Expression Of Toll Like Receptor 4 In Monocyte-derived Macrophages And The Role In Production Of IL-6 In Patients With Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a common autoimmune disorder .Recently,there were many reports about dysfounction of the immune cells and unbalance in immune molecule's production,which are greatly involved in the lupus pathology.Many cytokines could participate in development of lupus.An elevated level of certain cytikines has been well recognized. Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities that plays an important role in immune regulation and inflammation. Among other actions, it induces terminal differentiation of B lymphocytes into antibody-forming cells and the differentiation of T cells into effector cells. IL-6 also has multiple potent proinflammatory effects. An association between IL-6 and lupus was demonstrated in murine models of SLE and blocking IL-6 improved lupus in all models tested. Data from several studies suggest that IL-6 plays a critical role in the B cell hyperactivity and immunopathology of human SLE, and may have a direct role in mediating tissue damage, IL-6 has been found spontaneous highly expressed in SLE patients'periphery blood or kidney tissue,and correlate with disease severity.The exact mechanism for this is unclear.Infection,especially Gram-negative bacteria,or virus can initiate and aggravate lupus's clinical symptom.The exact relationship between SLE and Infection and how could pathogens induce dysfounction of the immune response system in patients with systemic lupus erythematosus are not very clear.Mammals sence pathogens through pattern-recoginition receptors(PRRs).The most important in PRRs is toll-like receptors. TLR4 is a member of the recently identified Toll-like receptor family of proteins which recognizes Gram-negative bacteria'product-lipopolysaccbaride(LPS).TLR4-CD14 complex recognizes LPS-LPS binding protein,then initiate multiple intracellular signaling events (4), including the activation of NF-κB, which ultimately leads to the synthesis and release of a number of proinflammatory mediators,including IL-1, IL-6, IL-8, and tumor necrosis factor-a.So TLR4 is considered playing a critical role in immune adjuvant effect on the brige of innate immunity against pathogens and body's adaptive immunity. The purpose of this study is to study the inflammatory response caused by SLE through regulating monocyte-derived macophages TLR4 and evaluating the role of TLR4 in the inflammatory response in SLE. I .The study of the stimulation of NF-κB by LPS through TLR4.Objective: to explore the signal pathway of the stimulation of NF-κB through LPS in monocyte-derived macrophages. Methods: Monocytes isolated from human peripheral blood mononuclear cells(PBMC) were cutrued to monocyte-derived macrophages in vitro, then we departed cells into 3 group:1.medium.Cells in only medium of DMEM.2.Medium+LPS.Cells were stimulated with LPS (10ng/ml)overnignt.3.Medium+LPS+anti-TLR4.Before stimulated with LPS overnght,human monocyte-derived macrophages were preincubated with various concentrations of anti-TLR4(5μg/mL, 20μg /mL, 30μg /mL) for 2h.IL-6 production in supernatants and NF-κB p65 in nuclear protein were measured by ELISA respectively. Results: In human monocytes-derived macrophages, LPS enhanced NF-κB activation, which was clearly inhibited by anti-TLR4. Moreover, LPS-induced IL-6 production by human monocytes-derived macrophages was inhibited by anti-TLR4. Conclusions:In this study, TLR4 appeared to be involved in an LPS-mediated activation of NF-κB.II. Expression changes of Toll-like receptor 4 in peripheral blood monocyte-derived macrophages in SLE.Objective: Since toll-like receptors 4 (TLR4) play an important role in infection immunity,mediating synthesis of inflammatory cytokines,the main aims of this study were to explore the difference in monocyte-derived macrophages TLR4 expression in patients with SLE between healthy control persons. Methods: seventeen patients with SLE were included in this study. Using the same method, monocyte-derived macrophages were prepared.The expression of TLR4 mRNA in SLE monocyte-derived macrophages were detected by RT-PCR and compared with healthy control persons.The level of IL-6 in supernants were detected by ELISA. Result: monocyte-derived macrophages TLR4 expression were upregulated in SLE patients. LPS can significantly increase TLR4 mRNA in SLE monocyte-derived macrophages and the level of IL-6 in supernatants.Conclusion: TLR4 appear to be involved in the inflammatory response in SLE , The quantity of macrophages TLR4 may be associated with immunity situation of the body.III .The study of the blocking agent of TLR4 blocking the stimulation of NF-κB and IL-6 level in patients with systemic lupus erythematosus.Objective: to study the effect of the blocking agent of TLR4 blocking the stimulation of NF-κB and production of IL-6 in SLE. Methods: Preincubated with or without anti-TLR4 for 2h, peripheral blood mononuclear cells from SLE were stimulated with LPS (1Ong/mL) overnight. Relative activity of NF-κB p65 in neuclear protein were detected by ELISA.IL-6 level in supernatants were measured by ELISA. Result: compared with control group,relative activity of NF-κB in anti-TLR4 groups were not different significantly.But the production of IL-6 were greatly inhibited by anti-TLR4.Conclusions:Antibodies against TLR-4 did not inhibit NF-κB activity in patients with SLE.IL-6 level in SLE were decreased after blocking TLR4 pathway,induction of IL-6 is TLR4-dependent in SLE monocyte-derived macrophages.