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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↗

PubMed1996

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Animals↗

PubMed2001

Simultaneous analysis of 1176 gene products in normal human aorta and abdominal aortic aneurysms using a membrane-based complementary DNA expression array.

BACKGROUND: A number of changes in gene expression have been described in abdominal aortic aneurysms (AAAs), but the spectrum of molecular alterations in this disease is unknown. The purpose of this study was to characterize the expression of approximately 1000 gene products in human AAA tissue and to compare the profile of genes expressed in AAAs with that observed in normal aorta. MATERIALS AND METHODS: Total RNA was isolated from abdominal aortic wall tissues (4 AAAs and 4 normal aortas), and array-specific [(32)P]-labeled complementary DNA (cDNA) probes were created with reverse transcription. The cDNA probes were hybridized with nylon membranes containing an array of 1176 cDNA clones (AtlasArray Human 1.2 I; Clontech, Palo Alto, Calif), and autoradiographs were scanned to identify the patterns of gene expression characteristic of each tissue type. Densitometric analysis was used to standardize the expression of individual genes to a panel of housekeeping controls, and differential gene expression was defined by a signal ratio of at least 2:1. RESULTS: One hundred forty-five (12.3%) of the 1176 genes were consistently expressed in aortic tissue. Thymosin beta-4 was the most abundant of 101 transcripts detected in both AAAs and normal aorta, whereas 44 genes exhibited differential patterns of expression (39 predominant in AAAs and 5 in normal aorta). Densitometric analysis confirmed differences in expression for 20 of these gene products between AAAs and normal aorta, with the greatest increases seen for myeloid cell nuclear differentiation antigen (31-fold), cathepsin H (30-fold), platelet-derived growth factor-A (23-fold), apolipoprotein E (13-fold), gelatinase B/matrix metalloproteinase-9 (12-fold), and interleukin-8 (11-fold). The only gene products substantially decreased in AAAs were myosin light chain kinase (39-fold) and beta-1 integrin (twofold). AAA tissues thereby exhibited a distinct pattern of gene expression reflecting chronic inflammation, extracellular matrix degradation, atherosclerosis, and smooth muscle cell depletion. CONCLUSIONS: cDNA expression arrays provide a powerful new approach to help identify the molecular mechanisms responsible for aneurysmal degeneration. Further studies will be needed to elucidate the functional and pathophysiologic significance of the individual genes that exhibit altered levels of expression in AAA tissue.

Aged↗

PubMed2002

Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxin-induced cytolysis.

Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to beta(2) integrins on the surface of bovine leukocytes. beta(2) integrins have a common beta subunit, CD18, that associates with three distinct alpha chains, CD11a, CD11b, and CD11c, to give rise to three different beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three beta(2) integrins, suggesting that the common beta subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.

Animals↗

PubMed1998

Adhesion and signaling mediated by the cytoplasmic tails of leucocyte integrins.

Integrins not only mediate cell adhesion but also contribute to a variety of other cellular processes including proliferation, cytokine production, cytotoxicity and apoptosis. They act as bi-directional signal transducers, mediating signaling from inside-to-outside the cell and from outside-to-inside the cell. Evidence is emerging that signaling through leukocyte integrins (beta 2 and beta 7) is distinct from signaling by the more widely distributed beta 1 integrins. Here we discuss the role of the cytoplasmic domains of leukocyte integrins and that of cytosolic proteins that bind integrins in mediating signal transduction. Distinct sites in the alpha as well as the common beta chain contribute to this process. We also show that beta 2 integrin distribution on the cell surface is of particular relevance for leukocytes to rapidly alter their adhesive state and display their highly dynamic adhesive behavior. From these and recently published findings the picture is arising that particular cell functions may be supported by integrin-specific signaling pathways.

Amino Acid Sequence↗

PubMed1992

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.

Cell Adhesion↗

PubMed1989

Intercellular contacts between germinal center cells. Mechanisms of adhesion between lymphoid cells and follicular dendritic cells.

Intercellular connections exist between germinal center cells especially between lymphoid cells and follicular dendritic cells (FDC). Even after isolation, FDC remain associated to lymphocytes and are able, in a cell suspension, to establish new connections with others. Using human tonsillar cells or mouse lymph node cells we analysed these connections which were shown to be species-specific. Low temperature as well as absence of Ca++ and Mg++ in the culture medium reduced the adherence of fluorochrome-labeled lymphoid cells to FDC. Colchicine treatment did not impair the adherence, whereas cytochalasin B dit it; this was the first observation underlining the importance of microfibrils in FDC. Antibodies directed towards integrin molecules (LFA-1 alpha or beta chain, CD11a and CD18 respectively) reduced the adherence, others (anti-CR3 or anti-gp 150/95, CD11b and c respectively) did not influence it. Antibodies directed against MHC class II exerted no inhibitory action on the lymphoid cell adhesion to FDC. As, at ultrastructural level, gold-labeled immune complexes can be found between FDC and lymphoid cells, we examined the effect on cell adhesion of the addition of immune complexes to the cell suspensions. It only impaired the lymphoid cell adhesion when complement components were present. IgM complexes were then more inhibitory than IgG complexes. When antibodies against Fc IgG receptors (CD16) were added, the adhesion was strongly reduced whereas antibodies to Fc IgE (CD23) receptors had no influence. The antibody DRC1, specifically recognizing an antigen on human FDC reduced the attachment of cells to FDC. This antigen thus seems to play a role in the intercellular contacts; this is the first function ascribed to this FDC specific antigen.

Animals↗

PubMed1993

The Peyer's patch high endothelial receptor for lymphocytes, the mucosal vascular addressin, is induced on a murine endothelial cell line by tumor necrosis factor-alpha and IL-1.

The specificity of lymphocyte homing from the blood into a tissue is determined in part by complementary pairs of adhesion receptors on lymphocytes and endothelial cells termed homing receptors and vascular addressins, respectively. The mucosal vascular addressin involved in lymphocyte homing to Peyer's patches is a 66-kDa glycoprotein, MAdCAM-1. Investigation of the regulation and molecular genetics of MAdCAM-1 have been hampered by the lack of a murine cell line expressing this adhesion molecule. We show herein using indirect immunofluorescence studies that MAdCAM-1 can be induced on a murine endothelial cell line, bEnd.3, by cytokines and LPS. Western blot analysis of MAdCAM-1 purified by affinity column chromatography from TNF-alpha-treated bEnd.3 cells demonstrates a 66-kDa protein that comigrates in SDS-PAGE with the MAdCAM-1 constitutively found on high endothelial venules in murine mesenteric lymph nodes. Comparison of MAdCAM-1 expression on the bEnd.3 cells was made to the expression of adhesion molecules ICAM-1 and VCAM-1. MAdCAM-1 and VCAM-1 are not constitutively expressed on the bEND.3 surface but can be induced in a concentration-dependent manner by LPS, TNF-alpha, and IL-1. ICAM-1 is constitutively expressed on the endothelioma surface and expression is increased by TNF-alpha, IL-1, LPS, and IFN-gamma. Surface expression of MAdCAM-1 peaks 12 to 18 h after exposure to TNF-alpha and remains elevated at 48 h, whereas expression of VCAM-1 peaks at 4 h and inducible ICAM-1 peaks between 4 and 18 h. Interestingly, IFN-gamma has differential effects on expression of these three adhesion receptors. IFN-gamma alone induces VCAM-1 and enhances ICAM-1 expression, but does not induce MAdCAM-1. Furthermore, although, preincubation of bEND.3 cells with IFN-gamma modestly increases the induction of ICAM-1 and VCAM-1 in response to TNF-alpha and IL-1, it dramatically reduces the TNF-alpha, IL-1, and LPS-induced expression of MAdCAM-1. MAdCAM-1 on bEnd.3 cells is functional as the murine T lymphoma TK1, known to bind MAdCAM-1, also binds to TNF-alpha-stimulated endothelioma but not to unstimulated cells. This binding is blocked by the antibodies against MAdCAM-1 and against the alpha 4-chain of its integrin receptor, alpha 4 beta 7, on TK1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1996

Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.

Neurons in organotypic hippocampal slice cultures (OHSCs) are known to preserve morphological and physiological features of the in vivo situation; however, little is known about the properties of microglial cells under these in vitro conditions. In this study, we addressed the question whether microglial cells in OHSCs are initially activated following explantation but return to a resting state during in vitro cultivation. Thus, we analyzed a) microglial cell morphology, b) microglial cell distribution, and c) expression of integrin adhesion molecules as putative markers of microglial activation. Hippocampal slices fixed immediately following explantation showed only resting microglial cells, mainly located in the paraventricular regions. After 3 days in vitro (div) OHSC surfaces were covered by activated microglia, whereas intermediate layers contained fewer microglial cells, giving the slices a sandwich-like appearance with the intact hippocampal formation being surrounded by glial tissue. After 3 div, microglial cells in intermediate layers of OHSCs showed activated morphology with ovaloid cytoplasm and no or merely few cytoplasmic processes; after 6 div, however, an increasing degree of ramification could be observed. After 9 div, microglia in intermediate layers had almost regained the morphological appearance of resting cells with filigrane cytoplasmic processes extended in all directions. The integrin adhesion molecules LFA-1 (alpha and beta chains) and VLA-4 were expressed on most microglial cells with activated morphology, as verified by co-localization with double immunofluorescence labeling for LFA-1 or VLA-4 and Griffonia simplicifolia isolectin B4 (GFS-B4). In contrast, only low levels of integrin adhesion molecule expression were also found on reactive astrocytes along slice surfaces. However, LFA-1 or VLA-4 were never found on ramified microglial cells, and double immunofluorescence labeling of LFA-1 or VLA-4 with ramified GFS-B4+ microglia never occurred. We conclude that a) originally resting microglial cells activated in an early phase of in vitro culture but regain a resting status after at least 6 div; and b) integrin adhesion molecules LFA1 and VLA-4 are potential markers of microglial activation, as they were found on activated but never on resting microglial cells. This enables further investigations on immunological and electrophysiological features of resting and activated microglial cells under in vitro conditions.

Animals↗

PubMed1991

Characterization of integrin chains in normal and neoplastic human pancreas.

Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1-6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗

PubMed2000

Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins.

Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 beta 11 and alpha 6 beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 beta 1 transfectants. Upon stimulation with the activating anti-integrin beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 beta 1 and alpha 6 beta 1 integrins with high avidity and also to alpha 6 beta 4 integrin.

Antigens, CD↗

PubMed1994

CD9 antigen is an accessory subunit of the VLA integrin complexes.

The CD9 antigen is a cell surface glycoprotein of unknown function which belongs to the tetraspans family. We demonstrate here, by precipitation, Western blotting and co-capping experiments, that this molecule is associated with a large fraction of beta 1 integrins in two cell lines, the pre-B cell line NALM-6 and the megakaryocytic cell line HEL. In HEL cells, CD9 antigen is only associated with VLA-4. In contrast, in NALM-6 cells, CD9 antigen is associated with both VLA-4 and VLA-5. On the other hand, only the beta 1 chain is co-precipitated with the CD9 antigen in transfected L cells. These data show that the CD9 antigen is associated with the beta 1 chain rather than with a particular integrin. CD9 monoclonal antibodies (mAb) did not modify the binding of HEL and NALM-6 cells to fibronectin, laminin or collagen. The association of CD9 antigen to VLA integrins is strengthened by the fact that both CD9 and anti-VLA mAb induce aggregation of the two cell lines and inhibit their migration in Transwell chambers. Because the aggregating effect, but not the inhibition of migration, is observed in CEM or CD9-transfected CEM cells, these two effects are likely to be mediated by different mechanisms.

Amino Acid Sequence↗

PubMed1994

Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells.

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

Adhesins, Bacterial↗

PubMed1996

Integrin upregulation on sputum neutrophils in smokers with chronic airway obstruction.

To determine the relationship between the expression of leukocyte-specific integrins in the airways and the airway obstruction in smokers, we analyzed hypertonic saline-induced sputum in 33 male subjects, age 64.7 +/- 0.5 yr (mean +/- SEM), with a smoking history of 12 to 94 pack-years, at the end of a 15-yr follow-up study. Average FEV1/VC ratio was 69 +/- 1% at the beginning of the study and 66 +/- 2% at the end of the follow-up period, and annual decline of FEV1 was 20 +/- 3 ml/yr. Fourteen individuals exhibited airway obstruction as assessed by a FEV1/VC ratio lower than 63.3%. Differential leukocyte count was performed on cytospin preparations and the expression of integrin alpha (CD11a, CD11b, CD11c) and beta (CD18) chains was assessed on granulocytes and mononuclear cells by immunocytology. The numbers of neutrophils expressing CD11b and CD18, but not CD11c or CD11a, were increased in the subjects with airway obstruction compared with those without airway obstruction. CD11b- and CD18-positive neutrophils were negatively correlated with FEV1/VC ratio (p < 0.01). No significant correlations were found between CD11a-, CD11b-, CD11c-, CD18-positive mononuclear cells and lung function measurements. In conclusion, our results suggest that leukocyte-specific integrin CD11b/CD18 expressed on sputum polymorphonuclear leukocytes represents a marker for the smokers who develop chronic airway obstruction.

Aged↗

PubMed1992

Expression and role of integrin receptors in Sézary syndrome.

The potential role of integrins in the epidermotropism of the atypical lymphocytes of Sézary syndrome was studied by monitoring the expression of alpha and beta chains and their major ligands in skin biopsies and peripheral blood cells in patients at different progression stages. Most mononuclear cell integrins were also detected on infiltrating cells including the leukocyte complex CD11/CD18, alpha 4 beta 1, and their ligands, ICAM-1 and VCAM-1. Conversely, alpha 6 and beta 4 were present only in epidermal basal cells. Mononuclear infiltrates of SS were positive for both alpha 3 and alpha 5 chains, whereas in inflammatory cutaneous diseases only alpha 5 was expressed, indicating that a major feature of Sézary cells is the unique expression of alpha 3 beta 1. Significant changes of alpha 3 beta 1 were monitored in the follow-up of Sézary patients and correlated with the results of the therapy. The heterodimer alpha 1 beta 1 was absent from mononuclear cells except in one case. Among matrix molecules, laminin and type IV collagen displayed a pattern similar to that of the controls, whereas fibronectin and tenascin deposition were apparently increased. Circulating Sézary cells, both at diagnosis and during follow-up, were alpha 3 and alpha 5 negative and failed to acquire these adhesion molecules after mitogenic stimulation. We propose that the expression of alpha 3 beta 1 is a distinguishing feature of skin-infiltrating Sézary cells and may be related to their epidermotropism. It could also be adopted as an additional parameter of the progression and therapeutic stage of Sézary syndrome.

Antigens, CD↗

PubMedDate not supplied

Leukocyte adhesion deficiency disorder in an infant.

Leukocyte adhesion defect (LAD) is an inherited disorder of phagocytic function. The three different alpha-chains of the leukocyte integrin family (CD 11a, 11b, 11c) are all dependent on the common beta chain (CD18) for proper insertion into the cell membrane. Deficiency of CD 18 thus causes an inability to express any of these proteins and results in the syndrome of LAD type 1. Clinical features include delayed separation of the umbilical cord, persistent leukocytosis and recurrent infection. A case of severe LAD occurred in a 1 month-old infant. The immunological pattern of the baby showed pathological values of CD11b and CD18 (1.3% and 0.2% respectively) when compared with normal values (50-70%) and lack of random (1.0 nm vs normal of 10±5) and casein-induced (22.0 nm vs normal of 60-120nm) chemotaxis. The baby is now waiting for bone marrow transplantation. Although LAD is a rare form of congenital immune deficiency this disease should be considered when delayed wound healing and recurrent bacterial skin infections are present in a newborn.

Journal Article↗

PubMed1993

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF.

Cell Adhesion↗

PubMed1996

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Animals↗