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Infection and Immunity, April 2005, p. 2554-2555, Vol. 73, No. 4
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.4.2554-2555.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Medicine,1 Department of Microbiology and Immunology,2 Arizona Cancer Center, University of Arizona Health Sciences Center,4 Medical and Research Services, Southern Arizona Veterans Affairs Medical Center, Tucson, Arizona,3 Department of Microbiology and Immunology, University of California at Davis, Davis, California5
Received 30 June 2004/ Returned for modification 3 August 2004/ Accepted 2 December 2004
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ABSTRACT |
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TEXT |
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CD206), a monoclonal antibody directed against MR (4), to block the surface expression of MR on adherent peripheral blood mononuclear cells (PBMC) and examined the effect of this on the cellular immune response induced by T27K, a glycosylated coccidioidal antigen preparation (1-3).
PBMC, derived from the blood of healthy human donors of known coccidioidal immunity, were resuspended in RPMI 1640 (GIBCO, Grand Island, Mich.) with 10% autologous serum, added to 35-mm flat-bottom wells (Falcon, Becton Dickinson Labware), and incubated at 37°C in 95% air-5% CO2. For the first 30 min, mannan (from Sacchyromyces cerevisiae; Sigma Chemical Company, St. Louis, Mo.) or CD206 (from clone 19.2; BD Biosciences Pharmingen, San Diego, Calif.) was added to wells. In some experiments, immunoglobulin G1
(IgG1) (no. 555748; BD Biosciences Pharmingen), the isotype of CD206, was used. Control wells received nothing. After 30 min, 10-µg/ml T27K was added to cells and further incubated for 72 h. Adherent cells were removed and incubated with fluorescein isothiocyanate (FITC)-labeled CD206 or FITC-labeled IgG1 for 30 min at 22°C in the dark. Cell viability just prior to flow cytometry was >90%, as determined by trypan blue exclusion. A gate was set around viable nonlymphocytes and 4,000 events were collected. MR surface expression was measured as the ratio of the geometric mean fluorescent intensity (MFI) of samples stained with FITC-labeled CD206 divided by the geometric MFI of cells stained with labeled isotype. Interleukin-2 (IL-2) and gamma interferon (IFN-
) concentrations in harvested supernatant were measured using a flow cytometry-based immunoassay (CBA; BD Immunosciences, San Jose, Calif.) or by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.).
Monosaccharide analysis of T27K was performed by the Glycotechnology Core Resource of the University of California at San Diego by using high-pH anion-exchange chromatography with pulsed amperometric detection after protein denaturization and desalting (A. Datta, personal communication). The Wilcoxon signed-rank test for paired data was employed for statistical analysis. All work was approved by the Human Subjects Protection Program of the University of Arizona.
To assess mannan blocking of MR, 3.0 x 106 PBMC in 2 ml of RPMI 1640 with 10% autologous serum were incubated for 72 h with or without 3 mg of mannan/ml. Subsequently, 1 mg of FITC-labeled dextran (Sigma)/ml was added for 1 h at either 37 or 4°C. After this, the adherent cells were removed using a sterile rubber scraper, resuspended in phosphate-buffered saline, and then analyzed by flow cytometry as described above. The geometric MFI in the FL-1 channel was used as a measure of cell association.
Mannose was the principal monosaccharide of T27K and was present at a concentration of 1,151 nM/mg. Other monosaccharides detected were glucose (375 nM/mg), galactose (163 nM/mg), N-acetylgalactosamine (58 nM/mg), and N-acetylglucosamine (24 nM/mg).
Among nine immune donors, incubation of PBMC with 3 mg of mannan/ml and T27K for 72 h resulted in significant reduction of MR surface expression on adherent PBMC (P = 0.01) as well as reduced IL-2 (P = 0.01) and IFN- release (P = 0.05) compared to incubation with T27K alone (Fig. 1). In another seven immune donors, incubation of PBMC with 20 µg of CD206/ml and T27K also resulted in reduction in MR surface expression (P = 0.02). Incubation with 20 µg of IgG1/ml, the isotype of CD206, did not reduce MR expression (data not shown). Incubation of cells with CD206 and T27K decreased IL-2 concentrations significantly below those seen in control samples (P = 0.02) and those for samples incubated with IgG1 (P = 0.03).
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Pathogen-associated molecular patterns expressed on microorganisms play critical roles both in inducing the innate immune response and in modulating subsequent acquired immunity (6). Among the ligands for these pathogen-associated molecular patterns, MR is of particular interest since it binds terminal mannoses present on fungi.
Several studies have shown that the immune response to fungal pathogens is mediated through MR. Mansour and colleagues demonstrated that blockade of MR with mannan and methyl--D-mannopyranoside resulted in diminished production of IL-2 by a T-cell hybridoma responsive to mannoprotein derived from Cryptococcus neoformans (7). Syme and colleagues demonstrated that blocking MR with an anti-MR antibody resulted in diminished phagocytosis of C. neoformans by human dendritic cells (13). It is not clear from this study whether the addition of mannan or CD206 simply blocked expression of MR on the cell surface through binding or whether it diminished production of MR. Our assumption is that preincubation of cells with mannan and CD206 led to these moieties binding to surface MR, thus making it unavailable to T27K. The fact that mannan inhibited the association of FITC-dextran with cells suggests that it functionally blocked MR rather than simply interfering with CD206 binding, since pinocytosis of dextran is known to occur through MR (11).
Analysis of T27 revealed that mannose is its predominant monosaccharide. Other studies (5, 8-10, 12, 14) have demonstrated that Coccidioides spp. contain significant amounts of mannose and 3-O-methylmannose. Based on this, it is clearly feasible that MR could bind T27K or even whole Coccidioides isolates and mediate a specific cellular immune response.
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ACKNOWLEDGMENTS |
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We thank John Galgiani for his insightful comments.
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FOOTNOTES |
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REFERENCES |
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, P = 0.05.