RESUMO
AIM: To compare the therapeutic effect of α(2) and α(4) integrin-blocking antibodies to conventional inflammatory bowel disease drugs methotrexate, 5-aminosalicylic acid and azathioprine in the dextran sulphate sodium mouse colitis model. METHODS: Colitis was induced in balb/c mice with 2.5-3.0% dextran sulphate sodium. Treatment was given daily for 7 days after the onset of colitis, by rectal installation. Clinical signs of disease were assessed daily using a disease activity index. After 19 days, all animals were killed and colon samples collected for histological grading and mRNA/protein analysis. All treatment groups were compared with an untreated control group and a treatment group receiving dextran sulphate sodium alone to monitor the potential degree of clinical remission. RESULTS: Treatment with anti-α(2) antibodies and methotrexate reduced the body weight loss. At the end of treatment, anti-α(2) antibodies reduced rectal bleeding, while methotrexate reduced the disease activity index score. Histological evaluation showed that anti-α(2) antibodies, methotrexate, 5-aminosalicylic acid and azathioprine treatment reduced the acute inflammation; methotrexate was the only treatment with effect on the crypt score. Compared with the dextran sulphate sodium alone group, the methotrexate group showed down-regulation of IL-1ß at the mRNA level, while the anti-α(2) antibody group displayed decreased protein expression of iNOS and IL-1ß. CONCLUSIONS: Specific blocking of extravascular trafficking of leucocytes with α(2)-antibodies could be a new beneficial drug target in inflammatory bowel disease.
Assuntos
Anticorpos Bloqueadores/farmacologia , Colite/tratamento farmacológico , Integrina alfa2/metabolismo , Integrina alfa4/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Colite/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by approximately 20-50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 microm in diameter), but not small (<45 microm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.
Assuntos
Arteriosclerose/etiologia , Inflamação/etiologia , Selectina L/fisiologia , Leucócitos/patologia , Animais , Artérias/patologia , Artérias/fisiopatologia , Arteriosclerose/patologia , Arteriosclerose/fisiopatologia , Adesão Celular , Movimento Celular , Inflamação/patologia , Inflamação/fisiopatologia , Selectina L/genética , Leucócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Vídeo , Vênulas/patologia , Vênulas/fisiopatologiaRESUMO
Leukocyte infiltration in atherosclerosis has been extensively investigated by using histological techniques on fixed tissues. In this study, intravital microscopic observations of leukocyte recruitment in the aorta of atherosclerotic mice were performed. Interactions between leukocytes and atherosclerotic endothelium were highly transient, thereby limiting the ability for rolling leukocytes to firmly adhere. Leukocyte rolling was abolished by function inhibition of P-selectin (P<0.001, n=8), whereas antibody blockage of E-selectin (n=10) decreased rolling leukocyte flux to 51 +/- 9.9% (mean+/-SE, P<0.01) and increased leukocyte rolling velocity to 162 +/- 18% (P<0.01) of pretreatment values. Notably, function inhibition of the integrin alpha(4) subunit (n=5) had no effect on rolling flux (107+/-25%, P=0.782) or rolling velocity (89+/-6.1%, P=0.147), despite endothelial expression of vascular cell adhesion molecule 1 (VCAM-1). Leukocytes interacting with atherosclerotic endothelium were predominantly neutrophils, because treatment with antineutrophil serum decreased rolling and neutrophil counts in peripheral blood to the same extent. In conclusion, we present the first direct observations of atherosclerosis in vivo. We show that transient dynamics of leukocyte-endothelium interactions are important regulators of arterial leukocyte recruitment and that leukocyte rolling in atherosclerosis is critically dependent on the endothelial selectins. This experimental technique and the data presented introduce a novel perspective for the study of pathophysiological events involved in large-vessel disease.
Assuntos
Aorta/patologia , Arteriosclerose/fisiopatologia , Movimento Celular/fisiologia , Selectina E/metabolismo , Endotélio Vascular/patologia , Leucócitos/fisiologia , Microscopia/métodos , Selectina-P/metabolismo , Animais , Anticorpos Anticitoplasma de Neutrófilos/farmacologia , Antígenos CD/metabolismo , Aorta/metabolismo , Aorta/ultraestrutura , Arteriosclerose/patologia , Selectina E/imunologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Integrina alfa4 , Leucócitos/efeitos dos fármacos , Leucócitos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Induction of beta1 integrin (CD49/CD29) expression in polymorphonuclear leukocytes (PMN) has been shown to be associated with transendothelial migration recently. Yet, beta1 integrin expression is relatively insensitive to cell activation with soluble agonists, such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). We hypothesized that beta2 integrins (CD11/CD18), critically involved in PMN adhesion and extravasation, may play a role in regulating 1 integrin expression in PMN. Antibody cross-linking of CD18, mimicking adhesion-dependent engagement of beta2 integrins, resulted in rapid, tyrosine kinase-dependent upregulation of beta1 integrins. This response was potentiated by simultaneous chemoattractant (fMLP) stimulation of PMN. Moreover, upregulation of beta1 integrins evoked by CD18 cross-linking was found to support adhesion of fMLP-stimulated PMN to matrix proteins and also was critical for the ability of PMN to migrate in collagen gels in response to a gradient of fMLP. Taken together, these data demonstrate that engagement of beta2 integrins in human PMN induces beta1 integrin expression in these cells of significance for their migration in the extravascular tissue. Thus, beta2 integrins may serve the function to regulate PMN locomotion in extravascular tissue via receptor crosstalk with beta1 integrins.
Assuntos
Antígenos CD11/fisiologia , Antígenos CD18/fisiologia , Adesão Celular/fisiologia , Integrina beta1/biossíntese , Neutrófilos/metabolismo , Transporte Biológico , Adesão Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Colágeno , Fibronectinas , Géis , Humanos , Integrina beta1/genética , Microscopia Confocal , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Peroxidase/análise , Regulação para Cima/efeitos dos fármacosRESUMO
We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)-2, we isolated and characterized several antibacterial peptides/proteins from the supernatant-alpha-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B-although other active components were also present. We then used reverse transcriptase-polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, gammadelta T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several alphabeta T-cell lines. The alpha-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, gammadelta T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-gamma. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human alpha-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Linfócitos/fisiologia , Monócitos/fisiologia , alfa-Defensinas/genética , Antibacterianos/farmacologia , Linfócitos B/fisiologia , Proteínas de Transporte/farmacologia , Catelicidinas , Linhagem Celular , Quimiotaxia de Leucócito , Clonagem Molecular , Histonas/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Interferon gama/farmacologia , Interleucina-6/farmacologia , Células Matadoras Naturais/fisiologia , Ativação Linfocitária , Linfócitos/efeitos dos fármacos , Muramidase/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/fisiologia , alfa-Defensinas/farmacologia , alfa-Defensinas/fisiologiaRESUMO
The molecular mechanisms underlying leukocyte recruitment in large arteries have been extensively studied using histological techniques on fixed tissues. However, there are no reports that address the dynamics of leukocyte recruitment in large arteries in vivo. We developed an intravital microscopy technique for direct observation of leukocyte-endothelium interactions in the mouse aorta. Circulating leukocytes were labeled intravasally with rhodamine 6G and microscopically visualized within the aorta, allowing direct analysis of leukocyte rolling and adhesion. In untreated vessels, leukocyte-endothelium interactions were virtually absent. However, local pretreatment with cytokines interleukin-1beta and tumor necrosis factor-alpha induced clear-cut leukocyte rolling and adhesion, compatible with normal blood flow and wall shear rate. High shear decreased rolling leukocyte flux and increased leukocyte rolling velocity, thus decreasing the tendency for firm adhesion. Leukocyte rolling was almost abolished by an antibody blocking the function of P-selectin, whereas function-blocking antibodies against E-selectin and the alpha(4)-integrin subunit decreased rolling leukocyte flux to 51+/-34% (mean +/- SD) and 59+/-11% of the value before antibody treatment, respectively. In addition, inhibition of E-selectin function, but not of alpha(4) integrin, resulted in increased leukocyte rolling velocity (from 48+/-32 to 71+/-32 microm per second). Taken together, we introduce the first model for direct studies of leukocyte-endothelium interactions in a large artery in vivo and demonstrate cytokine-induced shear-sensitive leukocyte rolling that is critically dependent on P-selectin and modulated by E-selectin and alpha(4) integrin.
Assuntos
Antígenos CD/metabolismo , Arteriosclerose/imunologia , Endotélio Vascular/citologia , Interleucina-1/farmacologia , Leucócitos/imunologia , Selectina-P/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Aorta/citologia , Arteriosclerose/patologia , Velocidade do Fluxo Sanguíneo/fisiologia , Pressão Sanguínea/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Tamanho Celular/imunologia , Endotélio Vascular/química , Endotélio Vascular/imunologia , Frequência Cardíaca/fisiologia , Integrina alfa4 , Leucócitos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência/métodos , Selectina-P/imunologia , Estresse MecânicoRESUMO
Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the alpha(2)beta(1) (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the alpha(2)beta(1) integrin reduced PMN migration velocity in the extravascular tissue by 73% +/- 3% and 70% +/- 10%, respectively (means +/- SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-alpha(2) mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the alpha(4)beta(1) or alpha(5)beta(1) integrins. Further evidence for a critical role of expression of alpha(2)beta(1) integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-alpha(2) mAb treatment. Thus, expression of alpha(2)beta(1) integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated. (Blood. 2000;95:1804-1809)
Assuntos
Quimiotaxia de Leucócito/fisiologia , Integrinas/fisiologia , Neutrófilos/fisiologia , Adulto , Animais , Anticorpos Monoclonais/farmacologia , Vesícula/patologia , Adesão Celular , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Colágeno , Gelatina , Géis , Humanos , Inflamação , Integrina beta1/fisiologia , Integrinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Vídeo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Cavidade Peritoneal/citologia , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Wistar , Receptores de ColágenoRESUMO
Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.