Increased Expression of Proatherogenic Proteins in Immune Cell Subtypes in Tobacco Cigarette Smokers But Not in Electronic Cigarette Vapers.

It is unclear how oxidative stress triggered by smoking and vaping may alter specific immune cell subsets. In this study, we showed that tobacco cigarette smoking, but not electronic-cigarette vaping, is associated with increased expression of major proteins in the toll-like receptor 4 (TLR4) inflammasome-interleukin (IL)-6 signalling axis in monocyte subtypes and T cells. TLR4 senses oxidative stress in immune cells caspase-1 is a key protein of inflammasome activation, and IL-6R-α is the receptor for IL-6 that drives proatherogenic IL-6 signalling. These findings implicate the non-nicotine, pro-oxidant toxicants in tobacco cigarette smoke as instigators of increased expression of key proteins in the TLR4-inflammasome-IL-6 axis that contribute to atherogenesis.

A Novel Role for IL-6 Receptor Classic Signaling: Induction of RORγt+Foxp3+ Tregs with Enhanced Suppressive Capacity.

BACKGROUND: New therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown. METHODS: To learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell-specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations. RESULTS: Lack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra-/- Tregs resulted in severe aggravation of GN in mice. CONCLUSIONS: Our data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell-intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6-directed therapies for GN need to be cell-type specific.

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma.

OBJECTIVE: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. METHODS: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. RESULTS: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL-KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. CONCLUSIONS: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.

Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide in which the vast majority of cases exhibit little genetic risk but are associated with a sedentary lifestyle and obesity. Although the mechanisms underlying CRC and colitis-associated colorectal cancer (CAC) remain unclear, we hypothesised that obesity-induced inflammation predisposes to CAC development. Here, we show that diet-induced obesity accelerates chemically-induced CAC in mice via increased inflammation and immune cell recruitment. Obesity-induced interleukin-6 (IL-6) shifts macrophage polarisation towards tumour-promoting macrophages that produce the chemokine CC-chemokine-ligand-20 (CCL-20) in the CAC microenvironment. CCL-20 promotes CAC progression by recruiting CC-chemokine-receptor-6 (CCR-6)-expressing B cells and γδ T cells via chemotaxis. Compromised cell recruitment as well as inhibition of B and γδ T cells protects against CAC progression. Collectively, our data reveal a function for IL-6 in the CAC microenvironment via lymphocyte recruitment through the CCL-20/CCR-6 axis, thereby implicating a potential therapeutic intervention for human patients.

Effects of Interleukin-6 on posterior capsular opacification.

The purpose of this work was to determine the effects of interleukin-6 (IL-6) on the development of posterior capsular opacification (PCO) in vitro and in vivo. Western blot and real-time PCR were used to test the IL-6-induced epithelial-mesenchymal transition (EMT) marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM) markers fibronectin (Fn) and type I collagen (COL-1), transforming growth factor ß2 (TGF-ß2), and the activation and role of the JAK/STAT3 signaling pathway in human lens epithelial cells (HLECs). Immunocytofluorescence staining was performed to detect gp130 and IL-6Rα expression in HLECs. Rat PCO models were then established to examine the impact of STAT3 knockdown by shRNA adeno-associated virus on PCO development, and immunohistochemical staining was performed to detect the expression of Fn in the anterior and posterior capsule in vivo. We found that IL-6 promotes the expression of Fn, COL-1, TGF-ß2, p-JAK2 and p-STAT3 in HLECs but exerts little effect on α-SMA. The JAK/STAT3 inhibitor WP1066 effectively suppressed the IL-6-induced expression of Fn and COL-1 in lens epithelial cells. STAT3 knockdown effectively inhibited the development of PCO in rats and significantly reduced the expression of Fn in the anterior and posterior capsule. These data suggest that IL-6 contributes to the development of PCO by promoting TGF-ß2 activation and ECM synthesis through a JAK/STAT3 signaling-dependent mechanism. Furthermore, inhibiting JAK/STAT3 signaling effectively impairs both PCO development in rats and ECM synthesis in the lens capsule.

Functional interleukin-6 receptor-α is located in tanycytes at the base of the third ventricle.

Interleukin (IL)-6- /- mice develop mature onset obesity, whereas i.c.v. injection of IL-6 decreases obesity in rodents. Moreover, levels of IL-6 in cerebrospinal fluid (CSF) were reported to be inversely correlated with obesity in humans. Tanycytes lining the base of the third ventricle (3V) in the hypothalamus have recently been reported to be of importance for metabolism. In the present study, we investigated whether tanycytes could respond to IL-6 in the CSF. With immunohistochemistry using a well characterised antibody directed against the ligand binding receptor for IL-6, IL-6 receptor α (IL-6Rα), it was found that tanycytes, identified by the two markers, vimentin and dopamine- and cAMP-regulated phosphoprotein of 32 kDa, contained IL-6Rα. There were fewer IL-6Rα on another type of ventricle-lining cells, ependymal cells, as identified by the marker glucose transporter-1. To demonstrate that the immunoreactive IL-6Rα were responsive to IL-6, we injected IL-6 i.c.v. This treatment increased immunoreactive phosphorylated signal transducer and activator of transcription-3 (pSTAT3) in tanycytes after 5 minutes and in cells in the medial part of the arcuate nucleus after 5 and 15 minutes. Intracerebroventricular injection of leptin exerted similar effects. As expected, i.p. injection of leptin also induced pSTAT3 staining in the hypothalamus, whereas i.p. IL-6 injection had little effect on this parameter. Intracerebroventricular or i.p. injection of vehicle only had no effect on pSTAT3-immunoreactivity. In summary, there are functional IL-6Rα on tanycytes at the bottom of the 3V, in agreement with the possibility that ventricular administration of IL-6 decreases obesity in mice via an effect on this cell type.

Molecular Dynamics Simulations Reveal Key Roles of the Interleukin-6 Alpha Receptor in the Assembly of the Human Interleukin-6 Receptor Complex.

Human interleukin-6 (hIL-6) is a pleiotropic cytokine with three distinct receptor epitopes, termed sites I, II, and III, which function to assemble a signaling complex. hIL-6 signals via a glycoprotein 130 (gp130) homodimer after initially forming a heterodimer with the nonsignaling α-receptor (IL-6Rα). The molecular description of the assembly of the hIL-6 signaling complex remains elusive because available structures provide descriptions of hIL-6 in its free and fully bound receptor forms, but not for intermediate steps that are crucial in the stepwise assembly of the signaling complex. In this report, molecular dynamics simulations provide atomic details describing the functional role of the initial hIL-6/IL-6Rα complex in facilitating subsequent interactions with gp130, which have not been previously shown. IL-6Rα binding to hIL-6 rigidifies the flexible N-terminus of the hIL-6 AB-loop through interactions with the D2 domain of IL-6Rα. This rigidification combined with repositioning of residues involved in gp130 receptor recognition promotes gp130 binding at site III. Binding of gp130 receptors at sites II and III is coupled with the release of the hIL-6 N-terminal AB-loop interaction and a pivoting of IL-6Rα around the hIL-6 helix bundle to the state of the hIL-6/IL-6Rα/gp130 complex.

Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

Interleukin-6 receptor alpha blockade improves skin lesions in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by antinuclear autoantibodies (ANA) and immunocomplexes, commonly affecting kidneys, skin, heart, lung or even the brain. We have shown that JunB(Δep) mice develop a SLE phenotype linked to increased epidermal Interleukin (IL)-6 secretion. Blocking of IL-6 receptor alpha (IL-6Rα) is considered as therapeutic strategy for the treatment of SLE. JunB(Δep) and wild-type mice were treated for short (5 weeks) or long term (21 weeks) with the IL-6Rα-blocking antibody MR16-1. Skin and kidney of mice were investigated by histology and immunofluorescence, and in addition, kidneys were analysed by electron microscopy. Furthermore, soluble IL-6R (sIL-6R), antihistone and antinucleosome antibodies levels were measured and associated with disease parameters. Treatment with MR16-1 resulted in significant improvement of SLE-like skin lesions in JunB(Δep) mice, compared to untreated mice. The sIL-6R amount upon long-term treatment with MR16-1 was significantly higher in JunB(Δep) versus untreated JunB(Δep) (P = 0.034) or wild-type mice (P = 0.034). MR16-1 treatment over these time spans did not significantly improve kidney pathology of immunoglobulin deposits causing impaired function. Significantly higher antihistone (P = 0.028) and antinucleosome antibody levels (P = 0.028) were measured in MR16-1-treated JunB(Δep) mice after treatment compared to levels before therapy. In conclusion, blockade of IL-6Rα improves skin lesions in a murine SLE model, but does not have a beneficial effect on autoimmune-mediated kidney pathology. Inhibition of IL-6R signalling might be helpful in lupus cases with predominant skin involvement, but combinatorial treatment might be required to restrain autoantibodies.

Remodeling of plasma lipoproteins in patients with rheumatoid arthritis: Interleukin-6 receptor-alpha inhibition with tocilizumab.

PURPOSE: Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin-6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation-induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin-6 receptor-alpha inhibitor. EXPERIMENTAL DESIGN: Size-exclusion chromatography and reverse-phase protein arrays using 29 antibodies against 26 proteins were applied at baseline and after tocilizumab treatment to analyze the distributions of apolipoproteins, enzymes, lipid transfer proteins, and other associated proteins in plasma lipoprotein fractions from 20 women with RA. RESULTS: A 30% reduction in high-density lipoprotein (HDL)-associated serum amyloid A4 and complement C4 occurred with tocilizumab. Levels of C-reactive protein, associated or comigrating with HDL and low-density lipoprotein (LDL) peaks, were reduced on treatment by approximately 80% and 24%, respectively. Reductions in lipoprotein-associated phospholipase A2, lipoprotein (a), and cholesteryl ester transfer protein in the LDL fraction suggest reductions in LDL-associated proatherogenic factors. Elevations in very low-density lipoprotein (VLDL) enriched with apolipoprotein E were equally observed. CONCLUSIONS AND CLINICAL RELEVANCE: Tocilizumab treatment led to reductions in proinflammatory components and proatherogenic proteins associated with HDL. Whether changes in the proteome of VLDL, LDL, and HDL induced by anti-inflammatory tocilizumab treatment in RA patients modify cardiovascular disease risk requires further investigation.

Modular organization of Interleukin-6 and Interleukin-11 α-receptors.

Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.

Interleukin-6 (IL-6) mediated the increased contraction of distal colon in streptozotocin-induced diabetes in rats via IL-6 receptor pathway.

Colonic dysmotility occurs in diabetes and blood plasma interleukin (IL)-6 levels are significantly elevated in type 1 diabetes mellitus. The aim of this study was to investigate whether IL-6 and the IL-6 receptor pathway mediates colonic dysfunction in type 1 diabetes mellitus. Male SD rats were treated with a single intraperitoneally injected dose of streptozotocin (STZ), and those displaying sustained high blood glucose were selected as diabetes mellitus models. Longitudinal muscle strips of colon were prepared to monitor colonic contraction in vitro. Contractile responses of strips of colon were recorded following treatment with IL-6 in control animals, and following anti IL-6 antibody treatment in STZ-induced diabetes in rats. Concentration of IL-6 in plasma and colon were determined by ELISA. Expressions of IL-6 α-receptor and IL-6 ß-receptor in colon tissues were determined by immunohistochemistry or Western blot analysis. The non-diabetes rats treated with IL-6 and the untreated diabetes rats showed increased contraction of distal colon, whereas the diabetes rats treated with anti-IL-6 antibody showed decreased contraction of distal colon compared with the untreated diabetes rats. The IL-6 levels of plasma but not colon increased in diabetes rats. The expression of IL-6 α-receptor increased in diabetes rats. These results indicate that diabetes rats show an increase in the contractions of distal colon partly via the IL-6-IL-6 receptor pathway.

The expression of interleukin-6 and its receptor in various brain regions and their roles in exploratory behavior and stress responses.

We examined the involvement of interleukin-6 (IL-6) and its receptor IL-6Rα on behavior and stress responses in mice. In the open field, both wild-type (WT) and IL-6 deficient mice displayed similar levels of locomotor activity; however, IL-6 deficient mice spent more time in the central part of the arena compared to control WT mice. After behavioral testing, mice were subjected to stress and then sacrificed. The levels of IL-6 and its receptor in their brains were determined. Immunohistochemical labeling of brain sections for IL-6 showed a high level of expression in the subventricular zone of the lateral ventricles and in the border zone of the third and fourth ventricles. Interestingly, 95% of the IL-6-expressing cells had an astrocytic phenotype, and the remaining 5% were microglial cells. A low level of IL-6 expression was observed in the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, cerebellum, midbrain and several brainstem structures. The vast majority of IL-6-expressing cells in these structures had a neuronal phenotype. Stress increased the number of IL-6-immunoreactive astrocytes and microglial cells. The levels of the IL-6Rα receptor were increased in the hypothalamus of stressed mice. Therefore, in this study, we describe for the first time the distribution of IL-6 in various types of brain cells and in previously unreported regions, such as the subventricular zone of the lateral ventricle. Moreover, we provide data on regional distribution and expression within specific cell phenotypes. This highly differential expression of IL-6 indicates its specific roles in the regulation of neuronal and astrocytic functions, in addition to the roles of IL-6 and its receptor IL-6Rα in stress responses.

An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model.

INTRODUCTION: Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells. METHODS: Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b(+)Gr-1(+) MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in in vivo metastasis, we blocked the interactions between MDSCs and cancer cells. RESULTS: Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis. CONCLUSION: In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Ra, which, in turn, triggered a persistent increase of pSTAT3 in tumor cells. This potential tumor-MDSC axis involving IL-6 trans-signaling directly affected breast cancer cell aggressiveness, leading to spontaneous metastasis.

Flavanoids induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene and suppress IL-6-activated signal transducer and activator of transcription 3 (STAT3) activation in vascular endothelial cells.

The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs (vascular endothelial cells) by activating the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 (suppressor of cytokine signalling 3). Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr7°5) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK (extracellular-signal-regulated kinase)-dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.

Susceptibility to autoimmune myocarditis is associated with intrinsic differences in CD4(+) T cells.

A.SW and B10.S mice share the same major histocompatibility complex (MHC) haplotype (H-2(s)). However, A.SW mice are susceptible to experimental autoimmune myocarditis (EAM) and develop severe disease after immunization with myosin, whereas B10.S mice are resistant. We found that naive A.SW mice have intrinsically increased total CD4(+) T cell counts and increased proportions of CD4(+) T cells in their spleens compared to B10.S mice. Among total CD4(+) T cells, naive A.SW mice have a lower relative frequency of forkhead box protein 3 (FoxP3(+))CD25(+) regulatory T cells (T(regs)). A.SW mice also had a higher proportion of CD4(+) T cells and a lower proportion of T(regs) in their hearts and spleen during EAM, with greater T cell activation and proliferation, compared to B10.S mice. These differences in the T cell compartment were not antigen-specific, as ovalbumin/complete Freund's adjuvant (OVA/CFA) or CFA immunization elicited the same differences in CD4(+) T cells and T(regs) between A.SW and B10.S mice. Moreover, A.SW mice had more T helper type 17 (Th17) cells and B10.S had more Th1 cells in their hearts. The higher percentage of CD4(+) T cells and their enhanced potential to differentiate towards the Th17 pathway was also observed in naive A.SW mice. Interleukin (IL)-6 is required for Th17 induction. Interestingly, IL-6Rα expression was greater on naive A.SW CD4(+) T cells, compared to B10.S CD4(+) T cells, indicating that this intrinsic difference, together with a relatively lower T(reg) proportion of CD4(+) T cells, might lead to heightened Th17 responses and greater susceptibility to autoimmunity in A.SW mice.

Dampened ERK signaling in hematopoietic progenitor cells in rheumatoid arthritis.

In rheumatoid arthritis (RA), hematopoietic progenitor cells (HPC) have age-inappropriate telomeric shortening suggesting premature senescence and possible restriction of proliferative capacity. In response to hematopoietic growth factors RA-derived CD34(+) HPC expanded significantly less than age-matched controls. Cell surface receptors for stem cell factor (SCF), Flt 3-Ligand, IL-3 and IL-6 were intact in RA HPC but the cells had lower transcript levels of cell cycle genes, compatible with insufficient signal strength in the ERK pathway. Cytokine-induced phosphorylation of ERK1/2 was diminished in RA HPC whereas phosphorylated STAT3 and STAT5 molecules accumulated to a similar extent as in controls. Confocal microscopy demonstrated that the membrane-proximal colocalization of K-Ras and B-Raf was less efficient in RA-derived CD34(+) cells. Thus, hyporesponsiveness of RA HPC to growth factors results from dampening of the ERK signaling pathways; with a defect localized in the very early steps of the ERK signaling cascade.

Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): salmonid IL-6Rα possesses a polymorphic N-terminal Ig domain with variable numbers of two repeats.

Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we have cloned both the IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-D6) and presence of intracellular motifs important for signal transduction to tetrapod gp130s. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine-binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, a phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra large D1 domain of the salmonid IL-6Rα molecules results partially from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in the cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness.

The response of T cells to interleukin-6 is differentially regulated by the microenvironment of the rheumatoid synovial fluid and tissue.

OBJECTIVE: Interleukin-6 (IL-6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL-6 receptor (IL-6R; CD126) or via trans-signaling, in which soluble IL-6R/IL-6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL-6 in the joints of patients with rheumatoid arthritis (RA). METHODS: Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients. RESULTS: Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans-signaling by soluble IL-6R/IL-6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down-regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL-6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up-regulated locally. Among a range of cytokines tested, only IL-10 induced CD130 expression in T cells. CONCLUSION: The inflamed microenvironment in the synovial tissue maintains responsiveness to IL-6 trans-signaling through the up-regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL-10.