RESUMO
Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.
Assuntos
Receptores de Superfície Celular/genética , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Northern Blotting , Clonagem Molecular , DNA/genética , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Receptores do Fator de Necrose TumoralRESUMO
Human platelet-derived growth factor (PDGF) consists of two distinct but related polypeptide chains designated PDGF-A and PDGF-B. The gene encoding PDGF-B has given rise to the v-sis oncogene. In the present study the transforming activities of PDGF-A and PDGF-B genes are compared. The PDGF-A chain gene is markedly less efficient in inducing transformation than the PDGF-B gene under the influence of the same promoter. There are significant differences in the secretory and growth stimulating properties of the two chains. These properties appear to account for the much more potent transforming ability of the PDGF-B gene. These findings provide insights into biologic properties of a growth factor responsible for potent autocrine stimulation of abnormal cell proliferation.
Assuntos
Transformação Celular Neoplásica , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Compartimento Celular , Linhagem Celular , Regulação da Expressão Gênica , Técnicas Imunológicas , Camundongos , Peso Molecular , Receptores do Fator de Crescimento Derivado de Plaquetas , SolubilidadeRESUMO
The amino acid sequences of several, recently cloned cytokine receptors show significant homologies, primarily in their extracellular, ligand-binding domains. With one exception, their cognate cytokines mediate biological activities on a variety of hematopoietic cell types; thus we have designated the receptors as the hematopoietic receptor superfamily.
Assuntos
Fatores Biológicos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Citocinas , Humanos , Dados de Sequência Molecular , Conformação Proteica , Receptores de Superfície Celular/classificação , Receptores Imunológicos/classificação , Homologia de Sequência do Ácido NucleicoRESUMO
There have been several recent developments in the detection of cytokines, their receptors, and the genes that encode them. New methods for characterizing cytokines and lymphokines include the use of molecular hybridization techniques, bioassays and antibody-mediated detection systems. The most common techniques used for identifying and characterizing cytokine receptors rely upon the use of radiolabeled, fluoresceinated or biotinylated ligands.
Assuntos
Citocinas/análise , Linfocinas/análise , Receptores Imunológicos/análise , Animais , Bioensaio , Clonagem Molecular , Citocinas/genética , DNA/análise , Corantes Fluorescentes , Humanos , Imunoensaio , Linfocinas/genética , Hibridização de Ácido Nucleico , Receptores Imunológicos/genéticaRESUMO
We have developed rabbit polyclonal antibodies to the C-terminus of the flt3-encoded protein, which is a member of the receptor tyrosine kinase family. Immunoprecipitation using this antiserum brings down two protein bands, a major band of 143 kDa and a less abundant, more diffuse, band of 158 kDa. Pulse-chase analysis of flt3 protein from transfected COS-7 cells shows that the larger band is derived from the smaller one and presumably represents maturation of the protein from a glycosylated high-mannose form to a complex carbohydrate form. N-glycosidase F digestion confirmed the presence of N-linked carbohydrates, and cell-surface labeling of flt3-transfected cells indicated that the 158-kDa glycoprotein is the species found on the cell surface. A mutated form of the flt3 protein that was defective in its glycosylational processing was identified. Western blotting of the immunoprecipitated flt3 protein showed that it is heavily phosphorylated on tyrosine, and that this phosphorylation probably occurs in the absence of ligand. In this regard, the flt3 protein resembles the c-erbB2 protein, which is also highly phosphorylated in the absence of ligand. These data suggest that the flt3 receptor regulates the growth and differentiation of cells via an as yet unknown ligand.
Assuntos
Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/farmacologia , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Peptídeos/imunologia , Fosfotirosina , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Tirosina/análogos & derivados , Tirosina/química , Tirosina Quinase 3 Semelhante a fmsRESUMO
We have recently described a novel hematopoietic growth factor, referred to as the flt3 ligand, that stimulates the proliferation of sub-populations of hematopoietic cells that are enriched for stem and progenitor cells. This factor is a transmembrane protein that undergoes proteolytic cleavage to generate a soluble form of the protein. We have isolated additional flt3 ligand isoforms by PCR that contain an extra exon and encode what are predicted to be either a soluble form of the ligand or a longer version of the transmembrane protein. We have also isolated cDNAs from murine T cell libraries that encode an isoform of the flt3 ligand that has an unusual C-terminus. This isoform results from a failure to splice out an intron during mRNA processing. The protein encoded by this cDNA is expressed on the cell surface, where it is biologically active. However, this novel isoform does not appear to give rise to a soluble form of the protein. Regulation of mRNA splicing is likely to control the generation of cell bound or soluble forms of this hematopoietic growth factor. Genetic mapping studies localize the gene encoding the flt3 ligand to the proximal portion of mouse chromosome 7 and to human chromosome 19q13.3.
Assuntos
Processamento Alternativo , Substâncias de Crescimento/análise , Proteínas de Membrana/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , DNA Complementar , Feminino , Substâncias de Crescimento/genética , Humanos , Íntrons , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/genética , SolubilidadeRESUMO
We previously described the cloning of a human myeloid cell surface receptor for the Fc region of immunoglobulin A (Fc alpha R). In the present study, a soluble version of the Fc alpha R (solFc alpha R) was generated by removing the transmembrane and cytoplasmic coding regions from full-length Fc alpha R cDNA and ligating into a mammalian expression vector. COS-7 cells transfected with the solFc alpha R plasmid secreted a protein that inhibited both immunoglobulin A (IgA) and anti-Fc alpha R monoclonal antibody (mAb) binding to Fc alpha R+ U937 cells. Furthermore, the solFc alpha R bound specifically to and could be eluted from an anti-Fc alpha R mAb-immunoaffinity column, retaining biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the recombinant full-length Fc alpha R migrates over a molecular mass range of approximately 40-60 kd, consistent with the reported size and heterogeneity of the naturally occurring myeloid cell surface Fc alpha R. The solFc alpha R ran on SDS-PAGE as a smaller band (37-55 kd) that reduced to two bands of 23 and 25 kd following N-glycanase treatment, indicating that the Fc alpha R is a heavily glycosylated protein. The biochemical data, coupled with flow cytometry studies showing that the recombinant Fc alpha Rs bind to five different anti-Fc alpha R mAbs, clearly demonstrate that the cloned Fc alpha R corresponds directly to the major Fc alpha R species expressed on human monocytes, neutrophils, and myeloid cell lines. The generation of soluble receptor protein will permit investigations of the role of Fc alpha R in IgA-mediated immunoregulation, effector functions, and disease.
Assuntos
Antígenos CD , Receptores Fc/biossíntese , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade , DNA , Humanos , Dados de Sequência Molecular , Receptores Fc/química , Receptores Fc/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SolubilidadeRESUMO
Interleukin-8 (IL-8), neutrophil activating peptide-2 (NAP-2), and growth regulated gene (GRO, also known as melanoma growth stimulatory activity) are members of a family of peptides which are chemotactic agents for inflammatory cells such as neutrophils. Receptors have been identified for IL-8, GRO and NAP-2 on human neutrophils and granulocytic cell lines, and it has been observed that these cytokines can cross-compete for binding to a common receptor. Using the recently characterized rabbit IL-8 receptor as a probe, two classes of cDNAs, termed type 1 and type 2, were isolated from a human neutrophil library. The type 1 receptor binds only IL-8 while the type 2 receptor binds IL-8, GRO and NAP-2 at high affinity when respective cDNAs are expressed in COS-7 cells. The two cDNAs encode proteins that have an amino acid sequence identity of 77% while the type 1 and 2 receptors have an identity of 84 and 74% with the rabbit IL-8 receptor. These receptors also show significant homology with receptors for other chemotactic agents and with potential coding regions from the human cytomegalovirus genome.
Assuntos
Quimiocinas CXC , Fatores Quimiotáticos/metabolismo , Substâncias de Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Receptores de Citocinas , Receptores Imunológicos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Quimiocina CXCL1 , DNA/isolamento & purificação , Relação Dose-Resposta a Droga , Granulócitos/metabolismo , Humanos , Interleucina-8/metabolismo , Leucemia Mieloide , Camundongos , Dados de Sequência Molecular , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Interleucina-8A , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , beta-TromboglobulinaRESUMO
Human recombinant interleukin 4 (IL-4) and interleukin 7 (IL-7) have been modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 and IL-7 receptors (R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA) or immobilized CD3 monoclonal antibody the intensity of biotinylated IL-4 staining increased approximately twofold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused a considerable increase in IL-4 receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R although it was able to cause a significant increase in receptor expression on T cells co-cultured with PHA or CD3. Freshly isolated T cells expressed high levels of IL-7R, as determined by biotinylated IL-7 binding and flow cytometry, which did not change significantly with culture in medium alone. Stimulation with PHA, Concanavalin A (Con A) or CD3 had little effect on the intensity of staining. In contrast, activation with phorbol ester resulted in a decrease in IL-7R expression. Similarly, in the presence of IL-4 or IL-7, but not IL-2, the intensity of staining with biotinylated IL-7 was lowered. Analysis of purified T-cell populations showed that IL-7R were present, and IL-4R could be induced, on both CD4+ and CD8+ populations. Analysis of IL-4 receptor expression by this flow cytometric technique was supported by results from 125I-labeled IL-4 binding and by Northern blot analysis of mRNA levels. Taken together, the results of these studies show that the use of biotinylated cytokines and flow cytometry provides a very sensitive method with which to study the expression and regulation of cytokine receptors.
Assuntos
Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Linfócitos T/imunologia , Animais , Antígenos CD4 , Antígenos CD8 , Humanos , Técnicas In Vitro , Interleucina-4/metabolismo , Interleucina-7/metabolismo , Fito-Hemaglutininas/imunologia , RNA Mensageiro/biossíntese , Ratos , Receptores de Interleucina-4 , Receptores de Interleucina-7 , Subpopulações de Linfócitos T/imunologiaAssuntos
Receptores de Interleucina/química , Receptores de Interleucina/fisiologia , Sequência de Aminoácidos , Humanos , Interleucina-8/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Receptores de Interleucina/genética , Receptores de Interleucina-8A , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de SequênciaRESUMO
We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.
Assuntos
Fibroblastos/citologia , Fatores de Crescimento Transformadores/fisiologia , Northern Blotting , Contagem de Células , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Replicação do DNA , Humanos , Mitógenos/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/metabolismo , Fatores de Crescimento Transformadores/genéticaRESUMO
The purpose of this study was to determine whether or not membrane-bound and soluble forms of IL-4 receptors are expressed by isolated subsets of murine lung fibroblasts and to evaluate the potential functional consequences of IL-4 receptor triggering. Recent studies demonstrate that IL-4-synthesizing Th2 cells and mast cells are present in increased numbers in the lung during inflammation and fibrosis, suggesting that IL-4 may play a regulatory role in these events. We hypothesize that pulmonary fibroblasts and subsets thereof are intimately involved in this inflammatory response and that IL-4 is an active player in stimulating fibroblast collagen synthesis and hyperproliferation, creating a fibrotic environment in the lung. The fibroblast subsets used in these experiments differ not only in surface expression of the thymocyte-1 (Thy-1) Ag, but also in function and morphology. We now report the novel finding that IL-4 receptors are present at discordant levels on Thy-1+ and Thy-1- lung fibroblasts. IL-4R level and affinity were analyzed using a monoclonal anti-IL-4R Ab and equilibrium binding analysis with 125I-labeled IL-4. Reverse transcriptase PCR demonstrated the presence of mRNA for membrane-bound and soluble IL-4R. Lung fibroblast subsets secrete soluble IL-4R protein at dramatically different levels, as detected by an ELISA. Thy-1+ and Thy-1- lung fibroblasts were treated with IL-4 to determine whether this cytokine was profibrotic. Thy-1+ fibroblasts responded to IL-4 by proliferating and up-regulating collagen production. In contrast, Thy-1- fibroblasts proliferate to a lesser degree than Thy-1+ fibroblasts and were not stimulated to secrete increased levels of collagen. Overall, these results suggest that elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis.
Assuntos
Colágeno/biossíntese , Fibroblastos/metabolismo , Receptores Mitogênicos/metabolismo , Animais , Antígenos de Superfície/análise , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Primers do DNA/química , Fibroblastos/citologia , Expressão Gênica , Técnicas In Vitro , Interleucina-4/farmacologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Receptores de Interleucina-4 , Receptores Mitogênicos/química , Solubilidade , Antígenos Thy-1RESUMO
The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.
Assuntos
Receptores de Superfície Celular/isolamento & purificação , Útero/análise , Animais , Membrana Celular/análise , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas de Imunoadsorção , Octoxinol , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Polietilenoglicóis , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Suínos , Aglutininas do Germe de TrigoRESUMO
Human recombinant interleukin 4 (IL-4) was modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 receptors (IL-4R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA), or immobilized CD3 mAb the intensity of biotinylated IL-4 staining increased approximately 2-fold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused an increase in receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R, although it was able to induce a significant increase in receptor expression on T cells co-cultured with PHA or CD3 mAb. Flow cytometric analysis of purified T cell subsets confirmed that the up-regulation of IL-4R occurred on both CD4+ and CD8+ subpopulations. Two-color staining of T cells activated with PHA and IL-7 revealed that this increase in IL-4R expression occurred almost exclusively on cells expressing the p55 IL-2Ra subunit, although a significant number of cells expressing p55 do not express IL-4R. Analysis of IL-4R expression by this flow cytometric technique was substantiated by 125I-labeled IL-4 binding data and Northern blot analysis of IL-4R mRNA levels, suggesting that use of biotinylated human IL-4 for ligand binding and its detection by flow cytometry provides a very sensitive method for the study of IL-4R regulation.
Assuntos
Interleucina-4/metabolismo , Receptores Mitogênicos/metabolismo , Linfócitos T/imunologia , Biotina/análogos & derivados , Citometria de Fluxo , Humanos , Técnicas In Vitro , Ativação Linfocitária , RNA Mensageiro/metabolismo , Receptores de Interleucina-4 , Succinimidas , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.
Assuntos
Interleucina-4/metabolismo , Receptores Mitogênicos/metabolismo , Animais , Anticorpos Monoclonais , Sequência de Bases , Linhagem Celular , Reagentes de Ligações Cruzadas , Camundongos , Dados de Sequência Molecular , Peso Molecular , Oligonucleotídeos/química , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase , Testes de Precipitina , RNA Mensageiro/genética , Receptores de Interleucina-4 , Receptores Mitogênicos/químicaRESUMO
The effects of interleukin (IL) 4 on B cell growth and differentiation are mediated through binding of IL 4 to a specific cell surface receptor. The murine T cell IL 4 receptor (IL 4R) has recently been cloned and monoclonal antibodies (mAb) which bind specifically to the IL 4R have been developed. The ability of two of these anti-IL 4R mAb (M1 and M2) to inhibit IL 4-induced B cell functions in vitro was examined. The M1 mAb inhibited the ability of IL 4 to induce B cell proliferation in a dose-related fashion. The inhibition was specific for proliferation induced by IL 4 in that the antibody did not affect induction of proliferation by IL 1. Similarly, M1 inhibited IL 4-dependent B cell differentiation as measured by induction of IgG1 and IgE secretion, decreased IgG3 secretion, increased Ia expression, and increased Fc epsilon R (CD23) expression. In contrast, the anti-IL 4R-specific mAb M2 had no effect upon any of these activities. The ability of M1 but not M2 to inhibit IL 4-induced B cell growth and differentiation correlated with the inhibition of binding of radiolabeled IL 4 by M1. These reagents should be valuable tools with which to analyze the involvement of IL 4 in immune responses.
Assuntos
Linfócitos B/imunologia , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Superfície/biossíntese , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/biossíntese , Imunoglobulinas/análise , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Receptores Fc/biossíntese , Receptores de IgERESUMO
Macrophage inflammatory protein (MIP)-1 alpha, part of a family termed chemokines, has been implicated in suppression of hemopoietic stem and progenitor cell proliferation. The chemokine family has been organized into two subgroups with MIP-1 alpha, MIP-1 beta, macrophage chemotactic and activating factor (MCAF) and RANTES belonging to one subgroup, and GRO-alpha, MIP-2 alpha (GRO-beta), MIP-2 beta (GRO-gamma), platelet factor 4 (PF4), IL-8, and neutrophil activating peptide (NAP)-2 belonging to the other. These molecules were evaluated for effects on colony formation by human bone marrow multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. None of the chemokines stimulated colony formation in the absence of CSF, or influenced colony formation stimulated by a single growth factor such as granulocyte-macrophage-CSF or erythropoietin. However, MIP-1 alpha, MIP-2 alpha, PF4, IL-8, and MCAF suppressed in dose-response fashion colony formation of immature subsets of myeloid progenitor cells stimulated by GM-CSF plus steel factor. Effects were apparent on low density and CD34 HLA-DR(+)-sorted marrow cells in which up to 88.4% of the cells were composed of progenitor cells, suggesting direct effects on the progenitors themselves. Up to 2500-fold less of each chemokine could be used to demonstrate synergistic suppression when any two of these five chemokines were used together at low concentrations, effects also apparently directly on the progenitors. In contrast, MIP-1 beta, MIP-2 beta, GRO-alpha, NAP-2, and RANTES were not suppressive nor did they synergize with MIP-1 alpha, MIP-2 alpha, PF4, IL-8, or MCAF to suppress. However, a fivefold excess of MIP-1 beta blocked the suppressive effects of MIP-1 alpha. Similarly, a fivefold excess of either MIP-2 beta or GRO-alpha blocked the suppressive effects of IL-8 and PF4. These suppressing, synergizing and blocking effects may be of relevance to blood cell regulation.
Assuntos
Medula Óssea/efeitos dos fármacos , Citocinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Monocinas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL4 , Quimiocina CCL5 , Quimiocina CXCL2 , Fatores Quimiotáticos/farmacologia , Interações Medicamentosas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-8/farmacologia , Linfocinas/farmacologia , Proteínas Inflamatórias de Macrófagos , Camundongos , Proteínas Quimioatraentes de Monócitos , Fator Plaquetário 4/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
The potential use of soluble cytokine receptors as therapeutics in disease states when excessive or prolonged cytokine expression leads to pathogenesis is just beginning (Van Brunt, 1989). The inhibitory effects of soluble receptors have been found to be highly potent and specific for their respective cytokines (Maliszewski and Fanslow, 1990; Maliszewski et al., 1990). Recent in vivo data have shown that exogenously administered soluble receptors can function as cytokine antagonists and suppress autoimmune inflammatory responses (Jacobs et al., 1991a), allograft rejection, and alloreactivity (Fanslow et al., 1990b). The proposed frequency of administration and dosage of a therapeutic agent is dependent on the half-life of the agent and the route of administration. The elimination or half-life of a drug usually depends on its physiochemical properties (molecular size, glycosylation, isoelectric point, and hydrophobic/hydrophilic properties) (DiPalma and DiGregorio, 1990; Katzung, 1984). The half-life will also depend on the mechanism of clearance for that specific receptor. Once pharmacokinetic data are available for soluble receptors, the therapeutic potential of these molecules can be better evaluated. Only limited pharmacokinetic data are currently available for soluble cytokine receptors (Jacobs et al., 1991b). For sIL-1R, the majority of an intravenously administered dose was cleared in the second elimination phase, with a reasonably long half-life (6.3 hr), such that the entire dose was not eliminated until 35 hr. If administration is by subcutaneous injection, the half-life was even more prolonged. One explanation for the prolonged half-life is the minimal distribution to liver and kidneys and thus low levels of clearance by these organs. In contrast, elimination of intravenously administered sIL-4R was relatively rapid, with a short half-life (2.3 hr). This appeared mainly due to liver distribution and clearance, which has been the highest observed for any soluble cytokine receptor. Administering sIL-4R by subcutaneous injection significantly prolonged the half-life. This was most likely due to delaying the rate of liver distribution by slowing the rate of sIL-4R absorption into the circulation. Thus, subcutaneous injection would be the recommended route of administration for this receptor. Construction of a larger dimeric sIL-4R fusion protein did not prolong the i.v. half-life compared to that of the monomer, as the sIL-4R fusion protein was distributed to, and was cleared by, the liver to a greater degree.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Interleucina-1 , Interleucina-4 , Receptores de Superfície Celular , Receptores de Interleucina-1 , Receptores Mitogênicos , Fator de Necrose Tumoral alfa , Animais , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos BALB C/metabolismo , Farmacocinética , Receptores de Interleucina-4 , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/farmacocinética , Solubilidade , Distribuição TecidualRESUMO
CD27 is a disulfide-linked 120-kDa transmembrane glycoprotein expressed on the majority of T cells, B cells, and NK cells; it has homology to a family of molecules that includes the receptors for nerve growth factor and TNF. Previous studies strongly suggest that the CD27 molecule plays a key role in the process of T cell activation. To further determine its role in T cell activation, a recombinant soluble molecule composing only the extracellular domain of CD27 was produced by transfection of Chinese hamster ovary cells. We have defined the binding properties of recombinant soluble CD27 (rsCD27) to CD27 ligand (CD27L) cDNA transfected NIH 3T3 cells and have determined its functional effects on in vitro T cell activation as well as on PWM-driven B cell IgG synthesis. rsCD27 bound specifically to CD27L and the binding was inhibited by one of our anti-CD27 mAbs, anti-1A4, suggesting that the 1A4 epitope of CD27 plays a role in the binding to CD27L. Functionally, rsCD27 inhibited T cell proliferation induced by various stimuli, such as PHA, tetanus toxoid, and anti-CD2, as well as PWM-driven B cell IgG synthesis, similar to the effects of adding anti-1A4. Determination of CD27L expression showed that CD27L mRNA is induced rapidly on activated T and B cells. Taken together, these results provide direct evidence that CD27-CD27L interaction plays a critical role in T cell activation as well as in T cell-dependent B cell IgG synthesis, suggesting that the CD27-CD27L interaction may constitute a component of the T cell-T cell or T cell-B cell interaction seen after activation with Ag or mitogen.
Assuntos
Antígenos CD , Ativação Linfocitária , Proteínas de Membrana/fisiologia , Linfócitos T/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Formação de Anticorpos , Sequência de Bases , Ligante CD27 , Primers do DNA/química , Expressão Gênica , Ligantes , Cooperação Linfocítica , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes , SolubilidadeRESUMO
CD27 is a lymphocyte-specific member of the TNF/NGF-R family that is highly induced on T cells after TCR stimulation, primarily on lymphocytes belonging to the unprimed CD45RA+ subset. However, after prolonged activation both in vivo and in vitro, CD27 expression is gradually switched off. In search of physiologic signals that influence CD27 expression, we found that B cell lines can mediate down-regulation of CD27 surface expression on activated T cells via direct cell-to-cell contact and that preincubation of activated T cells with CD27 mAbs prevented this modulation. Based on these observations, we hypothesized that a ligand for CD27L is expressed on B cell lines and that ligand interaction would result in down-modulation of the molecule from the T cell surface. To identify this putative CD27L, mAbs were raised against the Burkitt cell line Ramos and screened for their ability to block down-modulation of CD27 on activated T cells. Using this approach, we isolated a mAb, designated 2F2, that inhibits CD27 down-regulation in a dose-dependent manner. We demonstrate that this mAb recognizes CD27L as it 1) reacted to mouse fibroblasts expressing the recently cloned CD27L, a novel surface-expressed member of the TNF gene family, and 2) prevented binding of a soluble CD27-Fc construct to a CD27L expressing cell line. Down-regulation of CD27 from the T cell surface by recombinant CD27L was shown to be at least partially caused by release of soluble CD27. The 2F2 mAb enabled us to begin to analyze the biochemical properties, tissue distribution, and function of the CD27L on human cells. From cell lysates of 125I surface-labeled Burkitt cells a complex immunoprecipitation pattern with dominant bands of 29, 55, and 122 to 127 kDa was obtained using the 2F2 mAb. Phenotypical analyses showed that freshly isolated lymphocytes lacked CD27L expression, but that expression of the molecule could be induced after in vitro stimulation of T and B cells. No expression was found on cells of the myeloid lineage or on endothelial cells. Finally, blocking of naturally expressed CD27L by the 2F2 mAb exerted a potent inhibitory effect on the proliferation of T cells in response to allogeneic B cells and PHA. The data presented in this paper identify CD27 and its ligand as potentially important structures involved in cellular interactions between T and B lymphocytes.