Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein

Abstract

We have shown previously that specific receptors on PBMCs and a serum factor other than Ab and complement are involved in the TNF-alpha response to cryptococcal mannoprotein (MP2). To characterize the mechanism of MP2 recognition by PBMCs, 10(6) PBMCs were incubated with 25 microg of FITC-labeled MP2 in 10% normal human serum (1 h). The cells were analyzed by flow cytometry. FITC-MP2 binding was CaCl2 and temperature dependent and was enhanced by prestimulating PBMCs with unlabeled MP2. Binding to PBMCs was specific, since unlabeled MP and mannan produced dose-dependent inhibition. Beta-Glucan laminarin produced background inhibition. mAbs against CD14, CD11b, and CD18 did not prevent FITC-MP2 binding to PBMCs, implying that these receptors are not involved in MP2 recognition by PBMCs. mAb against CD14 blocked (>90%) MP2-induced TNF-alpha release by PBMCs, while mAbs against CD11b/CD18 caused no inhibition. Removal of human mannose binding protein (hMBP) by preincubation of serum with a specific mAb abrogated TNF-alpha induction by MP2 and strongly inhibited its binding to PBMCs. Recombinant hMBP enhanced TNF-alpha induction by MP2 as well as binding of FITC-MP2 to PBMCs. In addition, incubation of serum with MP2-coated beads and analysis by SDS-PAGE resulted in the detection of a protein of approximately 33/34 kDa that could be partially removed by preincubating the serum with hMBP mAb. We conclude that hMBP is involved in the binding of MP2 to PBMCs and the release of TNF-alpha.