Human platelets stimulate mesangial cells to produce monocyte chemoattractant protein-1 via the CD40/CD40-ligand pathway and may amplify glomerular injury

Background and objective

Depletion of platelets reduces influx of hemopoietic cells into the glomeruli, decreases resident cell proliferation and improves renal function in animal models of glomerulonephritis. However, the precise mechanisms by which platelets promote glomerular injury are poorly understood. We explored whether platelets induce production of monocyte chemoattractant protein-1 (MCP-1) by cultured human mesangial cells (MCs) and the mechanisms involved.

Methods

Platelets were isolated from normal human donors and cocultured with MCs at various ratios. The contribution of cell-to-cell contact was examined by a double-chamber culture system. MCP-1 synthesis in cocultures was determined by semiquantitative reverse transcription/real-time PCR and ELISA. Signal transduction was studied by mobility gel-shift assays and phosphotyrosine immunoblots.

Results

Platelets, at 1:100 ratio (MC: platelets), induced a more than 10-fold increase in mesangial MCP-1 production, both in mRNA and protein levels. Using a double-chamber culture system, we demonstrated that the direct cell-to-cell contact between platelets and MC is indispensable for MCP-1 production. Importantly, blockade of the CD40-CD40 ligand (CD40L) pathway with neutralizing antibodies decreased MCP-1 production by approximately 60%. Immunostaining confirmed the expression of CD40 by cultured MCs. When added into MC cultures, recombinant CD40L in combination with recombinant IFN-γ, enhanced MCP-1 production twofold (relative to the basal levels in response to recombinant IFN-γ alone), confirming that CD40 was functionally expressed on MCs. Using gel-shift assay and a specific inhibitors, we showed that the contact of MCs with platelets induced NF-κB activation, which was essential for MCP-1 synthesis. In additional experiments, we found that activation of p38 mitogen-activated protein kinase (MAPK) and protein tyrosine kinases (PTKs) are involved in platelet-mediated MCP-1 induction by MCs.

Conclusion

Platelet/MC contact stimulates the production of MCP-1 and may contribute to glomerular inflammatory responses by recruiting leukocytes from the peripheral blood.