RESUMEN
A model of the tertiary structure of human IL-6, derived from the crystal-structure of granulocyte-colony stimulating factor, reveals a 5th helical region in the loop between the first and second alpha-helix. To investigate the importance of this region for biological activity of IL-6, residues Glu-52, Ser-53, Ser-54, Lys-55, Glu-56, Leu-58, and Glu-60 were individually replaced by alanine. IL-6.Leu-58Ala displayed a 5-fold reduced biological activity on the IL-6 responsive human cell lines XG-1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL-6 receptor-alpha-chain-dependent binding to the IL-6 signal transducer, gp130.
Asunto(s)
Antígenos CD , Interleucina-6/fisiología , Leucina/fisiología , Glicoproteínas de Membrana/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , División Celular , Receptor gp130 de Citocinas , Humanos , Hibridomas , Interleucina-6/química , Interleucina-6/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Alineación de Secuencia , Células Tumorales CultivadasRESUMEN
Mutagenesis of a region of human interleukin (IL)-6 which is important for triggering signal transduction via the IL-6 receptor beta-chain (gp130) has lead to the isolation of a variant of human IL-6 (IL-6.Q160E/T163P), which could antagonize the biological activity of wild type IL-6 on the human EBV transformed B cell line CESS and the human hepatoma cell line HepG2. Surprisingly this antagonistic IL-6 variant had an agonistic effect on the human myeloma cell line XG-1, albeit at a 1000-fold higher concentration than wild type IL-6. This residual activity of the mutant arose from triggering gp130, because it could be inhibited by a gp130 specific mAb. Extensive mutagenesis of residues between Q153 and H165 of human IL-6, a region which is partly homologous in cytokines which also signal via gp130 (oncostatin M, ciliary neurotrophic factor, leukaemia inhibitory factor, IL-11), did result in the isolation of a second antagonist for IL-6 activity on CESS and HepG2 cells. However on XG-1 cells this variant was active as well. These results suggest that (an) additional region(s) of the IL-6 molecule might be involved in gp130 triggering. Recently we indeed found that residues Lys42-Ala57 are also important for gp130 triggering. Inhibition experiments with neutralizing IL-6R alpha-chain specific mAb show that this region can be functionally separated from the Q153-H165 region. These findings have important implications for the development of receptor antagonists of IL-6 and IL-6 family members.
Asunto(s)
Antígenos CD/química , Interleucina-6/genética , Mieloma Múltiple/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Receptores de Interleucina/química , Transducción de Señal/efectos de los fármacos , Alanina/química , Anticuerpos Monoclonales , Biblioteca de Genes , Pruebas Genéticas , Glutamina/química , Histidina/química , Humanos , Interleucina-6/química , Receptores de Interleucina-6 , Treonina/química , Células Tumorales CultivadasRESUMEN
We have shown that through mutagenesis of IL-6 it is possible to separate receptor binding from signal transduction of the cytokine. Mutations in residues important for signal transduction via gp130 result in IL-6 variants that can competitively inhibit wtIL-6 activity in vitro. The differential effects of these signaling deficient mutants on various cell lines of human origin suggest that receptor composition and/or signal transduction pathways may vary between cells of different origin. The observations that three sites have been identified which are important for gp130 interaction raises the question what the role of each region is in the stepwise formation of the active IL-6 receptor complex. The overall tertiary conformation of the beta-site mutants is intact, as judged from their binding characteristics to conformation specific mAbs and IL-6R alpha. As can be deduced from Figure 1, beta-site mutations may therefore affect a direct interaction with gp130, dimerization of IL-6, or maybe a conformational change in IL-6R alpha, important for gp130 interaction. A future challenge will therefore be to determine the function of each of the beta-sites in IL-6 receptor interaction.
Asunto(s)
Interleucina-6/fisiología , Receptores de Interleucina/antagonistas & inhibidores , Humanos , Estructura Terciaria de Proteína , Receptores de Interleucina-6 , Transducción de Señal , Relación Estructura-ActividadAsunto(s)
Antígenos CD , Interleucina-6/química , Receptores de Interleucina/metabolismo , Animales , Sitios de Unión , Receptor gp130 de Citocinas , Humanos , Interleucina-6/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ratones , Receptores de Interleucina-6 , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The pleiotropic cytokine interleukin-6 (IL-6) interacts with the specific ligand binding subunit (IL-6R alpha) of the IL-6 receptor, and this complex associates with the signal-transducing subunit gp130 (IL-6R beta). Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a region (residues 43-55) within the human IL-6 protein, which is important for the interaction with gp130. Subdividing this region shows that mainly residues 50-55 of the human IL-6 are necessary for this interaction. Recently, another human IL-6 double mutant (Q159E and T162P) showed reduced affinity to gp130 but residual activity on the human myeloma cell line XG-1. Into this IL-6 mutant we introduced the murine residues 43-49 or 50-55 together with two point mutations, F170L and S176A, which had been reported to increase the affinity of IL-6 to the IL-6R alpha. The resulting IL-6 molecule, which contained the murine residues 50-55, was inactive on human myeloma cells and in addition completely inhibited wild type IL-6 activity on these cells. Such an antagonist may be used as a specific inhibitor of IL-6 activity in vivo.
Asunto(s)
Antígenos CD , Interleucina-6/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/antagonistas & inhibidores , Animales , Secuencia de Bases , Receptor gp130 de Citocinas , Humanos , Interleucina-6/química , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Oligodesoxirribonucleótidos , Mutación Puntual , Conformación Proteica , Receptores de Interleucina-6 , Células Tumorales CultivadasRESUMEN
The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.
Asunto(s)
Interleucina-6/farmacología , Mieloma Múltiple/inmunología , Receptores de Interleucina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Secuencia de Bases , Carcinoma Hepatocelular , División Celular , Línea Celular Transformada , Cartilla de ADN , Herpesvirus Humano 4/genética , Humanos , Interleucina-6/análogos & derivados , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Cinética , Neoplasias Hepáticas , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple/patología , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Receptores de Interleucina/fisiología , Receptores de Interleucina-6 , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.
Asunto(s)
Receptores de Interleucina/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Línea Celular , Humanos , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ratones , Mutación , Unión Proteica , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Transducción de SeñalRESUMEN
In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.
Asunto(s)
Inhibición de Migración Celular , Indometacina/farmacología , Monocitos/inmunología , Dinoprostona/farmacología , Humanos , Inmunización , Factores Inhibidores de la Migración de Leucocitos/biosíntesis , Leucotrieno B4/farmacología , Tuberculina/inmunologíaRESUMEN
The level of Interleukin-6 (IL-6) in the aqueous humor of 24 patients with 2 types of uveitis was measured with a specific bioassay using the murine hybridoma cell line B9. Sixteen patients had Fuchs' heterochromic cyclitis (FHC) and 8 had toxoplasma uveitis (TU). Sixty-three percent of each of the FHC and TU groups had raised levels of IL-6 in their aqueous (mean: 543 and 19,228 units/ml respectively). Thirteen control aqueous samples, obtained at surgery for senile cataract, showed IL-6 levels of less than 10 units/ml. Serum obtained at the same time as each aqueous humor sample also showed IL-6 levels of less than 10 units/ml, indicating that the raised levels of IL-6 found in the aqueous of uveitis patients did not result from serum leakage, but from local production. This is the first report on intraocular IL-6 levels, and indicates that IL-6 may play a role as an inflammatory mediator in uveitis.