RESUMO
Hair cortisol might better represent chronic stress than salivary cortisol in piglets. To test this hypothesis, 24 female, 7-day old piglets were allocated to two groups and artificially reared. The piglets in the stressed group were exposed to overcrowding (0.10m2/piglet) and frequent mixing with unfamiliar piglets until the age of 28 days. The control group remained in an unchanging group at a density of 0.29m2/piglet. After 3 weeks, stressed animals had gained significantly less weight (median, here and throughout, 7.58kg) than the control animals (6.43kg; P=0.021). Additionally, hair from the stressed group contained significantly higher cortisol concentrations (87.29 vs. 75.60pg/mg hair; P=0.005), whereas salivary cortisol concentrations did not significantly differ between groups (0.30 vs. 0.25 µg/dL saliva; P=0.447). Weight gain and hair cortisol concentrations were significantly correlated (P=0.036, r=-0.430), but neither of these parameters were correlated with salivary cortisol concentrations (P=0.929, r=0.019 and P=0.904, r=0.026, respectively).
Assuntos
Criação de Animais Domésticos , Cabelo/metabolismo , Hidrocortisona/metabolismo , Saliva/metabolismo , Estresse Fisiológico , Doenças dos Suínos/diagnóstico , Animais , Animais Recém-Nascidos , Feminino , Masculino , SuínosRESUMO
To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.
Assuntos
Quimera , Epitopos/análise , Mutação , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Genes , Variação Genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Plasmídeos , Conformação Proteica , Mapeamento por Restrição , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.
Assuntos
Linfotoxina-alfa/metabolismo , Triptofano , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sobrevivência Celular , Clonagem Molecular , DNA/genética , DNA Recombinante , Escherichia coli/genética , Humanos , Linfotoxina-alfa/genética , Camundongos , Dados de Sequência Molecular , Mutação , Fenilalanina , Plasmídeos , Relação Estrutura-Atividade , Transformação Genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three-dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.
Assuntos
Linfotoxina-alfa , Fator de Necrose Tumoral alfa , Sequência de Aminoácidos , Animais , Gráficos por Computador , Cristalografia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas RecombinantesRESUMO
The induced expression of xanthine-guanine phosphoribosyl transferase (XGPRT) by low concentrations (-2 pg/ml) of interferon-alpha (IFN-alpha) or IFN-beta, in the 2fTPGH cell line caused a 50% cytotoxicity when these cells were grown in medium containing 6-thioguanine. We extended the application of this sensitive, reliable, and easy bioassay to other members of the cytokine family. To activate the IFN signaling pathway, we made receptor chimeras, consisting of the IFN type I receptor intracellular and transmembrane domains, fused to either the interleukin-5 (IL-5) receptors or erythropoietin (Epo) receptor extracellular domains as model systems. 2fTGH cells, stably transfected with these receptor chimeras, responded to very low concentrations of IL-5 or Epo (IC50 values of approximately 15 pg and 3 pg/ml, respectively) and thus can be used as a very sensitive bioassay for both ligands. Background activity of IL-5, Epo, tumor necrosis factor (TNF), IL-6, or leptin on cells that did not carry the receptor chimeras was very low. This methodology can in principle be extended to any ligand that acts via clustering of its receptors.
Assuntos
Bioensaio , Eritropoetina/análise , Interferon beta/farmacologia , Interleucina-5/análise , Pentosiltransferases/biossíntese , Agregação de Receptores , Transdução de Sinais , Corantes , Meios de Cultura/farmacologia , Indução Enzimática/efeitos dos fármacos , Eritropoetina/farmacologia , Fibrossarcoma/patologia , Violeta Genciana , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Interleucina-5/farmacologia , Interleucina-6/farmacologia , Leptina/farmacologia , Ligantes , Proteínas de Membrana , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Pentosiltransferases/genética , Agregação de Receptores/efeitos dos fármacos , Receptor de Interferon alfa e beta , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores de Interferon/efeitos dos fármacos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes , Seleção Genética , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Tioguanina/toxicidade , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The genes for a number of proteins, potentially useful in cancer therapy and collectively called "biological response modifiers", have been cloned and expressed in micro-organisms in recent years. These recombinant proteins, which are now available in pure form in nearly unlimited quantities, include interferons, interleukins and cytotoxins such as Tumor Necrosis Factor (TNF) and lymphotoxin. Most often the human gene has been cloned and expressed, with view to possible applications in medicine, but usually the mouse equivalent gene was also characterized in order to carry out syngeneic animal model experiments. TNF is selectively toxic for many transformed cell lines, either alone or in combination with interferon or inhibitors of RNA or protein synthesis. Cells sensitive to the cytotoxic action of TNF and cells unaffected by it nonetheless usually carry about an equal number of TNF receptors; hence it is the secondary, intracellular signal which makes the difference between a transformed cell and a normal, diploid cell. TNF can induce a number of different genes in a variety of cells; for example, endothelial cells express a surface antigen responsible for adherence of leucocytes. Another gene which is induced by TNF is interleukin 6 (also called 26 kDa protein or BSF-2). This interleukin, IL-6, is a growth and differentiation factor for B cells as well as for T cells; it is responsible for functions previously ascribed to hepatocyte-stimulating factor, but has no interferon activity. The toxic action of TNF on tumor cells must involve the release of arachidonic acid as phospholipase inhibitors block the TNF-induced effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Produtos Biológicos/farmacologia , Genes , Interleucinas/genética , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular Transformada , Clonagem Molecular , Citocinas , Citotoxicidade Imunológica , Regulação da Expressão Gênica , Humanos , Interleucina-6 , Interleucinas/farmacologia , Camundongos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
To test the hypothesis that a low molecular weight fraction of colostral whey could affect the morphology and barrier function of the small intestine, 30 3-d-old piglets (normal or low birth weight) were suckled (n = 5), artificially fed with milk formula (n = 5), or artificially fed with milk formula with a low molecular weight fraction of colostral whey (n = 5) until 10 d of age. The small intestine was sampled for histology (haematoxylin and eosin stain; anti-KI67 immunohistochemistry) and enzyme activities (aminopeptidase A, aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase, and sucrase). In addition, intestinal permeability was evaluated via a dual sugar absorption test and via the measurement of occludin abundance. Artificially feeding of piglets reduced final BW (P < 0.001), villus height (P < 0.001), lactase (P < 0.001), and dipeptidylpeptidase IV activities (P < 0.07), whereas crypt depth (P < 0.001) was increased. No difference was observed with regard to the permeability measurements when comparing artificially fed with naturally suckling piglets. Supplementing piglets with the colostral whey fraction did not affect BW, enzyme activities, or the outcome of the dual sugar absorption test. On the contrary, the small intestines of supplemented piglets had even shorter villi (P = 0.001) than unsupplemented piglets and contained more occludin (P = 0.002). In conclusion, at 10 d of age, no differences regarding intestinal morphology and permeability measurements were observed between the 2 BW categories. In both weight categories, the colostral whey fraction affected the morphology of the small intestine but did not improve the growth performances or the in vivo permeability. These findings should be acknowledged when developing formulated milk for neonatal animals with the aim of improving the performance of low birth weight piglets.
Assuntos
Animais Recém-Nascidos , Colostro/química , Dieta/veterinária , Suplementos Nutricionais , Mucosa Intestinal/efeitos dos fármacos , Proteínas do Leite/farmacologia , Sus scrofa/crescimento & desenvolvimento , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/veterinária , Feminino , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/anatomia & histologia , Intestino Delgado/enzimologia , Proteínas do Leite/análise , Permeabilidade/efeitos dos fármacos , Gravidez , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Proteínas do Soro do LeiteRESUMO
When the tumour necrosis factor-alpha (TNF-alpha) gene was cloned the protein became available for use in clinical trials as an antineoplastic agent. However, side effects have severely limited its application in cancer treatment. Studies on the species specificity of TNF have indicated that the p75 TNF receptor (TNFR75) may play an important role in the generation of these side effects in humans. Using human TNF mutants with selective receptor-binding properties it has been demonstrated in neutrophils and endothelium that TNFR75 is involved in the mediation of the proinflammatory activity of TNF by facilitating the p55 TNF receptor (TNFR55). However, only TNFR55 appears to be involved in mediating TNF cytotoxicity. Therefore the potential exists for the successful reintroduction of TNF-alpha, in the form of TNFR55-selective mutants, into the clinical arena with the promise of reduced side effects.
Assuntos
Antígenos CD/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Antígenos CD/química , Antígenos CD/genética , Ensaios Clínicos como Assunto , Humanos , Ligantes , Camundongos , Estrutura Molecular , Mutagênese , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Sistemas do Segundo Mensageiro , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
Previously, we reported that the cytotoxic activity of human (h) tumor necrosis factor (TNF) on murine (m) L929 cells requires the integrity of three loops (positions 30-36, 84-88 and 138-150) which cluster around the interface between each two subunits of the trimeric hTNF structure. The collection of hTNF mutants was further characterized by their activity on various human cell systems as well as by their binding to the two types of hTNF receptor (R), R55 and R75. It turned out that two amino acids (Leu29 and Arg32) were specifically involved in hR75 binding, as Leu29-->Ser (L29S) and Arg32-->Trp (R32W) mutant molecules had largely lost binding to hR75, but not to hR55. In order to screen for more highly R55-specific mutants, nine other amino acids were inserted at these two positions; only the substitutions L29G and L29Y showed an increased differential binding as compared to L29S, while no further improvement was found with mutations at position 32 compared to R32W. Biological assays mediated either by hR55 or hR75 confirmed the results obtained by physical binding to purified receptors. A similar substitution in mTNF, Arg32-->Tyr, also resulted in a preferential loss of binding to hR75 and a large decrease in mR75-mediated bioactivity. Except for the double mutant L29S-R32W, all other tested amino acid substitutions in the loops at positions 30-36 or 84-88 of hTNF led to a substantial loss of affinity for both receptors and a concomitant reduction of biological activity. In the loop at positions 138-150, the non-conservative replacement of Glu by Lys at position 146 (E146K) resulted in an even lower binding to R75 as compared to R32W, while binding on and bioactivity through R55 was only slightly reduced. Remarkably, a reversed differential binding was observed after substitution at position 143 in hTNF; replacing Asp by non-conservative residues such as Tyr, Phe or Asn resulted in a much larger decrease in binding to R55 than to R75. In conclusion, receptor-specific mutants such as R32W, E146K and D143N can be used to study the function either of R55 or R75 on different human cell types. In vivo, we presume that the R55-specific mutants will retain antitumor activity in the absence of R75-dependent, severe side effects.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Sequência de Bases , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Receptores do Fator de Necrose Tumoral/química , Relação Estrutura-AtividadeRESUMO
In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32----Trp, Leu36----Phe, Ser86----Phe and Ala84----Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84----Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29----Ser and Glu146----Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val----Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.
Assuntos
Mutagênese Sítio-Dirigida , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células L/citologia , Células L/efeitos dos fármacos , Substâncias Macromoleculares , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Mapeamento por Restrição , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The mechanism by which tumour necrosis factors (TNF and lymphotoxin, also called TNF alpha and TNF beta respectively) exert their cytotoxic activity on many malignant cells, remains largely unknown. Furthermore, the broad array of differentiation (gene induction) and mitogenic activities towards many primary cells is still a subject of intensive investigation. TNF is an important mediator in inflammation, immune responses and infection-related phenomena and these activities contribute to the severe toxicity seen when TNF is used as an anticancer agent. The first step in the mechanism of action is the specific binding of the ligand to its receptors and dissection of the molecular mechanism involved in this interaction is the subject of this review. The reasons for the interest in this aspect are obvious: first, the development of strong antagonistic TNF analogues can be useful in dampening the potentially lethal or debilitating effects of an overproduction of the cytokine (as in septic shock or rheumatoid arthritis). Secondly, since two distinct TNF receptors exist, construction of TNF muteins that distinguish between both types may lead to derivatives of this pleiotropic agent with a more restricted biological activity pattern. Ideally, one would like to develop a TNF mutant that has retained its cytotoxic action on tumour cells without inducing the deleterious systemic toxicity. Such an optimized TNF molecule could become a potent anticancer agent.
Assuntos
Conformação Proteica , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Mamíferos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The human interleukin-5 (IL-5) receptor consists of an alpha-chain that specifically binds the ligand with intermediate affinity, and a beta c-chain, that associates with the IL-5/IL-5R alpha complex, leading to a high-affinity, signal transducing receptor complex. Structure-function studies showed that modification of the putative beta c-chain binding site in IL-5 (E13Q mutein) converted the molecule into an antagonist. However, analysis of the effect of this mutant IL-5 on COS-1 cells transfected with both receptor subunits, did not show reduced interaction with the beta c subunit [Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Van der Heyden, J., Guisez, Y. & Oefner, C. (1995) Proc. Natl Acad. Sci. USA 89, 7041-7045]. To gain more insight into the mechanism of IL-5 antagonism by E13Q, we tested its biological activity on two FDC-P1 subclones that express clearly different numbers of alpha-subunits yet an almost constant number of murine beta c-subunits. Here we show that E13Q has a biological activity comparable to wild-type IL-5 only when a high number of alpha-chains is present on the cells. Confirming the critical role of the IL5R alpha cell-surface expression level, treatment with suboptimal doses of a neutralising anti-IL-5R alpha antibody results in reduced activity of the mutant but not of wild-type IL-5.
Assuntos
Regulação da Expressão Gênica/genética , Receptores de Interleucina/genética , Animais , Anticorpos Monoclonais/farmacologia , Células COS , Divisão Celular/genética , Humanos , Ligação Proteica/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina-5 , Transdução de Sinais/genética , Transfecção/genéticaRESUMO
TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.
Assuntos
Citotoxicidade Imunológica , Tolerância Imunológica , Fator de Necrose Tumoral alfa/fisiologia , Animais , Northern Blotting , Linhagem Celular , Retroalimentação , Fibrossarcoma/imunologia , Citometria de Fluxo , Expressão Gênica , Macrófagos/imunologia , Camundongos , Mutagênese Sítio-Dirigida , RNA/análise , Receptores de Superfície Celular/análise , Receptores do Fator de Necrose Tumoral , TransfecçãoRESUMO
Identifying novel targets for therapy in allergic disease: protein interactions inside the cell Therapy of allergic disease currently relies on pharmacological manipulation of mediators or immunotherapy. Drugs have been developed to target specific mediators and their receptors: for example antihistamines blocking the H1 receptor have been refined to maximize antagonism and reduce central side-effects or adverse effects of activity on other receptors such as muscarinic cholinergic receptors. Traditional pharmacological approaches identify new surface receptors against which chemists will then design or screen compounds for activity: examples are H3 or H4 histamine receptors. With the advent of the sequenced human genome we are faced with a vast array of genes and proteins that interact to define normal physiology or indeed pathology. A major challenge to biotechnology is to evolve novel techniques to understand the function and interaction of these myriad proteins. One particular area of current interest is the signalling cascades downstream of surface receptors. For many years pathways have appeared overlapping and to offer little chance of specific intervention. However, greater understanding of the complexity and integration of signalling, together with the possibility of directing drugs to specific cells has aroused considerable interest in this area for novel therapeutics. Indeed, targeting events within the cell has been done for many years with steroids. Here, Jan Tavernier and colleagues describe some signalling pathways relevant to allergic disease and potential methods for understanding protein interactions that allow mapping of the cascades. In particular they describe an elegant new system of analysis of protein-protein interactions in a mammalian system, which they have developed, termed MAPPIT. The basis of the system is an engineered receptor with JAK kinase but which lacks STAT activation sites. To the cytoplasmic end of the receptor is added a bait protein of interest, and the cell line can then be transduced with plasmid containing 'prey' cDNA from a library of interest linked to an active STAT binding site. If this cDNA encodes a protein which, upon expression, is activated and recruited to the membrane complex, it will bind to the receptor via the bait, then STAT activation will occur and activate a reporter gene system such as luciferase or puromycin resistance. This novel system allows study of known protein-protein interactions by targeted mutagenesis, or screening for novel interactions. It has the advantage over existing systems such as yeast 2 hybrid that it uses mammalian cells and thus can reproduce the physiological conditions for protein processing or activation. As new genes and proteins are linked to the atopic phenotypes, systems such as this hold promise of rapidly defining their function and interacting proteins and may be important in linking genomics and proteomics with function and pharmacology in the future.
Assuntos
Citocinas/metabolismo , Mamíferos , Receptores de Citocinas/genética , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Animais , Humanos , Hipersensibilidade/metabolismoRESUMO
In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference.
Assuntos
Regulação para Baixo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Animais , Western Blotting , Linhagem Celular , Separação Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Citometria de Fluxo , Humanos , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Janus Quinase 1 , Cinética , Ligantes , Luciferases/metabolismo , Proteínas de Membrana , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Receptor de Interferon alfa e beta , Receptores da Eritropoetina/metabolismo , Receptores de Interferon/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , TYK2 Quinase , Transcrição Gênica , Transfecção , Tirosina/metabolismoRESUMO
We constructed chimeric receptors wherein the extracellular domain of the erythropoietin receptor (EpoR) was fused to the transmembrane and intracellular domains of the interferon (IFN) type I receptor subunits, IFNaR1 or IFNaR2-2. Transfection into 2fTGH and Tyk2-deficient 11,1 cells showed that EpoR/IFNaR2-2 alone was able to transduce a signal upon stimulation with erythropoietin (Epo), as judged by induction of the interferon type I-inducible 6-16 promoter. In contrast, protection against infection with encephalomyocarditis virus or vesicular stomatitis virus was reduced or absent, respectively. To further investigate the role of IFNaR1 in the induction of an antiviral state, we analyzed the Epo- versus IFNalpha-induced transcription of a set of genes, involved in antiviral protection. Up to 24 h after stimulation with Epo or IFNalpha, comparable transcription of the p56, dsRNA-dependent protein kinase, 2'-5'A synthetase, and MxA genes was seen. However, at later time points, only in the case of Epo induction, a sharp decrease of mRNA levels was observed. Western blotting analysis of dsRNA-dependent protein kinase showed a similar pattern at the protein level. Taken together, our results imply a role for IFNaR1 in the induction of sustained mRNA and protein levels that are likely required for optimal antiviral activity.
Assuntos
Regulação da Expressão Gênica , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Receptores de Interferon/metabolismo , Fosfatase Alcalina/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Fibrossarcoma/metabolismo , Humanos , Proteínas de Membrana , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta , Receptores da Eritropoetina/metabolismo , Receptores de Interferon/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Temperatura , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-alpha (TNF-alpha), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-alpha inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4, and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-alpha was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-alpha neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-alpha to accelerate apoptosis was lost if neutrophils were primed with 1 micromol/L PAF or aged for 6 hours before TNF-alpha addition. The TNFR55-selective TNF-alpha mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-alpha. Although the TNFR75-selective mutant (D143F) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-alpha-stimulated apoptosis. Hence, TNF-alpha has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.
Assuntos
Antígenos CD/fisiologia , Apoptose/fisiologia , Neutrófilos/citologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Cinética , Leucotrieno B4/farmacologia , Lipopolissacarídeos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Ácido Fítico/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mutação , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Sítios de Ligação , Moléculas de Adesão Celular/biossíntese , Sobrevivência Celular , Células Cultivadas , Selectina E , Endotélio Vascular/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Inflamação , Interleucina-8/biossíntese , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genéticaRESUMO
The remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines. But clinical trials in cancer patients have on the whole been disappointing, and it has been estimated that a TNF dose would be effective only at 5-25 times the maximum tolerated dose. High TNF concentrations give a much more pronounced antitumour activity in mice, in which murine TNF is about 50-fold more systemically toxic than human TNF. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines. This dual action of TNF may be explained by the existence of two types of receptor for TNF: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF; the larger, TNF-R75, is also present on many cell types, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that sill interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Mutação Puntual , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Cicloeximida/farmacologia , Feminino , Humanos , Cinética , Células L , Camundongos , Camundongos Nus , Transplante de Neoplasias , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genéticaRESUMO
The receptor for interleukin 5 (IL-5) consists of a cytokine-specific alpha chain (IL-5Ralpha) and a signaling beta chain, which is shared with interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). These 3 cytokines can act in eosinophil development and activation in vitro, but gene deletion or antibody blocking of IL-5 largely ablates eosinophilic responses in models of allergic disease or helminth infection. We investigated factors acting in differential IL-5Ralpha gene splicing to generate either the membrane-anchored isoform (TM-IL-5Ralpha) which associates with the common beta chain to allow IL-5 responsiveness, or a secreted, antagonist variant (SOL-IL-5Ralpha). In a murine myeloid cell line (FDC-P1), transfected with minigenes allowing expression of either IL-5Ralpha variant, IL-5 itself, but not IL-3 or GM-CSF, stimulated a reversible switch toward expression of TM-IL-5Ralpha. A switch from predominantly soluble isoform to TM-IL-5Ralpha messenger RNA (mRNA) expression was also seen during IL-5-driven eosinophil development from human umbilical cord blood-derived CD34(+) cells; this was accompanied by surface expression of IL-5Ralpha and acquisition of functional responses to IL-5. IL-3 and GM-CSF also supported eosinophil development and up-regulation of TM-IL-5Ralpha mRNA in this system, but this was preceded by expression of IL-5 mRNA and was inhibited by monoclonal antibody to IL-5. These data suggest IL-5-specific signaling, not shared by IL-3 and GM-CSF, leading to a switch toward up-regulation of functional IL-5Ralpha and, furthermore, that IL-3 and GM-CSF-driven eosinophil development is dependent on IL-5, providing an explanation for the selective requirement of IL-5 for expansion of the eosinophil lineage. (Blood. 2000;95:1600-1607)