Integrin activation: the link between ligand binding and signal transduction.

A major function of the integrin family of receptors is to provide a physical connection between extracellular adhesion proteins and intracellular cytoskeletal/signalling molecules. These linkages are dynamic and are influenced in a bidirectional manner by changes in the microenvironment of the plasma membrane that occur both inside and outside of cells. The mechanisms employed by integrins to transduce information are complex, but a series of recent studies has clarified their molecular basis. In particular, explanations for the interdependence of ligand binding, occupancy by divalent cations and receptor conformation have been obtained, and some of the key sites responsible for each property have been localized within the integrin heterodimer. These insights now permit a better visualization of the intricate molecular switch that controls the adhesive phenotype.

VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin.

We previously showed that fibronectin (FN) synergized with anti-CD3 in induction of CD4+ T cell proliferation, and that VLA-5 acted as a functional FN receptor in a serum-free culture system. In the present study, we showed that VLA-4 is also involved in this CD3-dependent CD4 cell activation through its interaction with the alternatively spliced CS1 domain of FN. When highly purified CD4 cells were cultured on plates coated with anti-CD3 plus synthetic CS1 peptide-IgG conjugate, significant proliferation could be observed. Neither CS1 alone nor anti-CD3 alone induced this activation. This proliferation was completely blocked by anti-VLA beta 1 (4B4) and anti-VLA-4 (8F2), while anti-VLA-5 (monoclonal antibody [mAb] 16 and 2H6) had no effect. These data indicate that VLA-4 mediates CD3-dependent CD4 cell proliferation via the CS1 domain of FN. Anti-VLA-4 also partially (10-40%) inhibited CD4 cell proliferation induced by native FN plus anti-CD3, implying that the CS1 domain is active in the native plasma FN. However, this native FN-dependent proliferation was entirely abolished by addition of anti-VLA-5 alone. Moreover, when native FN-coated plates were pretreated with anti-FN (mAb 333), which blocks RGDS sites but not CS1 sites, no CD4 cell activation could be observed. These results strongly suggest that CD4 cell activation induced by plasma FN/anti-CD3 may be dependent on both VLA4/CS1 and VLA5/RGDS interactions, although the latter interaction may be required for function of the former.

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1.

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

The structure of cell-adhesion molecules.

In recent years, following the identification and molecular cloning of many key adhesion molecules, the three-dimensional structures of some of the domains that mediate adhesive interactions have been determined. This review discusses how these studies have helped explain the unique functional roles of the different families of adhesion molecules.

Collagen can modulate cell interactions with fibronectin.

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.

Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment.

Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system.

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.

Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin.

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin.

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.

Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment.

The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells.

Adhesive interactions between cells and the extracellular matrix occur at several stages of metastasis. Such interactions might be inhibited by synthetic peptide probes derived from the cell-binding regions of matrix molecules. Gly-Arg-Gly-Asp-Ser (GRGDS) is a pentapeptide sequence that appears to be critical for cell interaction with fibronectin. Coinjection of GRGDS with B16-F10 murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Two closely related control peptides, in which specific amino acids within the GRGDS sequence were transposed or substituted, displayed little or no activity. Inhibition by GRGDS was dose-dependent, noncytotoxic, and did not result from an impairment of cellular tumorigenicity. GRGDS may function by inhibiting tumor cell retention in the lung since radiolabeled B16-F10 tumor cells injected with the peptide were lost at a substantially greater rate than control cells.

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

Swainsonine inhibition of spontaneous metastasis.

We have previously shown that swainsonine, administered systemically to C57BL/6 mice, inhibited the pulmonary metastasis of iv injected B16-F10 melanoma cells by a mechanism involving interleukin-2 production and augmentation of natural killer cell activity. From this finding, which uses an "experimental metastasis" model system, we considered: (a) whether swainsonine would be effective in the inhibition of authentic or spontaneous metastasis; (b) whether the drug would also inhibit metastasis formation in organs other than the lungs; and (c) whether the drug would block the metastasis of tumor cells of different histological type or origin. Our data indicated that swainsonine effectively inhibited the spontaneous metastasis of B16-BL6 melanoma (by 88%) and M5076 reticulum sarcoma (by 95%) murine tumor cells to the lung and liver, respectively. In both cases, the antimetastatic activity of the drug increased as a function of the concentration in drinking water up to 3 micrograms/mL. These findings indicate that the antimetastatic activity of swainsonine is not limited to artificial or experimentally induced metastasis nor to a single tumor type or specific organ. The inhibition of metastasis is likely due to a combination of events, which are currently under investigation.

Inhibition of experimental metastasis by castanospermine in mice: blockage of two distinct stages of tumor colonization by oligosaccharide processing inhibitors.

The extent of maturation of the oligosaccharide subunits of tumor cell glycoproteins appears to correlate with malignant potential, suggesting that modification of oligosaccharide structures may alter metastatic capacity. Castanospermine, a recently discovered inhibitor of glucosidase I, was tested for its effect on experimental metastasis of B16-F10 murine melanoma cells and was compared to treatment with swainsonine and tunicamycin. All three drugs block different steps in the pathway of glycoprotein processing yet each was a potent inhibitor of pulmonary colonization after i.v. injection of treated cells into C57BL/6 mice (greater than or equal to 80% inhibition). This result indicates a generality of inhibition of experimental metastasis by blockage of protein glycosylation or oligosaccharide processing and strongly implicates carbohydrate residues in at least one critical step of the metastatic cascade. Cytotoxic side effects could not account for the inhibitory activity. In order to identify a possible mechanism of inhibition of colonization, the adhesive behavior and pulmonary retention properties of B16-F10 cells treated with the above inhibitors were examined. Tunicamycin-treated B16-F10 cells exhibited poor adhesion to substrate-adsorbed fibronectin and laminin, whereas both castanospermine- and swainsonine-treated cells possessed near normal adhesive capacity; furthermore, the initial rate of loss of tunicamycin-treated cells from the lungs of mice was substantially greater than either control, castanospermine- or swainsonine-treated cells. These data suggest that these processing inhibitors can block experimental metastasis by at least two different mechanisms. The antimetastatic effect of tunicamycin may be related to interference in tumor cell-extracellular matrix interactions, whereas treatment with castanospermine or swainsonine appears to block at a stage distal to initial tumor cell arrest.

Augmentation of murine natural killer cell activity by swainsonine, a new antimetastatic immunomodulator.

Swainsonine, an indolizidine alkaloid, has been found to inhibit the experimental metastasis of B16-F10 melanoma cells when administered systemically to syngeneic C57BL/6 mice. The inhibition was both potent and dose dependent with greater than or equal to 80% reduction in pulmonary colonization being observed after only 24-h exposure to 3 micrograms/ml of swainsonine in drinking water. In contrast, the inhibitory activity of swainsonine was completely abrogated when assays were performed in mice depleted of their natural killer (NK) cell activity either experimentally (anti-asialo-GM1 antibody- or cyclophosphamide-treated C57BL/6 mice) or as a result of genetic mutation (homozygous C57BL/6bg/bg beige mice). Swainsonine elicited a 32.0% increase in spleen cell number 2 days after administration and induced a concomitant 2- to 3-fold increase in splenic NK cell activity. These results indicate (a) an absolute requirement for a functional NK cell population in order for swainsonine to exert its inhibitory effects on experimental metastasis, and (b) that the antimetastatic activity of swainsonine is mediated primarily through the ability of the drug to augment NK cell reactivity. On the basis of these findings, swainsonine can be classified as a new immunomodulator that has the ability, at least in a prophylactic setting, to block tumor metastasis.

Integrin cell adhesion receptors and the concept of agonism.

Most cells are adherent and rely on adhesive interactions to regulate their shape, motility and growth. These interactions are critical for tissue integrity and homeostasis but they also contribute to many of the most common diseases in humans. The integrins are a key family of cell-surface receptors that mediate the downstream consequences of cell adhesion and are therefore prime targets for the development of therapeutic agents. In addition to their adhesive activity, integrins also exhibit several other classical features of signalling receptors. Sufficient evidence is now available to pose the question of whether integrins should be classified as true signalling receptors; this article both reviews this evidence and attempts to identify remaining gaps.

Regulation of macrophage phagocytosis of apoptotic neutrophils by adhesion to fibronectin.

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.

Enhancing electron affinity and tuning band gap in donor-acceptor organic semiconductors by benzothiadiazole directed C-H borylation.

Electrophilic borylation using BCl3 and benzothiadiazole to direct the C-H functionalisation of an adjacent aromatic unit produces fused boracyclic materials with minimally changed HOMO energy levels but significantly reduced LUMO energy levels. In situ alkylation and arylation at boron using Al(alkyl)3 or Zn(aryl)2 is facile and affords boracycles that possess excellent stability towards protic solvents, including water, and display large bathochromic shifts leading to far red/NIR emission in the solid state with quantum yields of up to 34%. Solution fabricated OLEDs with far red/NIR electroluminescence are reported with EQEs > 0.4%.

The integrin beta subunit.

The integrin family of cell adhesion receptors plays a fundamental role in the processes involved in cell division, differentiation and movement. The extracellular domains of integrin alpha/beta heterodimers mediate cell-matrix and cell-cell contacts while their cytoplasmic tails associate with the cytoskeleton. Integrins are capable of transducing information in a bidirectional manner and the beta subunit is now recognised to play an important role in this process. Recent studies have led to the identification of a ligand-binding region on the beta subunit similar to that already characterised on some alpha subunits, and sequences in the cytoplasmic tails of the beta subunits that interact with cytoskeletal and signalling components. Adhesive events can also play a role in the progression of all four major classes of human disease--neoplastic, inflammatory, traumatic and infectious--and the specific nature of integrin adhesion mechanisms make them an attractive target for therapy.