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PubMed2005

Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons.

The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.

Adolescent↗
PubMed1996

Interaction and immunological effect of very late antigen-4, 5, and fibronectin in tonsillar lymphocytes and their relation to age.

We examined the expressions of alpha-subunits of very late antigen (VLA)-4 (alpha4) and VLA-5 (alpha5) on tonsillar lymphocytes and the interaction between these integrin receptors and their ligand, fibronectin (FN). Immunohistological and flow cytometric analyses showed that alpha4 and alpha5 were expressed in the lymphoid follicle and were positive on about 10% each of T cells and on 55% and 35% of B cells. When tonsillar B cells were separated by a discontinuous Percoll gradient, the number of alpha4- and 5-positive cells decreased as the cell density went down, while the number of activated cells went up. After in vitro activation of tonsillar B cells by Staphylococcus aureus Cowan I strain (SAC), the expression of alpha5 and the adhesiveness to FN decreased. The increased proliferation of B cells was observed when tonsillar B cells were cultured with immobilized FN. The expressions of alpha-subunits of VLA-4 and VLA-5 on tonsillar T and B lymphocytes increased with age. These results suggest that: i) B cell activation may cause decreased expressions of VLA-4 and -5, which gives a costimulatory effect on B cell activation itself again in cooperation with FN, ii) Increased expressions of VLA-4 and -5 on tonsillar lymphocytes with age may be related to regional immune response of the palatine tonsils.

Adolescent↗
PubMed1998

Beta 1 integrin-dependent binding of Jurkat cells to fibronectin is regulated by a serine-threonine phosphatase.

We investigated the effects of signaling molecule inhibitors on the expression and function of beta1 integrins in Jurkat cells. Jurkat cells expressed alpha4beta1 and alpha5beta1, with significant levels of constitutively activated beta1 integrins as assessed by labeling with mAb 15/7 that distinguishes between activation states. Adhesion to fibronectin (Fn) was mediated equally through alpha4 and alpha5 subunits, and was potentiated by the beta1 integrin activating mAb 8A2. Fn adhesion was decreased by okadaic acid through effects on both alpha4beta1, and alpha5beta1. Tyrphostin A23 also decreased adhesion but was less potent. Neither inhibitor had any effect on the surface expression of total or activated beta1 integrins. The effect of tyrphostin was completely reversed by 8A2; the effect of okadaic acid was only partially reversed. Using Calyculin A, we determined that Jurkat adhesion to Fn was regulated via protein phosphatase 1, independent of the levels of integrins or integrin activation epitopes. Activation of Jurkat cells with a CD3-stimulating mAb enhanced adhesion to Fn and was partially blocked by okadaic acid. These data demonstrate different regulatory pathways for constitutive versus activation-dependent adhesion via beta1 integrins, and implicate both tyrosine kinases and serine-threonine phosphatases in integrin function.

CD3 Complex↗
PubMed2000

Fibronectin-bound TNF-alpha stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-alpha avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-alpha complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-alpha by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-alpha into ECM-FN. FN-bound TNF-alpha (FN/TNF-alpha) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-alpha activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Cheniotaxis through ECM gels in the presence of soluble or bound TNF-alpha was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-alpha required functional activated beta1 integrins, FN/TNF-alpha-induced MMP-9 secretion was independent of binding to beta1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing nAb to alpha4, alpha5, and beta1 subunits, which blocked cell adhesion; (2) a mAb that stimulated beta1 integrin-mediated adhesion; and (3) binding TNF-alpha to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-alpha-ECM interactions may serve to limit the cytokine's availability and bioactivity to target areas of inflammation.

Animals↗
PubMed2002

A fragment of paxillin binds the alpha 4 integrin cytoplasmic domain (tail) and selectively inhibits alpha 4-mediated cell migration.

The alpha(4) integrins play important roles in embryogenesis, hematopoiesis, cardiac development, and the immune responses. The alpha(4) integrin subunit is indispensable for these biological processes, possibly because the alpha(4) subunit regulates cellular functions differently from other integrin alpha subunits. We have previously reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule, and this interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion. We also have identified a conserved 9-amino acid region (Glu(983)-Tyr(991)) in the alpha(4) cytoplasmic domain that is sufficient for paxillin binding, and an alanine substitution at either Glu(983) or Tyr(991) within this region disrupted the alpha(4)-paxillin interaction and reversed the effects of the alpha(4) cytoplasmic domain on cell spreading and migration. In the current study, we have mapped the alpha(4)-binding site within paxillin using mutational analysis, and examined its effects on the alpha(4) tail-mediated functional responses. Here we report that sequences between residues Ala(176) and Asp(275) of paxillin are sufficient for binding to the alpha(4) tail. We found that the alpha(4) tail, paxillin, and FAT, the focal adhesion targeting domain of pp125(FAK), could form a ternary complex and that the alpha(4)-binding paxillin fragment, P(Ala(176)-Asp(275)), specifically blocked paxillin binding to the alpha(4) tail more efficiently than it blocked binding to FAT. Furthermore, when expressed in cells, this alpha(4)-binding paxillin fragment specifically inhibited the alpha(4) tail-stimulated cell migration. Thus, paxillin binding to the alpha(4) tail leads to enhanced cell migration and inhibition of the alpha(4)-paxillin interaction selectively blocks the alpha4-dependent cellular responses.

Animals↗
PubMed2005

Expression of adhesion molecules on peripheral lymphocytes predicts future lesion development in MS.

The expression of adhesion molecules (alpha4beta1-integrin, LFA-1, ICAM-1) on T cells, measured by flow cytometry, was compared in different subtypes of multiple sclerosis (MS) and related to future lesion development as seen as delta T1 and T2 lesion load per year on magnetic resonance imaging (MRI). LFA-1 and alpha4beta1-integrin showed higher expression on CD4 and CD8 T lymphocytes in the secondary progressive compared to the relapsing-remitting (CD4: p<0.01, p=ns, p<0.05; CD8: p<0.001, p<0.001, p<0.001, respectively) and primary progressive MS phase (CD4: p<0.001, p<0.01, p<0.05; CD8: p<0.01, p<0.01, p<0.001, respectively). The adhesion molecule expression of alpha4- (r=0.31; p<0.05) and beta1-integrin (r=0.38; p<0.01) on CD4+ cells and of LFA-1beta on both CD4+ and CD8+ (r=0.28, p<0.05) and r=0.29; p<0.05, respectively) cells was significantly related to increase in T2 lesion load. Our study provides further evidence for the involvement of integrins in lesion development, shown as T2 lesions on MRI in MS.

Adult↗
PubMed2004

Molecular regulation of osteoblasts for tissue engineered bone repair.

The use of biodegradable polymers in medicine and biomedical research is increasing. A key growth area has been the use of these materials in tissue engineering, especially for guided regeneration of bone and cartilage. Our interest has been in determining the mechanisms by which cellular attachment and growth occurs on these materials. In the current study, we examined human osteoblast cell adhesion, growth, and morphologic changes on polymeric scaffolds composed of polylactic-co-glycolic acid and polylactic acid materials. We examined these characteristics in association with measurements of levels of key adhesion integrin receptors in the presence and absence of antibodies against alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits, and the adhesion ligand peptides RGD (Arg-Gly-Asp) and RGE (Arg-Gly-Ser). At 2 hours, results showed initial cell adhesion was considerably decreased on polylactic-co-glycolic acid and polylactic acid in the presence of the alpha2 and beta1, antibodies with a 70% adhesion rate difference observed among the groups evaluated. Higher levels of inhibition were observed on polylactic-co-glycolic acid relative to polylactic acid, which may be correlated to a higher number of cells being able to interact with the surface initially. The presence of known competitive peptide (RGD) at 2 hours, revealed its ability to block cellular adhesion to these matrices relative to the control and noncompetitive peptide RGE on polylactic-co-glycolic acid matrices. Overall adhesion rate was affected by the presence of the integrin antibodies to the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits with highest differences among polylactic-co-glycolic acid relative to its control, therefore suggesting that initial osteoblastic cell adhesion to commonly used biomaterials is regulated through integrin binding.

Biocompatible Materials↗
PubMed1998

The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells.

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10(-7) m dexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five alphaintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. alpha2, alpha5 and alphaV integrin chains showed consistent dexamethasone-induced changes in expression, while alpha3 and alpha4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.

Blotting, Western↗
PubMed1998

T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

Carbohydrate Conformation↗
PubMed1999

Modulation of integrin expression on mesotheliomas: the role of different histotypes in invasiveness.

The in vitro and in vivo integrin expression in human pleural malignant mesothelioma (MM) of three different histotypes was studied. Cell lines from MM of epithelioid (E1), fibrous (F1), byphasic histotype (B1) and normal mesothelial cells (NM) were analysed for the surface expression of alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 subunits and alphavbeta5 integrins. We found that alpha6, beta4 subunits and alphavbeta5, weakly detectable on NM cells, were expressed on MM cells. The beta3 subunit, well expressed on NM cells, was absent on MM cells. Differential expression among histotypes was observed, the MM-E1 was the least and the MM-B1 the most positive. Specimens for each MM histotype, were analysed by immunohistochemistry. The alpha6 and alphav subunits were more evident on the epithelioid histotype. Intense staining for beta3 and beta4 subunits, was found in all MM, particularly in invading cells, while the alpha5, and alphavbeta5 integrins were variously expressed. The different histotypes can affect the in vitro integrin expression and may indicate a preferential involvement of some subunits in vivo during MM tumor progression.

Humans↗
PubMed2000

Activation of mitogen-activated protein kinase through alpha5/beta1 integrin is required for cell cycle progression of B progenitor cell line, Reh, on human marrow stromal cells.

OBJECTIVE: Attachment to bone marrow (BM) stromal cells is crucial for the normal growth and development of B-cell progenitors (pro-B). However, the molecular mechanisms by which contact facilitates the proliferation of pro-B cells are not completely understood. This study was performed to investigate this interaction. MATERIALS AND METHODS: A model pro-B cell line (Reh) and a human BM stromal cell line (KM102) were used. Flow cytomery was used for cell cycle analysis. Western Blotting and immunoprecipitation were utilized to examine the levels of cyclin-dependent kinase (cdk) and p27(Kip1). RESULTS: Attachment to both KM102 and normal BM stromal cells significantly promoted the growth of Reh cells. Pretreatment of Reh cells with anti-integrin beta1 or alpha5 monoclonal antibody (mAb), but not alpha4 or ICAM-1 mAb, abrogated this enhancement of proliferation. Furthermore, stroma attachment resulted in shortening of the G(1) phase of cell cycle, significant increases cdk2 activity, degradation of cdk inhibitor p27-GST protein, and decrease in levels of p27(Kip1) protein. In addition, solid-phase cross-linking of alpha5 via immobilized antibody also resulted in extracellular signal-regulated (ERK)-2 kinase phosphorylation, increase in cdk2 activity, decrease in levels of p27(Kip1) protein, and enhanced proliferation that was inhibited by treatment with PD98059, a specific ERK inhibitor. CONCLUSION: Integrin alpha5beta1-mediated stroma contact promotes the proliferation of B-cell progenitors through the activation of ERK-2, which in turn modulates cell cycle regulation machinery including induction of cdk2 activity and degradation of p27(Kip1) and contributing to acceleration of the G(1) phase of cell cycle progression.

B-Lymphocytes↗
PubMed1997

Distribution of integrins and the extracellular matrix proteins in the baboon endometrium during the menstrual cycle and early pregnancy.

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.

Animals↗
PubMed1999

Characterization of the integrin and activation steps mediating human eosinophil and neutrophil adhesion to chronically inflamed airway endothelium.

We have used the Stamper-Woodruff frozen-section assay (FSA) to characterize the integrin and activation steps involved in adhesion of peripheral blood eosinophils and neutrophils to nasal polyp endothelium (NPE). Eosinophil and neutrophil adhesion was significantly inhibited by monoclonal antibodies (mAbs) against CD18 (beta2) and CD11a-c. Eosinophil adhesion was also inhibited to a lesser extent by mAbs against CD29 (beta1), CD49d (alpha4), and vascular cell adhesion molecule-1. The involvement of integrins raised the possibility of an activation step being involved in the adhesion process. Although stimulation of the cells with granulocyte macrophage colony-stimulating factor (GM-CSF) before the assay failed to modulate adhesion, binding was inhibited by up to 50% by treatment of the leukocytes with azide. In addition, neutrophil adhesion was completely abrogated by pertussis toxin (PT) and inhibited by about 50% by the platelet-activating factor antagonist WEB 2086 and antibodies against interleukin (IL)-8 and the two IL-8 receptors IL8RA and IL8RB (C-X-CR1 and -CR2). In contrast, eosinophil adhesion was unaffected by PT, WEB 2086, or anti-IL8R mAbs. mAbs against CCR-3, IL-3, IL-5, and GM-CSF also had no effect. This study demonstrates that eosinophil and neutrophil adhesion to NPE in the FSA conforms to the multistep paradigm for leukocyte adhesion and can be used to model the molecular basis for adhesion to endothelium in the context of chronic inflammatory disease. Using this assay, we have observed significant differences in integrin usage between eosinophils and neutrophils and a striking difference in the mechanism of integrin activation. These differences could explain, in part, the preferential accumulation of eosinophils in diseases such as asthma.

CD11 Antigens↗
PubMed1998

Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.

Animals↗
PubMed1998

Divalent cations and ligands induce conformational changes that are highly divergent among beta1 integrins.

Here we show striking differences in conformational regulation among beta1 integrins. Upon manganese stimulation, a beta1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha4beta1), moderately (on alpha5beta1), weakly (on alpha2beta1), or was scarcely detectable (on alpha6beta1 and alpha3beta1). Comparable results were seen for the beta1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha4beta1), moderate (alpha5beta1), weak (alpha2beta1, alpha6beta1), or minimal (alpha3beta1) induction of the 9EG7 epitope. Exchange or deletion of alpha chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), except that EGTA reduced rather than induced 9EG7 expression on alpha4beta1. Thus in contrast to other beta1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha4beta1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha4beta1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha4beta1 was eliminated by a D698E mutation in alpha4, suggesting that Asp-698 may play a key role in maintaining atypical alpha4beta1 response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha4beta1 > alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), (iii) allow us to hypothesize that beta1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha4beta1 response to calcium that requires alpha4 Asp-698.

Amino Acid Sequence↗
PubMed1996

Widespread expression of beta1 integrins in the developing chick retina: evidence for a role in migration of retinal ganglion cells.

During extension of axons, critical neuronal interactions with extracellular matrix (ECM) and other cells are thought to be mediated in part by heterodimeric beta1 integrin receptors. In this report, we examine the expression and function of beta1 integrins in the developing chick retina. Expression of the beta1 subunit, assayed by in situ hybridization and antibody staining of dissociated cells, was widespread in undifferentiated neuroepithelial cells, before the initiation of axons. Expression persisted in most retinal cell layers throughout embryonic development, during and after axon extension. The repertoire of beta1-associated alpha subunits was examined using reverse transcription-polymerase chain reaction. In addition to the alpha6 and alpha8 subunits previously reported, chick homologues of the alpha2 and alpha4 subunits were detected. Developmental Northern blots revealed varying patterns of integrin subunit expression and showed that expression of beta1 and the mRNAs of its associated alpha subunits are not always coregulated during retinal development. The timing and distribution of expression suggested that beta1 integrins may be involved in other developmental events in addition to axon extension. To address functions carried out by beta1 integrins in the early retina, explanted eye cups were incubated in the presence of function blocking anti-beta1 antibody and migration of newly born retinal ganglion cells (RGCs) was assessed. RGC migration from the ventricular zone to the vitreal border was significantly inhibited, suggesting that beta1 integrins play a role in neuroblast migration in the retina.

Amino Acid Sequence↗
PubMed1998

The lymphocytic alveolitis in sarcoidosis is associated with increased amounts of soluble and cell-bound adhesion molecules in bronchoalveolar lavage fluid and serum.

OBJECTIVES: Pulmonary sarcoidosis is a chronic inflammatory disorder of unknown aetiology accompanied by a lymphocytic alveolitis. It is likely that a selective and temporal expression of adhesion molecules plays a crucial role in the recruitment of cells to the inflammatory site. We investigated the expression of adhesion molecules on alveolar T-lymphocytes and in bronchoalveolar lavage (BAL) fluid and serum to elucidate mechanisms behind the accumulation of cells in the lung in sarcoidosis. DESIGN: In a cross-sectional study in patients with active and inactive sarcoidosis and in healthy volunteers, we examined, in serum and in BAL fluid, the soluble adhesion molecules, VCAM-1, ICAM-1, and P-, E- and L-selectin. In addition, the expressions of alpha4-beta1 (VLA-4) and alpha5-beta1 (VLA-5) integrins on alveolar T-lymphocytes were analysed. SETTING: The subjects attended the outpatient clinic at the Division of Respiratory Medicine, Karolinska Hospital, Stockholm, Sweden. SUBJECTS: Nineteen sarcoidosis patients, nine with clinically active disease, and 13 healthy volunteers were included in the study. The sarcoidosis diagnosis was based on a typical histological and/or clinical (symptoms, radiograph, lung function) picture. RESULTS: In sarcoidosis patients, particularly in those with active disease, an increase of the expressions of beta1-integrins was accompanied by elevated concentrations in BAL fluid of soluble VCAM-1. In serum, the levels of E-selectin and ICAM-1 were significantly higher in patients with active disease than in those with inactive disease and controls. CONCLUSIONS: The findings offer some mechanistic explanations as to how the cell-rich alveolitis in sarcoidosis occurs, and furthermore suggest additional markers, such as s-ICAM-1, for assessment of disease activity.

Adult↗
PubMed2000

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells.

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.

Antibodies, Monoclonal↗