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PubMed1992

Adhesion receptors are differentially expressed on developing thymocytes and epithelium in human thymus.

The thymic microenvironment consists of a network of interrelated cells of epithelial, fibroblastic, endothelial, and hemopoietic origin. Within this environment, the development of specific T-lymphocyte subpopulations partially depends on the selective interaction of T-cell precursors with such cells. Human thymic epithelial cell strains, generated with a defective retroviral vector containing simian virus 40 (SV40) large T antigen and the neomycin resistance gene or by transfection with an SV40 plasmid defective in the origin of replication, provide useful tools for understanding the mechanisms contributing to the control of T-cell maturation. Because interepithelial, epithelial-macrophage, and lymphocyte-epithelial cell interactions are important for thymocyte differentiation, the distribution of integrin and nonintegrin adhesion receptors on these cells and on developing thymocytes in vivo and in vitro has been examined in detail. Our results indicate that the transformed human thymic epithelial cell strains express the common very late antigen (VLA)-beta 1 receptor and unique alpha chains VLA-2, VLA-3, and VLA-6. The cells are also positive for LFA-3 and ICAM-1 and weakly express beta 3, beta 4, and VNR alpha. They do not express the Leu-cellular adhesion molecules (CAM). This phenotypic profile on cultured thymic epithelium generally corresponds to the distribution of integrin and other receptor molecules on thymic epithelial cells in tissue sections. The majority of thymocytes also express the integrin VLA-beta 1 and -beta 2 chains as well as VLA-4, VLA-6, and LFA-1 alpha(L). Three-color flow cytometric analyses show differential levels of expression of these adhesion receptors on human thymocyte subsets. Taken together with the immunohistochemical localization of extracellular matrix molecules, these studies suggest that both the distribution of receptor-ligand pairs and the level of expression of adhesion molecules may influence T-cell development within the thymus.

Antigens, Polyomavirus Transforming↗
PubMed1991

Expression of collagen binding integrins during cardiac development and hypertrophy.

The interaction between components of the extracellular matrix and the cell surface of cardiac myocytes appears to be regulated in part by receptors belonging to the integrin superfamily. The expression of the integrins was investigated at different stages of development of the heart as well as during cardiac hypertrophy. The characterization of the membrane proteins showed that a beta 1-integrin and associated alpha-chains were responsible for the interaction with collagen, laminin, and fibronectin. Immunoprecipitation data indicated that the presence of specific alpha-chains varied with development. These data were correlated with the ability of the isolated myocytes to attach to specific components of the extracellular matrix. The expression of the alpha 1-chain was prominently associated with the recognition of interstitial collagens. The presence of the alpha 1-chain was also associated with stages when collagen synthesis was increased, especially during fetal and neonatal growth and cardiac hypertrophy. Immunohistochemical localization with the antiserum against beta 1-integrin demonstrated its specific localization near the Z lines of cardiac myocytes. The localization both in vitro and in vivo indicated that the beta 1-integrin may play a role in myofibrillogenesis during development. The present immunohistochemical, cell adhesion, and biochemical data clearly indicate that integrins play a major role in the regulation of the interaction between cardiac myocytes and the extracellular matrix during development and disease.

Animals↗
PubMed1990

Epithelial integrins.

We have undertaken the study of integrins specifically or predominantly expressed in epithelial cells, as they may be involved in establishing and maintaining properties peculiar to epithelia, such as polarization and morphogenetic movements. We describe here recent results regarding two such integrins. One of these contains the novel beta chain, beta 6, whose structure was deduced from cDNA clones. Some initial results on distribution and possible alpha chain associated to beta 6 are discussed. Structural data are then summarized on another epithelial integrin, alpha 6 beta 4. The alpha 6 subunit and a variant form of the beta 4 subunit were recently cloned in our laboratory. Adhesion assay results indicate that alpha 6 beta 4 may mediate attachment of epithelial cells to basement membranes.

Amino Acid Sequence↗
PubMed1991

Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia.

We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.

Animals↗
PubMed1994

Conformation-dependent platelet adhesion to collagen involving integrin alpha 2 beta 1-mediated and other mechanisms: multiple alpha 2 beta 1-recognition sites in collagen type I.

Platelet adhesion has been measured to type-I monomeric collagen, collagen fibres, alpha 1(I) and alpha 2(I) chains and the chain fragments alpha 1(I)CB3, alpha 1(I)CB6, alpha 1(I)CB7 and alpha 1(I)CB8, and alpha 2(I)CB3,5 and alpha 2(I)CB4. Little if any adhesion occurred to any denatured species at 37 degrees C, demonstrating the importance of the collagen helix. However, on coating at 4 degrees C to promote helix formation, and assaying at room temperature to avoid denaturation, adhesion was observed to both alpha-chain types and all fragments, the exact level of which depended on the identity of the species in question. Adhesion was strongly Mg(2+)-dependent. Antibodies against the integrin alpha 2 beta 1 partially inhibited adhesion to alpha-chains and all fragments except alpha 1(I)CB6, indicating a wide distribution of alpha 2 beta 1-binding sites in the collagen molecule. 'Activation-dependent' adhesion to monomeric collagen, totally secondary to alpha 2 beta 1-mediated adhesion, involved at least two mechanisms, one mediated by integrin alpha IIb beta 3 and insensitive to prostaglandin E1, the other inhibitable by prostaglandin E1 but independent of integrin alpha IIb beta 3. alpha IIb beta 3-mediated adhesion to fragments was, at least in part, independent of the alpha 2 beta 1-mediated adhesion. Adhesion to fibres was largely bivalent-cation-independent with only minor involvement of integrin alpha 2 beta 1. Some alpha IIb beta 3-mediated adhesion occurred but was independent of any alpha 2 beta 1-initiated adhesion. Total 'activation-dependent' adhesion to fibres was less than to monomeric collagen. Affinity chromatography revealed bivalent-cation-independent binding to fibres of three main platelet surface proteins, 90, 150 and 190 kDa in size.

Amino Acid Sequence↗
PubMed1997

Integrin and basement membrane normalization in mouse grafts of human keratinocytes--implications for epidermal homeostasis.

Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains alpha 2, alpha 3, beta 1 and alpha 6, while beta 4 already showed a preferential basal location; (2) de novo expression of alpha 5 and alpha v; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular alpha 2 beta 1 and alpha 3 beta 1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin alpha 6 beta 4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.

Animals↗
PubMed1990

The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.

Hybridoma fusions with hamster hosts were undertaken to generate mAbs to mouse spleen dendritic cells. Two mAb were obtained and used to uncover the distinct integrins of these APC. One, 2E6, bound a determinant common to all members of the CD11/CD18 family, most likely the shared 90 kD CD18 beta chain. 2E6 immunoprecipitated the characteristic beta 2 integrin heterodimers from lymphocytes (p180, 90; CD11a) and macrophages (p170,90; CD11b), but from dendritic cells, a p150,90 (presumably CD11c) integrin was the predominant species. 2E6 inhibited the binding function of the CD11a and CD11b integrins on B cells and macrophages in appropriate assays, but 2E6 exerted little or no inhibition on the clustering of dendritic cells to T cells early in primary MLR, suggesting a CD11/CD18-independent mechanism for this binding. The second mAb, N418, precipitated a 150, 90 kD heterodimer that shared the 2E6 CD18 epitope. This N418 epitope may be the murine homologue of the previously characterized human CD11c molecule, but the epitope was only detected on dendritic cells. N418 did not react with peritoneal macrophages, anti-Ig-induced spleen B blasts, or bulk lymph node cells. When used to stain sections of spleen, N418 stained dendritic cells in the T-dependent areas, much like anti-class II mAbs that were also generated in these fusions. In addition, N418 revealed nests of dendritic cells that punctuated the rim of marginal zone macrophages between red and white pulp. This localization positioned most dendritic cells at regions where arterial vessels and T cells enter the white pulp. We conclude that the p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and we speculate that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.

Animals↗
PubMed1994

Adhesive properties and integrin expression profiles of two colonic cancer populations differing by their spreading on laminin.

The mostly undifferentiated parental HT29 (HT29p) human colonic adenocarcinoma cell line and a differentiated subpopulation selected by the anti-cancer drug 5-fluorouracil (HT29-Fu) (Lesuffleur et al. (1991) Int. J. Cancer 49, 721-730) display strikingly different behavior when grown on laminin coatings: the former grows as aggregates while the latter grows as monolayers. In an attempt to explain this difference, we performed a comparative study of cell adhesion properties and of expression, involvement and localization of the alpha 6, beta 1 and beta 4 subunits constituting the integrin family among the two cell populations. HT29p and HT29-Fu cells exhibited a similar adhesion pattern to laminin and laminin fragments E8 and P1. In both cell lines, cell adhesion could be blocked at about 90% with anti-alpha 6 subunit antibodies and around 30-50% with anti-beta 1 antibodies; no inhibition of the cell adhesion was obvious when using anti-beta 4 antibodies. Immunoprecipitations of iodinated membrane-solubilized proteins and immunoblotting experiments showed that all alpha 6 chains expressed in both HT29p and HT29-Fu cell populations exist as alpha 6 beta 4 integrins; beta 1 subunits are associated with alpha 2 and alpha 3 chains. When HT29p or HT29-Fu cells were injected subcutaneously in nude mice, a similar expression pattern of alpha 6, beta 4 and beta 1 integrin subunits was noticeable in the resulting tumors: alpha 6 and beta 4 subunits were localized at the basal surface of the tumor cells facing the stromal elements, and to a lesser extent at the cell-cell contacts within the tumor-cell clumps; beta 1 subunits were mainly found within the cytoplasm of the tumor cells. Despite these overall similarities among the two cell lines, the following changes could account for their different behavior on laminin: less proteolytic processing of the beta 4 integrin subunit occurred in HT29-Fu cells yielding peptidic fragments of 175 kDa, which are absent from the parental cells; the immunostaining pattern of the various subunits demonstrated a segregation of alpha 6, beta 4 and beta 1 integrin subunits on the basal side of the HT29-Fu cells when cultured on laminin to the detriment of their lateral location, a phenomenon that was not obvious in the parental cells. Altogether, these results suggest that the distinct behavior of the undifferentiated versus differentiated HT29 cell populations on laminin is not related to altered adhesion properties of the cells but rather to a deficient stabilization of the adhesion leading to cell spreading.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Non-peptide alpha(v)beta(3) antagonists: identification of potent, chain-shortened RGD mimetics that incorporate a central pyrrolidinone constraint.

Antagonists of the integrin receptor alpha(v)beta(3) are expected to have utility in the treatment of osteoporosis through inhibition of bone resorption. A series of potent, chain-shortened, pyrrolidinone-containing alpha(v)beta(3) receptor antagonists is described. Two sets of diasteromeric pairs of high-affinity antagonists demonstrated marked differences in log P values, which translated into differing dog pharmacokinetic properties. One member of this set was demonstrated to be effective in reducing bone resorption in rats.

Adrenergic alpha-Antagonists↗
PubMed1995

Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals.

We have shown previously that the expression of collagenase is upregulated in rabbit synovial fibroblasts cultured on a substrate of antibody to the alpha 5 chain of the alpha 5 beta 1 integrin fibronectin receptor or on the 120-kD cell-binding chymotryptic fragment of plasma fibronectin, but remains at basal levels in cells plated on intact plasma fibronectin. We now have identified some of the components of a signaling pathway that couples the fibronectin receptor to the induction of collagenase transcription. We studied the control of collagenase gene expression in cells adhering to the 120-kD fragment of fibronectin, to antifibronectin receptor antibody, or to plasma fibronectin by transiently introducing promoter-reporter constructs into rabbit synovial fibroblasts before plating cells on these matrices. The constructs contained segments of the human collagenase promoter regulating transcription of chloramphenicol acyl transferase. Expression of constructs containing the -1200/-42-bp segment or the -139/-42-bp segment of the collagenase promoter inserted upstream from the reporter gene was induced to similar extents in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in fibroblasts plated on fibronectin. The expression of the construct containing the -66/-42-bp segment of the promoter was not regulated and was similar to that of the parent pBLCAT2 plasmid, suggesting that the -139/-67 region of the collagenase promoter, which contains PEA3- and AP1-binding sites, regulates the transcription of collagenase caused by integrin-derived signals. Expression of a reporter construct containing only the PEA3 and AP1 sites in the collagenase promoter (-90/-67) also increased in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. Mutations in either the AP1 or PEA3 site of this minimal promoter abrogated its activity in cells plated on these inductive ligands. Expression of c-fos mRNA increased within 1 h of plating cells on the 120-kD fibronectin fragment or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. c-Fos protein accumulated in the nuclei of fibroblasts within 10 min of plating on the 120-kD fibronectin fragment. The increase in c-Fos was required for the increase in collagenase in cells plated on the 120-kD fibronectin fragment: incubation of cells with antisense, but not sense, c-fos oligonucleotides diminished both basal and induced expression of the -139/-42 collagenase promoter-reporter construct and decreased expression of the endogenous collagenase gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Altered glycosylation of integrin adhesion molecules in colorectal cancer cells and decreased adhesion to the extracellular matrix.

The integrin mediated interactions between tumour cells and the surrounding extracellular matrix are thought to play crucial parts in the complex process of invasion and metastasis. It has been previously shown that the expression of integrins is differently diminished in a chain-specific manner in human colorectal cancer. To further characterise the integrins still expressed in colorectal carcinomas, immunoblots with monoclonal antibodies against the beta 1 integrin subunit have been performed. In isolated cell membranes of colorectal cancers a second smaller beta 1 chain (105 kD non-reduced) was found as well as the mature beta 1 chain (116 kD non-reduced) present in normal mucosa of the colon. This smaller beta 1 chain comigrates with the diminished glycosylated precursor form of the beta 1 chain. The role of N-glycosylation for the function and expression of integrins in vitro was therefore investigated, with deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) as specific inhibitors of N-glycan processing. Pretreatment of human colon adenocarcinoma derived HT-29 cells with DMJ resulted in an expression of the 105 kD beta 1 precursor chain and of smaller forms of the alpha 1, alpha 3, alpha 6, and alpha v integrin subunits in a time and dose dependent manner. HT-29 cells treated with DMJ adhered poorly to laminin (8% of untreated controls), collagen type IV (40%), and fibronectin (35%). Pretreatment of the cells with DNJ did not alter the molecular weight of the integrin chains expressed and reduced HT-29 adhesion to laminin and fibronectin only to 68% and 49% respectively. Adhesion to collagen type IV was increased to 124% by DNJ. These results show that N-glycan processing is essential for the function and expression of integrins in human colorectal cancer cells. An altered glycosylation of these adhesion receptors may contribute to a more invasive or metastatic phenotype in colorectal cancer.

Adenocarcinoma↗
PubMed2006

Folate deficiency induced hyperhomocysteinemia changes the expression of thrombosis-related genes.

Hyperhomocysteinemia (HH) is an independent risk factor for thrombosis although the precise pathogenesis is still unresolved. Previous studies have demonstrated that HH changes whole blood coagulation by increasing the velocity, increasing the firmness of the formed clot, and by prolonging the initiation phase of the coagulation. With the aim of elucidating the genetic pathogenesis which might be responsible for the changes in whole blood coagulation, we applied oligo-array technology to RNA from buffycoat-cells comparing animals suffering from hyperhomocysteinemia (42 micromol/l) with controls (6 micromol/l). Data mining identified a number of relevant genes, and the expression pattern was validated by real time reverse transcriptase-polymerase chain reaction. An upregulation of integrin beta-3, Rap 1b, glycoprotein V, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) led us to deduce increased platelet activation/aggregation. Coagulation factor XIIIa was upregulated and may contribute in increasing the firmness of the formed clot. Impaired fibrinolysis was anticipated, since an upregulation of plasminogen activator inhibitor-1 (PAI-1) and a downregulation of tissue-type plasminogen activator (t-PA) were detected. Reduced spontaneous contact activation was anticipated due to a downregulation of the kallikrein gene. Upregulation of selectins may contribute to increased tethering and rolling of leukocytes. In conclusion, folate deficiency induced hyperhomocysteinemia changes in the gene expression of buffy coat cells which was characterized by increased platelet activation, impaired fibrinolysis and a reduced contact activation of the coagulation. These changes may contribute to explain the increased risk of thrombosis seen in hyperhomocysteinemia individuals. This pattern of the hyperhomocysteinemia-affected genes may represent a reference for further studies at the protein level to define the folate depletion effects in blood cells.

Animals↗
PubMed2000

Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans.

The ability of leukotoxin from Actinobacillus actinomycetemcomitans to induce release of lysosomal constituents was studied with human polymorphonuclear leukocytes (PMNL). Leukotoxin purified from A. actinomycetemcomitans or bacterial cells of a leukotoxic strain were mixed with human PMNL and the suspension was incubated under anaerobic conditions. Samples were taken at certain time intervals to examine the cell morphology of PMNL by electron microscopy and the extracellular concentrations of the granule components lactoferrin and elastase by enzyme-linked immunosorbent assay (ELISA). Electron microscopy revealed that within 10 min of exposure to leukotoxin, the number of intracellular granules was markedly reduced and the remaining granules were translocated to the periphery in PMNL. At the same time, the extracellular concentrations of lactoferrin and elastase were elevated, while that of the cytosolic enzyme lactate dehydrogenase, an indicator of cell lysis, remained low. The lysosome molecules CD63 and CD66b were also exposed on the PMNL surface, indicating fusion of lysosomes with the plasma membrane. These effects were completely abolished by the addition of anti-leukotoxin serum. Pre-incubation of PMNL with monoclonal antibodies to CD11a and CD18 that recognize alpha- and beta-chains of the LFA-1 integrin, a leukotoxin receptor on PMNL, inhibited the cytolysis, but not the release of granule components. The present results demonstrate the ability of A. actinomycetemcomitans leukotoxin to trigger a rapid release of lysosomal compounds in human PMNL. The release is due to an active process stimulated by the interaction of PMNL with the toxin or toxin-carrying bacteria.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Tumor necrosis factor-alpha augments the pro-inflammatory interaction between PMN and GBM via a CD18 dependent mechanism.

Acute glomerulonephritis is frequently associated with intraglomerular neutrophil (PMN) accumulation and the intensity of the inflammatory reaction is correlated with elevated concentrations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha). PMN are thought to damage glomeruli due to a combination of reactive oxygen species and proteolytic enzymes. Using an in vitro model of anti-GBM nephritis the effects of TNF alpha on GBM damage by PMN were evaluated. The interaction of GBM and PMN resulted in a low grade respiratory burst that was significantly augmented by the addition of TNF alpha. Luminol dependent chemiluminescence (LCL) was increased from 2.4 x 10(6) to 48.1 x 10(6) (P < 0.05). The GBM induced LCL could be > 85% inhibited by blocking with monoclonal antibodies (mAbs) to the common beta chain of the PMN beta 2 integrin family (CD18), but was unaffected by mAbs to CD11a or CD11b subunits. Degradation of GBM, however, was not influenced by either TNF alpha priming of PMN or anti-beta 2 integrin mAbs. When PMN were incubated with GBM-anti-GBM IgG complex they underwent an increase in LCL from 2.4 x 10(6) to 31.1 x 10(6). They also degraded more GBM than controls (10.1% vs. 1.8%). These aspects of PMN activation were Fc receptor mediated, dependent upon anti-GBM IgG being bound to GBM and inhibited by mAb to the PMN Fc receptor. These studies show that TNF alpha can modulate the inflammatory response of PMN in contact with GBM in a CD18 dependent manner. In contrast, Fc receptor mediated events are uninfluenced by TNF alpha.

Basement Membrane↗
PubMed1995

VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells.

The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CEC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against beta 1-, alpha 4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the alpha 5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as BI-CFC, remains to be determined.

Antibodies, Monoclonal↗
PubMed1992

The role of CD18 in IL-8 induced dermal and synovial inflammation.

1. The intradermal administration of endothelial IL-8 (IL-8(1-77) or monocyte derived IL-8 (IL-8(1-72) to rabbits produced a concentration-dependent increase in plasma extravasation and an accumulation of polymorphonuclear leukocytes (PMNs) when measured over a 3 h time period. When plasma extravasation and PMN accumulation were measured over a 30 min time period no significant increases in PMN accumulation or plasma extravasation were observed in response to IL-8 alone. However, under these conditions, the addition of prostaglandin E2 (100 pmol) produced a significant potentiation of IL-8-induced plasma extravasation. There was no significant difference between the biological activities of IL-8(1-77) and IL-8(1-72). 2. Plasma extravasation and PMN accumulation induced by IL-8 were inhibited in rabbits pretreated with the monoclonal antibody designated IB4 (1 mg kg-1, i.v.) directed against the common beta chain (CD18) of the leukocyte integrins. 3. The intra-articular administration to rabbits of IL-8(1-77) (1 nmol) resulted 24 h later in the appearance of a mixed population of leukocytes (PMNs and mononuclear cells) in synovial lavage fluid. Biochemical analyses revealed the presence of an increased level of sulphated proteoglycans (sPG) and of the metalloproteinase stromelysin. Pretreatment of rabbits with IB4 (3 mg kg-1, i.v.) inhibited the accumulation of PMNs but had no effect on the mononuclear infiltrate nor on the levels of sPG or stromelysin. 4. The intradermal or intra-articular injection of E. coli-derived endotoxin induced similar inflammatory changes to those observed with IL-8.The possibility that the biological activities of IL-8 were attributable to minor contamination with endotoxin is unlikely for two reasons. Firstly, biological effects of endotoxin were observed at levels greater than that contained in the IL-8 preparation. Secondly,reduction of the endotoxin content of the IL-8 preparation by a factor of 10 did not produce a concomitant reduction in the observed biological activity of the IL-8.

Animals↗
PubMed1999

Generation of multinucleated giant cells in vitro by culture of human monocytes with Mycobacterium bovis BCG in combination with cytokine-containing supernatants.

Multinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself. Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products. The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro. Stimulation of monocytes with Mycobacterium bovis bacillus Calmette-Guérin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27%. In contrast, stimulation with one component alone induced only low fusion rates of up to 10%. Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria. BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation. Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria. MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the beta chain (CD18) of beta2-integrins. These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.

Adult↗
PubMed1997

Protection from apoptosis in human neutrophils is determined by the surface of adhesion.

Recent work suggests that various neutrophil agonists affect the rate of apoptosis in these cells. On the basis of these observations, we hypothesized that signals triggered in neutrophils via their adhesion receptors might also modify their life span. This hypothesis has been tested using human neutrophils adherent to tissue culture plastic, either untreated or coated with extracellular matrix (ECM) proteins or with monolayers of human umbilical vein endothelial cells. To detect and quantitate apoptotic changes in adherent cells, we developed a microtiter plate assay using a cell-permeable DNA-binding fluorescent dye, Hoechst 33342. Use of this assay demonstrated that 1) the number of apoptotic cells among neutrophils adherent to plastic after 6-20 h of incubation was significantly lower than that among neutrophils adherent to the ECM proteins fibronectin or laminin; 2) adhesion to interleukin-1-activated endothelial cells delayed apoptosis, whereas adhesion to nonactivated endothelium accelerated neutrophil death; and 3) monoclonal antibodies directed against intercellular adhesion molecule 1 or against the common beta 2-chain of the leukocyte integrins abolished the protective effect of interleukin-1-activated endothelial cells on apoptosis of adherent neutrophils. These results suggest that the life span of adherent neutrophils. depends on the activating signals triggered by the surface of adhesion.

Apoptosis↗