Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

β_2整合素-ICAM-1相互作用的动力学测试

<正>β_2-整合素α_Lβ_2(LFA-1)和α_Mβ_2(MAC-1)是白细胞表面的一类重要粘附分子,与其配体ICAM-1的相互作用在炎症反应和肿瘤转移过程中起着重要作用。在炎症反应发生时,β_2-整合素和ICAM-1的相互作用介导了白细胞与内皮细胞的稳态粘附,是选择素介导滚动粘附的后续反应,使白细胞渗出血管到达炎症部位。在肿瘤转移过程中,白细胞表面的β_2-整合素分别与黑色素瘤细胞及内皮细胞上的ICAM-1作用,使黑色素瘤细胞外渗进入组织,形成继发肿瘤。

不同长度β_2整合素_M亚基与ICAM-1的亲和性比较

国家自然科学基金项目(30730032/10702075)

整合素-ICAM-1相互作用的二维反应动力学研究

国家自然科学基金项目(30730032)

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

不同长度β_2整合素_M亚基与ICAM-1的亲和性比较

<正>整合素作为细胞表面糖蛋白受体,介导细胞-细胞、细胞-胞外基质以及细胞-病原体间粘附和信息传递,在免疫应答、凝血反应、炎症反应、肿瘤转移和创伤愈合等许多病理生理过程中起重要作用。整合素是由α、β两个亚基非共价结合而成的异源二聚体,其结构类似于两条近平行的腿部支撑着一个球形的头部。研究表明,β_2整合素_M亚基头部的.domain为与配体直接作用的结构域,并可通过"open"或"close"的构象变化

整合素-ICAM-1相互作用的二维反应动力学研究

<正>_2整合素(LFA-1和Mac-1)与其配体细胞间粘附分子-1(ICAM-1)的相互作用在诸如肿瘤转移、炎症反应等许多病理生理过程中起着重要的作用。研究表明,中性粒细胞(PMN)在特定环境下可通过_2整合素与ICAM-1的相互作用而增强黑色素瘤细胞的转移能力,但是其动力学调控机制还不清楚。受体-配体键结合和解离的二维反应动力学定量描述了分子结合的快慢和强弱,是回答_2整合素与ICAM-1相互作用如何调

β_2整合素-ICAM-1配体相互作用的生物力学与生物物理

<正>细胞粘附在诸如炎症反应、肿瘤转移、血栓形成等病理生理过程中起着至关重要的作用。在血流作用下,表达于细胞表面的特异性粘附分子(如整合素、ICAM-1配体)间如何介导细胞粘附、怎样定量描述受体-配体相互作用及其反应动力学、外力如何调控受体-配体键强度和寿命、分子键结合与解离的结构基础是什么等,均是亟待认识的基本科学问题。

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis

Autoimmune vasculitis is characterized by the presence of autoantibodies, particularly anti-neutrophil cytoplasmic antibodies (ANCA) and anti-nuclear antibodies (ANA), in patient sera. These autoantibodies have an incompletely understood role in development of vascular injury. The expression or up-regulation of cell adhesion molecules is an early phase in the development of an inflammatory vascular lesion. Autoantibody-positive sera from patients with vasculitis were assessed for their ability to modulate adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). Autoantibody-positive serum samples from 11 out of 21 patients with primary vasculitis produced substantial up-regulation of ICAM-1 on HUVEC. Autoantibody-negative samples did not produce adhesion molecule up-regulation. Up-regulation of adhesion molecules on HUVEC was observed with samples positive for ANA, a phenomenon not previously reported. Preincubation of the sera with purified antigens recognized by ANCA failed to block this activation. In addition, MoAbs to ANCA antigens were ineffective at inducing ICAM-1 up-regulation, suggesting that activation is independent of the molecular specificity of the antibody. This capacity of ANCA- and ANA-positive sera to up-regulate adhesion molecules on endothelial cells may be a factor in the vessel wall inflammation seen in ANCA-associated vasculitis.

Requirements for ICAM-1 immunogene therapy of lymphoma.

Intercellular cell adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein capable of eliciting bidirectional signals that activate signalling pathways in leukocytes, endothelial, and smooth muscle cells. Gene transfer of xenogeneic ICAM-1 into EL-4 lymphomas causes complete tumor rejection; however, it is unknown whether the mechanism responsible involves the "foreignness" of the ICAM-1 transgene, bidirectional signalling events, ICAM-1-receptor interaction, or a combination of the latter. To begin to address this question, we constructed four different therapeutic expression vectors encoding full-length ICAM-1, and forms in which the N-terminal ligand-binding domains and cytoplasmic tail had been deleted. Mouse EL-4 tumors (0.5 cm in diameter), which actively suppress the immune response, were significantly inhibited in their growth following injection of expression plasmids encoding either full-length xenogenic (human) ICAM-1, or a functional cytoplasmic domain-deficient form that retains ligand-binding activity. Efficacy of ICAM-1-mediated antitumor immunity was significantly augmented by administration of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which suppressed blood supply to the tumor, leading to enhanced leukocyte infiltration, and complete tumor eradication in a gene dosage and CD8(+) T cell and NK cell-dependent fashion. Generation of potent cytotoxic T cell (CTL)-mediated antitumor immunity was reflected by ICAM-1-facilitated apoptosis of tumor cells in situ. In contrast, nonfunctional ICAM-1 lacking the N-terminal ligand-binding Ig domain failed to generate antitumor immunity, even in the presence of DMXAA. These studies demonstrate that ICAM-1-stimulated antitumor immunity can overcome tumor-mediated immunosuppression, particularly when employed in combination with an attack on the tumor vasculature. The ligand-binding domain of ICAM-1 is essential for generating antitumor immunity, whereas the cytoplasmic domain and bidirectional activation of tumor signalling pathways are not essential.

Perfil plasmático da ICAM-1 e da VCAM-1 no pós-operatório cardíaco de lactentes submetidos à circulação extracorpórea

Introdução – Grande parte da população de crianças portadoras de defeitos cardíacos congênitos torna-se criticamente doente durante o primeiro ano de vida e necessita tratamento cirúrgico. No entanto, durante cirurgias cardíacas, ocorre uma resposta inflamatória intensa desencadeada pelo período de circulação extracorpórea (CEC), o que provoca disfunção de órgãos e tecidos. Os neutrófilos assumem um papel importante e complexo neste período, envolvendo a ligação às células endoteliais, ativação e liberação de mediadores inflamatórios. Esse processo é iniciado e mantido através de moléculas de adesão específicas, como a molécula de adesão intercelular-1 (ICAM-1) e a molécula de adesão celular-vascular-1 (VCAM-1). Formas solúveis destas moléculas apresentam variações de seus níveis durante cirurgias cardíacas com uso de CEC. Há poucos dados na literatura relacionados ao comportamento destas moléculas após cirurgia cardíaca com uso de CEC em lactentes, estando seu significado no plasma ainda por ser decifrado. Objetivos – Mensurar os níveis plasmáticos das moléculas de adesão solúveis ICAM-1 e VCAM-1 em condições basais e após exposição ao circuito de CEC em lactentes submetidos à cirurgia cardíaca para correção de defeitos cardíacos congênitos. Comparar os níveis plasmáticos destas moléculas entre pacientes acianóticos e cianóticos. Métodos – Foram estudados 21 lactentes, durante o período de junho de 1998 a março de 1999, submetidos à cirurgia cardíaca com uso de CEC. Foram analisados os níveis plasmáticos da ICAM-1 e da VCAM-1 solúveis pelo método de ELISA, na indução anestésica, ao término da CEC e 8 e 26 horas após o término da CEC. Resultados – As patologias cardíacas congênitas mais comuns foram defeito do septo atrioventricular e tetralogia de Fallot. As médias de idade e de peso foram 6,6 meses e 5,8 Kg. As medianas dos tempos de CEC e de clampeamento de aorta foram, respectivamente, 87 e 53 minutos. Todos os lactentes utilizaram inotrópicos. As medianas dos tempos de intubação e de internação foram 72 horas e 21 dias. A taxa de mortalidade dos pacientes foi de 9,5%. Os níveis basais da ICAM-1 e da VCAM-1 foram significativamente mais elevados do que aqueles considerados normais (P<0,0001). Os níveis da ICAM-1 diminuíram significaticamente ao término da CEC (P<0,001), voltando a aumentar significativamente 8 horas após o término deste período (P<0,005), sem no entanto, alcançar os valores basais 26 horas depois. A VCAM-1 apresentou comportamento semelhante. No entanto, 26 horas após CEC houve diminuição significativa de seus níveis em relação ao valor identificado 8 horas após tal período (P<0,005). Não houve diferença estatisticamente significativa quanto aos valores das moléculas entre pacientes acianóticos e cianóticos (P>0,1). Não houve associação entre os valores das moléculas e as variáveis perioperatórias e os desfechos clínicos. Conclusões – Os níveis plasmáticos das moléculas de adesão solúveis ICAM-1 e VCAM-1 são aumentados em lactentes com cardiopatias congênitas acianóticas e cianóticas no seu estado basal. Os níveis plasmáticos de ICAM-1 e VCAM-1 solúveis variam após exposição ao circuito de CEC em lactentes submetidos à cirurgia cardíaca para correção de cardiopatias congênitas, apresentando um comportamento característico nestes pacientes. Não há diferenças no comportamento das moléculas entre pacientes acianóticos e cianóticos.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.