Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose

Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1β, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1β was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.     

IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes

Inflammation occurs in adipose tissue in obesity. We have examined whether IL-33, a recently identified IL-1 gene family member, and its associated receptors are expressed in human adipocytes. IL-33, IL-1RL1 and IL-1RAP gene expression was observed in human visceral white fat, in preadipocytes and in adipocytes (SGBS cells). Treatment with TNFα for 24 h induced a 6-fold increase in IL-33 mRNA level in preadipocytes and adipocytes. Time-course studies with adipocytes showed that the increase in IL-33 mRNA with TNFα was maximal (>55-fold) at 12 h. This response was markedly different to IL-1β (peak mRNA increase at 2 h; 5.4-fold) and 1L-18 (peak mRNA increase at 6 h; >1500-fold). Exposure of adipocytes to hypoxia (1% O₂, 24 h) did not alter IL-33 mRNA level; in preadipocytes, however, there was a 3-fold increase. Human adipocytes and preadipocytes express IL-33, but the various IL-1 family members exhibit major differences in responsiveness to TNFα.     

Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines.

Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.     

THE NEUROPEPTIDE CALCITONIN GENE-RELATED PEPTIDE INHIBITS TNF-α BUT POORLY INDUCES IL-6 PRODUCTION BY FETAL RAT OSTEOBLASTS

Tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) are potent inflammatory cytokines produced by osteoblasts and whose contribution to bone loss occurring in oestrogen deficiency is well documented. Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphyses and periosteum suggesting that it controls bone homeostasis locally. Since CGRP was shown to inhibit TNF-α production by T cells and stimulate IL-6 expression by fibroblasts, this study was designed to investigate whether CGRP regulated TNF-α and IL-6 production by osteoblasts. We show that CGRP inhibits the production of TNF-α by both lipopolysaccharide (LPS)- and IL-1-stimulated fetal rat osteoblasts. Like CGRP, the cAMP agonists prostaglandin E₂(PGE₂), dibutyryl cAMP (Bt₂cAMP) and forskolin inhibit TNF-α production by osteoblasts. Exposure of osteoblasts to a high dose of phorbol myristoyl acetate (PMA) to deplete PKC activity abolished CGRP-mediated TNF-α suppression. In contrast with its potent inhibition of TNF-α production, we show that CGRP is a weak inducer of IL-6 when compared to PGE₂, Bt₂cAMP and forskolin. However, in presence of isobutylmethylxanthine (IBMX) CGRP stimulates the production of IL-6. Collectively, these data suggest that the inhibition of TNF-α CGRP is cAMP dependent and PMA sensitive and that the concentration of intracellular cAMP may be a regulatory mechanism for IL-6 expression in osteoblasts.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1β, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1β in a dose-dependent manner. IL-1β stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis.     

Interleukin-18 up-regulates osteoprotegerin expression in stromal/osteoblastic cells

Osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) are crucial regulators of osteoclastogenesis. To determine the biological role of interleukin (IL)-18 produced by stromal/osteoblastic cells in osteoclastogenesis, we examined the effects of IL-18 on the OPG and ODF mRNA levels in these cells. When bone marrow stromal ST2 cells, osteoblastic MC3T3-E1 cells, and mouse calvarial osteoblasts were stimulated with IL-18, the expression of OPG mRNA, but not ODF mRNA, was transiently increased, its expression reaching a maximal level at 3 h after the beginning of the culture. In accordance with this observation, all these cells expressed the mRNAs of two IL-18 receptor components and MyD88, an adapter molecule involved in IL-18 signaling. Moreover, in these cells, mitogen-activated protein kinase was phosphorylated after stimulation with IL-18. These results suggest that stromal/osteoblastic cells are IL-18-responsive cells and that IL-18 may inhibit osteoclastogenesis by up-regulating OPG expression, without stimulation of ODF production, in stromal/osteoblastic cells.     

Secretory leukocyte protease inhibitor reduces inflammation and alveolar bone resorption in LPS-induced periodontitis in rats and in MC3T3-E1 preosteoblasts

In periodontitis, alveolar bone resorption is induced by excessive host immune and inflammatory response against bacterial infection. Secretory leukocyte protease inhibitor (SLPI) has anti-bacterial and anti-inflammatory activity in inflammatory responses. SLPI inhibits joint inflammation and bone destruction, but the function of SLPI in periodontitis is unclear. Therefore, this study investigated whether SLPI inhibits the inflammatory response and alveolar bone resorption in LPS-induced periodontitis of rats. Micro-computed tomography and histological analysis showed that SLPI inhibited alveolar bone resorption by LPS-induced periodontitis. Immunohistochemistry revealed that SLPI decreased tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) expression in periodontitis tissue, and decreased mRNA and protein expression of TNF-α and IL-1β in LPS-stimulated MC3T3-E1 cells. The results indicated that SLPI reduced alveolar bone resorption in LPS-induced periodontitis and inhibited inflammatory cytokine, such as TNF-α and IL-1β, expression in LPS-stimulated MC3T3-E1 preosteoblasts. Therefore, SLPI could be a regulatory molecule by inhibiting alveolar bone resorption through the reduction of inflammatory cytokines, and inducing osteoblast activation for bone formation.     

类风湿关节炎中白三烯B4诱导TNF-α和IL-1β的表达

为了探讨类风湿关节炎的发病过程中白三烯B4(leukotriene,LTB4)对TNF-α和IL-1β表 达的影响.加入外源性LTB4或者在LIT存在的情况下,加入苯丁抑制素(bestatin,LTA4水解酶 抑制剂)和MK-886(5-脂氧合 酶激动蛋白抑制剂)后, 采用实时PCR和酶联免疫吸附分析法来检测原代培养的类风湿滑膜细胞及培养上清液中TNF-α和IL-1β在mRNA及蛋白水平的表达.外源性的LTB4 10^-8mol/L使TNF-α和IL-1β mRNA水平表达分别增加了14倍和1倍, 蛋白水平分别增加了3倍.加入LIT刺激内源性的LTB4增加了14倍后,使TNF-α和IL-1βmRNA水平表达分别增加了145倍和12倍, 蛋白水平分别增加了3倍.在LIT存在的情况下, MK-886 10 μmol/L使LTB4合成降低了62%(P<0.000 1), 使TNF-α和IL-1β mRNA水平表达分别降低了66%(P<0.05)和71%(P<0.001),它们的蛋白水平分别降低了75%和70%(P<0.01). 100 μg/ml苯丁抑制素使LTB4合成降低了78%(P<0.000 1), 使TNF-α和IL-1β mRNA水平表达分别降低了86%(P<0.001)和79%(P<0.01), 它们的蛋白水平分别降低了84%和76%(P<0.05). 在类风湿关节炎中,LTB4诱导TNF-α和IL-1β的表达. 这一结果为类风湿关节炎发病机制进一步探讨提供了一条新思路.     

Role of TGF-β1 and TNF-α in IL-1β mediated activation of proMMP-9 in pulmonary artery smooth muscle cells: involvement of an aprotinin sensitive protease

We investigated the role of TGF-β1 and TNF-α in mediating the effect of IL-1β in activating proMMP-9 and proMMP-2, and the involvement of an aprotinin sensitive protease in this scenario in bovine pulmonary artery smooth muscle cells. IL-1β induces TGF-β1 mediated stimulation of 92 kDa proMMP-9 and 72 kDa proMMP-2 mRNA and protein expression; whereas, the elevated level of TNF-α promotes activation of proMMP-9 and proMMP-2. Interestingly, TNF-α induced activation of proMMP-9 appeared to be mediated via a 43 kDa aprotinin sensitive protease. TNF-α inhibited aprotinin and TIMP-1 mRNA and protein expression, which apparently facilitated the proteolytic conversion of proMMP-9 to MMP-9 with the involvement of the aprotinin sensitive protease. The aprotinin sensitive protease did not activate proMMP-2 under IL-1β stimulation, albeit a marked inhibition of TIMP-2 mRNA and protein expression were elicited by TNF-α. Thus, IL-1β induced stimulation of the two progelatinases occurs via different mechanisms.     

New insights into the epigenetics of osteoporosis

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.     

IL-11 expression in retinal and corneal cells is regulated by interferon-γ

Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-α + IL-1 induced IL-11 secretion and this production was inhibited by NFκB pathway inhibitors. IFN-γ significantly inhibited TNF-α and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-β induced IL-11 secretion that was blocked by TGF-β receptor 1 inhibitor but not by IFN-γ. RT-PCR analysis confirmed the effects of IL-1, TNF-α, IFN-γ and TGF-β on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-γ is a physiological inhibitor of IL-11 expression.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.