RESUMO
Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24â¯h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40â¯kDa dextran tracer within 1â¯h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.
Assuntos
Barreira Hematoencefálica/patologia , Toxina da Cólera/farmacologia , Inflamação/patologia , Interferon gama/farmacologia , Interleucina-17/farmacologia , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Impedância Elétrica , Haptoglobinas , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Permeabilidade , Precursores de Proteínas , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
The activation of STAT3 by tyrosine phosphorylation, essential for normal development and for a normal inflammatory response to invading pathogens, is kept in check by negative regulators. Abnormal constitutive activation of STAT3, which contributes to the pathology of cancer and to chronic inflammatory diseases such as rheumatoid arthritis, occurs when negative regulation is not fully effective. SOCS3, the major negative regulator of STAT3, is induced by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphorylation about 2 h after initial exposure of cells to members of the IL-6 family of cytokines by binding cooperatively to the common receptor subunit gp130 and JAKs 1 and 2. We show here that when the epidermal growth factor receptor (EGFR) is present and active, STAT3 is rephosphorylated about 4 h after exposure of cells to IL-6 or oncostatin M and remains active for many hours. Newly synthesized IL-6 drives association of the IL-6 receptor and gp130 with EGFR, leading to EGFR-dependent rephosphorylation of STAT3, which is not inhibited by the continued presence of SOCS3. This second wave of STAT3 activation supports sustained expression of a subset of IL-6-induced proteins, several of which play important roles in inflammation and cancer, in which both IL-6 secretion and EGFR levels are often elevated.
Assuntos
Receptor gp130 de Citocina/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-6/biossíntese , Fator de Transcrição STAT3/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Receptor gp130 de Citocina/genética , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/farmacologia , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oncostatina M/metabolismo , Oncostatina M/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Fator de Transcrição STAT3/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
The mechanism of IFN-ß therapy in relapsing-remitting multiple sclerosis (RRMS) is not well understood, but induction of apoptosis in specific leukocyte subsets is likely to be important. Enhanced expression of TNFSF10 or TNF-related apoptosis-inducing ligand (TRAIL) mRNA in unseparated leukocytes has been put forward as a therapeutic response marker, but it is unclear which leukocyte subsets express TRAIL. We investigated the basis of TRAIL expression in response to IFN-ß by studying activation of STATs 1, 3, and 5, p38 MAPK, and NF-κB in different leukocyte subsets of patients with RRMS. Monocytes, B cells, and T cells showed substantial differences in the activation of p38 and the STATs in response to i.m. injection of IFN-ß1a or stimulation in vitro. Induction of cell-surface TRAIL, analyzed in nine leukocyte subsets, was observed only on monocytes and granulocytes and correlated with the activation of p38 and/or NF-κB in these subsets only, in agreement with previous work in fibroblasts showing that the induction of TRAIL in response to IFN-ß depends on the activation of p38 and NF-κB as well as STATs 1 and 2. We propose that, in myeloid cells, the differential activation of p38 and NF-κB and induction of TRAIL, which sensitizes cells to apoptosis, can help to explain differences in responsiveness to IFN-ß therapy among patients with RRMS and, furthermore, that such differential patterns of activation and expression may also be important in understanding the therapeutic responses to IFN-α/ß in hepatitis and cancer.
Assuntos
Granulócitos/efeitos dos fármacos , Interferon beta/uso terapêutico , Monócitos/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Adulto , Antineoplásicos/uso terapêutico , Citometria de Fluxo , Granulócitos/metabolismo , Humanos , Pessoa de Meia-Idade , Monócitos/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-ß and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-ß, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-ß activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-ß increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Interferon beta/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Apoptose/imunologia , Linfócitos B/metabolismo , Western Blotting , Linhagem Celular , Separação Celular , Citometria de Fluxo , Expressão Gênica/imunologia , Humanos , Fator Gênico 3 Estimulado por Interferon , Interferon beta/metabolismo , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/imunologia , Fator de Transcrição STAT5/metabolismo , Linfócitos T/metabolismoRESUMO
In this longitudinal study, we examined the expression of Fas, FasL, CCR3, CCR5 and CXCR3 mRNA in peripheral blood mononuclear cells (PBMCs) of secondary progressive (SP) and relapsing-remitting (RR) multiple sclerosis (MS) patients. In RR patients, FasL, CCR3 and CCR5 mRNA levels were increased prior to the exacerbations, but these decreased during clinical activity, while mRNA levels of Fas increased. SP patients have increased the levels of Fas and FasL mRNA; the latter was particularly increased during lesional activity. Our data support the hypothesis that changes in Fas and FasL mRNA related to clinical activity are due to the migration of inflammatory cells to the central nervous system (CNS).
Assuntos
Regulação para Baixo/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Esclerose Múltipla Recidivante-Remitente/imunologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Regulação para Cima/imunologia , Receptor fas/biossíntese , Receptor fas/sangue , Adulto , Apoptose/imunologia , Antagonistas dos Receptores CCR5 , Estudos Transversais , Regulação para Baixo/genética , Proteína Ligante Fas , Humanos , Ligantes , Estudos Longitudinais , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/sangue , Esclerose Múltipla Crônica Progressiva/imunologia , Esclerose Múltipla Crônica Progressiva/patologia , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/patologia , Receptores CCR3 , Receptores CCR5/genética , Receptores CXCR3 , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Regulação para Cima/genética , Receptor fas/genéticaRESUMO
The type I interferons (IFNs) are pleiotropic cytokines that regulate many different cellular functions. The major signaling pathway activated by type I IFNs involves sequential phosphorylation of the tyrosine residues of the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) proteins, providing the primary mechanism through which gene expression is induced. Recent work has shown that the responses are quite complex, as shown by different responses to specific subtypes of type I IFN, activation of kinases in addition to JAKs, patterns of activation of all seven STATs in different cells, and activation of transcription factors other than STATs. The type I IFNs use this complexity to regulate many different biological functions in different types of cells, by activating different specific signals and patterns of gene expression.