Cellular targets and receptors for interleukin-6 II. Characterization of IL-6 binding and receptors in peripheral blood cells and macrophages (original) (raw)
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Journal of Biological Chemistry, 1991
From the Institut f 6 r ~~o c h e m~e , Rheinisch-W e s~f~~s c h Technische Hochschule Aachen, ~~i n~~u~~ P a u w e~s~~s e 30, and Slnstitut fur ~n a~o m~e f z e~l b i o~~~e~, Rheinisch-Wes~f~isch T e c h n~c~ ~ochschu& Aachen, ~e~i 2 i n~c h-~e c~n i s c h e ~~t~ W e~l i~~e~ 2, W-~~~~ Aachen, ~e d e r a l ~e p u b~~ of ~e r r n a n~ and the Vnstitute for ~~~c u~r and C e~~u~r ~~o~~ Osaka ~n i u e r s i~, 565 Osaka, ~~n A cDNA coding for the human interle~k~n-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for lz6I-la~1ed recombinant human interleukin-6 (l2"1-rhIL-6) (& = 440 p~, 20,000 receptors per celf). Affinity cross-linking of '"'I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the deteetion of three lz61-rhIL-6-cont~ning protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex o r~~i z a t i o n of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIHf3T3 cells exerted growth inhibition. This antigrowth effect was observed by the measurement of cell numbers and ornithine decarboxyla~ mRNA expression. IL-6-R overexpressing fibroblasts internalized '261-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel e l~t~p h o r~~s. A possible implicat~on of skin fibrobl~ts in the catabolism of IL-6 is discussed. Many d~fferent cells s~t h e s~z e inter~eukin.6 (It-6)' after appropriate st~mulation (for recent reviews, see Refs. 1-3). Monoc~es/macroph~es, endothetial cells, and fibroblasts are believed to be the major IL-6-pr~ucing cells in mammals. IL-6 is a d~fferent~ation and growth factor involved in the immune response, in hematopo~esis, and in the acute-phase reaction (1-3). The main activities exerted by IL-6 are induc-" .~ ~ *This work was supported by grants from the Deutsche For-schun~gemeinschaft and the Fonds der Chemischen Industrie. The costs of publ~cation of this articIe were defrayed in part by the payment of page charges. This article must therefore be hereby marked "~d~e r~~e m e n~~' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
High- and low-affinity receptors for murine interleukin 6. distinct distribution on b and t cells
European Journal of Immunology, 1989
High-and low-affinity receptors for murine interleukin 6. Distinct distribution on B and T cells* The distribution of the murine receptor for interleukin (IL) 6 was examined on a variety of cells. Binding sites for IL6 were found on many cell lines including fibroblasts and bone marrow-derived macrophages. The highest density of binding sites was found on B cell hybridomas and plasmacytomas, irrespective of their IL6 dependence for growth. Scatchard analysis carried out with these cells identified about 1000 high-affinity sites (Kd = 25 PM) and 10 000 low-affinity sites (Kd = 2.5 nM). The binding was specific for IL6 and led to the internalization of the ligand. Receptors for IL6 were also present on other B cell lines, but binding on normal resting or activated B cells was below the limit of detection (< 3 molecules/cell). In contrast, receptors were found on mature thymocytes and on peripheral Tcells. However, unlike plasmacytomas and hybridomas,Tcells expressed only high-affinity binding sites. The difference in the relative numbers of high-and low-affinity receptors on different cells suggests that IL 6 interacts with several proteins, the expression of which varies from one cell type to another.
FEBS Letters, 1991
Ilowcytomctry usintl ph~eoerytltrin (PE)-conjujatcd lijand, Treatment of PMNL with rh//.r~mulocyt¢.macroplt=lB~ colony.=climulatinl~t factor (GM.CSF) led to the inability of PMNL to bind MT I1~ monoclona| antibody (moAb) and to display binding site~ for PE-¢onjug-'~ted rh IL.6, l=~vels of IL.6.R transcripts in PMNL exposed to GM.CSF were aboa| .~.fold t~low those of PMNL cultured in rneditm; only, Though a definitive role for IL-6 to modulate the function of PMNL w~ts not found, treatment of PMNL with rh IL-6 clearly resulted in an enh=Lncement of Iranscript levels of the e~trly response tlenes ¢-fo.~ lind ¢.jun in these cells, thus indicatinl~ that IL.6 bindinf, is followed by sit, hal tr=msduction, Neutrophii; IL-6 receptor; GM-CSF; Receptor downre~ulation I. INTRODUCTION lnterleukin.6 is one of the central mediators of the complex reaction of the host to injury or infection referred to as the acute phase response ([1,2] for review). Important sites of" IL-6 synthesis include monocytes, blood vessel endothelials, smooth muscle cells, connective tissue fibroblasts, chondrocytes, osteoblasts, keratinocytes, mesangial cells, astrocytes, anterior pituitary cells, and stromal endometrium cells ([3] for review). We have recently shown that PMNL may also be able to produce IL-6, upon activation with GM-CSF or tumor necrosis factor [4]. The physiological ef,fects exerted by IL.6 include induction of fever, induction of immunoglobulin synthesis in activated Bcells, activation of T-cells and natural killer cells, stimulation of megakaryopoiesis, induction of acute phase response synthesis by the liver, and corticotropin release by cells of the pituitary gland ([3] for review), IL-6 acts on its target cells via a specific dimeric receptor. A 80 kDa membrane protein serves as IL-6 binding site [5]. IL-6 binding is followed by association of the 80 kDa protein with a second 130 kDa transmembrane protein providing signal transduction [6]. Given the previous demonstration by several investigators, ineluding ourselves, of biosynthetic activity of human PMNL upon exposure to cytokines [4,7-9], and the
FEBS Letters, 1994
We have studied the expression and regulation of the interleukin-6 receptor (gp80) and its signal transducer gp130 in primary human blood monocytes. Here, we show that freshly isolated human monocytes express mRNAs for gp80 and gp130. In contrast to a previous report [(1989) FEBS L&t. 249, 27-301 we find that neither lipopolysaccharide nor interleukin-6 (IL-6) lead to a down-regulation of IL-6 receptor mRNA in monocytes. Also in the human monocytic cell line Mono Mac 6 no effect of IL-6 on receptor mRNA levels was observed. For signal transducer gp130 mRNA in monocytes a small and transient up-regulation by IL-6 was found.
European Journal of Biochemistry, 1993
The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in v i m folding procedure using L-arginine and the glutathione-redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column. The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145-345) of the human interleukin-6 receptor (IL-6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL-6 binding, it could be used to establish a cell-free IL-6-binding assay, In this assay, the srhIL-6R bound IL-6 with an affinity of Kd = 1.5 nM as measured by Scatchard-plot analysis. When '"I-IL-6 was chemically cross-linked to the purified srhIL-6R and analyzed by SDSPAGE, several '251-IL-6-containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL-6/IL-6R complex. The srhIL-6R was shown to exhibit biological acitivity, i.e. it stimulated acute-phase protein synthesis in the recently established human hepatoma cell line HepG2-IL-6 which does not express the IL-6-binding subunit of the IL-6R complex on the cell surface. Interleukin-6 (IL-6) is a multifunctional cytokine with a pleiotropic spectrum of action. The major biological effects of TL-6 are stimulation of (a) B-cell and neuronal-cell differentiation, (b) proliferation of hybridoma and plasmacytoma cells, (c) maturation of cytotoxic T cells and (d) synthesis of acute-phase proteins in the liver (Heinrich et al., 1990; Hirano and Kishimoto, 1990; Van Snick, 1990). IL-6 acts via a specific cell-surface receptor consisting of two subunits, an a chain of 80 kDa (gp80) which binds IL-6 and a p chain of 130 kDa (gp130) which is involved in signal transduction (Yamasaki et al., 1988; Hibi et al., 1990). Both subunits belong to the recently recognized hematopoietic receptor superfamily which also includes the receptors for IL-2 (the p and y subunit), IL-3, IL-4, IL-5, IL-7, IL-9, granulocyte-macrophage colony-stimulating factor, prolactin, growth hormone, erythropoietin, leukemia inhibitory factor and ciliary neurotropic factor (CNTF;
The EMBO Journal, 1997
IL-6 plays an important role in post-menopausal bone loss. IL-6 belongs to a family which share remarkably similar Interleukin 6 (IL-6) has many biological activities structural features both for the cytokines and the receptors in vivo, and deregulation has been implicated in many to which they bind. These similarities also extend to the disease processes. IL-6, a 185 amino acid polypeptide sequential clustering events leading to signal transduction. was refolded, purified and crystallized. The crystals The closest members of the family include leukemia diffracted to beyond 1.9 Å and the structure was solved inhibitory factor (LIF), cillary neurotrophic factor (CNTF), using single isomorphous replacement. The X-ray oncostatin M and IL-11 (Yamasaki et al., 1988; Davis structure of IL-6 is composed of a four helix bundle et al., 1991; Gearing et al., 1991; Kishimoto et al., 1992, linked by loops and an additional mini-helix. 157 out 1994; Miyajima et al., 1992). IL-6 receptor consists of 185 residues are well defined in the final structure, of two polypeptides: the α chain (IL-6r), an 80 kDa with 18 N-terminal and 8 A-B loop amino acids transmembrane glycoprotein that binds IL-6 with low displaying no interpretable electron density. The threeaffinity, and the β chain (gp130), a 130 kDa transmembrane dimensional structure has been used to construct a glycoprotein that binds to the IL-6-IL-6r heterodimer to model of IL-6 interacting with the IL-6 receptor form the high affinity signal transducing complex (Taga (α-chain) and gp130 (β-chain) that gives new insight et al., 1989). The IL-6r is a transmembrane protein into the process of molecular recognition and signaling. composed of a cytokine binding type I domain (necessary Based on this model, we predict a fourth binding site and sufficient for binding IL-6; Yawata et al., 1993), an on IL-6, a low affinity IL-6-IL-6 interaction, which Ig-like domain and a short cytosolic domain (Yamasaki may be necessary for the sequential assembly of a et al., 1988) that is not required for signaling (Taga et al., functional hexameric IL-6 receptor complex. 1989). gp130 is also a transmembrane protein composed Keywords: crystal structure/interleukin 6/receptor/ of an Ig-like domain, cytokine type I domain, a contactinsignaling like region, a transmembrane domain and a cytosolic domain necessary for signaling, containing a motif known as box1, box2 (Murakami et al., 1991). gp130 is a signal transduction component of not only the IL-6 receptor but
Interleukin 6 and its receptor in the immune response and hematopoiesis
International journal of cell cloning, 1990
Interleukin 6 (IL-6) plays critical roles in the immune response and hematopoiesis. It is a potent B cell differentiation factor inducing antibody-forming plasma cells. It enhances interleukin 3-induced proliferation of hematopoietic stem cells. Furthermore, IL-6 induces maturation of megakaryocytes. In IL-6 transgenic mice, a massive polyclonal plasmacytosis and an increase in the number of mature megakaryocytes in the bone marrow were observed. The data indicated that deregulated expression of the IL-6 gene induced a polyclonal plasmacytosis and could be involved in the oncogenesis of plasma cell neoplasias. IL-6 receptor (IL-6R) was molecularly cloned and found to be an immunoglobulin superfamily having an MW of 80 kDa. Upon the binding of IL-6 to its 80 kDa IL-6R, a second non-binding molecule, gp130 was shown to associate with IL-6R. The complex of IL-6 and soluble IL-6R lacking both transmembrane and cytoplasmic domains could bind gp130 and transduce the signal. The results in...
Cytokine, 1993
Nano-to picomolar concentrations of high affinity IgG antibody to interleukin 6 (ILdab) were detected in sera of 71 of 467 normal Danish blood donors (15%). IL-6 bound to the Fab fragments of ILdab, and the antibody specifically and competitively interfered with enzyme-linked immunosorbent assays for human IL-6. Only ILdab-positive sera interfered with these ELISAs. A statistically positive correlation was found between total IL-6 binding and specific IL-6 binding to serum (P < O.OOOl), suggesting that IL-6ab dominates as IL-6 binding factors in normal human serum. The ILdab also inhibited binding of IL-6 to receptors on the human U-937 macrophage-like cell line, the human U-266 myeloma cell line and the mouse hybridoma cell line B13.29, clone B9 (B9 cells). IL-6-induced proliferation of the B9 cells was competitively inhibited by ILdab. We conclude that sera of normal individuals may contain appreciable levels of autoantibodies to IL-6. These IgG autoantibodies may be physiologically and pathophysiologically important because they are potent inhibitors of IL-6 in vitro. Cells involved in immunoinflammatory reactions communicate by direct contact and through elaboration of soluble, hormone-like peptides termed cytokines. Cytokines act by binding to specific receptors on lymphoid and non-lymphoid cells and many of these mediators, including interleukin 6 (IL-6), are centrally involved in antigen-induced activation of T-and B-lymphocytes and in several aspects of cellular and humoral inflammatory responses.i-4 IL-6 participates in the terminal differentiation of activated B cells into antibody-producing cells.58 It induces or participates as a permissive factor in hybridoma and myeloma growth,%11 cytotoxic T-cell differentiation,i2,13 monocyte colony formation,i4 maturation of megakaryocytes,i5,16 nerve cell differentation,iT fever18 and increased production of From the tDepartment of Infectious Diseases and 2Medicine TTA,