Expression Of High Mobility Group Box Chromosomal Protein 1 And The Modulating Effects On Downstream Cytokines In Systemic Lupus Erythematosous

Part One Study on Expression of High Mobility Group Box Chromosomal Protein 1(HMGB1) in Skin Lesions in Patients with systemic Lupus Erythematosus and Dicoid Lupus ErythematosusObjectiveTo study the expression of lesional skin high mobility group box chromosomal protein-1(HMGB-1) in patients with systemic lupus erythematosus and dicoid lupus erythematosus,respectively;and investigate the role of HMGB-lin the pathogenesis of lupus erythematosus.MethodsImmunohistochemistry and Western-blot were used to test the expression of HMGB-1 in skin lesions from 20 DLE patients,25 SLE patients and 20 healthy controls.ResultsIn healthy control,HMGB-1 mainly expressed in the nucleus of keratinocytes.In skin lesions of DLE patients,HMGB-1 mainly expressed in the involved in the inflammatory of lupus erythematosus mononuclear cells of dermis,but the percentage of positive keratinocytes in epidermis of lesional parts was decreased than that of healthy control (t=11.315,P＜0.01).In nonlesional parts,HMGB-1 also expressed in the nucleus of keratinocytes and there was no difference on the percentage of positive keratinocytes between DLE and healthy control(P＞0.05).By Western-blot,there was no statistical significance in total protein between DLE and healthy control(t=0.681,P＞0.05).In SLE,besides the mononuclear cells,HMGB-1 could be detected both in the cytoplasmic and extracellular space in dermis,while the HMGB-1 nuclear expressions in keratinocytes of epidermis were decreased than those of DLE. (t=6.821,P＜0.01),and in nonlesional parts,HMGB-1 also expressed in the nucleus of keratinocytes and there was no difference on the percentage of positive keratinocytes with healthy control(P＞0.05).The total protein was increased in SLE than those of healthy control and DLE patients(t=15.494,P＜0.01;t=13.221,P＜0.01,respectively).The intensity of HMGB-1 was correlated with SLEDAI and proteinuria(r=0.565,P＜0.01,OR=1.027,P＜0.05,respectively.).ConclusionCompared with healthy control,there were HMGB-1 translocation and alteration of expression levels in patients with lupus erythematosus, which indicates that HMGB-1 may be involved in the inflammatory of lupus erythematosus.Part Two Expression and Translocation of High Mobility Group Box Chromosomal Protein-1(HMGB-1) in Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus ErythematosusObjectiveTo investigate the expression and the translocation of HMGB1 in peripheral blood mononuclear cells(PBMCs) from Patients with Systemic Lupus Erythematosous.MethodsHMGB-1 concentrations in sera from 39 SLE patients and 39 healthy controls were measured by immunoblot analysis.HMGB-1 messenger RNA(mRNA) expression in peripheral blood mononuclear cells(PBMCs) unstimulated or stimulated with lipopolysaccharide(LPS, 200ng/ml) were detected by real time reverse transcription-polymerase chain reaction(real time RT-PCR).Immunofluorescence assay was employed to observe the translocation of HMGB-1 in mononuclear cells after endotoxin stimulation.ResultsHMGB-1 concentrations were significantly higher in serum of SLE patients than in that of the healthy subjects and HMGB-1mRNA expression was up-regulated in unstimulated SLE PBMCs.The pattern and localization of HMGB1 translocation in PBMCs were similar in both SLE patients and healthy controls.LPS-induced HMGB-1 production was elevated in SLE patients,especially in those with proteinuria.ConclusionThe up-regulation of HMGB-1 in SLE patients indicated that HMGB-1 is involved in the inflammatory process of the disease,and this up-regulation may correlate with a negative feedback mechanism. HMGB-1 exerts its damaging effects mainly at site of the stimulation.Part three The Modulating Effects of HMGB-1 on TNFαand IL-6 production by PBMCs from Patients with Systemic Lupus ErythematosousObjectiveTo investigate the modulating effects on proinflammatory cytokines TNF-αand IL-6 in Systemic Lupus Erythematosous.MethodsPBMCs were isolated from SLE patients and healthy individuals,and were stimulated with HMGB-1(1μg/ml)or LPS(100ng/ml)or medium alone.The levels of tumor necrosis factor-α(TNF-α) and interlukin-6(IL-6) produced in the supernatants of the cells were measured via enzyme-linked immunosorbent assay.The expression of TNFαmRNA was measured using RT-PCR.ResultsAfter stimulation with HMGB-1,TNF-αlevel was continuously low in SLE,while IL-6 level was greater and delayed to reach peak in SLE patients compared with healthy controls.The expression of TNFαmRNA was down-regulated in SLE patients when stimulated with HMGB-1.ConclusionPBMCs from SLE patients may have defects in their capacity of TNFαsecretion.The imbalance of TNF-αand IL-6 induced by HMGB-1 suggests that HMGB-1 acts in an autocrine- paracrine loop to constitute a unique cytokine milieu involved in the chronic inflammation in SLE.