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Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans
W Chaka1, A F Verheul, V V Vaishnav
1Division of Vaccines, University Hospital Utrecht, The Netherlands.
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.
Insights
Human mannose-binding protein (hMBP) mediates the binding of cryptococcal mannoprotein (MP2) to peripheral blood mononuclear cells (PBMCs), triggering TNF-alpha release. This interaction is crucial for the immune response to fungal components.
Area of Science:
- Immunology
- Mycology
- Molecular Biology
Background:
- Previous studies indicated specific receptors and a serum factor, distinct from antibodies and complement, mediate TNF-alpha response to cryptococcal mannoprotein (MP2).
- The precise mechanism of MP2 recognition by PBMCs and subsequent immune activation remained unclear.
Purpose of the Study:
- To elucidate the mechanism by which PBMCs recognize MP2.
- To identify the specific serum factor involved in MP2-PBMC interaction and TNF-alpha induction.
Main Methods:
- Flow cytometry was used to analyze FITC-labeled MP2 binding to PBMCs.
- Monoclonal antibodies (mAbs) against specific cell surface markers (CD14, CD11b, CD18) and human mannose-binding protein (hMBP) were employed.
- Serum depletion and recombinant protein assays were performed to assess hMBP's role.
- SDS-PAGE was used to identify proteins interacting with MP2.
Main Results:
- MP2 binding to PBMCs was specific, temperature and CaCl2 dependent, and enhanced by pre-stimulation.
- CD14 was critical for MP2-induced TNF-alpha release, but not for MP2 binding.
- Depletion of hMBP from serum abrogated TNF-alpha induction and inhibited MP2 binding.
- Recombinant hMBP enhanced both MP2 binding and TNF-alpha induction.
- A ~33/34 kDa protein, identified as potentially hMBP, was detected interacting with MP2.
Conclusions:
- Human mannose-binding protein (hMBP) plays a significant role in the recognition and binding of MP2 to PBMCs.
- hMBP is essential for MP2-induced TNF-alpha release from PBMCs.
- The findings identify hMBP as a key mediator in the immune response to cryptococcal mannoprotein.