Upregulation of chemokine CXCL1/KC by leptospiral membrane lipoprotein preparation in renal tubule epithelial cells

We have previously shown that leptospiral membrane lipoprotein preparation (LMLP) extracted from pathogenic Leptospira santarosai serovar Shermani stimulates the secretion of pro-inflammatory mediators in renal tubule epithelial cells, and implicated its role in the initiation of tubulointerstitial nephritis. Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. Initial studies by cytokine protein array analysis revealed significant upregulation of neutrophil-chemoattractant keratinocyte-derived chemokine (CXCL1/KC) at nanogram range of LMLP stimulation in cultured murine proximal tubule cells (PTCs). As PTCs express Toll-like receptors (TLRs), this study investigated the roles of TLR signaling pathways in PTCs stimulated by LMLP and its relation to CXCL1/KC secretion. The LMLP stimulated the early secretion of CXCL1/KC and enhanced the level of TLR2 mRNA expression in PTCs through time- and dose-dependent effect. The LMLP-stimulated secretion of human growth-related oncogene alpha, a functional homolog to murine KC, in TLR-defective human embryonic kidney 293 cells transiently transfected with TLR2-expressing plasmids and the response was augmented by coexpression of TLR1 and TLR2. Moreover, silencing of TLR2, myeloid differentiation factor 88, and TNF receptor-associated factor 6 with specific small interfering RNA significantly reduces the response caused by LMLP in PTCs. The LMLP-stimulated CXCL1/KC secretion was also significantly reduced by pre-incubating PTCs with a specific p38 inhibitor. These results indicate that LMLP stimulates the production of CXCL1/KC to recruit polymorphonuclear neutrophils at the site of inflammation through a TLR2-mediated pathway in renal tubule cells.