Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.
Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis. (+info)
Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor.
The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226. (+info)
Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34(+) cells through glycoprotein (gp)130 signaling.
We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo. (+info)
Anticytokine therapy in autoimmune diseases.
Autoimmune reaction and inflammation observed in autoimmune diseases may be caused by the deregulated production of cytokines. Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities such as support of hematopoiesis, regulation of acute phase reactions, and generation of immune responses. Uncontrolled hyperproduction of IL-6 causes plasmacytosis, hyper-gamma-globulinemia, thrombocytosis, mesangial cell proliferation of the kidney as well as inflammatory symptoms which are frequently observed in autoimmune diseases. Thus, interference with IL-6 signal transduction may be useful for autoimmune disease therapy. The pathogenic significance of IL-6 in autoimmune disorders and new therapeutic approaches involving blocking of IL-6 signal transduction are discussed. (+info)
Natural variability of circulating levels of cytokines and cytokine receptors in patients with heart failure: implications for clinical trials.
OBJECTIVES: The purpose of this study was to examine the variability in cytokines and cytokine receptors in patients with heart failure in comparison with a group of healthy control subjects who were free of cardiovascular disease. BACKGROUND: Despite increasing interest in cytokines as mediators of disease progression in heart failure and the recent interest in suppressing cytokines in clinical studies, the extent of variability in cytokines and cytokine receptors is largely unknown. This information is important for interpreting the results of studies in which changes in cytokine levels are measured in response to a specific form of therapy. METHODS: Circulating levels of tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptors (types 1 and 2), as well as interleukin (IL)-6 and IL-6 receptor were measured on a daily, weekly and monthly basis in heart failure patients (New York Heart Association class IIIa and IIIb; n = 10) and healthy volunteer subjects (n = 10). Measurements of cytokines and cytokine receptors were performed on plasma samples by enzyme-linked immunoassay. The daily, weekly and monthly degree of variability in cytokine and cytokine receptor levels was assessed by determining the coefficient of variation each point in time. RESULTS: The coefficient of variation for TNF-alpha and IL-6 levels increased over time in patients with heart failure; moreover, the coefficient of variation in heart failure subjects was significantly greater for IL-6 than for TNF-alpha. The coefficient of variation in cytokine receptor levels was minimal, and did not differ significantly between heart failure and control subjects. CONCLUSIONS: In patients with heart failure the degree of natural variability in circulating cytokine levels increases with time, and is greater for IL-6 than for TNF-alpha. Accordingly, the results of the present study suggest that the sample size needed to show a statistically significant change in the circulating level of a given cytokine will vary depending on the specific cytokine that is being measured, as well as the time period over which that cytokine is being assayed. (+info)
A rhesus macaque rhadinovirus related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 encodes a functional homologue of interleukin-6.
The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi's sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6). To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathione S-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays. Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner. Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6. Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells. These results demonstrate that RRV encodes an accessory protein with IL-6-like activity. (+info)
Response to IL-6 of HPV-18 cervical carcinoma cell lines.
The human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) controls cell type-specific expression of the viral oncoproteins E6 and E7. The HPV-18 URR is active in the cervical carcinoma cell line HeLa but inactive in the hepatoma cell line HepG2. C/EBPss (NF-IL-6) was shown to participate as an important regulator in HPV transcription dependent on the cell type. The finding that C/EPBss is critical for HPV-18 URR activity and that C/EPBss is induced by IL-6 offers the opportunity of manipulating HPV activity by specific cytokine treatment. In this report, we show that treatment with IL-6 results in activation of HPV-18 URR activity in HepG2 cells. In contrast, the HPV-18 URR is not inducible by IL-6 in three cervical carcinoma cell lines. In all three cell lines we found decreased expression of the IL-6 receptor compared to the IL-6-responsive HepG2 cells, whereas the level of expression of the signal transduction component gp130 is present in all cells. These results suggest that cervical carcinoma cells may circumvent the IL-6-induced cellular defense mechanism through downregulation of the IL-6-receptor. (+info)
Effect of interleukin-6 on the growth of human lung cancer cell line.
OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the growth of human lung cancer in vivo as well as in vitro. METHODS: To examine the mRNA level of IL-6 receptor (IL-6R) in high-metastatic human lung giant cell carcinoma cell line PG by means of reverse transcription polymerase chain reaction (RT-PCR). To assess the existence of IL-6 receptor complex (including IL-6R and gp130) with the treatment of PG cells by use of recombinant human IL-6 (rhIL-6), recombinant human oncostatin M (rhOSM), and recombinant human leukemia inhibitory factor (rhLIF), respectively. To detect the expression of IL-6 by Northern blotting hybridization and bioactive assay. To identify the effect of IL-6 secreted by PG cells by use of IL-6 and IL-6R antisense oligodeoxynucleotides (ODNs), and specific neutralizing antibody to IL-6. To document the influence of IL-6 on PG cells growth in vivo through the strategy of the transfection of expression vector inserted antisense IL-6 cDNA. RESULTS: RT-PCR analysis revealed that PG cells expressed IL-6R mRNA. Any one of the recombinant cytokine IL-6, OSM and LIF stimulated the growth of PG cells in vitro in a concentration-dependent manner. These results demonstrated IL-6 receptor complex exist in PG cells. At the same time, PG cells expressed IL-6 mRNA and secreted bioactive IL-6. Both IL-6 antisense ODNs and IL-6R ODNs inhibited PG cells proliferation. Treatment of PG cells with IL-6 antibodies reduced the growth of PG cells in vitro. PG cells transfected with IL-6 antisense expression vector showed a decreased growth in nude mice. CONCLUSION: IL-6 functions as an autocrine growth stimulator for PG cells in vivo as well as in vitro. (+info)