The Mannose Receptor Mediates the Cellular Immune Response in Human Coccidioidomycosis

Abstract

Mannose is the predominant monosaccharide in the coccidioidal antigen preparation T27K. Mannan and anti-CD206 antibody significantly decreased the surface expression of mannose receptor (MR) on adherent peripheral blood mononuclear cells and reduced the interleukin-2 (IL-2) release induced by T27K. These data suggest that MR mediates IL-2 release by T27K.The mannose receptor (MR) is found on the surface of macrophages and dendritic cells, can bind terminal mannoses found on fungi and other pathogens (6), and has been shown to mediate the in vitro cellular immune response to fungal antigens (7). We used mannan, an MR ligand (11), and anti-CD206 (␣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% CO 2 . 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-g/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 Ͼ90%, 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 ϫ 10 6 PBMC in 2 ml of RPMI 1640 with 10% autologous serum were incubated for 72 h with or without 3 mg of mannan/ml. Subsequent...

Functional Interaction of Cockroach Allergens and Mannose Receptor (CD206) in Human Circulating Fibrocytes

“…The relative binding activity was expressed as optical density (OD) for each test antigen after subtracting the values from the background. To confirm the binding specificity, inhibition assays were performed with serial dilution of anti-hMR blocking antibody (3.0 µg/ml, clone 19.2, BD Pharmingen, Mississauga, Ontario), mannan (3.0 mg/ml, Sigma), a CD206 ligand [43], [44], and mannose (100.0 µg/ml, Sigma, Oakville, Ontario) added and incubated for 1 h before the allergens were added to the plates for binding assays.…”

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

“…Because we observed a high expression of CD206 in the presence of miR-27a-loaded EVs, we reasoned that the increase in CD206 mediated by miR27a might be responsible for the increased phagocytosis. We used mannan, a CD206 ligand, and anti-CD206 (␣-CD206), a monoclonal antibody directed against CD206, to block the surface expression of CD206 on the monocytes and examined the effect of miR-27a-loaded EVs on naive monocytes (50). We observed that both mannan and ␣-CD206 treatment led to a significant decrease in the phagocytic activity of monocytes (Fig.…”

Preliminary Evaluation of Whole-Blood Gamma Interferon Release for Clinical Assessment of Cellular Immunity in Patients with Active Coccidioidomycosis

“…However, prior studies using T27K as the antigen preparation have demonstrated specificity. For example, we found that the number of CD3 ϩ lymphocytes producing intracellular IFN-␥ was significantly higher from samples from previously identified healthy immune donors than from nonimmune healthy donors (8) and was higher than with donors with active, disseminated coccidioidomycosis (4). In addition, we have been able to induce a specific in vitro coccidioidal cellular immune response in lymphocytes from both nonimmune healthy donors and from nonreactive individuals with active coccidioidomycosis using mature dendritic cells incubated with T27K (25).…”

“…Currently, there are no defined coccidioidal antigens available that have been shown to be immunogenic in humans. Moreover, defined antigens, particularly those that are genetically cloned, may be less immunogenic than preparations derived directly from the organism, such as T27K, because the latter are highly glycosylated, allowing for attachment to innate receptors, such as that for mannose (8).…”