Figure 3

Identification of the main molecule attracting THP-1 cells among NTHI-induced molecules derived from SLFs. Viral CC chemokine inhibitor (CCI) blocks migration of THP-1 cells responding to the NTHI-induced SLF-derived molecules (A). Migration assays were performed using THP-1 cells and the conditioned medium of NTHI-exposed SLFs with and without treatment of CCI (0.02, 0.1 and 0.5 μg/ml) for 12 h and 36 h. Silencing of MCP-1/CCL2 inhibits migration of THP-1 cells attracted by the conditioned medium of NTHI-exposed SLFs, which is restored by addition of the recombinant MCP-1/CCL2 (rMCP-1, 0.1 and 1 ng/ml) (B). SLFs were transfected with either non-specific siRNA (NC) or MCP-1-specific siRNA (MCP-1) and culture medium was collected after 12 h with and without exposure to the NTHI lysate. Results were expressed relative to the fold change of mRNA levels, taking the value of the NC siRNA-treated group as 1. THP-1 cell migration in response to the conditioned medium of NTHI-exposed SLFs is inhibited more than 60% by MCP-1/CCL2 deficiency, but not by TNF-α deficiency (C). Primary SLFs were cultured in the lower compartment of the migration chamber and were exposed to the NTHI lysate. THP-1 cells were co-cultured in the upper compartment, and migrated cells to the lower chamber were quantitated using a hemocytometer. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p < 0.05.