RESUMO
A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Ralpha monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Ralpha selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Ralpha mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Ralpha restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity.
Assuntos
Apoptose , Colo/citologia , Células Epiteliais/citologia , Receptores de Interleucina/biossíntese , Receptores de Interleucina/química , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Northern Blotting , Western Blotting , Caspase 9 , Caspases/metabolismo , Divisão Celular , Linhagem Celular , Células Cultivadas , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Interleucina-11/metabolismo , Subunidade alfa de Receptor de Interleucina-11 , Interleucina-8/metabolismo , Janus Quinase 1 , Glicoproteínas de Membrana/metabolismo , Mucosa/patologia , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores de Interleucina-11 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3 , Fatores de Tempo , Transativadores/metabolismo , Tirosina/metabolismoRESUMO
Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.
Assuntos
Comunicação Autócrina/fisiologia , Quimiocina CCL2/metabolismo , Quimiocinas CXC , Quimiotaxia de Leucócito/fisiologia , Endotélio Vascular/efeitos dos fármacos , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-6/metabolismo , Neutrófilos/efeitos dos fármacos , Receptores de Interleucina-6/fisiologia , Trombina/farmacologia , Doença Aguda , Animais , Coagulação Sanguínea/fisiologia , Células Cultivadas , Quimiocina CXCL1 , Fatores Quimiotáticos/farmacologia , Doença Crônica , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Substâncias de Crescimento/farmacologia , Humanos , Interleucina-1/farmacologia , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Substâncias Macromoleculares , Camundongos , Modelos Animais , Neutrófilos/metabolismo , Receptores de Interleucina-6/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Trombina/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Veias UmbilicaisRESUMO
IL-6 exerts its biological activities through interaction with specific receptors expressed on the surface of target cells. IL-6 binds first to a low-affinity (10(-9) M) subunit, a 80 kDa glycoprotein also called gp80 or IL-6R alpha. The IL-6/IL-6R alpha complex recruits the signal-transducing b subunit, a 130 kDa glycoprotein called gp130. The association of gp130 with IL-6 and IL-6R alpha leads to the formation of the high-affinity IL-6 receptor complex, to the linkage of two gp130 subunits and to signal transduction. Soluble forms of both receptors have been described and found in biological fluids. Soluble cytokine receptors are generated by either proteolytic cleavage of their membrane moiety or by alternative splicing. Both mechanisms have been described for sIL-6R and sgp130 formation. Interestingly, the association of IL-6 with the soluble form of IL-6R alpha is capable of eliciting a biological response in cells that express only the membrane gp130. This type of activation, called "trans-signalling", renders virtually all cells capable of responding to IL-6/sIL-6R alpha complexes, making for a large new spectrum of IL-6 activities, ranging from the control of the immune response to involvement in pathological states. In this review the biological activities of IL-6 will be considered in the light of new knowledge concerning the association of IL-6 and the soluble IL-6 receptors.
Assuntos
Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/fisiologia , Receptor gp130 de Citocina , Humanos , Interleucina-6/fisiologia , Substâncias Macromoleculares , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Transdução de Sinais , SolubilidadeRESUMO
A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Ralpha) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Ralpha. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Ralpha fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Ralpha and most also recognized the denatured receptor on immunoblots after SDS-PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ralpha. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ralpha expressed in gp130/hIL-11Ralpha-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Ralpha, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Ralpha. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Ralpha monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Ralpha on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.
Assuntos
Anticorpos Monoclonais , Citometria de Fluxo/métodos , Leucócitos Mononucleares , Receptores de Interleucina/imunologia , Receptores de Interleucina/isolamento & purificação , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Reações Cruzadas , Epitopos , Humanos , Subunidade alfa de Receptor de Interleucina-11 , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina-11 , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Distribuição TecidualRESUMO
Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (31 atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. Interleukin-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.
Assuntos
Asma/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/sangue , Hipersensibilidade Respiratória/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Asma/diagnóstico , Feminino , Citometria de Fluxo , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Hipersensibilidade Respiratória/diagnósticoRESUMO
On the basis of the finding of alternatively spliced mRNAs, the alpha-subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha) and a soluble form (solGMRalpha). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha-specific band by Western blot, whereas a tmGMRalpha-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMRalpha. The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha. A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 +/- 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Neutrófilos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Processamento Alternativo , Animais , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562 , Cinética , Leucaférese , Neutrófilos/citologia , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/biossíntese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Regressão , Transfecção , Células U937RESUMO
Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.
Assuntos
Antígenos CD/metabolismo , Inibidores do Crescimento , Interleucina-11/genética , Linfocinas , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Divisão Celular , Receptor gp130 de Citocina , Citocinas/agonistas , Citocinas/antagonistas & inibidores , Humanos , Interleucina-11/química , Interleucina-6/metabolismo , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores de Citocinas/metabolismo , Receptores de OSM-LIF , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/genética , Células Tumorais CultivadasRESUMO
RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Récepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/análise , Especificidade de Anticorpos , Linhagem Celular , Cães , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Ensaio RadioliganteRESUMO
Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.
Assuntos
Anticorpos Monoclonais/farmacologia , Interleucina-6/imunologia , Receptores de Interleucina-6/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Antígenos CD/farmacologia , Linhagem Celular , Receptor gp130 de Citocina , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Modelos Moleculares , Testes de Neutralização , Testes de Precipitina , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismoRESUMO
gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.
Assuntos
Antígenos CD/fisiologia , Citocinas/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Melanoma/fisiopatologia , Glicoproteínas de Membrana/fisiologia , Receptores de Interleucina-6/fisiologia , Animais , Antígenos CD/biossíntese , Fator Neurotrófico Ciliar , Receptor gp130 de Citocina , Feminino , Inibidores do Crescimento/antagonistas & inibidores , Humanos , Interleucina-11/antagonistas & inibidores , Fator Inibidor de Leucemia , Linfocinas/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oncostatina M , Peptídeos/antagonistas & inibidores , Receptores de Interleucina-6/antagonistas & inibidores , Transdução de Sinais , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Long-term stable Ba/F3 transfectants (B13R alpha1 and B13R alpha2) expressing two isoforms of the human IL-IIR alpha receptor (alpha1 full length or alpha2 lacking the cytoplasmic domain) in combination with human gp130 were established. IL-11R alpha1 and IL-11R alpha2 were each expressed and detected as three bands upon Western blot analysis, with apparent molecular masses in agreement with those of the polypeptide backbone (47 and 44 kDa, respectively) with no, one or two N-linked sugars. B13R alpha1 and B13R alpha2 bound IL-11-thioredoxin with similar efficiencies and proliferated with superimposable dose-response curves to IL-11, demonstrating that the intracellular domain of IL-11R alpha has no significant contribution on ligand binding and signaling. Analysis of a set of anti-human gp130 mAbs confirmed the similar responsiveness of B13R alpha1 and B13R alpha2 transfectants.
Assuntos
Interleucina-11/farmacologia , Receptores de Interleucina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-11 , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Interleucina/genética , Receptores de Interleucina-11 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6-inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their alpha-subunit receptors suggests that the structural location of certain binding sites are conserved.
Assuntos
Antígenos CD/química , Interleucina-6/química , Conformação Proteica , Receptores de Interleucina/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Melanoma/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Macrophage stimulating protein (MSP) is a ligand for the RON receptor protein tyrosine kinase. Activation of RON in murine resident macrophages results in cell shape change and migration. We studied cell movement induced by MSP in different types of human epithelial cells and the possible role of phosphatidylinositol-3 (PI-3) kinase in RON-mediated signal transduction. We observed specific and saturable binding of 125I-MSP to RON on several epithelial cell lines. In addition to activation and phosphorylation of RON, MSP also induced tyrosine phosphorylation of the PI-3 kinase p85 subunit in a time-dependent manner, with a peak at 15 min. Moreover, phosphorylated RON formed a complex with PI-3 kinase in both HK-NOC keratinocyte and RON cDNA-transfected MDCK cells. An in vitro protein interaction assay confirmed that PI-3 kinase from a lysate of MSP-activated cells bound to pure RON protein. MSP, at a concentration range of 1 to 5 nM, induced migration of three epithelial cell lines. This effect was inhibited by wortmannin, a specific inhibitor for PI-3 kinase, with an IC50 of 10 nM. MSP-induced shape change in murine resident peritoneal macrophages was also abolished by wortmannin. These data suggest that activation of PI-3 kinase is required for MSP-induced epithelial cell migration. The stimulation by MSP of epithelial cell movement may have implications for tissue repair, wound healing, and tumor metastasis.
Assuntos
Movimento Celular , Substâncias de Crescimento/farmacologia , Fator de Crescimento de Hepatócito , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Células Epiteliais , Substâncias de Crescimento/metabolismo , Humanos , Fosfatidilinositol 3-Quinases , Fosforilação , Transdução de Sinais , Tirosina/metabolismoRESUMO
The alpha-subunit of interleukin-6 (IL-6) receptor is a member of the hematopoietin receptor family. The alignment of its amino acid sequence with those of other members of this family (human somatotropin receptor/murine IL-3 receptor beta and human IL-2 receptor beta) has suggested that amino acids included in two SSFY repeats found in each of its hematopoietin receptor domains, contribute to the binding of the ligand. The involvement of these amino acids in IL-6 binding and signal transduction was studied by site-directed mutagenesis and molecular modelling. We present a computer-derived three-dimensional model of the IL-6/IL-6 receptor complex based on the structure of the human somatotropin/human somatotropin receptor complex. This model allowed the location of distinct regions important for IL-6 and gp130 binding. We show that some of the residues included in the SSFY repeats located in our IL-6 receptor model in the loops between beta-strands E and F of domain-I and B' and C', of domain-II, participate in the formation of a major IL-6-binding site. These residues are necessary for IL-6 and gp130 binding and for signal transduction. Using our IL-6 receptor mutants we mapped the epitopes of our anti-(IL-6 receptor) neutralising monoclonal antibodies to these residues. Our results demonstrate that a generic hematopoietin receptor family structural module can be used for the study of both alpha and beta receptor subunits belonging to this family.
Assuntos
Antígenos CD/química , Antígenos CD/fisiologia , Interleucina-6/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Mapeamento de Epitopos , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Coelhos , Receptores de Interleucina/efeitos dos fármacos , Receptores de Interleucina-6 , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Pgp 170 is the main integral membrane protein involved in acquired or de novo multidrug resistance (MDR), frequently implicated in chemotherapeutic failure. Because there is at present no method for quantitating Pgp 170 levels, a new and convenient assay, using flow cytometry with a standard fluorescence curve and MRK 16, a mAb recognizing an external epitope of human Pgp 170, was developed. Assuming a 1:1 stoichiometry, we calculated for the first time the apparent number of Pgp 170 molecules per cell. The method was applied successfully to cells in suspension or grown as monolayers and their mixtures. All quality criteria were checked and proved the suitability of the method for quantifying Pgp 170.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Citometria de Fluxo , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The use of inhibiting cytokine-binding-proteins (CBPs) such as soluble cytokine receptors and anticytokine antibodies is considered for the treatment of cytokine-dependent diseases. The pleiotropic cytokine interleukin-6 (IL-6) is a target for immunointervention in numerous pathologic situations, including multiple myeloma, B-cell lymphoma, and rheumatoid arthritis. An antitumor response was obtained in the treatment of a patient with multiple myeloma. A controversial issue is to evaluate whether the carrier effect of the CBPs might limit their efficiency in blocking the target cytokine. We analyzed the pharmacokinetics of radiolabeled IL-6 in mice treated with various combinations of anti-IL-6 antibodies. We show that injection of one or two antibodies led to the stabilization of the cytokine. Conversely, simultaneous treatment with three anti-IL-6 antibodies, binding to three distinct epitopes, induced the rapid uptake of the trimeric immune complexes by the liver and the elimination of IL-6 from the central compartment. The use of cocktails of three antibodies binding simultaneously to a cytokine thus provides a new means of enhancing the clearance of the target molecule and should help in the design of antibody-based clinical trials by overcoming the problem of the accumulation of the cytokine in the form of monomeric immune complexes.
Assuntos
Anticorpos Monoclonais/farmacocinética , Interleucina-6/imunologia , Animais , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/análise , Cromatografia em Gel , Feminino , Humanos , Radioisótopos do Iodo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA/imunologia , Técnica de Diluição de Radioisótopos , Distribuição TecidualAssuntos
Antígenos CD/fisiologia , Interleucina-6/fisiologia , Receptores de Interleucina/fisiologia , Complexo Antígeno-Anticorpo , Antígenos CD/análise , Antígenos CD/sangue , Antígenos CD/química , Proteínas de Transporte , Receptor gp130 de Citocina , Humanos , Hipergamaglobulinemia/sangue , Interleucina-6/sangue , Interleucina-6/farmacocinética , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/fisiologia , Receptores de Interleucina/análise , Receptores de Interleucina/química , Receptores de Interleucina-6 , Transdução de SinaisRESUMO
Soluble forms of most cytokine receptors, able to bind effectively to their respective ligands, have now been described. A soluble interleukin-6-binding molecule derived from the gp80 component of the multichain IL-6 receptor can be detected in biological fluids, and can act as an agonist of IL-6 activity. The clinical significance of the soluble receptor levels still remains to be explored. We took advantage of the characterization of an anti-IL-6 monoclonal antibody and of an anti-IL-6R monoclonal antibody that both bound to IL-6/IL-6R complexes to design an immunometric assay for the measurement of soluble IL-6R complexed to IL-6. This reaction scheme was designated as ELIA (enzyme-ligand immunoassay). When exogeneous IL-6 was added in excess to an sIL-6R containing sample, all sIL-6R was present in a complexed form. Thus, the reaction scheme could also be used to determine total sIL-6R concentrations. A recombinant sIL-6R standard was prepared from the supernatant of murine thymoma cells transfected with a gene coding for an extracellular portion of the IL-6 receptor. The assay permitted the precise and reproducible measurement of sIL-6R in serum or plasma. This approach is of general relevance for the determination of soluble cytokine receptors in biological fluids, provided that adequate anti-cytokine and anti-receptor antibodies are available.
Assuntos
Técnicas Imunoenzimáticas , Interleucina-6/imunologia , Receptores de Interleucina/análise , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Primers do DNA/química , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Receptores de Interleucina-6 , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Solubilidade , Transfecção , Células Tumorais CultivadasRESUMO
Determinations of total cytokine concentration in biological fluids by immunoassays face two major problems: the biochemical heterogeneity of the analyte and the interference of cytokine-binding proteins. We developed an ultrasensitive enzyme immunoassay for interleukin-6 (IL-6), using monoclonal antibodies and acetylcholinesterase as the tracer enzyme. The antibodies recognized recombinant and glycosylated forms of IL-6 equally. The antibodies measured dimeric recombinant IL-6, yet we could not detect IL-6 oligomers in plasma samples. We investigated the potential interference of soluble IL-6 receptor (sIL-6R), which is present at high concentrations in plasma samples (1 to 2 nmol/L). Heat treatment of the sample obviated the sIL-6R interference. Using calibrators in a plasma matrix, we demonstrated by fractionation, dilution, and recovery experiments that the immunoassay accurately measured total IL-6 in both normal and pathological serum and plasma samples.