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PubMed2003

Redox regulation of surface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics.

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin alpha-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and beta-actin. NAC pretreatment augmented integrin alpha-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.

Acetylcysteine↗

PubMed1995

Molecular mapping of functional antibody binding sites of alpha 4 integrin.

Integrin alpha 4 beta 1 is a leukocyte receptor for fibronectin and vascular cell adhesion molecule-1 (VCAM-1). It is important in inflammatory recruitment of leukocytes, lymphopoiesis, and a number of development events. Here we have mapped a panel of functional monoclonal antibodies (mAbs) recognizing the integrin alpha 4 chain, using murine/human chimeric constructs expressed in COS7 cells. We find that: 1) mAbs that induce homotypic aggregation (epitope A mAbs) map to the most N-terminal 100 amino acids of the human alpha 4 chain; 2) mAbs that block adhesion of alpha 4 beta 1 to VCAM-1 and fibronectin (epitope B mAbs) map to a 52-amino-acid region between residues 152 and 203 of human alpha 4; 3) epitope B mAbs that do or do not induce aggregation (epitope B2 and B1 mAbs, respectively) map to the same regions and are therefore indistinguishable by this analysis; 4) mAbs that neither induce homotypic aggregation nor block adhesion (epitope C mAbs) map to a distinct region of the molecule comprising amino acids 422-606. The N-terminal region of the alpha 4 chain identified by functional A and B epitope mAbs does not correspond to ligand binding sites identified in other alpha subunits, such as cation binding sites or the "I-domain," which alpha 4 lacks, and thus represents a novel site for epitope functionality among the integrins.

Animals↗

PubMed2000

Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain.

alpha(4)beta(1) integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other beta(1) integrins. Paxillin, a signaling adapter protein, binds tightly to the alpha(4) cytoplasmic domain and is implicated in alpha(4) integrin signaling. We now report the mapping of a paxillin-binding site in the alpha(4) cytoplasmic domain and an assessment of its role in the alpha(4) tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu(983)-Tyr(991)) within the alpha(4) cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr(991) and Glu(983) as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length alpha(4) tail sequence. Y991A or E983A substitution disrupted the interaction of alpha(4) integrins with paxillin. These same two point mutations reversed the effects of the alpha(4) tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all alpha(4) integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of alpha(4) integrins.

Amino Acid Motifs↗

PubMed2006

The Wilms tumor suppressor Wt1 promotes cell adhesion through transcriptional activation of the alpha4integrin gene.

Cell-matrix interaction through specific adhesion molecules is a critical step during organ development. In addition, down-regulation of cell adhesion receptors may promote tumor invasion and metastasis. We show here that the Wilms tumor suppressor Wt1, which is necessary for normal development of the epicardium, coronary vessels, genitourinary system, and other tissues, activates transcription of the alpha4integrin gene. Binding of the Wt1(-KTS) form, which is transcriptionally active, to the proximal alpha4integrin promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. A reporter construct harboring approximately 1.9 kb of the human alpha4integrin gene promoter was activated significantly by transient co-transfection of a Wt1(-KTS) expression plasmid. Introducing mutations in two identified Wt1(-KTS) binding motifs in the proximal promoter of the alpha4integrin gene abrogated this stimulatory effect. Endogenous alpha4integrin transcripts were increased more than 3-fold in human embryonic kidney 293 cells with stable expression of the Wt1(-KTS) protein. Wt1-overexpressing cells showed augmented adhesion to the alpha4integrin ligand vascular cell adhesion molecule-1 that was abolished upon incubation with an inhibitory alpha4integrin antibody. Double immunofluorescent staining revealed co-localization of Wt1 and alpha4integrin in the developing epicardium of mouse embryos. Cardiac expression of alpha4integrin was reduced significantly in embryos with a homozygous Wt1 defect (Wt1-/-). These findings demonstrate that Wt1 can support cell adhesion through enhanced expression of alpha4integrin. This transcriptional activation of the alpha4integrin gene by Wt1(-KTS) might contribute to normal formation of the epicardium and other tissues in the developing embryo.

Alleles↗

PubMed2002

Increased CD4+ T cells during acute attack of Ménière's disease.

OBJECTIVE: We aimed to investigate if there is an alteration in CD4 and CD8 T-cell populations and in CD25 and CD49d activation antigens linked to these cells during an acute attack of Ménière's disease (MD). MATERIAL AND METHODS: Patients with MD who met the American Academy of Otolaryngology-Head and Neck Surgery criteria for the disease and healthy controls were enrolled in this study. Blood samples were collected during an acute attack of MD and when the disease was in its quiescent phase. RESULTS: 16 patients with MD and nine healthy controls were enrolled. The percentage of CD4 (T helper) cells was significantly increased during an acute attack of MD compared to that in healthy controls and in patients in the quiescent phase (71% vs 58% and 59%, respectively; p < 0.05). The increase in activation antigens was not statistically significant. CONCLUSION: To our knowledge this is the first demonstration of an increase in CD4 cells during an acute attack of MD. Further investigation is required to clarify the role of T cells in the pathogenesis of the disease.

Acute Disease↗

PubMed1996

Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle.

It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.

Animals↗

PubMed1996

The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo.

A role for the alpha 4-integrin (alpha 4 beta 1 or alpha 4 beta 7), has been implicated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the alpha 4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the alpha 4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for approximately 50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the alpha 4-integrin. Anti-alpha 4 therapy also eliminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 (beta 2-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-alpha 4 mAb demonstrated that alpha 4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the alpha 4-integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the alpha 4-integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and beta 2-integrins.

Animals↗

PubMed1996

The genomic structure of the murine alpha 4 integrin gene.

The VLA-4 (alpha 4 beta 1) integrin is a leukocyte glycoprotein involved in both cell-extracellular matrix and cell-cell interactions. We report here the cloning of the murine alpha 4 gene whose protein product is antigenically related to the human VLA-4 alpha chain. The alpha 4 m gene is about 75 kb long and consists of 28 exons, ranging in size from 46 bp (exon 13) to 437 bp (exon 1). The introns varied from 79 bp (intron 8) to more than 17 kb (intron 2). Three mRNA transcripts from this alpha 4 m gene can be visualized on Northern blot. After cloning the 3' untranslated region (3' UTR), four polyadenylation sites could be identified, presumably responsible for the presence of three to four transcripts of the alpha 4 gene, differing substantially in length.

Animals↗

PubMed1996

Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells.

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negative cis-acting regulatory elements that are needed to modulate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of a cis-acting DNA sequence designated the alpha 4.1 element, centered at position -45 relative to the human alpha 4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays. The apparent molecular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively. Electrophoretic mobility-shift assays (EMSAs) indicated that only Bp2 also possesses the ability to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. alpha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on alpha 4 gene basal expression.

Animals↗

PubMed2000

The role of alpha(4) and LFA-1 integrins in selectin-independent monocyte and neutrophil migration to joints of rats with adjuvant arthritis.

Monocytes and neutrophils are chronically recruited to joints in rheumatoid arthritis. In the joints of rats with adjuvant arthritis, this is mediated, in part, by selectin-dependent and selectin-independent mechanisms. To define the selectin-independent mechanisms, (51)Cr-labeled blood monocytes, (111)In-labeled neutrophils and function blocking mAb to the selectins and integrins were utilized. Integrins contributed to the selectin-independent monocyte migration to arthritic joints with 58-70% inhibition of this recruitment by anti-alpha(4) or anti-LFA-1 mAb, relative to selectin blockade alone. alpha(4) plus P-selectin blockade was as effective as combined blockade of alpha(4), P-, E- and L-selectin, mediating approximately 83% of the overall monocyte migration to the joints. In contrast, LFA-1 was the predominant selectin-independent mechanism for neutrophil recruitment to the joints. LFA-1 together with P-selectin had essential roles in the talar joint. In dermal inflammation in the arthritic rats, LFA-1 accounted for most (69%) of the selectin-independent monocyte migration to the chemoattractant C5a(desArg) (zymosan-activated serum), whereas LFA-1 and Mac-1 both contributed to selectin-independent neutrophil recruitment to C5a(desArg). alpha(4) integrin and P-selectin in concert mediated monocyte recruitment to lipopolysaccharide and IFN-gamma lesions (81%). Thus: (1) either alpha(4) or LFA-1 can mediate monocyte migration to arthritic joints in the absence of selectin function and alpha(4) together with P-selectin is particularly important; (2) LFA-1 is the predominant mechanism of selectin-independent migration of neutrophils to inflamed joints; and (3) in arthritic rats, selectin-independent migration of monocytes and neutrophils to dermal inflammation is mediated by alpha(4) or LFA-1 or both LFA-1 and Mac-1, depending on the leukocyte type, and inflammatory stimulus.

Animals↗

PubMed2000

Roles of alpha(4) integrins/VCAM-1 and LFA-1/ICAM-1 in the binding and transendothelial migration of T lymphocytes and T lymphoblasts across high endothelial venules.

Several cell adhesion molecules that mediate the binding of lymphocytes to high endothelial venules (HEV) from flowing blood have been identified but the regulation of lymphocyte migration across the HEV wall into the lymph node (LN) is far from understood. In this study we have used an in vitro model of lymphocyte migration across HEV, and analysed the roles of two integrins in the binding and transendothelial migration of T lymphocytes and T lymphoblasts. The adhesion of T lymphocytes to high endothelial cells (HEC) cultured from rat LN HEV differed from that of T lymphoblasts since the percentage of T lymphoblasts that adhered and transmigrated was higher and was not increased by IFN-gamma pretreatment of HEC. Antibodies to alpha(4) integrins, VCAM-1 or LFA-1 maximally inhibited T lymphocyte adhesion by 40-50%, whereas antibodies to ICAM-1 were less effective (<20% inhibition). The effects of alpha(4) integrin and LFA-1 antibodies were additive, giving >90% inhibition. T lymphocytes which adhered in the presence of LFA-1 antibody showed reduced levels of transmigration and, in the presence of alpha(4) integrin antibody, slightly increased transmigration. Antibodies to alpha(4) integrins, VCAM-1, LFA-1 or ICAM-1 had little effect on T lymphoblast adhesion (maxima of 10-30% inhibition) and T lymphoblasts transmigrated normally in the presence of either alpha(4) integrin or LFA-1 antibodies. However, the effects of alpha(4) integrin and LFA-1 antibodies on T lymphoblast adhesion were synergistic, giving >90% inhibition of adhesion. These results suggest that the majority of T lymphoblasts use either alpha(4) integrins or LFA-1 to bind and transmigrate HEV, and the roles of these integrins on activated T cells are overlapping and redundant. In contrast, either integrin supports half-maximal binding of unactivated T lymphocytes to the surface of HEV and LFA-1 makes a larger contribution than alpha(4) integrins to transendothelial migration.

Animals↗

PubMed2002

BCR signal through alpha 4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation.

alpha 4 potentially mediates BCR signals through a rapamycin-sensitive TOR pathway. To investigate a potential role for alpha 4 in B cell activation, the alpha 4 gene was disrupted conditionally in B cells by mating male CD19-Cre mice with female alpha 4-floxed mice. CD19-Cre+/alpha 4flox mice showed loss of alpha 4 protein in B lineage cells and a decreased number of phenotypically normal mature B cells. Compared to normal B cells, alpha 4(-) B cells showed a decreased proliferation in response to the B cell stimulants (anti-IgM antibody plus IL-4, anti-CD40 mAb and lipopolysaccharide), and a reduced S6 kinase activation and rapamycin sensitivity. While CD19-Cre+/alpha 4flox mice showed impaired antibody responses to both T cell-independent and T cell-dependent (TD) antigens, the TD antigen response was markedly impaired as demonstrated by reduced isotype switching, reduced germinal center formation and reduced V region somatic hypermutation. These results show that alpha 4 plays a pivotal role in antigen-specific signal transduction during B cell activation and differentiation in vivo.

Animals↗

PubMed1995

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via alpha 4 integrin-VCAM-1 interaction.

The present investigation examines the localization and migration of purified T cell subsets in comparison with B cells, CD8 T cells and CD4+ CD8- single-positive thymocytes. CD4 T cell subsets in the rat are defined by mAb MRC OX22 (anti-CD45RC), which distinguishes resting CD4 T cells (CD45RC+) from those (CD45RC-) which have encountered antigen in the recent past--subpopulations often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 T cells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to the spleen was followed by a redistribution to mesenteric lymph nodes (MLN) and cervical lymph nodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization of these subpopulations to the small or large intestine [Peyer's patches (PP) excluded]. In contrast, CD45RC- CD4 T cells (indistinguishable in size from the CD45RC+ subset) localized in large numbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for at least 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC- CD4 T cells in the MLN could be accounted for entirely by afferent drainage from the intestine. Unexpectedly, CD45RC- CD4 T cells (but not other subsets) localized and accumulated in the thymus. In vivo treatment with mAb HP2/1 against the integrin alpha 4 subunit inhibited almost entirely CD45RC- CD4 T cell migration into the PP (98.1%), intestine (87.1%), MLN (89.1%) and thymus (93.5%); migration into the CLN was only reduced by half. To distinguish between recognition of MAdCAM-1 and VCAM-1 by alpha 4-containing integrins, recipients were treated with mAb 5F10 against rat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC- CD4 T cells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC- CD4 T cells alone showed enhanced localization to the gut and PP, probably via alpha 4 beta 7-MAdCAM-1 interaction; (ii) that many CD45RC- cells entered non-mucosal LN independently of alpha 4 integrin or VCAM-1; and (iii) that entry of mature recirculating CD45RC- CD4 T cells into the thymus across thymic endothelium was apparently regulated by alpha 4 integrin-VCAM-1 interaction.

Animals↗

PubMed1995

Homing of lymphocytes into islets of Langerhans in prediabetic non-obese diabetic mice is not restricted to autoreactive T cells.

Non-obese diabetic mice spontaneously develop a type 1 diabetes. The entry of leukocytes in the islets of Langerhans was studied in untreated and in irradiated mice. FITC-labeled cells from spleen, lymph nodes or bone marrow of healthy or diabetic donors did home to the inflamed islets of unmanipulated recipients. B and T cells migrated equally well, whereas rare neutrophils entered the islets. Lymphocyte homing was blocked by anti-L-selectin and anti-alpha 4 integrin antibodies. Insulitis transfer experiments using mice congenic at the Thy-1 locus showed that anti-alpha 4 integrin treatment totally inhibited the migration of donor type T cells in the islets, whereas anti-L-selectin only had an early and transient effect. The expression of vascular addressins in the islets was linked to the presence of mononuclear cells. Thus, in the developing islet infiltrate, the entry of cells appears continuous and restricted to lymphocytes, whether autoreactive or not, and involves the L-selectin. This mechanism rather promotes the migration of naive-type cells. Conversely, during the adoptive transfer of insulitis the entry of L-selectin- diabetogenic T cells is highly favored, to the detriment of L-selectin+ naive type cells.

Animals↗

PubMed1995

Involvement of alpha 1 and alpha 4 integrins in gut mucosal injury of graft-versus-host disease.

We have investigated the involvement of adhesion molecules in the lymphocyte infiltration associated with acute intestinal graft-versus-host disease (GVHD) induced by injection of C3H lymph node cells into irradiated (C3H x DBA/2)F1 mice. First we analyzed the expression profile of adhesion molecules including alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, alpha L and beta 7 integrins, CD44 and L-selectin of lymphocytes from lymph nodes and gut mucosa in normal mice. In normal mice, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) uniquely showed increased expression of alpha 1, alpha 2 and beta 7 integrins, and decreased expression of L-selectin compared with that of lymphocytes of the lymph nodes and Peyer's patches. In mice with GVHD, IEL and LPL of donor lymph node cells origin underwent phenotypic changes characterized by the increased expression of alpha 1, alpha L and beta 7 integrins, and the loss of L-selectin. The expression profile of adhesion molecules on IEL and LPL of GVHD mice resembled that of normal mice except for the lack of alpha 2 integrin. Treatment of GVHD mice with anti-alpha 1, -alpha 4 or -beta 7 integrin antibody alone partially prevented the mucosal pathology of intestinal GVHD, whereas only mice treated with anti-alpha 1 showed reduced donor lymphocytic infiltration into the intestinal mucosa. In contrast, treatment with anti-alpha L or anti-CD44 antibody did not affect the intestinal GVHD. Furthermore, dual blockade of both alpha 1 and alpha 4 integrins completely inhibited the mucosal pathology and donor lymphocyte infiltration of intestinal GVHD.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen.

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (>or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

Animals↗

PubMed2001

Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins.

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.

Animals↗

PubMed2000

Cloning of porcine intercellular adhesion molecule-1 and characterization of its induction on endothelial cells by cytokines.

BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.

Amino Acid Sequence↗