RESUMO
Recently, infections have been implicated in the pathogenesis of Alzheimer's disease. Apart from the direct effects of pathogens, it can be hypothesized that inflammatory mechanisms, such as the production of pro-inflammatory mediators by resident glia, may result in neurotoxicity. Here, we examined the inflammatory responses in murine microglial cell (MMC) and murine astrocyte cell (MAC) lines following infection with Chlamydia pneumoniae (Cpn), a pathogen that has recently been associated with Alzheimer's disease. Furthermore, we determined whether these inflammatory responses are sufficient to cause neuronal cell death in vitro. MMCs and MACs were infected with Cpn. Subsequently, various chemo- and cytokines were determined in the culture supernatant fluid of infected/control cells at different time points post-infection. Significantly higher levels of monocyte chemoattractant protein 1, interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and IL-1beta were found in supernatant fluids of infected MMCs compared with controls. In contrast, in the supernatant fluid of infected MACs, only monocyte chemoattractant protein 1 and IL-6 displayed significantly higher levels compared with controls. Moreover, neurotoxicity was examined up to 72 h after transferring the conditioned supernatant fluid to a neuronal cell layer. No neuronal cell death was observed when supernatant fluids from infected/mock-treated MACs were transferred. However, when neurones were exposed to conditioned supernatant fluid from infected MMCs, a significant increase in cell death was observed compared with mock. Furthermore, adding neutralizing antibodies against IL-6 and TNF-alpha to that conditioned supernatant fluid prevented neuronal cell death by approximately 50%. In conclusion, these data suggest that Cpn infection results in a pro-inflammatory milieu, particularly by activating MMCs, that ultimately results in neurodegeneration with prominent roles for both IL-6 and TNF-alpha.
Assuntos
Astrócitos/imunologia , Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae , Microglia/imunologia , Animais , Anticorpos/farmacologia , Apoptose/imunologia , Astrócitos/citologia , Astrócitos/microbiologia , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Humanos , Interleucina-6/imunologia , Interleucina-6/metabolismo , Neoplasias Hepáticas , Microglia/citologia , Microglia/microbiologia , Muridae , Necrose , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The atypical protein kinase C (PKC) isoforms (aPKC) have been implicated in the regulation of a number of essential signaling events. Early studies using dominant-negative mutants suggested that they are important intermediaries in the activation of the canonical nuclear factor (NF)-kappaB pathway. More recent data using knockout mice genetically demonstrate that in fact the PKCzeta isoform is essential for the adequate activation of this cascade both upstream and downstream the IkappaB kinase complex. In this review, we summarize the mechanistic details whereby the aPKC pathway regulates important cellular functions and how this is achieved by the ability of these kinases to interact with different protein regulators and adapters, as well as to impinge in NF-kappaB-independent signaling cascades such as the Janus kinase-1/signal transducer and activator of transcription 6 system, which plays a critical role in T-cell-mediated hepatitis and asthma.
Assuntos
NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Apoptose , Proliferação de Células , Drosophila , Imunidade , Interleucina-4/farmacologia , Janus Quinase 1 , Fígado/imunologia , Camundongos , Dados de Sequência Molecular , Proteína Quinase C/genética , Homologia de Sequência de Aminoácidos , Células Th2/metabolismo , Ativação TranscricionalRESUMO
Here we have addressed the role that zetaPKC plays in NF-kappaB activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-beta receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected which requires the previous degradation of IkappaB. Although in zetaPKC-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited.
Assuntos
Acetilcisteína/análogos & derivados , Fibroblastos/fisiologia , Marcação de Genes , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteína Quinase C/genética , Transcrição Gênica/genética , Acetilcisteína/farmacologia , Animais , Apoptose/fisiologia , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Genes Reporter/genética , Quinase I-kappa B , Interleucina-1/farmacologia , Pulmão/enzimologia , Pulmão/fisiologia , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/metabolismo , Fenótipo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/metabolismo , Fator de Transcrição RelA , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-beta receptor-Ig fusion protein (LTbetaR-Ig) in utero, a treatment that blocked the formation of both Peyer's and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTbetaR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-gamma gene knockout (IFN-gamma(-/-)) mice. Four weekly treatments with LTbetaR-Ig resulted in deletion of Peyer's and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-gamma(-/-) mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTbetaR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-gamma. Thus, LTbetaR-Ig may offer a possible treatment for the Th2-dominant form of colitis.
Assuntos
Colite/prevenção & controle , Imunoglobulinas/farmacologia , Receptores do Fator de Necrose Tumoral/imunologia , Células Th2/imunologia , Animais , Anticorpos Monoclonais , Antígenos CD/imunologia , Colite/etiologia , Colite/imunologia , Colite/patologia , Citocinas/biossíntese , Feminino , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Receptor beta de Linfotoxina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/patologia , Gravidez , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologiaRESUMO
Lymph nodes (LNs) are important sentinal organs, populated by circulating lymphocytes and antigen-bearing cells exiting the tissue beds. Although cellular and humoral immune responses are induced in LNs by antigenic challenge, it is not known if LNs are essential for acquired immunity. We examined immune responses in mice that lack LNs due to genetic deletion of lymphotoxin ligands or in utero blockade of membrane lymphotoxin. We report that LNs are absolutely required for generating contact hypersensitivity, a T cell-dependent cellular immune response induced by epicutaneous hapten. We show that the homing of epidermal Langerhans cells in response to hapten application is specifically directed to LNs, providing a cellular basis for this unique LN function. In contrast, the spleen cannot mediate contact hypersensitivity because antigen-bearing epidermal Langerhans cells do not access splenic white pulp. Finally, we formally demonstrate that LNs provide a unique environment essential for generating this acquired immune response by reversing the LN defect in lymphotoxin-alpha(-/)- mice, thereby restoring the capacity for contact hypersensitivity.
Assuntos
Dermatite de Contato/etiologia , Linfonodos/fisiologia , Linfotoxina-alfa/fisiologia , Animais , Movimento Celular , Células Dendríticas/fisiologia , Feminino , Células de Langerhans/fisiologia , Linfotoxina-beta , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3HRESUMO
Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut-associated lymphoid tissue (GALT), which includes Peyer's patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) alpha-/- mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTbeta-/- mice and LTalpha/LTbeta+/- animals do not have PP but could be orally tolerized, as measured by IFN-gamma production and delayed-type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTbeta-receptor-IgG-fusion-protein (LTbetaRigG)-treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high- or low-dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.
Assuntos
Tolerância Imunológica/imunologia , Imunidade nas Mucosas/imunologia , Nódulos Linfáticos Agregados/anormalidades , Nódulos Linfáticos Agregados/imunologia , Administração Oral , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Fezes/química , Deleção de Genes , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Imunoglobulina A/imunologia , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-2/metabolismo , Linfonodos/anormalidades , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfotoxina-alfa/antagonistas & inibidores , Linfotoxina-alfa/genética , Linfotoxina-alfa/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Nódulos Linfáticos Agregados/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.
Assuntos
Comunicação Celular , Células-Tronco Hematopoéticas/citologia , Mesoderma/citologia , Nódulos Linfáticos Agregados/embriologia , Animais , Moléculas de Adesão Celular/biossíntese , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Separação Celular , Células Cultivadas , Quimiocina CXCL13 , Quimiocinas/biossíntese , Quimiocinas/farmacologia , Quimiocinas CXC/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Receptor beta de Linfotoxina , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/imunologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/metabolismo , Receptores CXCR5 , Receptores de Quimiocinas , Receptores de Citocinas/deficiência , Receptores de Citocinas/genética , Receptores de Interleucina-7/deficiência , Receptores de Interleucina-7/genética , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling.
Assuntos
Antígenos CD/metabolismo , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Quinase I-kappa B , Receptor beta de Linfotoxina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Inibidor de NF-kappaB alfa , Nódulos Linfáticos Agregados/embriologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais/imunologia , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Quinase Induzida por NF-kappaBRESUMO
To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin beta-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4(+) T-cell-derived IFN-gamma, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.
Assuntos
Tolerância Imunológica/imunologia , Ovalbumina/imunologia , Nódulos Linfáticos Agregados/imunologia , Administração Oral , Animais , Linfócitos B/imunologia , Citocinas/biossíntese , Feminino , Haptenos/imunologia , Receptor beta de Linfotoxina , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/sangue , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Linfócitos T/imunologia , Células Th1/imunologia , Células Th2/imunologia , Ácido Trinitrobenzenossulfônico/imunologiaRESUMO
A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B/fisiologia , Transformação Celular Neoplásica , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3 , Animais , Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Proteínas de Transporte/metabolismo , Divisão Celular , Linhagem Celular Transformada , Células HT29 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Neoplasias/terapia , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Proteína Transmembrana Ativadora e Interagente do CAML , Células Tumorais Cultivadas , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genéticaRESUMO
Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/)- mice correlates with a significant reduction in lymphotoxin (LT)alphabeta(+)alpha(4)beta(7)(+)CD45(+)CD4(+)CD3(-) cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/)- mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTalphabeta expression on CD45(+) CD4(+)CD3(-) cells, as LNs could not be induced in LTalpha(-/)- mice. LTalpha(-/)- mice also showed defects in the fate of CD45(+)CD4(+)CD3(-) cells similar to TRANCE(-/)- mice. Thus, we propose that both TRANCE and LTalphabeta regulate the colonization and cluster formation by CD45(+) CD4(+)CD3(-) cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Linfonodos/crescimento & desenvolvimento , Glicoproteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linfócitos B , Complexo CD3 , Antígenos CD4 , Antígenos Comuns de Leucócito , Camundongos , Camundongos Transgênicos , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , BaçoRESUMO
The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.
Assuntos
Sistema Digestório/imunologia , Imunoglobulina A/biossíntese , Mucosa Intestinal/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Sistema Digestório/metabolismo , Epitopos de Linfócito T/imunologia , Imunidade nas Mucosas , Switching de Imunoglobulina/imunologia , Mucosa Intestinal/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfotoxina-alfa/fisiologia , Linfotoxina-beta , Proteínas de Membrana/fisiologia , Mesentério , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nódulos Linfáticos Agregados/metabolismo , Nódulos Linfáticos Agregados/patologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.
Assuntos
Artrite/prevenção & controle , Adesão Celular/fisiologia , Colágeno/metabolismo , Dermatite Alérgica de Contato/prevenção & controle , Hipersensibilidade Tardia/prevenção & controle , Integrinas/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Artrite/imunologia , Artrite/patologia , Colágeno/toxicidade , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/patologia , Dermatite Irritante/imunologia , Dermatite Irritante/patologia , Dermatite Irritante/prevenção & controle , Edema/etiologia , Edema/prevenção & controle , Feminino , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Integrina alfa1beta1 , Integrinas/imunologia , Leucócitos/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de ColágenoRESUMO
TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-kappaB and c-Jun NH(2)-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-beta receptor. To investigate the functional role of TRAF5 in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-kappaB or c-Jun NH(2)-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated in traf5(-/-) mice. However, traf5(-/-) B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover, in vitro Ig production of traf5(-/-) B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired in traf5(-/-) T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.
Assuntos
Antígenos CD40/imunologia , Ativação Linfocitária , Proteínas Quinases Ativadas por Mitógeno , Proteínas/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Ligante de CD40 , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fator 5 Associado a Receptor de TNFRESUMO
Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTbetaR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTalpha and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTbetaR-signaling molecule expressed by the stroma of the lymphoid organs.
Assuntos
Compartimento Celular/imunologia , Quimera/imunologia , Tecido Linfoide/anormalidades , Tecido Linfoide/embriologia , Linfotoxina-alfa/genética , Animais , Transplante de Medula Óssea/imunologia , Compartimento Celular/genética , Quimera/genética , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/imunologia , Teste de Complementação Genética , Receptor beta de Linfotoxina , Linfotoxina-alfa/biossíntese , Linfotoxina-alfa/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Receptores do Fator de Necrose Tumoral/fisiologiaRESUMO
To investigate the potential involvement of T helper (Th)2-type responses in murine models of intestinal inflammation, we used trinitrobenzene sulfonic acid (TNBS)-hapten to induce inflammatory bowel disease in situations where Th1-type responses with interferon (IFN)-gamma synthesis are either diminished or do not occur. Intracolonic administration of TNBS to either normal (IFN-gamma+/+) or Th1-deficient IFN-gamma knockout (IFN-gamma-/-) BALB/c mice resulted in significant colitis. In IFN-gamma-/- mice, crypt inflammation was more severe than in IFN-gamma+/+ mice and was accompanied by hypertrophy of colonic patches with a lymphoepithelium containing M cells and distinct B and T cell zones resembling Peyer's patches. Hapten-specific, colonic patch T cells from both mouse groups exhibited a Th2 phenotype with interleukin (IL)-4 and IL-5 production. TNBS colitis in normal mice treated with anti-IL-4 antibodies or in IL-4(-/-) mice was less severe than in either IFN-gamma+/+ or IFN-gamma-/- mice. Our findings now show that the Th2-type responses in TNBS colitis are associated with colonic patch enlargement and inflammation of the mucosal layer and may represent a model for ulcerative colitis.
Assuntos
Colite Ulcerativa/imunologia , Colo/patologia , Doenças Inflamatórias Intestinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th2/imunologia , Animais , Anticorpos/imunologia , Colite Ulcerativa/induzido quimicamente , Colo/imunologia , Modelos Animais de Doenças , Haptenos , Hipertrofia/patologia , Doenças Inflamatórias Intestinais/induzido quimicamente , Interferon gama/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Interleucina-5/imunologia , Interleucina-5/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/patologia , Células Th1/imunologia , Ácido TrinitrobenzenossulfônicoRESUMO
We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTbeta-R and TNF-R signaling. Lymph nodes developed in LTalpha-/- mice treated in utero with agonist anti-LTbeta-R monoclonal antibody. Thus, LTbeta-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTalphabeta/LTbeta-R interaction were generated. Normal mice treated in utero with LTbeta-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTalpha-/- mice had disrupted cellular organization. Therefore, LTbeta-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.
Assuntos
Linfonodos/embriologia , Linfotoxina-alfa/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Cricetinae , Feminino , Ligantes , Receptor beta de Linfotoxina , Linfotoxina-alfa/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Fator de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose TumoralRESUMO
For a brief period during fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules surprisingly express the Peyer's patch addressin MAdCAM-1. This expression allows initial seeding of this incipient structure by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. We show here that CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1 and that can become natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. Since the necessity of lymphotoxin in lymphoid organ development has been shown, we propose that the novel subset of CD4+CD3-LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.
Assuntos
Células Apresentadoras de Antígenos/citologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/citologia , Células Matadoras Naturais/citologia , Leucopoese , Linfonodos/embriologia , Subpopulações de Linfócitos/citologia , Linfotoxina-alfa/metabolismo , Proteínas de Membrana/metabolismo , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Moléculas de Adesão Celular , Citotoxicidade Imunológica , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunidade Celular , Imunoglobulinas/metabolismo , Integrinas/metabolismo , Interleucina-2/farmacologia , Antígenos Comuns de Leucócito/análise , Linfonodos/citologia , Linfotoxina-beta , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mucoproteínas/metabolismo , RNA Mensageiro/genética , Receptores CXCR5 , Receptores de Quimiocinas , Receptores de Citocinas/genética , Baço/embriologia , Baço/imunologia , Linfócitos T/citologiaRESUMO
The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines. Cells that bound LT beta R also bound anti-LT alpha and anti-LT beta mAbs in a FACS analysis. The ability of these reagents to discriminate between surface TNF and LT was verified by analysis of surface TNF-positive, LPS-activated murine RAW 264.7 monocytic cells. Primary mouse leukocytes from spleen, thymus, lymph node, and peritoneum were activated in vitro, and CD4+ and CD8+ T cells as well as B cells expressed surface LT ligand but not the LT beta R. Conversely, elicited peritoneal monocytes/macrophages were surface LT negative yet LT beta R positive. This study shows that on mononuclear cells, surface LT complexes and receptor are expressed similarly in mice and man, and the tools described herein form the foundation for study of the functional roles of the LT system in the mouse.
Assuntos
Linfócitos/química , Linfócitos/metabolismo , Linfotoxina-alfa/química , Proteínas de Membrana/química , Fator de Necrose Tumoral alfa/química , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Linfócitos B/química , Linhagem Celular , Citometria de Fluxo , Humanos , Hibridomas , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Linfócitos/imunologia , Linfoma de Células T , Linfotoxina-alfa/imunologia , Linfotoxina-beta , Macrófagos , Proteínas de Membrana/imunologia , Camundongos , Ratos , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Especificidade da Espécie , Linfócitos T/química , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
B7-1 (CD80) and B7-2 (CD86) are genetically and structurally related molecules expressed on antigen-presenting cells. Both bind CD28 to co-stimulate T lymphocytes, resulting in proliferation and cytokine production. The extracellular portions of B7-1 and B7-2 which bind to CD28 and CTLA-4 are related to Ig variable (V) and Ig constant (C) domain sequences. Recent reports have described splice variant forms of B7 proteins which occur in vivo and are of unknown function. Here we describe soluble recombinant forms of B7-1 and B7-2 containing either both of the Ig-like extracellular domains or the individual IgV or IgC domains coupled to an Ig Fc tail. Soluble B7-1 and B7-2 bind to CD28 and CTLA-4, and effectively co-stimulate T lymphocytes resulting in their proliferation and the secretion of cytokines. Furthermore, the IgV domain of B7-2 binds CD28 and CTLA-4, competes with B7-1 and B7-2 for binding to these receptors, and co-stimulates T lymphocytes. Cross-linked soluble B7-2v was the most potent co-stimulatory molecule tested and was active at a concentration approximately 100-fold lower than cross-linked soluble B7-1 or B7-2 proteins. When bound to tosyl-activated beads, B7-2v was capable of sustaining multiple rounds of T cell expansion. These data complement the description of naturally occurring variants to suggest that T cell co-stimulation in vivo may be regulated by soluble or truncated forms of B7 proteins.