Evaluation of integrin molecules involved in substrate adhesion

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.     

Expression of adhesion molecules and costimulatory molecules in inflammatory myopathies

To clarify the local immune responses in inflammatory myopathies, we examine recent studies on expression of adhesion and costimulatory molecules. Adhesion molecules participate in MNC influx from blood vessels into muscle tissues and interactions of inflamed muscle cells with surrounded MNC. These antigens may work as costimulatory molecules in antigen presenting process by muscle cells, as well as in augmentation of inflammation by infiltrating MNCs.     

Endothelial cell integrin laminin receptor expression in multiple sclerosis lesions

A model that explains natal dispersal in resident screech-owls is presented and examined. The model is based on interactions among hormonal changes, body condition and social stimuli. It predicts that corticosterone, an adrenal glucocorticoid known to stimulate locomotor and foraging activity, increases in blood plasma prior to dispersal through a combination of endogenous and exogenous events. This mediates the locomotor activity that underlies dispersal behaviour. Juveniles in good body condition (i.e. those with sufficient fat reserves) will disperse when corticosterone increases. Birds in poor body condition will not, but they will increase their foraging activity under the influence of corticosterone. Dispersal of siblings will reduce aggression and/or competition for food, enabling the remaining juveniles to improve their body condition and disperse. Initial studies on screech-owls, Otus asio and O. kennicottii, have produced results that are generally consistent with the model. For example, captive juvenile screech-owls showed increased locomotor activity in the weeks leading up to the time when free-living juveniles are dispersing, and activity levels declined thereafter. Peaks in corticosterone corresponded with periods of high locomotor activity (i.e. at the time of dispersal) in captive owls. Finally, field studies indicate that dominant juveniles, which are presumably in better physical condition, initiated dispersal before their more subordinate siblings. Copyright 1998 The Association for the Study of Animal Behaviour.     

Expression of the beta 7 integrin by human endothelial cells

Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells.     

Expression of adhesion molecules and HLA-DR by macrophages and dendritic cells in aphthoid lesions of Crohn's disease: an immunocytochemical study

The phenotypes and ultrastructure of macrophages and dendritic cells in aphthoid lesions of the colon were immunocytochemically observed in patients with Crohn's disease. Biopsy specimens were endoscopically obtained from both aphthoid and advanced lesions in Crohn's disease patients. Biopsy specimens obtained from patients with infectious colitis and from normal individuals served as controls. Aphthoid lesions contained densely aggregated CD68+ macrophages, which were surrounded by numerous ID-1+ dendritic cells. In the normal controls and infectious colitis patients, however, a few scattered CD68+ macrophages and ID-1+ dendritic cells were noted beneath the surface epithelium. CD3+ lymphocytes were significantly increased in both aphthoid and advanced lesions of Crohn's disease, but the CD4/CD8 ratio was similar in all groups studied. The double immunoperoxidase staining method revealed that both CD68+ macrophages and ID-1+ dendritic cells in the aphthoid lesions simultaneously expressed ICAM-1 and HLA-DR antigens. Electronmicroscopic observation revealed that CD68+ macrophages had numerous vesicles and lysosomal granules and few projections, and that ID-1+ dendritic cells had appreciable cytoplasmic protrusions with a few vacuoles. These findings suggested that the colonic mucosa in Crohn's disease contained two types of macrophage/dendritic cells in the same lineage that expressed intercellular adhesion molecules and class-II MHC antigens. It also appeared that the aphthoid lesions of Crohn's disease featured an increase in macrophages and dendritic cells consistent with immunological activation.     

Expression of MHC and cell adhesion molecules in muscular sarcoidosis

Biopsied skeletal muscles from 5 patients with muscular sarcoidosis (nodular type; 1, and myopathic type; 4) were immunocytochemically examined. All biopsies presented granulomatous changes. Atrophic or regenerating muscle fibers adjacent to granuloma demonstrated compression or ischemic changes. In the center of the granuloma, CD68+ epitheloid cells and giant cells, and CD4+ T cells were localized. At the periphery of the granuloma, CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages were found. Expression of HLA-A,B,C was diffuse in the muscle fibers. Expression of HLA-DR and ICAM-1 was more prominent near the granuloma or perifascicular fibers, and that of LFA-3 was moderate in those lesions. VCAM-1 was expressed in endothelial cells and macrophages near the granuloma. Those findings indicate that interferon-gamma or TNF-alpha produced by infiltrating inflammatory cells may induce expression of these immunologic markers or adhesion molecules. Immunocytochemical differences between the nodular and myopathic forms of sarcoidosis are not evident, but either localization or abundance of granuloma in muscle bulks is relevant to weakness or atrophy of clinically affected muscle.     

Expression of integrin family of cell adhesion receptors in rheumatoid synovium. Alpha 6 integrin subunit in normal and hyperplastic synovial lining cell layer

Integrins are heterodimeric cell adhesion receptors. The beta 1 integrin subunit can be in a complex with multiple a subunits and form receptors for collagen, laminin, fibronectin, and vitronectin. We have characterized the distribution of eight integrin subunits in rheumatoid synovium, with special interest in the lining cell layer. The beta 1 integrin subunit was found in abundance in synovial stroma and in lining cells. The only alpha subunit seen constantly in lining cells was alpha 6. In complex with alpha beta subunit, alpha 6 forms a laminin receptor usually seen in epithelial or endothelial cells or in macrophages. The fact that laminin was found in the extracellular matrix around synovial cells suggests the importance of alpha 6 integrin in the adhesion of synovial lining cells. Furthermore, alpha 6 expression was noticeably weaker in strongly proliferative lining cell layers, indicating that the inflammatory process may regulate integrin expression. A potential connection between altered expression of cell adhesion receptors and the pathological behavior of rheumatoid lining cells is suggested.     

Human herpesvirus 8 variants in sarcoid tissues

BACKGROUND: The cause of sarcoidosis is unknown, although mycobacteria have been implicated. We examined sarcoid tissues for human herpesvirus 8 (HHV-8) in addition to mycobacterial genomic sequences. METHODS: Biopsy samples from 17 patients with sarcoidosis were studied (eight transbronchial, 27 lymph node, two skin, and two oral mucosa). We used tissues (n = 137) from 96 patients without sarcoidosis as negative controls. A nested PCR was applied to amplify a segment of open reading frame (ORF) 26 of the HHV-8 genome, and a heminested PCR was to amplify a segment of ORF 25 of HHV-8 and of the 16 S rRNA gene of mycobacteria. Differences in base sequences of the amplified fragments were resolved with single-strand conformation polymorphism and dideoxy sequencing. FINDINGS: HHV-8 ORF 26 DNA was detected in significantly higher proportions of sarcoid than of non-sarcoid tissue samples from lung (8/8 vs 0/54; p < 0.0001), lymph nodes (26/27 vs 6/29; p < 0.0001), skin (2/2 vs 0/17; p = 0.006), and oral tissues (2/2 vs 1/13; p = 0.029). 31 (82%) of the 38 ORF 26 DNA-positive sarcoid specimens were also positive for ORF 25 DNA. For mycobacteria-like 16 S rRNA DNA, the proportion positive was significantly higher in sarcoid than non-sarcoid tissues for lymph node samples (11/27 vs 2/29; p = 0.003) but not for other tissues (lung 3/8 vs 22/54; skin 2/2 vs 15/17; and oral tissues 1/2 vs 0/13). Overall, the prevalence of HHV-8 ORF 26 sequences was higher in sarcoid tissues than in non-sarcoid tissues (p < 0.0001). When patients whose tissues were included in a masked phase of the study were treated as units of analysis, eight of eight sarcoidosis patients were positive for HHV-8 ORF 26 DNA, compared with three of 56 control patients (p < 0.0001); for mycobacteria-like sequences, three of eight sarcoidosis patients were positive, compared with four of 56 controls (p = 0.0464). The HHV-8 ORF 26 sequences, ten of which were unique, could be segregated into four groups according to peptide motifs. In seven of nine patients from whom biopsy samples were taken from various sites, different sequences were recovered. The mycobacterial sequences amplified from sarcoid tissues were also varied, but none was homologous to those of known species. INTERPRETATION: Variant HHV-8 DNA sequences are found in a wide range of sarcoid but not non-sarcoid tissues. Mycobacteria-like 16 S rRNA sequences are more frequently present in sarcoid lymph nodes and not in other tissue types, but do not indicate infection by a particular mycobacterial species.     

Expression of integrin and organization of F-actin in epithelial cells depends on the underlying surface

PURPOSE: To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS: Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS: The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS: This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.     

Expression of vascular adhesion molecules in saphenous vein coronary bypass grafts

BACKGROUND: Adhesion of blood elements to the endothelium is an important step in the development of vein graft disease. This study examines the expression of vascular adhesion molecules on explanted saphenous vein bypass grafts. METHODS: Immunocytochemical staining was performed using explanted saphenous vein grafts from 28 patients. Antibodies against the endothelial markers CD31, von Willebrand factor, intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin were used. RESULTS: Staining for CD31 and von Willebrand factor demonstrated the presence of endothelial cells in the lumen and the vasa vasorum. Expression of intercellular adhesion molecule-1 was variable between grafts, whereas vascular adhesion molecule-1 and E-selectin were almost always absent on the luminal endothelium. In contrast, the endothelium of the vasa vasorum stained positively for intercellular adhesion molecule-1 and vascular adhesion molecule-1, and was also seen on nonendothelial cells within the vessel wall. Expression of these adhesion molecules did not vary with the severity of vein graft disease. CONCLUSIONS: This study highlights the blood vessels in the adventitia as possible sites for the adhesion and migration of cells into the vessel wall.     

Expression of fibronectin and its integrin receptor alpha 5 beta 1 in canine mammary tumours

Fibronectin and its integrin receptor alpha 5 beta 1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the alpha 5 beta 1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of alpha 5 beta 1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of alpha 5 and beta 1 was diminished in metastatic tumours but there were some alpha 5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of alpha 5 beta 1 and the expression of fibronectin in canine mammary tumours.     

Expression of vascular endothelial growth factor in human gallbladder lesions

Pregnancy-induced hypertension has been suggested to be mediated by several mechanisms, including reduced nitric oxide (NO) synthesis. In this study, the effects of chronic treatment with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure and the underlying changes in vascular reactivity were investigated in virgin and late-pregnancy Sprague-Dawley rats. The systolic blood pressure was 120+/-2, 124+/-5, 116+/-4, and 171+/-5 mm Hg in untreated virgin, virgin treated with L-NAME, untreated pregnant, and pregnant treated with L-NAME rats, respectively. The rats were killed, and the thoracic aorta was cut into strips for measurement of active stress in response to alpha1-adrenergic stimulation with phenylephrine and membrane depolarization by high KCl. In pregnant rats, the maximal active stress to phenylephrine (0.31+/-0.03 x 10(4) N/m2) and the high-KCl-induced active stress (0.55+/-0.09 x 10(4) N/m2) were smaller than those in virgin rats. By contrast, in the L-NAME-treated pregnant rats, the maximal phenylephrine-induced active stress (0.76+/-0.1 x 10(4) N/m2) was greater than that in virgin rats (0.52+/-0.1 x 10(4) N/m2), whereas the high-KCl-induced active stress (1.08+/-0.14 x 10(4) N/m2) was indistinguishable from that in virgin rats (1.03+/-0.14 x 10(4) N/m2). Treatment with L-NAME did not affect the phenylephrine-releasable Ca2+ stores in pregnant rats and had minimal effect on active stress in virgin rats. Thus, reduction of NO synthesis during late pregnancy is associated with a significant increase in blood pressure and vascular responsiveness to alpha-adrenergic stimulation, which can possibly be explained in part by enhanced Ca2+ entry from extracellular space. However, other mechanisms such as increased myofilament force sensitivity to Ca2+ and/or activation of a completely Ca2+-independent mechanism cannot be excluded.     

Expression pattern of integrin beta 1 subunit in Purkinje cells of rat and cerebellar mutant mice

We found that integrin beta 1 subunit (INT beta 1)-immunoreactive Purkinje cells first appeared caudally at postnatal day (PD) 6 of rat and most Purkinje cells gradually became positive by PD 12. The expression of INT beta 1 was then suppressed in some of these cells, so that the positive Purkinje cells in the adult were organized into parasagittal bands interposed by negative cells throughout the vermis and hemispheres. When Purkinje cells were deprived of their climbing fiber innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the subsequent patterning of INT beta 1-positive Purkinje cells was not changed. In both reeler and weaver mice, the INT beta 1-positive parasagittal bands were observed, however, the Purkinje cells in the staggerer mice did not express INT beta 1 at any stage. These data suggest that the expression of INT beta 1 in Purkinje cells is genetically programmed in the developing cerebellum, and that the afferent synaptic inputs by climbing and parallel fibers are not prerequisites for INT beta 1 expression in Purkinje cells. Therefore, the unique distribution patterns of INT beta 1-positive Purkinje cells provides a new marker for postnatal development of rodent cerebella.     

Involvement of the IP-10 chemokine in sarcoid granulomatous reactions

The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in the series of events that lead to granuloma formation in sarcoidosis. In this study, we evaluated the pulmonary production of IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells. Striking levels of IP-10 were demonstrated in the bronchoalveolar lavage (BAL) fluid of 24 patients with pulmonary sarcoidosis and lymphocytic alveolitis, as compared with patients with inactive disease or control subjects. A positive correlation was demonstrated between IP-10 levels and the number of sarcoid CD45R0+/CD4+ cells in the BAL. Immunochemistry, performed with an anti-human IP-10 polyclonal Ab in lymph nodes displaying prominent sarcoid granulomas, showed that cells bearing IP-10 were mainly epithelioid cells and CD68+ macrophages located inside granulomatous areas. Macrophages recovered from the BAL of sarcoid patients stained positive for IP-10 protein. Furthermore, alveolar macrophages isolated from sarcoid patients with T cell alveolitis and cultured for 24 h in presence of IFN-gamma secreted definite levels of IP-10 capable of inducing T cell chemiotaxis. Interestingly, alveolar lymphocytes recovered from patients with active sarcoidosis were CD4+ T cells expressing Th1 cytokines (IL-2 and IFN-gamma) and high levels of CXCR3. Taken together, these data suggest the potential role of IP-10 in regulating the migration and activation of T cells toward sites of sarcoid inflammatory process and the consequent granuloma formation.     

Expression of adhesion molecules on circulating PMN during hyperdynamic endotoxemia

The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), was increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. The inhibitory capability of a specific EP. 24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of naturally-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results suggest that the binding of synthetic analogs to the enzyme may differ significantly from the binding, and thus hydrolysis, of natural peptide substrates and caution against drawing conclusions about substrate interactions with the active site from data obtained with modified peptide ligands.     

Changes in T-cell phenotype and adhesion molecules expression in psoriatic lesions after low-dose cyclosporin therapy

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).