Tratamiento con tocilizumab en amiloidosis AA: informe de casos y revisión de la literatura / Tocilizumab treatment in aa amyloidosis: case report and literature review

Introducción: la amiloidosis AA puede ser una complicación de ciertos trastornos inflamatorios crónicos, aunque entre el 21% y 50% puede ser idiopática. No existe un tratamiento específico. El tocilizumab, dirigido contra el receptor de IL-6 y orientado a disminuir la producción de SAA, podría ser eficaz. Métodos: en este estudio informamos datos de 6 pacientes con amiloidosis AA tratados con tocilizumab monoterapia subcutáneo en el período 2011-2018. Los criterios de valoración principales fueron la mejora clínica y bioquímica de los órganos afectados y los parámetros bioquímicos marcadores de inflamación. Resultados: el riñón estaba afectado en todos los pacientes, manifestándose con caída del filtrado glomerular y síndrome nefrótico. La hemorragia digestiva se presentó en un paciente y otro tenía afectación pulmonar en la biopsia. Luego del posterior al tratamiento, todos mejoraron el hematocrito, la albúmina sérica y el índice de masa corporal. El SAA disminuyó en 5 pacientes. Un paciente mejoró su función renal, mientras 4 se mantuvieron estables. Tres pacientes disminuyeron los valores de proteinuria. Conclusión: el tratamiento con tocilizumab podría ser eficaz en el tratamiento de los pacientes con amiloidosis AA. (AU)

Introduction: AA amyloidosis can be a complication of certain chronic inflammatory disorders, although between 21% and 50% can be idiopathic. There is no specific treatment. Tocilizumab, directed against the IL-6 receptor and aimed at decreasing SAA production, could be effective. Methods: in this study, we report data from 6 patients with AA amyloidosis treated with subcutaneous tocilizumab monotherapy between the period 2011-2018. The main endpoints were the clinical and biochemical improvement of the affected organs and the biochemical parameters markers of inflammation. Results: the kidney was affected in all patients, manifesting with a fall in glomerular filtration rate and nephrotic syndrome. Gastrointestinal bleeding occurred in one patient and another had lung involvement on biopsy. After treatment, all improved hematocrit, serum albumin, and body mass index. SAA decreased in 5 patients. One patient improved his kidney function, while 4 remained stable. Three patients decreased proteinuria values. Conclusion: treatment with tocilizumab could be effective in the treatment of patients with AA amyloidosis. (AU)

The expression profile of genes encoding tumor necrosis factor-alpha, interleukin-6 and their receptor in benign adrenal tumors.

In the process of neoplasia, during which benign adrenal tumors are formed, stimulators of new blood vessel growth as well as growth of tumor cells are cytokines, especially tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). We analyzed the expression profile of genes coding: TNF-α, tumor necrosis factor receptor 1 (TNF-R1), TNF-R2, IL-6, interleukin 6 receptor (IL-6R) in sections of adrenocortical tumor tissue, rated on the Weiss point scale, in patients with clinically diagnosed Conn's and Cushing's syndrome, and the usefulness of determining the examined genes as markers differentiating individual clinical units. There was no correlation between the expression of the examined genes and clinical parameters such as age, BMI or blood pressure, both in the entire study group and in individual subgroups. Elevated expression of the genes coding TNF-α, TNF-R2 and IL-6R was observed, whereas genes encoding TNF-R1 and IL-6 showed relatively low expression. The highest statistically significant differences in the expression of the examined genes were observed between IL-6 and IL-6R. High positive correlation was found in the subgroup of patients with Conn's clinical syndrome, between genes encoding both types of receptors for TNF-α, IL-6 and TNF-R2, TNF-α and IL-6 receptor, and between TNF-R2 and IL6-R receptors, which may suggest the mutual influence of these cytokines and their receptors on their own expression.

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1in juvenile diabetes.

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.

AT-rich-interactive domain-containing protein 5A functions as a negative regulator of retinoic acid receptor-related orphan nuclear receptor γt-induced Th17 cell differentiation.

OBJECTIVE: The proinflammatory cytokines tumor necrosis factor α and interleukin-6 (IL-6) and the Th17 cell cytokine IL-17A are implicated in the pathogenesis of rheumatoid arthritis (RA), and the blockade of these cytokines by biologic agents provides clinical benefits for RA patients. We undertook this study to clarify the mechanisms underlying the efficacy of IL-6 blockade in RA and to find a novel target for treatment of RA. METHODS: We examined gene expression profiles of CD4+ T cells by DNA microarray analysis before and after treatment with an anti-IL-6 receptor antibody, tocilizumab (TCZ), in RA patients who exhibited good clinical responses to the treatment. Using murine CD4+ T cells, we then examined the roles of a newly identified molecule whose expression was significantly reduced in CD4+ T cells by TCZ therapy. We also examined the effect of the forced expression of the molecule on retinoic acid receptor-related orphan nuclear receptor γt (RORγt)-induced IL-17A production in CD4+ T cells and on RORγt-induced IL-17A promoter activation. RESULTS: We identified AT-rich-interactive domain- containing protein 5A (ARID-5A) as a new molecule down-regulated by IL-6 blockade in the form of TCZ therapy. IL-6 induced the expression of ARID-5A in CD4+ T cells during Th17 cell differentiation by a STAT-3-dependent mechanism, whereas IL-6-induced ARID-5A expression was not affected by the absence of RORγt, a lineage-specifying transcription factor of Th17 cells. Furthermore, ARID-5A physically associated with RORγt through its N-terminal region and inhibited RORγt-induced Th17 cell differentiation. CONCLUSION: ARID-5A is a lineage-specific attenuator of Th17 cell differentiation and may be involved in the pathogenesis of RA.

Interleukin-6 in combination with the interleukin-6 receptor stimulates glucose uptake in resting human skeletal muscle independently of insulin action.

AIM: To examine if the physiological concentrations of both interleukin-6 (IL-6), in combination with IL-6 receptor (IL-6R), are able to stimulate glucose uptake in human skeletal muscle and to identify the associated signalling pathways. METHODS: Skeletal muscle tissue (~60 mg) obtained from healthy female volunteers via muscle biopsy was subjected to incubation in the absence or presence of insulin (60 µU/ml), recombinant human IL-6 (rhIL-6) (4 ng/ml) or a combination of rhIL-6 (4 ng/ml) and rhIL-6R (100 ng/ml) for 30 min, with glucose transport measured for each incubation. Western blot analysis was conducted on key signalling proteins, protein kinase B (PKB/Akt), adenosine monophosphate kinase (AMPK) and mammalian target of rapamycin (mTOR) to gain an early insight into any differing transport mechanisms. RESULTS: Human skeletal muscle exhibited increased glucose uptake with insulin (1.85-fold; p < 0.05) and stimulated phosphorylation of PKB/Akt and AMPK (0.98 ± 0.23 and 1.49 ± 0.13, respectively, phosphorylated: total; p < 0.05). IL-6/IL-6R increased phosphorylation of mTOR (fourfold, p < 0.05) compared to insulin, IL-6 alone and basal control. IL-6 did not stimulate glucose uptake but combined with IL-6R, induced 1.5-fold increase in glucose uptake (p < 0.05) and phosphorylation of AMPK (0.95 ± 0.19; phosphorylated: total, p < 0.05). CONCLUSIONS: IL-6 in combination with IL-6R and not IL-6 alone increased glucose uptake in human skeletal muscle. IL-6/IL-6R-mediated glucose uptake occurred independently of PKB/Akt phosphorylation, showing that IL-6/IL-6R-induced glucose uptake is dependent on a divergent pathway.

Efficacy and safety of anti-interleukin 6 receptor monoclonal antibody (tocilizumab) in Colombian patients with Takayasu arteritis.

PURPOSE: The aim of this study was to describe the efficacy and safety of anti-interleukin 6 receptor antibody (tocilizumab [TCZ]) in patients with severe or refractory Takayasu arteritis (TA). METHODS: We describe 8 Colombian patients with severe and/or refractory TA treated with TCZ during a period of at least 9 months. Clinical, radiological, biological, and associated treatments were evaluated before, during, and after TCZ infusions. RESULTS: The median age at evaluation was 31 years (12-43 years). All patients were female and experienced clinical and biological improvement, in addition to a corticosteroid-sparing effect from a median dose of 50 mg/d at baseline (30-60 mg/d) to 6.25 mg/d (2.5-10 mg/d) at 9 months. In 4 cases, in which imaging studies were available, an improvement was observed. The median duration of TCZ infusions was 18 months (9-36 months). Major adverse effects related to TCZ were not evidenced during a period of at least 9 months of treatment. One relapse was observed. Tocilizumab was continued in all cases until the last follow-up. CONCLUSIONS: This study shows a clinical, biological, and radiological response in patients with refractory TA treated with TCZ.

Interleukin-6 receptor blockade enhances CD39+ regulatory T cell development in rheumatoid arthritis and in experimental arthritis.

OBJECTIVE: The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti-IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti-IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. METHODS: Mice with collagen-induced arthritis (CIA) were treated with a mouse anti-IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. RESULTS: In MR16-1-treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell-sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. CONCLUSION: In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti-IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

Dietary fish oil reduces the acute inflammatory response and enhances resolution of antigen-induced peritonitis.

Dietary n-3 polyunsaturated fatty acids (PUFA) influence the inductive phase of inflammation but less is known about their effects on the resolution phase. This study examined the effects of dietary fish oil on induction and resolution of antigen-induced inflammation in mice. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Prior to and at different time points after mBSA administration, peritoneal cells were analyzed and expression of surface molecules determined by flow cytometry. Concentration of chemokines, cytokines and soluble cytokine receptors was determined by ELISA. Mice fed the fish oil diet had fewer peritoneal neutrophils, shorter resolution interval and lower levels of pro-inflammatory cytokines and chemokines than mice fed the control diet. In mice fed the fish oil diet there was an early peak in peritoneal levels of the immunosuppressive molecules sIL-6R and TGF-ß, that was not seen in mice fed the control diet. In the resolution phase, peritoneal macrophages from mice fed the fish oil diet expressed more of the atypical chemokine receptor D6 and peritoneal TGF-ß levels were higher than that in mice fed the control diet. Furthermore, in the late-resolution phase there were more peritoneal eosinophils and macrophages in mice fed the fish oil diet than in mice fed the control diet. These results demonstrate a suppressive effect of n-3 PUFA on the inductive phase of inflammation and indicate an enhancing effect of n-3 PUFA on resolution of inflammation.

Successful effect of tocilizumab in anti-TNF-α-induced palmoplantar pustulosis in rheumatoid arthritis.

Anti-tumour necrosis factor-alpha (TNF-α) agents are effective drugs used in several chronic inflammatory diseases such as rheumatoid arthritis (RA). Psoriasiform lesions, including palmoplantar pustulosis, have been described following anti-TNF-α therapy. These lesions often resolve with topical therapy with or without discontinuation of these drugs. However, in some cases, psoriasiform lesions may persist despite anti-TNF-α withdrawal. We report on two RA patients treated with adalimumab (ADA) who developed palmoplantar pustular despite dermatological treatment and ADA discontinuation. Tocilizumab (TCZ) therapy was initiated because of persistence of skin lesions and flare of the disease. Following treatment with this drug, complete resolution of the dermatological lesions and induction of remission of RA was achieved. To the best of our knowledge, management of palmoplantar pustulosis due to TNF-α agents with TCZ leading to both improvement of the disease and resolution of the cutaneous lesions has not previously been reported.

Brief report: inhibition of interleukin-6 function corrects Th17/Treg cell imbalance in patients with rheumatoid arthritis.

OBJECTIVE: From an immunologic standpoint, the mechanisms by which treatment with tocilizumab (TCZ), a humanized anti-interleukin-6 (anti-IL-6) receptor antibody, results in improvement in rheumatoid arthritis (RA) patients are still not fully understood. In vitro studies and studies in mouse models have demonstrated the critical role of IL-6 in Th17 cell differentiation. Th17 lymphocytes have been shown to be strongly involved in RA pathogenesis, and the purpose of this study was to investigate the effect of IL-6 blockade on the balance between Th17 cells and Treg cells in patients with active RA. METHODS: Patients with active RA for whom TCZ had been prescribed by a rheumatologist were enrolled in this study. Phenotypic analyses of T cell populations were performed, and the Disease Activity Score in 28 joints (DAS28) was assessed. Serum cytokine levels and other parameters of inflammation were measured before the first infusion and after the third infusion of TCZ (8 mg/kg). RESULTS: Compared to controls, levels of Th17 cells (CD4+IL-17+) were increased and Treg cells (CD4+CD25(high) FoxP3+) were decreased in the peripheral blood of patients with active RA. The suppressive function of circulating Treg cells was not impaired in patients with active RA. TCZ treatment induced a significant decrease in the DAS28 associated with a significant decrease in the percentage of Th17 cells (from a median of 0.9% to 0.45%; P = 0.009) and an increase in the percentage of Treg cells (from a median of 3.05% to 3.94%; P = 0.0039) in all patients. CONCLUSION: This study demonstrates for the first time that inhibition of IL-6 function by TCZ corrects the imbalance between Th17 cells and Treg cells in patients with RA.

Systematic review of tocilizumab for rheumatoid arthritis: a new biologic agent targeting the interleukin-6 receptor.

BACKGROUND: Tocilizumab (TCZ), a humanized anti-interleukin-6 receptor monoclonal antibody, represents a new treatment strategy for patients with rheumatoid arthritis (RA) and is currently approved in the United States for RA patients who have failed to improve with at least one anti-tumor necrosis factor therapy. OBJECTIVE: The goal of this study was to summarize the efficacy and safety profile of TCZ. METHODS: A systematic literature review was conducted to identify English-language articles within PubMed and the Cochrane Library from January 1989 to August 2011 reporting results from Phase III TCZ double-blind, randomized controlled trials (RCTs), noncontrolled clinical trials, and open-label extensions with a duration ≥6 months. Study outcomes had to include at least one of the following: American College of Rheumatology (ACR) 20, 50, or 70 response rates; tender/swollen joint count; Health Assessment Questionnaire-Disability Index; radiographic outcomes and drug persistence. Phase II RCTs were included only if they contained relevant information not available in Phase III RCTs. Relevant studies were selected to evaluate TCZ's pharmacokinetics and pharmacodynamics. RESULTS: Ten published clinical trials (7 Phase III, 3 Phase II) for TCZ were retrieved (7833 articles initially identified) from PubMed and 31 from the Cochrane library. Compared with methotrexate (MTX) monotherapy, TCZ 8 mg/kg IV monotherapy had higher rates of ACR20 (P < 0.001), ACR50 (P = 0.002), and ACR70 (P < 0.001) scores at week 24. TCZ 8 mg/kg IV plus oral MTX had a higher ACR20 response rate than oral MTX plus placebo in patients with RA who failed to respond to MTX or anti-tumor necrosis factor therapy (P < 0.001). Patients receiving TCZ 8 mg/kg had less radiographic progression on the Genant-modified Sharp score (85% had no progression) than the control group (67% had no progression) (P < 0.001). The rate of serious infections was 4.7 events/100 patient-years of exposure in the TCZ groups. A greater frequency of neutropenia, thrombocytopenia, hyperlipidemia, and transaminitis was observed with TCZ compared with placebo. CONCLUSION: The short-term efficacy and safety profile of TCZ is promising. Additional long-term safety data are needed to better characterize the risk-benefit profile of this agent.

High habitual dietary alpha-linolenic acid intake is associated with decreased plasma soluble interleukin-6 receptor concentrations in male twins.

BACKGROUND: alpha-Linolenic acid (ALA) is associated with a low risk of cardiovascular disease; however, the underlying mechanism is not completely known. OBJECTIVE: The objective was to examine whether habitual dietary ALA intake is associated with plasma concentrations of inflammatory biomarkers after control for shared genetic and common environmental factors. DESIGN: We cross-sectionally studied 353 middle-aged male twins. Habitual diet was assessed with the Willett food-frequency questionnaire. Fasting plasma concentrations of interleukin-6 (IL-6) and its soluble receptor (sIL-6R), high-sensitivity C-reactive protein (hsCRP), and tumor necrosis factor-alpha (TNF-alpha) were measured. Linear mixed-effect regression analysis was used to partition the overall association into within- and between-pair associations. RESULTS: A 1-g increment in habitual dietary ALA intake was associated with 11.0% lower concentrations of sIL-6R (P = 0.004) but not of IL-6 (P = 0.31), TNF-alpha (P = 0.16), or hsCRP (P = 0.36) after adjustment for energy intake, nutritional factors, known cardiovascular disease risk factors, and medications. After further control for shared genetic and common environmental factors by comparison of brothers within a twin pair, a twin with a 1-g higher ALA intake was likely to have 10.9% (95% CI: 3.7%, 17.6%; P = 0.004) lower sIL-6R concentrations than his co-twin with a low intake, whereas ALA intake was not significantly associated with plasma concentrations of IL-6, TNF-alpha, or hsCRP. These results were validated by using 1000 bootstrap samples. CONCLUSIONS: Habitual dietary ALA intake is inversely associated with plasma sIL-6R concentrations independent of shared genetic and common environmental influences. Lowering sIL-6R may be a mechanism underlying the cardioprotective properties of habitual dietary ALA. This study was registered at clinicaltrials.gov as NCT00017836.

Interleukin-6 leads to interleukin-10 production in several human multiple myeloma cell lines. Does interleukin-10 enhance the proliferation of these cells?

UNLABELLED: Multiple myeloma (MM) is characterised as a malignant plasma cell proliferation. Interleukin-6 (IL-6) is an important cytokine in the proliferation of the multiple myeloma cells. Interleukin-10 (IL-10) is involved in the terminal differentiation of B cells into plasma cells and enhances the proliferation of B cells. Elevated IL-10 levels were detected in patients with MM, relating to the clinical manifestation of the disease. In this study it was investigated the effect of exogenous IL-6 on the IL-10 production and the proliferative effect of IL-6 and IL-10 in human multiple myeloma cell lines. The ten cell lines had different sources: blood, ascitic fluid, bone marrow, peritoneum and lymph node. RESULTS: (1) Four cell lines produced IL-10 (up to 179 pg/ml) spontaneously. IL-6 increased the IL-10 production up to 1626 pg/ml in 6/10 cell lines. With IL-6 receptor antagonist (IL-6RA) the values of IL-10 were in the range of untreated samples (spontaneous cytokine production). IL-6RA counteracted the increased IL-10 production induced by IL-6 treatment and decreased the high values of IL-10 significantly. (2) IL-6 enhanced the proliferation (up to 160%) in 9/10 cell lines, IL-10 enhanced the proliferation (up to 170%) in 7/10 cell lines. There was a significant correlation between the proliferative effects of IL-6 and IL-10. CONCLUSION: It is reported for the first time that IL-6 leads to a marked production of IL-10 and that this cytokine is an IL-6 related growth factor for MM cells. The findings of this study can be important in the therapeutic modalities of multiple myeloma.

Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth.

Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.

Interleukin-6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells.

Although abundant evidence indicates mutual regulation between the immune and the central nervous systems, how the immune signals are transmitted to the brain is still an unresolved question. In a previous study we found strong expression of proinflammatory cytokine receptors, including interleukin (IL)-1 receptor I and IL-6 receptor alpha in the rat carotid body (CB), a well-known arterial chemoreceptor that senses a variety of chemostimuli in the arterial blood. We demonstrated that IL-1 stimulation increases intracellular calcium ([Ca(2+)](i)) in CB glomus cells, releases ATP, and increases the discharge rate in carotid sinus nerve. To explore the effect of IL-6 on CB, here we examine the effect of IL-6 on [Ca(2+)](i) and catecholamine (CA) secretion in rat CB glomus cells. Calcium imaging showed that extracellular application of IL-6 induced a rise in [Ca(2+)](i) in cultured glomus cells. Amperometry showed that local application of IL-6 evoked CA release from glomus cells. Furthermore, the CA secretory response to IL-6 was blocked by 200 microM Cd(2+), a well-known Ca(2+) channel blocker. Our experiments provide further evidence for the responsiveness of the CB to proinflammatory cytokines and indicate that the CB might play a role in inflammation sensing and transmission of such information to the brain.

Epigenetic modification of SOCS-1 differentially regulates STAT3 activation in response to interleukin-6 receptor and epidermal growth factor receptor signaling through JAK and/or MEK in head and neck squamous cell carcinomas.

Signal transducer and activator of transcription 3 (STAT3) has been reported to be activated by interleukin-6 receptor (IL-6R) or epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), which may have important implications for responsiveness to therapeutics targeted at EGFR, IL-6R, or intermediary kinases. Suppressor of cytokine signaling-1 (SOCS-1) has been implicated recently in the negative regulation of IL-6R/Janus-activated kinase (JAK)-mediated activation of STAT3, suggesting that SOCS-1 could affect alternative activation of STAT3 by EGFR, IL-6R, and associated kinases. We investigated whether epigenetic modification of SOCS-1 affects STAT3 activation in response to IL-6R-, EGFR-, JAK-, or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-mediated signal activation. STAT3 was predominantly activated by IL-6R via Jak1/Jak2 in HNSCC lines UMSCC-9 and UMSCC-38 in association with transcriptional silencing of SOCS-1 by hypermethylation. In UMSCC-11A cells with unmethylated SOCS-1, STAT3 activation was regulated by both EGFR and IL-6R via a JAK-independent pathway involving MEK. Pharmacologic inhibitors of JAK and MEK and expression of SOCS-1 following demethylation or transient transfection inhibited STAT3 activation and cell proliferation and induced cell apoptosis in corresponding cell lines. Hypermethylation of SOCS-1 was found in about one-third of human HNSCC tissues, making it a potentially relevant marker for STAT-targeted therapy in HNSCC patients. We conclude that SOCS-1 methylation status can differentially affect STAT3 activation by IL-6R and EGFR through JAK or MEK in different HNSCC and response to pharmacologic antagonists. Identifying the potential factors and the regulatory pathways in STAT3 activation has important implications for the development and selection of molecularly targeted therapy in HNSCC.

Multilevel regulation of IL-6R by IL-6-sIL-6R fusion protein according to the primitiveness of peripheral blood-derived CD133+ cells.

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied, in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines, but the role of glycoprotein 130 activation has not yet been investigated. In this study, CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry, 2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction, 3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay, 4) analyze cell-cycle status, and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs, independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1, an inhibitor of a disintegrin and metalloprotease proteases, suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion, expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment.