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Background: Idiopathic multicentric Castleman disease (iMCD) is a rare inflammatory disorder mediated by excessive proinflammatory cytokine signaling, most notably by interleukin 6 (IL‐6). IL‐6‐induced extramedullary hematopoiesis (EMH) has been reported in murine models of iMCD. Herein we present four cases of iMCD with EMH in humans. Case Series: The index case is a 24‐year‐old white woman who presented with pancytopenia, hepatosplenomegaly, and diffuse lymphadenopathy (LAD) with EMH in core lymph node biopsies. We then searched ACCELERATE, a Castleman disease (CD) natural history registry, and identified three additional CD cases with EMH reported in biopsies: A 23‐year‐old Asian man with fatigue, edema, LAD, and splenomegaly; a 20‐year‐old white man with fever, dyspnea, LAD, and hepatosplenomegaly; and a 50‐year‐old white man with constitutional symptoms, LAD, and myelodysplastic syndrome in bone marrow with a KRAS mutation. Results: All four patients presented with thrombocytopenia and fever and/or markedly elevated C‐reactive protein. Patient 1 had iMCD‐NOS (not otherwise specified) with severe thrombocytopenia, reticulin fibrosis in bone marrow, small volume LAD and organomegaly but no anasarca. The other three patients had iMCD‐TAFRO (thrombocytopenia, anasarca, reticulin fibrosis, renal dysfunction, organomegaly). Two had mixed CD and two had hypervascular CD in lymph nodes. All four had bone marrow hypercellularity and megakaryocyte hyperplasia and two had reticulin fibrosis. Conclusions: This case series demonstrates that EMH can be seen in CD, particularly in iMCD‐TAFRO. Given the similarity of this finding to previous murine models of IL‐6‐induced marrow and lymph node changes we hypothesize that this is an IL‐6‐mediated phenomenon. [ABSTRACT FROM AUTHOR]

IL-6 is one of the major mediators of the hyper-inflammatory responses with complex biological functions as it can signal via different modes of action. IL-6 by classical signalling has anti-inflammatory and antibacterial activities, while trans-signalling mediates pro-inflammatory effects. The net biological effect of IL-6 is established by multiple factors beyond its absolute concentration. Here, we assess the relationship between IL-6 signalling variables [IL-6, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130)] and outcomes in a cohort of 366 COVID-19 patients. The potential trans-signalling was evaluated by a ratio between the pro-inflammatory binary IL-6:sIL-6R complex and the inactive ternary IL-6:sIL-6R:sgp130 complex (binary/ternary complex) and the fold molar excess of sgp130 over sIL-6R (FME). Our data provide new evidence that high levels of IL-6, sIL-6R, sgp130, binary/ternary complex ratio, and low FME are independent predictors of COVID-19 severity in survivor patients (without death), and the combination of IL-6 + sIL-6R + sgp130 exhibited the most robust classification capacity. Conversely, in a subgroup of patients with a very poor prognosis, we found that high levels of IL-6 and low levels of sIL-6R, sgp130, and binary/ternary complex ratio were predictors of death. In this context, the highest predictive capacity corresponded to the combined analysis of IL-6 + FME + lymphopenia + creatinine. Herein, we present IL-6 signalling variables as a helpful tool for the early identification and stratification of patients with clear implications for treatment and clinical decision-making. [ABSTRACT FROM AUTHOR]

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 þ sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions. [ABSTRACT FROM AUTHOR]

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines. [ABSTRACT FROM AUTHOR]

One of the gold standards for animal models of rheumatoid arthritis is the murine collagen-induced arthritis model. Native type II collagen together with CFA is injected into susceptible mouse strains. Unfortunately, only mice with H-2q or H-2r MHC haplotypes are susceptible, making the widely used C57BL/6 mouse strain, which carries the H-2b haplotype, resistant against the disease. In this issue of the European Journal of Immunology, Stevanin et al. [Eur. J. Immunol. 2017. 47: 637-645] now convincingly show that although WT C57BL/6 mice are resistant to collagen-induced arthritis, mice with a homozygous deletion of CD11b on the same genetic background are fully susceptible in this important animal model of rheumatoid arthritis. They clearly demonstrate that the injection of type II collagen together with CFA leads to early onset of the disease with high incidence and with sustained severity. The authors further characterize this disease with an increase of leukocyte infiltration and enhanced TH17 differentiation. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is among the five most frequent causes for cancer-related deaths in Europe. One of the most important tumor-associated antigens for CRC is carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), which is involved in cell adhesion, migration, anoikis, tumor invasion and metastasis. Its family member CEACAM6 is also upregulated in adenomas and carcinomas of the colon and an independent predictor of poor survival. Previous studies have reported a link between upregulation of CEACAM5 and interleukin-6 (IL-6). IL-6 plays an important role in CRC progression, and signaling is mediated via two pathways (classic and trans-signaling). However, this link could not be confirmed by other studies, and the role of IL-6 trans-signaling in the CEACAM5 upregulation has not been elucidated. Moreover, the impact of IL-6 on the expression of CEACAM6 has not yet been examined.Methods: The expression of IL-6, IL-6 receptor (IL-6R), glycoprotein (gp) 130, CEACAM5 and CEACAM6 was analyzed by RT-PCR, Western blot, flow cytometry or qPCR. Colon cell lines were incubated with IL-6 or Hyper-IL-6 (mediating IL-6 trans-signaling), and subsequently, the expression of CEACAMs was determined by qPCR or Western blot. FLLL31, an inhibitor of the phosphorylation of signal transducer and activator of transcription-3 (STAT3), was used to determine the role of STAT3 phosphorylation.Results: We confirmed that colon carcinoma cell lines express IL-6 and IL-6R. We observed only a weak upregulation of CEACAM5 and CEACAM6 by classic IL-6 signaling, but a strong increase by IL-6 trans-signaling. This upregulation depended on the phosphorylation of STAT3.Conclusions: Our data show the upregulation of the tumor-associated antigens CEACAM5/6 by trans-signaling of the pro-inflammatory cytokine IL-6. This mechanism may contribute to the tumor-promoting role of IL-6 and could therefore be a target for therapeutic intervention in particular by specific inhibitors such as sgp130Fc. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human tumors, with radical surgical resection as the only curative treatment option. However, resection is only possible in a small fraction of patients, and about 80% of the patients develop recurrencies. PDAC development is facilitated by the cytokine interleukin-6 (IL-6), which acts via classic and trans-signaling. Both pathways are inhibited by the anti-IL-6-receptor antibody tocilizumab, whereas the fusion protein sgp130Fc specifically blocks trans-signaling. Here, we show that conservative or adjuvant therapy with both inhibitors reduces tumor growth in an orthotopic model of human Colo357 cells in SCID/bg mice. In the conservative setting, median primary tumor weight was reduced 2.4-fold for tocilizumab and 4.4-fold for sgp130Fc. sgp130Fc additionally led to a decrease in microvessel density, which was not observed with tocilizumab. In the adjuvant therapeutic setting after surgical resection of the primary tumor, treatment with tocilizumab or sgp130Fc decreased the local recurrence rate from 87.5% in the control group to 62.5 or 50%, respectively. Furthermore, the median weight of the local recurrent tumors was clearly diminished, and both inhibitors reduced the number of distant metastases. A significant reduction of tumor weight and metastases-comparable to gemcitabine treatment-was also observed with both inhibitors in another model using the poorly differentiated PancTuI cells. Our findings demonstrate the inhibition of IL-6 as a new treatment option in PDAC. [ABSTRACT FROM AUTHOR]

Interleukin 6 (IL-6) and, hence, activation of the IL-6 receptor signalling subunit glycoprotein 130 (gp130; also known as interleukin-6 receptor subunit β, IL6ST), has been linked to inflammation and tumour formation. Recently, deletion mutations in gp130 have been identified in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope and rendered gp130 constitutively active in a ligandindependent manner. Here, we show that gp130 deletion mutants, but not wild-type gp130, localise predominantly to intracellular compartments, notably the endoplasmic reticulum (ER) and early endosomes. One of the most frequent mutants, gp130 Y186- Y190del (∆YY) is retained in the ER quality control system because of its association with the chaperone calnexin. Furthermore, we can show that gp130 ∆YY induces downstream signalling from both ER and endosomes, and that both signals contribute to ligand-independent cell proliferation. We also demonstrate that the endosomal localisation of gp130 ∆YY is crucial for fully fledged STAT3 activation. Therefore, aberrant signalling from intracellular compartments might explain the tumorigenic potential of naturally occurring somatic mutations of gp130. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6) is a cytokine largely induced during infection, inflammation, and cancer. In the liver, IL-6 induces the synthesis of acute-phase proteins, which are believed to support the response of the body during infection and inflammation. Moreover, IL-6 has been reported to be a growth factor in multiple myeloma. IL-6 on cells binds to an IL-6 receptor (IL-6R) forming an IL-6/IL-6R complex, which associates with a homodimer of a second receptor subunit, gp130, to initiate signaling. Gp130 is present on all cells of the body, whereas IL-6R is only expressed on hepatocytes, some leukocytes, and some epithelial cells. Since gp130 has no measurable affinity for IL-6, cells which do not express IL-6R are unresponsive to the cytokine. A soluble form of IL-6R, which is found in the blood, can still bind IL-6 and the complex of IL-6/sIL-6R can bind to cellular gp130 also on cells without IL-6R expression. This signaling mechanism has been called trans-signaling. Interestingly, a soluble form of gp130 (sgp130) blocks IL-6 trans-signaling without affecting classic IL-6 signaling via the membrane-bound IL-6R. We used a dimerized version of sgp130 fused to the Fc portion of an IgG1 antibody (sgp130Fc) to discriminate between classic and trans-signaling of IL-6. It turned out that proinflammatory activities of IL-6 are mediated via trans-signaling, whereas anti-inflammatory or regenerative activities are mediated via classic signaling. These results are important for strategies to inhibit IL-6 signaling in autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and inflammation-associated colorectal cancer. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background & Aims: The anti–tumor necrosis factor (TNF) antibodies infliximab, adalimumab, and certolizumab pegol have proven clinical efficacy in Crohn''s disease. Here, we assessed the effects of anti-TNF antibodies on apoptosis in inflammatory bowel disease (IBD). Methods: CD14+ macrophages and CD4+ T cells were isolated from peripheral blood and lamina propria mononuclear cells from patients with IBD and control patients. Cell surface markers and apoptosis were assessed by immunohistology and fluorescence-activated cell sorting techniques. Results: Lamina propria CD14+ macrophages showed significantly more frequent and higher membrane-bound TNF (mTNF) expression than CD4+ T cells in IBD, whereas mTNF-dependent signaling proteins such as TNF receptor (TNFR) 2, TNFR-associated factor (TRAF) 2, and nuclear factor κB were induced in IBD mucosal CD4+ T cells. Most anti-TNF antibodies did not induce T-cell apoptosis in purified peripheral or mucosal CD4+ T cells. However, in contrast to etanercept, administration of all clinically effective anti-TNF antibodies resulted in a significant induction of T-cell apoptosis in IBD when lamina propria CD4+ T cells expressing TNFR2+ were cocultured with mTNF+ CD14+ intestinal macrophages. In contrast, no effects in control patients were noted. T-cell apoptosis in IBD occurred in vivo after treatment with adalimumab and infliximab, was critically dependent on TNFR2 signaling, and could be prevented via interleukin-6 signal transduction. Blockade of interleukin-6R signaling augmented anti-TNF–induced T-cell apoptosis in IBD. Conclusions: Clinically effective anti-TNF antibodies are able to induce T-cell apoptosis in IBD only when mucosal TNFR2+ T cells are cocultured with mTNF-expressing CD14+ macrophages. The finding that anti-TNF antibodies induce apoptosis indirectly by targeting the mTNF/TNFR2 pathway may have important implications for the development of new therapeutic strategies in IBD. [ABSTRACT FROM AUTHOR]

Tumour growth is not solely driven by tumour cell-intrinsic mechanisms, but also depends on paracrine signals provided by the tumour micro-environment. These signals comprise cytokines and growth factors that are synthesized as trans-membrane proteins and need to be liberated by limited proteolysis also termed ectodomain shedding. Members of the family of A disintegrin and metalloproteases (ADAM) are major mediators of ectodomain shedding and therefore initiators of paracrine signal transduction. In this review, we summarize the current knowledge on how ADAM proteases on tumour cells but also on cells of the tumour micro-environment contribute to the formation of gastrointestinal tumours, and discuss how these processes can be exploited pharmacologically. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent dementia in the elderly people. Obesity and insulin resistance, which may cause major health problems per se, are risk factors for AD, and cytokines such as interleukin-6 (IL-6) have a role in these conditions. IL-6 can signal either through a membrane receptor or by trans-signaling, which can be inhibited by the soluble form of the co-receptor gp130 (sgp130). We have addressed the possibility that blocking IL-6 trans-signaling in the brain could have an effect in the triple transgenic 3xTg-AD mouse model of AD and/or in obesity progression, by crossing 3xTg-AD mice with GFAP-sgp130Fc mice. To serve as control groups, GFAP-sgp130Fc mice were also crossed with C57BL/6JOlaHsd mice. Seventeen-month-old mice were fed a control diet (18% kcal from fat) and a high-fat diet (HFD; 58.4% kcal from fat). In our experimental conditions, the 3xTg-AD model showed a mild amyloid phenotype, which nevertheless altered the control of body weight and related endocrine and metabolic factors, suggestive of a hypermetabolic state. The inhibition of IL-6 trans-signaling modulated some of these traits in both 3xTg-AD and control mice, particularly during HFD, and in a sex-dependent manner. These experiments provide evidence of IL-6 trans-signaling playing a role in the CNS of a mouse model of AD. [ABSTRACT FROM AUTHOR]

Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin β, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease characterized by a progressive decline in lung function. Fibrotic diseases, such as IPF, are characterized by uncontrolled activation of fibroblasts. Since the microenvironment is known to affect cell behavior, activated fibroblasts can in turn activate healthy neighboring cells. Thus, we investigated IPF paracrine signaling in human lung fibroblasts (HLFs) derived from patients with IPF.Methods: Primary human fibroblast cultures from IPF (IPF-HLF) and control donor (N-HLF) lung tissues were established and their supernatants were collected. These supernatants were then added to N-HLFs for further culture. Protein and RNA were extracted from IPF/ N-HLFs at baseline. Interleukin-6 (IL-6) and TGF-β-related signaling factors (e.g. STAT3, Smad3) were evaluated by western blot and qPCR. IL-6 levels were measured by ELISA. IL-6 signaling was blocked by Tocilizumab (TCZ) (10 ng/ml).Results: IPF-HLFs were found to significantly overexpress IL-6 receptor (IL-6R), suppressor of cytokine signaling 3 (SOCS3), phospho-STAT3-Y705 and phospho-Smad3 in comparison to N-HLFs (p < 0.05). In addition, they were found to proliferate faster, secrete more IL-6 and express higher levels of the soluble IL-6R. IPF-HLF increased proliferation was inhibited by TCZ. Moreover, IPF-HLF derived supernatants induced both direct and indirect STAT3 activation that resulted in Smad3 phosphorylation and elevated Gremlin levels in N-HLFs. These effects were also successfully blocked by TCZ.Conclusions: IPF-HLF paracrine signaling leads to IL-6R overexpression, which in turn, affects N-HLF survival. The IL-6/STAT3/Smad3 axis facilitates cellular responses that could potentially promote fibrotic disease. This interplay was successfully blocked by TCZ. [ABSTRACT FROM AUTHOR]

• AD-induced mortality was reversed by the inhibition of IL-6 trans-signaling. • The inhibition of IL-6 trans-signaling modulated some AD-induced behavioral traits. • Sgp130Fc in the CNS reduced amyloid plaque load in Tg2576 females. • Sgp130Fc in the CNS reduced monomeric Aβ levels in cortex from Tg2576 females. Alzheimer's disease (AD) is the most commonly diagnosed dementia but its underlying pathological mechanisms still unclear. Neuroinflammation and secretion of cytokines such as interleukin-6 (IL-6) accompany the main hallmarks of the disease: amyloid plaques and neurofibrillary tangles. In this study, we analyzed the role of IL-6 trans-signaling in two mouse models of AD, Tg2576 and 3xTg-AD mice. The inhibition of IL-6 trans-signaling partially rescued the AD-induced mortality in females of both models. Before amyloid plaques deposition, it reversed AD-induced changes in exploration and anxiety (but did not affect locomotion) in Tg2576 female mice. However, after plaque deposition the only behavioral trait affected by the inhibition of IL-6 trans-signaling was locomotion. Results in the Morris water maze suggest that cognitive flexibility was reduced by the blocking of the IL-6 trans-signaling in young and old Tg2576 female mice. The inhibition of IL-6 trans-signaling also decreased amyloid plaque burden in cortex and hippocampus, and Aβ 40 and Aβ 42 levels in the cortex, of Tg2576 female mice. The aforementioned changes might be correlated with changes in blood vessels and matrix structure and organization rather than changes in neuroinflammation. 3xTgAD mice showed a very mild phenotype regarding amyloid cascade, but results were in accordance with those of Tg2576 mice. These results strongly suggest that the inhibition of the IL-6 trans -signaling could represent a powerful therapeutic target in AD. [ABSTRACT FROM AUTHOR]

The role of the pro-inflammatory cytokine interleukin-6 (IL-6) in the etiology of stress-induced synaptic plasticity is yet unknown. We took advantage of a genetically modified mouse (TG) in which IL-6 trans-signaling via the soluble IL-6 receptor was blocked, to determine the role of IL-6 trans-signaling in the effects of a Social Defeat protocol (SD) on synaptic function of the medial prefrontal cortex (mPFC). Synaptic function in stress-sensitive (S) and stress-resilient (R) animals was studied in a mPFC slice preparation with whole-cell patch-clamp recording. SD altered numerous synaptic properties of the mPFC: R WT (but not TG) displayed a decreased ratio between N methyl-D-aspartate receptor (NMDAR-) dependent and amino propionic acid receptor (AMPAR-) dependent-current (I NMDA /I AMPA), while S WT animals (but not TG) showed a reduced ratio between AMPA and γ-amino-butyric acid receptor type A (GABA A R)-dependent currents (I AMPA /I GABA). Also, SD induced an increase in the frequency but a decrease in the amplitude of excitatory action-potential dependent PSCs (sEPSCs), both in an IL-6 dependent manner, as well as a generalized (S/R-independent) decrease in the frequency of action potential independent (miniature) excitatory (IL-6 dependent) as well as inhibitory (IL-6 independent) postsynaptic current frequency. Interestingly, corner preference (measuring the intensity of social defeat) correlated positively with I NMDA /I AMPA and eEPSC frequency and negatively with I AMPA /I GABA. Our results suggest that SD induces behaviorally-relevant synaptic rearrangement in mPFC circuits, part of which is IL-6 dependent. In particular, IL-6 is necessary to produce synaptic plasticity leading to stress resilience in some individuals, but to stress sensitivity in others. • The prefrontal cortex is critically affected by social stress. • Social defeat (SD) induces extensive synaptic changes in the prefrontal cortex. • Some of these changes differ between stress-resilient and stress-sensitive individuals. • Stress-resilient and stress-sensitive specific synaptic modifications induced by SD are IL-6 dependent. [ABSTRACT FROM AUTHOR]

Purpose To evaluate the effect of an intravitreally applied anti-IL-6 antibody for the treatment of experimental autoimmune uveitis (EAU). Methods EAU was induced in female B10.RIII mice by Inter-Photoreceptor-Binding-Protein (IRBP) in complete Freund’s adjuvant, boosted by Pertussis toxin. Single blinded intravitreal injections of anti-IL-6 antibody were applied 5–7 days as well as 8–10 days (3 day interval) after EAU induction into the randomized treatment eye and phosphate buffered saline (PBS) into the fellow control eye. Clinical and fluorescein angiography scoring (6 EAU grades) was done at each injection day and at enucleation day 14. Enucleated eyes were either scored histologically (6 EAU grades) or examined by ELISA for levels of IL-6, IL-17 and IL-6 soluble Receptor (sIL-6R). Results Uveitis developed in all 12 mice. Clinical uveitis score was significantly reduced (p = 0.035) in treated eyes (median 2.0, range 0–4.0, n = 12) compared to the fellow control eyes (median 3.0, range 1.0–4.0, n = 12). Angiography scores were reduced in 9/12 treated eyes and histological scores in 3/4 treated eyes compared to the fellow control eyes. Cytokine levels were determined in 8 mice, of which 4 responded to anti-IL-6 treatment and 4 did not respond. All mice responding to treatment had a significant reduction of IL-6 (p < 0.01) and IL-17 (p = 0.01) levels in treated eyes compared to the fellow control eyes. This difference was not seen in non-responding mice. Conclusions Intravitreal anti-IL-6 treatment significantly attenuates experimental autoimmune uveitis in mice. EAU activity correlates with ocular IL-6 and IL-17 levels. [ABSTRACT FROM AUTHOR]

Background & Aims: Interleukin-6 (IL-6) is a crucial factor in liver regeneration following partial hepatectomy (PH); however, the role of IL-6 and IL-6 trans-signaling in particular, in hepatocyte mitosis remains controversial. IL-6 trans-signaling relies upon the release of the soluble IL-6R (sIL-6R), which binds IL-6 to form an agonistic IL-6/sIL-6R complex. Herein we have examined the hypothesis that IL-6 trans-signaling plays a crucial and distinct role in liver regeneration following PH. Methods: The specific IL-6/sIL-6R antagonist, sgp130Fc, was expressed in mice and analyzed for its effect on hepatocyte mitosis following PH. Alternatively, we examined the effect of the IL-6/sIL-6R super-agonist, Hyper-IL-6, or IL-6 expressed either alone or in combination with hepatocyte growth factor (HGF) on hepatocyte mitosis in the absence of PH. Results: Following PH, the dramatic rise of circulating IL-6 levels is accompanied by a concurrent ∼2-fold increase in circulating sIL-6R levels. Ectopic expression of sgp130Fc reduced hepatocyte mitosis by about 40% at early times following PH, while substantially reducing AKT, but not STAT3, activation. But, ectopic Hyper-IL-6 expression in mice without PH was not mitogenic to hepatocytes in vivo. Rather, Hyper-IL-6, but not IL-6, markedly increased HGF-induced hepatocyte mitosis. This cooperative effect correlated with greater resistance of HIL-6 than IL-6 to HGF-mediated reduction of AKT activation, rather than changes in STAT3 or MAPK signaling, and was completely blocked by PI3K inhibition. Conclusions: Following PH, IL-6/sIL-6R cooperates with growth factors, through a PI3K/AKT-dependent mechanism to promote entry of hepatocytes into the cell cycle. [Copyright &y& Elsevier]

Abstract: The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4×106 cells/ml v. 0.35×106 cells/ml in controls) (p <0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62×106 cells/ml v. 0.007×106 cells/ml in controls) (p <0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122pg/ml v. 64pg/ml in controls, p <0.01 and MCP-1; median: 692pg/ml v. 182pg/ml in controls, p <0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p <0.01), was weakly expressed (median: 50pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung. [Copyright &y& Elsevier]

Abstract: Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). It exerts its cellular effects by a membrane-bound IL-6 receptor (IL-6R), or, alternatively, by forming a complex with the soluble IL-6R (sIL-6R), a process named IL-6 transsignalling. Here we investigate the role of IL-6 transsignalling in myelin basic protein (MBP)-induced EAE in the Lewis rat. In vivo blockade of IL-6 transsignalling by the injection of a specifically designed gp130-Fc fusion protein significantly delayed the onset of adoptively transferred EAE in comparison to control rats injected with PBS or isotype IgG. Histological evaluation on day 3 after immunization revealed reduced numbers of T cells and macrophages in the lumbar spinal cord of gp130-Fc treated rats. At the same time, blockade of IL-6 transsignalling resulted in a reduced expression of vascular cell adhesion molecule-1 on spinal cord microvessels while experiments in cell culture failed to show a direct effect on the regulation of endothelial adhesion molecules. In experiments including active EAE and T cell culture, inhibition of IL-6 transsignalling mildly increased T cell proliferation, but did not change severity of active MBP-EAE or regulate Th1/Th17 responses. We conclude that IL-6 transsignalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the blood-brain barrier. [Copyright &y& Elsevier]

Abstract: Soluble gp130 is the natural inhibitor of trans-signaling mediated by the soluble IL-6/IL-6R complex. In mouse models, recombinant sgp130 has been successfully applied for the treatment of diseases that are triggered and maintained by soluble IL-6R like Crohn’s disease, peritonitis, rheumatoid arthritis, and colon cancer. The novel heterodimeric cytokine IL-27 (p28/EBV-induced gene 3) has been shown to act via a heterodimeric receptor complex of gp130 and the WSX-1 receptor, and to co-regulate the Th1 immune response after infection. Therefore, we have tested the potential of the recombinant sgp130-Fc protein to also inhibit signaling-mediated IL-27. Here we show that sgp130-Fc does not interfere with IL-27 signaling. We therefore conclude that IL-27 does not bind with high affinity to gp130. [Copyright &y& Elsevier]

The proinflammatory cytokine IL-6 binds to the membrane bound or soluble IL-6 receptor (IL-6R) and activates an intracellular signaling cascade after complex formation with two gp130 molecules. These mediate general homeostasis and orchestrates the immune response during disease. Trans-signalling via the soluble IL-6R has importance for the development and maintenance of human diseases like Crohn''s disease, peritonitis and rheumatoid arthritis. We have developed an enzyme-linked immunoabsorbent assay (ELISA) that detects the binding of gp130 to the IL-6/sIL-6R complex. Competitive binding of sgp130-Fc, viral IL-6, and the inhibitory drug Suramin to gp130 has been demonstrated. The assay can be used for high-throughput screening of peptide and chemical compound libraries for the identification of new agonists and antagonists of the binding between gp130 and IL-6/sIL-6R. [Copyright &y& Elsevier]

Interleukin-6 (IL-6) is involved in the growth and differentiation of numerous cell types. In the skin it is produced primarily by keratinocytes. The transcription factor STAT3 is activated by cytokines of the IL-6 family. In this study, we examined the involvement of IL-6, soluble IL-6-receptor, and STAT3 in epidermal barrier repair after injury to the stratum corneum by tape-stripping. After barrier disruption in wild-type mice we found an increased immunostaining of IL-6 and IL-6R on epidermal keratinocytes at 15 min to 5 h after treatment. The increase in IL-6 and IL-6R was confirmed by western blotting using epidermal homogenates and was partially prevented by occlusion immediately after barrier disruption. In IL-6-deficient mice, epidermal barrier repair was reduced at 3–24 h after treatment. Topical application of IL-6 or Hyper-IL-6, a complex of IL-6 linked to the soluble IL-6 receptor, enhanced epidermal barrier repair in wild-type mice. Application of the fusion protein gp130-FC, a specific inhibitor of the agonist IL-6/sIL-6 receptor complex, delayed barrier repair in wild, but not in IL-6-deficient mice. STAT3 tyrosine phosphorylation was induced after barrier disruption in wild-type, but markedly reduced in IL-6-deficient mice. Our results indicate that the IL-6 cytokine system, particularly transsignalling via the soluble IL-6R, is critically involved in barrier repair after skin injury. [ABSTRACT FROM AUTHOR]

Background: Cytokine-dependent activation of signalling pathways is tightly orchestrated. The spatiotemporal activation of signalling pathways dictates the specific physiological responses to cytokines. Dysregulated signalling accounts for neoplastic, developmental, and inflammatory diseases. Grb2-associated binder (Gab) family proteins are multi-site docking proteins, which expand cytokine-induced signal transduction in a spatial- and time-dependent manner by coordinating the recruitment of proteins involved in mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) and phosphatidyl-inositol-3-kinase (PI3K) signalling. Interaction of Gab family proteins with these signalling proteins determines strength, duration and localization of active signalling cascades. However, the underlying molecular mechanisms of signal orchestration by Gab family proteins in IL-6-induced signalling are only scarcely understood. Methods: We performed kinetic analyses of interleukin-6 (IL-6)-induced MAPK activation and analysed downstream responses. We compared signalling in wild-type cells, Gab1 knock-out cells, those reconstituted to express Gab1 mutants, and cells expressing gp130 receptors or receptor mutants. Results: Interleukin-6-induced MAPK pathway activation can be sub-divided into an early Gab1-independent and a subsequent Gab1-dependent phase. Early Gab1-independent MAPK activation is critical for the subsequent initiation of Gab1-dependent amplification of MAPK pathway activation and requires binding of SH2 domain-containing phosphatase 2 (SHP2) to the interleukin-6 receptor complex. Subsequent and coordinated recruitment of Grb2 and SHP2 to Gab1 is essential for Gab1-dependent amplification of IL-6-induced late MAPK pathway activation and subsequent gene expression. Conclusions: Overall, we elaborated the molecular requirements for Gab1-dependent, spatiotemporal orchestration of interleukin-6-dependent MAPK signalling. We discriminated IL-6-induced Gab1-independent, early activation of MAPK signalling and Gab1-dependent, sustained activation of MAPK signalling. [ABSTRACT FROM AUTHOR]