RESUMO
The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Mesentério/irrigação sanguínea , Receptores Notch/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/enzimologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Masculino , Inibidores de Proteínas Quinases/farmacologia , Ratos Wistar , Vênulas/efeitos dos fármacos , Vênulas/enzimologiaRESUMO
Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²âº. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.
Assuntos
Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Ativadores de Enzimas/farmacologia , Heme Oxigenase-1/deficiência , Hidrocarbonetos Fluorados/farmacologia , Inflamação/prevenção & controle , Intestino Delgado/irrigação sanguínea , Isquemia/tratamento farmacológico , Proteínas de Membrana/deficiência , Oclusão Vascular Mesentérica/tratamento farmacológico , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Traumatismo por Reperfusão/prevenção & controle , Doenças Vasculares/tratamento farmacológico , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Guanilato Ciclase/metabolismo , Heme Oxigenase-1/genética , Sulfeto de Hidrogênio/metabolismo , Inflamação/enzimologia , Inflamação/genética , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Proteínas de Membrana/genética , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/enzimologia , Artéria Mesentérica Superior/cirurgia , Isquemia Mesentérica , Oclusão Vascular Mesentérica/enzimologia , Oclusão Vascular Mesentérica/genética , Oclusão Vascular Mesentérica/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Doenças Vasculares/enzimologia , Doenças Vasculares/genética , Doenças Vasculares/fisiopatologia , Vasodilatação/efeitos dos fármacos , Vênulas/efeitos dos fármacos , Vênulas/enzimologiaRESUMO
RATIONALE: Intravascular neutrophil recruitment and activation are key pathogenic factors that contribute to vascular injury. Intravenous immunoglobulin (IVIG) has been shown to have a beneficial effect in systemic inflammatory disorders; however, the mechanisms underlying IVIG's inhibitory effect on neutrophil recruitment and activation are not understood. OBJECTIVE: We studied the mechanisms by which IVIG exerts protection from neutrophil-mediated acute vascular injury. METHODS AND RESULTS: We examined neutrophil behavior in response to IVIG in vivo, using real-time intravital microscopy. We found that an antibody that blocks both FcγRIII and its inhibitory receptor counterpart, FcγRIIB, abrogated the inhibitory effect of IVIG on leukocyte recruitment and heterotypic red blood cell (RBC) interactions with adherent leukocytes in wild-type mice. In the context of sickle cell disease, the blockade of both FcγRIIB and III abrogated the protective effect of IVIG on acute vaso-occlusive crisis caused by neutrophil recruitment and activation. Analysis of FcγRIIB- and FcγRIII-deficient mice revealed the predominant expression of FcγRIII on circulating neutrophils. FcγRIII mediated IVIG-triggered inhibition of leukocyte recruitment, circulating RBC capture, and enhanced Mac-1 activity, whereas FcγRIIB was dispensable. In addition, FcγRIII-induced IVIG anti-inflammatory activity in neutrophils was mediated by recruitment of Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1). Indeed, the protective effect of IVIG on leukocyte recruitment and activation was abrogated in SHP-1-mutant mice. CONCLUSIONS: FcγRIII, a classic activating receptor, has an unexpected inhibitory role on neutrophil adhesion and activation via recruitment of SHP-1 in response to IVIG. Our results identify SHP-1 as a therapeutic target in neutrophil-mediated vascular injury.
Assuntos
Anemia Falciforme/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , Músculo Liso/irrigação sanguínea , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de IgG/metabolismo , Doenças Vasculares/prevenção & controle , Anemia Falciforme/enzimologia , Anemia Falciforme/genética , Anemia Falciforme/imunologia , Anemia Falciforme/patologia , Animais , Constrição Patológica , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Antígeno de Macrófago 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Vídeo , Neutrófilos/enzimologia , Neutrófilos/imunologia , Receptores de IgG/deficiência , Receptores de IgG/genética , Fatores de Tempo , Doenças Vasculares/enzimologia , Doenças Vasculares/genética , Doenças Vasculares/imunologia , Doenças Vasculares/patologia , Vênulas/efeitos dos fármacos , Vênulas/enzimologia , Vênulas/imunologiaRESUMO
It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show that modulation of CB1 function has anti-inflammatory effects and suggests that inverse agonism of CB1 block signal transduction mechanisms controlling encephalitogenic T cells adhesion in inflamed brain venules by a PKA-dependent mechanism.
Assuntos
Encéfalo/irrigação sanguínea , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Adesão Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Camundongos , Subpopulações de Linfócitos T/enzimologia , Vênulas/enzimologia , Vênulas/imunologia , Vênulas/patologiaRESUMO
It has been suggested that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent mechanisms in the ocular vasculature; however, the importance of the pathway in regulating retinal circulation in vivo remains to be elucidated. Therefore, we investigated the role of COX-dependent mechanisms in NO-induced vasodilation of retinal blood vessels in thiobutabarbital-anesthetized rats with and without neuronal blockade (tetrodotoxin treatment). Fundus images were captured with a digital camera that was equipped with a special objective lens. The retinal vascular response was assessed by measuring changes in diameter of the retinal blood vessel. The localization of COX and soluble guanylyl cyclase in rat retina was examined using immunohistochemistry. The NO donors (sodium nitroprusside and NOR3) increased the diameter of the retinal blood vessels but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a nonselective COX inhibitor, or SC-560, a selective COX-1 inhibitor, markedly attenuated the vasodilation of retinal arterioles, but not the depressor response, to the NO donors. However, both the vascular responses to NO donors were unaffected by the selective COX-2 inhibitors NS-398 and nimesulide. Indomethacin did not change the retinal vascular and depressor responses to hydralazine, 8-(4-chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (a membrane-permeable cGMP analog) and 8-(4-chlorophenylthio)-adenosine-3', 5'-cyclic monophosphate (a membrane-permeable cAMP analog). Treatment with SQ 22536, an adenylyl cyclase inhibitor, but not ODQ, a soluble guanylyl cyclase inhibitor, significantly attenuated the NOR3-induced vasodilation of retinal arterioles. The COX-1 immunoreactivity was found in retinal blood vessels. The retinal blood vessel was faintly stained for soluble guanylyl cyclase, although the apparent immunoreactivities on mesenteric and choroidal blood vessels were observed. These results suggest that NO exerts a substantial part of its dilatory effect via a mechanism that involves COX-1-dependent pathway in rat retinal vasculature.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico/metabolismo , Vasos Retinianos/enzimologia , Vasodilatação , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Anestésicos Locais/administração & dosagem , Animais , Arteríolas/enzimologia , Pressão Sanguínea , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Inibidores de Ciclo-Oxigenase/administração & dosagem , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Frequência Cardíaca , Masculino , Proteínas de Membrana/antagonistas & inibidores , Doadores de Óxido Nítrico/administração & dosagem , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Vasos Retinianos/efeitos dos fármacos , Guanilil Ciclase Solúvel , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Vênulas/enzimologiaRESUMO
After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.
Assuntos
5'-Nucleotidase/imunologia , Adenosina/imunologia , Movimento Celular/imunologia , Endotélio Vascular/imunologia , Linfonodos/imunologia , Linfócitos T/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adenosina/genética , Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/imunologia , Monofosfato de Adenosina/metabolismo , Aminopiridinas/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Endotélio Vascular/enzimologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Selectina L/imunologia , Selectina L/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Receptor A2B de Adenosina/imunologia , Receptor A2B de Adenosina/metabolismo , Linfócitos T/enzimologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologia , Vênulas/enzimologia , Vênulas/imunologiaRESUMO
Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.
Assuntos
Aspirina/análogos & derivados , Plaquetas/efeitos dos fármacos , Doenças Cardiovasculares/prevenção & controle , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Hipercolesterolemia/tratamento farmacológico , Proteínas de Membrana/metabolismo , Doadores de Óxido Nítrico/farmacologia , Pirazóis/farmacologia , Animais , Aspirina/farmacologia , Aspirina/uso terapêutico , Plaquetas/enzimologia , Transplante de Medula Óssea , Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Adesão Celular/efeitos dos fármacos , Quimera/metabolismo , Colesterol na Dieta , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/genética , Inibidores de Ciclo-Oxigenase/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Hipercolesterolemia/induzido quimicamente , Hipercolesterolemia/complicações , Hipercolesterolemia/enzimologia , Hipercolesterolemia/fisiopatologia , Leucócitos/efeitos dos fármacos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Microscopia de Vídeo , Doadores de Óxido Nítrico/uso terapêutico , Adesividade Plaquetária/efeitos dos fármacos , Pirazóis/uso terapêutico , Vênulas/efeitos dos fármacos , Vênulas/enzimologiaRESUMO
During inflammation, leukocytes roll along the wall of postcapillary venules scanning the surface for immobilized CXCL1, a chemokine that triggers firm adhesion by activating CXCR2 on the neutrophil. PI-3K are signaling molecules important in cellular processes, ranging from cellular differentiation to leukocyte migration. PI-3Kgamma can be activated directly by the betagamma dimer of heterotrimeric G proteins coupled to CXCR2. Here, we used in vivo and ex vivo intravital microscopy models to test the role of PI-3Kgamma in leukocyte arrest. PI-3Kgamma null mice showed an 80% decrease in CXCL1-induced leukocyte adhesion in venules of the exteriorized mouse cremaster muscle. In wild-type mice, rolling leukocytes showed rapid and sustained adhesion, but in PI-3Kgamma(-/-) mice, adhesion was not triggered at all or was transient, suggesting that absence of PI-3Kgamma interferes with integrin bond strengthening. Wild-type mice reconstituted with PI-3Kgamma null bone marrow showed a 50% decrease in CXCL1-induced leukocyte adhesion. In a blood-perfused micro-flow chamber, leukocytes from PI-3Kgamma(-/-) mice showed a defect in adhesion on a P-selectin/ICAM-1/CXCL1 substrate, indicating that leukocyte PI-3Kgamma was required for adhesion. The adhesion defect in PI-3Kgamma(-/-) mice was as severe as that in mice lacking LFA-1, the major integrin responsible for neutrophil adhesion. We conclude that the gamma isoform of PI-3K must be functional in leukocytes to allow efficient adhesion from rolling in response to chemokine stimulation.
Assuntos
Quimiocinas CXC/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Leucócitos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Quimiocina CXCL1 , Quimiocinas CXC/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Isoenzimas/deficiência , Isoenzimas/metabolismo , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/citologia , Camundongos , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Selectina-P/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Vênulas/citologia , Vênulas/enzimologiaRESUMO
Lymphocyte migration into the brain represents a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms controlling the recruitment of lymphocytes to the CNS via inflamed brain venules are poorly understood, and therapeutic approaches to inhibit this process are consequently few. In this study, we demonstrate for the first time that human and murine Th1 lymphocytes preferentially adhere to murine inflamed brain venules in an experimental model that mimics early inflammation during EAE. A virtually complete inhibition of rolling and arrest of Th1 cells in inflamed brain venules was observed with a blocking anti-P-selectin glycoprotein ligand 1 Ab and anti-E- and P-selectin Abs. Th1 lymphocytes produced from fucosyltransferase (FucT)-IV(-/-) mice efficiently tethered and rolled, whereas in contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VII(-/-) or Fuc-VII(-/-)FucT-IV(-/-) mice was drastically reduced, indicating that FucT-VII is critical for the recruitment of Th1 cells in inflamed brain microcirculation. Importantly, we show that Abs directed against cutaneous lymphocyte Ag (CLA), a FucT-VII-dependent carbohydrate modification of P-selectin glycoprotein ligand 1, blocked rolling of Th1 cells. By exploiting a system that allowed us to obtain Th1 and Th2 cells with skin- vs gut-homing (CLA(+) vs integrin beta(7)(+)) phenotypes, we observed that induced expression of CLA on Th cells determined a striking increase of rolling efficiency in inflamed brain venules. These observations allow us to conclude that efficient recruitment of activated lymphocytes to the brain in the contexts mimicking EAE is controlled by FucT-VII and its cognate cell surface Ag CLA.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/patologia , Movimento Celular/imunologia , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Fucosiltransferases/biossíntese , Glicoproteínas de Membrana/biossíntese , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos T , Antígenos de Neoplasias , Encéfalo/enzimologia , Comunicação Celular/genética , Comunicação Celular/imunologia , Movimento Celular/genética , Células Cultivadas , Selectina E/fisiologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Humanos , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/fisiologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologia , Vênulas/enzimologia , Vênulas/imunologia , Vênulas/patologiaRESUMO
L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.
Assuntos
Endotélio Linfático/enzimologia , Selectina L/fisiologia , N-Acetilglucosaminiltransferases/fisiologia , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Endotélio Linfático/imunologia , Endotélio Linfático/metabolismo , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Selectina L/metabolismo , Leucócitos/enzimologia , Leucócitos/imunologia , Leucócitos/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Microscopia de Vídeo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Músculo Esquelético/imunologia , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/enzimologia , Nódulos Linfáticos Agregados/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vênulas/enzimologia , Vênulas/imunologia , Vênulas/patologiaRESUMO
Leukocyte capture and rolling are mediated by selectins expressed on leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To investigate the role of core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I) for synthesis of functional selectin ligands in vivo, leukocyte rolling flux and velocity were studied in venules of untreated and tumor necrosis factor-alpha (TNFalpha)-pretreated autoperfused cremaster muscles of C2GlcNAcT-I-deficient (core 2(-/-)) and littermate control mice. In untreated core 2(-/-) mice, leukocyte rolling was dramatically reduced with markedly increased rolling velocities (81 +/- 4 microm/s vs 44 +/- 3 microm/s). The reduced rolling in core 2(-/-) mice was due mainly to severely impaired binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1). Some rolling remained after blocking PSGL-1 in controls but not in core 2(-/-) mice. In TNFalpha-pretreated mice, rolling was markedly reduced in core 2(-/-) mice owing to impaired P-selectin- and E-selectin-mediated rolling. Rolling velocities in core 2(-/-) mice treated with an E-selectin-blocking monoclonal antibody (59 +/- 4 microm/s) were significantly higher than in controls (14 +/- 1 microm/s), which provides further evidence for the severe impairment in P-selectin-mediated rolling. In conclusion, P-selectin ligands including PSGL-1 are largely C2GlcNAcT-I dependent. In addition, E-selectin-mediated rolling in vivo is partially dependent on the targeted C2GlcNAcT-I. (Blood. 2001;97:3812-3819)
Assuntos
Adesão Celular/efeitos dos fármacos , N-Acetilglucosaminiltransferases/farmacologia , Vênulas/enzimologia , Animais , Células da Medula Óssea/química , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Selectina E/efeitos dos fármacos , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Inflamação/induzido quimicamente , Inflamação/patologia , Ligantes , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/deficiência , Neutrófilos/química , Selectina-P/efeitos dos fármacos , Selectina-P/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.
Assuntos
Endotélio Vascular/metabolismo , Glutamina/metabolismo , Ornitina-Oxo-Ácido Transaminase/metabolismo , Ornitina/biossíntese , Alanina/metabolismo , Amônia/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Aorta/citologia , Aorta/enzimologia , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Azasserina/farmacologia , Dióxido de Carbono/metabolismo , Bovinos , Células Cultivadas , Diazo-Oxo-Norleucina/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Enzimas/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Microcirculação/citologia , Microcirculação/enzimologia , Veias Umbilicais/citologia , Veias Umbilicais/enzimologia , Vênulas/citologia , Vênulas/enzimologiaRESUMO
OBJECTIVES: The distribution of the intestinal vascular lesions and their relation with the fibrinolysis process are poorly known in Crohn's disease (CD). The mediators of the plasminogen activator system, namely urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1), are a key complex involved in fibrinolysis. The aims of this study were: (1) to further define vascular lesions and their distribution in the intestine; and (2) to study concomitantly the qualitative in situ expression and the levels of u-PA, t-PA and PAI-1 in the ileum of patients with CD. PATIENTS AND METHODS: Histological, immunohistochemical and ultrastructural studies of vascular lesions in the resected ileum of 27 patients with CD were performed and compared with 36 control patients. Levels of u-PA, t-PA and PAI-1 measured by ELISA methods were compared in healthy and inflamed ileal tissues of 17 patients with CD. RESULTS: Acute vascular lesions involving mainly serosal venules and capillaries were present in 63% of patients with CD vs 3/36 controls and were associated with PAI-1 expression. They were prominent on the mesenteric border beneath macroscopically normal mucosa. In contrast, chronic vascular lesions were present in all layers beneath mucosal ulcerations, where a significant increase of PAI-1 levels was found. CONCLUSIONS: These results suggest that vascular involvement associated with abnormalities of PAI-1 expression is an early and widespread event in CD. Their prominence on the mesenteric border might explain the characteristic location of CD ulceration along the mesenteric margin.
Assuntos
Doença de Crohn/patologia , Íleo/enzimologia , Íleo/patologia , Inflamação/patologia , Ativadores de Plasminogênio/metabolismo , Adolescente , Adulto , Idoso , Biópsia , Capilares/enzimologia , Capilares/patologia , Capilares/ultraestrutura , Criança , Doença de Crohn/enzimologia , Feminino , Humanos , Íleo/irrigação sanguínea , Imuno-Histoquímica , Inflamação/enzimologia , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Vênulas/enzimologia , Vênulas/patologia , Vênulas/ultraestruturaRESUMO
High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. Studies with chlorate, a metabolic inhibitor of sulfation, had previously revealed that production of PAPS (3'-phosphoadenosine-5'-phosphosulfate), the high-energy donor of sulfate, is required for sulfation and high-affinity recognition of HEV sialomucins GlyCAM-1 and CD34 by the lymphocyte homing receptor L-selectin. Here, we report the molecular characterization of a novel 2.5 kb human cDNA from MECA-79+ HEV-derived endothelial cells that encodes the target of chlorate, PAPS synthetase, a multifunctional enzyme containing domains for both ATP sulfurylase and adenosine-5'-phosphosulfate kinase. Functional expression of the isolated cDNA in Chinese hamster ovary cells results in high levels of PAPS synthesis, which is abolished by treatment of the transfected cells with chlorate. Northern blot analysis reveals a wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may be important for sulfation not only of HEV sialomucins, but also of many other molecules, including mucins such as the P-selectin ligand PSGL-1, proteoglycans, hormones, neurotransmitters, drugs, and xenobiotics.