Popovic Milan, Laumonnier Yves, Burysek Ladislav, Syrovets Tatiana, Simmet Thomas
Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Helmholtzstr. 20, D-89081 Ulm, Germany.
J Leukoc Biol. 2008 Jul;84(1):215-23. doi: 10.1189/jlb.0907652. Epub 2008 Apr 24.
CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-alpha. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-kappaB-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-kappaB signaling pathway by the IkappaB kinase inhibitor acety-11-keto-beta-boswellic acid or by transient overexpression of a transdominant-negative form of IkappaBalpha. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.
CX3CL1（趋化因子、神经趋化素）是唯一的CX3C趋化因子。它以裂解形式诱导单核细胞运动，但在其膜锚定形式下，它也作为一种黏附分子发挥作用。促炎细胞因子如IL-1或TNF-α可上调内皮细胞中CX3CL1的表达。在此，我们研究了丝氨酸蛋白酶凝血酶对内皮细胞CX3CL1诱导的影响及其与单核细胞功能的潜在相关性。在人脐静脉内皮细胞（HUVEC）中，凝血酶在mRNA和蛋白质水平上触发了CX3CL1的时间和浓度依赖性表达，这通过逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹和流式细胞术分析得以证实。如使用PAR1激活肽和PAR1特异性拮抗剂SCH 79797所证明的，凝血酶通过激活蛋白酶激活受体1（PAR1）诱导CX3CL1。如电泳迁移率变动分析（EMSA）、酶联免疫吸附测定（ELISA）以及通过IκB激酶抑制剂乙酰基-11-酮-β-乳香酸抑制NF-κB信号通路或通过瞬时过表达IκBα的显性负性形式所表明的，凝血酶诱导的CX3CL1表达依赖于NF-κB。当人单核细胞与HUVEC共培养时，HUVEC中凝血酶依赖性的膜锚定CX3CL1诱导触发了单核细胞黏附，并增强了单核细胞释放单核细胞趋化蛋白-1/CCL2（MCP-1/CCL2），并增强了单核细胞跨内皮迁移。相应地，CX3CL1的重组细胞外结构域诱导单核细胞释放CCL2。因此，凝血酶诱导的由CX3CL1表达增加介导的单核细胞/内皮细胞相互作用增强了CCL2趋化因子的产生，这可能有助于将单核细胞募集到炎症区域。
