| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 105 === Helicobacter pylori neutrophil-activating protein (HP-NAP), a virulence factor of Helicobacter pylori, induces production of reactive oxygen species (ROS) and cytokines by neutrophils and monocytes. HP-NAP activates PTX-sensitive G protein-coupled receptor (GPCR) to produce ROS in neutrophils. HP-NAP-induced activation of TLR2 is considered to be related to cytokines secretion in monocytes. However, it is not clear whether TLR2 is involved in HP-NAP-induced secretion of interleukin-8 (IL-8) in neutrophils. In this study, all-trans retinoic acid (ATRA)- and dimethyl sulfoxide (DMSO)-induced differentiated HL-60 cells were employed to serve as a neutrophil model cell line to determine whether they are appropriate for studying the function of HP-NAP. Both differentiated HL-60 cells not only show a similar morphology to neutrophils but also express CD11b, a mature myeloid cell marker. However, HP-NAP was capable of inducing ROS production in ATRA-induced differentiated HL-60 cells but not in DMSO-induced differentiated HL-60 cells. The production of ROS induced by HP-NAP depended on PTX-sensitive heterotrimeric G protein but not on TLR2 in ATRA-induced differentiated HL-60 cells. PTX-sensitive heterotrimeric G protein signaling leading to activation of ERK1/2 and p38-MAPK was also contributed to HP-NAP-induced production of ROS in this differentiated HL-60 cells. Thus, HP-NAP-induced production of ROS in ATRA-induced differentiated HL-60 cells and neutrophils is through the same molecular mechanism. I then investigated whether TLR2 was involved in HP-NAP-induced secretion of IL-8 in ATRA-induced differentiated HL-60 cells. HP-NAP was able to induce ATRA-induced differentiated HL-60 cells to secrete IL-8 in a time- and dose-dependent manner. The secretion of IL-8 induced by HP-NAP was significantly inhibited by the treatment of TLR2-neutralizing antibody. However, PTX treatment resulted in a nearly complete inhibition of HP-NAP-induced secretion of IL-8 in ATRA-induced differentiated HL-60 cells. Similarly, the secretion of IL-8 induced by Pam3CSK4, a TLR2 ligand, was also inhibited by PTX. The results imply that the secreted IL-8 induced by HP-NAP and Pam3CSK4 could activate CXCR1/2, IL-8 receptors, coupled to PTX-sensitive heterotrimeric G protein. Treatment of reparixin and SB225002, inhibitors of CXCR1 and CXCR2, was able to inhibit secretion of IL-8 induced by HP-NAP and Pam3CSK4 in ATRA-induced differentiated HL-60 cells, indicating that autocrine and paracrine signaling played an essential role in HP-NAP- and Pam3CSK4-induced secretion of IL-8. Thus, both TLR2 and autocrine and paracrine signaling are involved in induction of IL-8 secretion by HP-NAP and Pam3CSK4 in ATRA-induced differentiated HL-60 cells. However, combination treatment of CXCR1/2 inhibitors and TLR2-neutralizing antibody did not completely inhibit secretion of IL-8 in ATRA-induced differentiated HL-60 cells, suggesting that PTX-sensitive G protein might be a downstream signaling molecule of TLR2 for HP-NAP-induced secretion of IL-8. Taken together, in ATRA-induced differentiated HL-60 cells, PTX-sensitive G protein-coupled receptor is involved in HP-NAP-induced production of ROS, while TLR2 is participated in HP-NAP-induced secretion of IL-8. The autocrine and paracrine signaling is also involved in HP-NAP- and Pam3CSK4-induced secretion of IL-8. Here, ATRA-induced differentiated HL-60 cells may serve as a model cell line for studying the function of HP-NAP in neutrophils.
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