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We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Purpose: Bacillus Calmette-Guerin (BCG) binds to the tumor cell as a result of mycobacterial receptors for fibronectin (FN). Cell surface bound FN serves as a bridge through which BCG attaches to the tumor cell. Despite the importance of FN studies have demonstrated an idiosyncratic decrease in BCG adherence in response to exogenous FN. We evaluated the effect of exogenous and autocrine FN on the ability of BCG to adhere to the tumor cell surface and initiate cellular signaling., Materials and Methods: BCG adherence to parental 253J and FN over expressing 253JTGFbeta1-8 cells as well as to the intrinsic FN expressing cell line 647V was quantified using green fluorescent protein-BCG. Experiments were performed to assess the effect of FN on BCG initiated signal transduction through nuclear factor kappaB and AP1. Finally, the integrity of the BCG activated signaling pathway in transforming growth factor-beta1/FN over expressors was assessed using antibody mediated cross-linking of the FN receptor., Results: BCG adherence was decreased in cell lines with high autocrine expression of FN. Exogenous FN prevented BCG induced transactivation of nuclear factor kappaB and AP1 reporter constructs. No BCG stimulated signaling to these reporters could be detected in FN over expressing 253J cells. NonFN dependent alpha5beta1 cross-linking initiated signal transduction in FN over expressing cells., Conclusions: We propose that by saturating cellular and BCG receptors excess FN expression decreases the ability of cellular or mycobacterial bound FN to bind vacant receptors on BCG or on the cell. Excess FN inhibits BCG adherence and BCG initiated signal transduction.

Integrins of the very late antigen (VLA) family mediate leucocyte traffic to lymphoid organs under physiological conditions and in chronic inflammatory situations such as autoimmunity. Accordingly, the current thinking is of a positive correlation between VLA expression and capability of the generation of autoimmunity. Herein we discuss recent findings on the defective expression of integrin-type fibronectin receptors alpha4beta1 (VLA-4) and alpha5beta1 (VLA-5) in the non-obese diabetic (NOD) mouse, a murine model of autoimmune insulin-dependent diabetes mellitus. As compared with normal animals, NOD thymocytes (including the CD4+CD25+ regulatory T cells) exhibit a decrease in the membrane expression of alpha5beta1, resulting in a functional impairment of fibronectin-mediated interactions, including cell migration. Interestingly, thymocytes that are trapped within the giant perivascular spaces seen in NOD thymus are consistently alpha5beta1 negative, suggesting that the progressive arrest of mature cells can be related to the alpha5beta1 defect. Peripheral T cells also exhibit decreased alpha5beta1 membrane expression and impaired fibronectin-driven migration. Additionally, we observed a defect in alpha4beta1 fibronectin receptor expression in NOD macrophages. Peritoneal, bone marrow-derived-precursor, as well as thymic macrophages of NOD mice showed an impaired upregulation of alpha4-integrin chain expression, dependent on the level of macrophage maturation. Overall these data lead to the notion that NOD leucocytes bear distinct fibronectin receptor-mediated cell migration defects, which may be involved in the pathogenesis and/or pathophysiology of the autoimmune events seen in NOD mice. Further studies will be helpful to define whether or not this concept can be applied for other autoimmune diseases.

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Adhesion molecules on respiratory epithelial cells play a critical role in inflammatory cell recruitment and accumulation at sites of inflammation. Bordetella pertussis colonizes the human respiratory tract by infecting epithelial cells, leading to an inflammatory response. In this study, the role of bacterial factors in the expression of intercellular adhesion molecule-1 (ICAM-1) on human respiratory epithelial cells was investigated in response to B. pertussis. Flow cytometry and real time RT-PCR analysis showed that BEAS-2B human bronchial epithelial cells expressed increased levels of ICAM-1 mRNA and surface protein in response to B. pertussis infection. Filamentous hemagglutinin (FHA) played a role in this response because of the impaired capability of a FHA-deficient isogenic strain. A mutant strain in which an Arg-Gly-Asp (RGD) site of FHA had been changed to Arg-Ala-Asp had diminished ability to up-regulate ICAM-1 expression. RGD sequence-associated up-regulation of ICAM-1 expression was also observed in primary normal human bronchial epithelial cells. Pretreatment of cells with integrin antagonists such as RGD-containing peptide and antibody against very late antigen-5 (VLA-5) inhibited the up-regulation of ICAM-1 expression, suggesting the participation of VLA-5 integrin in this response. Pertussis toxin (PT) prevented the up-regulation of ICAM-1 expression because a PT-deficient mutant strain induced higher levels of ICAM-1 mRNA and surface protein than the parental strain. Consistent with this, purified PT suppressed the up-regulation of epithelial ICAM-1 expression. These findings demonstrate that B. pertussis FHA up-regulates ICAM-1 expression on respiratory epithelial cells through interaction of its RGD site with host cell VLA-5 integrin, and that PT impairs this response.

Objective: Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors., Methods: Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis., Results: Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the alpha5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the alpha5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 microg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of alphav and alpha1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of alpha5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to alpha5 inhibition was associated with apoptosis., Conclusion: These results demonstrate that chondrocyte survival signals are transmitted via the alpha5beta1 FN receptor. Inhibition of matrix survival signals mediated by alpha5beta1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from alpha5beta1.

Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-alpha and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-alpha nor IL-5, a known eosinophil-priming agent. Priming caused activation of alpha(M)beta(2) integrin; mAb against either the common beta(2) integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against beta(1) or alpha(5), but not alpha(4) integrin subunit, together with anti-beta(2) integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking alpha(5)beta(1) integrin increased alpha(M)beta(2) integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of alpha(5)beta(1) integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for alpha(5)beta(1) integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-beta(2) integrin mAb, reduced adhesion to basal levels. In conclusion, alpha(5)beta(1) integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-alpha, potentiated C5a activation of alpha(M)beta(2) integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for alpha(5)beta(1) integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.

Tumor necrosis factor-alpha (TNF-alpha) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-alpha. TNF-alpha also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-alpha treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in alpha(5)beta(1) or total beta(1) integrin expression on the surface of the CPAE cells after TNF-alpha. Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of beta(1)-integrin function since the beta(1)-integrin blocking antibody mAb 13 significantly (P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-alpha-treated cells. In addition, antibodies which activate beta(1) integrins restored (P < 0.05) adhesion of TNF-alpha-treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-alpha-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of (125)I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-alpha was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-alpha. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-alpha via a non-beta(1)- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.

It has been hypothesized that Candida albicans possesses integrin-like receptors on its cell surface. This is because C. albicans binds numerous fluid-phase extracellular matrix (ECM) proteins on its cell surface and adheres to the same ECM proteins when immobilized. In addition, numerous antibodies to human integrins (receptors for ECM proteins) bind to the fungal cell surface and in so doing inhibit the binding of the respective proteins. To demonstrate the presence of such a cell surface integrin, a cDNA library of C. albicans yeast cells was screened with polyclonal antiserum to the human fibronectin receptor (alpha5beta1 integrin). Clones isolated by this screening technique also reacted specifically to antiserum against the human vitronectin receptor (alpha(v)beta3 integrin). DNA sequence analysis of the cloned insert predicted a 350 aa protein (37 kDa). This predicted protein showed 75% homology at the nucleotide sequence level to alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae. In vitro transcription/translation of the cloned inserts yielded a 37 kDa protein that was immunoprecipitated with antibodies to the alpha5beta1 and alpha(v)beta3 integrins and an antibody to a C. albicans fibronectin receptor. These antibodies and an mAb to the human vitronectin receptor demonstrated an antigen of -37 kDa present in the cell-wall preparations of C. albicans and in spent growth medium. All four antibodies reacted with authentic ADH. The possible significance of these results in relation to C. albicans adherence is discussed.

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)-rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP-inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking-antibodies to the FN receptor alpha 5 beta 1 integrin and by the proline analog 3,4-dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up-regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up-regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin alpha 5 beta 1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN., (Copyright 2001 Wiley-Liss, Inc.)

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against alpha5beta1 integrins or by a peptide (GRGDSP) that binds to beta1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of beta1 or beta2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, alpha5beta1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by alpha5beta1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human alpha5beta1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell alpha5beta1 integrin., (Copyright 2001 Academic Press.)

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.

MCF-7 breast cancer cells grow as adherent cells, but following overexpression of protein kinase C-alpha these cells (MCF-7-PKC-alpha cells) become anchorage-independent and exhibit increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells are also sensitized to apoptosis in response to phorbol ester but not serum starvation. Flourescence-activated cell sorting revealed that several integrin subunits were down-regulated in MCF-7-PKC-alpha cells, however, the fibronectin receptor alpha 5 beta 1 was upregulated. MCF-7-PKC-alpha cells growing under non-adherent conditions underwent cell death when antibodies to alpha 5 beta 1 were added to growth media lacking serum but not when serum was present. Addition of soluble fibronectin to cells incubated without serum suppressed apoptosis triggered by anti-alpha 5 beta 1 antibodies but not by phorbol esters. MCF-7-PKC-alpha cells also were shown to express more fibronectin on their cell surface than MCF-7V cells (MCF-7 cells transfected with pSV(2)M(2)6 vector only). This study indicates that the survival of MCF-7-PKC-alpha cells under non-adherent conditions in the absence of serum results from the ligation of alpha 5 beta 1 with surface-bound fibronectin, which may account, in part, for the increased aggressiveness of these cells.

Previously, experimental in vivo results showed that the productively and persistently human cytomegalovirus (HCMV)-infected neuroblastoma cell line UKF-NB-4AD169 exhibits a more malignant phenotype than the non-infected variant UKF-NB-4. To prove the assumption that enhanced malignancy may be due to enhanced invasive potential of the infected cells we studied interactions of both lines with monolayers of cultured endothelial cells. UKF-NB-4AD169 cells adhered to and transmigrated through endothelial monolayer to a significantly higher extent compared with UKF-NB4. Furthermore, the adhesion of UKF-NB-4AD169 but not of UKF-NB4 resulted in focal disruption of the monolayer integrity which facilitates tumor cell transmigration. Blocking antibodies directed against the beta1 integrin chain as well as beta1alpha5 on the tumor cells specifically inhibited adhesion in a concentration-dependent manner. When UKF-NB-4 were pretreated with a beta1 integrin activating antibody, focal disruption of the endothelial integrity also occurred. These findings lead us to suggest that HCMV infection activates beta1alpha5 in the host neuroblastoma cell which in turn enables these cells to tightly adhere to endothelial cells. In the presence of the protease inhibitor phenantroline, beta1alpha5-mediated adhesion was not impaired whereas UKF-NB4AD169-mediated endothelial monolayer permeabilization was dose dependently inhibited. We conclude that human cytomegalovirus infection contributes to augmented neuroblastoma invasiveness via adhesion of activated beta1alpha5 and subsequent matrix digestion by proteases.

Recent data have revealed the involvement of the alpha(v)beta(3) integrin in angiogenesis. However, few studies to date have provided a convincing role for this receptor in in vitro assays of endothelial cell morphogenesis where defined steps can be examined. Here, we present data showing that two integrins, alpha(v)beta(3) and alpha(5)beta(1), regulate human endothelial cell vacuolation and lumen formation in three-dimensional fibrin matrices. Cells resuspended in fibrin formed intracellular vacuoles that coalesced into lumenal structures. These morphogenic events were completely inhibited by the simultaneous addition of anti-alpha(v)beta(3) and anti-alpha(5) integrin antibodies. Complete blockade was also accomplished with a combination of the cyclic Arg-Gly-Asp (cRGD) peptide and anti-alpha(5) integrin antibodies. No blockade was observed with the control Arg-Gly-Glu (RGE) peptide alone or in combination with control antibodies. Finally, we were able to demonstrate regression of vacuoles and lumens several hours after the addition of cRGD peptides combined with anti-alpha(5) integrin antibodies. These effects were not observed with control peptides alone or in combination with control antibodies. We report here the novel involvement of both the alpha(v)beta(3) and alpha(5)beta(1) integrins in vacuolation and lumen formation in a fibrin matrix, implicating a role for multiple integrins in endothelial cell morphogenesis.

The integrins are protein heterodimers consisting of noncovalently associated alpha and beta subunits. The adhesive interactions mediated by integrins are necessary for cellular survival and proliferation. In this study we investigated the effects of three different integrin antibodies on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Human Tenon's capsule fibroblasts were cultured into 96 well plates and treated with different concentrations (ranging from 10(-6) to 1 microg ml(-1)) of three different integrin antibodies: human integrin alpha-2 antibody, human integrin alpha-3 antibody and human integrin alpha-5/FnR (fibronectin receptor) antibody. Coulter counter, hexosaminidase, and 3H-thymidine assays were used to determine the inhibitory effects of these integrin antibodies on ocular fibroblasts on days 0 (attachment), 1,3 and 7 following antibody treatment. The concentration of each antibody required to produce a proliferation 50% less than the control (ID50) was calculated for each assay. With respect to attachment, all three antibodies studied displayed some inhibitory activity. All three antibodies also displayed dose-dependent antiproliferative properties, especially at the highest concentration tested after 7 days of exposure. The integrin alpha-2 antibody was the most potent of the inhibitors, followed by the integrin alpha-3 antibody, with the integrin alpha-5 antibody being the least potent antibody tested. In addition, the anti-proliferative activities of the integrin alpha-2 and integrin alpha-3 antibodies increased with increasing incubation time. In conclusion, these integrin antibodies demonstrated some inhibitory effects on the attachment and proliferation of human Tenon's capsule fibroblasts in culture. Further investigation will be required to determine whether integrin antibodies can significantly limit scar formation in vivo without significant toxicity.

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors alpha 5 beta 1 and alpha v beta 3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of alpha 5 beta 1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by alpha 5 beta1 integrin ligation is mediated via induction and autocrine production of IL-1 beta, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, alpha v beta 3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1 beta, NO, and PGE2 (triggered by alpha 5 beta 1, IL-1 beta, IL-18, or IL-1 beta, TNF-alpha, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated "outside in" and "inside out" signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.

The expression of alphavbeta6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of alphavbeta6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed alphavbeta6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of alphavbeta6 integrin. In addition to alphavbeta6, all 3 cell lines expressed alpha5beta1 and alphavbeta1 fibronectin receptor integrins. Function-blocking experiments with inhibitory anti-integrin antibodies showed that all these three integrins were functional in SCC cell spreading on fibronectin. Integrin alphavbeta6, however, was not used as a primary but as an alternative fibronectin receptor by SCC cells, as the inhibitory anti-beta6 integrin antibody alone had no effect on spreading. In migration, however, alphavbeta6, alpha5beta1, and alphavbeta1 integrins were all used in cooperation. The presence of alphavbeta1 integrin in SCC cells is a novel finding as is its contribution to SCC cell migration. When one or two of these three receptors were blocked, the cells demonstrated an adaptive ability to remain migratory using integrins that were not targeted by antibodies. Utilization of a combination of receptors of different affinities may be beneficial for SCC cell migration versatility., (Copyright 2000 Academic Press.)

The arrector pili (AP) muscle is a small band of smooth muscle that attaches proximally to the bulge area of the pilosebaceous apparatus in the reticular dermis and extends up toward the epidermis. The distal anatomy of the AP and the anchorage mechanism allowing hair erection have not been previously described. Integrins are likely candidates mediating this attachment. Immunohistochemical techniques were used to determine the distribution of the following integrins: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 as well as fibronectin. Frozen human scalp tissue was sectioned in traditional planes, obliquely and horizontally to visualize microanatomy in three dimensions. Histological examination revealed that the distal portions of smooth muscle fibers splay extensively between collagen bundles of the upper dermis. Integrin subunits alpha1, alpha5 and beta1 were expressed by the AP muscle. Analysis of the relative density of immunoreactivity in digitized sections revealed increased alpha5 subunit expression at the extracellular matrix (ECM)-muscle interface. These data suggest that anchorage of the AP muscle to the ECM is via alpha5beta1 integrin and alpha1beta1 integrin functions in muscle cell-cell adhesion. Extensive splaying of smooth muscle fibers may allow increased surface area contact between the ECM and smooth muscle cells expressing peripherally situated alpha5 integrin.

We have found previously that disaccharides (DS) enzymatically generated from heparin or heparan sulphate can modulate tumour necrosis factor-alpha (TNF-alpha) secretion from immune cells in vitro and cell-mediated immune reactions in vivo. Here, we show that such DS can modulate the adhesion and migration of human T cells. We found that certain heparin- and heparan sulphate-derived DS induced, in a dose-dependent manner, the adhesion of human T cells to both extracellular matrix (ECM) and immobilized fibronectin (FN); maximal T-cell adhesion occurred with 1 ng/ml of DS. The levels of T-cell adhesion to ECM that were induced by the tested DS molecules resembled those induced by the prototypic chemokine, macrophage inflammatory protein 1beta (MIP-1beta). However, the kinetics of DS-induced T-cell adhesion to FN resembled that induced by phorbol myristate acetate (PMA), but not that induced by MIP-1beta. This adhesion appeared to involve beta1 integrin recognition and activation, and was associated with specific intracellular activation pathways. Although a first exposure of T cells to certain DS molecules appeared to result in cell adhesion, a subsequent exposure of T cells to pro-adhesive chemokines, such as MIP-1beta or RANTES, but not to other pro-adhesive stimuli, for example interleukin-2 or CD3 cross-linking, resulted in inhibition of T-cell adhesion to and chemotactic migration through FN. Hence, we propose that the breakdown products of tissues generated by inflammatory enzymes are part of an intrinsic functional programme, and not necessarily molecular waste. Moreover, because the DS molecules exert their modulatory functions within a limited time, it appears that the historical encounters of the tissue-invading cells with the constituents of inflamed loci may dictate the cells' behaviour upon subsequent exposure to proinflammatory mediators.

The high-affinity interaction of integrin alpha5beta1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha5beta1, identified by phage display, which blocks alpha5beta1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha5 were replaced with the corresponding residues in mouse alpha5 by site-directed mutagenesis; wild-type or mutant human alpha5 was expressed on the surface of alpha5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha5beta1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.

Purpose: We investigated the interactions of hyaluronan with extracellular matrix proteins (fibronectin, laminin, and type IV collagen) on corneal epithelial migration by using an organ-culture system of rabbit corneas., Methods: Rabbit corneal blocks were cultured in the absence or presence of various reagents for a total of 20 h. The corneal blocks were then fixed, dehydrated, embedded in paraffin, and stained by hematoxylineosin, and the length of the path of epithelial migration was measured., Results: The addition of hyaluronan, fibronectin, or type IV collagen alone stimulated epithelial migration in a dose-dependent manner, but laminin did not. When corneal blocks were pretreated with extracellular matrix proteins (fibronectin, laminin, or type IV collagen) for 5 h and then were cultured for 15 h with hyaluronan, only pretreatment with fibronectin demonstrated a synergistic effect with hyaluronan on corneal epithelial migration. In contrast, when corneal blocks were pretreated with hyaluronan for 5 h and then cultured for 15 h with extracellular matrix proteins, the additive effects of hyaluronan with fibronectin or hyaluronan with type IV collagen were observed. The synergistic effects of hyaluronan and fibronectin on corneal epithelial migration were partially inhibited by the addition of antiserum against fibronectin. In contrast, the addition of antiserum against fibronectin receptor completely inhibited the synergistic effects of hyaluronan and fibronectin on epithelial migration., Conclusion: These results suggest that hyaluronan and fibronectin have a synergistic effect, with fibronectin pretreatment augmenting hyaluronan-stimulated corneal epithelial migration.

Bordetella pertussis interacts with very-late antigen-5 (VLA-5) receptors on the human monocyte resulting in cross-linking of these receptors followed by activation of complement receptor 3 (CR3) and firm adhesion of B. pertussis to these monocytes. In the present study we investigated whether protein tyrosine kinases are involved in the activation of CR3 on monocytes, which was assessed by the binding of C3bi-coated erythrocytes (EC3bi). Pre-incubation of monocytes with tyrphostin-A47, a specific protein tyrosine kinase inhibitor, before adherence of the cells to an anti-VLA-5 monoclonal antibody-coated surface, or addition of tyrphostin-A47 within 10 min of the adherence to such surface, reduced the binding of EC3bi to monocytes significantly. Pre-incubation of monocytes with tyrphostin-A47 reduced the binding of B. pertussis to such monocytes as well. Inhibitors of protein kinase A and/or C had no effect on EC3bi binding to monocytes. Cross-linking of VLA-5 on monocytes resulted in tyrosine phosphorylation of several proteins. Together, these results indicate that protein tyrosine kinases are involved in the VLA-5-induced activation of CR3 on human monocytes.

Vitronectin (VN) is an extracellular matrix (ECM) protein, the synthesis of which in vivo by glioma cells correlates with tumor grade. Although the role of VN as a permissive substrate for glioma migration has been well characterized, its role in conferring a survival advantage for tumor cells has not been addressed previously. By using an in vitro assay of DNA fragmentation as a quantitative measure of apoptotic cell death, we sought to determine whether the sensitivity of two human glioma cell lines (D54 and U251) to drug-induced apoptosis could be inhibited by VN. As well, the extent to which apoptosis could be inhibited was correlated with the levels of the Bcl-2 family of proteins that are known to modulate apoptosis and chemoresistance. Results of the study were: (a) VN coatings, in a dose-dependent manner, inhibited topoisomerase (Topo)-induced apoptosis by up to 50% (optimal coating density, 500 ng/cm2); in contrast, fibronectin (FN), an ECM protein present in abundance in the brain, demonstrated no protection; (b) in a dose-response study, VN clearly conferred a survival advantage (LD50 of Topo: on VN, 120 ng/ml; on FN, 35 ng/ml); (c) the protective effect of VN was not due to enhanced cell adhesion or alterations in the cell cycle distribution; (d) both of the classic integrin receptors that bind VN (alpha(v)beta3, alpha(v)beta5) were capable of mediating this protective effect, because ligation of either of the two classic integrins conferred chemoresistance to Topo; and (e) chemoresistance observed with VN was associated with an increase in expression of two antiapoptotic proteins, Bcl-2 and Bcl-X(L), with a consequent increase in the ratios for Bcl-2:Bax and Bcl-X(L):Bax. VN, an ECM protein preferentially expressed at the tumor-brain interface in vivo, may confer a survival advantage to glioma cells at the advancing tumor margin and may thus, in part, underlie the high level of tumor recurrence at this interface.

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.

The mechanism whereby some lymphomas invade liver extensively has not been fully investigated. There is no basement membrane under the sinusoidal endothelium of the liver, and hepatocytes produce fibronectin (FN); therefore, adhesion to this FN may be particularly important for liver infiltration by lymphoma cells. A mouse lymphoma cell line, RL-male1, adhered to FN. However, this cell line did not express classical FN receptors such as very late antigen (VLA)-4 and VLA-5, as estimated by immunofluorescent staining. We have generated monoclonal antibodies (mAbs) that inhibit adhesion of RL-male1 cells to FN. Western blot and immunoprecipitation analyses showed that the new mAbs recognize a protein with an approximate molecular weight of 55,000 (p55). This antigenic protein was highly purified by immunoprecipitation and processed for microsequencing. From NH2-terminal sequence results, the p55 antigen was not identical to known FN receptors. Radioisotope-labeled RL-male1 cells, when injected i.v. into mice, rapidly infiltrated the liver (30-35% of injected cells), as measured by a gamma counter. Intravenous injection of the new mAbs partially (20%) blocked the infiltration of i.v.-injected lymphoma cells into the liver, whereas control rat IgG and an anti-CD11a mAb did not. These results demonstrate that the mouse lymphoma cell line RL-male1 nses a novel FN receptor for liver infiltration.

Metargidin (ADAM-15) is a type I transmembrane glycoprotein belonging to the ADAM (A Disintegrin and Metalloprotease Domain) family of proteins and is widely expressed in different tissues and cell types. Members of this family contain an amino-terminal metalloprotease domain followed by a disintegrin domain, a cysteine-rich region and a membrane proximal EGF-like domain. The disintegrin domain of metargidin contains an RGD tripeptide sequence, suggesting that it may potentially interact with the integrin family of proteins. Here we identify integrin ligands for metargidin on haemopoietic cells, by using a chimeric protein containing the extracellular domain of metargidin fused to the Fc portion of human IgG. Binding activity to a panel of human cell lines was analysed by solid-phase cell-adhesion assays. Metargidin bound to a monocytic cell line, U937, and a T cell line, MOLT-4, in a specific manner. Adhesion was divalent cation- and temperature- dependent and strongly enhanced by Mn2+, all features of integrin-mediated binding. Using a panel of anti-integrin antibodies we show that alphavbeta3 is a ligand for metargidin on U937 cells. In contrast, for MOLT-4 cells, the integrin alpha5beta1 contributes to cell binding. Adhesion was mediated by the disintegrin domain of metargidin as RGD-based peptides inhibited cell binding to both cell lines. The specificity of the interaction between both alphavbeta3 and alpha5beta1 and metargidin was further confirmed by solid-phase adhesion assays using purified recombinant integrins. These results together indicate that metargidin can function as a cell adhesion molecule via interactions with alphavbeta3 and alpha5beta1 integrins.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

During human thymic differentiation, interactions between fibronectin (Fn)/beta1 integrins and hyaluronan (HA)/RHAMM control motility and Fn/beta1 integrins mediate spontaneous Fn-dependent adhesion. Multinegative (MN, CD3-4-8-) thymocytes exhibit strong spontaneous adherence to Fn (75%) that was efficiently inhibited by anti-alpha5beta1 and only weakly inhibited by anti-alpha4beta1. The relatively weak adherence of unfractionated thymocytes to Fn required both alpha4beta1 and alpha5beta1. Video time-lapse microscopy indicates that a subset of thymocytes also undergo spontaneous Fn-dependent motility mediated by alpha5beta1, alpha4beta1, and the HA-receptor RHAMM, but not by CD44. The loss of motility after hyaluronidase treatment of thymocytes indicated that motility is strongly dependent on HA. Of motile cells, 55% were DP, 19% were DN, and 24% were CD4+SP, but only 1% were CD8+SP. Overall, for MN thymocytes, beta1 integrin mediated Fn-adhesion, but after expression of CD4/CD8, beta1 integrins mediated Fn-dependent motility. Treatment with the activating anti-beta1 mAb QE.2E5 inhibited thymic motility and converted otherwise nonadherent thymocytes to an adherent state. High-avidity interactions via integrins appear to supercede the motogenicity of RHAMM and HA, suggesting that integrin avidity may regulate RHAMM. During thymic development, changes in adhesion or motility appear to be mediated by integrin avidity modulation.

The potential for leukocyte-mediated host tissue damage during resolution of inflammatory responses is influenced by the rate at which extravasated apoptotic leukocytes are cleared from inflammatory sites. Regulation of macrophage capacity for clearance of apoptotic granulocytes is likely to be an important factor determining whether inflammation ultimately resolves or progresses to a chronic state. In this study we have investigated the molecular basis for rapid augmentation of macrophage phagocytosis of apoptotic neutrophils, which was observed following macrophage adhesion to fibronectin. We used a combination of monoclonal antibodies, blocking peptides, and recombinant fibronectin fragments to investigate the role of beta1 integrins in mediating the fibronectin effects. Blockade of alpha5beta1 or alpha4beta1 alone did not attenuate fibronectin-augmentation of phagocytosis. In addition, adhesion of macrophages to recombinant fibronectins lacking alpha4beta1 recognition motifs failed to promote phagocytosis of apoptotic neutrophils. Our results would be consistent with a model in which multiple fibronectin receptors, including beta1 integrins, act co-operatively to augment macrophage phagocytic responses. Together, these data suggest that the extracellular matrix environment of macrophages may provide regulatory signals that act indirectly to rapidly alter the potential for removal of apoptotic cells and influence the process of resolution of inflammation.

Trophoblast growth and invasion of the uterus are tightly regulated by locally produced factors. Since insulin-like growth factor (IGF)-II is produced by the invasive human extravillous trophoblast (EVT) cells and IGF-binding protein (IGFBP)-1 by the decidual cells in situ that are in proximity to each other, we examined the possible influence of these molecules on proliferation, migration, and invasiveness of first-trimester EVT cells in culture. These EVT cell functions were respectively measured by 3H-TdR uptake, in vitro migration, and invasion assays. Secretion of invasion-associated enzymes was assessed by zymography, and IGF-binding moieties on the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cells was stimulated by addition of serum but unaffected by a wide range of IGF-I, IGF-II, and IGFBP-1 concentrations. IGF-II and IGFBP-1 or their combination stimulated EVT cell invasiveness and migration, when assays were conducted in serum-reduced media. Affinity cross-linking studies failed to detect the type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules including type 2 IGF receptor were identified on the EVT cell surface. IGF-II enhancement of invasion was unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVT cell invasion, indicating that type 1 IGF receptor was not responsible for the IGF-II effects. Secretion of gelatinases or plasminogen activators was unaltered by IGF-II or IGFBP-1. We conclude that trophoblast-derived IGF-II and decidua-derived IGFBP-1 provide autocrine/paracrine enhancement of trophoblast invasiveness largely by stimulating migration, an essential step in invasion., (Copyright 1998 Academic Press.)

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alphavbeta3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha5, beta1, and alpha5/beta1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alphavbeta3, anti-alphav, anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha5beta1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.

Dendritic cells (DC) are migratory cells which exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). The underlying mechanisms involved in these processes are still ill defined. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and ECM. DC were differentiated from monocytes by in vitro exposure to granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days. In adhesion assays a considerable proportion of DC bound to resting EC monolayers: (17% +/- 4%, mean +/- SE of eight experiments). Adhesion to tumor necrosis factor (TNF)-activated EC was increased to 29% +/- 5% (n = 8). Binding to resting EC was strongly inhibited by anti-CD11a and CD11b, but not by CD11c monoclonal antibodies (MoAbs); on TNF-activated EC, anti-VLA-4 in concert with anti-CD18 inhibited adhesion by more than 70%. Binding to a natural ECM, derived from cultured EC, or to purified fibronectin was high: 52% +/- 6% (n = 8) involved VLA-4 and VLA-5 integrins. In a transmigration assay, 10% +/- 2% (n = 6) of input cells were able to cross the EC monolayer. Unlike adhesion, transendothelial migration was significantly reduced by anti-CD31 MoAb. The amount of DC transmigrated through a monolayer of EC was increased twofold to threefold by a defined set of C-C chemokines including RANTES, MIP1alpha, MIP5, and, to a lesser extent, by MIP1beta and MCP-3. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC (13% +/- 4% of input cells seeded) was able to migrate across the endothelial basement membrane and, subsequently, across the endothelial barrier (reverse transmigration). The adhesion molecules and chemoattractants characterized herein are likely to underlie the complex trafficking of DC in vivo.

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.

Entamoeba histolytica trophozoites do interact with extracellular matrix components in order to invade and finally destroy tissue. An important step in this interaction involves the binding of a 140-kDa membrane protein that binds to fibronectin. The similarity of this amoebic receptor to fibronectin receptors from higher eukaryotic cells was defined by indirect immunofluorescence, western blot and immunohistochemistry, using polyclonal monospecific antibodies raised against the amoebic protein. These results suggest that lower eukaryotic cells have and use a beta 1 integrin-like molecule as well as mechanisms similar to those present in higher eukaryotic cells during interaction with extracellular matrix components.

Objective: To investigate factors regulating fibronectin fibrillar matrix formation by synovial fibroblasts., Methods: Basal and cytokine stimulated extracellular matrix (ECM) fibronectin produced by synovial fibroblasts was identified by immunofluorescence and Western blot. Alternative mRNA splicing of fibronectin was studied by reverse transcription polymerase chain reaction. The integrin receptor responsible for supporting fibronectin fibrillar matrix was identified by blocking antibodies and receptor levels studied by Western blot., Results: Transforming growth factor-beta (TGF-beta) or platelet derived growth factor (PDGF), but not interleukin 1 or exogenous fibronectin, induced ECM fibronectin. ECM fibronectin was blocked by the addition of antibody to the alpha5beta1 integrin. Cytokines did not significantly change alternative mRNA splicing of fibronectin or levels of alpha5beta1 integrin expression., Conclusion: Synovial cell production of a fibrillar fibronectin matrix is induced by growth factors, including TGF-beta and PDGF. This induction is mediated by the alpha5beta1 integrin. Since fibrillar fibronectin formation was not strongly dependent on increased fibronectin or alpha5beta1 integrin levels, this effect may be mediated by growth factor induced changes in receptor affinity.

Insulin-like growth factor binding protein-1 (IGFBP-1) can bind to the alpha5beta1 integrin and stimulate cellular migration in Chinese hamster ovary (CHO) cells. IGFBP-1 is a major product of the endometrium of pregnancy (decidua), and may interact with invading cytotrophoblasts expressing alpha5beta1 integrin to modulate their invasion. The present study investigated IGFBP-1 interaction with cytotrophoblast alpha5beta1 integrin, and its effects on trophoblast attachment to fibronectin and invasion into decidualized endometrial stromal cell multilayers. IGFBP-1 incubated with cytotrophoblast extracts was co-precipitated by an antibody to the alpha5 integrin subunit. Up to 55% of radiolabeled IGFBP-1 bound to cytotrophoblasts was displaced by excess non-radioactive IGFBP-1, but not by IGFBP-3. Cytotrophoblast attachment to fibronectin was inhibited by an RGD-containing octapeptide, by antibodies to the alpha5 subunit or the alpha5beta1 heterodimer, and by IGFBP-1. Cytotrophoblasts showed limited invasion into endometrial stromal multilayers decidualized in vitro secreting abundant IGFBP-1, but invaded multilayers when IGFBP-1 production was inhibited by insulin. Invasion into insulin-treated multilayers was prevented by addition of exogenous IGFBP-1 but not by IGFBP-3. These findings suggest IGFBP-1 may modulate trophoblast invasiveness.

Our previous study showed that human peritoneal conditioned medium (CM) increased the matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of ovarian cancer cells (NOM1). In an effort to identify this MMP-9-stimulating factor, we examined the effects of extracellular matrix components, such as type IV collagen, laminin, and fibronectin, on ovarian cancer cells. We found that fibronectin increased the MMP-9 activity of NOM1 cell CM in a concentration-dependent manner and that the peritoneal CM contained high level of fibronectin. An increase of MMP-9 activity in NOM1 cell CM by the peritoneal CM was almost completely blocked by 20 microg/ml of anti-integrin alpha5/FnR antibody and RGD polypeptides. Furthermore, after immunoprecipitation by antifibronectin antibody supernatant of the peritoneal CM did not increase MMP-9 activity in NOM1 cells. Fibronectin and the peritoneal CM also increased MMP-9 activity and expression in NOM1 cell lysate, and these effects were blocked by anti-integrin alpha5/FnR antibody. Invasiveness of NOM1 cells was enhanced by fibronectin and the peritoneal CM in a concentration-dependent manner, and anti-integrin alpha5/FnR antibody blocked these effects. These results suggested that fibronectin secreted from peritoneum increased MMP-9 activity and expression, and, in turn, invasiveness of ovarian cancer cells.

Recent evidence indicates that integrin ligation results in activation of focal adhesion kinase (pp125FAK), the prototype of a new subfamily of nonreceptor protein tyrosine kinase (PTK), including FAKB and the proline-rich tyrosine kinase 2 (PYK-2), also termed cell adhesion kinase-beta or related adhesion focal tyrosine kinase. We have previously shown that cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors on human NK cells stimulates tyrosine phosphorylation of two proteins migrating at 105 and 115 kDa. Here we report that cross-linking of beta 1 integrins on human NK cells stimulates tyrosine phosphorylation and PTK activity of PYK-2. PYK-2 tyrosine phosphorylation was maximal at 1 min and started to decline 20 min after stimulation. Engagement of alpha 4 beta 1 and alpha 5 beta 1 either with specific mAbs or after cell adhesion to fibronectin or its 120- and 40-kDa fragments also triggered PYK-2 tyrosine phosphorylation. Stimulation of PYK-2 tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by EGTA, indicating that PYK-2 tyrosine phosphorylation is PTK, but not calcium, dependent. We also demonstrate that PYK-2 is constitutively associated with paxillin, which undergoes tyrosine phosphorylation with the same kinetics of PYK-2 upon beta 1 integrin ligation.

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the beta2 integrin leucocyte function-associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by alpha4beta1 and, to a lesser extent, alpha5beta1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg2+, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases alpha4beta1 integrin-mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of beta1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by alpha5beta1, and is increased when the alpha4beta1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of alpha4-blocking mAb. The ability of mAb 24 Fab' fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and beta1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.

Nonspecific cross-reacting antigen (NCA), a member of the carcinoembryonic antigen (CEA) family, shows increased expression levels in colorectal cancer tissues. To elucidate the mechanism, we observed the effect of interferon (IFN)-gamma on the expression level of NCA mRNA in colon cancer cell lines by quantitative reverse transcriptase-polymerase chain reaction assay. IFN-gamma induced NCA mRNA in three of four cell lines tested. The effect of anti-fibronectin receptor (FnR) antibody on the expression of NCA mRNA was then examined in the same manner. Colo201 and DLD-1 cells showed an increased expression level of NCA mRNA after stimulation with the antibody. On flow cytometry, FnR was expressed in only two, Colo201 and DLD-1, of the five cell lines tested. These findings indicate that IFN-gamma and anti-FnR antibody induce NCA mRNA in cultured colon cancer cell lines, suggesting that inflammatory response and cell-to-extracellular matrix interaction may be related to the increased expression of NCA mRNA in colorectal cancers in vivo.

Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.

VLA-5 recognizes the GRGDSP sequence of fibronectin (FN) in the extracellular matrix (ECM). We examined the role of beta1 integrin in the spreading of the human plasma cell line, FR4ds, induced by FN and laminin (LN). We first examined the role of VLA-5 in the spreading induced by FN. Anti-alpha4 antibody induced 46.4% inhibition, whereas anti-alpha5 had no effect. A combination of anti-alpha4 and anti-alpha5 enhanced the inhibition of spreading significantly. Complementary inhibition was also demonstrated using the GRGDSP peptide plus anti-alpha4 and the GRGDNP peptide of LN plus anti-alpha4. The results suggested that VLA-5 is a regulator of VLA-4 and that it is involved in the recognition of GRGDNP. We then examined the role of VLA-5 in the spreading induced by LN. Anti-alpha6 induced 53.1% inhibition. Anti-alpha5 alone had no effect. A combination of alpha5 and anti-alpha6, however, significantly enhanced the inhibition of spreading. The combination of GRGDSP plus anti-alpha6 and GRGDNP plus anti-alpha6 resulted in complete inhibition. These results suggested that VLA-5 participates in the recognition of LN cooperatively with VLA-6 and that VLA-5 is a common regulator of VLA-4 and the LN receptor, VLA-6.