Influence of humanized anti-IL-6R antibody, tocilizumab on the activity of soluble gp130, natural inhibitor of IL-6 signaling

Tocilizumab, humanized anti-IL-6R antibody is a novel anti-rheumatic drug. In the present study, we examined the influence of tocilizumab on the inhibitory activity of soluble gp130 (sgp130) against IL-6 signaling. BAF-h130 cells, human gp130 transfected mouse pro-B cell line, were cultured with the mixture with IL-6, sIL-6R and sgp130 for 72 h. BAF-h130 cells proliferated by the addition of both IL-6 and sIL-6R, but not IL-6 or sIL-6R alone. The proliferation induced by IL-6/sIL-6R complex was inhibited by sgp130 concentration-dependently. Tocilizumab inhibited IL-6/sIL-6R-induced cell proliferation. On the other hand, it did not affect the suppressive property of sgp130. To examine if tocilizumab can dissociate IL-6/sIL-6R/sgp130 complex, we established an ELISA system to detect IL-6/sIL-6R/sgp130 complex using anti-IL-6R coated ELISA plate. The results clearly indicated that ELISA system established was detectable IL-6/sIL-6R/sgp130 complex in a concentration dependent manner. Tocilizumab was added to IL-6/sIL-6R/sgp130 complex-fixed plate and then remaining IL-6/sIL-6R/sgp130 complexes on the plate were measured. The amount of IL-6/sIL-6R/sgp130 complexes on the plate was not changed by the addition of tocilizumab. In conclusion, tocilizumab exerts its inhibitory effect through the inhibition of IL-6R directly without affecting natural inhibitor sgp130.     

Soluble gp130 is up-regulated in the implantation window and shows altered secretion in patients with primary unexplained infertility

Members of the IL-6 family of cytokines, which includes leukemia inhibitory factor (LIF) and IL-11, play important roles in implantation. The activity of these cytokines is modified by soluble receptors such as the IL-6 receptor (sIL-6R). gp130 is a signal transduction molecule common to the receptor complexes of this family, and its soluble form (sgp130) antagonizes their actions. The purpose of this study was to determine whether secretion of IL-6, LIF, sIL-6R, and sgp130 was different in the endometrium of women with primary unexplained infertility compared with normal fertile women. Endometrial biopsies were taken between d LH+6 and +13 and cultured in serum-free medium for 4 h. Secretion of IL-6, LIF, sIL-6R, and sgp130 was measured in the supernatant by ELISA. We also measured the secretion of IL-6, sIL-6R, and sgp130 by endometrial biopsies taken throughout the menstrual cycle in normal fertile women. Secretion of sgp130 increased 20-fold between d 20 and 26 of the cycle, coinciding with the implantation window (proliferative phase, median, 27.0 pg/ml.mg; range, 23-36; d 20-26, median, 501.5 pg/ml x mg; range, 26.1-1344; P = 0.03). RT-PCR showed that none of the known splice variants of gp130 were present in endometrium, indicating that sgp130 is produced by proteolytic cleavage of the membrane-bound form. IL-6 secretion varied considerably between patients and was greatest during the secretory phase and at menstruation. No significant change was seen in sIL-6R during the cycle. Between LH+6 and +13, secretion of sgp130 was significantly reduced in the infertile group (median, 93.1 pg/ml.mg; range, 28.5-256; compared with the fertile group, median, 223 pg/ml x mg; range, 63-534; U-statistic = 37; P = 0.017). Secretion of IL-6, LIF, and sIL-6R did not differ between the two groups. Immunolocalization of gp130, IL-6R, and the LIF receptor showed that the glandular epithelium and also endothelial cells are targets for IL-6 and LIF. These findings show that during a normal menstrual cycle, sgp130 secretion is greatly increased between d LH+6 and +13, due to proteolytic cleavage of membrane-bound gp130. Infertile patients show reduced secretion of sgp130 compared with fertile controls during this period, which coincides with the implantation window.     

Circulating interleukin-6 family cytokines and their receptors in patients with congestive heart failure

gp130 is a common signal-transducing receptor subunit for the interleukin (IL)-6 cytokine family. Studies in genetically engineered animal models have demonstrated a critical role for the gp130-dependent cardiomyocyte survival pathway in the transition to heart failure. In the present study, we examined plasma levels of the IL-6 family of cytokines and the soluble form of their receptors in patients with congestive heart failure (CHF). Circulating levels of the IL-6 family of cytokines, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) were examined in 48 patients with various degrees of CHF, including dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), and valvular cardiomyopathy (VCM). Circulating levels of IL-6, leukemia inhibitory factor (LIF), and sgp130 significantly increased in association with the severity of CHF. No significant difference was observed in the circulating levels of sIL-6R and IL-11 among these patients. Interestingly, DCM patients showed higher circulating sgp130 levels than patients with ICM or VCM. Our findings suggest that gp130 expression in the heart is likely to be dynamic, and that the IL-6 family of cytokines and their common receptor gp130 participates in the pathogenesis of CHF, especially in DCM.     

Receptor engagement by viral interleukin-6 encoded by Kaposi sarcoma-associated herpesvirus.

Receptor usage by viral interleukin-6 (vIL-6), a virokine encoded by Kaposi sarcoma- associated herpesvirus, is an issue of controversy. Recently, the crystal structure of vIL-6 identified vIL-6 sites II and III as directly binding to glycoprotein (gp)130, the common signal transducer for the IL-6 family of cytokines. Site I of vIL-6, however, comprising the outward helical face of vIL-6, where human IL-6 (hIL-6) would interact with the specific alpha-chain IL-6 receptor (IL-6R), is accessible and not occupied by gp130. This study examined whether this unused vIL-6 surface is available for IL-6R binding. By enzyme-linked immunosorbent assay, vIL-6 bound to soluble gp130 (sgp130) but not to soluble IL-6R (sIL-6R). Using plasmon surface resonance, vIL-6 bound to sgp130 with a dissociation constant of 2.5 microM, corresponding to 1000-fold lower affinity than that of hIL-6/sIL-6R complex for gp130. sIL-6R neither bound to vIL-6 nor affected vIL-6 binding to gp130. In bioassays, vIL-6 activity was neutralized by 4 monoclonal antibodies (mAbs) recognizing a domain within vIL-6 site I, mapped to the C-terminal part of the AB-loop and the beginning of helix B. The homologous region in hIL-6 participates in site I binding to IL-6R. In addition, binding of vIL-6 to sgp130 was interfered with specifically by the 4 neutralizing anti-vIL-6 mAbs. Based on the vIL-6 crystal structure, the vIL-6 neutralizing mAbs map outside the binding interface to gp130, suggesting that they either produce allosteric changes or block necessary conformational changes in vIL-6 preceding its binding to gp130. These results document that vIL-6 does not bind IL-6R and suggest that conformational change may be critical to vIL-6 function.     

Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130

The interleukin-6 (IL-6) signal is transduced through membrane-anchored gp130, which is associated with IL-6 receptor (IL-6R) in the presence of IL-6. Soluble forms of gp130 (sgp130) with molecular weights of 90 and 110 Kd were found in human serum. In the presence of recombinant IL- 6 (rIL-6), serum sgp130 were capable of associating with serum sIL-6R. By the sandwich enzyme-linked immunosorbent assay, healthy human sera was shown to contain 390 +/- 72 ng/mL of sgp130. A mouse pro-B-cell line-derived transfectant, BAF-130, expressing human gp130 was used to examine the function of serum sgp130. When supplemented with rIL-6, human serum induced DNA synthesis in BAF-130 cells, whereas the serum deprived of sIL-6R did not. In contrast, the DNA synthesis induced in BAF-130 cells by rIL-6-supplemented serum was increased when the serum was deprived of sgp130. These results indicated that serum sgp130 could negatively regulate the IL-6 signal. Recently, gp130 has been shown to be involved in the signaling processes of oncostatin M, leukemia inhibitory factor, and ciliary neurotropic factor, in addition to those of IL-6. Recombinant sgp130 showed inhibitory effect on the biologic function of such cytokines. This work implies physiologic roles of naturally produced serum sgp130 in modulating signals through gp130.     

Elevated maternal soluble Gp130 and IL-6 levels and reduced Gp130 and SOCS-3 expressions in women complicated with preeclampsia

Increased inflammatory response plays a significant role in the vascular pathophysiology in preeclampsia. However, the mechanism for increased inflammatory response in preeclampsia is largely unknown. Interleukin (IL)-6 levels are elevated in women with preeclampsia. IL-6 and its receptors, IL-6R and glycoprotein (gp)130, play a critical role in mediating antiinflammatory response via induction of SOCS-3 (suppressor of cytokine signaling-3). However, IL-6 receptor levels and expressions have not been studied in preeclampsia. In this study, we measured IL-6 and its 2 soluble receptors, soluble IL-6R and soluble gp130, in maternal plasma from normal and preeclamptic pregnant women and found that not only IL-6 but also soluble gp130 levels were significantly higher in preeclamptic women than in normotensive pregnant controls. We further examined IL-6R, gp130, and SOCS-3 expressions in maternal vessels and leukocytes and found that gp130 and SOCS-3 expressions were downregulated in both vessel endothelium and leukocytes from preeclampsia. Different patterns for IL-6R and gp130 expressions were found. IL-6R expression was also downregulated in leukocytes from preeclampsia. Our results suggest that increased plasma soluble gp130/soluble IL-6R/IL-6 ratio and reduced membrane transsignaling gp130 expression could contribute to decreased SOCS-3 expression and subsequent reduction in SOCS-3 antiinflammatory activity in women with preeclampsia. Thus, reduced gp130 and SOCS-3 expressions may offer, at least in part, a plausible explanation of reduced antiinflammatory protection in the maternal vascular system in preeclampsia.     

High-dose glucocorticoids increase serum levels of soluble IL-6 receptor alpha and its ratio to soluble gp130: an additional mechanism for early increased bone resorption

OBJECTIVE: Glucocorticoids (GCs) at pharmacological doses stimulate bone resorption. Mechanisms of this action are unclear. The osteoclastogenic cytokine interleukin (IL)-6 acts through an oligomeric receptor consisting of two subunits, gp80 (or IL-6 receptor alpha, IL-6Ralpha) and gp130; both exist in membrane and soluble forms. Soluble IL-6Ralpha (sIL-6Ralpha) enhances, while sgp130 inhibits IL-6 signalling. In vitro, GCs enhance many effects of IL-6 by up-regulation of IL-6Ralpha. The aim of the present study was to assess acute changes of IL-6 system in the peripheral blood of patients given high-dose GCs. SUBJECTS AND METHODS: Serum levels of IL-6, sIL-6Ralpha, sgp130 and bone turnover markers were assessed before and each day during treatment in 24 multiple sclerosis (MS) patients undergoing high-dose (prednisolone, 15 mg/kg per day), short-term (3 to 5 days) intravenous GC therapy for relapse at the Regional Multiple Sclerosis Centre. RESULTS: An immediate and marked fall of osteocalcin and an early increase of C-terminal telopeptide of type I collagen were already noticed at day 2 (P < 0.001 and P < 0.02, respectively); both became more apparent in the subsequent days. IL-6 was always below or near the detection limit of our ELISA. sgp130 showed a slight increase. sIL-6Ralpha significantly increased, peaking at day 4 (P < 0.01). However, inter-individual variability of response was noticed. Four patients showed a slight decrease, while no change was observed in one patient and an increase was noticed in the remaining nineteen (maximum change ranging from +10% to +67% with respect to baseline). In these patients, a significant increase of sIL-6Ralpha/sgp130 ratio was apparent. No correlation was found between bone turnover markers and any measured component of the IL-6 system. CONCLUSIONS: sIL-6Ralpha and sIL-6Ralpha/sgp130 ratio are precociously increased in the peripheral blood of the vast majority of patients given high-dose, intravenous GCs. The increase of systemically available sIL-6Ralpha conceivably results in the enhancement of IL-6-dependent osteoclastogenesis. The role of such a mechanism in the bone loss observed in inflammatory and immune-mediated diseases (where abundancy of IL-6 in the bone microenvironment is expected) requires further investigation.     

Interleukin-6 Signaling,Soluble Glycoprotein 130, and Inflammation in Heart Failure

Both experimental and clinical evidence accumulated over the last couple of decades has linked inflammatory activation to the initiation and progression of chronic heart failure (HF). Circulating levels of inflammatory mediators are associated with cardiac function and inform risk prediction in patients, but the effect of anti-inflammatory therapy in HF remains uncertain. Interleukin (IL)-6 type cytokines are central to the inflammatory response, and convey their signals through the ubiquitously expressed glycoprotein (gp) 130 receptor subunit. IL-6-type/gp130 signaling therefore represents an inflammatory nexus, with inherent potential for disease modification. This review focuses on the current knowledge of IL-6/gp130 signaling in relation to HF, with a particular emphasis on the role of soluble gp130 (sgp130), a signaling pathway modulator. Biological aspects of sgp130 and IL-6 signaling are discussed, as are potential novel therapeutic approaches to modulate this central inflammatory signaling pathway.     

Glycoprotein 130 polymorphism predicts soluble glycoprotein 130 levels

ObjectiveInterleukin-6 (IL-6) is a key cytokine in inflammatory diseases. It exerts its biological function via binding to a homodimer of its signal transducer glycoprotein 130 (gp130). Soluble gp130 (sgp130) is the natural inhibitor of IL-6 trans-signalling. The aim of this study was to test a possible influence of the gp130 genotype on sgp130 serum levels.Material and methodsIn two separate populations, subjects were genotyped for the gp130 polymorphism G148C. Sgp130, IL-6 and soluble interleukin-6 receptor (sIL-6R) levels were measured. The OSLO population consisted of 546 male subjects at high risk for CAD. The VIENNA population consisted of 299 male subjects with angiographically proven CAD.ResultsIn the OSLO population, 124 (22.7%) subjects were hetero- or homozygote for the rare C allele. Individuals carrying the polymorphism had significantly higher levels of sgp130. In a multivariate linear regression model this association remained significant (adjusted p = 0.001). In the VIENNA population, 48 (16.1%) subjects were hetero- or homozygote for the rare C allele. Consistent with the former study, sgp130 levels were significantly higher in carriers of the polymorphism compared to wildtype carriers (adjusted p = 0.038). In the VIENNA population, sgp130 levels were significantly higher in diabetic patients. In the OSLO population, sgp130 was higher in patients with increased body mass index and in smokers (p < 0.05).ConclusionsSgp130 serum levels are significantly higher in subjects carrying the gp130 polymorphism G148C compared to wildtype carriers. This finding proposes a possible genetical influence on sgp130 levels which may alter individual coping mechanisms in inflammatory diseases.     

The balance between soluble receptors regulating IL-6 trans-signaling is predictive for the RANKL/osteoprotegerin ratio in postmenopausal women with rheumatoid arthritis

The objective of this study is to investigate the relationship between soluble components of the interleukin 6 (IL-6) system mediating and modifying IL-6 trans-signaling and the RANKL–RANK–osteoprotegerin system in postmenopausal women with rheumatoid arthritis (RA). The following parameters were investigated in 126 postmenopausal women with RA: IL-6, soluble IL-6-receptor (sIL-6R), soluble glycoprotein 130 (sgp130), sRANKL, osteoprotegerin (OPG), osteocalcin, erythrocyte sedimentation rate and C-reactive protein in sera, pyridinolin and desoxypyridinolin crosslinks in the morning urine. Bone mineral density (BMD) was measured by dual X-ray absorptiometry at the lumbar spine (BMD-LS) and at the femoral neck (BMD-FN). Predictors of RANKL/OPG ratio and BMD were evaluated by multiple linear regression analysis. The following determinants of the RANKL/OPG ratio were identified: sIL-6R/sgp130 ratio and daily glucocorticoid (GC) dose as positive determinants in the whole group (R ² = 0.56; P = 0.001), sIL-6R/sgp130 ratio as the exclusive positive determinant in patients with GC therapy (R ² = 0.48; P = 0.001) and sgp130 as negative determinant in patients without GC (R ² = 0.42; P = 0.031). Sgp130 was highly significantly positively correlated with OPG in the whole group (P < 0.001) as well as in patients with (n = 70; P < 0.05) and without GC therapy (n = 56; P < 0.01). sIL-6R was the main negative predictor of BMD-LS (R ² = 0.41; P = 0.019). High sIL-6R/sgp130 ratio and/or low sgp130 are associated with a high sRANKL/OPG ratio in sera of postmenopausal women with RA indicating the critical significance of IL-6 trans-signaling for an increase in the RANKL/OPG ratio and of bone resorption. Inhibition of IL-6 trans-signaling may be an effective bone-protecting principle in postmenopausal women with RA.     

Antiinflammatory role of endomorphins in osteoarthritis, rheumatoid arthritis, and adjuvant-induced polyarthritis

OBJECTIVE: Pain sensitization and the related secretion of neuropeptides from sensory nerve terminals are proinflammatory in osteoarthritis (OA), rheumatoid arthritis (RA), and adjuvant-induced polyarthritis. In contrast, endogenous opioids such as the recently discovered endomorphins (EMs) are antiinflammatory. However, the role of endogenous EMs such as EM-1 and EM-2 has never been investigated in OA and RA. METHODS: We established a highly sensitive radioimmunoassay to detect EM-1 and EM-2. In patients with RA and patients with OA, immunohistochemistry for EM-1 and EM-2 was performed, and double-staining was used to identify EM-positive cells. The effects of EM-1 and EM-2 on the secretion of interleukin-6 (IL-6) and IL-8 from human synovial tissue were studied by tissue superfusion, and the therapeutic effects of EM-1 were tested in a rat model of adjuvant-induced polyarthritis. RESULTS: EM-positive cells were located in the sublining area and vessel walls but were particularly evident in the highly inflamed lining area. Human macrophages, T cells, and fibroblasts stained positive for EMs. The synovial density of EM-positive cells was higher in patients with OA than in those with RA. EM-1 inhibited synovial secretion of IL-6 in patients with RA and secretion of IL-8 in patients with RA and those with OA (maximum 10(-10)M). EM-2 inhibited IL-8 secretion only from RA tissue (maximum 10(-10)M). In rats with adjuvant-induced polyarthritis, thymus, spleen, and synovial tissue contained significantly more EM-1 than was observed in controls. Rats with adjuvant-induced polyarthritis benefited from EM-1 treatment. CONCLUSION: EM-1 had antiinflammatory effects in patients with OA or RA and in a model of adjuvant-induced polyarthritis. Local enhancement of EM-1 might be an interesting therapeutic option in different forms of arthritis.     

In vivo inhibition of angiogenesis by interleukin-13 gene therapy in a rat model of rheumatoid arthritis

OBJECTIVE: Interleukin-13 (IL-13) is a pleiotropic cytokine that can affect vessel formation, an important component of the rheumatoid arthritis (RA) synovial tissue pannus. The purpose of this study was to use a gene therapy approach to investigate the role of IL-13 in angiogenesis in vivo, using a rat adjuvant-induced arthritis model of RA. METHODS: Ankle joints of female rats were injected preventatively with an adenovirus vector containing human IL-13 (AxCAIL-13), a control vector with no insert (AxCANI), or phosphate buffered saline (PBS). Joints were harvested at the peak of arthritis, and histologic and biochemical features were evaluated. RESULTS: AxCAIL-13-treated joint homogenates had lower hemoglobin levels, suggesting reduced joint vascularity, and both endothelial cell migration and tube formation were significantly inhibited (P < 0.05). Similarly, AxCAIL-13 inhibited capillary sprouting in the rat aortic ring assay and vessel growth in the Matrigel plug in vivo assay. IL-13 gene delivery resulted in up-regulation and association of phosphorylated ERK-1/2 and protein kinase Calpha/betaII, suggesting a novel pathway in IL-13-mediated angiostasis. The angiostatic effect of AxCAIL-13 was associated with down-regulation of proangiogenic cytokines (IL-18, cytokine-induced neutrophil chemoattractant 1/CXCL1, lipopolysaccharide-induced CXC chemokine/CXCL5) and up-regulation of the angiogenesis inhibitor endostatin. The expression and activity of matrix metalloproteinases 2 and 9, which participate in angiogenesis, was impaired in response to IL-13 as compared with AxCANI and PBS treatment. CONCLUSION: Our findings support a role for IL-13 as an in vivo antiangiogenic factor and provide a rationale for its use in RA to control pathologic neovascularization.     

Serum levels of soluble interleukin 6 receptor and soluble gp130 are elevated in patients with localized scleroderma

OBJECTIVE: To investigate the clinical relevance of serum soluble interleukin 6 receptor (sIL-6R) and soluble gp130 (sgp130) in localized scleroderma. METHODS: Serum levels of sIL-6R and sgp130 were examined by ELISA in 45 patients with localized scleroderma. Twenty patients with systemic sclerosis (SSc) and 20 healthy individuals served as controls. RESULTS: Serum levels of both sIL-6R and sgp130 were significantly elevated in patients with localized scleroderma compared with healthy controls. Moreover, serum sgp130 levels in patients with localized scleroderma were significantly higher than in patients with SSc. In patients with localized scleroderma, elevated sIL-6R levels significantly correlated with levels of IgM antihistone antibodies, the presence of rheumatoid factor, the number of linear lesions, and the number of body areas involved. Elevated sgp130 levels were significantly associated with levels of IgG antihistone antibodies, the number of plaque lesions, the total number of lesions, and the number of body areas involved. CONCLUSION: These results suggest that elevated serum sIL-6R and sgp 30 may reflect activation of the IL-6 system that may be associated with the development of sclerotic lesions and autoantibody production in localized scleroderma.     

可溶性糖蛋白130与稳定型冠心病患者冠状动脉粥样硬化严重程度之间关系

目的: 评估稳定型冠状动脉疾病（stable coronary artery disease, CAD）患者血清白细胞介素-6（IL-6）、可溶性IL-6受体（sIL-6R）和可溶性糖蛋白130（sgp130）浓度及与冠状动脉粥样硬化严重程度间的关系。方法：纳入2017年1月到2019年1月间于惠州市第六人民医院心内科具有动脉造影适应症疑似冠心病患者89例,根据冠状动脉造影结果将患者分成两组：存在冠状动脉粥样斑块CAD组,即粥样斑块组,共64例;不存在冠状动脉粥样斑块CAD组,即非粥样斑块组,共25例。采用ELISA法检测两组患者血清IL-6、sIL-6R和sgp130浓度,Spearman相关分析sgp130浓度与受累冠脉数目及Gensini评分的相关性,多因素logistic回归分析冠状动脉粥样硬化斑块病变的预测因子。结果： 粥样斑块组与非粥样斑块组在年龄、BMI、高血压、糖尿病、血脂参数上无统计学差异（P>0.05）, 粥样斑块组患者男性吸烟者居多（P<0.05）。粥样斑块组血清sgp130浓度显著低于非粥样斑块组（314.97±84.39 VS 399.08±79.99 ng/ml, P<0.001）,粥样斑块组血清IL-6浓度显著高于非粥样斑块组（P<0.05）, 粥样斑块组血清sIL-6R浓度和C-反应蛋白浓度(CRP)与非粥样斑块组比较差异无统计学意义。多因素logistic回归分析示血清sgp130浓度是冠状动脉粥样硬化斑块病变存在的预测因子（P=0.018）。血清sgp130浓度与受累冠状动脉数目间呈负相关（r=-0.310,P=0.007）,Gensini评分指数与血清sgp130浓度呈负相关（r=-0.410, P=0.001）,稳定型CAD患者sgp130浓度是Gensini评分指数独立危险因素。结论：稳定型CAD患者血清sgp130浓度与冠状动脉损伤严重程度呈负相关,血清sgp130水平是冠状动脉粥样硬化严重程度血清标志物。     

Serum interleukin-6, soluble interleukin-6 receptor and soluble gp130 exhibit different patterns of age- and menopause-related changes

Growing evidence suggests that interleukin-6 (IL-6) may play a pathogenetic role in postmenopausal bone loss and in other age-related pathological conditions. In this study, we have examined the age-related changes in the serum levels of IL-6 and the soluble receptors that modulate its biological activity--soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130)--in 220 women (from 25 to 104yr old), including 22 centenarians. Serum IL-6 rose exponentially with age (r=0.74, p<0.0001). The median level of IL-6 increased almost ten-fold with age, from 1.16pg/ml in premenopausal women to 10.27pg/ml in centenarians. Serum sIL-6R and sgp130 showed an increase until the seventh decade and a progressive decrease in older ages (r=0.39, p<0.0001 and r=0.26, p=0.008, respectively). IL-6, sIL-6R and sgp130 were significantly higher in women within 10yr of menopause as compared to premenopausal subjects (1.51 vs. 1.16pg/ml, p=0.012; 41.9 vs. 35.7ng/ml, p=0.002; and 253.4 vs. 230.7ng/ml, p=0.008, respectively). In postmenopausal women, a negative correlation was found between sIL-6R and the lumbar bone mineral density (BMD) (r=-0.28, p=0.002) even after adjusting for age and weight. Furthermore, sIL-6R levels were higher in osteoporotic compared to normal women (47.9 vs. 39.5ng/ml, p=0.001). In conclusion, our results show that the serum levels of IL-6, sIL-6R and sgp130 exhibit different patterns of age- and menopause-related changes, and that the biological activity of IL-6 may be increased with age with potential implications in the age-related diseases such as osteoporosis.     

IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3+ infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130DeltaSTAT/DeltaSTAT). T cell migration was related to STAT3 activity, because monoallelic deletion of Stat3 in gp130(Y757F/Y757F) mice (gp130Y757F/Y757F:Stat3+/-) corrected the exaggerated responses observed in gp130Y757F/Y757F mice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.     

Enhanced production of interleukin-6 (IL-6), oncostatin M and soluble IL-6 receptor by cultured peripheral blood mononuclear cells from patients with systemic sclerosis.

OBJECTIVES: To determine whether the spontaneous production of interleukin 6 (IL-6), oncostatin M (OSM), soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) from peripheral blood mononuclear cells (PBMC) is increased in patients with systemic sclerosis (SSc). METHODS: The culture supernatants of PBMC from patients with SSc (n = 33) and healthy controls (n = 20) were examined by enzyme-linked immunosorbent assay. RESULTS: The production levels of IL-6, OSM and sIL-6R were significantly higher in patients with SSc than in controls. However, sgp130 levels in supernatants from patients with SSc were not significantly elevated when compared with those from controls. Soluble IL-6R levels correlated significantly with the severity of pulmonary fibrosis in patients with SSc. CONCLUSIONS: The enhanced production of IL-6, OSM and sIL-6R from PBMC may cooperatively contribute to the disease process in SSc. In particular, enhanced sIL-6R production from PBMC may be related to the development of pulmonary fibrosis via enhancement of IL-6 signal transduction in SSc, since sIL-6R can act as an agonist of IL-6.     

(-)-表没食子儿茶素没食子酸酯对类风湿关节炎的免疫调节机制研究

目的 ①研究(-)-表没食子儿茶素没食子酸酯(EGCG)对类风湿关节炎(RA)患者外周血和滑液中T细胞增殖的影响及对RA相关细胞因子的免疫调节作用.②观察EGCG对RA滑膜成纤维细胞(FLS)增殖的影响.方法 ①取RA患者外周血(30份)、滑液(23份)分离单个核细胞,并分设空白对照组,阴性对照组,甲氨蝶呤阳性对照组,EGCG低、中、高剂量组,通过同位素(3H)掺入法,在体外研究EGCG对RA患者外周血及滑液中T细胞增殖的影响;酶联免疫吸附试验(ELISA)法检测EGCG对RA外周血及滑液中肿瘤坏死因子(TNF)-α、干扰素-γ、白细胞介素(IL)-1、IL-6和IL-17A的影响.②取RA患者滑膜组织(8例)进行原代培养,并分设空白对照组,甲氨蝶呤组(阳性对照),EGCG低、中、高剂量组,通过四甲基偶氮唑蓝(MTT)比色法,在体外研究EGCG对RA患者FLS增殖的影响.统计学处理采用方差分析和SNK-q检验.结果 ①RA患者外周血及滑液中EGCG高剂量组每分钟脉冲数(cpm)值[分别是(15 136±2910),(11587±3135)],较阴性对照组(42 856±2127)(35 748±4512)下降,差异均有统计学意义(P均＜0.01);外周血中ECCG高剂量组TNF-α、干扰素-γ、IL-1、IL-6和IL-17A吸光度值分别是[(321±13)、(298±20)、(132±12)、(197±7)、(59±8)pg/ml],均较阴性对照组[(458±28)、(505±26)、(346±28)、(405±25)、(109±13)pg/ml]下降,差异有统计学意义(P＜0.05或P＜0.01);滑液中EGCG高剂量组TNF-α、干扰素-γ、IL-1、IL-6和IL-17A吸光度值分别是[(41.4±2.9)、(182±16)、(56.3±11.0)、(34.2±1.9)、(44±8)pg/ml],均较阴性对照组[(388.3±19.3)、(469±20)、(104.2±17.8)、(114.5±4.8)、(104±11)pg/ml]下降,差异有统计学意义(P＜0.05或P＜0.01).②EGCG高剂量组FLS吸光度值(0.080±0.017),较空白对照组(0.274±0.041)下降,差异有统计学意义(P＜0.05).结论 EGCG可体外抑制RA患者外周血和滑液中T细胞增殖,抑制TNF-α、干扰素-γ、IL-1、IL-6和IL-17A分泌;可体外抑制RA FLS增殖.     

Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1beta (IL-1beta)-induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene alpha (GROalpha/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROalpha produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-kappaB. RESULTS: EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 microM or 20 microM significantly inhibited IL-1beta-induced ENA-78, RANTES, and GROalpha, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 microM caused a complete block of IL-1beta-induced production of RANTES, ENA-78, and GROalpha, and reduced production of MCP-1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1beta-induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCdelta and inhibited the activation and nuclear translocation of NF-kappaB in IL-1beta-treated RA synovial fibroblasts. CONCLUSION: These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.