Shear force-induced activation of integrin LFA-1 by chemokine CXCL12 under flows / 医用生物力学

Objective To explore the activate process of lymphocyte function-associated antigen 1 (LFA-1) triggered by chemokine under shear stresses. Methods Jurkat cells were perfused over ICAM-1 in the parallel-plate flow under 10-30 mPa shear stresses. The effects of soluble and immobilized Chemokines on transient adhesion behavior of Jurkat cells were observed and analyzed to obtain their tether characteristics. Results The immobilized CXCL12 could mediate brief tether (0.13-0.2 s) of Jurkat cells under flow. Only immobilized CXCL12 could effectively activate LFA-1 on Jurkat cells to bind ICAM-1, and then enhance cell adhesion fraction and greatly prolong the tether time (0.8-1.2 s). Two distinct activation states of LFA-1/ICAM-1 were reflected by their dissociation rate k1 (1.09-1.24 s-1) and k2 (0.28-0.7 s-1), respectively. The shear stress would affect the transient adhesion behavior of cells through regulation of k2 and β (the contribution ratio of high affinity to total tether time). Conclusions Shear stress can rapidly trigger LFA-1 activation in 0.2 s through G protein coupled receptors induced by chemokine CXCL12, and further regulate the whole adhesion process of leukocyte. These research findings will contribute to further understanding the integrin activation mechanism of chemokine-force cooperative regulation.

Shear force-induced activation of integrin LFA-1 by chemokine CXCL12 under flows / 医用生物力学

Objective To explore the activate process of lymphocyte function-associated antigen 1 (LFA-1) triggered by chemokine under shear stresses.Methods Jurkat cells were perfused over ICAM-1 in the parallel-plate flow under 10-30 mPa shear stresses.The effects of soluble and immobilized Chemokines on transient adhesion behavior of Jurkat cells were observed and analyzed to obtain their tether characteristics.Results The immobilized CXCL12 could mediate brief tether (0.13-0.2 s) of Jurkat cells under flow.Only immobilized CXCL12 could effectively activate LFA-1 on Jurkat cells to bind ICAM-1,and then enhance cell adhesion fraction and greatly prolong the tether time (0.8-1.2 s).Two distinct activation states of LFA-1/ICAM-1 were reflected by their dissociation rate k1 (1.09-1.24 s-1) and k2 (0.28-0.7 s-1),respectively.The shear stress would affect the transient adhesion behavior of cells through regulation of k2 andβ (the contribution ratio of high affinity to total tether time).Conclusions Shear stress can rapidly trigger LFA-1 activation in 0.2 s through G protein coupled receptors induced by chemokine CXCL12,and further regulate the whole adhesion process of leukocyte.These research findings will contribute to further understanding the integrin activation mechanism of chemokine-force cooperative regulation.

Mechanism of force-induced activation of integrin LFA-1 under circulating flows / 医用生物力学

Objective To reveal the mechanical regulation mechanism for activation of lymphocyte function-associated antigen 1 (LFA-1). Methods The LFA-1 expressed on Jurkat cell surface was pre-activated by Mg2+ from quiescent-to intermediate-affinity state, and the tether events of Jurkat cells under different wall shear stresses (4.5-10 mPa) were observed and analyzed by flow chamber experiment. Meanwhile, a probabilistic model of integrin affinity jumping was established. Results The affinity jumping model was well fitted with the data obtained from flow chamber experiment. Under flowing loads, LFA-1 from intermediate to high-affinity state was observed, with prolonging of the adhesion bonds. The probability of tether event was 15%-26%. LFA-1 at high-affinity state contributed a significant fraction (about 26%-40%) of the bond lifetime. The off-rate of LFA-1 at high-affinity state was slower by 19%-65% as compared to that at intermediate-affinity state. Dissociating of ICAM-1 from LFA-1 was force-dependent and governed either by slip-bond at intermediate-affinity state or by catch-slip bond at high-affinity state. Conclusions The force-induced activation of LFA-1 mediates the slower rolling and firm adhesion of the cells. This research finding will further the understanding of inflammatory response events of circulating leukocytes, and contribute to the discovery of new antibody drug targets for the associated diseases.

Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients

Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain β1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the β2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.

Effects of Xuebijing on the expression of ICAM-1 and its counterreceptor LFA-1 in acute necrotizing pancreatitis of rats / 中国基层医药

Objective To investigate the effects of Xuebijing injection on the production of ICAM-1 and its counterreceptor(LFA-1)in ANP of rats at different time points.Methods ANP was induced by multiple points in- jection of 5% chenodeoxycholic acid into the pancreas,followed by ligation of pancreatic duct.Expressions of ICAM- 1 at different time points were assessed by immunohistochemistry method,the expressions of LAF-1 on neutrophils at different time points were detected by flow cytometer.The effect of Xuebijing injection was also evaluated by de- tecting the output of ascitic fluid,amylase level and histological changes in pancreas and lungs.Results The expres- sions of ICAM-1 in pancreas and lung of Xuebijing injection group decreased significantly than those in ANP group at most time points during the research period(P

Expression of ICAM-1,LFA-1 and VEGFR-3 in Brest Cancer and its Relationship with Lymphatical Metastasis / 医学研究杂志

Objective To explore the influence of ICAM-1 and LFA-1 on the breast cancer metastasis and provide scientific base for studying the molecular mechanism of breast cancer metastasis by observing the expression of ICAM-1(intercellular adhension molecule1) and LFA-1(Lymphocyte function related antigen 1) in different time different metastasis stage of breast cancer. Methods We observed the expression of ICAM-1 and LFA-1 in breast cancer tissue as well as the expression of ICAM-1、LFA-1 and VEGFR-3 in Lymphatic vessel endothelial cell of nearby the breast cancer tissues from the patients of the operation in the defferent stages with the methods of HE staining and immunohistochemistry staining. Results The positive rate of ICAM-1 and LFA-1 expression in cancer cell and cancer nest of breast cancer was increased gradually along with the increase of clinical stages and the occurrence of lymph node metastasis. And the positive rate of ICAM-1、LFA-1 and VEGFR-3 expression in lymphatic vessel endothelial cell of nearby the breast cancer tissues was increased gradually too along with the increase of clinical stages and the occurrence of lymph node metastasis,which has the positive correlation,and it has the positive correlation that the ICAM-1 and LFA-1 expressed in Lymphatic vessel endothelial cell of nearby the breast cancer tissues(P0.05).Conclusion The ICAM-1 and LFA-1 were related with breast cancer lymph metastasis,and the ICAM-1 and LFA-1 promoted breast cancer lymph metastasis through synergism each other.The increase or decrease of ICAM-1 and LFA-1 expression in the breast cancer tissue and lymphatic vessel endothelial cell of nearby the breast cancer tissues could as the data target to display certain instruction function for prevention and treatment of breast cancer lymph metastasis.The Lymphatic vessel quantity of nearby the breast cancer tissues was increased along with tumor progress, which may be related with breast cancer lymph metastasis.VEGFR-3 has the high specificity in the lymphatic vessel endothelial cell expression,and may act as the lymphatic vessel as the lymphatic vessel endothelial cell specific marker.

Expression of ICAM-1 and LFA-1 in Lymphatic Metastasis of Breast Cancer and its Clinical Significance at Present / 医学研究杂志

Objective To explore the biological function of ICAM-1and LFA-1 in tumor metastasis,we observed the expression of ICAM-1(Intercellular Adhension Molecule-1)and LFA-1(Lymphocyte function related antigen-1)in cancer tissue and normal tissue.Methods A total of the samples were obtained from hospital,it includes 60 cases of breast cancer and 60 cases of normal mammary tissues.We observed the expression of ICAM-1 and LFA-1 by SP immunohistochemistry.We analyzed the relationship between the expression of ICAM-1,LFA-1and lymphatic vessel metastasis of breast cancer.Results The expression positive rate of ICAM-1and LFA-1 in breast cancer was obviously higher than that in normal mammary tissues(P

Reduced Incidence and Severity of Collagen-induced Arthritis in Mice Lacking LFA-1 / 中国生物工程杂志

Lymphocyte function associated antigen-1 (LFA-1) is a member of integrin family, that plays an important role in the adhesion of lymphocytes with other cells and matrix. To investigate the role of LFA-1 in collagen-induced arthritis (CIA), the incidence of CIA, histological and radiological assessments in the LFA-1 deficient (LFA-1~ -/- ) mice and control mice were examined. LFA-1~ -/- mice and control mice were immunized with 100?g collagen type II(CII) emulsified with an equal volume of Freund’s complete adjuvant (CFA), followed by the booster injection of the same amount of CII in CFA on day 21. Then, clinical, histological and radiological assessments were done. It showed that 57% control mice developed arthritis and apparently changed in the histological and radiological assessment, whereas the all of LFA-1~ -/- mice had the normal histological and radiographic response and none developed arthritis. These results suggeste that LFA-1 is indispensable for the onset of CIA.

Cytoplasmic Domain of Intercellular Adhesion Molecule-1(ICAM-1)is Important for the ICAM-1 Mediated Membrane Projection of Endothelial Cells / 대한해부학회지

Intercellular adhesion molecule-1 (ICAM-1)has been shown to enhance leukocyte adhesion, thereby inducing migration through blood endothelial cells. However, the molecular event during the process of adhesion is largely unknown. To examine the role of ICAM-1 cytoplasmic domain in SDF-1 alpha-induced T lymphocyte migration and adhesion, mutant human ICAM-1 molecules were expressed in COS-7 cell line. COS-7 cells expressing ICAM-1_GFP mutant without alpha-actinin revealed no association with the actin cytoskeleton, while wild-type ICAM-showed clear association with the actin, as observed by confocal microscopy, suggesting that actinin binding motif in the cytoplasmic domain of ICAM-1 is important for the proper localization of ICAM-1 on the cell membrane. However, based on adhesion assay, we found that the cytoplasmic domain of ICAM-1 is not essential for the binding of lymphocytes which were activated by SDF-1alpha. On the other hand, ICAM-1-mediated receptor-ligand clustering event was significantly inhibited in the cells expressing ICAM-1 mutants without alpha-actinin or whole cytoplasmic domain. Taken together, these results suggest that ICAM-1 cytoplasmic domain is not essential for the adhesion but important for the ligand-receptor-mediated membrane projection of endothelial cells before trans-endothelial migration of lymphocytes.

The study of genome DNA methylation in rheumatoid arthritis / 中华风湿病学杂志

Objective To study the genome DNA methylation in rheumatoid arthirits(RA)and the re- lated factors of DNA methylation.Methods Twenty-first cases with RA and 20 controls were recruited to par- ticipate the study.Plasma Hcy,SAM,SAH,the MTHFR gene C677T polymorphism and the expression of LFA-1 in CD4~+T cells was measured in all patients and controls.Results①The SAM levels were lower sig- nificantly in RA groups than in controls.The SAH levels were higher significantly in RA groups than in con- trols.②There was significant inverse correlation between plasma Hcy level and SAM level(r=-0.932,P＜0.01). There was significant positive correlation between plasma Hcy level and SAH level(r=0.924,P＜0.01).③The expression of LFA-1 in CD4~+T cells was higher significantly in RA groups than in controls.There was a signif- icant positive correlation between LFA-1 expression level and Hcy level(r=0.557,P＜0.01),a significant in- verse correlation between LFA-1 expression level and SAM level(r=-0.651,P＜0.01).④The MTHFR gene mu- tation lead to dramatically increase of Hcy,SAH level and the expression of LFA-1 level in CD4~+T cells and genome DNA hypomethylation.Conclusion①Hypomethylation of genome DNA is found in most RA pa- tients.②The factors associated with genome DNA hypomethylation include MTHFR gene mutation and hyper- homocysteinemia.③The expression of LFA-1 in CD4~+ T cells is higer in RA groups than in controls,which re- lates to the DNA methylation level and the MTHFR gene C677T polymorphism.

The Function of Dimeric Structure of Soluble Intercellular Adhesion Molecule - 1 ( sICAM-1 ): Inhibition of Leukocyte Function Associated Antigen - 1 ( LFA-1 ) Binding to ICAM-3

Intercellular adhesion molecule-1 (ICAM-1) is a membrane protein, exists as a dimer on the cell surface, and interacts with leukocyte function associated antigen-1 (LFA-1), a member of beta2-integrin family. A soluble circulating form of ICAM-1 (sICAM-1) is also detected in human serum, and has been implicated as a regulator for LFA-1-dependent cell-cell interaction in vivo. However, previous reports demonstrated that sICAM-1 shows little inhibitory effect on LFA-1 binding to ICAM-l, indicating that sICAM-1 is unlikely to antagonize LFA-1/ICAM-1-mediated cellular events in vivo. Here, we investigated the property of the dimeric sICAM-1 as an inhibitor of LFA-1 interaction with ICAM-3, since the lower avidity of LFA-1 for ICAM-3 compared with ICAM-1 or ICAM-2 had been speculated. Using recently constructed heterodimeric sICAM-1 joined at the C terminus via an a-helical coiled coil (ACID-BASE) (Jun, CD. et al., 2001, Proc Natl Acad Sci 98, 6830-6835), we also tested whether the structural integrity in dimer could affect the inhibitory action of sICAM-1. Engineered sICAM-1 dimer that contained intact ectodomain (E34/E34) significantly blocked SKW3 cell (LFA-1+) binding to ICAM-3, but not to ICAM-1 and ICAM-2, indicating the lower avidity of LFA-1 to ICAM-3 than that of both ICAM-1 and ICAM-2. A one binding site knock out mutant (E34/K34) showed -2-fold reduction in efficiency compared with E34/E34 to inhibit cell binding. Interestingly, a one binding domain deletion mutant (E34/deltaD1-D2) showed significant reduction (~5-fold) compare with E34/K34, suggesting that structural integrity, which is precluded in E34/deltaD1-D2, is necessary for optimal binding of dimeric sICAM-1 to LFA-1, thereby inhibiting LFA-1/ICAM-3-dependent adhesion. Furthermore, BIAcore affinity measurements revealed that E34/deltaD1-D2 bound to immobilized soluble open LFA-1 I domain with an -3-fold reduced affinity compared with E34/K34. Overall, our results demonstrate that maintaining the structural integrity in dimer is necessary for optimal binding of sICAM-1 to LFA-1, and further suggest the therapeutic potential of dimeric sICAM-1 to antagonize LFA-1/ICAM-3-mediated cellular events in vivo.

Regulation of homotypic aggregation of myeloma-derived cell line IM-9 by CD82 / 대한해부학회지

Although reduced expression of CD82 transmembrane protein facilitates metastasis of cancer cells, little is known about its biological function. Here we have investigated the role of CD82 in B cell lymphocyte adhesion. When IM-9 cells were engaged with anti-CD82 monoclonal antibody, they formed homotypic aggregates in a short time. This adhesion was inhibited by anti-CD11a monoclonal antibody that has been known to block LFA-1-mediated cell adhesion. The cell surface expression of LFA-1 has not been changed by CD82 engagement. Homotypic aggregation was decreased in the cells in which the level of CD82 expression was low, and it was not recovered by anti-CD99 monoclonal antibody or PMA that has been known to stimulate cell adhesion. In addition, it was recovered by Mg++ treatment that induces conformational change of LFA-1 moleucles, but not by Ca++ treatment that leads to clustering of LFA-1 on the cell surface. CD82-induced cell aggregation was dramatically abrogated by addition of the phos-phatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Taken together, these results suggest that CD82 molecule may fascilitate adhesion of lymphocytes by inducing conformational change of LFA-1 to pro-adhesive structure through PI3-K or p38 MAPK signal pathway.

Immunohistochemical Study of CD4 , CD8 ICAM-1 and LFA-1 in Experimental Uveitis

We have reported an animal model of human anterior uveitis in Lewis rats with bovine melanin associated antigen[BMAA] which has been named as experimental autoimmune anterior uveitis[EAAU]. Immunopathogenesis in this model during different clinical stages was examined by the immunohistochemical detection of CD4 , CD8 , ICAM-1 and LAF-1 which play an important role in immune and inflammatory response. After the separation of eyes, these eyes were sectioned by cryostat at 6micrometer thick sections and stored at -70degreesC. For immunohistochemical study monoclonal antibodies to CD4, CD8, ICAM-1, and LAF-1 were used. An increase of ICAM-1 expression was shown on the epithelial cells of uveal tissues before clinical sign of uveitis and followed by LFA-1 expression. And there was no staining in retinal pigment epithelium and cornea. In conclusion, the immunopathogenesis of EAAU appears to be mediated by T cells and cells expressing ICAM-1, LFA-1.

Endotoxin Induced Uveitis Model

Endotoxin-induced uveitis model was produced in Lewis rats by footpad injection of Salmonella endotoxin(lipopolysaccharide, LPS). The clinical course and histological examination were observed at intervals of two hours. Immunohistochemical staining of intercellular adhesion molecule-1(ICAM-1) and lymphoyte function associated antigen-1(LFA-1) were also peformed. Initial intraocular inflammatory signs were observed at 8-10 hours after injection. Clinical and histological abnormalities peaked at 24-48 hours and were resolving after 72 hours. Histological changes were limited to anterior uvea. ICAM-1 expression was first noted on cells of iris and ciliary body and LFA-1 was expressed on infiltring inflammatory cells 8 hours after the injection and increased by 24 hours and disappeared after 72 hours. LPS induced uveitis in the rat provides a simple, reproducible model for anterior uveitis. ICAM-1 and LFA-1 expression in uveal tract are increased during early phase of uveitis and may enhance the adherence of inflammatory cells with the subsequent initiation of inflammation.

Effect of cytokines on the expression of cell adhesion molecule and on the adhesion of melanoma cells to endothelial cells

We examined the role of cell adhesion molecules (CAM) by which tumor cells bind to the endothelial cells using human umbilical vein endothelial cells (HUVEC) and cultured melanoma cells. Endothelial cells from human umbilical veins were isolated and examined for CAM expression and its modulation by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6) or interferon-gamma (IFN-gamma). The expression of intercellular adhesion molecule 1 (ICAM-1) on HUVEC was increased by TNF-alpha, IL-1 and IFN-gamma when measured by ELISA or flow cytometric (FACS) analysis. IL-6 did not increase ICAM-1 expression on HUVEC. Two melanoma cell lines, Malme-3M and SK-Mel-28, showed increased expression of ICAM-1 after treatment with TNF-alpha, IL-1 and IFN-gamma in FACS analysis. IFN-gamma induced increased expression of HLA-DR only in SK-Mel-28 melanoma cells, not in Malme-3M melanoma cells. Neither HUVEC nor melanoma cells expressed lymphocyte function-associated antigen 1 (LFA-1) in either the basal (i.e., cytokine untreated) condition or the cytokine treated condition. Melanoma cells showed minimal increment in adhesion to TNF-alpha or IL-1 treated HUVEC than to cytokine untreated HUVEC. HUVEC and melanoma cells did not express LFA-1 and increased ICAM-1 expression by TNF-alpha, IL-1 and IFN-gamma treatment in FACS analysis did not coincide with minimal increase of melanoma cells adhesion to cytokine treated HUVEC. These results suggest that adhesion between melanoma cells and HUVEC is probably mediated by molecular interaction other than ICAM-1/LFA-1.

Possible roles of LFA-1 and Fc gamma receptors on the functional immaturities of cord blood polymorphonuclear leukocytes

The functional immaturity of PMNs is one of the major causes of overwhelming sepsis in newborns. In this study, we observed functions and surface markers of PMNs to investigate what causes the functional immaturity of PMNs in newborns. As results, the percentage of EA rosette forming PMNs (58.5 +/- 15.5%) and the chemotactic movement (0.14 +/- 0.09 mm) of cord blood PMNs were significantly lower than those of adult peripheral blood PMNs (70.8 +/- 9.9%, 0.60 +/- 0.34 mm). Cord blood PMNs showed decreased glass adherence and ADCC activity. The expression of Fc gamma RII or Fc gamma RIII was a little lower than those of adult peripheral blood PMNs, but the expression of Fc gamma RI (43.1 +/- 26.8%) was significantly higher than that of adult peripheral blood PMNs (3.2 +/- 1.8%). There was a significant difference in LFA-1 expression between EA rosette forming PMNs (92.9 +/- 9.1%) and EA rosette non-forming PMNs (25.6 +/- 22.6%). From these results, it is assumed that neonatal PMNs may consist of heterogeneous populations. And the relatively high percentage of EA rosette non-forming PMNs which express a low level of LFA-1 may be responsible for the functional immaturity of cord blood PMNs.