159 Thrombin Stimulates Interleukin-8 Production in Human Macrophages Through a JNK and NFκB Pathway

Abstract

Wound healing is a complex biological event orchestrated by various resident and infiltrating cell types spanning multiple stages. Thrombin, a multifunctional serine protease released upon wounding, stimulates fibroblasts and macrophages at the sites of wounds to express, chemokines, such as interleukin-8 (IL-8). We have used cellular and molecular approaches to investigate the mechanisms involved in thrombin induces IL-8 expression in human macrophages. Western blot analysis and RT-PCR show that thrombin stimulates IL-8 in a rapid and dose-dependent manner. Various inhibitors for signal transduction proteins were used to delineate the pathways involved. We found that, at nanomolar concentrations, inhibitors for both c-Jun N-terminal Kinase (JNK) and NFɻB strongly inhibited the thrombin-induced production of IL-8 suggesting that they are major regulators of this stimulation. JNK was shown to be phorsphorylated in a time-dependent manner in response to thrombin treatment confirming its involvement. In order to identify the mediator of JNK and the thrombin receptor, we use affinity pull down and found that Rho GTPase was activated by thrombin suggesting that Rho may be the mediator of JNK activation. In addition, we found that PKC is a negative regulator of thrombin-induced IL-8 expression. Our study broadens the horizon of how IL-8 is regulated in wound healing and may help develop pharmaceutical methods to manipulate this process in pathological conditions.