RESUMO
The effect of potent sheep anti-mouse interferon globulin was investigated in several different experimental virus diseases of mice. In anti-interferon globulin-treated mice infected intraperitoneally with herpes simplex virus (HSV) type I, the latent period was shortened, and the overall LD50 was increased several hundredfold compared to virus-infected control mice. When HSV was inoculated subcutaneously all anti-interferon globulin-treated mice died, whereas only 5% of virus-infected control mice died. Subsequent treatment with anti-interferon globulin of previously HSV-infected mice did not result in reactivation of HSV. Treatment of adult mice with anti-interferon globulin resulted in an earlier appearance of MSV-induced tumors, a greater number of mice bearing tumors, an increase in tumor size, and an increase in the duration of tumors. All tumors eventually regressed despite reinjection of anti-interferon globulin. Anti-interferon globulin treatment resulted in a rapid onset of disease and death in adult mice inoculated (intranasal) with VSV and in newborn mice infected with NDV. Anti-interferon globulin exerted no effect on the course of influenza virus infection of mice. We conclude that the early production of interferon is an importane element in the response of the mouse to several viruses exhibiting different pathogeneses.
Assuntos
Interferons/fisiologia , Viroses/microbiologia , Animais , Animais Recém-Nascidos , Reações Antígeno-Anticorpo , Vírus do Sarcoma Aviário/crescimento & desenvolvimento , Herpes Simples/microbiologia , Vírus da Influenza A/crescimento & desenvolvimento , Interferons/imunologia , Camundongos , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Sarcoma Experimental/microbiologia , Simplexvirus/crescimento & desenvolvimento , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimentoRESUMO
Friend erythroleukemia cells (FLC) (H-2d) injected intravenously into adult syngeneic DBA/2 or allogeneic C57B1/6 (H-2b) or C3H (H-2k) mice lodge in the liver but only multiply in the liver of syngeneic mice. Our results indicated that endogenous IFN-alpha/beta was a crucial factor in preventing the multiplication of FLC in the liver of adult allogeneic mice. (a) Treatment of allogeneic adult C57B1/6 or C3H mice with polyclonal antibody to mouse IFN-alpha/beta (but not antibody to IFN-gamma) completely abrogated the resistance to the multiplication of FLC in the liver and 87% of tumor-injected, antibody-treated C57B1/6 mice died with extensive tumor involvement of the liver. In contrast, after intravenous inoculation FLC do not multiply at all (or very rarely) in the liver of adult C57B1/6 mice left untreated or treated with a variety of control globulins, and no deaths occurred. (b) 8 h after intravenous inoculation of FLC, poly(A)+ RNA hybridizable with specific DNA probes for mouse IFN-alpha or -beta (but not -gamma) was present in the liver of injected C57B1/6 mice. Using the expression of the Mx protein as an indicator of the presence of IFN-alpha/beta, we showed that Mx+ congenic C57B1/6 mice injected with FLC exhibited a marked increase in the expression of the Mx protein in the liver, spleen, kidney and lung, and this increase was blocked by treatment of mice with antibody to IFN-alpha/beta. The possibility that different host mechanisms are elicited depending on the site of tumor growth in allogeneic mice is discussed. IFN-alpha/beta appears to be of particular importance in determining the resistance of the liver to FLC in allogeneic mice.
Assuntos
Proteínas de Ligação ao GTP , Interferon Tipo I/imunologia , Leucemia Eritroblástica Aguda/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Fatores Etários , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Antivirais/genética , Antivirais/imunologia , Sondas de DNA , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/patologia , Neoplasias Hepáticas Experimentais/patologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Resistência a Myxovirus , Transplante de Neoplasias , Proteínas/genética , Proteínas/imunologia , RNA Mensageiro/análise , Soroglobulinas/imunologia , Células Tumorais CultivadasRESUMO
The role of interferon in the pathogenesis of encephalomyocarditis (EMC) virus infection was determined by treating mice with potent, partially purified sheep anti-mouse interferon globulin. In control mice, EMC virus was present in low titers in various visceral organs but attained high titers in the brain towards the 4th to 5th day, at which time mice died with signs of central nervous system disease. In mice treated with anti-mouse interferon globulin, virus was present in high titer in visceral organs 24--36 h after viral inoculation and virtually all mice were dead by 45 h. This rapid evolution of EMC virus infection was not observed in mice treated with the globulin fraction prepared from a normal sheep, from a sheep exhibiting a low anti-mouse interferon-neutralizing titer, nor from a sheep having a high titer of antibody to human leukocyte interferon. The experimental results indicated that anti-interferon globulin neutralized the interferon liberated by virus-infected cells, thus permitting extensive virus multiplication in several visceral organs. We conclude that interferon is an important early component of host resistance to this virus infection.
Assuntos
Vírus da Encefalomiocardite/crescimento & desenvolvimento , Infecções por Enterovirus/microbiologia , Interferons/fisiologia , Complexo Antígeno-Anticorpo , Reações Antígeno-Anticorpo , Interferons/análise , Interferons/imunologiaRESUMO
Injection of DBA/2, C57Bl/6, or BALB/c mice with antibody to mouse interferon alpha/beta enhanced the i.p. transplantability of six different murine tumors, as manifested by an increase in the percentage of tumor-bearing mice and a decrease in survival time. The effect was observed in mice injected with antibody to interferon raised in three sheep, a goat, and a rabbit, but not with sheep antibody to "impurities" present in the mouse interferon preparations or with normal sheep or goat globulins. The enhancement in transplantability was most marked when tumor cells had been previously passaged in vitro and were of low tumorigenicity. Analysis of some of the experimental conditions using interferon-sensitive and interferon-resistant lines of Friend erythroleukemia cells (FLC) showed that the enhancing effect was observed over a wide range of tumor cell inocula, was directly related to the amount of antibody to interferon injected and was most pronounced when antibody was administered at the time of tumor cell injection. Enhancement was also observed when FLC were injected subcutaneously (s.c.). Antibody did not act directly on the tumor cells in vitro. Although we were unable to demonstrate any biologically active interferon in mice before or after tumor cell inoculation, the results suggest that endogenous interferon is present and plays a role in inhibiting the transplantability of some murine tumors in immunocompetent mice.
Assuntos
Interferon Tipo I/fisiologia , Neoplasias Experimentais/imunologia , Animais , Anticorpos/administração & dosagem , Feminino , Sobrevivência de Enxerto , Interferon Tipo I/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Neoplasias Experimentais/mortalidade , Fatores de TempoRESUMO
To characterize the mechanisms underlying apoptosis induced by viral infection, transcriptional activation of genes encoding members of the 'BH3-only' family of proteins was analysed during the course of virus infection. Among these genes, only NOXA is transcriptionally activated by vesicular stomatitis virus (VSV), sendai virus (SV), measles virus, herpes simplex virus, or dsRNA and required for efficient apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53, but requires the presence of interferon regulatory factor 1 (IRF-1), IRF-3 and cAMP-responsive element binding protein (CREB). Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving different pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by Toll like receptor 3 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection correlates with a 10 kDa increase in its molecular weight, suggesting a covalent linkage with a previously unknown regulatory polypeptide.
Assuntos
Apoptose , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Vírus de RNA/fisiologia , RNA Viral/genética , Proteína Supressora de Tumor p53/metabolismo , Northern Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Vírus do Sarampo/fisiologia , Fosfolipases A/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vírus de RNA/classificação , Vírus Sendai/fisiologia , Transdução de Sinais , Simplexvirus/fisiologia , Receptor 3 Toll-Like/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Dimethyl sulfate (DMS) genomic footprinting revealed the presence of putative regulatory proteins attached to specific sequences of the promoter region of the interferon (IFN) alpha-1 gene in normal human tissue. The pattern of protein-DNA interactions observed for the human alpha-1 promoter in freshly isolated human spleen cells was identical to that seen in DNA from the B-cell line Namalwa. The protein interactions involving the human IFN alpha-1 promoter spanned a region from positions -38 to -174 relative to the cap site which encompasses that part of the IFN alpha-1 promoter previously shown by deletion analysis to confer virus inducibility on the IFN alpha-1 gene. DNase I footprinting performed on isolated nuclei revealed a pattern of protein-DNA interactions for the promoter region of the IFN alpha-1 gene similar to that obtained with DMS footprinting performed on whole cells, with the appearance or disappearance of only a few additional protected nucleotides outside the region identified by the use of DMS. These results provide the first direct evidence for the presence of proteins bound in vivo to those parts of the IFN alpha-1 promoter between positions -64 and -109 previously shown by deletion analysis to confer virus inducibility on the IFN alpha-1 gene. The pattern of protein-DNA interactions observed for the IFN alpha-1 promoter after virus induction was identical to that seen before induction, in keeping with the finding that many transcriptional activators are present in both induced and uninduced cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes , Interferon Tipo I/genética , Regiões Promotoras Genéticas , Sequência de Bases , Linhagem Celular , DNA/genética , DNA/isolamento & purificação , Desoxirribonuclease I , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Mapeamento de Nucleotídeos , Baço/imunologia , Ésteres do Ácido SulfúricoRESUMO
Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.
Assuntos
Regulação da Expressão Gênica , Genes , Interferon Tipo I/genética , Regiões Promotoras Genéticas , Linhagem Celular , Cicloeximida/farmacologia , Expressão Gênica , Células HeLa , Humanos , Interferon Tipo I/farmacologia , Vírus da Parainfluenza 1 Humana/genética , Poli I-C/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas Recombinantes , Transcrição Gênica/efeitos dos fármacosRESUMO
Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.
Assuntos
Interferon Tipo I/fisiologia , Oncogenes , Transcrição Gênica , Linhagem Celular , Células Clonais , Humanos , Cinética , RNA Mensageiro/genéticaRESUMO
A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.
Assuntos
Interferon Tipo I/genética , Camundongos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Códon/genética , Genes , Humanos , Sequências Repetitivas de Ácido Nucleico , Regiões Terminadoras GenéticasRESUMO
Affinity purified, freshly isolated CD34+ progenitors were shown to express low levels of type I interferon (IFN) receptors (740 +/- 60 binding sites/cell, K(d) 0.7 +/- 0.04 nM) determined by Scatchard's analysis using a radiolabelled, neutralizing, monoclonal antibody directed against the IFNAR1 chain of the human type I IFN receptor. Treatment of freshly isolated (day 0), highly purified (>95% pure) CD34+ cells with recombinant IFN-alpha resulted in rapid tyrosine phosphorylation and activation of STAT1, Tyk2 and JAK1 as shown by Western immunoblotting. Similarly, IFN treatment was shown by confocal microscopy to result in rapid nuclear localization of the transcription factors IRF1 and STAT2, demonstrating the presence of functional IFN receptors on freshly isolated (day 0) CD34+ cells. The number of specific type I IFN receptor binding sites expressed on hematopoietic progenitor cells increased to some 1440 +/- 40 per cell after 11 days of cultivation of CD34+ cells in vitrosuggesting that receptor expression increases with cell differentiation. IFN-mediated signal transduction and the inhibitory effect of IFN-alpha on 7 or 14 days CFU-GM and BFU-E colony formation was abrogated in the presence of the anti-IFNAR1 mAb, indicating that IFN-alpha acts directly on the proliferation of human hematopoietic progenitor cells via receptor activated signal transduction without excluding the induction of other cytokines or growth factors by residual accessory cells.
Assuntos
Antígenos CD34/análise , Células-Tronco Hematopoéticas/metabolismo , Receptores de Interferon/fisiologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Anticorpos Monoclonais/farmacologia , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/química , Humanos , Fator Regulador 1 de Interferon , Interferon-alfa/antagonistas & inibidores , Janus Quinase 1 , Cinética , Proteínas de Membrana , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/antagonistas & inibidores , Receptores de Interferon/imunologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , TYK2 Quinase , Transativadores/metabolismoRESUMO
Constitutive expression of IFN-alpha5 and IFN-beta was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase-polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon-induced transcription factor complex ISGF3 was also detected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in maintaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN-alpha5 and IFN-beta are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis.
Assuntos
Interferon-alfa/biossíntese , Interferon beta/biossíntese , Leucócitos/imunologia , Monócitos/imunologia , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Consenso , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes MHC Classe I , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Luciferases/biossíntese , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Type I interferons are potent immuno-modulatory cytokines that enhance expression of the major histocompatibility complex (MHC) class I antigens, T-cell cytotoxicity, and natural killer (NK) cell activity, all of which are implicated in graft rejection. A monoclonal antibody (mAb) directed against the extracellular domain of the human interferon gamma (IFN-gamma) receptor (IFN-alpha R), which inhibits both the binding and biological activity of all the type I IFNs tested, exerted a dose-dependent inhibition of the mixed lymphocyte reaction and induced permanent survival of skin allografts in MHC-divergent Cynomologus monkeys treated with a subeffective dose of cyclosporin A. Marked differences were observed in the composition of T lymphocyte subpopulations in anti-IFN-alpha R mAb-treated animals relative to the various control groups. Skin biopsies from animals treated with anti-IFN-R Mab + cyclosporin A revealed very low levels of MHC class I and class II antigen expression and the absence of histological signs of rejection, whereas skin biopsies from control animals exhibited high levels of MHC antigen expression and the histological signs of acute rejection, including a pronounced lymphocytic infiltrate, edema, and necrosis. No monkey antibodies (IgG) to the mouse anti-human IFN-alpha R mAb were detected in the serum of any of the animals treated with the anti-IFN-alpha R mAb either alone or together with cyclosporin A. Treatment of lethally irradiated Cynomologus monkeys with the anti-IFN-alpha R mAb together with a subeffective dose of cyclosporin A was also found to markedly enhance the survival of animals grafted with allogeneic bone marrow cells from donors differing in both MHC class I and class II antigens. These results show that selective and lasting immunosuppression can be obtained by the short-term administration of an IFN-alpha antagonist together with a subeffective dose of cyclosporin A, and may have important implications for the therapy of human allograft rejection.
Assuntos
Rejeição de Enxerto/imunologia , Interferon Tipo I/imunologia , Animais , Humanos , Macaca fascicularis , CamundongosRESUMO
Two monoclonal antibodies that recognize different epitopes of the extracellular domain of one of the proteins that constitute the type I interferon receptor were used to delineate the interferon binding site. Antibody 64G12 both inhibits the binding of radiolabeled interferon-alpha 2 and IFN-alpha 8 to their cell surface receptors and neutralizes the antiviral and antiproliferative actions of all the type I interferons tested, including IFN-beta, IFN-omega, and human leukocyte IFN, a mixture of different interferon-alpha isotypes. Antibody 34F10 recognizes the type I interferon receptor with an affinity similar to that of the MAb 64G12 but does not inhibit either the binding or the biologic activity of any of the type I interferons tested. Both antibodies recognize a protein of 105 +/- 5 kD from either Daudi or Ly28 cells. Immunoprecipitation following surface iodination demonstrated that the neutralizing MAb recognizes a protein of 105 kD and the nonneutralizing MAb a protein of 110 kD in extracts of Daudi cells. A second less intense band was also detected by both antibodies. Cross-linking of IFN-alpha 2 to its receptor before immunoprecipitation prevented the neutralizing antibody from immunoprecipitating the receptor protein, but the nonneutralizing MAb was still able to recognize a 140 kD protein corresponding to the cross-linked interferon-receptor protein complex. Thus, an interferon binding domain appears to be localized in a region between amino acids 23 and 229 of the extracellular domain of a transmembrane protein that forms part of the type I interferon receptor complex containing the epitopes recognized by each antibody.
Assuntos
Interferon Tipo I/química , Receptores de Interferon/química , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Western Blotting , Reagentes de Ligações Cruzadas , Epitopos/química , Epitopos/imunologia , Humanos , Interferon Tipo I/imunologia , Ligantes , Proteínas de Membrana , Testes de Neutralização , Testes de Precipitina , Receptor de Interferon alfa e beta , Receptores de Interferon/imunologia , Células Tumorais CultivadasRESUMO
Oromucosal administration of murine interferon-alpha/beta (IFN-alpha/beta) or individual recombinant species of murine IFN-alpha, IFN-beta, or IFN-gamma or recombinant human IFN-alpha1-8, which is active in the mouse, exerted a marked antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or varicella zoster virus (VZV). The effects observed were dose dependent and similar in magnitude to those observed following parenteral administration of the same dose of IFN. No antiviral activity was observed after oromucosal administration of murine IFN-alpha/beta in animals in which the IFN receptor had been inactivated by homologous recombination. In contrast to parenteral treatment, oromucosal IFN therapy was found to be ineffective when IFNs were administered before virus infection. Oromucosal administration of IFN-alpha also exerted a marked antitumor activity in mice injected i.v. with highly malignant Friend erythroleukemia cells or other transplantable tumors, such as L1210 leukemia, which has no known viral etiology, the EL4 tumor, or the highly metastatic B16 melanoma. These results show that high doses of IFN can be administered by the oromucosal route apparently without ill effect, raising the possibility that the oromucosal route will prove to be an effective means of administering high doses of IFN that are clinically effective but poorly tolerated.
Assuntos
Antineoplásicos/uso terapêutico , Antivirais/uso terapêutico , Interferon Tipo I/uso terapêutico , Administração Oral , Animais , Vírus da Encefalomiocardite/patogenicidade , Feminino , Vírus da Leucemia Murina de Friend/patogenicidade , Herpesvirus Humano 3/patogenicidade , Humanos , Dose Letal Mediana , Leucemia Experimental/tratamento farmacológico , Masculino , Camundongos , Mucosa Bucal , Neoplasias Experimentais/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/patogenicidadeRESUMO
Oromucosal (o.m.) administration of interferon-alpha (IFN-alpha) during either allergic sensitization (days 0-6) or the hypersensitive response (days 11 and 12) or both periods caused a dose-dependent reduction in allergen-specific IgE production and allergen-induced eosinophil recruitment in mice sensitized to ragweed pollen, a common allergen in humans. Treatment during the hypersensitive response period alone appeared to be most effective. Oromucosal treatment was as effective as intraperitoneal (i.p.) treatment, with maximum inhibition of both allergen-specific IgE production and allergen-induced eosinophil recruitment observed at a dose of a 1000 IU IFN-alpha. Treatment of animals with up to 10(5) IU murine IFN-alpha/beta (MuIFN-alpha/beta) by either the om. or i.p. route did not inhibit significantly allergen-specific IgG production, which may even have been increased at certain doses of IFN. Treatment of animals with up to 10(5) IU MuIFN-alpha/beta by either the o.m. or i.p. route did not affect significantly total serum IgE or IgG levels. Oromucosal administration of IFN-alpha reduced allergen-specific IgE production and allergen-induced eosinophil recruitment in the absence of detectable toxicity, the induction of H(2) antigen expression, and 2',5'-oligoadenylate synthetase activity associated with parenteral administration of IFN-alpha and thus may find application for the treatment of asthma and associated viral infections.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Imediata/imunologia , Imunização , Interferon Tipo I/administração & dosagem , Pólen/imunologia , 2',5'-Oligoadenilato Sintetase/metabolismo , Administração Intranasal , Alérgenos/administração & dosagem , Animais , Asma/enzimologia , Asma/imunologia , Asma/patologia , Movimento Celular/imunologia , Eosinófilos/imunologia , Eosinófilos/patologia , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Hidrocortisona/administração & dosagem , Hipersensibilidade Imediata/enzimologia , Hipersensibilidade Imediata/patologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Intraperitoneais , Interferon Tipo I/uso terapêutico , Interleucina-12/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
The specific IgM and IgG antibody responses to subcutaneous (s.c.) treatment of mice with recombinant human IFN-alpha2a (rHuIFN-alpha2a) or IFN-beta were inhibited in a dose-dependent manner by prior oromucosal (o.m.) administration of rHuIFN-alpha2a or IFN-beta, respectively. Pretreatment of animals once a day for 7 days by the o.m. route with the highest dose of IFN-alpha2a tested (10(7) IU) resulted in complete inhibition of the peak IFN-alpha2a-specific IgG antibody response detected 28 days after subsequent s.c. injection of IFN-alpha2a (p < 0.001). Similarly, prior o.m. administration of 1-10 microg rHuGM-CSF per day for 7 days resulted in a statistically significant (p < 0.001) inhibition of the peak GM-CSF-specific IgG antibody response detected 28 days after s.c. administration of GM-CSF. In contrast, prior o.m. treatment with a quantity of bovine serum albumin (BSA) (100 microg) or human serum albumin (HSA) (10 microg) equivalent, respectively, to the protein content of the highest dose of IFN-alpha2a or GM-CSF administered by the o.m. route, did not affect significantly the IFN-alpha2a-specific or GM-CSF-specific IgG antibody responses detected on subsequent s.c. administration of IFN-alpha2a or GM-CSF. Oromucosal administration of IFN-alpha2a, IFN-beta, or GM-CSF alone did not induce detectable IFN-alpha2a-specific, IFN-beta-specific, or GM-CSF-specific IgM or IgG antibody responses at any of the time points tested. These results suggest that short-term o.m. administration of a recombinant protein is an effective means of inducing peripheral tolerance to subsequent parenteral administration of a therapeutic protein.
Assuntos
Tolerância a Medicamentos , Proteínas Recombinantes/farmacologia , Administração Intranasal , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Injeções Subcutâneas , Interferon Tipo I/administração & dosagem , Interferon Tipo I/farmacologia , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêuticoRESUMO
The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa B enhancer and a NFIL-6 (C/EBP beta) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the kappa B element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1 beta or TNF-alpha. Gel-retardation analysis of nuclear extracts from IL-1 beta or TNF-alpha-treated cells using specific antibodies has shown that at least five retarded complexes bind to the IL-6 kappa B element in addition to NF-kappa B. Furthermore, apart from p50 (NF-kappa B1) and p65 (RelA), no other members of the Rel family are present in these complexes. Comparative analysis with the kappa B enhancer of the immunoglobulin kappa chain gene shows that three of these complexes bind specifically to the IL-6 kappa B enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in which NF-kappa B subunits, NFIL-6, and NFIL-6 beta (C/EBP delta), were overexpressed in cells transfected with mutated IL-6 enhancer elements linked to a reporter gene show that interaction between members of the two families of factors is required for activation of the IL-6 gene in the absence of the NFIL-6 binding site. We conclude that the kappa B enhancer of the IL-6 promoter is the IL-1 beta and TNF-alpha responsive element and that its activity is dependent on the direct interaction of NF-kappa B with non-Rel transcription factors.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Elementos Facilitadores Genéticos , Interleucina-1/farmacologia , Interleucina-6/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
In this work, we present evidence that enriched human peripheral blood T lymphocytes, depleted of contaminating monocytes, rapidly express tumor necrosis factor alpha (TNF-alpha) mRNA when exposed to low doses of gamma-irradiation. In total PBL, TNF-alpha mRNA accumulation increased threefold as early as 30 minutes following exposure to 4 Gy and then declined to the baseline level by 3-5 h, as measured by the reverse transcriptase-polymerase chain reaction (RT-PCR). The increase in TNF-alpha mRNA was also observed in populations of enriched T cells and decreased when the dose of irradiation was increased to 10 Gy, strongly suggesting that T lymphocytes, the most radiosensitive cells of the body, contributed directly to the increase of TNF-alpha mRNA. A good correlation was found between mRNA expression and TNF-alpha protein secretion. Interestingly, a eightfold increase in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA accumulation was also detected in both PBL and enriched T cells irradiated at 4 Gy for 3 h compared with unirradiated cells. This irradiation effect was almost completely abolished, however, following exposure to 10 Gy. Together these data suggest that T cells are responsible for the irradiation-induced expression of TNF-alpha and GAPDH.
Assuntos
Raios gama , Linfócitos T/efeitos da radiação , Fator de Necrose Tumoral alfa/biossíntese , Sequência de Bases , Células Cultivadas , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Valores de Referência , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Mucosal administration of the Th1 stimulatory cytokines interleukin-2 (IL-2), IL-12, IL-15, IL-18, or granulocyte-macrophage colony-stimulating factor (GM-CSF) induced antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV) similar to that observed following parenteral administration. In contrast, mucosal administration of the Th2 stimulatory cytokines IL-4, IL-5, IL-10, or IL-13 did not affect significantly the survival of EMCV-infected animals. Mucosal administration of IL-2 or IL-12 also exerted a marked antitumor activity in mice inoculated intravenously with Friend erythroleukemia cells. Recombinant IL-2 and IL-18, but none of the other recombinant cytokines tested, induced low levels of IFN in vitro. Polyclonal antibodies to both mouse and human interferon-alpha/beta (IFN-alpha/beta) abrogated the antiviral activity of IL-2 in vivo, even though the anti-human IFN-alpha/beta antibody did not neutralize mouse IFN-alpha/beta, and neither antibody bound to IL-2. IL-15 did not exhibit antiviral activity in IFN-alpha/beta R-/- mice, which are deficient in natural killer (NK) cell activity. These results suggest that mucosal Th1 cytokine therapy induces a soluble factor or activates a specific cell population in the lymphoid or epithelial tissue of the oropharyngeal cavity, which potentiates elimination of virus-infected or neoplasic cells systemically.
Assuntos
Antineoplásicos/uso terapêutico , Antivirais/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Interferons/uso terapêutico , Interleucinas/uso terapêutico , Animais , Vírus da Leucemia Murina de Friend/efeitos dos fármacos , Humanos , Dose Letal Mediana , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/patologia , Camundongos , Mucosa/efeitos dos fármacos , Transplante de Neoplasias , Proteínas Recombinantes/uso terapêutico , Taxa de Sobrevida , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacosRESUMO
Intraperitoneal (i.p.) administration of 20,000 IU recombinant murine IFN-alpha (rMuIFN-alpha) was highly effective in protecting mice challenged i.p. with doses of encephalomyocarditis virus (EMCV) ranging from 44 to 440 LD(50) (p<0.001). Oromucosal (o.m.) IFN therapy was also found to be effective in protecting mice challenged with a lethal dose of EMCV. Thus, 40% of animals infected with 44 LD(50) of EMCV and treated o.m. with 20,000 IU rMuIFN-alpha survived infection with a mean survival time of 12.0 +/- 2.46 days relative to a mean of 6.11 +/- 0.38 days in the control group (p<0.05). Oromucosal IFN therapy was found to be ineffective, however, in animals infected with higher doses of EMCV (88-440 LD(50)), even though intraperitoneal administration of the same dose of rMuIFN-alpha resulted in the survival of 90%, 50%, and 60% of animals infected with 88, 220, and 440 LD(50) of EMCV, respectively. These results suggest that oromucosal IFN therapy is effective at relatively low viral load only and that the mechanism of action of oromucosal IFN therapy may be different from that of parenterally administered IFN. Our results suggest that oromucosal IFN therapy may be most effective in chronic viral infections as an alternative to parenterally administered IFN, which is clinically effective but poorly tolerated.