High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation

Flagella from diverse gram-negative bacteria induce tumor necrosis factor a lpha (TNF-alpha) and interleukin-lp (IL-1 beta) synthesis by human monocyte s (F, Ciacci-Woolwine, P. F, McDermott, and S. B. Mizel, Infect. Immun, 67: 5176-5185, 1999), In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Ente ritidis, S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNP-alpha production by human monocytes and TH P-1 myelomonocytic cells, Fifty percent of maximal TNF-alpha production (EC ,,) was obtained with 1.5 x 10-(11) M flagellin (0.75 ng/ml), Mutagenesis s tudies revealed that the central hypervariable region of flagellin is essen tial for the TNF-alpha-inducing activity of the protein. Although less acti ve than the wild-type protein, a Salmonella flagellin mutant composed of on ly the central hypervariable region retained substantial TNF-alpha-inducing activity at nanomolar concentrations, In contrast, the conserved amino- an d carboxy-terminal regions are inactive. Mutational analysis of the hyperva riable region revealed that it contains two equally active TNF-alpha-induci ng domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken togethe r, our results demonstrate that the cytokine-inducing activity of flagellin s from gram-negative bacteria results from the interaction of these protein s with high-affinity cell surface polypeptide receptors on monocytes.