Assuntos
Alphapapillomavirus/fisiologia , Transformação Celular Viral/imunologia , Quimiocina CXCL12/imunologia , Interações Hospedeiro-Patógeno/imunologia , Queratinócitos/virologia , Receptores CXCR4/imunologia , Animais , Comunicação Autócrina , Divisão Celular , Movimento Celular , Suscetibilidade a Doenças , Epiderme/patologia , Epiderme/virologia , Humanos , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/virologia , Queratinócitos/patologia , Camundongos , Camundongos Nus , Mucosa/patologia , Mucosa/virologia , Doenças da Imunodeficiência Primária , Replicação Viral , Verrugas/imunologia , Verrugas/virologiaRESUMO
The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.
Assuntos
Quimiocina CXCL12/fisiologia , Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 18/fisiologia , Queratinócitos/virologia , Infecções por Papillomavirus/virologia , Receptores CXCR/biossíntese , Animais , Movimento Celular , Transformação Celular Viral , Quimiocina CXCL12/biossíntese , Proteínas de Ligação a DNA/biossíntese , Feminino , Humanos , Síndromes de Imunodeficiência/patologia , Síndromes de Imunodeficiência/virologia , Recém-Nascido , Masculino , Camundongos , Camundongos Nus , Proteínas Oncogênicas Virais/biossíntese , Proteínas E7 de Papillomavirus/biossíntese , Infecções por Papillomavirus/patologia , Doenças da Imunodeficiência Primária , Receptores CXCR/fisiologia , Receptores CXCR4/biossíntese , Proteínas Repressoras/biossíntese , Verrugas/patologia , Verrugas/virologiaRESUMO
The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.
Assuntos
Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica , Cálcio/metabolismo , Calorimetria , Linhagem Celular , Proliferação de Células , Chalconas/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL12/fisiologia , Humanos , Inflamação , Ligantes , Ligação Proteica , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Espectrometria de Fluorescência/métodosRESUMO
WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of CXC chemokine receptor 4 (CXCR4). Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immunohematologic clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on beta-arrestin (betaarr) recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced betaarr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4(1013) maintains association with betaarr2 and triggers augmented and prolonged betaarr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4(1013)-mediated chemotaxis critically requires betaarr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4(1013) abrogates betaarr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4(1013) spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between betaarr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12.
Assuntos
Arrestinas/metabolismo , Quimiocina CXCL12/farmacologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Receptores CXCR4/química , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Quimiotaxia de Leucócito , Dimerização , Humanos , Síndromes de Imunodeficiência/genética , Infecções/genética , Infecções/imunologia , Infecções/metabolismo , Mutação , Neutropenia/genética , Neutropenia/imunologia , Neutropenia/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Verrugas/genética , Verrugas/imunologia , Verrugas/metabolismo , beta-ArrestinasRESUMO
We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.
Assuntos
Apoptose , Proliferação de Células , Perfilação da Expressão Gênica , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Glicoproteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Glicoproteína da Espícula de Coronavírus , Transcrição Gênica , Transdução Genética , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
We reported the analytical interference of anti-Escherichia coli protein (EP) antibodies in human sera and residual EP in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay as a possible source of false positives in severe acute respiratory syndrome serodiagnosis. The rate of false positives was significantly reduced by adding mouse anti-EP antiserum in the blocking step.
Assuntos
Proteínas de Escherichia coli/imunologia , Proteínas do Nucleocapsídeo/imunologia , Síndrome Respiratória Aguda Grave/diagnóstico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Proteínas do Nucleocapsídeo de Coronavírus , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas do Nucleocapsídeo/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Testes Sorológicos/métodos , Síndrome Respiratória Aguda Grave/sangue , Síndrome Respiratória Aguda Grave/imunologiaRESUMO
The pro-apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus-mediated over-expression of SARS-CoV spike (S) protein and its C-terminal domain (S2) induce apoptosis in Vero E6 cells in a time- and dosage-dependent manner, whereas the expression of its N-terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS-CoV induced apoptosis and the molecular pathogenesis of SARS.
Assuntos
Adenoviridae/metabolismo , Apoptose , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Sobrevivência Celular , Chlorocebus aethiops , Vetores Genéticos , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus , Fatores de Tempo , Transdução Genética , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
Combined phylogenetic and chromosomal location studies suggest that the orphan receptor RDC1 is related to CXC chemokine receptors. RDC1 provides a co-receptor function for a restricted number of human immunodeficiency virus (HIV) isolates, in particular for the CXCR4-using HIV-2 ROD strain. Here we show that CXCL12, the only known natural ligand for CXCR4, binds to and signals through RDC1. We demonstrate that RDC1 is expressed in T lymphocytes and that CXCL12-promoted chemotaxis is inhibited by an anti-RDC1 monoclonal antibody. Concomitant blockade of RDC1 and CXCR4 produced additive inhibitory effects in CXCL12-induced T cell migration. Furthermore, we provide evidence that interaction of CXCL12 with RDC1 is specific, saturable, and of high affinity (apparent KD approximately 0.4 nM). In CXCR4-negative cells expressing RDC1, CXCL12 promotes internalization of the receptor and chemotactic signals through RDC1. Collectively, our data indicate that RDC1, which we propose to rename as CXCR7, is a receptor for CXCL12.