INDUCTION OF TNF-ALPHA IN HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS BYTHE MANNOPROTEIN OF CRYPTOCOCCUS-NEOFORMANS INVOLVES HUMAN MANNOSE-BINDING PROTEIN

We have shown previously that specific receptors on PBMCs and a serum factor other than Ab and complement are involved in the TNF-alpha resp onse to cryptococcal mannoprotein (MP2). To characterize the mechanism of MP2 recognition by PBMCs, 10(6) PBMCs were incubated with 25 mu g of FITC-labeled MP2 in 10% normal human serum (1 h). The cells were an alyzed by flow cytometry. FITC-MP2 binding was CaCl2 and temperature d ependent 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-MP 2 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 in hibition. Removal of human mannose binding protein (hMBP) by preincuba tion of serum with a specific mAb abrogated TNF-alpha induction by MP2 and strongly inhibited its binding to PBMCs. Recombinant hMBP enhance d 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 33/3 4 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 P BMCs and the release of TNF-alpha.