RESUMO
BACKGROUND: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn's disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-α4ß7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. METHODS: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. RESULTS: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-α4ß7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4ß1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of α4 and α4ß1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4ß1 integrins, but not α4ß7 integrins. CONCLUSIONS: Our findings suggest that Teff cell homing to the ileum through the axis α4ß1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.
Assuntos
Doença de Crohn/imunologia , Íleo/imunologia , Integrina alfa4beta1/imunologia , Receptores de Retorno de Linfócitos/imunologia , Linfócitos T/imunologia , Adulto , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Moléculas de Adesão Celular , Movimento Celular , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Feminino , Citometria de Fluxo , Fármacos Gastrointestinais/farmacologia , Humanos , Íleo/patologia , Imunoglobulinas/efeitos dos fármacos , Imunoglobulinas/imunologia , Imuno-Histoquímica , Integrina alfa4beta1/efeitos dos fármacos , Masculino , Camundongos , Mucoproteínas/efeitos dos fármacos , Mucoproteínas/imunologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) is best known for its activity as T-cell-active chemoattractant regulating the egress of T cells from the lymph node and, consequently, the availability of T cells for migration into peripheral tissues. This physiological role of S1P is exploited by the drug fingolimod, a first-line therapy for multiple sclerosis, which "detains" T cells in the lymph nodes. In recent year, it has been elucidated that S1P exerts regulatory functions far beyond T-cell egress from the lymph node. Thus, it additionally regulates, among others, homing of several immune cell populations into peripheral tissues under inflammatory conditions. In addition, evidence, mostly derived from mouse models, has accumulated that S1P may be involved in the pathogenesis of several inflammatory skin disorder and that S1P receptor modulators applied topically are effective in treating skin diseases. These recent developments highlight the pharmacological modulation of the S1P/S1P receptor system as a potential new therapeutic strategy for a plethora of inflammatory skin diseases. The impact of S1P receptor modulation on inflammatory skin diseases next requires testing in human patients.
Assuntos
Cloridrato de Fingolimode/farmacologia , Imunossupressores/farmacologia , Linfócitos/efeitos dos fármacos , Lisofosfolipídeos/fisiologia , Dermatopatias/tratamento farmacológico , Esfingosina/análogos & derivados , Animais , Cloridrato de Fingolimode/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Lisofosfolipídeos/efeitos dos fármacos , Dermatopatias/metabolismo , Esfingosina/fisiologia , Tiazóis/uso terapêuticoRESUMO
OBJECTIVE: Correlation between GALT homing markers on lymphocytes and the low blood CD4 T-cell reconstitution in immunological nonresponders (INRs) has been studied. DESIGN: Thirty-one INRs, 19 immunological responders (IRs), and 12 noninfected controls were enrolled in this study. INRs were defined by an undetectable plasma viral load RNA less than 40 copies per milliliter and CD4 T-cell count <500 cells per cubic milliliter in at least 3 years. METHODS: A complete peripheral and mucosal lymphocyte immunophenotyping was performed on these patients with a focus on the CCR9, CCR6, and α4ß7 gut-homing markers. RESULTS: A highly significant upregulation of α4ß7 on INRs peripheral lymphocytes compared with that of IRs has been observed. This upregulation impacts different lymphocyte subsets namely CD4, CD8, and B lymphocytes. The frequency of ß7 Th17 and Treg cells are increased compared with IRs and healthy controls. The frequency of ß7 CD8 T cells in the blood is negatively correlated with integrated proviral DNA in rectal lymphoid cells in contrast to ß7 CD4 T cells associated with HIV integration. CONCLUSIONS: Alteration of lymphocyte homing abilities would have deleterious effects on GALT reconstitution and could participate to HIV reservoir constitution. These results emphasize the great interest to consider α4ß7-targeted therapy in INR patients to block homing of lymphocytes and/or to directly impair gp120-α4ß7 interactions.
Assuntos
Terapia Antirretroviral de Alta Atividade , Neoplasias do Ânus/virologia , Infecções por HIV/imunologia , HIV-1/imunologia , Mucosa Intestinal/virologia , RNA Viral/imunologia , Receptores de Retorno de Linfócitos/imunologia , Neoplasias do Ânus/imunologia , Neoplasias do Ânus/patologia , Linfócitos T CD4-Positivos/imunologia , Progressão da Doença , Detecção Precoce de Câncer , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , Humanos , Imunofenotipagem , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Carga ViralRESUMO
The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF-ß1. RA independently caused only IgA switching, whereas TGF-ß1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA-secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF-ß1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4ß7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF-ß have important effects on the overall gut IgA antibody response.
Assuntos
Imunoglobulina A/imunologia , Switching de Imunoglobulina/efeitos dos fármacos , Fator de Crescimento Transformador beta1/imunologia , Tretinoína/farmacologia , Animais , Células Cultivadas/imunologia , Seleção Clonal Mediada por Antígeno , Endotoxinas/toxicidade , Genes de Imunoglobulinas , Imunidade nas Mucosas/efeitos dos fármacos , Imunoglobulina A/biossíntese , Imunoglobulina G/imunologia , Integrinas/biossíntese , Integrinas/genética , Linfonodos/imunologia , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/imunologia , Receptores CCR/biossíntese , Receptores CCR/genética , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/imunologia , Receptores do Ácido Retinoico/fisiologiaRESUMO
INTRODUCTION: Inflammation and infection are associated with premature birth and with activation of the fetal immune system. We hypothesized that exposure to microbial Toll-like receptor (TLR) ligands plays an important role in neonatal T-cell maturation and that early exposure to microbial products may result in early T-cell maturation and a tendency for these matured effector cells to change their homing receptor patterns. RESULTS: Expression of the CD45RO marker was induced in term neonatal T cells after in vitro exposure to TLR ligands for 7 days. Interestingly, naive T cells from adult blood were unaffected by TLR ligand exposure. In addition, neonatal T cells had more cells with decreased expression of the α4ß7 integrins and increased expression of CCR4 after in vitro exposure of TLR ligands-similar to the expression of these molecules in adult naive T cells. DISCUSSION: These findings are relevant for the understanding of neonatal T-cell maturation and may contribute to our understanding of multiorgan inflammatory complications of prematurity. METHODS: Cord blood was obtained from term and preterm infants. Using flow cytometry, we identified a mature (CD45RO(+)) phenotype in preterm infant cord blood (CB) T cells that had decreased expression of the α4ß7 integrins and increased expression of the C-C chemokine receptor 4 (CCR4) as compared with term infant CB.
Assuntos
Envelhecimento/imunologia , Recém-Nascido Prematuro/imunologia , Nascimento Prematuro/imunologia , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Adulto , Fatores Etários , Estudos de Casos e Controles , Células Cultivadas , Corioamnionite/imunologia , Corioamnionite/microbiologia , Feminino , Sangue Fetal/imunologia , Citometria de Fluxo , Idade Gestacional , Humanos , Fatores Imunológicos/farmacologia , Memória Imunológica , Imunofenotipagem , Recém-Nascido , Recém-Nascido Prematuro/sangue , Integrinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Ligantes , Ativação Linfocitária , Fenótipo , Gravidez , Nascimento Prematuro/sangue , Nascimento Prematuro/microbiologia , Receptores CCR4/metabolismo , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Receptores Toll-Like/agonistas , Estados UnidosRESUMO
BACKGROUND: 1,24-Dihydroxyvitamin D3 (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D3 inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells. OBJECTIVES: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells. METHODS: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells. RESULTS: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model. CONCLUSIONS: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels.
Assuntos
Antígenos de Diferenciação de Linfócitos T/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Di-Hidroxicolecalciferóis/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Pele/imunologia , Linfócitos T/efeitos dos fármacos , Adulto , Animais , Antígenos de Diferenciação de Linfócitos T/metabolismo , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Modelos Animais de Doenças , Regulação para Baixo , Selectina E/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Selectina-P/metabolismo , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/metabolismoRESUMO
BACKGROUND AND AIMS: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. METHOD: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and beta7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNF-alpha)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. RESULTS: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased beta7 positive cell number, and the increased mRNA levels of IL-1beta, IL-6, and TNF-alpha in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. CONCLUSION: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.
Assuntos
Proteínas de Bactérias/uso terapêutico , Colite/terapia , Colo , Mucosa Intestinal/metabolismo , Naftóis/uso terapêutico , Propionibacterium/fisiologia , Animais , Proteínas de Bactérias/farmacologia , Moléculas de Adesão Celular/análise , Colite/metabolismo , Colite/prevenção & controle , Colo/microbiologia , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextrana , Ácidos Graxos Voláteis/análise , Fezes/química , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Cadeias beta de Integrinas/análise , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mucoproteínas , Naftóis/farmacologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Taxa de SobrevidaRESUMO
OBJECTIVES: Thyroid hormones exert immunomodulatory activities and the thymus is one of their target organs. We previously showed that triiodothyronine (T(3)) modulates thymic hormone production and extracellular matrix (ECM) expression by mouse thymic epithelial cells (TEC). This concept is enlarged herein by studying the effects of T(3) in human TEC preparations including primary cultures derived from thymic nurse cell complexes, as well as human and murine TEC lines. METHODS AND RESULTS: We observed that in all cases, ECM ligands and receptors (such as fibronectin, laminin, VLA-5 and VLA-6) are enhanced in vitro, as ascertained by immunocytochemistry, ELISA and cytofluorometry. Moreover, thymocyte adhesion to these TEC preparations is augmented by T(3). Interestingly, TEC-thymocyte adhesion is also upregulated when thymocytes from T(3)-treated mice adhere to untreated TEC cultures. Such an enhancing effect of T(3) upon TEC-thymocyte interactions is likely due to the increase in the expression of ECM ligands and receptors, since it is prevented when T(3)-treated TEC cultures are incubated with anti-ECM antibodies prior to the adhesion assay. We then tested whether T(3) could modulate interactions between thymocytes and nonepithelial microenvironmental cells, exemplified herein by the phagocytic cells of the mouse thymic reticulum. In fact, in vitro treatment of these cells with T(3) increases ECM ligands and receptors and augments their ability to adhere to thymocytes. Lastly, using immunochemistry-based assays, we showed the presence of the nuclear T(3) receptor in all thymic microenvironmental cell preparations. CONCLUSION: Our data show that T(3) upregulates ECM-mediated heterocellular interactions of thymocytes with distinct thymic microenvironmental cells, in both humans and mice.
Assuntos
Comunicação Celular/imunologia , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco/metabolismo , Linfócitos T/metabolismo , Timo/metabolismo , Tri-Iodotironina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Timo/citologia , Timo/efeitos dos fármacos , Tri-Iodotironina/farmacologiaRESUMO
The germinal center (GC) is a pivotal site for the development of B cell memory. Whereas GC B cells do not chemotax to most chemokines and do not express the adhesion receptors L-selectin, alpha(4)beta(7), and cutaneous lymphocyte Ag (CLA), memory B cells respond to various chemotactic signals and express adhesion receptors. In this study, we show that CD40 ligand, IL-2, and IL-10 together drive this transition of GC B cells to memory phenotype in vitro, up-regulating memory B cell markers, chemotactic responses to CXC ligand (CXCL)12, CXCL13, and CCL19, and expression of adhesion receptors L-selectin, alpha(4)beta(7), and CLA. Moreover, addition of IL-4 modulates this transition, preventing chemotactic responses to CXCL12 and CXCL13 (but not to CCL19), and inhibiting the re-expression of L-selectin, but not of CLA or alpha(4)beta(7). CCR7 expression, responsiveness to CCL19, and L-selectin/alpha(4)beta(7) phenotype are coordinately regulated. Thus, IL-2/IL-10 and IL-4 play important and distinctive roles in developing the migratory capacities of memory B cells.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Citocinas/farmacologia , Memória Imunológica/efeitos dos fármacos , Ligante de CD40/farmacologia , Quimiocina CCL19 , Quimiocina CXCL12 , Quimiocina CXCL13 , Quimiocinas CC/farmacologia , Quimiocinas CXC/farmacologia , Criança , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Técnicas In Vitro , Interleucina-10/farmacologia , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismoRESUMO
Integrin activity on cells such as T lymphocytes is tightly controlled. Here we demonstrate a key role for lipid rafts in regulating integrin function. Without stimulation integrin LFA-1 is excluded from lipid rafts, but following activation LFA-1 is mobilised to the lipid raft compartment. An LFA-1 construct from which the I domain has been deleted mimics activated integrin and is constitutively found in lipid rafts. This correlation between integrin activation and raft localisation extends to a second integrin, alpha4beta1, and the clustering of alpha4beta1 is also raft dependent. Both LFA-1 and alpha4beta1-mediated adhesion is dependent upon intact lipid rafts providing proof of the functional relevance of the lipid raft localisation. Finally we find that non-raft integrins are excluded from the rafts by cytoskeletal constraints. The presence of integrin in lipid rafts under stimulating conditions that activate these receptors strongly indicates that the rafts have a key role in positively regulating integrin activity.
Assuntos
Membrana Celular/metabolismo , Integrinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Lipídeos de Membrana/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinógenos/farmacologia , Adesão Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Colesterol/deficiência , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Humanos , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Magnésio/metabolismo , Magnésio/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ésteres de Forbol/farmacologia , Ligação Proteica/fisiologia , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tiazóis/farmacologia , Tiazolidinas , Células Tumorais CultivadasRESUMO
We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.
Assuntos
Movimento Celular/fisiologia , Quimiocinas CXC/metabolismo , Genes ras/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas Serina-Treonina Quinases , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Endotélio/citologia , Endotélio/metabolismo , Genes ras/genética , Humanos , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Células Jurkat , Leucócitos/fisiologia , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/farmacologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The present paper reports the molecular modeling-based design and synthesis of an optically pure noncarbohydrate mimetic of sialyl Lewis X to inhibit E-selectin. Biological evaluation of the designed substance as well as that of its enantiomer gave, contrary to expectations, comparable IC50 values. Results are discussed in terms of receptor binding specificity and the molecular modeling protocol used.
Assuntos
Selectina E/efeitos dos fármacos , Oligossacarídeos/química , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Sequência de Carboidratos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Antígeno Sialil Lewis X , EstereoisomerismoRESUMO
The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) and its G-protein-linked receptor CXCR4 are involved in hematopoietic progenitor cell and lymphocyte migration. The integrin VLA-4 is a cell adhesion receptor for CS-1/fibronectin and VCAM-1 and constitutes one of the main adhesion receptors mediating myeloma cell adhesion to bone marrow (BM) stroma in multiple myeloma (MM). It is shown here that MM CD38(hi)CD45RA(-) BM cells and myeloma-derived cell lines expressed CXCR4 and displayed a moderate chemotactic response to SDF-1alpha. Because cell migration in response to SDF-1alpha might require a dynamic regulation of integrin function, it was investigated whether SDF-1alpha can modulate VLA-4 function on myeloma cells. SDF-1alpha rapidly and transiently up-regulated VLA-4-mediated myeloma cell adhesion to both CS-1/fibronectin and VCAM-1, which was inhibited by pertussis toxin and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Modulation of VLA-4-dependent myeloma cell adhesion by SDF-1alpha could contribute to the trafficking and localization of these cells in the BM microenvironment.
Assuntos
Proteínas de Transporte/metabolismo , Quimiocinas CXC/farmacologia , Fibronectinas/metabolismo , Integrinas/efeitos dos fármacos , Mieloma Múltiplo/patologia , Oligopeptídeos/metabolismo , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Adesão Celular/efeitos dos fármacos , Separação Celular , Quimiocina CXCL12 , Quimiocinas CXC/fisiologia , Quimiotaxia/efeitos dos fármacos , Citocalasina D/farmacologia , Humanos , Integrina alfa4beta1 , Integrinas/fisiologia , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/fisiopatologia , Proteínas do Mieloma/metabolismo , Proteínas do Mieloma/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Toxina Pertussis , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CXCR4/metabolismo , Receptores de Retorno de Linfócitos/fisiologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/fisiologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K(+) currents (I(ir)) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267-C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca(2+)](i)) produced by Ca(2+) store depletion with thapsigargin (TG) and readdition of external CaCl(2) in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca(2+)](i) in monocytes bound to glass for 5 h in response to TG and CaCl(2) addition, adherence to VCAM-1 produced a 5-fold increase in [Ca(2+)](i). Depolarization of monocytes adherent to VCAM-1 by I(ir) blockade or exposure to high [K(+)] abolished the enhancement of the peak [Ca(2+)](i) response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K(+) ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca(2+)](i). Adherence of monocytes to E-selectin produced a similar peak [Ca(2+)](i) to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca(2+) entry and may impact Ca(2+)-dependent monocyte function.
Assuntos
Cálcio/metabolismo , Integrinas/metabolismo , Monócitos/metabolismo , Canais de Potássio/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Monócitos/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Tapsigargina/farmacologia , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacosRESUMO
Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.
Assuntos
Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina , Doença das Coronárias/metabolismo , Tromboplastina/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Anti-Hipertensivos/farmacologia , Fatores de Coagulação Sanguínea/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Células CHO , Linhagem Celular , Doença das Coronárias/induzido quimicamente , Doença das Coronárias/mortalidade , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Cricetinae , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Inflamação/fisiopatologia , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Tetrazóis/farmacologia , Tromboplastina/genética , Tromboplastina/metabolismo , Fator de Transcrição AP-1/metabolismo , Valina/análogos & derivados , Valina/farmacologia , Valsartana , Resistência Vascular/efeitos dos fármacosRESUMO
BACKGROUND: The use of Dithiothreitol (DTT) to improve cell dispersion is an integral step in induced sputum examination, which has become an important noninvasive method of assessing airway inflammation. Several studies have shown that sputum treatment with DTT does not affect cell morphology, differential cell counts, and cytokine levels in the supernatant. However, the effect of DTT on cell surface marker expression has not been systematically studied. OBJECTIVE: We have investigated the effect of different DTT concentrations on antigen expression on peripheral blood cells compared with antigen expression on PBS-treated cells. METHODS: Peripheral blood from different healthy donors was incubated with either DTT or PBS, washed, and then incubated with different fluorescence-labeled antibodies. Analysis was performed after lysis of erythrocytes on a calibrated flow cytometer. Respective cell populations were identified, and the mean fluorescence intensity of surface-marker expression for each cell population was compared between DTT- and PBS-treated cells. RESULTS: We found that DTT decreased the expression of CD11a and CD49d on lymphocytes and eosinophils. The expression of CD11a on neutrophils was also decreased after DTT treatment. DTT increased CD11b expression on lymphocytes, neutrophils, and eosinophils. DTT might also have a mild effect on cell activation. It decreased the expression of CD2 on lymphocytes and variably affected the expression of EG2 in eosinophils, although it had no significant effect on HLA-DR expression on lymphocytes. CONCLUSION: Our findings show that DTT can affect antigen expression on lymphocytes, neutrophils, and eosinophils and suggest the need for further investigation of similar consequences on induced sputum analysis.
Assuntos
Antígenos de Superfície/biossíntese , Ditiotreitol/farmacologia , Ribonucleases , Antígenos de Superfície/efeitos dos fármacos , Proteínas Sanguíneas/biossíntese , Antígenos CD11/sangue , Relação Dose-Resposta a Droga , Proteínas Granulares de Eosinófilos , Eosinófilos/imunologia , Antígenos HLA-DR/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Integrina alfa4beta1 , Integrinas/biossíntese , Integrinas/efeitos dos fármacos , Linfócitos/imunologia , Neutrófilos/imunologia , Receptores de IgG/sangue , Receptores de IgG/efeitos dos fármacos , Receptores de Retorno de Linfócitos/biossíntese , Receptores de Retorno de Linfócitos/efeitos dos fármacosRESUMO
The suppression of apoptosis is one mechanism by which tumours become drug resitant. Extracellular signals from the germinal centre (GC) of secondary lymphoid tissue can rescue B cells from physiological- and chemotherapy-induced apoptosis. Such survival signals include CD40 receptor ligation, interleukin-4 (IL-4) receptor stimulation and the interaction of the integrin ligand VCAM-1 with its receptor. The GC environment was modelled in vitro by providing B lymphoma cells with these survival signals. JLP119 B lymphoma cells underwent apoptosis after exposure to the topisomerase II inhibitor etoposide and this was dramatically reduced when the cells were cultured in the GC system. CD40 receptor ligation resulted in increased levels of Bcl-XL. Etoposide diminished the binding between Bax and Bcl-XL but this was restored by IL-4 and VCAM-1 triggered signals. These data demonstrate combined effects of three microenvironmental signals on the Bcl-2 family and illustrate the potential importance of such signalling pathways in drug resistance of tumour cells.
Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD40/fisiologia , Sobrevivência Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Centro Germinativo/fisiologia , Integrinas/fisiologia , Interleucina-4/fisiologia , Linfoma de Células B/patologia , Proteínas de Neoplasias/fisiologia , Receptores de Interleucina-4/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Antígenos CD40/imunologia , Etoposídeo/farmacologia , Humanos , Imunoglobulina G/farmacologia , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Interleucina-4/farmacologia , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/patologia , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Camundongos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptores de Interleucina-4/efeitos dos fármacos , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/farmacologia , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos , Integrinas/metabolismo , Rim/metabolismo , Nefrite/imunologia , Receptores de Retorno de Linfócitos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Análise de Variância , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Integrinas/imunologia , Ativação Linfocitária/imunologia , Cloreto de Mercúrio , Nefrite/induzido quimicamente , Proteinúria/imunologia , Ratos , Ratos Endogâmicos BN , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/imunologia , Valores de Referência , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/imunologiaAssuntos
Anticorpos Monoclonais/farmacologia , Inibição de Migração Celular , Colite/imunologia , Imunoglobulinas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Integrina beta1/imunologia , Mucoproteínas/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/imunologia , Contagem de Linfócito CD4 , Moléculas de Adesão Celular , Doença Crônica , Colite/patologia , Imunização Passiva , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos SCID , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/imunologiaRESUMO
Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.