Opsonic Requirements for Dendritic Cell-Mediated Responses to Cryptococcus neoformans
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感染与免疫杂志 2005年第1期
Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, Massachusetts
ABSTRACT
The encapsulated pathogenic yeast Cryptococcus neoformans is poorly recognized by phagocytic cells in the absence of opsonins. Macrophages will bind and internalize complement- or antibody-opsonized C. neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism. Thus, we studied the opsonic requirements for binding to C. neoformans by cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release. Binding of unopsonized C. neoformans to human and murine DCs was negligible. Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components. PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains. Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity. Antifungal activity against C. neoformans opsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response. Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated with C. neoformans opsonized in PHS and/or antibody. However, opsonized C. neoformans failed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population. Thus, human and murine DCs show maximal binding to and antifungal activity against C. neoformans via a process highly dependent on opsonization.
INTRODUCTION
The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of morbidity and mortality in individuals with compromised T-cell-mediated immunity, especially those with AIDS (29). Initial control of cryptococcosis is highly dependent on phagocytosis and intracellular killing of the fungus (32). However, virtually all clinical isolates possess a polysaccharide capsule, and in the absence of opsonins, capsule is poorly recognized by host phagocytes.
Macrophage-mediated recognition of C. neoformans is largely dependent on complement components in serum (7, 22). Incubation of encapsulated C. neoformans in normal human serum leads to activation of the alternate pathway of the complement system and deposition of substantial amounts of opsonic fragments of C3 at the capsular surface, primarily in the form of iC3b (16-18, 48). In general, capsule elicits weak antibody responses, although the opsonic capacity of some anticapsular antibodies (14, 15, 30, 43, 45) has prompted investigation of active and passive immunization strategies against cryptococcosis (3, 5).
Dendritic cells (DC), like macrophages, express a variety of phagocytic receptors, including complement receptors (CRs) and Fc receptors (FcRs). In addition to their well-recognized role as potent antigen-presenting and cytokine-secreting cells, it has become increasingly apparent that DC also have the capacity for phagocytosis and killing of live microbes (31, 35, 44). Fungal pathogens for which DC phagocytosis and antifungal activity have been documented include Candida albicans (31), Histoplasma capsulatum (9), and Aspergillus fumigatus (4). In support of a role for DC in cryptococcal immunity, human DC are able to phagocytose and degrade C. neoformans for presentation to T cells (42, 45), and a mass influx of DC into regional lymph nodes after immunization with cryptococcal antigens is associated with a protective immune response to C. neoformans (1, 2).
In the present study, the opsonic requirements for binding, internalization, anticryptococcal activity, and cytokine production by human and murine DC were examined in vitro. We found that human and murine DC exerted maximal binding to and anticryptococcal activity against opsonized organisms. Human DC-mediated anticryptococcal activity appeared to be dependent on both oxidative and nonoxidative mechanisms. In addition, human but not murine DC incubated with opsonized C. neoformans released significant amounts of tumor necrosis factor alpha (TNF-). However, neither DC population released detectable levels of interleukin-12 p70 (IL-12p70) or IL-10.
MATERIALS AND METHODS
Reagents. Unless otherwise stated, chemical reagents of the highest quality available were obtained from Sigma Chemical Co. (St. Louis, Mo.), tissue culture media were from Gibco Life Technologies (Rockville, Md.), and plasticware was purchased from Fisher Scientific (Pittsburgh, Pa.). The medium used in all DC experiments was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 50 mM 2-mercaptoethanol. For human DC, the medium was additionally supplemented with 10 mM HEPES buffer. All cell culture incubations were performed at 37°C in a humidified environment supplemented with 5% CO2.
Opsonins. Pooled human serum (PHS) was obtained by pooling sera from a minimum of 10 healthy individuals under conditions preserving complement activity and was stored in aliquots at –80°C until use (25). Heat-inactivated PHS (PHS) was prepared by incubating PHS at 56°C for 30 min. The mouse anticapsular monoclonal antibody 3C2 (isotype immunoglobulin G1 [IgG1]) was a generous gift from Thomas Kozel, University of Nevada, Reno (40).
C. neoformans strains and opsonization conditions. The encapsulated serotype A strain 145 (ATCC 62070) was used in all experiments unless otherwise indicated. For the binding experiments for which results are shown in Fig. 2, the encapsulated serotype A strain H99 (ATCC 208821), the serotype B strain NIH 444 (ATCC 32609), the serotype C strain 18 (ATCC 24066), and the serotype D strain B3501 (ATCC 34874) were used. C. neoformans was cultured on Sabouraud dextrose agar (Remel, Lenexa, Kans.) at 30°C in unsupplemented air and was harvested as described elsewhere (28). To facilitate rapid, accurate identification of yeast cells, the binding experiments utilized fluorescein isothiocyanate (FITC)-labeled, heat-killed organisms. Yeast cells were heat killed by incubation at 56°C for 1 h and then labeled with FITC (100 μg/ml), as described previously (19, 20). In preliminary experiments, heat killing and FITC labeling did not affect results (data not shown). For binding and cytokine assays, C. neoformans organisms were preopsonized by incubation with either PHS, PHS, the anticapsular antibody (final concentration, 1 μg/ml), or PHS plus the anticapsular antibody at 37°C for 1 h. Yeast cells were washed twice with the medium, counted by using a hemocytometer, and then added to the DC. For anticryptococcal assays, C. neoformans organisms were preopsonized by incubation in PHS or PHS at 37°C for 1 h, and where indicated, the anticapsular antibody was added directly to wells at the start of incubation with DC.
Human monocyte-derived and murine bone marrow-derived DC. Human monocyte-derived DC were cultured essentially as described previously (38). Briefly, human blood was obtained by venipuncture from healthy volunteers, heparinized, and diluted 1:1 with warm Hanks balanced salt solution. Peripheral blood mononuclear cells were then purified by centrifugation over a cushion of Ficoll-Hypaque, washed, and resuspended in the medium. Cell suspensions were then divided evenly into 6-well plates and incubated for 2 h, after which nonadherent cells were gently washed off plates and discarded. Cells were resuspended at the desired concentration with fresh medium supplemented with 150 ng of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex Corp., Seattle, Wash.)/ml and 50 ng of recombinant human IL-4 (BD/Pharmingen, San Jose, Calif.)/ml. Cells were cultured for 6 to 10 days before challenge with C. neoformans. Variations in the culture period over this range did not affect results. This culture method produces immature DC expressing high levels of CD11c (38).
Murine DC were isolated as described previously (26). Briefly, bone marrow precursor cells were collected from femurs and tibiae of 8- to 12-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) and seeded in complete medium supplemented with 10% filter-sterilized supernatant from the J558L cell line (which stably produces murine GM-CSF) (33). DC were harvested after 10 to 12 days of culture. Variations in the culture period over this range did not affect results. This method of culture typically yielded 90 to 95% CD11c+ cells (DC) as assessed by flow cytometry (data not shown).
Binding assays. Human and murine DC were harvested and washed in medium. Then 105 DC were placed in 1.5-ml microcentrifuge tubes with 2 x 105 or 1 x 106 preopsonized, FITC-labeled C. neoformans organisms in a final volume of 500 μl. Tubes were then tumbled for 1 h at 37°C. After incubation, the mixture in each tube was apportioned into wells of 96-well polystyrene flat-bottom plates and fixed with buffered formaldehyde at a final concentration of 1%. Wells were then visualized under an inverted microscope equipped with epifluorescence. A minimum of 100 DC were assessed for the number of cell-associated C. neoformans organisms as described previously (22). Cell-associated fungi include those that are surface bound as well as those that are fully internalized. The binding index was defined as the average number of fungi per DC.
Visualization of internalized C. neoformans. In a final volume of 1 ml, 2 x 106 human monocyte-derived DC were challenged with 107 selectively opsonized, heat-killed C. neoformans organisms in 1.5-ml microcentrifuge tubes with rotation for 1 h. The cells were then centrifuged at 2,000 x g for 1 min and resuspended in 2.5% (vol/vol) glutaraldehyde in phosphate-buffered saline. After a second gentle spin, the cells were resuspended in 1 ml of 2.5% (vol/vol) glutaraldehyde in 0.5 M sodium cacodylate-HCl buffer (pH 7.0; 60 min at 30°C). The fixed samples were washed three times and postfixed for 1 h in 1% (wt/vol) osmium tetroxide in the same buffer. The fixed cells were then washed again in the same buffer, dehydrated through a graded series of ethanol to 100%, and transferred through a series of 100% ethanol and LX112-Araldite 502 epoxy resin mixtures (SPI Supplies, West Chester, Pa.) starting with 2 parts ethanol and 1 part epoxy resin, followed by a mixture of 1 part ethanol and 2 parts epoxy resin, and ending with three changes of full-strength epoxy resin.
Following overnight polymerization at 70°C, the ends of the tubes were cut off and attached to blank epoxy stubs with a drop of Super Glue, and 1-μm-thick sections were cut from the blocks and attached to glass slides. Slides were then stained with toluidine blue (SPI Supplies) and imaged with a Zeiss compound light microscope (Carl Zeiss, Inc.) equipped with a digital camera. Toluidine blue stains DC lightly and stains C. neoformans intensely, thus allowing differentiation between fungi located inside and outside of DC.
Growth inhibition and killing assays. Killing and growth inhibition were measured essentially as described previously (21, 28, 37) with slight modifications. Briefly, 1 x 104 selectively opsonized C. neoformans organisms were added to 96-well flat-bottom polystyrene plates containing 2 x 105 DC in 100 μl of medium. Control wells contained selectively opsonized C. neoformans with no DC. Where indicated, the anticapsular antibody 3C2 was added directly to the well (final concentration, 1 μg/ml) at the start of incubation, and plates were incubated for 2 or 24 h. Cells were then lysed in Triton X-100 (0.1% final concentration), and the contents of the wells were diluted in double-distilled water and spread onto duplicate Sabouraud dextrose agar plates. These conditions disperse phagocytosed C. neoformans without affecting fungal viability (37). Following 48 to 72 h of incubation at 30°C, CFU were counted and the percentage of anticryptococcal activity was calculated as [1 – (CFU from experimental wells/CFU from control wells)] x 100 (28). C. neoformans grown in tissue culture medium requires approximately 6 h for a cycle of cell division. Thus, at 2 h the percentage of anticryptococcal activity is predominantly a measure of direct DC-mediated killing of the fungus, whereas at 24 h it reflects both killing of the fungus and inhibition of its growth (21, 37). Negative values for the percentage of anticryptococcal activity indicate greater growth of C. neoformans in wells containing DC than in control wells without DC.
Respiratory burst inhibition assays. The effects of inhibitors and scavengers of respiratory burst oxidants on growth inhibition and killing of C. neoformans were assayed as described above except that DC were suspended in a medium containing a respiratory burst inhibitor cocktail consisting of 80 μg of bovine liver catalase (Sigma C 3155)/ml, 8 μg of bovine erythrocyte superoxide dismutase (Sigma S 5395)/ml, and 100 mM D-mannitol (Sigma M 1902) and were then allowed to equilibrate for 15 min before the addition of selectively opsonized C. neoformans (6, 28). This inhibitor cocktail has no direct effect on the viability of C. neoformans (28).
Cytokine secretion assays. Human DC (2 x 105) were stimulated with 2 x 106 selectively opsonized C. neoformans organisms for 18 h in 96-well polystyrene plates containing a final volume of 200 μl per well. For negative controls, C. neoformans was omitted (unstimulated), whereas for positive controls, 100 ng of lipopolysaccharide (LPS) per ml was substituted for C. neoformans. All wells except those containing LPS were supplemented with 20 μg of polymyxin B sulfate/ml to control for potential endotoxin contamination. Cell-free supernatants were removed and stored at –80°C until analysis by an enzyme-linked immunosorbent assay (ELISA) for TNF-, IL-12p70 (both with Duo Set ELISA kits from R&D Systems, Minneapolis, Minn.), and IL-10 (eBioscience, San Diego, Calif.). The lower limits of detection (in picograms per milliliter) in each ELISA were as follows: 20 for murine IL-12p70, 15 for murine IL-10, 30 for murine TNF-, 30 for human IL-12p70, 2 for human IL-10, and 15 for human TNF-.
Statistics. Means and standard errors of the means (SEM) were compared by using the two-tailed, two-sample Student t test, with a P value of <0.05 considered significant. For experiments in which multiple comparisons were made, adjustments for significance were made by using Bonferroni's correction.
RESULTS
Opsonic requirements for binding of C. neoformans to human and murine DC. Due to its antiphagocytic capsule, opsonization with complement and/or an antibody is generally a requirement for immune recognition of C. neoformans by neutrophils and macrophages (15). Therefore, in the first set of experiments, we examined the opsonic requirements for binding of encapsulated C. neoformans to cultured human monocyte-derived DC and murine bone marrow-derived DC. Similar results were obtained with the two DC populations (Fig. 1A and B). Levels of binding of unopsonized C. neoformans to DC were negligible. Opsonization with PHS significantly increased the level of binding by human DC over that to unopsonized C. neoformans at fungus-to-DC ratios of 10:1 and 2:1 (Fig. 1A). Murine DC also demonstrated greater binding of PHS-opsonized than of unopsonized C. neoformans (P < 0.05); however, significance was lost after application of the Bonferroni correction (Fig. 1B). Heat inactivation of PHS reduced C. neoformans binding to both human and murine DC to near-baseline levels, strongly suggesting a role for heat-labile serum components such as complement. In agreement with data from Vecchiarelli et al. (45), addition of an anticapsular monoclonal antibody also increased binding by both murine and human DC. Further, opsonization with both PHS and antibody had an additive effect on binding (Fig. 1A and B).
We next investigated whether bound organisms were internalized by human DC (Fig. 1C and D). Following a 1-h incubation, a fraction of the C. neoformans yeast cells opsonized with PHS alone (Fig. 1C) or with PHS plus antibody (Fig. 1D) were fully internalized by the DC. However, other fungal cells appeared bound to the surface of DC or not directly associated with DC.
To determine whether the results for strain 145 were applicable to other cryptococcal strains, we next compared binding by human DC to a strain from each of the four defined serotypes (A through D) of C. neoformans. Binding of PHS-opsonized C. neoformans to human DC was similar with strains from serotypes A, C, and D; however, there was minimal binding by human DC to the PHS-opsonized serotype B strain NIH 444 (Fig. 2). Interestingly, opsonization with both PHS and the anticapsular antibody 3C2 had an additive effect on binding for the serotype A, B, and D strains but not for the serotype C strain (Fig. 2).
Effects of varying opsonic conditions on anticryptococcal activities of human and murine DC. Binding of C. neoformans to DC in vivo may represent a critical initial step ultimately leading to killing of the fungus or inhibition of its growth. Accordingly, we next determined the capacities of human and murine DC to mediate fungal killing and growth inhibition of selectively opsonized C. neoformans in vitro. Anticryptococcal activity was quantified by comparing the numbers of CFU in wells incubated with and without DC. In the absence of opsonins, no significant antifungal activity was observed (Fig. 3). PHS opsonization resulted in substantial anticryptococcal activity by human DC at 24 h (Fig. 3A). In contrast, murine DC exhibited significant activity against PHS-opsonized C. neoformans at 2 h but not after 24 h of incubation. Heat inactivation of PHS markedly reduced this activity in both DC populations, suggesting a role for heat-labile complement components. Interestingly, unopsonized and PHS-opsonized organisms grew more efficiently in the presence of murine DC than in medium alone (Fig. 3B). Maximum antifungal activity of human and murine DC at both early (2-h) and late (24-h) time points was observed after incubation of DC with C. neoformans opsonized with an anticapsular antibody. The combination of PHS and the anticapsular antibody did not increase antifungal activity over that seen with antibody opsonization alone (Fig. 3).
Effects of inhibitors and scavengers of respiratory burst oxidants on human DC anticryptococcal activity. To examine the relative contributions of oxidative and nonoxidative mechanisms to DC-mediated anticryptococcal activity, human DC were incubated for 2 and 24 h with selectively opsonized C. neoformans in the presence or absence of superoxide dismutase, catalase, and mannitol. This cocktail inhibits the generation of reactive oxygen intermediates and scavenges oxygen metabolites (6, 28). In experiments with four individual donors, addition of inhibitors resulted in significant, albeit modest, decrements in 2- and 24-h antifungal activity against PHS-plus-antibody-opsonized C. neoformans (Fig. 4). However, these inhibitors had no significant effect on anticryptococcal activity against PHS-opsonized C. neoformans at either time point. These data suggest that both oxidative and nonoxidative mechanisms of growth inhibition or killing of C. neoformans are operative in human DC.
C. neoformans-stimulated cytokine secretion by DC. The magnitude and T helper cell bias of an initiated cell-mediated immune response can be greatly affected by cytokines produced by DC, including TNF-, IL-10, and IL-12 (12). We therefore investigated the effects of opsonization on C. neoformans-induced cytokine secretion from human and murine DC. Human DC secreted modest, although significant, amounts of TNF- when stimulated with PHS-opsonized C. neoformans. Antibody opsonization of the fungus with and without PHS further increased the release of TNF- (Fig. 5). Interestingly, and in contrast to the findings for human DC, C. neoformans failed to stimulate significant TNF- release from murine DC regardless of opsonic conditions. Moreover, IL-10 and IL-12 were undetectable in supernatants obtained from both human and murine DC after stimulation with C. neoformans. The positive control for these experiments, LPS, stimulated significant TNF-, IL-10, and IL-12p70 release from both human and murine DC (Fig. 5 and data not shown).
DISCUSSION
The data presented here illustrate the roles of heat-labile serum components and anticapsular antibody in binding to, and antifungal activity against, C. neoformans by cultured DC in vitro. Human and murine DC exhibited similar opsonic requirements for binding to C. neoformans. Thus, binding to unopsonized C. neoformans by both DC populations was negligible, and binding increased after opsonization with PHS. Heat treatment of PHS virtually abolished binding. Our binding data are consistent with results of previous studies which have strongly suggested that anticapsular antibody increases in vitro binding and uptake of encapsulated C. neoformans by human DC or a DC-like cell line (43, 45). In addition, the anticapsular monoclonal antibody 3C2, alone or in combination with PHS, has been shown to augment murine macrophage-mediated phagocytosis of C. neoformans (30). Data from the present study reveal similar additive effects on DC-mediated binding to C. neoformans opsonized with both PHS and anticapsular antibody.
Binding of encapsulated C. neoformans to DC was negligible in the absence of opsonins. In other studies, DC have been shown to readily take up unopsonized, acapsular strains of C. neoformans by a process dependent on mannose and -glucan receptors (42, 45). However, virtually all patient isolates of C. neoformans are encapsulated, so the clinical relevance of these results is uncertain. Nonopsonic binding of acapsular fungi by DC is not limited to acapsular C. neoformans; other groups have shown direct binding of human DC to H. capsulatum yeasts through the fibronectin receptor VLA-5 (very late Ag-5) (9), to C. albicans via receptors that recognize mannose and fucose (31), to A. fumigatus conidia via C-type lectin receptors, and to A. fumigatus hyphae via complement and Fc receptors (4). Nonopsonic receptors do not appear to play a role in recognition of encapsulated C. neoformans by DC, apparently because of the lack of corresponding ligands on the capsular surface.
CR3 (CD11b/CD18) and CR4 (CD11c/CD18) preferentially recognize iC3b, the predominant C3 fragment on PHS-opsonized C. neoformans (17). Transfection of CHO cells with either CR3 or CR4 enables the cells to bind to and phagocytose iC3b-coated C. neoformans (24). Macrophages typically express CR3, and binding and phagocytosis of PHS-opsonized C. neoformans by human macrophages is mediated primarily by this receptor (22, 24, 50). For DC, CR4 likely plays a larger role than CR3 in complement-mediated phagocytosis. Most DC express high levels of CD11c, although some DC populations are also CD11b positive (39). Taborda and Casadevall demonstrated that anticapsular antibodies can promote complement-independent phagocytosis of C. neoformans not only via FcR but also via CR3 (CD11b/CD18) and CR4 (CD11c/CD18) (43). Thus, two mechanisms, which are not mutually exclusive, could explain our data demonstrating enhanced binding of DC to C. neoformans in the presence of anticapsular antibody. First, the increased binding may result from interactions of antibody-coated fungi with FcRs on DC. Alternatively, the antibody could be exposing ligands on the fungal capsule that bind to CR3 and/or CR4 on DC.
The four serotypes of C. neoformans (A through D) differ in the structure of the major capsule polysaccharide, glucuronoxylomannan (GXM). However, differences in GXM composition can also be found among strains within each serotype (13, 27, 40). The anticapsular antibody utilized in this study, 3C2, was originally made against a serotype C strain. This antibody reacts with some, but not all, strains from each of the C. neoformans serotypes (40). The limited increase in the binding of antibody-opsonized cryptococcal strain 18 to human DC in this study likely reflects restricted expression of 3C2-specific epitopes on the capsular surface of this particular strain. Nevertheless, the data clearly show that an anticapsular antibody can greatly increase binding to C. neoformans by both human and murine DC, and they lend support to vaccination strategies aimed at eliciting anticapsular antibodies (5). This may be particularly useful in situations where complement levels are low, as can occur during overwhelming cryptococcal infection.
Opsonization with an antibody and/or PHS promoted antifungal activity of DC against C. neoformans. A similar study showed that murine bronchoalveolar macrophages exhibited antifungal activity against C. neoformans in the presence of an IgG1 anticapsular antibody in vitro (49). The partial decrement in DC-mediated anticryptococcal activity in the presence of inhibitors of the respiratory burst suggests that human DC employ both oxidative and nonoxidative mechanisms during an in vitro antifungal response. These data correlate with results from a previous study in our lab in which partial inhibition of human neutrophil-mediated anticryptococcal activity was seen in the presence of the same inhibitor cocktail (28). Future studies will examine the specific nonoxidative anticryptococcal mechanisms operative in DC.
DC can engulf and degrade pathogens for presentation to T cells (42, 45). Accordingly, we found that PHS- and PHS-plus-antibody-opsonized C. neoformans can be internalized by human DC in vitro. However, it is not known to what extent, if any, whole C. neoformans is phagocytosed by DC in vivo. An additional mechanism by which DC may initiate a T-cell immune response against C. neoformans is by taking up, processing, and presenting soluble antigens released by the fungus. In this regard, we have demonstrated that DC acquire the capacity for antigen-specific T-cell stimulation following incubation with soluble cryptococcal mannoproteins (M. K. Mansour and S. M. Levitz, unpublished data). In vivo, DC interactions with whole C. neoformans organisms as well as with soluble cryptococcal antigens may cooperatively facilitate the initiation and maintenance of a cell-mediated immune response against the fungus.
Both antibody- and PHS-opsonized C. neoformans stimulated release of modest though significant levels of TNF- from human but not murine DC. TNF- is a powerful activator of DC, and release of TNF- is a hallmark of DC maturation (11). TNF- is critical for the development of protective immunity against C. neoformans in a murine model (10) and is essential for the accumulation of "mature" DC in regional lymph nodes after immunization with cryptococcal antigens (1). Initial release of TNF- may also enhance the phagocytic functions of neighboring cells including alveolar macrophages (41). In addition to DC, other mononuclear phagocyte populations secrete TNF- following cryptococcal stimulation (23).
Interestingly, neither DC population released detectable amounts of IL-12p70, a sentinel Th1-inducing cytokine, or IL-10, an important Th2 cytokine (8, 35, 47). A Th1 response, including IL-12 release, appears to be critical for the development of protective immunity to C. neoformans, while IL-10 is generally associated with deleterious effects in murine cryptococcosis (12). The lack of IL-10 and IL-12 release in the present study could be a function of in vitro culture conditions, and it will be important to determine whether DC make these cytokines in vivo following C. neoformans infection. Alternatively, cells other than DC, such as monocytes, may act as primary sources of these cytokines in vivo, as has been demonstrated in vitro (36, 46). Vecchiarelli et al. recently demonstrated that human DC incubated with encapsulated C. neoformans and anticapsular antibody can efficiently stimulate T-cell proliferation and release of gamma interferon (45).
Comparison of human and murine DC responses to opsonized C. neoformans revealed some interesting differences in anticryptococcal activity and TNF- release. Such disparities may result from innate differences between murine and human DC. Alternatively, factors such as the origin of the DC precursors (i.e., blood versus bone marrow) and the culture method could be responsible. Various "subsets" of DC arising from unique precursors and possessing distinctive phenotypes and functions make it difficult to directly compare cultured DC of different species. In addition, a specific microbe can dictate the release of a panel of cytokines unique among members of particular DC subsets (34). Despite the complexity of comparing the DC systems of humans and mice, recent work has revealed much common ground (39), and in our studies, human and murine DC had similar requirements for binding to C. neoformans.
Exposure to C. neoformans is thought to be common, yet cryptococcosis is rare in immunocompetent hosts. This suggests that efficient recognition of C. neoformans is critical for initial control of infection and prevention of dissemination. Our data illustrate the ability of both human and murine DC to recognize, bind to, and exert antifungal activity against opsonized C. neoformans. Further characterization of the receptors on DC involved in recognition of C. neoformans, and the biochemical events triggered after receptor binding, should provide key insights into the pathogenesis of cryptococcosis and hopefully suggest rational vaccination strategies.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of Health grants RO1 AI25780, RO1 AI37532, and T32 AI07309.
Present address: Department of Pediatrics, The Cancer Center, University of Minnesota, Minneapolis, MN 55414.
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ABSTRACT
The encapsulated pathogenic yeast Cryptococcus neoformans is poorly recognized by phagocytic cells in the absence of opsonins. Macrophages will bind and internalize complement- or antibody-opsonized C. neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism. Thus, we studied the opsonic requirements for binding to C. neoformans by cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release. Binding of unopsonized C. neoformans to human and murine DCs was negligible. Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components. PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains. Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity. Antifungal activity against C. neoformans opsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response. Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated with C. neoformans opsonized in PHS and/or antibody. However, opsonized C. neoformans failed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population. Thus, human and murine DCs show maximal binding to and antifungal activity against C. neoformans via a process highly dependent on opsonization.
INTRODUCTION
The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of morbidity and mortality in individuals with compromised T-cell-mediated immunity, especially those with AIDS (29). Initial control of cryptococcosis is highly dependent on phagocytosis and intracellular killing of the fungus (32). However, virtually all clinical isolates possess a polysaccharide capsule, and in the absence of opsonins, capsule is poorly recognized by host phagocytes.
Macrophage-mediated recognition of C. neoformans is largely dependent on complement components in serum (7, 22). Incubation of encapsulated C. neoformans in normal human serum leads to activation of the alternate pathway of the complement system and deposition of substantial amounts of opsonic fragments of C3 at the capsular surface, primarily in the form of iC3b (16-18, 48). In general, capsule elicits weak antibody responses, although the opsonic capacity of some anticapsular antibodies (14, 15, 30, 43, 45) has prompted investigation of active and passive immunization strategies against cryptococcosis (3, 5).
Dendritic cells (DC), like macrophages, express a variety of phagocytic receptors, including complement receptors (CRs) and Fc receptors (FcRs). In addition to their well-recognized role as potent antigen-presenting and cytokine-secreting cells, it has become increasingly apparent that DC also have the capacity for phagocytosis and killing of live microbes (31, 35, 44). Fungal pathogens for which DC phagocytosis and antifungal activity have been documented include Candida albicans (31), Histoplasma capsulatum (9), and Aspergillus fumigatus (4). In support of a role for DC in cryptococcal immunity, human DC are able to phagocytose and degrade C. neoformans for presentation to T cells (42, 45), and a mass influx of DC into regional lymph nodes after immunization with cryptococcal antigens is associated with a protective immune response to C. neoformans (1, 2).
In the present study, the opsonic requirements for binding, internalization, anticryptococcal activity, and cytokine production by human and murine DC were examined in vitro. We found that human and murine DC exerted maximal binding to and anticryptococcal activity against opsonized organisms. Human DC-mediated anticryptococcal activity appeared to be dependent on both oxidative and nonoxidative mechanisms. In addition, human but not murine DC incubated with opsonized C. neoformans released significant amounts of tumor necrosis factor alpha (TNF-). However, neither DC population released detectable levels of interleukin-12 p70 (IL-12p70) or IL-10.
MATERIALS AND METHODS
Reagents. Unless otherwise stated, chemical reagents of the highest quality available were obtained from Sigma Chemical Co. (St. Louis, Mo.), tissue culture media were from Gibco Life Technologies (Rockville, Md.), and plasticware was purchased from Fisher Scientific (Pittsburgh, Pa.). The medium used in all DC experiments was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 50 mM 2-mercaptoethanol. For human DC, the medium was additionally supplemented with 10 mM HEPES buffer. All cell culture incubations were performed at 37°C in a humidified environment supplemented with 5% CO2.
Opsonins. Pooled human serum (PHS) was obtained by pooling sera from a minimum of 10 healthy individuals under conditions preserving complement activity and was stored in aliquots at –80°C until use (25). Heat-inactivated PHS (PHS) was prepared by incubating PHS at 56°C for 30 min. The mouse anticapsular monoclonal antibody 3C2 (isotype immunoglobulin G1 [IgG1]) was a generous gift from Thomas Kozel, University of Nevada, Reno (40).
C. neoformans strains and opsonization conditions. The encapsulated serotype A strain 145 (ATCC 62070) was used in all experiments unless otherwise indicated. For the binding experiments for which results are shown in Fig. 2, the encapsulated serotype A strain H99 (ATCC 208821), the serotype B strain NIH 444 (ATCC 32609), the serotype C strain 18 (ATCC 24066), and the serotype D strain B3501 (ATCC 34874) were used. C. neoformans was cultured on Sabouraud dextrose agar (Remel, Lenexa, Kans.) at 30°C in unsupplemented air and was harvested as described elsewhere (28). To facilitate rapid, accurate identification of yeast cells, the binding experiments utilized fluorescein isothiocyanate (FITC)-labeled, heat-killed organisms. Yeast cells were heat killed by incubation at 56°C for 1 h and then labeled with FITC (100 μg/ml), as described previously (19, 20). In preliminary experiments, heat killing and FITC labeling did not affect results (data not shown). For binding and cytokine assays, C. neoformans organisms were preopsonized by incubation with either PHS, PHS, the anticapsular antibody (final concentration, 1 μg/ml), or PHS plus the anticapsular antibody at 37°C for 1 h. Yeast cells were washed twice with the medium, counted by using a hemocytometer, and then added to the DC. For anticryptococcal assays, C. neoformans organisms were preopsonized by incubation in PHS or PHS at 37°C for 1 h, and where indicated, the anticapsular antibody was added directly to wells at the start of incubation with DC.
Human monocyte-derived and murine bone marrow-derived DC. Human monocyte-derived DC were cultured essentially as described previously (38). Briefly, human blood was obtained by venipuncture from healthy volunteers, heparinized, and diluted 1:1 with warm Hanks balanced salt solution. Peripheral blood mononuclear cells were then purified by centrifugation over a cushion of Ficoll-Hypaque, washed, and resuspended in the medium. Cell suspensions were then divided evenly into 6-well plates and incubated for 2 h, after which nonadherent cells were gently washed off plates and discarded. Cells were resuspended at the desired concentration with fresh medium supplemented with 150 ng of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex Corp., Seattle, Wash.)/ml and 50 ng of recombinant human IL-4 (BD/Pharmingen, San Jose, Calif.)/ml. Cells were cultured for 6 to 10 days before challenge with C. neoformans. Variations in the culture period over this range did not affect results. This culture method produces immature DC expressing high levels of CD11c (38).
Murine DC were isolated as described previously (26). Briefly, bone marrow precursor cells were collected from femurs and tibiae of 8- to 12-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) and seeded in complete medium supplemented with 10% filter-sterilized supernatant from the J558L cell line (which stably produces murine GM-CSF) (33). DC were harvested after 10 to 12 days of culture. Variations in the culture period over this range did not affect results. This method of culture typically yielded 90 to 95% CD11c+ cells (DC) as assessed by flow cytometry (data not shown).
Binding assays. Human and murine DC were harvested and washed in medium. Then 105 DC were placed in 1.5-ml microcentrifuge tubes with 2 x 105 or 1 x 106 preopsonized, FITC-labeled C. neoformans organisms in a final volume of 500 μl. Tubes were then tumbled for 1 h at 37°C. After incubation, the mixture in each tube was apportioned into wells of 96-well polystyrene flat-bottom plates and fixed with buffered formaldehyde at a final concentration of 1%. Wells were then visualized under an inverted microscope equipped with epifluorescence. A minimum of 100 DC were assessed for the number of cell-associated C. neoformans organisms as described previously (22). Cell-associated fungi include those that are surface bound as well as those that are fully internalized. The binding index was defined as the average number of fungi per DC.
Visualization of internalized C. neoformans. In a final volume of 1 ml, 2 x 106 human monocyte-derived DC were challenged with 107 selectively opsonized, heat-killed C. neoformans organisms in 1.5-ml microcentrifuge tubes with rotation for 1 h. The cells were then centrifuged at 2,000 x g for 1 min and resuspended in 2.5% (vol/vol) glutaraldehyde in phosphate-buffered saline. After a second gentle spin, the cells were resuspended in 1 ml of 2.5% (vol/vol) glutaraldehyde in 0.5 M sodium cacodylate-HCl buffer (pH 7.0; 60 min at 30°C). The fixed samples were washed three times and postfixed for 1 h in 1% (wt/vol) osmium tetroxide in the same buffer. The fixed cells were then washed again in the same buffer, dehydrated through a graded series of ethanol to 100%, and transferred through a series of 100% ethanol and LX112-Araldite 502 epoxy resin mixtures (SPI Supplies, West Chester, Pa.) starting with 2 parts ethanol and 1 part epoxy resin, followed by a mixture of 1 part ethanol and 2 parts epoxy resin, and ending with three changes of full-strength epoxy resin.
Following overnight polymerization at 70°C, the ends of the tubes were cut off and attached to blank epoxy stubs with a drop of Super Glue, and 1-μm-thick sections were cut from the blocks and attached to glass slides. Slides were then stained with toluidine blue (SPI Supplies) and imaged with a Zeiss compound light microscope (Carl Zeiss, Inc.) equipped with a digital camera. Toluidine blue stains DC lightly and stains C. neoformans intensely, thus allowing differentiation between fungi located inside and outside of DC.
Growth inhibition and killing assays. Killing and growth inhibition were measured essentially as described previously (21, 28, 37) with slight modifications. Briefly, 1 x 104 selectively opsonized C. neoformans organisms were added to 96-well flat-bottom polystyrene plates containing 2 x 105 DC in 100 μl of medium. Control wells contained selectively opsonized C. neoformans with no DC. Where indicated, the anticapsular antibody 3C2 was added directly to the well (final concentration, 1 μg/ml) at the start of incubation, and plates were incubated for 2 or 24 h. Cells were then lysed in Triton X-100 (0.1% final concentration), and the contents of the wells were diluted in double-distilled water and spread onto duplicate Sabouraud dextrose agar plates. These conditions disperse phagocytosed C. neoformans without affecting fungal viability (37). Following 48 to 72 h of incubation at 30°C, CFU were counted and the percentage of anticryptococcal activity was calculated as [1 – (CFU from experimental wells/CFU from control wells)] x 100 (28). C. neoformans grown in tissue culture medium requires approximately 6 h for a cycle of cell division. Thus, at 2 h the percentage of anticryptococcal activity is predominantly a measure of direct DC-mediated killing of the fungus, whereas at 24 h it reflects both killing of the fungus and inhibition of its growth (21, 37). Negative values for the percentage of anticryptococcal activity indicate greater growth of C. neoformans in wells containing DC than in control wells without DC.
Respiratory burst inhibition assays. The effects of inhibitors and scavengers of respiratory burst oxidants on growth inhibition and killing of C. neoformans were assayed as described above except that DC were suspended in a medium containing a respiratory burst inhibitor cocktail consisting of 80 μg of bovine liver catalase (Sigma C 3155)/ml, 8 μg of bovine erythrocyte superoxide dismutase (Sigma S 5395)/ml, and 100 mM D-mannitol (Sigma M 1902) and were then allowed to equilibrate for 15 min before the addition of selectively opsonized C. neoformans (6, 28). This inhibitor cocktail has no direct effect on the viability of C. neoformans (28).
Cytokine secretion assays. Human DC (2 x 105) were stimulated with 2 x 106 selectively opsonized C. neoformans organisms for 18 h in 96-well polystyrene plates containing a final volume of 200 μl per well. For negative controls, C. neoformans was omitted (unstimulated), whereas for positive controls, 100 ng of lipopolysaccharide (LPS) per ml was substituted for C. neoformans. All wells except those containing LPS were supplemented with 20 μg of polymyxin B sulfate/ml to control for potential endotoxin contamination. Cell-free supernatants were removed and stored at –80°C until analysis by an enzyme-linked immunosorbent assay (ELISA) for TNF-, IL-12p70 (both with Duo Set ELISA kits from R&D Systems, Minneapolis, Minn.), and IL-10 (eBioscience, San Diego, Calif.). The lower limits of detection (in picograms per milliliter) in each ELISA were as follows: 20 for murine IL-12p70, 15 for murine IL-10, 30 for murine TNF-, 30 for human IL-12p70, 2 for human IL-10, and 15 for human TNF-.
Statistics. Means and standard errors of the means (SEM) were compared by using the two-tailed, two-sample Student t test, with a P value of <0.05 considered significant. For experiments in which multiple comparisons were made, adjustments for significance were made by using Bonferroni's correction.
RESULTS
Opsonic requirements for binding of C. neoformans to human and murine DC. Due to its antiphagocytic capsule, opsonization with complement and/or an antibody is generally a requirement for immune recognition of C. neoformans by neutrophils and macrophages (15). Therefore, in the first set of experiments, we examined the opsonic requirements for binding of encapsulated C. neoformans to cultured human monocyte-derived DC and murine bone marrow-derived DC. Similar results were obtained with the two DC populations (Fig. 1A and B). Levels of binding of unopsonized C. neoformans to DC were negligible. Opsonization with PHS significantly increased the level of binding by human DC over that to unopsonized C. neoformans at fungus-to-DC ratios of 10:1 and 2:1 (Fig. 1A). Murine DC also demonstrated greater binding of PHS-opsonized than of unopsonized C. neoformans (P < 0.05); however, significance was lost after application of the Bonferroni correction (Fig. 1B). Heat inactivation of PHS reduced C. neoformans binding to both human and murine DC to near-baseline levels, strongly suggesting a role for heat-labile serum components such as complement. In agreement with data from Vecchiarelli et al. (45), addition of an anticapsular monoclonal antibody also increased binding by both murine and human DC. Further, opsonization with both PHS and antibody had an additive effect on binding (Fig. 1A and B).
We next investigated whether bound organisms were internalized by human DC (Fig. 1C and D). Following a 1-h incubation, a fraction of the C. neoformans yeast cells opsonized with PHS alone (Fig. 1C) or with PHS plus antibody (Fig. 1D) were fully internalized by the DC. However, other fungal cells appeared bound to the surface of DC or not directly associated with DC.
To determine whether the results for strain 145 were applicable to other cryptococcal strains, we next compared binding by human DC to a strain from each of the four defined serotypes (A through D) of C. neoformans. Binding of PHS-opsonized C. neoformans to human DC was similar with strains from serotypes A, C, and D; however, there was minimal binding by human DC to the PHS-opsonized serotype B strain NIH 444 (Fig. 2). Interestingly, opsonization with both PHS and the anticapsular antibody 3C2 had an additive effect on binding for the serotype A, B, and D strains but not for the serotype C strain (Fig. 2).
Effects of varying opsonic conditions on anticryptococcal activities of human and murine DC. Binding of C. neoformans to DC in vivo may represent a critical initial step ultimately leading to killing of the fungus or inhibition of its growth. Accordingly, we next determined the capacities of human and murine DC to mediate fungal killing and growth inhibition of selectively opsonized C. neoformans in vitro. Anticryptococcal activity was quantified by comparing the numbers of CFU in wells incubated with and without DC. In the absence of opsonins, no significant antifungal activity was observed (Fig. 3). PHS opsonization resulted in substantial anticryptococcal activity by human DC at 24 h (Fig. 3A). In contrast, murine DC exhibited significant activity against PHS-opsonized C. neoformans at 2 h but not after 24 h of incubation. Heat inactivation of PHS markedly reduced this activity in both DC populations, suggesting a role for heat-labile complement components. Interestingly, unopsonized and PHS-opsonized organisms grew more efficiently in the presence of murine DC than in medium alone (Fig. 3B). Maximum antifungal activity of human and murine DC at both early (2-h) and late (24-h) time points was observed after incubation of DC with C. neoformans opsonized with an anticapsular antibody. The combination of PHS and the anticapsular antibody did not increase antifungal activity over that seen with antibody opsonization alone (Fig. 3).
Effects of inhibitors and scavengers of respiratory burst oxidants on human DC anticryptococcal activity. To examine the relative contributions of oxidative and nonoxidative mechanisms to DC-mediated anticryptococcal activity, human DC were incubated for 2 and 24 h with selectively opsonized C. neoformans in the presence or absence of superoxide dismutase, catalase, and mannitol. This cocktail inhibits the generation of reactive oxygen intermediates and scavenges oxygen metabolites (6, 28). In experiments with four individual donors, addition of inhibitors resulted in significant, albeit modest, decrements in 2- and 24-h antifungal activity against PHS-plus-antibody-opsonized C. neoformans (Fig. 4). However, these inhibitors had no significant effect on anticryptococcal activity against PHS-opsonized C. neoformans at either time point. These data suggest that both oxidative and nonoxidative mechanisms of growth inhibition or killing of C. neoformans are operative in human DC.
C. neoformans-stimulated cytokine secretion by DC. The magnitude and T helper cell bias of an initiated cell-mediated immune response can be greatly affected by cytokines produced by DC, including TNF-, IL-10, and IL-12 (12). We therefore investigated the effects of opsonization on C. neoformans-induced cytokine secretion from human and murine DC. Human DC secreted modest, although significant, amounts of TNF- when stimulated with PHS-opsonized C. neoformans. Antibody opsonization of the fungus with and without PHS further increased the release of TNF- (Fig. 5). Interestingly, and in contrast to the findings for human DC, C. neoformans failed to stimulate significant TNF- release from murine DC regardless of opsonic conditions. Moreover, IL-10 and IL-12 were undetectable in supernatants obtained from both human and murine DC after stimulation with C. neoformans. The positive control for these experiments, LPS, stimulated significant TNF-, IL-10, and IL-12p70 release from both human and murine DC (Fig. 5 and data not shown).
DISCUSSION
The data presented here illustrate the roles of heat-labile serum components and anticapsular antibody in binding to, and antifungal activity against, C. neoformans by cultured DC in vitro. Human and murine DC exhibited similar opsonic requirements for binding to C. neoformans. Thus, binding to unopsonized C. neoformans by both DC populations was negligible, and binding increased after opsonization with PHS. Heat treatment of PHS virtually abolished binding. Our binding data are consistent with results of previous studies which have strongly suggested that anticapsular antibody increases in vitro binding and uptake of encapsulated C. neoformans by human DC or a DC-like cell line (43, 45). In addition, the anticapsular monoclonal antibody 3C2, alone or in combination with PHS, has been shown to augment murine macrophage-mediated phagocytosis of C. neoformans (30). Data from the present study reveal similar additive effects on DC-mediated binding to C. neoformans opsonized with both PHS and anticapsular antibody.
Binding of encapsulated C. neoformans to DC was negligible in the absence of opsonins. In other studies, DC have been shown to readily take up unopsonized, acapsular strains of C. neoformans by a process dependent on mannose and -glucan receptors (42, 45). However, virtually all patient isolates of C. neoformans are encapsulated, so the clinical relevance of these results is uncertain. Nonopsonic binding of acapsular fungi by DC is not limited to acapsular C. neoformans; other groups have shown direct binding of human DC to H. capsulatum yeasts through the fibronectin receptor VLA-5 (very late Ag-5) (9), to C. albicans via receptors that recognize mannose and fucose (31), to A. fumigatus conidia via C-type lectin receptors, and to A. fumigatus hyphae via complement and Fc receptors (4). Nonopsonic receptors do not appear to play a role in recognition of encapsulated C. neoformans by DC, apparently because of the lack of corresponding ligands on the capsular surface.
CR3 (CD11b/CD18) and CR4 (CD11c/CD18) preferentially recognize iC3b, the predominant C3 fragment on PHS-opsonized C. neoformans (17). Transfection of CHO cells with either CR3 or CR4 enables the cells to bind to and phagocytose iC3b-coated C. neoformans (24). Macrophages typically express CR3, and binding and phagocytosis of PHS-opsonized C. neoformans by human macrophages is mediated primarily by this receptor (22, 24, 50). For DC, CR4 likely plays a larger role than CR3 in complement-mediated phagocytosis. Most DC express high levels of CD11c, although some DC populations are also CD11b positive (39). Taborda and Casadevall demonstrated that anticapsular antibodies can promote complement-independent phagocytosis of C. neoformans not only via FcR but also via CR3 (CD11b/CD18) and CR4 (CD11c/CD18) (43). Thus, two mechanisms, which are not mutually exclusive, could explain our data demonstrating enhanced binding of DC to C. neoformans in the presence of anticapsular antibody. First, the increased binding may result from interactions of antibody-coated fungi with FcRs on DC. Alternatively, the antibody could be exposing ligands on the fungal capsule that bind to CR3 and/or CR4 on DC.
The four serotypes of C. neoformans (A through D) differ in the structure of the major capsule polysaccharide, glucuronoxylomannan (GXM). However, differences in GXM composition can also be found among strains within each serotype (13, 27, 40). The anticapsular antibody utilized in this study, 3C2, was originally made against a serotype C strain. This antibody reacts with some, but not all, strains from each of the C. neoformans serotypes (40). The limited increase in the binding of antibody-opsonized cryptococcal strain 18 to human DC in this study likely reflects restricted expression of 3C2-specific epitopes on the capsular surface of this particular strain. Nevertheless, the data clearly show that an anticapsular antibody can greatly increase binding to C. neoformans by both human and murine DC, and they lend support to vaccination strategies aimed at eliciting anticapsular antibodies (5). This may be particularly useful in situations where complement levels are low, as can occur during overwhelming cryptococcal infection.
Opsonization with an antibody and/or PHS promoted antifungal activity of DC against C. neoformans. A similar study showed that murine bronchoalveolar macrophages exhibited antifungal activity against C. neoformans in the presence of an IgG1 anticapsular antibody in vitro (49). The partial decrement in DC-mediated anticryptococcal activity in the presence of inhibitors of the respiratory burst suggests that human DC employ both oxidative and nonoxidative mechanisms during an in vitro antifungal response. These data correlate with results from a previous study in our lab in which partial inhibition of human neutrophil-mediated anticryptococcal activity was seen in the presence of the same inhibitor cocktail (28). Future studies will examine the specific nonoxidative anticryptococcal mechanisms operative in DC.
DC can engulf and degrade pathogens for presentation to T cells (42, 45). Accordingly, we found that PHS- and PHS-plus-antibody-opsonized C. neoformans can be internalized by human DC in vitro. However, it is not known to what extent, if any, whole C. neoformans is phagocytosed by DC in vivo. An additional mechanism by which DC may initiate a T-cell immune response against C. neoformans is by taking up, processing, and presenting soluble antigens released by the fungus. In this regard, we have demonstrated that DC acquire the capacity for antigen-specific T-cell stimulation following incubation with soluble cryptococcal mannoproteins (M. K. Mansour and S. M. Levitz, unpublished data). In vivo, DC interactions with whole C. neoformans organisms as well as with soluble cryptococcal antigens may cooperatively facilitate the initiation and maintenance of a cell-mediated immune response against the fungus.
Both antibody- and PHS-opsonized C. neoformans stimulated release of modest though significant levels of TNF- from human but not murine DC. TNF- is a powerful activator of DC, and release of TNF- is a hallmark of DC maturation (11). TNF- is critical for the development of protective immunity against C. neoformans in a murine model (10) and is essential for the accumulation of "mature" DC in regional lymph nodes after immunization with cryptococcal antigens (1). Initial release of TNF- may also enhance the phagocytic functions of neighboring cells including alveolar macrophages (41). In addition to DC, other mononuclear phagocyte populations secrete TNF- following cryptococcal stimulation (23).
Interestingly, neither DC population released detectable amounts of IL-12p70, a sentinel Th1-inducing cytokine, or IL-10, an important Th2 cytokine (8, 35, 47). A Th1 response, including IL-12 release, appears to be critical for the development of protective immunity to C. neoformans, while IL-10 is generally associated with deleterious effects in murine cryptococcosis (12). The lack of IL-10 and IL-12 release in the present study could be a function of in vitro culture conditions, and it will be important to determine whether DC make these cytokines in vivo following C. neoformans infection. Alternatively, cells other than DC, such as monocytes, may act as primary sources of these cytokines in vivo, as has been demonstrated in vitro (36, 46). Vecchiarelli et al. recently demonstrated that human DC incubated with encapsulated C. neoformans and anticapsular antibody can efficiently stimulate T-cell proliferation and release of gamma interferon (45).
Comparison of human and murine DC responses to opsonized C. neoformans revealed some interesting differences in anticryptococcal activity and TNF- release. Such disparities may result from innate differences between murine and human DC. Alternatively, factors such as the origin of the DC precursors (i.e., blood versus bone marrow) and the culture method could be responsible. Various "subsets" of DC arising from unique precursors and possessing distinctive phenotypes and functions make it difficult to directly compare cultured DC of different species. In addition, a specific microbe can dictate the release of a panel of cytokines unique among members of particular DC subsets (34). Despite the complexity of comparing the DC systems of humans and mice, recent work has revealed much common ground (39), and in our studies, human and murine DC had similar requirements for binding to C. neoformans.
Exposure to C. neoformans is thought to be common, yet cryptococcosis is rare in immunocompetent hosts. This suggests that efficient recognition of C. neoformans is critical for initial control of infection and prevention of dissemination. Our data illustrate the ability of both human and murine DC to recognize, bind to, and exert antifungal activity against opsonized C. neoformans. Further characterization of the receptors on DC involved in recognition of C. neoformans, and the biochemical events triggered after receptor binding, should provide key insights into the pathogenesis of cryptococcosis and hopefully suggest rational vaccination strategies.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of Health grants RO1 AI25780, RO1 AI37532, and T32 AI07309.
Present address: Department of Pediatrics, The Cancer Center, University of Minnesota, Minneapolis, MN 55414.
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