重症烧伤休克大鼠白细胞表面beta-2整合素的表达

目的 :阐明烧伤休克时 beta-2整合素与白细胞 -内皮细胞粘附间的关系。方法 :将 16只 SD大鼠随机均分为对照组和烧伤休克组。应用流式细胞技术检测中性粒细胞及单细胞表面 beta-2整合素CD11a( LFA-1)和 CD11b( Mac-1)的表达量变化 ,同时比较观察二组动物肠系膜微循环中微静脉内白细胞与内皮细胞粘附数量变化。结果 :二组自身对照 (烫伤前与烫伤后 3 h或开腹前与开腹后 3 h)及相互对比结果显示 ,中性粒细胞及单核细胞表面 CD11a表达量均无显著差异 ( P>0 .0 5 ) ;在烧伤组 ,单核细胞 CD11b有显著降低( P<0 .0 5 ) ,而中性粒细胞 CD11b有显著升高 ( P<0 .0 5 )。微循环观察结果显示 ,休克组动物烫伤后随时程延长附壁滚动及紧密粘附的白细胞数量明显增多 ,而对照组则无明显变化。结论 :烧伤休克状态下 ,白细胞表面 CD11a数量无显著变化 ,而 CD11b数量则有所改变。白细胞 -内皮细胞粘附力的增强可能有 beta-2整合素功能活性上调参与。     

Integrin-associated proteins as potential therapeutic targets

Summary: Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin–ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin α and β subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the α4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with β2 integrins may participate in adhesive and migratory events in leukocytes. The β integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates β1 and β3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.     

Integrin CD11c contributes to monocyte adhesion with CD11b in a differential manner and requires Src family kinase activity

Integrin-mediated adhesion of human monocytes to fibrinogen regulated by CD11b/CD18 and the closely related integrin CD11c/CD18, play a key role in inflammation. Peripheral blood monocytes isolated from human donors despite expressing CD11c primarily utilized CD11b to mediate adhesion to fibrinogen upon stimulation with granulocyte macrophage-colony stimulating factor (GM-CSF) and fMLP. Blocking with anti-CD11b resulted in 90% (p<0.001, n=3) inhibition of monocyte adhesion. Monocytes cultured in human serum showed a shift in the participation of integrins, adhesion to fibrinogen involving both CD11b and CD11c. The participation of CD11c in cultured monocytes corresponded to a 3.4-fold increase in expression in CD11c. Blocking cultured monocytes with anti-CD11b or anti-CD11c alone showed no significant effect on adhesion. Treatment with both anti-CD11b and anti-CD11c resulted in inhibition of adhesion by 85% (p<0.001, n=3). Abrogation in adhesion upon treatment with PP1 or PP2 showed that Src family kinase activity was required for CD11b and CD11c mediated adhesion of cultured monocytes to fibrinogen upon stimulation with GM-CSF and fMLP. The clustering of CD11c on cultured monocytes upon adhesion to fibrinogen was diminished on inhibition with PP2 indicating a role for Src family kinase activity in regulating CD11c avidity. CD11b was critical to cytoskeletal events leading to increased spreading and formation of actin foci in cultured monocytes following adhesion to fibrinogen. Blocking cultured monocytes with anti-CD11b or anti-CD11c alone showed that the increase in spread area was diminished by 67+/-3% and 36+/-9%, respectively. The differential involvement of CD11c and CD11b in adhesion and subsequent cytoskeletal changes in monocytes exposed to different conditions indicates the importance of each integrin in distinct responses during inflammation.     

CD11b Expression on circulating leukocytes increases in preparation for parturition

PROBLEM: Initiation of parturition is associated with migration of leukocytes to the reproductive tract. This migration is controlled in part by expression of adhesion molecules on the surface of leukocytes and vascular endothelial cells. Within the reproductive tract, certain endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), are up-regulated at the end of gestation. ICAM-1 binds to the beta(2) integrin CD11b on the leukocytes. In this study, we wanted to investigate whether complementary changes occur in expression of adhesion molecules on maternal leukocytes in preparation for parturition. METHOD OF STUDY: We used flow cytometry to ascertain changes in adhesion molecules expression on leukocytes throughout third trimester and labor. RESULTS: We found a significant increase in the expression of CD11b on monocytes and granulocytes in women at >37 weeks of gestation. CONCLUSIONS: CD11b may be a key molecule for leukocyte trafficking to the reproductive tract at the end of pregnancy.     

同型半胱氨酸增强血白细胞粘附分子的表达

用免疫荧光流式细胞仪检测同型半胱氨酸是否改变体外健康人血单核细胞,中性粒细胞,及淋巴细胞表达粘附分子CD11b,CD18和L－选择素（L-selectin)的表达。结果显示,同型半胱氨酸在低浓度20μmol/L时明显增加CD11b和CD18在各种白细胞表面的表达,同时也增加了CD14在单核及中性粒细胞的表达。这种作用随同型半胱氨酸浓度升高而增强,但当浓度升至等于或大于1mmol/L时,各种粘附分子的表达反而降低。同型半胱氨酸使各种细胞类型表面L-selectin降低。此结果表明同型半胱氨酸可改变白细胞表面粘附分子的表达,可能在体内通过此途径导致白细胞粘附并游出血管内皮。     

Engagement of beta2 integrins induces surface expression of beta1 integrin receptors in human neutrophils

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.     

Increased adhesion of human monocytes to IL-4-stimulated human venous endothelial cells via CD11/CD18, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1)-dependent mechanisms.

Expression of adhesion molecules on endothelial cells (EC) can be up-regulated or induced by cytokines. The aim of the present study was to investigate the effect of IL-4 on both the expression of adhesion molecules on EC and monocyte adhesion to EC. Flow cytometric analysis showed that VCAM-1 expression on EC was up-regulated after stimulation with IL-4 for 24 h, whereas the expression of E-selectin (formerly called endothelial leucocyte adhesion molecule-1 (ELAM-1)) was not enhanced, and that of intercellular adhesion molecule-1 (ICAM-1) only slightly. The adhesion of monocytes to EC increased to maximum values upon stimulation of EC with IL-4 for 24 h. Coating of monocytes with MoAb against the integrin beta 2-subunit (CD18) significantly inhibited their adhesion to IL-4-stimulated EC; maximal inhibition was found when monocytes were coated with anti-CD18 MoAb in combination with MoAb against CD49d (the alpha-chain of VLA-4), whereas no inhibition was found when monocytes were coated only with MoAb against CD49d. Monocyte adhesion was not significantly inhibited when IL-4-stimulated EC were coated with MoAbs against ICAM-1 or VCAM-1 alone or in combination. Adhesion of monocytes was inhibited to a greater extent when in addition to coating of monocytes with MoAb against CD18 the EC were coated with MoAb against VCAM-1. From these results we conclude that monocytes bind to IL-4-stimulated EC via interaction of CD11/CD18 molecules on the monocytes with an as yet unknown endothelial ligand, and interaction of VLA-4 on monocytes with VCAM-1 on EC.     

T cell integrin activation by chemokines in inflammation

The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated as "inside-out signaling". A large number of lymphocytes are recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequences of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by chemokines is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines during inflammatory processes.     

Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma

Eosinophilic inflammation is a characteristic feature of asthma. Integrins are highly versatile cellular receptors that regulate extravasation of eosinophils from the postcapillary segment of the bronchial circulation to the airway wall and airspace. Such movement into the asthmatic lung is described as a sequential, multistep paradigm, whereby integrins on circulating eosinophils become activated, eosinophils tether in flow and roll on bronchial endothelial cells, integrins on rolling eosinophils become further activated as a result of exposure to cytokines, eosinophils arrest firmly to adhesive ligands on activated endothelium, and eosinophils transmigrate to the airway in response to chemoattractants. Eosinophils express seven integrin heterodimeric adhesion molecules: alpha 4 beta 1 (CD49d/29), alpha 6 beta 1 (CD49f/29), alpha M beta 2 (CD11b/18), alpha L beta 2 (CD11a/18), alpha X beta 2 (CD11c/18), alpha D beta2 (CD11d/18), and alpha 4 beta 7 (CD49d/beta 7). The role of these integrins in eosinophil recruitment has been elucidated by major advances in the understanding of integrin structure, integrin function, and modulators of integrins. Such findings have been facilitated by cellular experiments of eosinophils in vitro, studies of allergic asthma in humans and animal models in vivo, and crystal structures of integrins. Here, we elaborate on how integrins cooperate to mediate eosinophil movement to the asthmatic airway. Antagonists that target integrins represent potentially promising therapies in the treatment of asthma.     

Interleukin-15 induces adhesion receptor redistribution in T lymphocytes

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polarization and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)-15, a newly described cytokine with chemotactic properties, is able to induce uropod formation on T lymphoblasts to which intercellular adhesion molecule (ICAM)-3, a leukocyte-restricted counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, is redistributed. Other adhesion molecules, such as ICAM-1, ICAM-2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL-15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascular cell adhesion molecule (VCAM)-1 and ICAM-1, but not to bovine serum albumin or poly-L -lysine. The effect of IL-15 was dose dependent and specifically inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti-IL-2 receptor β chain mAb showed an inhibitory effect on IL-15-mediated redistribution of ICAM-3, whereas no effect was observed in the presence of anti-IL-2 receptor α chain mAb. The uropod induced by IL-15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.     

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.     

Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues.     

A novel ITGB2 variant with long survival in patients with leukocyte adhesion defect type-I

Leukocyte adhesion deficiency is an autosomal recessive primary immunodeficiency that has been divided into three types: LAD1 (beta-2 integrin (CD18) family deficiency/defect), LAD2 (absence of fucosylated carbonhydrate ligands for selectins) and LAD3 (defective activation of all beta integrins). However, recently LAD4 has been described in cystic fibrosis patients, with a defect in integrin activation reported in monocytes. LAD-I is the most common type and prevalence of 1 in 1,000,000 live births. Clinical features of LAD patients are recurrent bacterial and fungal infections, omphalitis with delayed umbilical stump separation, significant leukocytosis especially neutrophilia during infection periods, impaired pus formation, and delayed traumatic or surgical wound healing. Flow cytometry is considered a useful tool for rapid diagnosis of the disease. The study of CD18 and CD11 (a, b, c) expression patterns in peripheral blood leukocytes helps to distinguish different phenotypes of LAD-I. In general, patients with ≥ 2% CD18 expression tend to have a less severe infection and often survive until adulthood, whereas < 2% CD18 expression often results in death in infancy. In this case report, three siblings, 10, 15, and 17 years old, diagnosed with leukocyte adhesion defect type 1 in adolescence age group, are presented.

     

A role for Syk-kinase in the control of the binding cycle of the beta2 integrins (CD11/CD18) in human polymorphonuclear neutrophils

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the endothelial cell monolayer is required to allow ordered emigration. To elucidate the molecular mechanisms that control this process, intracellular protein tyrosine signaling subsequent to beta(2) integrin-mediated ligand binding was studied by immunoprecipitation and Western blotting techniques. The 72-kDa Syk-kinase, which was tyrosine-phosphorylated upon adhesion, was found to coprecipitate with CD18, the beta-subunit of the beta(2) integrins. Moreover, inhibition of Syk-kinase by piceatannol enhanced adhesion and spreading but diminished N-formyl-Met-Leu-Phe-induced chemotactic migration. The enhancement of adhesiveness was associated with integrin clustering, which results in increased integrin avidity. In contrast, piceatannol had no effect on the surface expression or on the affinity of beta(2) integrins. Altogether, this suggests that Syk-kinase controls alternation of beta(2) integrin-mediated ligand binding with integrin detachment.     

From rolling to arrest on blood vessels: leukocyte tap dancing on endothelial integrin ligands and chemokines at sub-second contacts

In order to extravasate the bloodstream at specific sites of inflammation or antigen presentation, circulating leukocytes must rapidly translate specific adhesive and stimulatory signals into firm adhesion. Leukocyte arrest is nearly exclusively mediated by integrin receptors. Recent in vitro and in vivo evidence suggests that specialized integrins support reversible tethers that slow down selectin-initiated rolling of leukocytes prior to their arrest. In situ activation of integrin avidity by ligand and chemokine signaling can take place within fractions of seconds, resulting either in augmented reversible adhesions or immediate arrest on the vascular endothelium. The ability of leukocyte integrins to rapidly respond to these in situ avidity modulators appears to depend on preformed affinity and clustering states, which are internally regulated by cytoskeletal constraints on integrin conformation and mobility. We discuss potential regulatory mechanisms by which a given set of chemokine receptors and integrins may interact to rapidly generate high avidity, shear-resistant integrin-mediated leukocyte arrest on vascular endothelium.     

P-selectin primes leukocyte integrin activation during inflammation

Selectins mediate leukocyte rolling and prime leukocytes for integrin-mediated leukocyte adhesion. However, neither the in vivo importance of nor the signaling pathway by which selectin-mediated integrin activation occurs has been determined. We report here that P-selectin-deficient mice manifested impaired leukocyte adhesion, which was 'rescued' by soluble P-selectin. Mechanistically, the cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruited the phosphoinositide-3-OH kinase p85-p110delta heterodimer and resulted in activation of leukocyte integrins. Inhibition of this signal-transduction pathway diminished the adhesion of leukocytes to capillary venules and suppressed peritoneal infiltration of leukocytes. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by P-selectin glycoprotein ligand 1.     

Systemic upregulation of leukocyte integrins in response to lower body ischemia-reperfusion during abdominal aortic aneurysm repair

Ischemia and reperfusion in myocardial infarction and stroke are associated with upregulation of leukocyte adhesion molecules, which contributes to tissue injury by facilitating leukocyte adhesion and infiltration in the affected tissues. Surgical repair of the abdominal aortic aneurysm involves clamping and declamping of the aorta, which necessarily results in ischemia and reperfusion of the lower half of the body. Given the large volume of the affected tissues and unimpeded venous return during reperfusion, we hypothesized that the procedure may result in upregulation of leukocyte integrins in the systemic circulation. To test this hypothesis, we studied neutrophil and monocyte surface densities of CD11b and CD18 in patients undergoing elective infrarenal abdominal aortic aneurysm repair. Serial blood samples were collected from the radial artery and femoral vein during the operation and leukocyte CD11b and CD18 surface densities were quantified by flow cytometry. Following reperfusion, CD11b expression in neutrophils and monocytes increased significantly in femoral venous and arterial blood. The mean time to peak expression of CD11 b in neutrophils and monocytes during reperfusion was 34.4 and 31.4 minutes in venous and 38.5 and 36.4 minutes in arterial blood, respectively. Similar rises in CD18 expression on neutrophils and monocytes were observed in venous and arterial blood. The mean time to peak expression of CD18 in neutrophils and monocytes during reperfusion was 34.0 and 40.0 minutes in venous and 47.5 and 50.0 minutes in arterial blood, respectively.