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1.
J Neurosci ; 20(20): 7587-94, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11027218

RESUMO

Reactive microglia have been suggested to play a role in the Alzheimer's disease (AD) process, and previous studies have shown that expression of CD45, a membrane-bound protein-tyrosine phosphatase (PTP), is elevated in microglia in AD brain compared with controls. To investigate the possible role of CD45 in microglial responsiveness to beta-amyloid (Abeta) peptides, we first co-treated primary cultured microglia with a tyrosine phosphatase inhibitor [potassium bisperoxo (1,10-phenanthroline) oxovanadate (phen), 5 micrometer] and freshly solubilized Abeta peptides (1000 nm). Data show synergistic induction of microglial activation as evidenced by tumor necrosis factor alpha (TNF-alpha) production and nitric oxide (NO) release, both of which we show to be dependent on activation of p44/42 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with phen and Abeta peptides results in microglia-induced neuronal cell injury. Stimulation of microglial CD45 by anti-CD45 antibody markedly inhibits these effects via inhibition of p44/42 MAPK, suggesting that CD45 is a negative regulator of microglial activation. Accordingly, primary cultured microglia from CD45-deficient mice demonstrate hyper-responsiveness to Abeta, as evidenced by TNF-alpha release, NO production, and neuronal injury after stimulation with Abeta peptides. As a validation of these findings in vivo, brains from a transgenic mouse model of AD [transgenic Swedish APP-overexpressing (Tg APP(sw)) mice] deficient for CD45 demonstrate markedly increased production of TNF-alpha compared with Tg APP(sw) mice. Taken together, these results suggest that therapeutic agents that stimulate the CD45 PTP signaling pathway may be effective in suppressing microglial activation associated with AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Microglia/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Animais , Anticorpos/farmacologia , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese
2.
Exp Hematol ; 28(9): 1062-70, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11008019

RESUMO

OBJECTIVE: Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. METHODS: The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. RESULTS: Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. CONCLUSIONS: These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.


Assuntos
Antígenos CD/fisiologia , Antígenos Comuns de Leucócito/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de IgG/fisiologia , Actinas/metabolismo , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Interleucina-6/metabolismo , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/fisiologia , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/farmacologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de IgG/efeitos dos fármacos , Receptores de IgG/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos
3.
J Biol Chem ; 275(47): 37224-31, 2000 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-10978311

RESUMO

It has been reported that ligation of CD40 with CD40 ligand (CD40L) results in microglial activation as evidenced by p44/42 mitogen-activated protein kinase (MAPK) dependent tumor necrosis factor alpha (TNF-alpha) production. Previous studies have shown that CD45, a functional transmembrane protein-tyrosine phosphatase, is constitutively expressed at moderate levels on microglial cells and this expression is greatly elevated on activated microglia. To investigate the possibility that CD45 might modulate CD40L-induced microglial activation, we treated primary cultured microglial cells with CD40L and anti-CD45 antibody. Data show that cross-linking of CD45 markedly inhibits CD40L-induced activity of the Src family kinases Lck and Lyn. Further, co-treatment of microglia with CD40L and anti-CD45 antibody results in significant inhibition of microglial TNF-alpha production through inhibition of p44/42 MAPK activity, a downstream signaling event resulting from Src activation. Accordingly, primary cultured microglial cells from mice deficient in CD45 demonstrate hyper-responsiveness to ligation of CD40, as evidenced by increased p44/42 MAPK activation and TNF-alpha production. Taken together, these results show that CD45 plays a novel role in suppressing CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK cascade.


Assuntos
Ligante de CD40/farmacologia , Antígenos Comuns de Leucócito/farmacologia , Microglia/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Proteína Tirosina Quinase CSK , Células Cultivadas , Ativação Enzimática , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 3 Ativada por Mitógeno , Esclerose Múltipla/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Quinases da Família src
4.
Int Immunol ; 8(12): 1947-53, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8982779

RESUMO

In this study the effects of CD45 engagement on CD3-TCR-driven stimulation of CD1+ human immature thymocytes have been analyzed. Simultaneous cross-linking of CD45 and CD3 antigens on highly purified CD1+ thymocytes reduced the number of cells undergoing apoptosis after 16 h of in vitro culture. This cell population might represent immature thymocytes committed in vivo to die by programmed cell death (PCD). CD45 engagement could also increase the number of cycling CD1+ thymocytes; of note, the large majority (> 95%) of dividing cells expressed the CD1 molecule at the cell surface, indicating that proliferating cells were actually represented by immature thymocytes. These data suggest that the CD45 molecule might play a role in the rescue of immature thymocytes from PCD during differentiation. Along this line, we found that activation of CD1+ thymocytes via the CD3-TCR complex could be enhanced by CD45, both in terms of transcription and surface expression of IL-2R. These effects might be explained by the finding that the CD45 molecule (but not its isoforms CD45RO and RA) was physically associated with the CD3-TCR complex at the cell surface of CD1+ human thymocytes, as shown by co-precipitation and co-capping experiments. Finally, cross-linking of CD45 and CD3 antigens led to the expansion of CD3+ thymocytes co-expressing CD4 and CD8, indicating that simultaneous engagement of CD45 and CD3 molecules can block CD1+ cells at the double-positive (CD3+CD4+CD8+) differentiation stage. On the other hand, stimulation through CD3 resulted in the expansion of thymocytes showing a mature phenotype (CD3+CD4+ or CD3+CD8+). Altogether, these findings suggest that the CD45 molecule is involved both in early activation and in the regulation of CD1+ thymocyte differentiation.


Assuntos
Antígenos CD1/análise , Apoptose/efeitos dos fármacos , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/farmacologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Timo/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Antígenos CD4/análise , Antígenos CD8/análise , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Ligação Proteica/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia
5.
Int Immunol ; 8(10): 1529-35, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8921432

RESUMO

Immature CD4+CD8+ double-positive (DP) thymocytes are positively selected for further development if they express TCR reacting with thymic ligands of low affinity. However, the majority of DP thymocytes express low TCR levels. This low level of TCR may be insufficient to recognize thymic ligands. To understand the basis for the low expression of TCR on DP thymocytes, we determined the density of TCR expression at various stages of their development using TCR transgenic (TCR-Tg) mice. We found that TCR expression was high in the thymocytes that had recently transited into the DP stage but then gradually decreased on DP cells if they were not selected by TCR interaction with MHC molecules. However, such TCR suppression was not observed in positively selected DP cells and in the non-selected DP cells obtained from CD45 deficient mice or from mice receiving anti-CD4 mAb. These findings suggest that the once highly expressed TCR at the DP stage is suppressed by CD45 and/or CD4 on non-selected thymocytes. Furthermore, TCR suppression is prevented by TCR-mediated signals. The maintenance of high TCR levels on positively selected DP thymocytes may facilitate their selection.


Assuntos
Antígenos CD4/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Regulação para Baixo/imunologia , Antígenos Comuns de Leucócito/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/efeitos dos fármacos , Timo/metabolismo , Animais , Antígenos CD4/imunologia , Diferenciação Celular/imunologia , Cruzamentos Genéticos , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timo/citologia
6.
Cell ; 86(5): 787-98, 1996 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-8797825

RESUMO

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , NF-kappa B/fisiologia , Ativação Transcricional/fisiologia , Tirosina/metabolismo , Alcaloides/farmacologia , Sequência de Bases , Calcimicina/farmacologia , Hipóxia Celular , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Genisteína , Humanos , Ionóforos/farmacologia , Isoflavonas/farmacologia , Antígenos Comuns de Leucócito/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , Fosforilação , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Estaurosporina , Linfócitos T , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Vanadatos/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/fisiologia
7.
Scand J Immunol ; 44(1): 37-44, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8693290

RESUMO

The CD45 glycoprotein isoforms exhibit a receptor-like composition and display intracellular protein tyrosine phosphatase (PTPase) activity. The present study links CD45 to the regulation of L-selectin (CD62L), a leucocyte glycoprotein important for extravasation and homotypic aggregation. Monoclonal antibodies (MoAbs) IOL1b and AICD45.2, but not GAP8.3, all of which are directed against common CD45 epitopes, were found to elicit lymphocyte L-selectin down-regulation. Lymphocyte L-selectin down-regulation in response to anti-CD45 MoAbs was enhanced by high cell density and partially antagonized by the protein tyrosine kinase (PTK) inhibitor, herbimycin A. The MoAbs IOL1b, AICD45.2 and GAP8.3 recognized granulocyte-expressed CD45 but did not induce loss of L-selectin expression of granulocytes. In contrast, the CD45 PTPase inhibitor, vanadate, induced L-selectin down-regulation both in lymphocytes and granulocytes. The PTPase activation by nitric oxide (NO) or the NO-generating compound, sodium nitroprusside, did not affect L-selectin surface expression. Increased concentrations of soluble L-selectin were detected after anti-CD45 or vanadate-induced down-regulation of L-selectin surface expression. While activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces rapid L-selectin down-regulation of L-selectin surface expression in both lymphocytes and granulocytes, the PKC inhibitor, H 7, was also found to down-regulate lymphocyte and granulocyte L-selectin surface expression. The inhibitor H 7 synergized with vanadate in down-regulating lymphocyte L-selectin surface expression, but partially inhibited vanadate-induced granulocyte L-selectin down-regulation. The results suggest that in a cell type-specific fashion the PKC system and tyrosine phosphorylation and dephosphorylation cascades are involved in the regulation of L-selectin surface expression.


Assuntos
Regulação para Baixo/imunologia , Selectina L/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/farmacologia , Adulto , Anticorpos Monoclonais/farmacologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Selectina L/efeitos dos fármacos , Selectina L/imunologia , Antígenos Comuns de Leucócito/imunologia , Óxido Nítrico/farmacologia , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/fisiologia , Vanadatos/farmacologia
8.
J Immunol ; 156(8): 2743-53, 1996 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8609392

RESUMO

Studies have shown an essential role for the CD45 protein tyrosine phosphatase in regulating Ag receptor-derived signals in lymphocytes. Co-ligating CD45 with the Ag receptor, however, can also inhibit receptor signaling, as manifest in T cells by the lack of IL-2 production and proliferation. We report that CD45 ligation alone induces apoptosis in normal T cells, and that death was greatly potentiated by cross-linking CD3. Normal B cells and T and B cell lines were also induced to die with insoluble CD45 mAb. CD45-induced cell death was blocked by inhibitors of protein tyrosine kinases and protein tyrosine phosphatases, but not by inhibitors of RNA or protein synthesis or by cyclosporin A. Morphologically, CD45-mediated apoptosis resembled death induced via CD95 (Fas), as evidenced by nuclear condensation and membrane blebbing, but did not cause DNA fragmentation into oligonucleosomes. Co-ligating CD45 and CD95 either enhanced or inhibited CD45-induced cell death, depending on the degree of CD45 and CD95 cross-linking. Finally, CD45 cross-linking induced its rapid association with the detergent-insoluble cell fraction, suggesting that it becomes linked to the cytoskeleton during CD45-induced apoptosis. These data show a novel role for CD45 in regulating lymphocyte death as well as proliferation.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Apoptose/imunologia , Linfócitos B/imunologia , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/farmacologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Especificidade de Anticorpos , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Complexo CD3/imunologia , Linhagem Celular , Sinergismo Farmacológico , Inibidores do Crescimento/farmacologia , Humanos , Imunoglobulina M/imunologia , Antígenos Comuns de Leucócito/imunologia , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Linfoma de Células T/imunologia , Camundongos , Polietilenoglicóis , Solubilidade , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas , Receptor fas/imunologia
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