Recruitment of CCR6-expressing Thl7 cells by CCL20 secreted from plasmin-stimulated macrophages

In the present study, monocyte-derived human macro-phages were differentiated from buffy coats. Naive CD^4＋ T-cells enriched from peripheral blood mononuclear cells using anti-CD4 magnetic beads and the autoMACS separ-ation system were polarized under T-helper 17 （Thl7）-pro-moting conditions for 6 days to get Thl7 cells. The frequency of Thl7 cell differentiation and the expression of C-C chemokine receptor type 6 （CCR6） on Thl7 cells were investigated by flow cytometry. Plasmin-triggered induc-tion of macrophage inflammatory protein-3alpha/C-C che-mokine ligand 20 （CCL20） genes in macrophages was assessed by reverse transcription-polymerase chain reac-tion, and secreted protein levels were measured by enzyme-linked immunosorbent assay. Thl7 cell migration induced by CCL20 secreted from plasmin-stimulated macrophages was tested in vitro by chemotaxis using a transwell system. These results demonstrate that plasmin triggers the expres- sion of chemokine CCL20 messenger RNA and the release of CCL20 protein in human monocyte-derived macro-phages, which critically depend on the proteolytic activity of plasmin and activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways. Expression of CCR6 was detected on 87.23 ± 8.6% of Thl7 cells in vitro. Similar to chemotaxis triggered by recombinant human CCL20, supernatants collected from plasmin-stimu- lated macrophage-induced chemotactic migration of Thl7 cells, which could be inhibited by an anti-CCL20 neutraliz-ing antibody. These results suggest that plasmin generated in inflamed tissues might elicit production of chemokine CCL20 by human macrophages leading to the recruitment of CCR6 positive Thl7 cells to the inflammatory sites.