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The Journal of Immunology, 2000, 165: 1541-1547.
Copyright © 2000 by The American Association of Immunologists

Inhibition of Human Endothelial Cell Chemokine Production by the Opportunistic Fungal Pathogen Cryptococcus neoformans1

Neelufar Mozaffarian*, Arturo Casadevall*,{dagger} and Joan W. Berman2,*,{ddagger}

* Department of Microbiology and Immunology, Division of Infectious Diseases, {dagger} Department of Medicine, and {ddagger} Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cryptococcus neoformans is an encapsulated fungal pathogen commonly acquired by inhalation. Extrapulmonary dissemination can lead to infection of the bloodstream and various organs, most commonly resulting in meningoencephalitis. However, infection with C. neoformans is often characterized by a scant inflammatory response. The leukocyte response to infection depends in part upon a gradient of chemotactic factors and adhesion molecules expressed by the host vascular endothelium, yet the inflammatory response of human endothelial cells (EC) to C. neoformans has not been previously investigated. We found that incubation of primary human EC with C. neoformans did not induce chemokine synthesis, and resulted in differential inhibition of cytokine-induced IL-8, IFN-{gamma}-inducible protein-10, and monocyte chemoattractant protein-1. In contrast, C. neoformans had little effect on EC surface expression of the leukocyte ligand, ICAM-1, as determined by flow cytometry. Modulation of chemokine production was dependent on the chemokine under study, the inoculum of C. neoformans used, fungal viability, and cell-cell contact, but independent of cryptococcal strain or encapsulation. These observations suggest a novel mechanism whereby C. neoformans can affect EC function and interfere with the host inflammatory response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C;-4qryptococcus neoformans var. neoformans is a fungus that is ubiquitous in the environment and infects humans via the respiratory tract. If not contained in the lung, C. neoformans can disseminate and cause a fatal meningoencephalitis. Although the mechanisms of extrapulmonary dissemination are poorly understood, there is considerable evidence that C. neoformans invades the bloodstream. In one prospective series, C. neoformans was the most common bloodstream infection in febrile, HIV+ adults (1). In animal models of cryptococcosis, i.v. inoculation leads to dissemination and intravascular granuloma formation (2), and C. neoformans is detectable in the blood for prolonged periods of time (3). In vitro studies have shown that C. neoformans adheres to and is internalized by human endothelial cells (EC),3 suggesting a potential mechanism for entry into and exit from the vascular compartment (4). Hence, there is both direct and circumstantial evidence for contact between C. neoformans and endothelium in human infection, although the contribution of this interaction to the pathogenesis of infection is not known.

It has been reported that cryptococcal infections often elicit little or no inflammation (5). The phenomenon is not well understood, but is generally assumed to be the result of fungal-induced immune suppression. Vascular EC express chemotactic cytokines (chemokines) and leukocyte adhesion molecules that mediate leukocyte activation, migration, and diapedesis (6). Many infectious organisms induce expression of chemokines and adhesion molecules in human EC, including Candida albicans (7), Staphylococcus aureus (8, 9), Trypanosoma cruzi (10), Listeria monocytogenes (11), dengue virus (12), Helicobacter pylori (13), CMV (14), Borrelia burgdorferi (15), Rickettsia conorii (16), and Chlamydia pneumoniae (17, 18). Given the central role of EC in mediating inflammatory responses and the fact that C. neoformans is often found in the vascular space, fungal-induced EC dysfunction may contribute to the inadequate host response commonly associated with disseminated cryptococcosis.

Therefore, we investigated the effects of C. neoformans on the expression of IL-8, IFN-{gamma}-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), and the leukocyte ligand ICAM-1, in primary HUVEC in the presence and absence of proinflammatory cytokines. IL-8 belongs to the CXC family of chemokines and is the prototypic neutrophil chemoattractant (19, 20), but also activates monocytes for firm adhesion to EC (21). IP-10 differs from other CXC chemokine family members in that it is chemotactic for activated T cells (22, 23). MCP-1 is a well-characterized member of the CC chemokine family, and is chemotactic for monocytes and activated T cells (24). Remarkably, we found that C. neoformans failed to induce chemokine or adhesion molecule expression in resting EC, and differentially inhibited chemokine production in cytokine-stimulated EC. Our results demonstrate that C. neoformans has the ability to interfere with inflammatory signaling in human EC, and suggest that C. neoformans may alter leukocyte activation and trafficking in the infected host.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of EC

Primary human EC were isolated from umbilical cords, as previously described (25). Briefly, umbilical veins were rinsed with sterile saline and digested with 0.1% collagenase (Worthington Biochemical, Freehold, NJ). EC were grown on gelatin-coated tissue culture plates (Falcon, Cockeysville, MD) at 37°C in a humidified chamber with 5% CO2. When confluent, EC were passaged using trypsin (Life Technologies, Grand Island, NY) digestion. For all experiments, EC were used at passages 3–4. EC medium consisted of: M199 (Life Technologies) supplemented with 0.16% bicarbonate, 11.1 mM HEPES (Calbiochem-Behring, La Jolla, CA), 1.6 mM L-glutamine (Life Technologies), 50 µg/ml ascorbate (Fisher, Fairlawn, NJ), 25 µg/ml heparin (Sigma, St. Louis, MO), 7.5 µg/ml endothelial cell growth factor (Sigma), 2.78 µl/ml bovine brain extract (Clonetics, San Diego, CA), 0.05 U/ml penicillin with 0.05 µg/ml streptomycin (Life Technologies), 20% newborn calf serum (Life Technologies), and 5% heat-inactivated human serum (Biocell Laboratories, Rancho Dominguez, CA).

Culture of C. neoformans

Two encapsulated strains of C. neoformans (B-3501 and SB4) and one acapsular strain (Cap 67) were used in this study. C. neoformans B-3501 (ATCC 34873; American Type Culture Collection, Manassas, VA) is a serotype D strain that was chosen because it is the parent strain for the acapsular mutant Cap 67 (ATCC 52817) (26), which has been shown to be complemented to the encapsulated strain by a single gene (27). Serotype D strains are pathogenic and are common clinical isolates in Europe (28, 29, 30). The serotype A strain SB4 was chosen because it is a recent clinical isolate that has been extensively studied (31, 32, 33), and represents the most common serotype in clinical infection worldwide (34). Flasks of Sabouraud’s agar broth (Difco Laboratories, Detroit, MI) were inoculated in sterile fashion from frozen stocks (stored at -70°C), and grown to late stationary phase in a rotating shaker at 30°C for 3 days. To minimize endotoxin contamination, all work was conducted in a biohazard hood. Fungal cells were washed three times in sterile PBS, counted using a hemacytometer, and diluted to the desired concentration in fresh EC medium. For experiments requiring dead fungi, organisms were grown as above, pelleted, and resuspended in sterile PBS. One-half of this aliquot was subjected to autoclaving, while the other half remained at room temperature. All samples were then washed twice in PBS, counted, diluted in EC medium, and used in parallel for treatment of EC monolayers. For other experiments, preparation of C. neoformans was conducted in an identical fashion, except that fungal cultures were killed by exposure to 24 µjoules of UV light (Stratalinker; Stratagene, La Jolla, CA) in place of autoclaving. Aliquots of fungal cultures were streaked on plates of Sabouraud’s dextrose agar (Difco Laboratories) to check for viability and to confirm colony morphology.

Treatment of EC for chemokine protein production

Confluent monolayers of EC were treated with cytokines and/or C. neoformans in a final volume of 0.6 ml/well in 24-well tissue culture plates (Falcon). Human rTNF-{alpha} (R&D Systems, Minneapolis, MN) was used at 40 U/ml, human rIFN-{gamma} (Genzyme, Cambridge, MA) at 100 U/ml, and human rIL-1ß (National Cancer Institute, Frederick, MD) at 3 U/ml. In some experiments, polyethylene cell culture inserts (Falcon) were used to prevent contact between EC and fungi. These inserts allowed cell supernatant to flow freely, but the 0.4 µm pore size was too small to allow passage of fungi. At the end of each time interval, EC supernatants were separated from cells by centrifugation, transferred to new tubes, and stored at -20°C until assayed.

Determination of chemokine protein levels

Chemokine levels in EC supernatants were measured by ELISAs developed using flat-bottom 96-well plates (Costar, Corning, Corning, NY) and paired anti-chemokine Abs, as directed by the manufacturer (R&D Systems). Plates were coated with capture Ab overnight and blocked with 1% BSA/PBS. Samples were incubated overnight and followed by biotinylated detection Ab and avidin-conjugated alkaline phosphatase. Wells were developed using tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and the reaction stopped with 1 M phosphoric acid (Sigma). Absorbance was measured at 450 nm, and the concentration of chemokine in each sample was calculated from a standard curve generated using known amounts of recombinant human chemokine (R&D Systems). Each sample was tested in duplicate, and results were averaged to obtain the final concentration (ng/ml) of chemokine. The limit of detection for these ELISAs is 15 pg/ml.

Flow-cytometric analysis of EC

EC cultures in six-well plates (Falcon) were treated with C. neoformans and/or TNF-{alpha} for the times indicated. EC were rinsed, removed from plates with 0.5 mM EDTA/PBS, and fixed using cold 2% formaldehyde/PBS. Cells were stained with 2 µg/ml of murine mAb to ICAM-1 (IgG1 anti-human CD54; Dako, Carpenteria, CA) or nonspecific mouse IgG1 myeloma (ICN/Cappel, Aurora, OH) in 1% BSA/PBS. FITC-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL) was used as the secondary Ab in 5% nonspecific goat serum/PBS. EC staining was evaluated by flow cytometry using Lysis II software (FACScan; Becton Dickinson, Mountain View, CA).

Localization of endothelial NF-{kappa}B

For visualization of NF-{kappa}B translocation, EC were treated in 24-well plates (Falcon) for 30 min, rinsed, and fixed in 100% cold methanol. Monolayers were incubated with 1% BSA/PBS to block nonspecific Ab binding. EC were incubated with rabbit Ab to NF-{kappa}B (polyclonal IgG anti-human Rel A/p65; Santa Cruz Biotechnology, Santa Cruz, CA) or nonspecific rabbit IgG (Southern Biotechnology Associates) in 1% BSA/PBS. Wells were washed and incubated with biotinylated goat anti-rabbit Ig (Vector Laboratories, Burlingame, CA) in 1% nonspecific goat serum/PBS (Vector Laboratories), and followed by avidin-conjugated cy3 (Sigma). Nuclei were counterstained with DAPI (5 µg/ml in PBS; Molecular Probes, Eugene, OR) before visualization under a fluorescence microscope (Olympus IX70, Tokyo, Japan).

EC viability

At the end of some experiments, EC monolayers were gently rinsed to remove unattached/extracellular fungi and examined by light microscopy for confluence, morphology, and trypan blue exclusion. EC damage or death after exposure to C. neoformans was measured by assessing the release of lactate dehydrogenase (LDH). Cell-free EC supernatants were incubated with a chromogenic substrate (Promega, Madison, WI) in duplicate wells of a 96-well plate (Falcon) for 30 min at room temperature. After the addition of 1 M acetic acid, absorbance was measured at 492 nm. Results were compared with a standard curve generated using an LDH-positive control made from lysed L929 fibroblasts (Promega) and are reported as arbitrary units.

Statistical analyses

To examine the effect of varying cryptococcal inocula, statistical analyses were conducted using one-way ANOVA, followed by Bonferroni correction (Primer of Biostatistics; McGraw-Hill, New York, NY). To examine the effect of C. neoformans on EC chemokine production under various conditions, data were analyzed using Wilcoxon Signed Rank (StatView; Abacus Concepts, Berkeley, CA). For all other comparisons, paired, two-tailed t test was used (Excel, Redmond, WA). For all tests, significance was assigned in which p < 0.05. To standardize for the different baseline levels of chemokine production across EC obtained from different donors, values in some experiments were normalized to the mean values obtained in the absence of C. neoformans, and are represented as the pooled means ± SEM. Normalization of protein concentrations revealed that relative changes in EC chemokine production in response to C. neoformans remained remarkably constant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C. neoformans fails to induce chemokine production in human EC and inhibits cytokine-induced chemokine expression

To determine whether C. neoformans affects EC chemokine production, we measured protein levels of IL-8, IP-10, and MCP-1 in EC supernatants at times ranging from 6 to 48 h. EC supernatants were also assayed for LDH activity to monitor cell damage/death. Treatment of EC with cytokines induced chemokine synthesis, as evidenced by accumulation of IL-8, IP-10, and MCP-1 in supernatants over time (Fig. 1Go). Surprisingly, C. neoformans strain B-3501 (2.4 x 107 cells/well, an E:T ratio of 250:1) did not induce EC chemokine production, either with or without cytokines. Because chemokine levels were followed for 48 h, it is unlikely that there was merely a delay in induction of chemokines by C. neoformans. In fact, C. neoformans inhibited TNF-{alpha}- and IFN-{gamma}-induced EC chemokine production (Fig. 1Go), without significantly increasing LDH levels. C. neoformans inhibition of EC chemokine production was evident by 24 h, and persisted through the 48-h study period (Fig. 1Go). Because inhibition of chemokine production was optimally observed at the later time point, the following experiments were conducted for 48 h, unless otherwise indicated.



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  		FIGURE 1. Effects of C. neoformans on EC production of IL-8, IP-10, and MCP-1. Primary human EC were treated with or without cytokines in the absence ({diamondsuit}) or in the presence ({square}) of C. neoformans (Cn), and supernatants were analyzed for chemokine production at 6, 24, and 48 h. Treatment of EC with C. neoformans did not induce chemokine production, and inhibited cytokine-induced production of all three chemokines, without significantly increasing the levels of LDH in these supernatants (p = 0.34 (TNF vs TNF + Cn) and p = 0.39 (IFN vs IFN + Cn)). Chemokine values were normalized to the mean protein values obtained in the absence of C. neoformans, and represent the pooled means ± SEM (n = 3, except at 6 h, where n = 1).

 
Inhibition of chemokine expression is dependent upon the inoculum of C. neoformans

To determine whether the fungal inhibition of chemokine production was inoculum dependent, EC were treated with TNF-{alpha} and various inocula of C. neoformans B-3501 (ranging from 9.6 x 106/well to 9.6 x 104/well, E:T ratios of 100:1 to 1:1). Significant decreases in EC MCP-1 protein production were observed in the presence of C. neoformans >9.6 x 105 fungi/well (an E:T ratio of 10:1). In these cultures, LDH release was not significantly different from control (Fig. 2Go), implying a noncytotoxic mechanism for reduction of MCP-1 levels. One inoculum of C. neoformans, which was effective in significantly lowering MCP-1 expression (2.4 x 106 cells/well, an E:T ratio of 25:1), was selected for further study.



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  		FIGURE 2. Influence of C. neoformans inoculum on EC MCP-1 and LDH release. EC were treated with TNF-{alpha} and varying inocula of C. neoformans (Cn) (ranging from 0 to 9.6 x 106 fungi/well), and supernatants were analyzed for MCP-1 protein and LDH activity after 48 h. C. neoformans inhibited production of MCP-1 from TNF-{alpha}-treated EC in an inoculum-dependent fashion, but did not significantly alter LDH release. Data were analyzed using one-way ANOVA followed by Bonferroni correction. Significant decreases in MCP-1 production were observed at Cn:EC ratios >=10:1 (*, represents p < 0.05). LDH activity was not statistically different between EC with and without C. neoformans. Values shown represent the pooled means ± SEM, and were normalized to the mean values of protein and LDH activity obtained in the absence of C. neoformans (n = 2).

 
Cryptococci were incubated with confluent monolayers of EC (with medium, TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma}), and 48-h supernatants were analyzed for chemokine expression by ELISA. TNF-{alpha} induced IL-8 and MCP-1 and synergized with IFN-{gamma} for IP-10 production. In EC treated with TNF-{alpha} + IFN-{gamma}, C. neoformans reduced protein levels ~30% for IL-8, 60% for IP-10, and 50% for MCP-1. Reduction of cytokine-induced chemokine protein by C. neoformans was statistically significant for all three chemokines (Fig. 3Go). This inoculum was used for all of the following experiments, unless otherwise indicated.



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  		FIGURE 3. Differential inhibition of cytokine-induced chemokine production by C. neoformans. EC were treated with or without cytokines and/or C. neoformans (Cn) for 48 h, and EC supernatants were analyzed for chemokine production. Cytokine treatment increased EC synthesis of IL-8, IP-10, and MCP-1 proteins. Incubation of EC with C. neoformans resulted in statistically significant reductions in the levels of all three chemokines. In EC treated with TNF-{alpha} + IFN-{gamma}, inhibition levels were ~30% for IL-8, 60% for IP-10, and 50% for MCP-1. Values were normalized to the mean protein values (ng/ml) obtained in the absence of C. neoformans and represent the pooled means ± SEM (n = 7–12). Statistical analysis was performed using Wilcoxon Signed Rank on data values before normalization (*, represents p < 0.05; **, represents p < 0.005).

 
Inhibition of EC chemokine production is independent of cryptococcal strain

To determine whether fungal inhibition of EC chemokine production was strain dependent, we tested C. neoformans strain SB4 (serotype A). EC (n = 2) were incubated with SB4 (at E:T ratios of 25:1 and 0.25:1) with and without cytokines (TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma}). Supernatants were collected at 24 and 48 h and analyzed for IL-8, IP-10, and MCP-1 by ELISA. We found that SB4 inhibited EC chemokine production in a manner similar to C. neoformans strain B-3501, and did not significantly augment LDH release. As with B-3501, inhibition was optimally observed at 48 h, and was dependent upon the inoculum of C. neoformans used (data not shown).

Inhibition of EC chemokine production is independent of fungal encapsulation, but is dependent upon cell-cell contact and fungal viability

Encapsulation of C. neoformans is important for virulence in rodent models of infection. The cryptococcal capsule is composed predominantly (80–90%) of glucuronoxylomannan (GXM), and several acapsular mutants lack the ability to produce this polysaccharide (26). To determine whether capsular GXM is required for modulation of chemokine production, C. neoformans strain B-3501 was compared with its acapsular variant, Cap 67.

C. neoformans B-3501 was used to treat confluent monolayers of EC (in the presence of medium, TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma}), and 48-h supernatants were analyzed for chemokine expression by ELISA. C. neoformans did not induce MCP-1, but did diminish cytokine-induced chemokine production. The effects of Cap 67 on EC chemokine production were similar to those of B-3501 (Fig. 4GoA), suggesting that a fungal component other than GXM was responsible for inhibition of proinflammatory signaling in EC. Acapsular or encapsulated C. neoformans that were physically separated from EC (Fig. 4GoB), heat killed (Fig. 4GoC), or UV irradiated (Fig. 4GoD) did not inhibit MCP-1 production. These data suggest that cell-cell contact between EC and metabolically active fungi or a heat/UV-labile surface molecule were required.



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  		FIGURE 4. Effects of cryptococcal capsule, cell-cell contact, and fungal viability on EC MCP-1 production. EC were treated with encapsulated (B-3501) or acapsular (Cap 67) C. neoformans (Cn) for 48 h in the presence and absence of cytokines. The effect of live fungal cultures was compared with that using C. neoformans separated from EC by a 0.4-µm membrane, and heat- or UV-killed C. neoformans. EC production of MCP-1 was reduced by live C. neoformans (A), but not by C. neoformans that had been physically separated from EC (B), autoclaved (C), or UV irradiated (D). Values were normalized to the mean protein values obtained in the absence of C. neoformans, and represent the pooled means ± SEM (A, n = 7–9; B, n = 2–6; C, n = 2–4; and D, n = 4 in the absence of TNF, n = 2 in the presence of TNF).

 
Inhibition of cytokine-induced chemokine production by C. neoformans is not specific to TNF-{alpha}

To establish whether the inhibitory effect of C. neoformans was cytokine specific, EC cultures were treated with IL-1ß, with and without IFN-{gamma} and/or C. neoformans strain B-3501. Like TNF-{alpha}, IL-1ß induced IL-8 and MCP-1 protein synthesis and synergized with IFN-{gamma} for IP-10 production (data not shown). C. neoformans down-modulated IL-1ß-induced EC chemokine expression in a fashion comparable with that observed in the presence of TNF-{alpha}, and this inhibition was reversed by the use of cell culture inserts or by prior UV killing of the yeasts (data not shown). Baseline expression of MCP-1 by untreated EC was also inhibited by C. neoformans (Figs. 1Go, 3Go, and 4Go).

EC expression of ICAM-1 is not altered in the presence of C. neoformans

To determine whether the inhibitory effect of C. neoformans was specific for chemokine production, we examined EC expression of the leukocyte adhesion molecule ICAM-1 (CD54). ICAM-1 is a member of the Ig gene superfamily that mediates firm adhesion of activated leukocytes to EC before extravasation (35). Untreated EC constitutively express low levels of surface ICAM-1, which is up-regulated by proinflammatory cytokines such as TNF-{alpha}, and peaks after ~12 h of treatment (36). EC were treated with C. neoformans B-3501 in the presence and absence of TNF-{alpha} for 12 h, and examined for ICAM-1 staining by flow cytometry. Untreated EC expressed ICAM-1, which was increased by treatment with TNF-{alpha} and was not altered by C. neoformans (Fig. 5Go). Comparison of the geometric mean fluorescence intensities using paired, two-tailed, t tests confirmed that there were no significant differences between EC with and without C. neoformans (n = 3, data not shown).



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  		FIGURE 5. Effect of C. neoformans on ICAM-1 expression. EC from three donors were treated for 12 h with TNF-{alpha} and/or C. neoformans and analyzed for ICAM-1 staining by flow cytometry. Representative histograms from one set of EC are shown. A, Untreated EC expressed low levels of ICAM-1 (anti-ICAM-1, shaded; control IgG1, open). B, Treatment of EC with C. neoformans had no effect on ICAM-1 staining (untreated, shaded; C. neoformans treated, open). C, TNF-{alpha} up-regulated the expression of ICAM-1 as compared with untreated EC (untreated, shaded; TNF-{alpha} treated, open). D, Treatment of EC with C. neoformans had no effect on cytokine-induced ICAM-1 staining (TNF-{alpha} treated, shaded; C. neoformans treated, open).

 
Treatment of EC with C. neoformans does not cause nuclear translocation of NF-{kappa}B

Nuclear translocation of NF-{kappa}B is involved in the transcription of many proinflammatory genes in EC, including adhesion molecules and chemokines (37, 38). Activation of this pathway is rapid, and NF-{kappa}B movement to the nucleus can often be detected within minutes after treatment of EC. To determine whether C. neoformans activated translocation of NF-{kappa}B, EC were incubated with medium, TNF-{alpha}, or C. neoformans for 30 min, and stained for NF-{kappa}B p65. Immunofluorescence revealed that cells treated with C. neoformans or medium had prominent cytoplasmic staining, with little nuclear staining, suggesting a lack of NF-{kappa}B translocation (Fig. 6Go). In contrast, TNF-{alpha}-treated EC exhibited intense nuclear staining for p65 (Fig. 6Go). EC treated with TNF-{alpha} plus C. neoformans also showed nuclear staining. However, we were not able to determine whether this nuclear staining was significantly different from that seen in EC treated with TNF-{alpha} alone due to the qualitative nature of this assay (data not shown).



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  		FIGURE 6. Visualization of NF-{kappa}B. EC were treated for 30 min with medium, TNF-{alpha}, or C. neoformans, and NF-{kappa}B localization was determined by immunofluorescence. Untreated and C. neoformans-treated EC showed NF-{kappa}B reactivity located mainly in the cytoplasm, while in TNF-{alpha}-treated EC, NF-{kappa}B staining was predominantly nuclear. No reactivity was observed in EC stained with control rabbit IgG. Nuclei were counterstained with DAPI and exhibit normal morphology (original magnification, x200).

 
Because some stimuli require longer incubation times to effect nuclear translocation of NF-{kappa}B (12), EC were also examined after 24 h of treatment. These EC had NF-{kappa}B staining patterns similar to those obtained after 30 min of treatment (data not shown).

EC viability is not reduced by C. neoformans

At the end of some experiments, we examined EC for viability after the prolonged contact with C. neoformans. EC were rinsed to remove unattached/extracellular fungi and examined by light microscopy. EC cultures maintained confluence and retained a normal cobblestone appearance. Several EC in C. neoformans-treated wells were in contact with the yeasts and/or contained intracellular organisms. Trypan blue was excluded from >99% of EC in every well, regardless of treatment. DAPI staining revealed nonapoptotic nuclei (Fig. 6Go), and analysis of EC supernatants revealed no increase in LDH release over controls. In agreement with our findings, EC cultured in 24-well plates and exposed to encapsulated C. neoformans (107 fungi/well) showed no signs of cell damage or death after 8 h, as measured by 51Cr release (4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
C. neoformans is a fungus that can cause life-threatening disease in immunocompromised hosts. The mechanisms of fungal dissemination are not well understood, but dissemination is believed to be inversely correlated with the host leukocyte response (32, 39, 40). Incubation of cryptococci with EC in our experiments did not lead to expression of IL-8, IP-10, MCP-1, lymphotactin, RANTES, MIP-1ß, MIP-1{alpha}, or I-309. In fact, C. neoformans inhibited cytokine-induced chemokine production from human EC. Inhibition of chemokine production occurred whether EC were treated with TNF-{alpha}, IL-1ß, IFN-{gamma}, or combinations of these cytokines, indicating a general down-modulatory effect on cytokine signaling.

C. neoformans caused greater reductions in EC expression of the mononuclear cell chemoattractants, IP-10 and MCP-1, than in the neutrophil chemoattractant IL-8. These data demonstrate that C. neoformans is capable of differential inhibition of CXC and CC chemokine synthesis from human EC, consistent with different signaling pathways for induction of these chemokines. The fact that C. neoformans only minimally reduces EC production of IL-8 is intriguing, as elevated IL-8 levels have been documented in the CSF of HIV-1+ patients with cryptococcal meningitis (41), and pulmonary cryptococcal infection in mice induces expression of neutrophil chemoattractants, but fails to induce MCP-1 or IP-10 (42).

Inhibition of cellular inflammatory mediators has been previously reported for C. neoformans. In vitro, C. neoformans down-modulated production of IL-12 by a murine macrophage cell line (43), NO by murine peritoneal macrophages (44), and TNF-{alpha} and GM-CSF by human NK cells (45). Cryptococcal inhibition of these factors was mediated by reduced transcription of these genes, was dependent on direct contact between the fungus and the leukocytes, and was not due to fungal killing of these leukocytes (43, 44, 45). In addition, reduction of macrophage NO was not mediated by cryptococcal capsular GXM, as two acapsular strains of C. neoformans also inhibited nitrite production (44).

ICAM-1 is a leukocyte adhesion molecule constitutively expressed by resting EC, and is up-regulated within hours after treatment with inflammatory stimuli (9, 11, 18, 46, 47). Binding to ICAM-1 allows for firm adhesion of leukocytes to the vessel wall, and is an important step before leukocyte extravasation (36, 48). We found that incubation of EC with C. neoformans did not induce ICAM-1 expression, and did not significantly alter baseline or TNF-{alpha}-induced ICAM-1 levels. However, C. neoformans-induced down-modulation of chemokine synthesis may affect the ability of leukocytes to adhere to adhesion molecules, as binding is triggered by chemoattractant-dependent activation of integrin counterreceptors on the leukocyte surface (49, 50, 51).

Activation of NF-{kappa}B signaling in EC and subsequent adhesion molecule and chemokine expression has been documented for S. aureus (our unpublished observations), C. albicans (7), T. cruzi (10), L. monocytogenes (11), B. burgdorferi (15), dengue virus (12), and respiratory syncytial virus (38), and may represent a common final pathway in the EC response to infectious agents. The failure of C. neoformans to induce nuclear translocation of NF-{kappa}B is consistent with the finding that C. neoformans does not induce expression of ICAM-1 or chemokines in human EC.

The conditions under which C. neoformans inhibited EC chemokine synthesis suggest that cell-bound and/or highly labile cryptococcal/EC products are involved in this process, such as those documented for other pathogens with viability- and contact-dependent effects on mammalian cells (52). Schistosomula of Schistosoma mansoni produce a lipophilic substance that interferes with NF-{kappa}B activity in EC, most likely via the activation of the cAMP/protein kinase A pathway (53). Treatment of human EC with cAMP-elevating agents did not affect TNF-{alpha}-induced expression of ICAM-1 (54), but did reduce EC chemokine synthesis (55). Therefore, the study of lipids and/or cAMP signaling in EC may be a promising avenue of investigation for understanding the immunomodulatory effects of C. neoformans.

In summary, our results indicate that C. neoformans fails to activate human EC for chemokine and ICAM-1 production and suppresses cytokine-induced chemokine synthesis, with greater inhibition of the mononuclear cell chemoattractants IP-10 and MCP-1, than the neutrophil chemoattractant, IL-8. These effects require fungal viability and cell-cell contact, and suggest a general down-modulatory effect of C. neoformans on EC activation. Reduced chemokine production could interfere with the ability of the host to mount an adequate inflammatory response at sites of cryptococcal infection. Future work will focus on the investigation of the signaling pathways responsible for the effects that we have described.


   Acknowledgments
 
We thank Dr. Tina Calderon for critical reading of this manuscript.


   Footnotes
 
1 N.M. is supported in part by the Department of Pathology, Albert Einstein College of Medicine, Yeshiva University. A.C. is supported in part by National Institutes of Health Grants AI-33774, AI-13142, and HL-59842. J.W.B. is supported in part by National Institutes of Health Grant PO1 NS 11920 and National Institute of Mental Health Grant RO1 MH 52974. Data in this paper are from a thesis to be submitted by N.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. Back

2 Address correspondence and reprint requests to Dr. Joan W. Berman, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Back

3 Abbreviations used in this paper: EC, endothelial cell; DAPI, 4',6'-diamidino-2-phenylindole; GXM, glucuronoxylomannan; IP-10, IFN-{gamma}-inducible protein-10; LDH, lactate dehydrogenase; MCP, monocyte chemoattractant protein. Back

Received for publication January 11, 2000. Accepted for publication May 16, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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