RESUMO
The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Citoesqueleto/metabolismo , Integrina alfa3/metabolismo , Selectina L/metabolismo , Neutrófilos/fisiologia , Tetraspanina 25/metabolismo , Animais , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Migração Transendotelial e TransepitelialRESUMO
This study supports a new concept where the opposing functions of the tetraspanins CD37 and CD82 may coordinate changes in migration and Ag presentation during dendritic cell (DC) activation. We have previously published that CD37 is downregulated upon monocyte-derived DC activation, promotes migration of both skin and bone marrow-derived dendritic cells (BMDCs), and restrains Ag presentation in splenic and BMDCs. In this article, we show that CD82, the closest phylogenetic relative to CD37, appears to have opposing functions. CD82 is upregulated upon activation of BMDCs and monocyte-derived DCs, restrains migration of skin and BMDCs, supports MHC class II maturation, and promotes stable interactions between T cells and splenic DCs or BMDCs. The underlying mechanism involves the rearrangement of the cytoskeleton via a differential activation of small GTPases. Both CD37(-/-) and CD82(-/-) BMDCs lack cellular projections, but where CD37(-/-) BMDCs spread poorly on fibronectin, CD82(-/-) BMDCs are large and spread to a greater extent than wild-type BMDCs. At the molecular level, CD82 is a negative regulator of RhoA, whereas CD37 promotes activation of Rac-1; both tetraspanins negatively regulate Cdc42. Thus, this study identifies a key aspect of DC biology: an unactivated BMDC is CD37(hi)CD82(lo), resulting in a highly motile cell with a limited ability to activate naive T cells. By contrast, a late activated BMDC is CD37(lo)CD82(hi), and thus has modified its migratory, cytoskeletal, and Ag presentation machinery to become a cell superbly adapted to activating naive T cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Movimento Celular , Células Dendríticas/imunologia , Proteína Kangai-1/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Tetraspaninas/imunologia , Animais , Separação Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da PolimeraseRESUMO
Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.
Assuntos
Actinas/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Citoesqueleto/imunologia , Neutrófilos/fisiologia , Tetraspaninas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígenos CD18/metabolismo , Adesão Celular , Movimento Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiotaxia/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Tetraspaninas/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Previous studies on the role of the tetraspanin CD37 in cellular immunity appear contradictory. In vitro approaches indicate a negative regulatory role, whereas in vivo studies suggest that CD37 is necessary for optimal cellular responses. To resolve this discrepancy, we studied the adaptive cellular immune responses of CD37(-/-) mice to intradermal challenge with either tumors or model antigens and found that CD37 is essential for optimal cell-mediated immunity. We provide evidence that an increased susceptibility to tumors observed in CD37(-/-) mice coincides with a striking failure to induce antigen-specific IFN-γ-secreting T cells. We also show that CD37 ablation impairs several aspects of DC function including: in vivo migration from skin to draining lymph nodes; chemo-tactic migration; integrin-mediated adhesion under flow; the ability to spread and form actin protrusions and in vivo priming of adoptively transferred naïve T cells. In addition, multiphoton microscopy-based assessment of dermal DC migration demonstrated a reduced rate of migration and increased randomness of DC migration in CD37(-/-) mice. Together, these studies are consistent with a model in which the cellular defect that underlies poor cellular immune induction in CD37(-/-) mice is impaired DC migration.
Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Tetraspaninas/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Adesão Celular/imunologia , Proliferação de Células , Células Dendríticas/patologia , Feminino , Expressão Gênica , Interferon gama/biossíntese , Interferon gama/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Transplante de Neoplasias , Pele/imunologia , Pele/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/transplante , Tetraspaninas/deficiência , Tetraspaninas/genéticaRESUMO
Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-ß levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.
Assuntos
Dermatite de Contato/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Pele/irrigação sanguínea , Pele/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Dermatite de Contato/genética , Dermatite de Contato/patologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/patologia , Linfócitos T Reguladores/metabolismo , Vênulas/imunologia , Vênulas/metabolismo , Vênulas/patologiaRESUMO
BACKGROUND & AIMS: Helicobacter pylori infection results in a diversity of pathologies, from asymptomatic gastritis to adenocarcinoma. The reason for these diverse outcomes is multifactorial and includes host factors that regulate severity of Helicobacter-induced gastritis. Protease-activated receptors (PAR) are environmental sensors that can detect tissue damage and pathogens. Whereas PAR-2 has proinflammatory activity and PAR-1 can protect the gastric mucosa against chemical damage, neither has previously been examined for their potential roles in regulating Helicobacter pathogenesis. METHODS: PAR-1(-/-), PAR-2(-/-), and wild-type mice were infected with H pylori for up to 2 months then colonization levels determined by colony-forming assay, gastritis by histology, and serum antibody levels by enzyme-linked immunosorbent assay. Responsiveness of primary epithelial cells to PAR-1 activation was assessed by calcium mobilization assay. Primary epithelial cells, macrophages, and dendritic cells were cocultured with H pylori and nuclear factor (NF)-kappaB, and cytokine secretion was determined by enzyme-linked immunosorbent assay. RESULTS: Two months postinfection, H pylori levels were significantly reduced in PAR-1(-/-) and increased in PAR-2(-/-) mice. This effect on colonization was inversely correlated with inflammation severity. Infection of PAR-1(-/-) mice induced an increased serum antibody response. Primary epithelial cells were activated by a PAR-1-activating peptide. H pylori stimulation of primary epithelial cells, but not macrophages or dendritic cells, from PAR-1(-/-) mice induced increased levels of NF-kappaB and the proinflammatory cytokine macrophage-inflammatory protein (MIP)-2. PAR-1 also down-regulated MIP-2 secretion in response to cag pathogenicity island activity. CONCLUSIONS: PAR-1 protects the host against severe Helicobacter-induced gastritis. This may be mediated by suppressing the production of proinflammatory cytokines such as MIP-2.
Assuntos
Gastrite/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/fisiologia , Imunidade Humoral/fisiologia , Inflamação/fisiopatologia , Receptor PAR-1/metabolismo , Animais , Células Cultivadas , Quimiocina CXCL2/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Epitélio/metabolismo , Epitélio/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrite/fisiopatologia , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor PAR-1/genética , Receptor PAR-2/genética , Receptor PAR-2/metabolismoRESUMO
Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)-gamma-chain. ceacam1(-/-) platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1(-/-) mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1(-/-) mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1(-/-) mice showed a reversal in the more stable thrombus growth phenotype. ceacam1(-/-) mice were more susceptible to type I collagen-induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.
Assuntos
Plaquetas/metabolismo , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Colágeno Tipo I/metabolismo , Trombose/metabolismo , Animais , Retroalimentação Fisiológica/fisiologia , Feminino , Hematopoese/fisiologia , Homeostase/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fosfolipase C beta/metabolismo , Fosforilação/fisiologia , Adesividade Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Embolia Pulmonar/metabolismo , Embolia Pulmonar/fisiopatologia , Trombose/fisiopatologiaRESUMO
Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.
Assuntos
Surtos de Doenças/veterinária , Infecções por Helicobacter/veterinária , Helicobacter/genética , Helicobacter/isolamento & purificação , Marsupiais , Zoonoses/epidemiologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana/veterinária , Feminino , Helicobacter/metabolismo , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/mortalidade , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA/veterinária , Urease/genética , Urease/metabolismo , Vitória , Zoonoses/microbiologia , Zoonoses/mortalidadeRESUMO
Tetraspanins regulate key processes in immune cells; however, the function of the leukocyte-restricted tetraspanin CD53 is unknown. Here we show that CD53 is essential for lymphocyte recirculation. Lymph nodes of Cd53-/- mice were smaller than those of wild-type mice due to a marked reduction in B cells and a 50% decrease in T cells. This reduced cellularity reflected an inability of Cd53-/- B and T cells to efficiently home to lymph nodes, due to the near absence of L-selectin from Cd53-/- B cells and reduced stability of L-selectin on Cd53-/- T cells. Further analyses, including on human lymphocytes, showed that CD53 stabilizes L-selectin surface expression and may restrain L-selectin shedding via both ADAM17-dependent and ADAM17-independent mechanisms. The disruption in lymphocyte recirculation in Cd53-/- mice led to impaired immune responses dependent on antigen delivery to lymph nodes. Together these findings demonstrate an essential role for CD53 in lymphocyte trafficking and immunity.
RESUMO
As the majority of human pathogens infect via a mucosal surface, delivery of killed vaccines by mucosal routes could potentially improve protection against many such organisms. Our ability to develop effective killed mucosal vaccines is inhibited by a lack of adjuvants that are safe and effective in humans. The Ulex europaeus agglutinin I (UEA-I) lectin specifically binds M cells lining the murine gastrointestinal tract. We explored the potential for M-cell-targeted vaccination of whole, killed Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer, and Campylobacter jejuni, the most common cause of diarrhea. Oral delivery of UEA-I-agglutinated H. pylori or C. jejuni induced a significant increase in both serum and intestinal antibody levels. This elevated response (i) required the use of whole bacteria, as it did not occur with lysate; (ii) was not mediated by formation of particulate clumps, as agglutination with a lectin with a different glycan specificity had no effect; and (iii) was not due to lectin-mediated, nonspecific immunostimulatory activity, as UEA-I codelivery with nonagglutinated bacteria did not enhance the response. Vaccination with UEA-I-agglutinated, killed whole H. pylori induced a protective response against subsequent live challenge that was as effective as that induced by cholera toxin adjuvant. Moreover, vaccination against C. jejuni by this approach resulted in complete protection against challenge in almost all animals. We believe that this is the first demonstration that targeting of whole killed bacteria to mucosal M cells can induce protective immunity without the addition of an immunostimulatory adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas/imunologia , Infecções por Campylobacter/prevenção & controle , Infecções por Helicobacter/prevenção & controle , Lectinas de Plantas/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Infecções por Campylobacter/patologia , Contagem de Colônia Microbiana , Feminino , Infecções por Helicobacter/patologia , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Mucosa Intestinal/química , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Lectinas de Plantas/administração & dosagem , Índice de Gravidade de Doença , Estômago/microbiologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
We have developed and validated a novel method to access efferent lymph draining the lung and gut of sheep. In this model, efferent lymph derived from the lung could be collected via cannulation of the thoracic duct just prior the thoracic duct-jugular vein junction. The thoracic duct was accessed in the neck region without needing to broach the thoracic cavity, thus avoiding extensive tissue damage to the animal and need for ventilation during surgery. In addition, this surgical approach allows for a second cannulation of an adjacent lymphatic draining the head/neck region, providing for an 'in-built' internal control with which to compare lymph parameters. To test the verity of cannulation procedure, a test protein ovalbumin (OVA) was infused into the left and right lungs via bronchoscopy. We found that OVA was recovered almost exclusively in the lymph draining the lungs compared to the lymph draining the head/neck where it was essentially non-existent. The method described here will be invaluable for optimizing intra-lung delivery of drugs or vaccines. In addition, access to lymph will also allow for analysis of immune responses to infections originating at this site.
Assuntos
Cateterismo/veterinária , Pulmão/anatomia & histologia , Linfa/metabolismo , Ovinos/cirurgia , Ducto Torácico/cirurgia , Animais , Cateterismo/métodos , Colesterol/metabolismo , Feminino , Pulmão/metabolismo , Linfa/química , Ovalbumina/farmacocinética , Corantes de Rosanilina/farmacocinética , Ducto Torácico/metabolismo , Triglicerídeos/metabolismoRESUMO
BACKGROUND: To overcome cell-mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3-dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo. METHODS: The PIEC stable lines expressing IDO, CD40Ig, and IL10 as single molecules were generated. In addition, PIEC lines expressing IDO with either CD40Ig, IL10 or CTLA4Ig were generated to produce cell lines expressing two molecules. BALB/c mice were primed with wild type PIEC, followed by harvesting splenocytes used as responder cells and PIEC expressing immunomodulatory molecules as stimulators, in proliferation and cytokine assays. In vivo effects of modified PIEC were examined by transplantation of PIEC lines expressing the immunomodulatory molecules under the renal capsule of naïve mice. PIEC grafts were harvested for histological evaluation at days 7 and 14. RESULTS: Proliferation of primed BALB/c splenocytes was inhibited most significantly by IDO compared with control cells (49%, P = 0.02). In addition both Th1 (interferon-gamma) and Th2 (IL4 and IL10) cytokines were markedly inhibited in vitro by IDO expression. IL10 expressing cells did not inhibit proliferation as potently (37%, P = 0.03) whilst CD40Ig lead to an increase in proliferative responses (59%, P = 0.02). Co-expression of CD40Ig, IL10, and CTLA4Ig with IDO resulted in further modest reductions in proliferation compared with IDO expression alone. When transplanted under the renal capsule of BALB/c mice, those grafts expressing IDO demonstrated significantly lower levels of lymphocyte infiltration at days 7 and 14 than control grafts and those expressing CD40Ig, CTLA4Ig or IL10 alone. Grafts co-expressing IDO and a second molecule were no better protected than those expressing IDO alone. Graft cell viability (PIECs) was reduced in some IDO expressing grafts suggesting high levels of IDO expression may inhibit PIEC viability, however, grafts co-expressing IDO-CTLA4Ig and IDO-IL10 were not affected in this way. CONCLUSION: Indoleamine 2,3-dioxygenase appears to be a potent molecule for protecting xenografts from cell-mediated rejection responses activated via the indirect pathway. Co-expression of IDO with both CTLA4Ig and IL10 warrants further investigation. Overall these findings support pursuing further studies, in larger animal models, to determine whether increased IDO activity within the graft itself can attenuate xenorejection responses.
Assuntos
Antígenos CD/imunologia , Antígenos CD40/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Imunoglobulinas/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-10/imunologia , Transplante Heterólogo/imunologia , Animais , Antígenos CD/genética , Antígenos CD40/genética , Antígeno CTLA-4 , Proliferação de Células , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Regulação da Expressão Gênica , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interleucina-10/genética , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Linfócitos T/imunologiaRESUMO
Homophilic engagement of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) induces 'outside-in' signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM-1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM-1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co-immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM-1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM-1 peptide (594-604) C595A containing the sequences (594)KAFYLRKAKAK(604). This CaM:PECAM-1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM-1 in a time- and dose-dependent manner. Furthermore, this membrane proximal portion of PECAM-1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM-binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM-1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM-1.
Assuntos
Plaquetas/metabolismo , Calmodulina/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrólise , Dados de Sequência Molecular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Homologia de Sequência de AminoácidosRESUMO
Pulmonary delivery of an influenza Iscomatrix adjuvant vaccine induces a strong systemic and mucosal antibody response. Since an influenza vaccine needs to induce immunological memory that lasts at least 1 year for utility in humans, we examined the longevity of the immune response induced by such a pulmonary vaccination, with and without antigen challenge. Sheep were vaccinated in the deep lung with an influenza Iscomatrix vaccine, and serum and lung antibody levels were quantified for up to 1 year. The immune memory response to these vaccinations was determined following antigen challenge via lung delivery of influenza antigen at 6 months and 1 year postvaccination. Pulmonary vaccination of sheep with the influenza Iscomatrix vaccine induced antigen-specific antibodies in both sera and lungs that were detectable until 6 months postimmunization. Importantly, a memory recall response following antigenic challenge was detected at 12 months post-lung vaccination, including the induction of functional antibodies with hemagglutination inhibition activity. Pulmonary delivery of an influenza Iscomatrix vaccine induces a long-lived influenza virus-specific antibody and memory response of suitable length for annual vaccination against influenza.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Colesterol/administração & dosagem , Memória Imunológica , Vacinas contra Influenza/imunologia , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Vacinação/métodos , Administração por Inalação , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Sangue/imunologia , Combinação de Medicamentos , Feminino , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza/administração & dosagem , Pulmão/imunologia , Ovinos , Fatores de TempoRESUMO
Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.
Assuntos
Interleucina-6/fisiologia , Linfonodos/fisiologia , Linfócitos/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Movimento Celular/imunologia , Movimento Celular/fisiologia , Feminino , Linfonodos/imunologia , Linfócitos/imunologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Ovinos/imunologia , Pele/imunologia , Fenômenos Fisiológicos da Pele/imunologiaRESUMO
Deep pulmonary delivery of an influenza ISCOMATRIX vaccine has previously been shown to induce a combined mucosal and systemic antibody response. To explore whether this combined response is influenced by intrinsic properties of the component antigen, we examined the efficacy of deep pulmonary delivery of ISCOMATRIX vaccines containing different recombinant antigens, specifically gB glycoprotein from cytomegalovirus and a fragment of catalase from Helicobacter pylori. Both these vaccines induced antigen-specific mucosal and systemic immunity, as well as antigen-specific proliferative cellular responses. Pulmonary immunisation with ISCOMATRIX vaccines may therefore be a generic way of inducing combined systemic and mucosal immunity.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Bactérias/imunologia , Antígenos Virais/imunologia , Colesterol/administração & dosagem , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Administração por Inalação , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Catalase/imunologia , Proliferação de Células , Combinação de Medicamentos , Feminino , Helicobacter pylori/enzimologia , Injeções Subcutâneas , Linfócitos/imunologia , Ovinos , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Platelet activation is crucial for normal hemostasis to arrest bleeding following vascular injury. However, excessive platelet activation in narrowed atherosclerotic blood vessels that are subject to high shear forces may initiate the onset of arterial thrombosis. When platelets come into contact with, and adhere to collagen exposed by damaged endothelium, they undergo morphological and functional changes necessary to generate a platelet-rich thrombus. This process is complex and involves precise co-ordination of various signaling pathways which lead to firm platelet adhesion to sites of tissue damage, release of granule contents from activated platelets, platelet shape change, platelet aggregation and subsequent thrombus formation and consolidation. Induction of tyrosine phosphorylation of key signaling molecules has emerged as a critical event central to stimulatory signaling pathways that generate platelet activation, but is an essential component associated with regulatory pathways that limit the extent of platelet activation. Understanding mechanisms that regulate platelet activation may contribute to the development of novel therapeutics that control common vascular diseases such as myocardial infarction and ischaemic stroke.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Fosfotirosina/fisiologia , Transdução de Sinais/fisiologia , Trombose/tratamento farmacológico , Trombose/metabolismo , Animais , Plaquetas/metabolismo , Humanos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Co-stimulatory blockade is known to inhibit lymphocyte responses and to prolong allograft and xenograft survival. The present study examines the effect of transgenic expression of cytotoxic T lymphocyte-associated molecule-4 immunoglobulin (CTLA4Ig) by a porcine endothelial cell line (PIEC) transduced by a lentiviral vector, on primed xenogeneic T-cell proliferative and cytokine responses. METHODS: Splenocytes from mice primed with PIEC were used as responder cells in a secondary proliferative assay. CTLA4Ig transduced and wild-type PIEC were used as stimulator cells. Responder cells were assayed for proliferation and cytokine production. RESULTS: Proliferation was profoundly inhibited by CTLA4Ig transduced cells compared with control cells. Cytokine analysis by enzyme linked immunospot demonstrated that production of interferon-gamma, IL4 (interleukin 4) and IL10 was inhibited by CTLA4Ig transduced cells compared with control cells. CONCLUSION: CTLA4Ig inhibited primed indirect xenogeneic T-cell proliferative and cytokine responses in vitro. Expression of immunomodulatory molecules by xenogeneic tissues has potential therapeutic applications for future xenotransplantation.
Assuntos
Citocinas/análise , Imunoconjugados/genética , Lentivirus/genética , Transfusão de Linfócitos , Linfócitos T/imunologia , Transplante Heterólogo/imunologia , Abatacepte , Animais , Animais Geneticamente Modificados , Linhagem Celular , Endotélio Vascular , Vetores Genéticos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , SuínosRESUMO
We investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin alpha(IIb)beta(3) complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin alpha(IIb)beta(3) expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal "inside-out" integrin alpha(IIb)beta(3) signaling (fluorescein isothiocyanate [FITC]-fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin alpha(IIb)beta(3) "outside-in" signaling events in platelets and is necessary for stability of arterial thrombi in vivo.
Assuntos
Plaquetas/fisiologia , Proteínas de Membrana/deficiência , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Animais , Retração do Coágulo/fisiologia , Hemostasia , Técnicas In Vitro , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Transdução de Sinais , Trombose/sangueRESUMO
Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.