RESUMO
Type III interferons (IFN-lambdas, IFN-λs) are important antiviral cytokines that can also modulate immune responses by acting through a heterodimeric receptor composed of the specific and limited expressed IFN-λR1 chain and the ubiquitous IL-10R2 chain, which is shared with IL-10 family cytokines. Conflicting data have been reported regarding which cells express the IFN-λR1 subunit and directly respond to IFN-λs. This is, in part, owing to transcript levels of the IFN-λR1 gene, IFNLR1, not always correlating with cell surface protein levels. In this study, we tested a panel of novel monoclonal antibodies (mAbs) that specifically recognize human IFN-λR1. Initially, antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA), from which a subset of antibodies was selected for additional flow cytometry and neutralization assays. We further characterized two antibodies based on their strong ELISA binding activity (HLR1 and HLR14) and found only HLR14 could reliably detect cell surface IFN-λR1 protein on a variety of cell lines by flow cytometry. HLR14 could also detect IFN-λR1 protein on certain primary human blood cells, including plasmacytoid dendritic cells and B cells from peripheral blood. Availability of the HLR14 mAb will enable the quantification of IFN-λR1 protein levels on cells and better characterization of the cell specificity of the IFN-λ response.
Assuntos
Interferons , Receptores de Interferon , Humanos , Receptores de Interferon/genética , Interferon lambda , Proteínas de Membrana , Anticorpos Monoclonais , CitocinasRESUMO
Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.
Assuntos
Antivirais , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Replicação Viral , Humanos , Substituição de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Endonucleases/genética , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Nucleotidiltransferases/genética , Tiepinas/farmacologia , Tiepinas/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Mutação , Linhagem CelularRESUMO
Influenza is an acute respiratory disease that can cause local annual epidemics and worldwide pandemics of different morbidity and mortality. Our understanding of host factors that modulate the frequency and severity of influenza virus infections is less than complete. In this study, we examined the inter-individual variations in the innate immune responses to H1N1 and H3N2 influenza A viruses (IAV) using primary cultures of normal human bronchial epithelial (NHBE) cells derived from two different donors (D1 and D2). Although IAV replication kinetics were similar in cultures derived from these two donors, the levels of type III interferons (IFNs) were significantly higher in D1 cells compared to D2 cells (Ë31-fold↑ in D1 cells versus D2 cells; P < 0.05). The levels of IFN-λ1 protein at individual time points as well as the total amounts of IFN-λ1 secreted over 72 h were also significantly higher in D1 than in D2 NHBE cells (0.7-7.7-fold↑, P < 0.05). The relative levels of IFN-stimulated gene (ISG) expression also differed significantly between D1 and D2 cells. Our data indicate that donor-specific differences can result in significant differences in IFN and ISG induction by human airway epithelium.
Assuntos
Células Epiteliais/imunologia , Imunidade Inata , Interferons/genética , Animais , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Cães , Células Epiteliais/virologia , Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Células Madin Darby de Rim Canino , Replicação ViralRESUMO
Macrophages coexpress both the interleukin (IL)-2Rγ chain (γ(c)) and IL-13Rα1. These receptor chains can heterodimerize with IL-4Rα to form type I or type II IL-4 receptor complexes, respectively. We used macrophages derived from Il2rg and Il13ra1 knockout (KO) mice to evaluate the requirements for these receptor chains for induction of the alternative macrophage activation (AMA) pathway by IL-4 and IL-13. Absence of γ(c) significantly decreased activation of STAT6 by IL-4 but not IL-13. However, although activation of STAT6 by IL-4 was markedly reduced in γ(c) KO macrophages, it was not abolished, indicating that IL-4 can still signal through type II IL-4 receptors via the IL-13Rα1 chain. IL-13 failed to activate STAT6 in macrophages derived from Il13ra1 KO mice; however, these cells remained fully responsive to IL-4. The inability of IL-13 but not IL-4 to signal in Il13ra1(-/-) macrophages correlated with the inability of IL-13 but not IL-4 to induce expression of genes such as Arg1, Retnla and Ccl11 that are characteristically expressed by alternatively activated macrophages. In addition, IL-13 but not IL-4 failed to induce membrane fusion and giant cell formation by Il13ra1 KO macrophages. These findings demonstrate that the IL-13Rα1 chain is essential for induction of the AMA pathway by IL-13 but not IL-4.
Assuntos
Via Alternativa do Complemento , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos/imunologia , Animais , Arginase/genética , Arginase/metabolismo , Antígenos CD11/genética , Antígenos CD11/metabolismo , Células Cultivadas , Via Alternativa do Complemento/genética , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subunidade gama Comum de Receptores de Interleucina/genética , Interleucina-13/genética , Subunidade alfa1 de Receptor de Interleucina-13/genética , Interleucina-4/genética , Fusão de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT6/metabolismoRESUMO
TLR agonists such as LPS and poly(I:C) induce expression of type I IFNs, such as IFN-α and -ß, by macrophages. To examine the role of IFN-ß in the induction of ISGs by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from WT and Ifnb1(-/-) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I), and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1(-/-) mice or Ifnar1(-/-) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1(-/-) macrophages correlated with the failure of LPS to induce activation of STAT1 and -2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 KO mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1(-/-) and Ifnar1(-/-) macrophages, activation of NF-κB and induction of NF-κB-responsive genes, such as Tnf (TNF-α) and Il1b (IL-1ß), were not affected by deletion of either the IFN-ß or IFN-αR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-ß and autocrine signaling through type I IFN receptors.
Assuntos
Regulação da Expressão Gênica , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon beta/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.
Assuntos
Cromossomos Humanos Par 19/genética , Hepatite C/prevenção & controle , Interleucinas/genética , Polimorfismo Genético/genética , Anticorpos Monoclonais , Perfilação da Expressão Gênica , Marcadores Genéticos/genética , Células Hep G2 , Hepatite C/imunologia , Humanos , Interleucinas/imunologia , Interleucinas/metabolismo , Desequilíbrio de Ligação , Microscopia Confocal , Modelos Biológicos , Fosforilação , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Análise de Sequência de RNA , Especificidade da Espécie , Estatísticas não ParamétricasRESUMO
This study compared the ability of IFN-α and IFN-λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN-α drug products are widely used to treat chronic HCV infection; however, IFN-α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN-λ1 is currently being tested as a potential alternative to IFN-α for treating chronic HCV. Although IFN-λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh-7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN-λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN-λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN-λ in hepatocytes was generally lower than that induced by IFN-α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN-α and IFN-λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN-α, IFN-λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN-λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN-α therapy.
Assuntos
Hepatócitos/imunologia , Interleucinas/imunologia , Linfócitos/imunologia , Monócitos/imunologia , Transdução de Sinais/fisiologia , Animais , Feminino , Células Hep G2 , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Humanos , Interferon-alfa/imunologia , Interferon-alfa/uso terapêutico , Interferons , Interleucinas/uso terapêutico , Janus Quinases/imunologia , Masculino , Camundongos , Especificidade de Órgãos/imunologia , Fatores de Transcrição STAT/imunologiaRESUMO
Interferon (IFN)-α, a type-I IFN, is widely used to treat chronic hepatitis C virus infection, but the broad expression of IFN-α receptors often leads to adverse reactions in many organs. Here, we examine IFN-λ, a type-III IFN, as a therapeutic alternative to IFN-α. Like IFN-α, IFN-λ also induces antiviral activity in hepatocytes, but might induce fewer adverse reactions because its receptor is largely restricted to cells of epithelial origin. We also discuss the recent discovery of single nucleotide polymorphisms (SNPs) near the human IFN-λ3 gene, IL28B, that correlate strongly with the ability to achieve a sustained virological response to therapy with pegylated IFN-α plus ribavirin in patients with chronic hepatitis C.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C Crônica , Imunoterapia/métodos , Interleucinas/imunologia , Interleucinas/farmacologia , Animais , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Cromossomos Humanos Par 19/química , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 19/imunologia , Quimioterapia Combinada , Regulação da Expressão Gênica/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/genética , Hepatite C Crônica/imunologia , Hepatite C Crônica/terapia , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interferon-alfa/uso terapêutico , Interferons , Interleucinas/química , Interleucinas/genética , Camundongos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Ribavirina/administração & dosagem , Ribavirina/uso terapêutico , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
IL-26 is classified as a member of the IL-10 cytokine family because it has limited sequence homology to IL-10 and the IL-10-related cytokines. The human IL-26 gene, IL26, is located on chromosome 12q15 between the genes for two other important class-2 cytokines, IFNG (IFN-γ) and IL22 (IL-22). IL-26 is often co-expressed with IL-22 by activated T cells, especially Th17 cells. It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-26 receptors are primarily expressed on non-hematopoietic cell types, particularly epithelial cells. Signaling through IL-26 receptor complexes results in the activation of STAT1 and STAT3 with subsequent induction of IL-26-responsive genes. The biological functions of IL-26 have only begun to be defined.
Assuntos
Interleucinas/biossíntese , Interleucinas/genética , Células Th17/imunologia , Humanos , Interleucina-10/biossíntese , Interleucina-10/genéticaRESUMO
Interleukin (IL)-4 and -13 are related cytokines sharing functional receptors. IL-4 signals through the type I (IL-4Ralpha/common gamma-chain [gammac]) and the type II (IL-4Ralpha/-13Ralpha1) IL-4 receptors, whereas IL-13 utilizes only the type II receptor. In this study, we show that mouse bone marrow-derived macrophages and human and mouse monocytes showed a much greater sensitivity to IL-4 than to IL-13. Lack of functional gammac made these cells poorly responsive to IL-4, while retaining full responsiveness to IL-13. In mouse peritoneal macrophages, IL-4 potency exceeds that of IL-13, but lack of gammac had only a modest effect on IL-4 signaling. In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts. Using levels of receptor chain expression and known binding affinities, we modeled the assemblage of functional type I and II receptor complexes. The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types. These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.
Assuntos
Regulação da Expressão Gênica/imunologia , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Monócitos/metabolismo , Transdução de Sinais/imunologia , Animais , Western Blotting , Citometria de Fluxo , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT6/genéticaRESUMO
The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [(3)H]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained alpha interferon (IFN-alpha; median, 43 pg/ml) and IFN-lambda (approximately 1 to 20 ng/ml). Neutralization of IFN-alpha with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-lambda with MAb against either of its receptor chains, IFN-lambdaR1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of interferons.
Assuntos
Antivirais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Citocinas/fisiologia , Terapia de Imunossupressão , Interferon-alfa/fisiologia , Interleucinas/fisiologia , Vírus Sincicial Respiratório Humano/imunologia , Adulto , Antígenos de Bactérias/farmacologia , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Proliferação de Células , Sistema Livre de Células/imunologia , Citocinas/metabolismo , Citocinas/farmacologia , Citomegalovirus/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Enterotoxinas/farmacologia , Humanos , Interferon-alfa/farmacologia , Interferons , Interleucinas/farmacologia , Monócitos/imunologia , Monócitos/metabolismoRESUMO
Recently discovered type III IFNs (IFN-lambda) exert their antiviral and immunomodulatory activities through a unique receptor complex composed of IFN-lambdaR1 and interleukin-10 receptor 2. To further study type III IFNs, we cloned and characterized mouse IFN-lambda ligand-receptor system. We showed that, similar to their human orthologues, mIFN-lambda2 and mIFN-lambda3 signal through the IFN-lambda receptor complex, activate IFN stimulated gene factor 3, and are capable of inducing antiviral protection and MHC class I antigen expression in several cell types including B16 melanoma cells. We then used the murine B16 melanoma model to investigate the potential antitumor activities of IFN-lambdas. We developed B16 cells constitutively expressing murine IFN-lambda2 (B16.IFN-lambda2 cells) and evaluated their tumorigenicity in syngeneic C57BL/6 mice. Although constitutive expression of mIFN-lambda2 in melanoma cells did not affect their proliferation in vitro, the growth of B16.IFN-lambda2 cells, when injected s.c. into mice, was either retarded or completely prevented. We found that rejection of the modified tumor cells correlated with their level of IFN-lambda2 expression. We then developed IFN-lambda-resistant B16.IFN-lambda2 cells (B16.IFN-lambda2Res cells) and showed that their tumorigenicity was also highly impaired or completely abolished similar to B16.IFN-lambda2 cells, suggesting that IFN-lambdas engage host mechanisms to inhibit melanoma growth. These in vivo experiments show the antitumor activities of IFN-lambdas and suggest their strong therapeutic potential.
Assuntos
Interferons/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Receptores de Interferon/imunologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Cricetinae , Feminino , Humanos , Interferons/genética , Ligantes , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Interferon/genética , Transdução de Sinais , TransfecçãoRESUMO
Autoantibodies against RNA polymerase I (RNAPI), DNA, La and ribosomal P proteins were detected in the urine of systemic lupus erythematosus (SLE) patients, many with normal protein excretion rates. In a number of cases, the antibodies were detectable in the urine but not the serum sample of the same patient. The presence and relative concentrations of the urinary autoantibodies correlated with disease activity. RNAPI antigens were detected in the urine of SLE patients by radioimmunoassay and immunoblotting using rabbit antisera prepared against the purified holoenzyme. Immunoaffinity purification of the rabbit anti-RNAPI with SLE urine proteins resulted in antibodies directed primarily against the largest RNAPI subunit (S1; 194 kDa). Antibodies prepared against recombinant fusion proteins representing the DNA binding regions of human RNAPI(S1) reacted with a 35 kDa SLE urinary protein, a putative fragment of RNAPI(S1). Ribosomal protein P0 was detected in SLE patients' urine by immunoblotting, using rabbit antiserum prepared against recombinant human P1 fusion protein. The relative quantities of urinary P0 correlated with disease status. Analysis of urinary autoantibodies and corresponding antigens in conjunction with analysis of serum autoantibodies may be of value for the purpose of monitoring disease activity.
Assuntos
Autoanticorpos/urina , Autoantígenos/urina , Lúpus Eritematoso Sistêmico/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Proteínas de Ciclo Celular , DNA/imunologia , Proteínas de Ligação a DNA/imunologia , Humanos , Lúpus Eritematoso Sistêmico/urina , Componente 3 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/imunologia , RNA Polimerase I/imunologia , Ribonucleoproteínas/imunologia , Fatores de Transcrição/imunologia , Antígeno SS-BRESUMO
Several novel interleukin (IL)-10-related cytokines have recently been discovered. These include IL-22, IL-26, and the interferon-lambda (IFN-lambda) proteins IFN-lambda1 (IL-29), IFN-lambda2 (IL-28A), and IFN-lambda3 (IL-28B). The ligand-binding chains for IL-22, IL-26, and IFN-lambda are distinct from that used by IL-10; however, all of these cytokines use a common second chain, IL-10 receptor-2 (IL-10R2; CRF2-4), to assemble their active receptor complexes. Thus, IL-10R2 is a shared component in at least four distinct class II cytokine-receptor complexes. IL-10 binds to IL-10R1; IL-22 binds to IL-22R1; IL-26 binds to IL-20R1; and IFN-lambda binds to IFN-lambdaR1 (also known as IL-28R). The binding of these ligands to their respective R1 chains induces a conformational change that enables IL-10R2 to interact with the newly formed ligand-receptor complexes. This in turn activates a signal-transduction cascade that results in rapid activation of several transcription factors, particularly signal transducer and activator of transcription (STAT)3 and to a lesser degree, STAT1. Activation by IL-10, IL-22, IL-26, or IFN-lambda can be blocked with neutralizing antibodies to the IL-10R2 chain. Although IL-10R2 is broadly expressed on a wide variety of tissues, only a subset of these tissues expresses the ligand-binding R1 chains. The receptors for these cytokines are often present on cell lines derived from various tumors, including liver, colorectal, and pancreatic carcinomas. Consequently, the receptors for these cytokines may provide novel targets for inhibiting the growth of certain types of cancer.
Assuntos
Citocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Animais , Humanos , Subunidade beta de Receptor de Interleucina-10 , Receptores de Interferon/genética , Receptores de Interleucina/metabolismo , Receptor de Interferon gamaRESUMO
Interleukin-19 (IL-19) is a newly discovered member of the IL-10 family of ligands whose function is presently undefined. We recently described its cloning and initial characterization and in so doing, noted that the induction of IL-19 by LPS in human monocytes was down-regulated by interferon-gamma (IFN-gamma) and up-regulated by IL-4. This preliminary observation led us to speculate that IL-19 may play a role in the Th1/Th2 system and we examined this hypothesis further. Our results suggested that IL-19 is able to influence the maturation of human T-cells. CD4+ T-cells resulting from SEB stimulation in the presence of IL-19 contained a higher proportion of IL-4 producing cells than those developing in the absence of IL-19. This observation was complimented by the observation that fewer IFN-gamma cells accrued in the presence of IL-19, thereby suggesting that IL-19 altered the balance of Th1/Th2 cells in favour of Th2. Furthermore, in whole PBMC cultures, IL-19 up-regulated IL-4 and down-regulated IFNgamma in a dose-dependent manner. These results are presented here in review format, in the context of an overall discussion of IL-19 and its receptor.
Assuntos
Interleucina-10/fisiologia , Receptores de Interleucina/metabolismo , Células Th2/imunologia , Humanos , Interleucina-10/metabolismo , Interleucinas , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Células Th2/metabolismoRESUMO
The receptor for IL-26 (AK155), a cytokine of the IL-10 family, has not previously been defined. We demonstrate that the active receptor complex for IL-26 is a heterodimer composed of two receptor proteins: IL-20R1 and IL-10R2. Signaling through the IL-26R results in activation of STAT1 and STAT3 which can be blocked by neutralizing Abs against IL-20R1 or IL-10R2. IL-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express IL-20R1. Therefore, the ability to respond to IL-26 is restricted by the expression of IL-20R1. IL-10, IL-19, IL-20, IL-22, and IL-24 fail to signal through the combination of IL-10R2 and IL-20R1 proteins, demonstrating that this receptor combination is unique and specific for IL-26.
Assuntos
Interleucina-10/metabolismo , Interleucinas/metabolismo , Interleucinas/fisiologia , Receptores de Interleucina/fisiologia , Transdução de Sinais/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Dimerização , Células HT29 , Humanos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores de Interleucina/biossíntese , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transativadores/metabolismoRESUMO
Leishmania donovani protozoan parasites, the causative agent of visceral leishmaniasis, establish an infection partly by interfering with cytokine signaling in the host macrophages. Therefore, we investigated the expression of the suppressor of cytokine signaling (SOCS) genes in human macrophages infected with L. donovani. The expression of SOCS3 mRNA was induced transiently after exposure to live or heat-killed parasites, but not purified lipophosphoglycan, while that of other SOCS genes remained unchanged. SOCS3 gene expression was not dependent on phagocytosis or on cytokines released by L. donovani-infected macrophages, such as interleukin-1beta or tumor necrosis factor alpha. In addition, Leishmania used a different signaling pathway(s) than bacterial lipopolysaccharide to induce SOCS3 mRNA, as indicated by the kinetics of induction and sensitivity to polymyxin B inhibition. Finally, phosphorylation of the STAT1 transcription factor was significantly reduced in L. donovani-infected macrophages and required de novo transcription. The induction of SOCS3 provides a potent inhibitory mechanism by which intracellular microorganisms may suppress macrophage activation and interfere with the host immune response.
Assuntos
Leishmania donovani/patogenicidade , Ativação de Macrófagos , Macrófagos/parasitologia , Proteínas/metabolismo , Proteínas Repressoras , Fatores de Transcrição , Animais , Citocinas/metabolismo , Humanos , Interferon gama/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de CitocinaRESUMO
Signal transducer and activator of transcription 6 (Stat6) plays an important role in interleukin (IL)-4-induced responses. To analyze the regulation of Stat6 phosphorylation, cells were cultured in the continuous presence of IL-4 or after a pulse and washout. In the continual presence of IL-4, Stat6 remained phosphorylated for an extended period. After IL-4 removal and inhibition of the Janus family kinase, tyrosine-phosphorylated Stat6 decayed at a rate dependent upon the length of IL-4 stimulation. The decay of tyrosine-phosphorylated Stat6 was similar in the presence or absence of either cycloheximide or actinomycin D. In the absence of functional Src homology-containing phosphatase-1 (SHP-1), the early loss of tyrosine-phosphorylated Stat6 was substantially reduced. Furthermore, the rate of loss of tyrosine-phosphorylated Stat6 in cells expressing a mutation of the human IL-4 receptor alpha in the immunoreceptor tyrosine-based inhibitory motif sequence (Y5F) was dramatically decreased compared with wild-type cells. The early rate of decay was similar in the presence or absence of MG132, an inhibitor of the proteasome, but the later rate of decay was decreased 5-fold. These results suggest that the loss of tyrosine phosphorylation of Stat6 is regulated by the action of SHP-1 and the proteasome but is not dependent on new protein synthesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Interleucina-4/metabolismo , Tirosina/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Camundongos , Fosforilação , Complexo de Endopeptidases do Proteassoma , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores de Interleucina-4/química , Fator de Transcrição STAT6 , Transativadores , Células Tumorais CultivadasRESUMO
We report here the identification of a ligand-receptor system that, upon engagement, leads to the establishment of an antiviral state. Three closely positioned genes on human chromosome 19 encode distinct but paralogous proteins, which we designate interferon-lambda1 (IFN-lambda1), IFN-lambda2 and IFN-lambda3 (tentatively designated as IL-29, IL-28A and IL-28B, respectively, by HUGO). The expression of IFN-lambda mRNAs was inducible by viral infection in several cell lines. We identified a distinct receptor complex that is utilized by all three IFN-lambda proteins for signaling and is composed of two subunits, a receptor designated CRF2-12 (also designated as IFN-lambdaR1) and a second subunit, CRF2-4 (also known as IL-10R2). Both receptor chains are constitutively expressed on a wide variety of human cell lines and tissues and signal through the Jak-STAT (Janus kinases-signal transducers and activators of transcription) pathway. This receptor-ligand system may contribute to antiviral or other defenses by a mechanism similar to, but independent of, type I IFNs.