Caporali Francesca, Capecchi Pier Leopoldo, Gamberucci Alessandra, Lazzerini Pietro Enea, Pompella Gerarda, Natale Mariarita, Lorenzini Sauro, Selvi Enrico, Galeazzi Mauro, Laghi Pasini Franco
Department of Clinical Medicine and Immunological Sciences, Section of Clinical Immunology, University of Siena, Policlinico Le Scotte, viale Bracci, Siena, Italy.
J Mol Med (Berl). 2008 Aug;86(8):937-49. doi: 10.1007/s00109-008-0365-8. Epub 2008 Jun 11.
Human type B synoviocytes are involved in joint injury during rheumatic diseases by producing inflammatory mediators such as interleukin-6 (IL-6). The increased level of purine and pirimidine nucleotides in the synovial fluid of rheumatoid arthritis (RA) patients could activate the large family of P2 receptors. Thus, we investigated the presence of P2 receptors in human type B synoviocytes from rheumatoid joints, also evaluating whether the P2X7 receptor is involved in IL-6 release. Reverse transcriptase polymerase chain reaction analysis revealed messenger ribonucleic acid (mRNA) expression for the P2X1, P2X2, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14 but not the P2X3, P2Y2, and P2Y6 receptors. The expression of the P2X7 receptor was confirmed by Western blot analysis. Adenosine triphosphate (ATP) and the P2X7 receptor agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) triggered an increase in intracellular calcium, thereby suggesting the expression of functional P2 receptors, including the P2X7 receptor. Moreover, BzATP treatment upregulated both IL-6 mRNA and protein expression. Synoviocytes spontaneously released low quantities of IL-6; the incubation with BzATP induced the release of larger amounts of the cytokine, and such a release was blunted by the P2X7 antagonist oxidized ATP. The selective P2X1 and P2X3 receptor agonist alpha,beta-methylene ATP did not affect IL-6 release. Finally, BzATP failed to induce a significant uptake of the large-molecule YO-PRO, thus suggesting the lack of pore formation after P2X7 receptor stimulation. In conclusion, among the different P2 receptors expressed on human RA type B synoviocytes, the P2X7 receptor may modulate IL-6 release but not inducing changes in cell membrane permeability.
人类B型滑膜细胞通过产生白细胞介素-6（IL-6）等炎症介质参与风湿性疾病期间的关节损伤。类风湿关节炎（RA）患者滑液中嘌呤和嘧啶核苷酸水平的升高可激活P2受体大家族。因此，我们研究了类风湿关节中人类B型滑膜细胞中P2受体的存在情况，同时评估P2X7受体是否参与IL-6的释放。逆转录聚合酶链反应分析显示P2X1、P2X2、P2X4、P2X5、P2X6、P2X7、P2Y1、P2Y4、P2Y11、P2Y12、P2Y13和P2Y14的信使核糖核酸（mRNA）表达，但未检测到P2X3、P2Y2和P2Y6受体的表达。通过蛋白质印迹分析证实了P2X7受体的表达。三磷酸腺苷（ATP）和P2X7受体激动剂2'-3'-O-（4-苯甲酰苯甲酰）ATP（BzATP）引发细胞内钙增加，从而提示包括P2X7受体在内的功能性P2受体的表达。此外，BzATP处理上调了IL-6 mRNA和蛋白表达。滑膜细胞自发释放少量IL-6；与BzATP孵育诱导释放大量细胞因子，而这种释放被P2X7拮抗剂氧化ATP所抑制。选择性P2X1和P2X3受体激动剂α，β-亚甲基ATP不影响IL-6的释放。最后，BzATP未能诱导大分子YO-PRO的显著摄取，因此提示P2X7受体刺激后缺乏孔形成。总之，在人类RA B型滑膜细胞上表达的不同P2受体中，P2X7受体可能调节IL-6的释放，但不会引起细胞膜通透性的改变。
