Interleukin-6 secretion is limited by self-signaling in endosomes

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood. In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.

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title: Acquiring signalling specificity from the cytokine receptor gp130.
abstract: Repeated use of a relatively small number of intracellular signalling molecules specifies tissue- and cell-type-specific responses to pleiotropic-acting growth factors and cytokines. Currently, gaining a better understanding of these mechanisms is a major challenge. The IL-6 family of cytokines shares a common receptor subunit called gp130. Phenotypic comparisons of mice with amino acid knock-in substitutions that disable individual signalling modules in gp130, with knockout mice lacking ligand-specific gp130 activation or transgenic mice with constitutive gp130 activation, has led to the identification of two molecular mechanisms. One mechanism is based on differential target-gene responsiveness to signalling threshold levels transduced by either the STAT1/3 or the SHP2/ERK cascade, which are under reciprocal negative regulation and together account for the majority of intracellular gp130 signalling. The second mechanism is based on the capacity of certain cell types to integrate the often-conflicting information transduced by these two pathways, and to prevent pathological responses.

title: Interleukin-6 and its receptors: a highly regulated and dynamic system.
abstract: Interleukin-6 (IL-6) is a multifunctional cytokine with well-defined pro- and anti-inflammatory properties. Although only small amounts in the picogram range can be detected in healthy humans, IL-6 expression is highly and transiently up-regulated in nearly all pathophysiological states. IL-6 induces intracellular signaling pathways after binding to its membrane-bound receptor (IL-6R), which is only expressed on hepatocytes and certain subpopulations of leukocytes (classic signaling). Transduction of the signal is mediated by the membrane-bound β-receptor glycoprotein 130 (gp130). In a second pathway, named trans-signaling, IL-6 binds to soluble forms of the IL-6R (sIL-6R), and this agonistic IL-6/sIL-6R complexes can in principle activate all cells due to the uniform expression of gp130. Importantly, several soluble forms of gp130 (sgp130) are found in the human blood, which are considered to be the natural inhibitors of IL-6 trans-signaling. Most pro-inflammatory roles of IL-6 have been attributed to the trans-signaling pathway, whereas anti-inflammatory and regenerative signaling, including the anti-bacterial acute phase response of the liver, is mediated by IL-6 classic signaling. In this simplistic view, only a minority of cell types expresses the IL-6R and is therefore responsive for IL-6 classic signaling, whereas gp130 is ubiquitously expressed throughout the human body. However, several reports point towards a much more complex situation. A plethora of factors, including proteases, cytokines, chemical drugs, and intracellular signaling pathways, are able to modulate the cellular expression of the membrane-bound and soluble forms of IL-6R and gp130. In this review, we summarize current knowledge of regulatory mechanisms that control and regulate the dynamic expression of IL-6 and its two receptors.

title: The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition.
abstract: Interleukin-6 has long been recognized as a prototypic pro-inflammatory cytokine that is involved in the pathogenesis of all inflammatory diseases. Activation of the gp130 homodimer by IL-6 leads to the initiation of Jak/STAT signaling, a pathway that is often constitutively switched on in inflammatory malignancies. However, a plethora of studies in the last decade has convincingly shown that only signaling via the soluble IL-6R (trans-signaling) accounts for the deleterious effects of IL-6, whereas classic signaling via the membrane-bound receptor is essential for the regenerative and anti-bacterial effects of IL-6 (classic signaling). In this review, we highlight recent developments in the field of IL-6 research, and specifically focus on advances towards a safe and specific inhibition of IL-6 trans-signaling.

title: Immunotherapeutic implications of IL-6 blockade for cytokine storm.
abstract: IL-6 contributes to host defense against infections and tissue injuries. However, exaggerated, excessive synthesis of IL-6 while fighting environmental stress leads to an acute severe systemic inflammatory response known as 'cytokine storm', since high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells but inhibit myocardial function. Remarkable beneficial effects of IL-6 blockade therapy using a humanized anti-IL-6 receptor antibody, tocilizumab were recently observed in patients with cytokine release syndrome complicated by T-cell engaged therapy. In this review we propose the possibility that IL-6 blockade may constitute a novel therapeutic strategy for other types of cytokine storm, such as the systemic inflammatory response syndrome including sepsis, macrophage activation syndrome and hemophagocytic lymphohistiocytosis.

title: IL-6 as a keystone cytokine in health and disease
abstract: Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.

title: Identification of a region within the cytoplasmic domain of the interleukin-6 (IL-6) signal transducer gp130 important for ligand-induced endocytosis of the IL-6 receptor.
abstract: Interleukin-6 (IL-6) exerts its action via a cell surface receptor complex consisting of two subunits, the IL-6 receptor and the signal transducer gp130. We have studied the role of both transmembrane proteins for IL-6 internalization and ligand-induced down-regulation of cell surface receptors. Co-expression of wild-type and mutant forms of both the IL-6 receptor and gp130 in transiently transfected COS-7 cells revealed that gp130 is essential for efficient endocytosis and receptor down-regulation. Whereas the cytoplasmic domain of the IL-6 receptor is not significantly involved in the internalization process, deletion of the corresponding domain of gp130 resulted in an almost complete loss of the ability to endocytose IL-6. Mutants with different truncations within the intracellular domain of gp130 revealed that a 10-amino acid sequence TQPLLDSEER is crucial for efficient internalization. Since this sequence contains a putative di-leucine internalization motif, we suggest that a di-leucine motif directs the receptor mediated endocytosis of the IL-6 receptor complex.

title: Multiple forms of IFN-beta 2/IL-6 in serum and body fluids during acute bacterial infection.
abstract: Many of the major alterations in plasma proteins characteristic of the hepatic acute phase response are regulated by IFN-beta 2/IL-6. Using a specific bioassay for IFN-beta 2/IL-6, which relies on the induction of the hepatic acute phase plasma protein alpha 1-antichymotrypsin in the human hepatoma cell line Hep3B clone 2 and its inhibition by anti-rIFN-beta 2/IL-6 antiserum, we have detected high levels of IFN-beta 2/IL-6 in the body fluids of patients with acute bacterial infections. Cerebrospinal fluid from four patients with acute bacterial meningitis (Streptococcus pneumoniae, Staphylococcus aureus, two cases of Listeria monocytogenes) all had high levels of IFN-beta 2/IL-6 (up to 500 ng/ml). Two of these patients with concomitant bacteremia had lower concentrations of IFN-beta 2/IL-6 in the serum (5 to 70 ng/ml). Three additional patients with Escherichia coli, Pseudomonas aeruginosa, and Neisseria meningitidis bacteremia had high levels of serum IFN-beta 2/IL-6, as did the ankle fluid of a patient with Streptococcus canis arthritis. Normal cerebrospinal fluid and serum had little detectable IFN-beta 2/IL-6. A combination of immunoaffinity chromatography and immunoblotting procedures were used to characterize the IFN-beta 2/IL-6 species present in a representative sampling of serum and cerebrospinal fluids. Multiple immunoreactive species of IFN-beta 2/IL-6 in the size range 23 to 30 kDa as well as immunoreactive complexes in the range 60 to 70 kDa were detected in human body fluids. This is the first demonstration that previous descriptions of heterogeneity in human IFN-beta 2/IL-6 species produced in cell culture correspond to observations in the infected host.

title: Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy.
abstract: In a phase 1 study of recombinant interleukin-6 (rIL-6) in patients with advanced solid tumors (n = 15), we discovered that the endogenous IL-6 levels, in pretreatment plasma or serum samples, were distributed into two groups. One set of patients (designated "type 1"; n = 9) was characterized by low plasma IL-6 levels (48 to 1,700 pg/mL) as measured using enzyme-linked immunosorbent assays (ELISA) for IL-6. In the second set of patients (designated "type 2"; n = 6), IL-6 ELISAs showed high levels of plasma IL-6 (50 to 600 ng/mL). Neither group had detectable B9 hybridoma cell growth factor activity associated with the IL-6 in their pretreatment plasma or serum. Plasma C-reactive protein (CRP) levels were markedly elevated in type II patients suggesting that the circulating IL-6 was biologically active in vivo. In both groups of patients there was a small but significant increase in B9 activity in the plasma within three hours after rIL-6 administration (n = 5). Gel filtration profiles showed that circulating IL-6 in type 1 patients, 15 to 120 minutes after rIL-6 administration was of approximate mass 20 to 40 kD, whereas in type 2 patients, the IL-6 before and after exogenous rIL-6 administration was indistinguishable and was of an approximate mass of 200 kD. IL-6 immunoaffinity purification of the 200 kD complexes showed these to contain multiple isoforms of IL-6 (14 to 31 kD) and the soluble IL-6 receptor (sIL-6R; 50 to 55 kD). A distinguishing clinical history was that all of the type 2 patients had been actively immunized with an anti-idiotypic monoclonal antibody (MoAb) (MK2-23) 3 to 12 months before initiation of this study for advanced melanoma. An analysis of the plasma IL-6 content in other melanoma patients (n = 16) during antiidiotypic MoAb immunization indicated that marked (up to 600 ng/mL) and sustained (several months) elevations of circulating "chaperoned" IL-6 were induced by active immunization regimens.

title: Distinct classes of chaperoned IL-6 in human blood: differential immunological and biological availability.
abstract: Transport of IL-6 in blood is fundamental to the biology of this cytokine. In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guerin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both). Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays. Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not. The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6. In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays. Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG. The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.

title: Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.
abstract: Psoriasis is a common papulosquamous skin disease. The histopathology is characterized by epidermal hyperplasia and inflammation. Recent studies suggest that keratinocyte proliferation and inflammation in psoriasis are manifestations of the same underlying pathological process. Interleukin 6 (IL-6), a cytokine that is a major mediator of the host response to tissue injury and infection, is produced by both keratinocytes and leukocytes in culture. IL-6 expression was studied in psoriatic plaques by immunoperoxidase staining with two different polyclonal anti-recombinant IL-6 antisera and by in situ nucleic acid hybridization with IL-6 cRNA probes. Epidermal and dermal cells in active psoriatic plaques from 35 psoriasis patients stained heavily for IL-6 as compared with nonlesional skin and with plaques after treatment with antimetabolic and antiinflammatory agents. Absorption of the anti-recombinant IL-6 antisera with purified fibroblast-derived IL-6 or with recombinant IL-6, but not bovine serum albumin, removed the immunostaining. Increased levels of IL-6 were detected in the plasma of patients with active psoriasis (mean 3 ng/ml) by using two different bioassays. IL-6 production by proliferating keratinocytes was suggested by IL-6-specific immunostaining in cultured normal and psoriatic keratinocytes and by the detection of mRNA specific for IL-6 in psoriatic epidermis by in situ hybridization. IL-6 stimulated the proliferation of cultured, normal human keratinocytes as assessed by two different assays. Thus, IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affect the function of dermal inflammatory cells.

title: The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome
abstract: Serum samples from patients with meningococcal disease were examined for the presence of IL-6, TNF-alpha, and LPS. Median serum concentration of IL-6 was 1,000 times higher in patients with septic shock (189 ng/ml) than in patients with bacteriaemia, meningitis, or combined septic shock and meningitis. 11 of 21 patients with serum levels greater than 3.0 ng/ml died, whereas all 58 patients with serum levels at less than or equal to 3.0 ng/ml, survived. All four patients with serum IL-6 levels greater than 750 ng/ml, died. IL-1 was detected in serum from three patients who also had high serum levels of IL-6, TNF-alpha, and LPS, and rapidly fatal courses. IL-6 appeared to be released into serum later than TNF-alpha, and was detected in serum for up to 36 h. The half-life of IL-6 and TNF-alpha was calculated to be 103 +/- 27 min and 70 +/- 11 min, respectively. These data indicate that a complex pattern of cytokines exists in serum from patients with meningococcal septic shock, and that the release of IL-6 and IL-1, in addition to TNF-alpha, is associated with fatal outcome.

title: Cytokine serum level during severe sepsis in human IL-6 as a marker of severity.
abstract: Forty critically ill surgical patients with documented infections were studied during their stay in an intensive care unit. Among these patients, 19 developed septic shock and 16 died, 9 of them from septic shock. Interleukin 1 beta (IL-1 beta), tumor necrosis factor (TNF alpha), and interleukin 6 (IL-6) were measured each day and every 1 or 2 hours when septic shock occurred. Although IL-1 beta was never found, TNF alpha was most often observed in the serum at a level under 100 pg/mL except during septic shock. During these acute episodes TNF alpha level reached several hundred pg/mL, but only for a few hours. In contrast, IL-6 was always increased in the serum of acutely ill patients (peak to 500,000 pg/mL). There was a direct correlation between IL-6 peak serum level and TNF alpha peak serum level during septic shock and between IL-6 serum level and temperature or C-reactive protein serum level. Moreover, IL-6 correlated well with APACHE II score, and the mortality rate increased significantly in the group of patients who presented with IL-6 serum level above 1000 pg/mL. Thus, IL-6 appears to be a good marker of severity during bacterial infection.

title: Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130
abstract: Studies in IL-6-deficient (IL-6−/−) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6−/− mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.

title: Role of cytokines as a double-edged sword in sepsis.
abstract: Sepsis is a deadly immunological disorder and its pathophysiology is still poorly understood. We aimed to determine if specific pro-inflammatory and anti-inflammatory cytokines can be used as diagnostic and therapeutic targets for sepsis.



Recent publications in the MEDLINE database were searched for articles regarding the clinical significance of inflammatory cytokines in sepsis.



In response to pathogen infection, pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, IL-18 and tumor necrosis factor-α (TNF-α)] and anti-inflammatory cytokine (IL-10) increased in patients with sepsis. Importantly, a decrease in IL-6 was associated with a better prognosis and overproduction of IL-10 was found to be the main predictor of severity and fatal outcome.



Both pro-inflammatory and anti-inflammatory cytokines constitute a double-edged sword in sepsis; on one hand they are critical to eliminate the infection while on the other, excessive production can cause tissue and organ damage. Increase in cytokines such as IL-6, Il-8, IL-10, IL-18 and TNF-α may have implications in diagnosis and treatment of sepsis.

title: Serum Interleukin 6 Before and After Therapy with Tocilizumab Is a Principal Biomarker in Patients with Rheumatoid Arthritis
abstract: Biologic treatments including the humanized anti-interleukin 6 (anti-IL-6) receptor antibody tocilizumab (TCZ) provide therapeutic options for patients with rheumatoid arthritis (RA). We investigated useful biomarkers to predict the responsiveness to TCZ by measurement of serum proinflammatory cytokine concentrations.  Serum samples were collected from 61 patients with RA before biologic treatment and at 4 weeks after initial administration of either TCZ (n = 32) or infliximab (IFX; n = 29) and from 13 healthy serum donor controls. Disease Activity Score of 28 joints (DAS28) was determined at baseline and after treatment.  Although IL-1β, IL-2, IL-6, IL-17A, IL-17F, interferon-α, and tumor necrosis factor-α (TNF-α) were all increased in sera from patients with RA compared with controls, only the IL-6 level was significantly correlated with DAS28 before treatment. The IL-6 level before treatment was positively correlated with DAS28 after TCZ treatment, and was significantly lower in TCZ-responsive patients (as judged by a post-treatment DAS28 < 3.2) than in TCZ-resistant patients (post-treatment DAS28 ≥ 3.2). DAS28 after TCZ was significantly lower than after administration of IFX in patients with low pretreatment IL-6 (< 51.5 pg/ml, the mean baseline value of IL-6 in all RA patients), but not in those with high pretreatment IL-6. These results indicate that low serum IL-6 is associated with a favorable response to TCZ.  Although both TNF-α and IL-6 are major targets of therapeutic intervention in RA, baseline serum IL-6 but not baseline TNF-α level is a potential biomarker reflecting disease activity. Measurement of serum IL-6 in RA before treatment may be useful to estimate residual disease activity after TCZ treatment and to predict responsiveness to TCZ treatment.

title: Circulating interleukin‐6 predicts survival in patients with metastatic breast cancer
abstract: Interleukin‐6 (IL‐6) is a multifunctional cytokine produced by macrophages, T cells, B cells, endothelial cells and tumour cells. Interleukin‐6 is able to promote tumour growth by upregulating anti‐apoptotic and angiogenic proteins in tumour cells. In murine models it has been demonstrated that antibodies against IL‐6 diminish tumour growth. Several reports have highlighted the prognostic importance of IL‐6 in e.g., prostate and colon cancer. We addressed prospectively the prognostic significance of serum IL‐6 (sIL‐6), measured at diagnosis of metastasis, in 96 unselected and consecutive patients with progressive metastatic breast cancer before the initiation of systemic therapy. The median sIL‐6 value for the breast cancer population was 6.6 ± 2.1 pg/ml. Patients with 2 or more metastatic sites had higher sIL‐6 values compared to those with only 1 metastatic site (respectively 8.15 ± 1.7 pg/ml and 3.06 ± 6.6 pg/ml; p < 0.001). Patients with liver metastasis (8.3 ± 2.4 pg/ml), with pleural effusions (10.65 ± 9.9 pg/ml) and with dominant visceral disease (8.15 ± 3.3 pg/ml) had significantly higher values compared to those without liver metastases (4.5 ± 3.4 pg/ml; p = 0.001), without pleural effusions (5.45 ± 1.5 pg/ml; p = 0.0077) and with dominant bone disease (4.5 ± 1.4 pg/ml; p = 0.007) respectively. No correlation between sIL‐6 and age, menopausal status, performance status, tumour grade, body‐mass index, histology and hormone receptor status was found. Multivariate analysis showed that high levels of serum IL‐6 have independent prognostic value. We conclude that circulating IL‐6 is associated with worse survival in patients with metastatic breast cancer and is correlated with the extent of disease.

title: Tumour immunology: Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment
abstract: Immune cells in the tumour microenvironment not only fail to mount an effective anti-tumour immune response, but also interact intimately with the transformed cells to promote oncogenesis actively. Signal transducer and activator of transcription 3 (STAT3), which is a point of convergence for numerous oncogenic signalling pathways, is constitutively activated both in tumour cells and in immune cells in the tumour microenvironment. Constitutively activated STAT3 inhibits the expression of mediators necessary for immune activation against tumour cells. Furthermore, STAT3 activity promotes the production of immunosuppressive factors that activate STAT3 in diverse immune-cell subsets, altering gene-expression programmes and, thereby, restraining anti-tumour immune responses. As such, STAT3 propagates several levels of crosstalk between tumour cells and their immunological microenvironment, leading to tumour-induced immunosuppression. Consequently, STAT3 has emerged as a promising target for cancer immunotherapy.

title: Diagnostic and Prognostic Value of Serum Interleukin-6 in Colorectal Cancer
abstract: AbstractThe application of serum interleukin-6 (IL-6) in the diagnosis and prognosis of colorectal cancer (CRC) has been evaluated in many studies, whereas the results were contradictive.The aim of this study was to systematically evaluate this issue.An original study was conducted to explore the diagnostic value of serum IL-6 in CRC. Pubmed, Embase, and Cochrane library databases were searched for eligible studies.For diagnostic meta-analysis, aggregate data (AD) and individual participant data (IPD) meta-analyses were both adopted. The sensitivity and specificity were pooled and a summary receiver-operating characteristic (ROC) curve was constructed. For prognostic meta-analysis, study-specific hazard ratios (HRs) of IL-6 for survival were summarized. Secondary analysis of survival data was performed to synthesize the Kaplan–Meier curves.Total 17 studies (including our study) were included in this meta-analysis. The pooled sensitivity, specificity, and area under curve (AUC) of serum IL-6 were 0.72 (95% CI: 0.46–0.88), 0.74 (95% CI: 0.56–0.86), and 0.79 (95% CI: 0.75–0.82) in CRC diagnosis, respectively. Further, IPD meta-analysis strengthened the diagnostic value of serum IL-6 (the AUC, sensitivity, and specificity were 0.794, 0.606, and 0.839, respectively). For prognostic analysis, the high serum level of IL-6 was inversely associated with overall survival (OS) (pooled HR = 1.76, 95% CI: 1.42–2.19, P < 0.001) and disease-free survival (DFS) (pooled HR = 2.97, 95% CI: 1.76–5.01, P < 0.001). The synthesized Kaplan–Meier curves indicated that CRC patients with higher serum IL-6 level had a worse OS (P = 0.0027) and DFS (P < 0.001), which further support the prognostic value of serum IL-6 in CRC patients.The present study confirmed that serum IL-6 may be a potential biomarker for CRC diagnosis, and the high serum IL-6 level was associated with poor prognosis for both CRC overall survival and disease-free survival.The study has been registered in an international registry of systematic reviews PROSPERO (CRD42013006485).

title: IL‐6: Regulator of Treg/Th17 balance
abstract: IL‐6 is a pleiotropic cytokine involved in the physiology of virtually every organ system. Recent studies have demonstrated that IL‐6 has a very important role in regulating the balance between IL‐17‐producing Th17 cells and regulatory T cells (Treg). The two T‐cell subsets play prominent roles in immune functions: Th17 cell is a key player in the pathogenesis of autoimmune diseases and protection against bacterial infections, while Treg functions to restrain excessive effector T‐cell responses. IL‐6 induces the development of Th17 cells from naïve T cells together with TGF‐β; in contrast, IL‐6 inhibits TGF‐β‐induced Treg differentiation. Dysregulation or overproduction of IL‐6 leads to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA), in which Th17 cells are considered to be the primary cause of pathology. Given the critical role of IL‐6 in altering the balance between Treg and Th17 cells, controlling IL‐6 activities is potentially an effective approach in the treatment of various autoimmune and inflammatory diseases. Here, we review the role of IL‐6 in regulating Th17/Treg balance and describe the critical functions of IL‐6 and Th17 in immunity and immune‐pathology.

title: Trans-presentation of interleukin-6 by dendritic cells is required for priming pathogenic TH17 cells
abstract: The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpα were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor α-chain (IL-6Rα), Sirpα+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Rα (called 'IL-6 cluster signaling' here) was needed to prevent premature induction of interferon-γ (IFN-γ) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.

title: Interleukin-6: Biology, signaling and strategies of blockade.
abstract: Interleukin-6 (IL-6) is one of the most important inflammatory cytokines. IL-6 is unique in signaling via a membrane bound and a soluble receptor. Intriguingly, these two pathways strongly differ in their biologic consequences. While classic IL-6 signaling via the membrane bound receptor is mainly regenerative and protective, IL-6 trans-signaling via the soluble IL-6R is rather pro-inflammatory. Intracellular signaling of IL-6 in response to receptor activation is through STAT-dependent and STAT-independent signaling modules, which are regulated by a complex regulatory network. The complex biology of IL-6 has consequences for therapeutic targeting of this cytokine. We hypothesize that specific inhibition of the trans-signaling pathway may be superior to global blockade of IL-6 activity with help of antibodies directed against IL-6 or IL-6R.

title: Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor.
abstract: Interleukin-6 (IL-6/BSF-2/IFN beta 2) is a multifunctional cytokine that regulates the growth and differentiation of various tissues, and is known particularly for its role in the immune response and acute phase reactions. A complementary DNA encoding the human IL-6 receptor (IL-6-R) has now been isolated. The IL-6-R consists of 468 amino acids, including a signal peptide of approximately 19 amino acids and a domain of approximately 90 amino acids that is similar to a domain in the immunoglobulin (Ig) superfamily. The cytoplasmic domain of approximately 82 amino acids lacks a tyrosine/kinase domain, unlike other growth factor receptors.

title: The hepatic interleukin‐6 receptor Down‐regulation of the interleukin‐6 binding subunit (gp80) by its ligand
abstract: Interleukin‐6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6‐binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp13O). We studied the role of gp80 in binding, internalization and down‐regulation of the hepatic IL6‐receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp8O with parental cells indicate that gp80 is responsible for low affinity binding (K d = 500 pM) of IL6. Furthermore, gp80 is rate‐limiting in internalization and degradation of IL6. Internalization resulted in a rapid down‐regulation (t ≈ 15–30 min) of IL6‐binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6‐binding sites at the cell surface required >8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down‐regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.

title: Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding
abstract: Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL‐6 may fold as a 4 α‐helix bundle structure with up‐up‐down‐down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL‐6 polypeptides display distinct species‐specific activities. Although human IL‐6 (hIL‐6) is active in both human and mouse cell assays, mouse IL‐6 (mIL‐6) is not active on human cells. Previously, we demonstrated that the 5 C‐terminal residues of mIL‐6 are important for activity, conformation, and stability (Ward LD et al., 1993, . To further probe the structure‐function relationship of this cytokine, we have constructed several human/mouse IL‐6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu‐62 (Lys‐65 in mIL‐6) in the putative connecting loop AB between helices A and B, at Arg‐113 (Val‐117 in mIL‐6) at the N‐terminal end of helix C, at Lys‐150 (Asp‐152 in mIL‐6) in the connecting loop CD between helices C and D, and at Leu‐178 (Thr‐180 in mIL‐6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity.

title: gp130 at the nexus of inflammation, autoimmunity, and cancer
abstract: Glycoprotein 130 (gp130) is a shared receptor utilized by several related cytokines, including IL‐6, IL‐11, IL‐27, Leukemia Inhibitory Factor (LIF), Oncostatin M (OSM), Ciliary Neurotrophic Factor (CNTF), Cardiotrophin 1 (CT‐1) and Cardiotrophin‐like Cytokine (CLC). Gp130 plays critical roles during development and gp130‐deficient mice are embryonically lethal. However, the best characterized facet of this receptor and its associated cytokines is the ability to promote or suppress inflammation. The aim of this review is to discuss the role of gp130 in promoting or preventing the development of autoimmunity and cancer, two processes that are associated with aberrant inflammatory responses.

title: Maximal activation of transcription by statl and stat3 requires both tyrosine and serine phosphorylation
abstract: Stat1 and Stat3 are latent transcriptional factors activated initially through phosphorylation on single tyrosine residues induced by cytokine and growth factor occupation of cell surface receptors. Here we show that phosphorylation on a single serine (residue 727) in each protein is also required for maximal transcriptional activity. Both cytokines and growth factors are capable of inducing the serine phosphorylation of Stat1 and Stat3. These experiments show that gene activation by Stat1 and Stat3, which obligatorily require tyrosine phosphorylation to become active, also depends for maximal activation on one or more of the many serine kinases.

title: STAT1 and STAT3 phosphorylation by porins are independent of JAKs but are dependent on MAPK pathway and plays a role in U937 cells production of interleukin-6.
abstract: A group of transcription factors, termed signal transducers and activators of transcription (STATs), appears to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Similarly, cytoplasmic Janus kinases (JAKs) seem to play a critical role in diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. In this work, we analysed the effects of the Salmonella enterica serovar Typhimurium porins on signaling by the JAK/STAT pathway and IL-6 release in U937 cells. Porins and LPS of membrane from Gram-negative bacteria are factors implicated in septic shock. In our assays porins induce interleukin-6 (IL-6) release (110+/-2.6pg/ml) 24h after stimulation and STAT1/STAT3 tyrosine (Tyr701/Tyr705) and serine (Ser727) phosphorylation after 15min. By using several selective inhibitors we demonstrate that porins modulate the activation of STAT1/STAT3 through mitogen activated protein kinases (MAPKs) and not JAKs. Furthermore, we demonstrated that STAT1 and STAT3 are not involved in the modulation of IL-6 release in U937 cells stimulated with porins. Inhibition of tyrosine/serine phosphorylation mediated by MAPKs of STAT1 and STAT3 decrease the IL-6 secretion following porin stimulation. Therefore, suggesting a key role of this pathway in phosphorylation of Ser 727 in STAT1 and STAT3. These results are confirmed by porin or LPS-induced nuclear translocation of STAT1 and STAT3 in U937 cells.

title: Identification of STAT3 and STAT5 proteins in the rat suprachiasmatic nucleus and the Day/Night difference in astrocytic STAT3 phosphorylation in response to lipopolysaccharide
abstract: Signal transducers and activators of transcription (STAT) proteins regulate many aspects of cellular physiology from growth and differentiations to immune responses. Using immunohistochemistry, we show the daily rhythm of STAT3 protein in the rat suprachiasmatic nucleus (SCN), with low but significant amplitude peaking in the morning. We also reveal the strong expression of STAT5A in astrocytes of the SCN and the STAT5B signal in nonastrocytic cells. Administration of lipopolysaccharide (LPS) acutely induced phosphorylation of STAT3 on Tyr705 during both the day and the night and induced phosphorylation on Ser727 but only after the daytime application. The LPS‐induced phospho‐STAT3 (Tyr705) remained elevated for 24 hr after the daytime application but declined within 8 hr when LPS was applied at night.

title: Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR
abstract: Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation.

title: Monocyte 15-Lipoxygenase Expression Is Regulated by a Novel Cytosolic Signaling Complex with Protein Kinase C δ and Tyrosine-Phosphorylated Stat31
abstract: Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)δ. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCδ with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCδ and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCδ association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCδ and subsequent PKCδ-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCδ with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.

title: Pattern of cytokine receptors expressed by human dendritic cells migrated‘qc from dermal explants
abstract: Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme‐sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short‐term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)‐DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)‐1R type 1 (monoclonal antibody (mAb) hIL‐1R1‐M1; and 6B5); IL‐1R type 2 (mAb hIL‐1R2‐M22); IL‐2R α chain (mAb anti‐Tac; and hIL‐2R‐M1) and IL‐2R γ chain (mAb 3B5; and AG14C). DDC did not stain for IL‐2R β chain using four mAbs recognizing two different epitopes of IL‐2Rβ (mAb 2R‐B; Mik‐β1; and CF1; Mik‐β3, respectively). DDC were also positive for the cytokine binding chains (α chains) of IL‐3R (mAb 9F5); IL‐4R (mAb ; and S456C9); and IL‐7R (mAb hIL‐7R‐M20; and R3434). DDC showed low levels of IL‐6R α chain (mAb B‐F19; B‐R6; and B‐E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon‐γ receptor (IFN‐γR) (mAb GIR‐208) and were negative for IL‐8R (mAb B‐G20; and B‐F25). All DDC were highly positive for granulocyte–macrophage colony‐stimulating factor receptor (GM‐CSFR) α chain (mAb hGM‐CSFR‐M1; SC06; SC04, and 8G6) and to a lesser extent for the common β chain of GM‐CSFR, IL‐3R and IL‐5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony‐stimulating factor receptor (G‐CSFR) (mAb hGCSFR‐M1) nor macrophage colony‐stimulating factor receptor (M‐CSFR) (mAb 7‐7A3‐17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75 000 MW (mAb utr‐1; hTNFR‐M1; and MR2‐1) but lacked TNFR 55 000 MW (mAb htr‐9; MR1‐1; and MR1‐2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL‐2Rαγ; IL‐6Rα/gp130; IL‐7Rαγ).

title: Differences in Wound Healing in Mice with Deficiency of IL-6 versus IL-6 Receptor
abstract: IL-6 modulates immune responses and is essential for timely wound healing. As the functions mediated by IL-6 require binding to its specific receptor, IL-6Rα, it was expected that mice lacking IL-6Rα would have the same phenotype as IL-6–deficient mice. However, although IL-6Rα–deficient mice share many of the inflammatory deficits seen in IL-6–deficient mice, they do not display the delay in wound healing. Surprisingly, mice with a combined deficit of IL-6 and IL-6Rα, or IL-6–deficient mice treated with an IL-6Rα–blocking Ab, showed improved wound healing relative to mice with IL-6 deficiency, indicating that the absence of the receptor contributed to the restoration of timely wound healing, rather than promiscuity of IL-6 with an alternate receptor. Wounds in mice lacking IL-6 showed delays in macrophage infiltration, fibrin clearance, and wound contraction that were not seen in mice lacking IL-6Rα alone and were greatly reduced in mice with a combined deficit of IL-6 and IL-6Rα. MAPK activation-loop phosphorylation was elevated in wounds of IL-6Rα–deficient mice, and treatment of wounds in these mice with the MEK inhibitor U0126 resulted in a delay in wound healing suggesting that aberrant ERK activation may contribute to improved healing. These findings underscore a deeper complexity for IL-6Rα function in inflammation than has been recognized previously.

title: Knockdown of the interleukin-6 receptor alpha chain of dendritic cell vaccines enhances the therapeutic potential against IL-6 producing tumors.
abstract: Tumor microenvironment has emerged as one of the major obstacles against the clinical efficacy of dendritic cell (DC) vaccines. Tumor-derived IL-6 may inhibit the differentiation of hematopoietic progenitor cells into DCs and suppress DC maturation, rendering DCs tolerogenic. We hypothesized that silencing the IL-6 receptor alpha chain (IL-6Rα) would restore the functional competence of DC vaccines in mice with an IL-6-producing TC-1 tumor, and eventually give rise to protective immunity. We found that the IL-6Rα knockdown-DC vaccine significantly enhanced the frequency of tumor-specific CD8(+) CTLs-producing effector molecules such as IFN-γ, TNF-α, FasL, perforin, and granzyme B, and generated more CD8(+) memory T cells, leading to the substantially prolonged survival of TC-1 tumor-bearing mice.

title: Pro‐inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum‐free conditions
abstract: Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte‐macrophage colony‐stimulating factor and interleukin (IL)‐4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte‐conditioned medium (MCM) and FCS‐free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)‐α, IL‐1β and IL‐6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF‐α/IL‐1/IL‐6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)‐γ and no IL‐4 or IL‐10. Production of IFN‐γ was significantly induced by addition of PGE2, while no effect on production of IL‐4 or IL‐10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN‐γlow, IL‐4neg cells, TNF‐α/IL‐1/IL‐6 promoted growth of IFN‐γintermediate, IL‐4neg CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN‐γ production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF‐α/IL‐1/IL‐6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF‐α/IL‐1, IL‐6 + PGE2‐cultured DC seem to be optimal for generation of IFN‐γ‐producing CD4/CD8+ T cells.

title: IL-6 Regulates In Vivo Dendritic Cell Differentiation through STAT3 Activation1
abstract: Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130F759/F759) or STAT3 (gp130FxxQ/FxxQ), and combined gp130 and IL-6 defects (gp130F759/F759/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130F759/F759 mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.

title: Dendritic Cell (DC)-Specific Targeting Reveals Stat3 as a Negative Regulator of DC Function
abstract: Dendritic cells (DCs) must achieve a critical balance between activation and tolerance, a process influenced by cytokines and growth factors. IL-10, which transduces signals through Stat3, has emerged as one important negative regulator of DC activation. To directly examine the role Stat3 plays in regulating DC activity, the Stat3 gene was targeted for deletion with a CD11c-cre transgene. Stat3 CKO mice developed cervical lymphadenopathy as well as a mild ileocolitis that persisted throughout life and was associated with impaired weight gain. Consistent with this, Stat3-deficient DCs demonstrated enhanced immune activity, including increased cytokine production, Ag-dependent T-cell activation and resistance to IL-10–mediated suppression. These results reveal a cell-intrinsic negative regulatory role of Stat3 in DCs and link increased DC activation with perturbed immune homeostasis and chronic mucosal inflammation.

title: Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells
abstract: Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. The mechanisms that block initiation of immune responses during cancer development are poorly understood. We report here that constitutive activation of Stat-3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators. Blocking Stat-3 in tumor cells increases expression of proinflammatory cytokines and chemokines that activate innate immunity and dendritic cells, leading to tumor-specific T-cell responses. In addition, constitutive Stat-3 activity induces production of pleiotropic factors that inhibit dendritic cell functional maturation. Tumor-derived factors inhibit dendritic cell maturation through Stat-3 activation in progenitor cells. Thus, inhibition of antitumor immunity involves a cascade of Stat-3 activation propagating from tumor to dendritic cells. We propose that tumor Stat-3 activity can mediate immune evasion by blocking both the production and sensing of inflammatory signals by multiple components of the immune system.

title: Inflammation-Induced IL-6 Functions as a Natural Brake on Macrophages and Limits GN.
abstract: IL-6 can mediate proinflammatory effects, and IL-6 receptor (IL-6R) blockade as a treatment for inflammatory diseases has entered clinical practice. However, opposing effects of IL-6 have been observed in models of GN. Although IL-6 is proinflammatory in murine lupus nephritis, protective effects have been observed for IL-6 in the nephrotoxic nephritis (NTN) model of acute crescentic GN. In light of the potential dangers of IL-6-directed treatment, we studied the mechanisms underlying the contradictory findings in GN. IL-6 can signal through the membrane-bound IL-6R, which is expressed only on hepatocytes and certain leukocytes (classic), or through the soluble IL-6R, which binds the ubiquitously expressed gp130 (alternative). Preemptive treatment of mice with anti-IL-6R or anti-IL-6 worsened NTN, whereas selective blockade of alternative IL-6 signaling by the fusion protein sgp130Fc did not. FACS analysis of mouse spleen cells revealed proinflammatory macrophages express the highest levels of IL-6Rα, and in vitro treatment with IL-6 blocked macrophage proliferation. Furthermore, proinflammatory macrophages were expanded during inflammation in IL-6(-/-) mice. Late application of anti-IL-6 after establishment of adaptive nephritogenic immunity was sufficient to aggravate NTN within 2.5 days, a period when macrophages are active. Finally, NTN was aggravated in mice with macrophage-specific impairment of IL-6 classic signaling, coincident with enhanced macrophage proliferation and accumulation in the kidney. Our data thus reveal a novel mechanism in which IL-6-mediated dampening of macrophage activation protects tissues from overshooting immune responses. This finding has important implications for potential IL-6-directed therapies and supports the careful choice of recipient patients and timing.

title: Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation
abstract: Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14+ monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients.

title: Distinct evolution of TLR-mediated dendritic cell cytokine secretion in patients with limited and diffuse cutaneous systemic sclerosis
abstract: Systemic sclerosis (SSc) is an autoimmune disease and accumulating evidence suggests a role for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs).  To map TLR-mediated cytokine responses of DCs from patients with SSc.  45 patients with SSc were included. Patients were stratified as having diffuse cutaneous SSc (dSSc) or limited cutaneous SSc (lSSc) according to the extent of skin involvement, and further divided into those with late (>3 years) or early disease (<2 years). DCs were stimulated with ligands for TLR2, TLR3, TLR4, TLR7/8 or combinations. Plasma samples were collected from patients with SSc (n=167) and measured for interleukin 6 (IL-6), tumour necrosis factor α (TNFα), IL-12, IL-10 and interferon γ.  Stimulation of DC subsets from patients with early lSSc and dSSc with ligands for TLR2, TLR3 or TLR4 resulted in higher secretion of IL-6 and TNFα compared with those having late disease or healthy controls. Remarkably, the production of IL-12 was lower upon stimulation with TLR ligands in most patients with SSc, whereas the secretion of IL-10 was very high in patients with the dSSc phenotype, particularly in those having early dSSc. The combination of various TLR ligands led to reduced cytokine secretion in all patients with SSc. Circulating levels of these cytokines further underscored the presence of differences between various SSc phenotypes.  The altered TLR-mediated activation of DCs may be responsible for Th2 skewed T-cell activation in SSc that may be orchestrated by fibrogenic T-cell cytokines, such as IL-4 and IL-13. DC targeting could thus offer new avenues for therapeutic intervention.

title: Kinetics of Intracellular, Extracellular and Production of Pro-Inflammatory Cytokines in Lipopolysaccharide- Stimulated Human Peripheral Blood Mononuclear Cells
abstract: To determine the detailed time course of intracellular, extracellular and production of proinflammatory cytokines (TNF-α, IL-1β, IL-1α, IL-6 and IL-8) in normal human peripheral blood mononuclear cells (PBMC).  PBMC were isolated from human whole blood, stimulated by 0.1 μg/ml lipopolysaccharide (LPS) and incubated at 37oC, 5% CO2. Samples were harvested at different time intervals (4, 8, 12, 16, 20 and 24 h) after stimulation. ELISA was employed for the measurement of the extracellular and intracellular cytokine levels of the samples.  The release of TNF-α, IL-6 and IL-8 on LPS-stimulated PBMC were significantly higher with concentrations in the range of 3161 ± 162.5 to 4027 ± 361.5 pg/ml (p < 0.001), 3921.5 ± 879.3 to 11628.3 ± 2647.3 pg/ml (p ≤ 0.030), and 4122.0 ± 382.9 to 5898.6±115.8 pg/ml (p ≤ 0.049), respectively compared to intracellular levels that were very low (TNF-α, 23.5 ± 5.0 to 69.5 ± 13.8 pg/ml; IL-6, 22.5 ± 16.5 to 96.5 ± 9.6 pg/ml; and IL-8, 501.1 ± 221.0 to 1452.5 ± 415.7 pg/ml) and remained unchanged during 24 h. In contrast, both IL-1α and IL-1β were secreted gradually. Secretion and production of IL-6 was significantly higher at 8 h (9394.4 ± 846.3 pg/ml; p = 0.002) and at 20 h (11628.3 ± 2647.3 pg/ml; p = 0.014) than other cytokines.  The differences in the characteristic kinetics of cytokines may be caused by different mechanisms of secretion and function. IL-1, TNF-α and IL-8 play a role as proinflammatory cytokines, whereas IL-6 consecutively plays a dual role as pro-inflammatory cytokine and anti-inflammatory.

title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFalpha.
abstract: Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-alpha (TNFalpha) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFalpha and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. ). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFalpha, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFalpha and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFalpha delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.

title: Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules
abstract: Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin‐6 (IL‐6) in human dendritic cells. By comparing this to the total secreted IL‐6, we estimate that secretory vesicles contain about 0.5–3 IL‐6 molecules, but with a large spread among cells/donors. Moreover, IL‐6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL‐6 production. IL‐6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs.

title: Membrane-associated STAT3 and PY-STAT3 in the Cytoplasm*
abstract: Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that ∼5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

title: The STAT3 beacon: IL-6 recurrently activates STAT 3 from endosomal structures.
abstract: Endocytic trafficking plays an important role in signal transduction. Signal transducer and activator of transcription 3 (STAT3) and mitogen-activate protein kinase (MAPK) have both been localized to endosomal structures and are dependent upon endocytosis for downstream function. While the dependence of MAPK signaling upon endosomes has been well characterized, the involvement of endosomes in regulating STAT3 signaling has not been defined. Consequently, this study evaluated the role of endosomes in the initiation, modulation, amplification and persistence of interleukin-6(IL-6)-induced STAT3 signal transduction and transcription, and utilized IL-6-induced MAPK signaling as a comparator. Using pharmacologic treatment and temperature control of endocytic trafficking, pulse-chase treatments and in vitro kinase assays, STAT3 was found to interact with endosomes in a markedly different fashion than MAPK. STAT3 was activated by direct interaction with internal structures upstream of the late endosome following IL-6 exposure and persistent STAT3 signaling depended upon recurrent activation from endocytic structures. Further, STAT3 subcellular localization was not dependent upon endocytic trafficking. Instead, STAT3 transiently interacted with endosomes and relocated to the nucleus by an endosome-independent mechanism. Finally, endocytic trafficking played a central role in regulating STAT3 serine 727 phosphorylation through crosstalk with the MAPK signaling system. Together, these data reveal endosomes as central to the genesis, course and outcome of STAT3 signal transduction and transcription.

title: SOCS3 negatively regulates IL-6 signaling in vivo
abstract: Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-γ (IFN-γ). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-γ is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3-deficient livers mimics that induced by IFN-γ. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-γ regulation and imply that SOCS3 has a role in preventing IFN-γ-like responses in cells stimulated by IL-6.

title: IL-6 promotes acute and chronic inflammatory disease in the absence of SOCS3
abstract: The lack of expression of the suppressor of cytokine signalling‐3 (SOCS3) or inactivation of the negative regulatory capacity of SOCS3 has been well documented in rheumatoid arthritis, viral hepatitis and cancer. The specific qualitative and quantitative consequences of SOCS3 deficiency on interleukin‐6 (IL‐6)‐mediated pro‐ and anti‐inflammatory responses remain controversial in vitro and unknown in vivo. Mice with a conditional deletion of SOCS3 in hematopoietic cells develop lethal inflammatory disease during adult life and develop gross histopathological changes during experimental arthritis, typified by elevated IL‐6 levels. To clarify the nature of the IL‐6 responses in vivo, we generated mice deficient in SOCS3 (SOCS3−/Δvav) or both SOCS3 and IL‐6 (IL‐6−/−/SOCS3−/Δvav), and examined responses in models of acute and chronic inflammation. Acute responses to IL‐1β were lethal to SOCS3−/Δvav mice but not IL‐6−/−/SOCS3−/Δvav mice, indicating that IL‐6 was required for the lethal inflammation induced by IL‐1β. Administration of IL‐1β to SOCS3−/Δvav mice induced systemic apoptosis of lymphocytes in the thymus, spleen and lymph nodes that was dependent on the presence of IL‐6. IL‐6 deficiency prolonged survival of SOCS3−/Δvav mice and ameliorated spontaneous inflammatory disease developing during adult life. Infection of SOCS3−/Δvav mice with LCMV induced a lethal inflammatory response that was dependent on IL‐6, despite SOCS3−/Δvav mice controlling viral replication. We conclude that SOCS3 is required for survival during inflammatory responses and is a critical regulator of IL‐6 in vivo.

title: SOCS3 regulates the plasticity of gp130 signaling
abstract: Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway. SOCS3 is upregulated by several signals in macrophages and has been implicated as a regulator of various signaling pathways. Here we show that phosphorylation of STAT3 is prolonged in mouse Socs3-deficient macrophages after stimulation with interleukin-6 (IL-6) but not IL-10, indicating that SOCS3 specifically affects signaling mediated by IL-6 and gp130. IL-6 induces a wider transcriptional response in Socs3-deficient macrophages than in wild-type cells; this response is dominated by interferon (IFN)-regulated genes owing to an excess of STAT1 phosphorylation. Thus, SOCS3 functions to control the quality of the response to IL-6 and prevents the activation of an IFN-induced program of gene expression.

title: The soluble interleukin‐6 receptor is generated by shedding
abstract: The ligand‐binding subunit (gp80) of the human interleukin‐6 receptor (IL‐6R) was transiently expressed in COS‐7 cells. The metabolically labeled protein was shown to be quantitatively released from the membrane within 20 h. We identified the protein released from the transfected COS‐7 cells after purification to homogeneity and N‐terminal sequencing as a soluble form of the gp80/IL‐6R. Shedding of the gp80 protein was strongly induced by 4β‐phorbol‐12‐myristate‐13‐acetate, indicating that the process was regulated by protein kinase C (PKC). This was further corroborated by the finding that co‐transfection of a PKC expression plasmid led to enhanced shedding of the gp80 protein. Since shedding of gp80 could not be prevented by treatment of the cells with inhibitors of all known classes of proteases, a novel protease seems to be involved. As a control, an unrelated membrane protein (vesicular stomatitis virus glycoprotein) was transfected into COS‐7 cells and analyzed for shedding. Since the turnover of this protein was not mediated by shedding, we conclude that the release of gp80 from COS‐7 cells is a specific process. The shed gp80 protein specifically binds IL‐6, and this complex shows biological activity on human hepatoma cells. Human peripheral blood monocytes released a soluble form of the gp80 protein into the culture medium upon PMA treatment indicating that PKC‐regulated shedding is the physiological mechanism of generation of the soluble IL‐6R.

title: The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site.
abstract: Like many proteins with a single transmembrane domain the IL-6R exists in a membrane-associated and soluble form. The soluble IL-6R is generated by limited proteolysis of the membranous receptor. This process, also called shedding, is drastically enhanced by PMA, an activator of protein kinase C. The soluble receptor protein was purified to homogeneity from supernatants of COS-7 cells transfected with a cDNA coding for the transmembrane IL-6R. The COOH-terminus of the shed receptor protein was analyzed by carboxypeptidase treatment and subsequent amino acid analysis. The established cleavage site Gln357/Asp358 was extensively altered by point mutations and small deletions to define the structural requirements for cleavage. Although point mutations around the cleavage site reduced shedding of the IL-6R up to fivefold, deletions of 5 or 10 amino acids almost completely abolished shedding. Deletion of the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site close to the proteolytic cleavage site of the IL-6R is used. However this N-glycosylation does not affect the efficiency of the shedding process. Furthermore, we demonstrate for the first time that the human IL-6R is constitutively phosphorylated and that this phosphorylation can be stimulated by PMA but is not correlated with shedding of the receptor protein. The knowledge of the mechanism by which the soluble IL-6R is generated will help to identify the processing enzyme involved and to analyze its regulation.

title: Cellular Cholesterol Depletion Triggers Shedding of the Human Interleukin-6 Receptor by ADAM10 and ADAM17 (TACE)*
abstract: Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-β-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-α-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

title: ADAM17 controls IL‐6 signaling by cleavage of the murine IL‐6Rα from the cell surface of leukocytes during inflammatory responses
abstract: The cytokine IL‐6 is part of a regulatory signaling network that controls immune responses. IL‐6 binds either to the membrane‐bound IL‐6 receptor‐α (classic signaling) or to the soluble IL‐6 receptor‐α (trans‐signaling) to initiate signal transduction via gp130 activation. Because classic and trans‐signaling of IL‐6 fulfill different tasks during immune responses, controlled shedding of the membrane‐bound IL‐6 receptor‐α from the surface of immune cells can be considered a central regulator of IL‐6 function. The results from cell culture‐based experiments have implicated both a disintegrin and metalloprotease 10 and a disintegrin and metalloprotease 17 in IL‐6 receptor‐α shedding. However, the nature of the protease mediating IL‐6 receptor‐α release in vivo is not yet known. We used hypomorphic a disintegrin and metalloprotease 17 mice and conditional a disintegrin and metalloprotease 10 knock‐out mice to identify the natural protease of the murine IL‐6 receptor‐α. Circulating homeostatic soluble IL‐6 receptor‐α levels are not dependent on a disintegrin and metalloprotease 10 or 17 activity. However, during Listeria monocytogenes infection, IL‐6 receptor‐α cleavage by the α‐secretase a disintegrin and metalloprotease 17 is rapidly induced from the surface of different leukocyte populations. In contrast, CD4‐Cre‐driven a disintegrin and metalloprotease 10 deletion in T cells did not influence IL‐6 receptor‐α shedding from these cells after L. monocytogenes infection. A disintegrin and metalloprotease 17 was also required for IL‐6 receptor‐α ectodomain cleavage and release during endotoxemia. These results demonstrate a novel physiologic role for a disintegrin and metalloprotease 17 in regulating murine IL‐6 signals during inflammatory processes.

title: The shedding protease ADAM17: Physiology and pathophysiology.
abstract: The disintegrin metalloprotease ADAM17 has been a matter of intense studies aiming to unravel structure, function and regulation of protease expression, maturation and activity. In this review, we summarize data on the physiological role of ADAM17 in health and disease. Here we provide an overview of ADAM17 substrates, mouse models of ADAM17-deficiencies and discuss recent findings of ADAM17 function in the immune system and central nervous system as well as in cancer. Whereas ADAM17 function in EGF-R-, in Interleukin-6 (IL-6)- and in TNFα-biology has been shown to play a decisive role in regulation of the immune system as well as cancer development, the role of ADAM17 in the central nervous system and neurodegeneration still remains elusive. We show ADAM17 expression in human dopaminergic neurons derived from induced pluripotent stem cells and we discuss how this state-of-the-art technology can be further exploited to study the function of this important protease in the brain and other tissues. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

title: Soluble interleukin‐6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism
abstract: To detect transcripts encoding the interleukin‐6 receptor (IL‐6R) molecule lacking the transmembrane (TM) domain, in various cell lines and peripheral blood mononuclear cells (PBMC), we used the polymerase chain reaction (PCR) with primer pairs that flank the TM domain and which were selected to generate a 398‐bp fragment. We detected 398‐bp and 304‐bp DNA molecules in the PCR products of the U1, J22HL60, MT‐2, MT‐4, U937 and HL60 cell lines and of PBMC isolated from several individuals. The sequencing analysis of both DNA molecules showed that a 94‐bp region consisting of the TM domain of IL‐6R was deleted in the 304‐bp molecule. Moreover, we detected a soluble (s) IL‐6R protein of 45 kDa in culture supernatants of the MT‐2, MT‐4 and U937 cell lines by radioimmunoprecipitation using specific antibodies against sIL‐6R. Our results indicate that active deletion of the TM domain by alternative splicing of mRNA represents one mechanism for release of sIL‐6R into the culture supernatants of cells, or into serum or urine.

title: Activation of STAT3 by IL-6 and IL-10 in Primary Human Macrophages Is Differentially Modulated by Suppressor of Cytokine Signaling 3 1
abstract: On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-α blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines.

title: The macrophage response towards LPS and its control through the p38(MAPK)-STAT3 axis.
abstract: In macrophages detection of gram-negative bacteria particularly involves binding of the outer-wall component lipopolysaccharide (LPS) to its cognate receptor complex, comprising Toll like receptor 4 (TLR4), CD14 and MD2. LPS-induced formation of the LPS receptor complex elicits a signaling network, including intra-cellular signal-transduction directly activated by the TLR4 receptor complex as well as successional induction of indirect autocrine and paracrine signaling events. All these different pathways are integrated into the macrophage response towards an inflammatory stimulus by a highly complex cross-talk of the pathways engaged. This also includes a tight control by several intra- and inter-cellular feedback loops warranting an inflammatory response sufficient to battle invading pathogens and to avoid non-essential tissue damage caused by an overwhelming inflammatory response. Several evidences indicate that the reciprocal cross-talk between the p38(MAPK)-pathway and signal transducer and activator of transcription (STAT)3-mediated signal-transduction forms a critical axis successively activated by LPS. The balanced activation of this axis is essential for both induction and propagation of the inflammatory macrophage response as well as for the control of the resolution phase, which is largely driven by IL-10 and sustained STAT3 activation. In this context regulation of suppressor of cytokine signaling (SOCS)3 expression and the recently described divergent regulatory roles of the two p38(MAPK)-activated protein kinases MK2 and MK3 for the regulation of LPS-induced NF-κB- and IRF3-mediated signal-transduction and gene expression, which includes the regulation of IFNβ, IL-10 and DUSP1, appears to play an important role.

title: Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF.
abstract: Interleukin-6 (IL-6) shares several biologic properties with IL-1, including hematopoietin-1 activity and stimulation of T cells. Because many of their biologic activities overlap, we developed and used a specific radioimmunoassay (RIA) for IL-6 to compare production of this cytokine on a molar basis with that of IL-1 alpha, IL-1 beta, and tumor necrosis factor (TNF)alpha. The RIA correlated well with the hybridoma bioassay for IL-6 (r = .87, P less than .001). Freshly isolated human peripheral blood mononuclear cells (PBMC) cultured in the absence of stimuli did not produce IL-6 in most cases. Kinetics of secretion and cell-association of IL-6 were studied. In contrast to IL-1 alpha but similar to TNF, IL-6 was almost entirely secreted into the extracellular fluid. Incubation with different stimuli (lipopolysaccharide [LPS], phytohemagglutinin [PHA], Staphylococcus epidermidis, or IL-1 alpha) resulted in production of IL-6. However, on a molar basis PBMC produced approximately two to three times less IL-6 than IL-1 alpha, IL-1 beta, or TNF, regardless of the stimulus. The amount of IL-6 produced from PBMC was consistent when measured in the same subjects six time during a 12-week period. In a cohort of 38 donors, the coefficient of variation for IL-6 production was .32, compared with .92 for IL-1 beta and .96 for TNF. Comparing cytokine production by PBMC, there was a significant correlation between IL-6 and IL-1 beta (r = .72) and between IL-6 and TNF (r = .66). IL-6 did not stimulate IL-1 beta or TNF production, but suppressed IL-1 beta and TNF production induced by LPS or PHA by 30% (P less than .01). This suppression of IL-1 beta and TNF by IL-6 appears to be on the level of transcription.

title: Oncogenic deletion mutants of gp130 signal from intracellular compartments
abstract: Interleukin 6 (IL-6) and, hence, activation of the IL-6 receptor signalling subunit glycoprotein 130 (gp130; also known as interleukin-6 receptor subunit &bgr;, 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 ligand-independent 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 (&Dgr;YY) is retained in the ER quality control system because of its association with the chaperone calnexin. Furthermore, we can show that gp130 &Dgr;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 &Dgr;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.

title: ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by gamma-secretase.
abstract: Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using gamma-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the gamma-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After gamma-secretase cleavage IL6R cell surface expression was lost and gamma-secretase cleavage product(s) of the IL6R were endocytosed. No GFP-fluorescence of a gamma-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded.

title: The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomes and late endosomes.
abstract: Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

title: A Role for the Phagosome in Cytokine Secretion
abstract: Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor α (TNFα) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFα and expands the membrane for phagocytosis.

title: Constitutive internalization and association with adaptor protein‐2 of the interleukin‐6 signal transducer gp130
abstract: The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin‐6 (IL‐6)‐type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal‐independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL‐6/IL‐6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL‐6/IL‐6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP‐2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

title: Cytokine‐Induced Slowing of STAT3 Nuclear Import; Faster Basal Trafficking of the STAT3β Isoform
abstract: The STAT3 signal transducer and activator of transcription is a key mediator of gene transcription in response to cytokines such as oncostatin M (OSM). We performed direct live cell imaging of GFP‐tagged STAT3 proteins for the first time, showing transient relocalization of STAT3α to the nucleus following OSM exposure, in contrast to sustained nuclear relocalization of the shorter STAT3β spliceform. To explore this further, we applied fluorescence recovery after photobleaching (FRAP) to determine the nuclear import kinetics of STAT3α and β, as well as of a C‐terminal truncation derivative STAT3ΔC comprising only the sequence shared by the spliceforms, in the absence or presence of OSM. The rates of basal nuclear import for STAT3β and STAT3ΔC were significantly faster than those for STAT3α. Strikingly, OSM slowed the import rates of all the three STAT3 proteins, whereas the import rates of GFP alone or a classical importin‐mediated cargo were unaffected, with analysis of Y705F mutant derivatives for all the three STAT3 constructs, or of a S727A mutant within the unique C‐terminus of STAT3α, reinforcing the contribution of specific phosphorylation to the cytokine‐stimulated changes. The results introduce a new paradigm where cytokine treatment prolongs nuclear retention simultaneous with decreasing rather than increasing the rate of nuclear import.

title: A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis
abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

title: The Epsins Define a Family of Proteins That Interact with Components of the Clathrin Coat and Contain a New Protein Module*
abstract: Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH2-terminal region, which represents a module (epsin NH2 terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

title: Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods.
abstract: In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.

title: SNAP‐23 and VAMP‐3 contribute to the release of IL‐6 and TNFα from a human synovial sarcoma cell line
abstract: Fibroblast‐like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE‐dependent. Here, the complement of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin‐6 (IL‐6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome‐associated protein of molecular mass 23 kDa (SNAP‐23) or the vesicle‐associated membrane protein 3 (VAMP‐3). Wild‐type SW982 cells expressed SNAP‐23, VAMP‐3, syntaxin isoforms 2–4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca2+‐dependent secretion of IL‐6 and TNFα when stimulated by interleukin‐1β (IL‐1β) or in combination with K+ depolarization. Specific knockdown of SNAP‐23 or VAMP‐3 decreased the exocytosis of IL‐6 and TNFα; the reduced expression of SNAP‐23 caused accumulation of SV2 in the peri‐nuclear area. A monoclonal antibody specific for VAMP‐3 precipitated SNAP‐23 and syntaxin‐2 (and syntaxin‐3 to a lesser extent). The formation of SDS‐resistant complexes by SNAP‐23 and VAMP‐3 was reduced upon knockdown of SNAP‐23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP‐23 and VAMP‐3 participate in IL‐1β‐induced Ca2+‐dependent release of IL‐6 and TNFα from SW982 cells.

title: Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
abstract: SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution.

title: Endosomes: A legitimate platform for the signaling train
abstract: Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.

title: Endocytosis and signaling.
abstract: Many cellular signaling processes are governed by endocytosis through the internalization of plasma membrane receptors. This receptor clearance defines the quality with which a cell can react to extracellular stimuli. However, growing evidence indicates that endocytosis also enables the formation of endosome-specific signal transduction complexes. Their activity is controlled by the balanced trafficking of receptors and signaling molecules through the endocytic compartments. These are commonly divided into early endosomes, recycling endosomes, and late endosomes. Recent progress has been made in the understanding of the biogenesis of these organelles, highlighting their dynamic interconversion, maturation and also the generation of heterogenous subdomains on their surface. These multifunctional compartments represent the physical basis for the assembly and turnover of signaling complexes, which in turn themselves can define specialized endosomal-signaling platforms.

title: Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.
abstract: Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

title: Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway.
abstract: The family of cytokines signalling through the common receptor subunit gp130 comprises interleukin (IL)-6, IL-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. These so-called IL-6-type cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation. The current knowledge on the signal-transduction mechanisms of these cytokines from the plasma membrane to the nucleus is reviewed. In particular, we focus on the assembly of receptor complexes after ligand binding, the activation of receptor-associated kinases of the Janus family, and the recruitment and phosphorylation of transcription factors of the STAT family, which dimerize, translocate to the nucleus, and bind to enhancer elements of respective target genes leading to transcriptional activation. The important players in the signalling pathway, namely the cytokines and the receptor components, the Janus kinases Jak1, Jak2 and Tyk2, the signal transducers and activators of transcription STAT1 and STAT3 and the tyrosine phosphatase SHP2 [SH2 (Src homology 2) domain-containing tyrosine phosphatase] are introduced and their structural/functional properties are discussed. Furthermore, we review various mechanisms involved in the termination of the IL-6-type cytokine signalling, namely the action of tyrosine phosphatases, proteasome, Jak kinase inhibitors SOCS (suppressor of cytokine signalling), protein inhibitors of activated STATs (PIAS), and internalization of the cytokine receptors via gp130. Although all IL-6-type cytokines signal through the gp130/Jak/STAT pathway, the comparison of their physiological properties shows that they elicit not only similar, but also distinct, biological responses. This is reflected in the different phenotypes of IL-6-type-cytokine knock-out animals.

title: Oncostatin M regulates the synthesis and turnover of gp130, leukemia inhibitory factor receptor alpha, and oncostatin M receptor beta by distinct mechanisms.
abstract: The cytokine receptor subunits gp130, leukemia inhibitory factor receptor alpha (LIFRalpha), and oncostatin M receptor beta (OSMRbeta) transduce OSM signals that regulate gene expression and cell proliferation. After ligand binding and activation of the Janus protein-tyrosine kinase/STAT and mitogen-activated protein kinase signal transduction pathways, negative feedback processes are recruited. These processes attenuate receptor action by suppression of cytokine signaling and by down-regulation of receptor protein expression. This study demonstrates that in human fibroblasts or epithelial cells, OSM first decreases the level of gp130, LIFRalpha, and OSMRbeta by ligand-induced receptor degradation and then increases the level of the receptors by enhanced synthesis. The transcriptional induction of gp130 gene by OSM involves STAT3. Various cell lines expressing receptor subunits to the different interleukin-6 class cytokines revealed that only LIFRalpha degradation is promoted by activated ERK and that degradation of gp130, OSMRbeta, and a fraction of LIFRalpha involves mechanisms that are separate from signal transduction. These mechanisms include ligand-mediated dimerization, internalization, and endosomal/lysosomal degradation. Proteosomal degradation appears to involve a fraction of receptor subunit proteins that are ubiquitinated independently of ligand binding.

title: IL-6 Production by Dendritic Cells Is Dispensable for CD8+ Memory T-Cell Generation
abstract: Following activation, naïve CD8+ T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8+ memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8+ effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8+ T-cell memory generation.

title: Fate of interleukin-6 in the rat. Involvement of skin in its catabolism.
abstract: Iodinated recombinant human interleukin-6 (125I-rhIL-6) was intravenously injected into rats and its fate was studied during 24 h. Between 10-20 min after a single-dose injection, 125I-rhIL-6 accumulated in liver as previously reported [Castell et al. (1988) ]. After 1 h, the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I-rhIL-6 5-8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I-albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiographic analysis of skin sections 5 h after 125I-rhIL-6 injection showed radioactivity in the interstitium. When the experiments were carried out with [35S]rhIL-6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection. In vitro experiments showed that 125I-rhIL-6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL-6.

title: Cellular targets and receptors for interleukin‐6 I. In vivo and in vitro uptake of IL‐6 in liver and hepatocytes
abstract: Interleukin‐6 (IL‐6) is a potent stimulator of the hepatic synthesis of acute‐phase proteins.  IL‐6 disappeared from the blood of rats with an overall half‐time of about 1.5 min; 41% of the injected tracer dose was recovered in the liver by 15 min. The clearance was biphasic. The simultaneous injection of tracer and an excess of unlabelled IL‐6 eliminated the initial rapid phase, and reduced the hepatic uptake to 14%. Light microscopic autoradio‐graphy showed 5% of the grains over non‐hepatocytes, and 80% over hepatocytes, accumulating in areas around the bile canaliculi. Thereafter, degradation products accumulated in the bile. At 4d̀C, isolated rat hepatocytes bound IL‐6 with an apparent Kd of 39 pmol 1‐1 to a uniform class of 4500 receptors per cell with an apparent molar mass of 115–120 kg mol‐1. The HepG2 human hepatocellular cell line bound IL‐6 with an apparent Kd of 21 pmol 1‐1 to a uniform class of 1200 receptors per cell with an apparent molar mass of 155–160 kg mol‐1. At 37d̀C, both cell types endocytosed the bound ligand slowly, and degradation products appeared in the medium after a relatively long lag period (40 min in hepatocytes and 1 h in HepG2 cells).,

title: Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion.
abstract: Although leukocytes of the innate immune system, including eosinophils, contain within their granules preformed stores of cytokines available for selective and rapid release, little is known about the mechanisms governing the mobilization and secretion of these cytokines. Here we show that a cytokine receptor, the IL-4 receptor alpha chain, mediates eotaxin-stimulated mobilization of preformed IL-4 from eosinophil granules into secretory vesicles. Eosinophils contain substantial intracellular quantities of several granule- and vesicle-associated cytokine receptors, including IL-4, IL-6, and IL-13 receptors as well as CCR3. Both IL-4 and IL-4 receptor alpha chain colocalized in eosinophil granules; and after eotaxin-stimulation, IL-4 receptor alpha chain, bearing bound IL-4, was mobilized into secretory vesicles. These findings indicate that intracellular cytokine receptors within secretory vesicles transport their cognate cytokines requisite for the secretion of cytokines preformed in innate immune leukocytes.

title: Cytokine Secretion in Macrophages: SNAREs, Rabs, and Membrane Trafficking
abstract: Macrophages have the capacity to rapidly secrete a wide range of inflammatory mediators that influence the development and extent of an inflammatory response. Newly synthesized and/or preformed stored cytokines and other inflammatory mediators are released upon stimulation, the timing, and volume of which is highly regulated. To finely tune this process, secretion is regulated at many levels; at the level of transcription and translation and post-translationally at the endoplasmic reticulum (ER), Golgi, and at or near the cell surface. Here, we discuss recent advances in deciphering these cytokine pathways in macrophages, focusing on recent discoveries regarding the cellular machinery and mechanisms implicated in the synthesis, trafficking, and secretion of cytokines. The specific roles of trafficking machinery including chaperones, GTPases, cytoskeletal proteins, and SNARE membrane fusion proteins will be discussed.

title: Dynamic imaging of the recycling endosomal network in macrophages.
abstract: Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.

title: Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130
abstract: Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R), the cDNA of which has already been cloned. We report here that an 80 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37 degrees C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0 degree C. Human IL-6-R can associate with a murine gp130 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.

title: Soluble human interleukin-6 receptor. Expression in insect cells, purification and characterization.
abstract: The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l.mol-1.cm-1 was calculated from the ultraviolet spectrum of the soluble IL-6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human 125I-labeled IL-6 with low affinity (Kd = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble 125I-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.

title: Both tumor necrosis factor receptors, TNFR60 and TNFR80, are involved in signaling endothelial tissue factor expression by juxtacrine tumor necrosis factor alpha.
abstract: We have investigated the role of the two distinct tumor necrosis factor (TNF) receptors (TNFR60 and TNFR80) in endothelial cell activation employing an in vitro model of tumor necrosis factor alpha (TNF-alpha)-dependent tissue factor production of human umbilical vein endothelial cells (HUVECs). In this model, tissue factor is produced either on addition of exogeneous TNF-alpha, or by induction of endogenous TNF-alpha via adhesion molecule-linked signal pathways. Under both conditions, tissue factor expression could be partially blocked by antagonistic antibodies against either TNFR60 or TNFR80 and was fully inhibited by simultaneous application of both antibodies. Selective inhibitors of either TNFR60 or TNFR80-induced signal pathways inhibited tissue factor expression, and selective triggering of either of the two TNF receptors by agonistic antibodies induced this response in HUVECs. Furthermore, a coculture system of HUVECs and Chinese hamster ovary transfectants expressing a noncleavable, exclusively membrane-bound form of TNF-alpha resulted in a potent activation of HUVECs with synergistic action of both TNF receptors. Together, these data underline the importance of juxtacrine pathways in endothelial cell activation of procoagulant functions and show that membrane TNF-alpha and both TNFR types play a critical role.

title: The interleukin‐15/interleukin‐15 receptor system as a model for juxtacrine and reverse signaling
abstract: Interleukin‐15 (IL‐15) is a pleiotropic cytokine of the 4 α‐helix bundle family, which binds to a receptor complex that displays common elements with the IL‐2 receptor and a unique high‐affinity α chain. This review focuses on juxtacrine and reverse signaling levels in the IL‐15/IL‐15R system. Specifically, we discuss how agonistic stimulation of membrane‐bound IL‐15 induces phosphorylation of members of the MAP kinase family and of focal adhesion kinase (FAK), thereby upregulating processes including cytokine secretion, cell adhesion and migration. In addition, we explore IL‐15 trans‐presentation and intracellular signaling, and define promising molecular targets for future pharmacological intervention in infectious diseases and immunological disorders. These frontiers in IL‐15/IL‐15Rα research serve as highly instructive examples for key concepts, unsolved problems and therapeutic opportunities in juxtacrine and reverse signaling in general. BioEssays 28: 362–377, 2006.

title: IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation
abstract: Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15Rα, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15Rα support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15Rα proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15Rα are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15Rα is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15Rα. We also find that soluble IL-15–IL-15Rα complexes are induced during inflammation, but membrane-bound IL-15–IL-15Rα complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15Rα specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine–receptor biosynthetic pathway in which IL-15Rα serves as a chaperone for IL-15, after which membrane-bound IL-15Rα–IL-15 complexes activate NK cells via direct cell–cell contact.

title: Proteolytic Cleavage Governs Interleukin-11 Trans-signaling.
abstract: Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11.

title: A metalloproteinase disintegrin that releases tumour-necrosis factor-α from cells
abstract: Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins1. Modification of the cell surface in this way can alter the cell's responsiveness to its environment2 and release potent soluble regulatory factors3. The release of soluble tumour-necrosis factor-α (TNF-α) from its membrane-bound precursor4,5 is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important6,7. The inhibition of TNF-α release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved3,8,9. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-α. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-α production. This enzyme (called the TNF-α-converting enzyme, or TACE) is a new member of the family of mammalian adama-lysins (or ADAMs)10, for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-α release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity*
abstract: We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor α (IL-15Rα) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Rα, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Rα expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Rα also increased the number of CD44high memory cells with effector phenotype (CD44highCD62L-). Thus, mutual stabilization of IL-15 and IL-15Rα leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor α either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Rα in the same cell and suggest that the IL-15Rα is part of the active IL-15 cytokine rather than part of the receptor.

title: How a Cytokine Is Chaperoned through the Secretory Pathway by Complexing with Its Own Receptor: Lessons from Interleukin-15 (IL-15)/IL-15 Receptor α
abstract: While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.

title: Mechanisms of cytokine secretion: a portfolio of distinct pathways allows flexibility in cytokine activity.
abstract: Since cytokines are potent immunoregulators that can determine the fate of an immune response, their expression is tightly regulated at the transcriptional level. Recent research, however, has also revealed complex post-translational mechanisms through which cytokine secretion, and thereby cytokine activity, is regulated. Here, we review the progress in our understanding of the portfolio of pathways that regulate cytokine intracellular storage, transport, and release. Like other secreted proteins, cytokines utilize canonical and non-canonical secretory pathways for extracellular release. Illustrated by IL-1β, IL-2, TNF-α, IL-12 and IL-15 secretion as selected examples, we discuss common and alternative cytokine secretion pathways and relate them to the consequences these distinct pathways have for cytokine function, mode of action and stability.

title: Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum.
abstract: IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.

title: Cytokines in Sepsis: Potent Immunoregulators and Potential Therapeutic Targets—An Updated View
abstract: Sepsis and septic shock are among the leading causes of death in intensive care units worldwide. Numerous studies on their pathophysiology have revealed an imbalance in the inflammatory network leading to tissue damage, organ failure, and ultimately, death. Cytokines are important pleiotropic regulators of the immune response, which have a crucial role in the complex pathophysiology underlying sepsis. They have both pro- and anti-inflammatory functions and are capable of coordinating effective defense mechanisms against invading pathogens. On the other hand, cytokines may dysregulate the immune response and promote tissue-damaging inflammation. In this review, we address the current knowledge of the actions of pro- and anti-inflammatory cytokines in sepsis pathophysiology as well as how these cytokines and other important immunomodulating agents may be therapeutically targeted to improve the clinical outcome of sepsis.

title: Increased plasma levels of interleukin-6 in sepsis.
abstract: Interleukin-6 (IL-6) is likely to be an important mediator of the inflammatory response. We measured levels of this cytokine in plasma samples from 37 patients with sepsis or septic shock obtained at the time of admission to the intensive care unit and related these levels to hemodynamic and biochemical parameters as well as to clinical outcome. In 32 of the 37 patients, increased levels of IL-6 were found, occasionally up to 7,500 times the normal level. The highest IL-6 levels were encountered in patients who suffered from septic shock (P value of the difference between patients with and without shock less than .0001). In addition, IL-6 significantly correlated with plasma lactate (P less than .0001), heart rate (P = .05) and, inversely, with mean arterial pressure (P = .01) and platelet counts (P = .0002). Significant correlations of IL-6 with the anaphylatoxins C3a (P = .0001) and C4a (P = .0002) and with the main inhibitor of the classical pathway of complement, C1-inhibitor (inverse correlation, P = .05), were also observed. IL-6 on admission appeared to be of prognostic significance: levels were higher in septic patients who subsequently died than in those who survived (P = .0003), in particular when only patients with septic shock were considered (P less than .0001). All nine septic patients with levels of less than 40 U/mL on admission survived, whereas 89% of the nine patients with levels exceeding 7,500 U/mL died. These data provide evidence for a role of IL-6 in the pathophysiology of septic shock. Further studies are needed to reveal whether IL-6 in sepsis is directly involved in mediating lethal complications or whether it is to be considered as an "alarm hormone" that reflects endothelial cell injury probably mediated by the anaphylatoxines.

title: Relationship between interleukin-6 plasma concentration in patients with sepsis, monocyte phenotype, monocyte phagocytic properties, and cytokine production.
abstract: Monocyte phenotype, their phagocytic capacity as well as the cytokine production from 10 patients with sepsis with low interleukin-6 (IL-6) serum concentrations (<1000 pg/mL) and 8 patients with sepsis with high IL-6 (> or = 1000 pg/mL) plasma concentrations were investigated within 24 hours of fulfilling the criteria for sepsis. Monocytes from patients with high IL-6 levels had higher levels of human leukocyte antigen (HLA)-DR, HLA-ABC, CD64, and CD71, and the production of tumor necrosis factor-alpha (TNF-alpha) and IL-8, as well as the capacity of monocytes to phagocytose, was significantly elevated. Of 8 patients with high levels of plasma IL-6, 4 patients died. In contrast, all 10 patients with low plasma IL-6 concentrations survived until day 28. Patients who died had constant high IL-6 concentrations during the first 3 days, whereas IL-6 levels in patients who survived decreased by 88%. Our data indicate that IL-6 levels are a better prognostic parameter in the early phase of sepsis than the monocyte HLA-DR expression.

title: IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses.
abstract: IL-6 is induced often together with the proinflammatory cytokines TNFalpha and IL-1 in many alarm conditions, and circulating IL-6 plays an important role in the induction of acute phase reactions. However, whether this endogenous IL-6 plays any additional pro- or antiinflammatory roles in local or systemic responses remains unclear. In this study, the role of IL-6 in acute inflammatory responses was investigated in animal models of endotoxic lung or endotoxemia by using IL-6+/+ and IL-6-/- mice. Aerosol exposure of endotoxin induced increased IL-6 and proinflammatory cytokines TNFalpha and MIP-2 and a neutrophilic response in the lung of IL-6+/+ mice. However, the levels of TNFalpha and MIP-2 and neutrophilia were significantly higher in the lung of IL-6-/- mice. The rate of neutrophil apoptosis in these mice was similar to that in IL-6+/+ mice. A low constitutive level of antiinflammatory cytokine IL-10 was not enhanced by endotoxin and remained similar in the lung in both IL-6+/+ and IL-6-/- mice. Systemically, intraperitoneal delivery of endotoxin resulted in much more pronounced circulating levels of TNFalpha, MIP-2, GM-CSF, and IFNgamma in IL-6-/- mice than in IL-6+/+ mice, and administration of recombinant IL-6 to IL-6-/- mice abolished these differences. In contrast, circulating IL-10 levels were induced to a similar degree in both IL-6+/+ and IL-6-/- mice. Thus, our studies reveal that endogenous IL-6 plays a crucial antiinflammatory role in both local and systemic acute inflammatory responses by controlling the level of proinflammatory, but not antiinflammatory, cytokines, and that these antiinflammatory activities by IL-6 cannot be compensated for by IL-10 or other IL-6 family members.

title: Role of Interleukin-6 in Mortality from and Physiologic Response to Sepsis
abstract: Previous studies have suggested that interleukin-6 (IL-6) serves as both a marker and a mediator for the severity of sepsis. We tested whether interleukin 6 knockout (IL-6KO) mice were more susceptible to sepsis mortality induced by cecal ligation and puncture. IL-6KO and wild-type (WT) mice were subjected to increasing degrees of sepsis severity. Physiologic support was given with fluids and appropriate antibiotics. Plasma IL-6 levels were determined 6 h after the onset of sepsis, and a complete hematologic profile was performed on day 2. As expected, increasing sepsis severity resulted in greater and more rapid mortality. However, the mortality was nearly identical in the IL-6KO and WT mice. All WT septic mice had high plasma levels of IL-6 6 h after the onset of sepsis, while IL-6KO were near or below the lower limit of detection. Among the WT mice, mortality was significantly higher in mice with plasma IL-6 >3,000 pg/ml. Both IL-6KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 h. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 h of severe sepsis routinely observed in the WT mice. These data demonstrate that IL-6 serves as a marker of disease severity in sepsis and does modulate some physiologic responses, but complete lack of IL-6 does not does not alter mortality due to sepsis.

title: Role of interleukin-6 in a non-septic shock model induced by zymosan.
abstract: In the present study, we used IL-6 knock-out mice (IL-6KO) to evaluate a possible role of IL-6 in the pathogenesis of non-septic shock induced by peritoneal injection of zymosan. A severe inflammatory response characterized by peritoneal exudation, high peritoneal levels of nitrate/nitrite, and leukocyte infiltration into peritoneal exudate was induced by zymosan administration in wild-type control (WT) mice. This inflammatory process coincided with the damage to the lung and small intestine, as assessed by histological examination. Lung, small intestine and liver myeloperoxidase (MPO) activity, indicative of neutrophil infiltration and lipid peroxidation, were significantly increased in zymosan-treated WT mice. Peritoneal administration of zymosan in the WT mice also induced a significant increase in the plasma levels of nitrite/nitrate and in the levels of peroxynitrite, 18 hours after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the lung of zymosan-treated WT mice. Zymosan-treated IL-6KO showed significantly decreased mortality and inhibition of the development of peritonitis. In addition, IL-6KO mice showed significant protection from the development of organ failure, since tissue injury and MPO was reduced in the lung, small intestine and liver. Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of IL-6KO mice subjected to zymosan-induced non-septic shock. In vivo treatment with anti-IL-6 (5,000 ng/day per mouse, 24 and 1 hour before zymosan administration) significantly reduced the inflammatory process. Taken together, the present study clearly demonstrates that IL-6 exerts a role in zymosan-induced non-septic shock.

title: Role of interleukin 6 in myocardial dysfunction of meningococcal septic shock
abstract: Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock.



We combined gene-expression profiling with protein and cellular methods to identify a serum factor causing cardiac dysfunction in meningococcal septic shock. We identified genes that were significantly upregulated in blood after exposure to meningococci. We then selected for further analysis those genes whose protein products had properties of a myocardial depressant factor--specifically a 12-25 kDa heat-stable protein that is released into serum shortly after onset of meningococcal infection.



We identified 174 significantly upregulated genes in meningococcus-infected blood: six encoded proteins that were of the predicted size and had characteristics of a myocardial depressant factor. Of these, interleukin 6 caused significant myocardial depression in vitro. Removal of interleukin 6 from serum samples of patients with meningococcaemia and from supernatants of inflammatory cells stimulated by meningococci in vitro abolished the negative inotropic activity. Furthermore, concentrations in serum of interleukin 6 strongly predicted degree of myocardial dysfunction and severity of disease in children with meningococcal septic shock.



Interleukin 6 is a mediator of myocardial depression in meningococcal disease. This cytokine and its downstream mediators could be a target for future treatment strategies.