to various concentrations of C. neoformans polysaccharide (GXM) in the presence or absence of 20 g of MAb/ml. (A) Microglia were exposed to medium alone (media); MAb against GXM alone (MAb); polysaccharide alone at 1 g/ml (GXM); or a combination of MAb and GXM at 0.1, 1, and 10 g/ml. Positive controls were microglia exposed to C. neoformans and MAb as described in the Fig. 1 legend. (B) Microglia were exposed to C. neoformans and MAb as described in the Fig. 1 legend, in the presence or absence of added soluble polysaccharide (GXM). All reagents were added simultaneously and washed out after a 90-min incubation. Cultures were treated with medium alone (media), MAb alone, or C. neoformans plus MAb (CN ⫹ MAb) in the presence of GXM at 0, 10, or 50 g/ml. After addition of fresh medium, chemokine levels were determined in 16-h culture supernatants. Values are means ⫾ standard deviations from triplicate wells. Similar results were found in three separate experiments. Asterisks in panel B denote P values of ⬍0.05 compared with cultures containing no GXM.
formans. Addition of 10 and 50 g of GXM/ml significantly inhibited the production of MIP-1␣ by C. neoformans plus MAb (Fig. 3B). Addition of GXM to LPS-stimulated microglia did not inhibit MIP-1␣ production (data not shown). Immunohistochemistry for GXM in these cultures demonstrated that phagocytosis of C. neoformans occurred in both cultures but was decreased in cultures treated with GXM (Fig. 4C and D). In cells incubated with both GXM and C. neoformans, light
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cytoplasmic staining for GXM was noted in addition to phagocytosed C. neoformans, indicating internalized GXM (Fig. 4D). mRNAs for multiple chemokines are induced by antibodyopsonized C. neoformans and inhibited by polysaccharide. The expression of RANTES, MIP-1␣, MIP-1, MCP-1, I-309, IP10, and IL-8 mRNA was measured in microglia using a multiprimer RNase protection assay (Fig. 5). Consistent with previous reports (22), we detected low levels of mRNA for RANTES, MIP-1, MIP-1␣, and MCP-1 and abundant mRNA for IL-8 in control microglia. IP-10 and I-309 were not detected. In microglia treated with either MAb, GXM at 50 g/ml, or C. neoformans alone, chemokine mRNA accumulation was not detected. Chemokine mRNA was not induced in cultures treated with GXM plus antibody, but MIP-1, MIP1␣, MCP-1, and IL-8 mRNA were abundantly expressed in microglia treated with C. neoformans plus MAb, and this was potently inhibited by addition of soluble GXM. In cultures treated with LPS, induction of all chemokine genes including those for RANTES and I-309 was detected as reported previously (22). Thus, the results of these experiments showed that antibody-opsonized C. neoformans can elicit accumulation of multiple chemokine mRNAs in microglia. Importantly, addition of GXM almost completely abrogated chemokine mRNA accumulation induced by opsonized C. neoformans in microglia. Cytochalasin D inhibits antibody-mediated phagocytosis of C. neoformans but does not reduce MIP-1␣ induction. To determine whether internalization of organisms was required for the chemokine induction associated with antibody-mediated phagocytosis of C. neoformans, we conducted experiments using cytochalasin D. Cytochalasin D inhibits actin polymerization and thereby interferes with phagocytosis, without interfering with the antibody-mediated attachment. In microglial cultures treated with cytochalasin D and challenged with opsonized C. neoformans, no internalized organisms were observed, although many organisms were attached to microglia (Fig. 6A and B). In spite of the striking differences in phagocytosis, no differences in MIP-1␣ levels were observed in association with cytochalasin D treatment compared to controls (Fig. 6C). Tyrosine kinase inhibitor herbimycin A abolishes chemokine induction by antibody-opsonized C. neoformans. Tyrosine phosphorylation is a key event involved in several signal transduction pathways, including FcR signaling (12, 25). Thus, we determined the role of tyrosine kinase in C. neoformans-plusMAb-mediated chemokine induction by use of a specific inhibitor, herbimycin A. MIP-1␣ induction by C. neoformans and MAb was potently inhibited by herbimycin A with the 50% inhibitory concentration being approximately 30 nM (Fig. 7). Herbimycin A did not affect phagocytosis (data not shown). The induction of MIP-1␣ by LPS was also inhibited by herbimycin A, with a similar 50% inhibitory concentration (Fig. 7). Herbimycin A did not induce microglial cell toxicity as determined by the trypan blue exclusion test and lactate dehydrogenase efflux (data not shown). These results support the idea that tyrosine phosphorylation is required for both C. neoformans-plus-MAb- and LPS-induced MIP-1␣ release in microglia.
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FIG. 4. GXM immunohistochemistry. Control microglia (A) or microglia exposed to MAb plus GXM (B), MAb plus C. neoformans (C), or MAb plus GXM plus C. neoformans (D) were examined by immunohistochemistry for GXM as described in Materials and Methods. GXM and MAb were added at 50 and 20 g/ml, respectively. Cultures were washed to remove unbound GXM or C. neoformans 90 min after incubation and were fixed after another 2 h. In the presence of MAb, there is uptake of GXM (B) or C. neoformans (C). The combination of GXM and C. neoformans resulted in a decrease in the number of intracellular C. neoformans fungal cells (D).
DISCUSSION The findings described in this paper are that C. neoformans can induce multiple chemokines in human fetal microglia in the presence of specific antibodies. We demonstrate by RNase protection assay the induction of mRNA for the -chemokines MIP-1␣, MIP-1 and MCP-1 and the ␣-chemokine IL-8. The induction of MIP-1␣ and MIP-1 was confirmed by ELISA. No increases in RANTES mRNA were detected by RNase protection assay, but a small increase in protein was detected by ELISA. C. neoformans-mediated induction of chemokines has been shown previously for monocytes. Huang and Levitz have shown that peripheral blood mononuclear cells release MIP-1␣, MIP1, and RANTES following exposure to C. neoformans (23). The induction of -chemokines required the presence of serum and was lost by heat inactivation, suggesting that complement-mediated phagocytosis was necessary for that process. Our measurements were conducted in the presence of heatinactivated serum, and they show a requirement for opsonizing antibody for chemokine induction. In our study, the involvement of FcR in the microglial chemokine response was suggested by several experiments. First, chemokine induction by C. neoformans required the presence of specific antibody. Second, occasional cultures treated with MAb alone demonstrated induction of MIP-1␣ and MIP-1. This is likely to be related to FcR cross-linking induced by the presence of antibody aggregates which can activate FcR in the absence of antigen. A similar phenomenon has been reported previously to account for the induction of IL-8 by monocytes by intravenous immunoglobulin (13). Third, microglia plated on immobilized immunoglobulin showed concentration-dependent chemokine production, with IgG2a being more efficient
than IgG1. This is consistent with the fact that mouse IgG2a is known to bind to human Fc␥RI with a higher affinity than that of IgG1 (33, 40, 41). Taken together, these results support a role for FcR in microglial chemokine expression. Involvement of FcRs in the induction of chemokines has also been demonstrated previously for mast cells (Fc⑀RI) and mesangial cells (Fc␣R) (17, 47). In monocytes, coengagement of Fc␥R (I or II) and intercellular adhesion molecule 3 has been shown previously to induce chemokine secretion (26). Although our study demonstrated that MAb-mediated MIP-1␣ induction was accompanied by MAb-mediated phagocytosis of C. neoformans, experiments with cytochalasin D demonstrated that these two events can be dissociated. While cytochalasin D completely inhibited MAb-mediated phagocytosis, it had no effect on MIP-1␣ induction. These results demonstrate that FcR-mediated C. neoformans internalization was dispensable for FcR signaling. In microglia, herbimycin A produced a dose-dependent inhibition of MIP-1␣ by opsonized C. neoformans, indicating a role for tyrosine kinase in this process. FcRs signal through the immunoreceptor tyrosinebased activation motifs that connect the ligand binding module with intracellular effectors of signal transduction pathways (12, 19, 25). Tyrosine kinases of the src and syk family have been shown to play crucial roles in signaling through the immunoreceptors (45). In addition, mitogen-activated protein kinases have been shown previously to be activated following FcR engagement (11). Thus, multiple kinases that could serve as a target for herbimycin A exist within the FcR signaling pathway. Elucidation of the FcRs and the signals involved in the induction of chemokine genes in microglia requires further study. While C. neoformans was able to induce chemokines in microglia in the presence of MAb, GXM at microgram-per-mil-
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FIG. 5. Induction of microglial chemokine mRNA by C. neoformans and MAb. Microglia were exposed to medium alone (control), antibody alone (MAb), GXM alone (GXM; 50 g/ml), C. neoformans alone (CN), GXM plus MAb, C. neoformans plus MAb, or C. neoformans plus MAb plus GXM for 90 min. Cultures were washed out to remove unbound C. neoformans or GXM and then further incubated for another 2 h. Sister microglial cultures treated with LPS (10 ng/ml) for 3.5 h were used as a control. Total RNA was analyzed for the expression of chemokine mRNAs with a human chemokine multiprimer RNase protection assay kit (A). Densitometric analysis was performed using GAPDH as the loading control (B). Results are representative of three independent experiments with similar findings.
liliter concentrations failed to induce chemokines. These results are interesting in light of our observations that GXM was internalized in microglia in the presence of antibody and that the same concentrations of GXM-MAb complexes have been shown previously to stimulate murine macrophage nitric oxide production (36). Furthermore, we found that GXM dose dependently inhibited the induction of chemokines in microglia by C. neoformans-MAb. We hypothesize that GXM-MAb elicits different signals than do C. neoformans-MAb complexes and
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FIG. 6. Cytochalasin D inhibits internalization of MAb-opsonized C. neoformans but does not alter MIP-1␣ induction. (A and B) Microglia were incubated with C. neoformans and MAb as described above in the absence (A) or presence (B) of 2 M cytochalasin D. After a 90-min incubation, cultures were washed to remove nonadherent organisms, suspended in medium with or without cytochalasin D, and allowed to incubate for 16 h. (C) MIP-1␣ levels in supernatants from these microglial cultures were determined by ELISA as described above.
that the observed decrease in chemokine production may have resulted from decreased binding of C. neoformans-MAb complexes to microglial FcRs. More importantly, the inhibition of microglial chemokine production by polysaccharide observed in our study suggests that the soluble GXM found in brain and cerebrospinal fluid in patients with cryptococcal meningoencephalitis may inhibit chemokine production by microglia and adds to the list of immunosuppressive effects of cryptococcal polysaccharide. Many patients with cryptococcal meningoencephalitis also harbor HIV-1 in the CNS (29, 30). Since some of the chemokines shown to be induced by C. neoformans in this study are also known to inhibit HIV-1 infection in microglia, our results suggest that C. neoformans and polysaccharide can modulate HIV-1 infection by affecting chemokine production. Our results showing suppression of microglial production
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FIG. 7. Herbimycin A (HA) inhibits MIP-1␣ production by MAbopsonized C. neoformans. Microglia were pretreated with herbimycin A for 1 h at 10 nM, 100 nM, and 1 M concentrations and then incubated with MAb and C. neoformans or LPS as described in the Fig. 1 legend. MIP-1␣ levels were determined by ELISA after 16 h. A dose-dependent inhibition of MIP-1␣ production by herbimycin A was observed for cultures treated with C. neoformans plus MAb or LPS. ⴱ, P ⬍ 0.05 compared with 0 nM herbimycin A.
of HIV-1-inhibitory chemokines such as MIP-1 by polysaccharide suggest a mechanism by which polysaccharide can enhance HIV-1 infection in the CNS. In summary, our results suggest a mechanism by which C. neoformans infections fail to induce strong inflammatory responses in patients with cryptococcal meningoencephalitis as a result of microglial nonresponsiveness to this fungus and its capsular polysaccharide. These in vitro observations suggest a mechanism by which cellular immunity can be enhanced by capsule-specific antibody. In support of this notion, Feldmesser and Casadevall have shown previously that mice treated with antibody to GXM have significantly enhanced granulomatous inflammation in response to infection with C. neoformans (18). A MAb (18B7) is presently undergoing clinical evaluation for adjunctive therapy for human cryptococcosis (5). These results provide encouragement for the development and use of antibody therapies for the treatment of cryptococcosis. ACKNOWLEDGMENTS We are grateful to Liwei Hua for help with the RNase protection assay. Tissue was obtained from the Einstein Fetal Tissue Repository. This work was supported by grants AI44641 and MH55477 (S.C.L.). D.G. and X.S. contributed equally to this work.
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