Primary Study On Molecular Mechanisms Of Nicotinamide, Tetracycline And Dapsone In The Treatment Of Bullous Pemphigoid

Bullous pemphigoid(BP) is an autoimmune inflammatory disease causing blister formation at the dermoepidermal junction. Several skin infiltrating inflammatory cells such as eosinophils, neutrophils and activated T lymphocytes, are involved in BP blister formation, suggesting that the migration and assemble of circulating leucocytes is crucial in the pathogenesis of the disease. While the accumulated evidence suggests that some cytokines are involved in the pathogenesis, there have been few reports about chemokine profiles in patients with BR Nicotinamide, tetracycline and dapsone are used effectively in the treatment of BP, but their molecular mechanisms are still unclear. In this thesis, serum levels of chemokines in the patients with BP, the secreted chemokines and apoptosis of cultured normal human keratinocytes treated with BP IgG, molecular mechanisms of some drugs in the treatment of BP are investigated. This study consisted of three chapters.Chapter I Serum Levels of Chemokines in the Patients With BPObjective To investigate serum levels of CC chemokines, including eotaxin, RANTES (regulated upon activation, normal T cell expressed and secreted), MCP(monocyte chemoattractant protein)-i and MIP(macrophage inhibitory protein)—1, and CXC chemokine IL(interleukin)-8 in the patients with bullous pemphigoid(BP). Methods Serum levels of eotaxin, RANTES, MCP--1, MIP-- 1αand IL-8 in 28 patients with BP and 30 healthy controls were measured by ELISA. Results Serum levels of eotaxin, MCP—1 and IL-8 were significantly increased in patients with BP than those in the controls. Conclusion There are abnormalities of serum levels of chemokines in patients with BP, eotaxin, MCP--1 and IL-8 may be involved in the pathogenesis of BP. ChapterⅡStudy on Release Chemokines and Apoptosis of Cultured Normal Human Keratinocytes Treated with BP IgGObjective To investigate whether treatment of cultured normal human keratinocytes with BP IgG lead to increased protein lever of eotaxin, MCP-land IL-8. To investigate the apoptosis of cultured normal human keratinocytes treated with BP IgG. Methods IgGs in normal plasma and blister fluid of patients were purificated by caprylic acid/ammonium sulfate precipitation. The level and purity of IgGs were tested by immunoturbidimetry and SDS-PAGE, respectively. The immunological activity of blist fluid before and after purification was analyzed by BP180 ELISA kit. Expression of eotaxin, MCP—1 and IL-8 in culture medium of normal human keratinocytes treated with BP IgG were measured by ELISA, the apoptosis of keratinocytes was detected by Annexin-V-FITC kit. Results The recovery of IgG from blister fluid was 51.4%, lower than that from normal serum, they both showed highly purity. Furthermore, the immunological activity of blist fluid was not impaired after purification. Increased protein lever of IL-8, but not eotaxin and MCP-1, was detected in the culture medium induced by BP IgG. This effect was concentration- and time-dependent. None apoptosis of keratinocytes treated with BP IgG was detected Conclusions Purification of IgG from blister fluid of patients with BP by caprylic acid/ammonium sulfate precipitation is simple and feasible, with highly purity. The IL-8 release form cultured human keratinocytes treated with BP IgG may be involved in the pathogenesis of BP.ChapterⅢPrimary Study on Molecular Mechanisms of Nicotinamide, Tetracycline and Dapsone in the treatment of BPObjective To investigate possible molecular mechanisms of anti-inflammatory action of nicotinamide, tetracycline and dapsone in the treatment of BR Methods Normal human keratinocytes were treated With 4mg/ml BPIgG for 24h. At the beginning of this incubation, nicotinamide,tetracycline and dapsone were added to the cultured medium. Cell proliferations were detected by MTT assay, protein lever of IL-8 in culture supernatant were measured by ELISA. Results In their phamacological range, nicotinamide, tetracycline and dapsone did not reduce viability of normal keratinocytes. Dapsone and tetracycline, but not nicotinamide, inhibit the IL-8 release from cultured human keratinocytes treated with BP lgG., in a dose-dependent fashion. Conclusion Dapsone and tetracycline exhibit their anti-inflammatory effect in the treatment of BP partly by inhibition of IL-8 release from cultured keratinocytes.