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(Journal of Leukocyte Biology. 2000;68:923-932.)
© 2000 by Society for Leukocyte Biology

Differential chemokine response of human monocytes to yeast and hyphal forms of Candida albicans and its relation to the ß-1,6 glucan of the fungal cell wall

Antonella Torosantucci, Paola Chiani and Antonio Cassone

Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome, Italy

Correspondence: Dr. Antonio Cassone, Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy. E-mail: cassone{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyphae formation from yeast cells is a virulence trait enabling the human opportunistic pathogen Candida albicans to invade host tissues. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, interleukin-8 (IL-8), and particularly, monocyte chemotactic protein-1 (MCP-1) by human monocyte. This different stimulation did not depend on the monocyte inability to ingest the hyphae nor did it imply hyphal resistance to the extracellular killing by the monocytes. Purified hyphal and yeast cell walls reproduced the differences shown by the intact cells, and chemical-enzymatic dissection of cell wall components suggested that cell wall ß-1,6 rather than ß-1,3 glucan was the main chemokine inducer. Coherently, immunofluorescence studies with an anti ß-1,6 glucan serum showed that the surface expression of this polysaccharide was much lower on hyphae than on yeast cells. By minimizing chemokine induction, the formation of hyphal filaments might facilitate C. albicans escaping from host immunity.

Key Words: phagocytes • macrophage inflammatory protein-1 {alpha} • macrophage inflammatory protein-1ß • monocyte chemotactic protein-1 • interleukin-8 • RANTES


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are chemoattractant cytokines that play crucial roles in host defense against infectious agents by their ability to induce and regulate magnitude and composition of leukocyte influx at the infected site. In addition, they may act as direct activators of the antimicrobial activity of leukocytes and also modulate lymphocyte differentiation into predominantly T-helper type 1 (Th1) or Th2 patterns [1 2 3 4 5 6 7 ]. Chemokines are grouped into different families on the basis of the arrangement of their highly conserved cysteine residues in the mature protein [1 ]. Upon appropriate stimulation, they are redundantly produced by a variety of cells of both leukocyte and non-leukocyte type.

The role of chemokines in fungal diseases has been poorly explored. In particular, a limited amount of information exists on chemokine response to Candida albicans, a major agent of opportunistic infections in AIDS patients and in many other categories of immunodepressed subjects [8 9 10 ]. Sironi et al. showed that human peripheral blood cells stimulated by the fungus were able to produce monocyte chemotactic protein-1 (MCP-1), a member of C-C chemokine family, and this production was inhibited by the anti-inflammatory agent benzydamine [11 ]. In their study of chemokine induction by oral microorganisms, Jiang et al. [12 ] also demonstrated that C. albicans was a potent stimulus for production of MCP-1 and interleukin-8 (IL-8), the first characterized chemokine belonging to the C-X-C family [1 ]. Hachicha et al. [13 ] showed that IL-8 and macrophage inflammatory protein (MIP)-1{alpha}, another member of C-C-chemokine family, were produced by human neutrophils stimulated in vitro with C. albicans, although this organism was a less potent stimulus than bacteria such as Salmonella typhimurium and Staphylococcus aureus. Finally, Huang and Levitz [14 ] have recently demonstrated that both C. albicans and, to a lesser degree, Cryptococcus neoformans are able to stimulate MIP-1{alpha}, MIP-1ß, and RANTES production by human peripheral blood mononuclear cells with no difference between HIV- and HIV+ subjects.

One common aspect of all previous investigations with C. albicans as chemokine stimulant is the narrow spectrum of chemokines being tested and/or the lack of studies aimed at identifying the critical fungal components or the mechanisms mediating chemokine induction in leukocytes. It is important to note that no attempts have been made to distinguish the stimulatory activity of yeast (Y) from that of hyphal (H) forms of the fungus, a key aspect in the interpretation of the chemokine response to C. albicans, which owes much of its pathogenicity to the transition from the Y to the H habit of growth [8 ].

Bearing this in mind, we have addressed the capacity of Y and H cells of C. albicans to stimulate human monocytes for production of C-X-C or C-C chemokines, with an effort to identify which Candida components could be responsible for this induction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fungal cells
Two strains of C. albicans were used throughout this study. C. albicans BP, serotype A, is a virulent, hyphal (H) conversion-competent strain from the established type collection of the Istituto Superiore di Sanità (Rome, Italy). C. albicans CA2 strain is a virulence-attenuated mutant unable to convert to H forms in vitro and in mouse internal organs [15 , 16 ].

Fungal cells were grown in the Y form in Winge medium for 24 h at 28°C, washed with saline, resuspended at 2 x 106 cells/mL in H growth-inducing buffered Lee’s medium, and incubated at 28 or 37°C for 90 min, as previously described [17 ]. At 28°C, both strains maintained the yeast form (Y) in this medium. At 37°C, the strain BP developed >90% H cells (i.e., short hyphal filaments emerging from Y cells and three to five times as long as the Y cell of origin) within 60–90 min of incubation, whereas strain CA2 maintained its Y form. Y or H cells were harvested by centrifugation, washed twice with endotoxin-free H2O, and resuspended in endotoxin-free phosphate-buffered saline (PBS) at the desired concentration. Inactivated Y and H cells were prepared from BP strain, by incubation in Lee’s medium at 28 or 37°C for 90 min as described above. Fungal cells were washed in H2O, resuspended in H2O, and heated at 70°C for 40 min. Inactivated cells were washed extensively with endotoxin-free PBS and resuspended in PBS at the desired cell density.

Monocyte cultures and cytokine assays
Leukocyte buffy coats from healthy human blood donors were diluted 1:4 in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) and separated by density gradient centrifugation onto Lympholyte-H solution (Cedarlane Laboratories, Hornby, Ontario, Canada). Monocytes were separated from cells at the interface layer by adherence. Briefly, these cells were washed twice with RPMI, resuspended in complete medium [CM; RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mmol L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO-BRL)] at 3–5 x 106 cells/mL, and incubated in tissue culture dishes (Becton Dickinson, Plymouth, UK) for 1 h at 37°C under 5% CO2 atmosphere. Nonadherent cells were removed by three washings with warm RPMI, and adherent monocytes were gently detached with a cell scraper. Cell recovery was evaluated by microscopic examination of Giemsa-stained cytosmears. Monocytes were resuspended in CM at 106/mL and cultured in multiwell microplates in the absence (unstimulated control) or in the presence of chemokine-inducing stimuli or inhibitors as specified in each single experiment. After 18 h of incubation (unless otherwise indicated), supernatants of cell cultures were harvested, spun free of cells and cellular debris, and assayed for cytokine content by indirect enzyme-linked immunosorbent assay (ELISA; R & D Systems, Minneapolis, MN), as per the manufacturer’s instructions. Monocyte viability was affected in no case by the stimulation with Candida cells or cell wall material (see below) as shown by dye (trypan blue) exclusion tests.

Phagocytosis was evaluated as inhibition of [3H]glucose uptake by live Candida cells upon a 2-h co-cultivation with monocytes, and also monitored by microscopic examination of PAS-stained samples of Candida-monocyte co-cultures [18 ]. To measure the killing of Y and H cells of C. albicans, monocytes (5 x 105 in 200 µL of CM) were co-cultured (duplicate samples) with 5 x 104 predifferentiated, live, Y or H cells from CA2 or BP strain, respectively. After 4 h of incubation (37°C, 5% CO2), monocytes were lysed by addition of Triton X-100 (final concentration 0.2%), and residual live fungal cells were enumerated by colony forming units (CFU) onto triplicate Sabouraud agar plates. Percent killing was calculated by comparing CFU counts from Candida monocytes co-cultures with CFU counts from duplicate control cultures with Candida Y or H cells alone.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine mRNA expression in Candida-stimulated monocytes
Monocyte cultures, either unstimulated or stimulated with Y or H cells at a Candida/monocyte ratio of 2:1 were prepared as described above. After 3 h of incubation, total RNA was extracted from monocytes (3 x 106) by the guanidinium-isothiocyanate method [19 ] and reverse-transcribed as previously described [18 ]. Aliquots of cDNA yielding equivalent amounts of a ß-actin amplified band, as estimated by densitometric analysis, were used for semiquantitative evaluation of cytokine mRNAs. PCR was performed in a 10 µL volume in a Perkin-Elmer 9600 thermal cycler running 25 (for ß-actin and MIP-1ß) or 28 (for MCP-1, MIP-1{alpha}, and MIP-1ß) cycles of 1-min denaturation at 94°C, 40 s annealing at 62°C [MCP-1, MIP-1{alpha}, MIP-1ß, tumor necrosis factor {alpha} (TNF-{alpha}), and ß-actin] or at 55°C (IL-8) and 1 min extension at 72°C. Cytokine-specific primer sequences were synthesized by GIBCO-BRL according to published sequences [20 21 22 ]. The PCR products were visualized by electrophoresis and ethidium bromide staining, identified by their predicted molecular sizes, and quantitatively evaluated by densitometric scanning.

Preparation of cell walls and cell wall fractions from C. albicans
Clean Y and H cell walls were prepared by mechanical breakage of cells with 0.45-mm glass beads followed by extensive washes in cold water, essentially as described by Angiolella et al. [23 ]. In addition, cell walls were subjected to a three-step lipid extraction with chloroform/methanol 2:1 (overnight, room temperature, twice), chloroform/methanol/H2O 10:10:1 (overnight, room temperature, twice) and chloroform/methanol/H2O 10:10:3 (overnight, room temperature, twice). Delipidized cell walls were washed with H2O and lyophilized.

The MP mannoprotein fraction (polysaccharide, >95% w/w, of which mannan >90%; protein, 5.0%) was obtained from Y cells and purified as previously described [24 , 25 ]. A pure ß-glucan fraction (GG; polysaccharide, >98% w/w, of which glucose >93%; protein <0.05%) was prepared by cycles of acid and alkali extraction at 100°C of Y cells, as described elsewhere [26 , 27 ]. Supernatants of acid extraction were pooled, dialyzed, and lyophilized to obtain a ß-1,6-enriched, acid-soluble glucan fraction, as reported by Cassone et al. [27 , 28 ]. ß-1,6 glycosylated mannoproteins were obtained from clean cell wall preparations as above by sodium dodecyl sulfate (SDS)-EDTA-ß-mercaptoethanol extraction followed by ß-1,3 endoglucanase (Zymoliase 100T) digestion, as described by Kapteyn et al. [29 ]. Finally, secreted mannoproteins from Y or H cells of C. albicans were separated from supernatants of 24-h fungal cultures by extensive dialysis/ultrafiltration through a low-adsorbance ultrafiltration membrane (molecular cutoff 10 kDa, Millipore, Bedford, MA), as described elsewhere [17 ]. All the above preparations were judged to be bacterial endotoxin-free because addition of polymxin B did not affect their biological activity.

Periodate oxidation of GG or MP extracts (2 mg) was performed at room temperature for 4 h with 1 mL of 200 mM sodium metaperiodate in 50 mM acetate buffer, pH 4.5. For ß-1,3 glucanase digestion, GG or MP extracts (2 mg) were treated for 2 h at 37°C with 1 mg/mL of enzyme (Zymoliase 100T, Seikagaku Kogyo, Tokyo, Japan) in PBS, then the enzyme was heat-inactivated (5 min, 100°C). Periodate or zymolase-treated GG fractions were repeatedly washed with PBS, whereas treated soluble MP fractions were extensively dialyzed by ultrafiltration in centrifuge ultrafiltration devices (Centricon, molecular cutoff 10 kDa, Millipore).

Dot-blot assays
Fractions to be analyzed were resuspended in carbonate buffer, pH 9.6. Aliquots containing equivalent amounts of polysaccharide material were adsorbed by gravity flow onto nitrocellulose membrane sheets (0.22-µm pore size, Bio-Rad Laboratories, Richmond, CA) in a microfiltration apparatus. Nitrocellulose sheets were reacted with either rabbit anti ß-1,6 glucan serum (see below) and alkaline phosphatase-conjugated anti-rabbit IgG antibodies or with digoxigenin-labeled concanavalin A (Roche) and alkaline phosphatase-conjugate anti-digoxigenin antibodies (Roche) and finally stained by incubation with 5-bromo-4-chloro-3-indolyl-phosphate and 4-nitroblue tetrazolium chloride solution as the enzyme substrate.

Immunofluorescence assays
Live Y or H cells of C. albicans strain BP, after adhesion on immunofluorescence microscope slides, were extensively washed with PBS containing 0.1% Tween 80 and blocked (1 h, 37°C) with 3% bovine serum albumin (BSA) in PBS. Spots were reacted (2 h, 37°C) with mouse anti ß-1,6 glucan serum (1:20 diluted in PBS-3% BSA), washed, and treated with fluorescein isothiocyanate (FITC)-conjugate-anti mouse IgM antibodies (1 h, 37°C). After extensive washings, the slides were mounted in glycerol, pH 9.6, and examined under a Leitz Diaplan fluorescence microscope.

Analytical determination
Total polysaccharide content of fractions was estimated by the phenol/sulfuric acid method of Dubois et al. [30 ], using glucose as the standard. Protein concentration was measured by a commercial assay (Bio-Rad).

Reagents
Escherichia coli lipopolysaccharide (LPS), cytochalasin D, and laminarin were purchased from Sigma Chemical (St. Louis, MO). Interferon-{gamma} was from R & D Systems. Pustulan was obtained from Calbiochem. Affinity-purified rabbit anti-ß-1,6 polyclonal antibodies were a kind gift of Dr. Frans M. Klis (Institute of Molecular Cell Biology, University of Amsterdam, The Netherlands). Preparation, affinity purification, and specificity of these antibodies have been described [31 ]. Mouse anti-ß-1,6 glucan serum was obtained by immunizing CD2F1 mice with pustulan. Mice received two subcutaneous injections, at weekly intervals, with 50 µg pustulan in incomplete Freund’s adjuvant (Sigma Chemical) followed by three further intraperitoneal injections, at weekly intervals, with 50 µg of pustulan without adjuvant. Seven days after the last injection, mice were bled by retroorbital puncture. The ELISA titer of pooled sera from immunized animals (pustulan as coating antigen) was 1:640.

All reagents employed in this study were prepared using endotoxin-free water and labware.

Statistics
Differences were evaluated by the non-parametric Mann-Whitney U test or by the parametric Student’s t test, as indicated in specific experiments. Significance was set at P < 0.05, two tailed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chemokine response to Y and H cells of C. albicans
We preliminarily examined whether live Y cells of virulent or avirulent C. albicans strains could induce production of cytokines (TNF-{alpha} and IL-12) and chemokines (MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8) by human monocytes. We noticed that the virulent strain of C. albicans was much less stimulatory, as compared to the avirulent strain, for all tested cytokines except for RANTES and TNF-{alpha}. Because the virulent strain rapidly (within 60–90 min) developed >90% H-cells in culture while the avirulent mutant maintained its initial Y form, we wondered whether the different stimulating ability of the two strains was indeed related to their different forms of growth during co-cultivation with monocytes. Thus, pre-differentiated Y and H cells of the virulent strain were assayed as chemokine inducers. Fungal cells were also heat-inactivated to eliminate any time-variable, possibly toxic, event in monocyte cultures.

In a typical dose-response experiment (Fig. 1 ), H cells were consistently less efficient than Y cells in eliciting a chemokine response by monocytes. In particular, MCP-1 production was very weakly or not stimulated at all by H cells. In contrast, Y and H cells were similarly capable of triggering TNF-{alpha} release, demonstrating that the fungal form-dependent effect on chemokine production was not a result of a general hyporesponsiveness of monocytes to H cells.



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  		Figure 1. Dose-response chemokine and TNF-{alpha} production by monocyte cultures stimulated with Y or H cells of C. albicans. Monocyte (106/mL) were co-cultured with heat-inactivated Y or H cells of C. albicans strain BP at the indicated ratios. Cytokines released in culture supernatants after an 18-h incubation were measured by ELISA. Contr, unstimulated monocytes.

 
The differential chemokine production described above was reproducibly observed in distinct experiments performed with monocyte cultures from 10 different donors (Fig. 2 ). In particular, H cell-stimulated monocytes showed no increase or even decrease of MCP-1 production, as compared with unstimulated controls. Overall, the mean (± SE) chemokine production (in ng/mL) by unstimulated and Y- or H-stimulated monocytes was 7.1 ± 2.1, 16.3 ± 2.6, and 6.3 ± 1.3 for MCP-1; 3.0 ± 0.8, 26.2 ± 2.4, and 18.3 ± 2.2 for MIP-1ß; 0.2 ± 0.05, 10.7 ± 1.8, and 5.9 ± 1.9 for MIP-1{alpha}; and 197 ± 21, 732 ± 68, and 481 ± 54 for IL-8. In pooled data, thus incorporating the expected high individual variations in chemokine production by the monocytes of the different donors, the difference between Y- and H-stimulated chemokine production was indeed significant at a P level of < 0.005, < 0.05, < 0.025, and < 0.05 for MCP-1, MIP-1ß, MIP-1{alpha}, and IL-8, respectively, while being not significant for RANTES and TNF-{alpha} (Mann-Whitney U test, two-tailed).



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  		Figure 2. Chemokine and TNF-{alpha} response of monocytes from different subjects to Y or H cells of C. albicans. Monocytes from different subjects were stimulated with heat-inactivated Y or H cells of C. albicans strain BP at a Candida/monocyte ratio of 2:1. Cytokines released in culture supernatants after an 18-h incubation were measured by ELISA. Contr, unstimulated monocytes. The difference between unstimulated and Y cell-stimulated chemokine and TNF-{alpha} production was always statistically significant (P from <0.05 to <0.005), as the differences in chemokine production after stimulation by Y and H cells (see text). All statistical assessments were made with both Student’s t test (paired data) and with Mann-Whitney U test (pooled data), two-tailed (see text).

 
Semiquantitative RT-PCR assays were also used to evaluate chemokine mRNA expression in monocyte cultures after a 3-h stimulation with fungal cells. Both forms of growth of the fungus could induce an early increase in the transcription of chemokine (MCP-1, MIP-1{alpha}, MIP-1ß, IL-8) and TNF-{alpha} genes, with respect to unstimulated control monocytes. Nevertheless, the levels of early chemokine gene expression were always lower in H-stimulated than in Y cell-stimulated monocytes, in contrast to similar ß-actin and TNF-{alpha} mRNA expression (Fig. 3 ). Thus, the differential ability of the two Candida growth forms was likely mediated by effects at the level of chemokine gene transcription and were not the result of preferential chemokine absorption to H cells or increased degradation in their presence.



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  		Figure 3. Analysis of chemokine gene expression in monocytes stimulated with Y or H cells of C. albicans. Monocytes (3x106) were co-cultured for 3 h with heat-inactivated Candida cells (BP strain) at a Candida/monocyte ratio of 2:1. Total RNA was extracted from the cultures and analyzed for the presence of specific chemokine mRNAs by semiquantitative RT-PCR assays, as descibed in Materials and Methods. Contr, unstimulated monocytes. Results are from one representative experiment out of two performed with similar results.

 
The different efficiency of Y and H cells in inducing chemokine production is not due to differences in phagocytosis of the two growth forms
Because Y cells of C. albicans are rapidly phagocytized by human monocytes while H cells remain mostly uningested [8 , unpublished observations], we examined whether phagocytosis could account for the differential fungal form-related chemokine response. To this aim, we measured Candida-stimulated chemokine production in the presence of a concentration (1 µg/mL) of cytochalasin D sufficient for a nearly 100% reduction of Y Candida cell ingestion by monocytes. LPS-stimulated chemokine production served as negative control, whereas the positive control was IL-12, the production of which had been shown to strictly depend on phagocytosis [58 ]. Table 1 shows that the blockade of phagocytosis with cytochalasin D, although almost completely abolishing Candida-stimulated IL-12 production, did not grossly affect chemokine production by monocytes in response to Candida cells to LPS. Only for MCP-1 was an appreciable reduction detected, which was not commensurate, however, with the almost total abolition of phagocytosis caused by the drug.


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  		Table 1. Effect of Phagocytosis Blockade with Cytochalasin D on the Chemokine Response of Human Monocytes to Candida Cells

 
Monocytes kill H and Y cells of C. albicans with similar efficiency
We next examined monocyte capability of killing the two forms of growth of the fungus. As shown in Figure 4 , H cells were as susceptible as, or even more susceptible than, Y cells to the antimicrobial action of monocytes. Phagocytosis blockade with cytochalasin D practically abolished the monocyte capacity of killing Y cells without affecting the killing of H cells. Thus, Y but not H cell killing required phagocytosis.



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  		Figure 4. Killing of Y and H cells by human monocytes. Monocytes were cocultured with predifferentiated, live, Y or H cells of C. albicans. After 4 h, surviving fungal cells were enumerated by CFU counts. Percent killing was calculated by comparison of CFU counted for Y or H cells cultured with monocytes with CFU counts of control cultures with Y or H cells alone. For additional details, see Materials and Methods. Values are means ± SE of three independent determinations. *Significant difference (P<0.01, two-tailed Student’s t test) between values of percent killing of Y cells by cytochalasin D-treated monocytes and those measured for each of the other three experimental conditions.

 
Cell wall components of C. albicans mediate chemokine induction
Because microbial cells express on their surface a number of potential cytokine inducers [for a review, see ref. 32 ], we addressed Candida cell wall (CW) in the search for specific chemokine-stimulating components. To this purpose, isolated CW from both Y and H forms of growth, as well as CW fractions enriched in their main chemical constituents, were assayed for chemokine-inducing activity compared with LPS. CWs were very efficient in stimulating chemokine production, as exemplified by MIP-1ß and MCP-1 production in Figure 5 . It is interesting that H-CW were about five and two times less efficient than Y-CW in inducing MCP-1 and MIP-1ß production, respectively, paralleling the differences observed with the intact cells.



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  		Figure 5. Chemokine induction by isolated Y- or GT-CW. Clean, delipidized, and lyophilized cell wall, obtained as described in Materials and Methods, were administered to monocyte cultures at the dose of 10 µg dry weight/mL. LPS was used at 0.1 µg/mL. MCP-1 and MIP-1ß production was measured after 18 h by ELISA. Control, unstimulated monocytes. Data are from one representative experiment out of three performed with similar results.

 
We next examined which one of the main cell wall constituents, e.g., mannoprotein (MP) or ß-glucan (which is present as 1-6 or 1-3 bound polymers) [33 34 35 36 ] could mediate chemokine induction. Table 2 shows that significantly increased levels of MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8, compared with unstimulated control monocytes, were measured by monocyte stimulation with both MP and ß-glucan (GG) components in a dose-dependent fashion. However, a 10- to 100-fold higher amount of MP was required to achieve the same level of stimulation as that achievable with GG. In particular, a significant increase of MIP-1{alpha}, MIP-1ß, and IL-8 production was detected in the presence of 1 µg/mL of GG, whereas the MP fraction was only effective at 50–100 µg/mL. The chemokine-inducing activity of Candida cell wall fractions was, however, less potent than that exerted by LPS. None of the fractions used contained LPS, as shown by the negative Limulus amebocyte lysate test, and no influence on the activity of the GG fraction was exerted by polymixin B, an LPS inhibitor (data not shown).


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  		Table 2. Comparison of Mannoprotein and ß-Glucan Fractions from the Cell Wall of C. albicans as Inducers of Chemokine Release by Human Monocytes

 
ß-1,6 glucan as a major stimulant of chemokine production
To further test the hypothesis that chemokine induction by C. albicans is mainly mediated by ß-glucan components, additional CW fractions enriched with ß-1,3 and ß-1,6 glucan or MP components, were used as stimulants. Table 3 shows that MIP-1ß production was consistently associated with the presence of ß-glucan rather than {alpha}-mannan, and in a dose-response fashion. It is noteworthy that the chemokine-inducing MP fraction was seen to contain ß-1,6 glucan in addition to mannan. In addition, treatments of both MP and GG fraction with sodium metaperiodate or with a purified ß-1,3 glucanase, strongly indicated that the presence of ß-1,6 glucan was essential for chemokine stimulation. In particular, treatment of alkali-acid insoluble glucan (a CW fraction that contains only ß-glucan), with periodate completely abolished the chemokine-inducing activity of this fraction, paralleling the disappearance of any anti-ß-1,6 serum-reactivity of the fraction. Similar findings were obtained with other chemokines instead of MIP-1ß (data not shown).


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  		Table 3. Chemokine-Inducing Activity of Different Cell Wall Fractions of C. albicans is Associated with the Presence of ß-1,6 Glucan Component

 
In a further attempt to discriminate between ß-1,6 and ß-1,3 glucans as chemokine stimulatory components, and because of the difficulties in obtaining Candida CW fractions of totally pure ß-1,6 or ß-1,3 molecular composition, we resorted to the use of well-characterized, low-molecular-weight, commercial glucan preparation. As shown in Table 4 , a ß-1,6 glucan (pustulan) but not a ß-1,3 glucan (laminarin) polymer exerted an appreciable stimulatory effect on the production of the two chemokines tested, MCP-1 and MIP-1ß.


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  		Table 4. A ß-1,6- But Not a ß-1,3-Soluble Glucan Polymer can Stimuluate Chemokine Production by Human Monocytes

 
ß-1,6 glucan is differentially expressed on the surface of Y and H cells
Having established a major role for cell wall ß-1,6 glucan in chemokine stimulation by C. albicans, we obviously wondered whether the different efficiency of the Y and H forms in chemokine induction could be accounted for by differential expression of ß-1,6 glucan on cell surface.

As shown in Figure 6 , ß-1,6 glucan constituents positively reacting with an anti-pustulan serum were indeed expressed, although in an uneven fashion, on the surface of Y cells. In contrast, H cells showed a very faint or no fluorescence at all on the hyphal filament, thus demonstrating a down-modulation of ß-1,6 glucan expression during Y to H cell transformation.



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  		Figure 6. Expression of ß-1,6 glucan constituents on Y and H cells. Y or H cells from C. albicans strain BP were allowed to adhere to immunofluorescence microscope slides and treated with mouse anti-ß-1,6 glucan serum followed by FITC-conjugated anti-mouse IgM antibodies. Ligation of anti-ß-1,6 glucan serum to the cell wall surface was monitored by microscopic observation. (A) Y cells; (B) H cells. For other technical information, see Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A key feature of C. albicans interaction with the host is the ability of the fungus to change its habit of growth from yeast (Y) to hyphal filament (H), a morphological transition that has long been considered to be critical for Candida pathogenicity [37 38 39 40 41 42 43 44 45 ]. We have here described a novel phenomenon which suggests that the pathogenicity associated with the above transition may impinge upon alteration of anti-Candida immune response. H forms were indeed significantly less stimulatory than Y forms for chemokine production by human monocytes. This differential capacity was not restricted to a single chemokine of a particular family but involved both C-C and C-X-C chemokines. IL-12, a key promoter of T helper type 1 protective responses [46 ] was also much less induced by the H than by the Y cells. To our knowledge, this is the first demonstration that a critical stage of fungal growth and pathogenicity such as H forms is significantly different from the Y cell in such an immunologically relevant aspect as the production of soluble mediators of leukocyte traffic, activation, and maturation of immune responses. It is interesting that the lower capacity of chemokine stimulation was not associated with a general hyporesponsiveness to cytokine production, as the two forms of growth were equally potent in stimulating the production of TNF-{alpha} and another chemokine such as RANTES was also equally produced by Y- and H-stimulated monocytes.

Particularly impressive was the substantial inability of the H forms to induce the production of MCP-1. This is a 9- to 13-kDa protein produced by various cellular sources, including epithelial and endothelial cells, and capable of recruiting both T cells and macrophages to the site of infection [1 2 3 ]. Jiang et al. [12 ] reported its induction by C. albicans in peripheral blood mononuclear cell cultures, but no attempts were made to identify the inductive fungal component (discussed below) or to define any potential difference between the two C. albicans growth forms. Production of MCP-1 has been shown to play a role in the recruitment of various inflammatory cells in the lungs of mice infected by another fungal pathogen, Cryptococcus neoformans [47 ]. In particular, chemokine induction did correlate well with CD4+ T cells in the lung granuloma and was considered to be crucial for host defense against this pathogen [47 ].

Recent studies have not only confirmed the role of monocytes as final anti-Candida effectors [48 49 50 ] but have also suggested that monocytes exert a critical influence on the induction of protective T-helper (Th) responses [50 51 52 53 54 ]. In this line, low IL-12 production and suboptimal recruitment of monocytes to the infection site, by differences in the stimuli provided by the infecting fungus, could evidently synergize in vivo, considering that natural immunoeffectors are devoted, among other things, to an early production of IL-12 in the absence of activated T cells. Both in serum and mucosa bathing fluids, C. albicans promptly differentiates from Y to H forms. Thus, the lower potency of these latter in inducing critical mediators of immune response may be of advantage to the fungus. It is noteworthy that H forms are as easily killed by the monocytes as the Y cells, though with extracellular rather than intracellular mechanisms [55, 56]. Thus, H cells can in theory resist these effectors only by diverting them from the site of infection.

The differences in chemokine production by the monocytes stimulated by the Y or the H forms of C. albicans were not a result of the differences in the ability of the immunoeffectors to ingest and phagocytize the two forms of growth of the fungus, as clearly shown by the experiments with the phagocytosis inhibitor cytochalasin D. Remarkably, phagocytosis proved essential for IL-12 production by monocytes, confirming previous findings [57 , 58 ]. This suggests that other signals in addition to phagocytosis are relevant for C. albicans-driven chemokine induction.

Among these putative signals, direct recognition of stimulatory cell wall components of the fungus may be critical. Human leukocytes may express both a mannose and a glucan receptor, and thus may bind the two main components of the C. albicans cell wall, also depending upon the differentiation stage of the phagocytic cell [50 , 59 60 61 62 ]. It is possible that the binding of cell wall constituents to these (or other receptor) releases a signal triggering chemokine production, associated with, or independently of, phagocytosis. It was therefore of particular interest to identify Candida constituent(s) capable of triggering chemokine production. From a diversified set of experimental approaches, the biologically active substance appeared to be a ß-1,6 glucan constituent. This conclusion was strengthened by the observation that a commercial non-Candida ß-1,6 (pustulan) but not a ß-1,3 glucan (laminarin) induced MIP-1ß and MCP-1 production by the monocytes. Overall, our data strongly suggest that chemokine induction occurs through direct recognition of the ß-1,6 glucan of Candida and its interaction with the ß-glucan receptor on the surface of the human monocyte. The identification of ß-glucan rather than mannan as chemokine inducer is also in line with the absence of mannose receptor in monocytes [50 , 59 ], with the demonstration given here that ß-1,6 glucan is expressed on Y cell surface, and the reported lack of involvement of mannose receptor in chemokine stimulation by the interaction of C. albicans with murine macrophages [63 ]. Other studies are, however, required to further validate this hypothesis and establishing the nature and extent of the interaction between Candida polysaccharides and its receptor(s).

Finally, we must wonder whether the identification of ß-1,6 glucan as the main if not the sole chemokine stimulant of C. albicans could account for the remarkable differences in the chemokine stimulatory potential of Y and H forms of the fungus. There are relatively few comparative studies of the biochemical differences in the cell wall of the two forms of growth, nonetheless, H-CW have been shown to contain about three times less ß-1,6 glucan than the yeast cell wall [64 , 65 ]. We have shown here that ß-1,6 glucan is consistently present on Y cell wall but scarce or absent on H cell wall. Whether this sole difference does indeed account for differential chemokine induction cannot be firmly stated at the moment.

In summary, the data reported here suggest that part of the virulence traits attributed to the filamentous form of C. albicans may eventually reside in its lower capacity for stimulating chemokine production, with the resultant lower degree of protective inflammatory and phagocytic response. Our studies also suggest that cell-surface-expressed ß-1,6 rather than ß-1,3 glucan or mannan is the principal chemokine inducer of this fungus.


   ACKNOWLEDGEMENTS
 
This research was in part supported by the National AIDS Program (Istituto Superiore di Sanità, Rome, Italy), contract No. 50 C/B. We wish to thank Mrs. A. Botzios, F. Girolamo, and C. Belotti for help in the preparation of the manuscript, Dr. G. Girelli (Clinical Immunology, University of Rome, La Sapienza) for the provision of blood from normal subjects, and Drs. F. M. Klis and J. C. Kapteyn for kindly donating a sample of anti-ß-1,6 glucan serum.

Received April 22, 2000; revised June 20, 2000; accepted June 22, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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