RESUMO
The TNF-receptor superfamily member CD30 is expressed on normal and malignant lymphocytes, including anaplastic large cell lymphoma (ALCL) cells. CD30 transmits multiple effects, including activation of NF-κB signaling, cell proliferation, growth arrest and apoptosis. How CD30 generates these pleiotropic effects is currently unknown. Herein we describe ALCL cells expressing truncated forms of the CD30 intracellular domain that allowed us to identify the key regions responsible for transmitting its biological effects in lymphocytes. The first region (CD30(519-537)) activated both the alternative and canonical NF-κB pathways as detected by p100 and IκBα degradation, IKKß-dependent transcription of both IκBα and the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and induction of cell cycle arrest. In contrast, the second region of CD30 (CD30(538-595)) induced some aspects of canonical NF-κB activation, including transcription of IκBα, but failed to activate the alternative NF-κB pathway or drive p21(WAF1/CIP1)-mediated cell-cycle arrest. Direct comparison of canonical NF-κB activation by the two motifs revealed 4-fold greater p65 nuclear translocation following CD30(519-537) engagement. These data reveal that independent regions of the CD30 cytoplasmic tail regulate the magnitude and type of NF-κB activation and additionally identify a short motif necessary for CD30-driven growth arrest signals in ALCL cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígeno Ki-1/genética , Antígeno Ki-1/farmacologia , Linfócitos/efeitos dos fármacos , Linfoma Anaplásico de Células Grandes/genética , NF-kappa B/genética , Motivos de Aminoácidos , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endonucleases , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Antígeno Ki-1/química , Linfócitos/metabolismo , Linfócitos/patologia , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição GênicaRESUMO
We have previously found that CD30 ligand (CD30L; CD153)/CD30 signaling executed by the T-T cell interaction plays a critical role in Th17 cell differentiation, at least partly via downregulation of IL-2 production. In this study, we investigated the role of CD30L in the development of colitis experimentally induced by dextran sulfate sodium (DSS), in which IL-17A is involved in the pathogenesis. CD30L(-/-) mice were resistant to both acute colitis induced by administration of 3 to ⼠5% DSS and to chronic colitis induced by administration of 1.5% DSS on days 0-5, 10-15, and 20-25 as assessed by weight loss, survival rate, and histopathology. The levels of IFN-γ, IL-17A, and IL-10 were significantly lower but the IL-2 level higher in the lamina propria T lymphocytes of CD30L(-/-) mice than those in lamina propria T lymphocytes of wild-type mice after DSS administration. Soluble murine CD30-Ig fusion protein, which was capable of inhibiting Th17 cell differentiation in vitro, ameliorated both types of DSS-induced colitis in wild-type mice. Modulation of CD30L/CD30 signaling by soluble CD30 could be a novel biological therapy for inflammatory diseases associated with Th17 responses.
Assuntos
Ligante CD30/antagonistas & inibidores , Ligante CD30/imunologia , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Colite/tratamento farmacológico , Colite/imunologia , Antígeno Ki-1 , Células Th17/imunologia , Doença Aguda , Animais , Ligante CD30/genética , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Doença Crônica , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Citocinas/imunologia , Sulfato de Dextrana/toxicidade , Feminino , Humanos , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Imunoglobulinas/farmacologia , Antígeno Ki-1/genética , Antígeno Ki-1/imunologia , Antígeno Ki-1/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Mucosa/imunologia , Mucosa/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th17/patologiaRESUMO
Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O(2)); culture and stimulation in hypoxia (1% O(2)); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia.
Assuntos
Mediadores da Inflamação/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Calcimicina/farmacologia , Degranulação Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Antígeno Ki-1/farmacologia , Lipopolissacarídeos/farmacologia , Mastócitos/efeitos dos fármacosRESUMO
Death ligands, including TNF-alpha, CD95L/FasL, and TRAIL, mediate safeguard mechanisms against tumor growth and critically contribute to lymphocyte homeostasis. We investigated death receptor-mediated apoptosis and CD30/CD95 crosstalk in four CD30-positive cell lines of cutaneous anaplastic large-cell lymphoma (cALCL). Whereas CD95 stimulation strongly induced apoptosis in cALCL cells, the pro-apoptotic pathways of TNF-alpha and TRAIL were completely blocked at an early step. Expression of TNF receptor 1 was lost in three of four cell lines, providing an explanation for TNF-alpha unresponsiveness. TRAIL resistance may be explained by the consistent overexpression of cellular flice inhibitory protein (c-FLIP) (four of four cell lines) and frequent loss of the pro-apoptotic Bcl-2 protein Bid (three of four cell lines). Changes at the receptor-expression level were largely ruled out. CD30/CD95 crosstalk experiments showed that CD30 ligation leads to NF-kappaB-mediated c-FLIP upregulation in cALCL cells, which in turn conferred enhanced resistance to CD95-mediated apoptosis. Knockdown of c-FLIP by a lentiviral approach enhanced basic apoptosis rates in cALCL cells and diminished the CD30-mediated suppression of apoptosis, thus proving the significance of c-FLIP in this context. These in vitro findings may be indicative of the clinical situation of cALCL. Further clarifying the defects in apoptosis pathways in cutaneous lymphomas may lead to improved therapies for these disorders.
Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Neoplasias Cutâneas/patologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Antígeno Ki-1/farmacologia , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/fisiopatologia , NF-kappa B/metabolismo , Interferência de RNA , Receptor Cross-Talk/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/fisiopatologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Receptor fas/metabolismoRESUMO
CD30, a non-death domain-containing member of the tumor necrosis factor receptor superfamily, triggers apoptosis in anaplastic large cell lymphoma cells. The CD30 signaling pathways that lead to the induction of apoptosis are poorly defined. Here, we show that the induction of apoptosis by CD30 requires concurrent inhibition of p38 mitogen-activated protein kinase, which itself is activated by engagement of CD30 with CD30 ligand. Treatment of anaplastic large cell lymphoma cells with CD30 ligand and pharmacologic inhibitors of p38 mitogen-activated protein kinase, but not with CD30 ligand or inhibitors alone, triggered the activation of caspase-8 and the induction of apoptosis. Caspase-8 activation occurred within a few hours (2.5-4 h) after receptor triggering, was unaffected by the neutralization of ligands for the death domain-containing receptors TNFR1, Fas, DR3, DR4, or DR5, but was abolished by the expression of a dominant-negative form of the adaptor protein FADD. Importantly, we show that expression of the caspase-8 inhibitor c-FLIP(S) is strongly induced by the CD30 ligand, and that this is dependent on the activation of p38 mitogen-activated protein kinase. Thus, we provide evidence that the induction of apoptosis by CD30 in anaplastic large cell lymphoma cells is normally circumvented by the activation of p38 mitogen-activated protein kinase. These findings have implications for CD30-targeted immunotherapy of anaplastic large cell lymphoma.
Assuntos
Apoptose/efeitos dos fármacos , Antígeno Ki-1/farmacologia , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Antígeno Ki-1/imunologia , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CD30 is a member of the tumor necrosis factor (TNF)-receptor superfamily, whose ligand (CD30L) has been identified on B cells, activated macrophages and a subset of activated T cells. We show here that infection in vitro with human immunodeficiency virus (HIV) of CD4+ T-cell clones generated from HIV-seronegative individuals can enhance the expression of CD30, which often preceeds and is associated with the death of clonal T cells. Furthermore, cross-linking CD30 with an agonistic CD30-specific monoclonal antibody potentiated HIV replication induced by an insolubilized anti-CD3 antibody in T-cell lines generated from HIV-infected individuals. More importantly, paraformaldehyde-fixed CD8+ T-cell clones expressing CD30L enhanced HIV replication in anti-CD3-stimulated allogeneic or autologous HIV-infected CD4+ T-cell lines and such a potentiating effect was inhibited by an anti-CD30L antibody. The anti-CD30L antibody also exerted a suppressive effect on the spontaneous HIV replication occurring in lymph node cells, freshly derived from an HIV-seropositive patient showing CD30 expression in B cells and in a proportion of CD8+ T lymphocytes. Thus, CD30 triggering may play an important role in both HIV replication and the death of HIV-infected CD4+ T cells.