In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin

Although dendritic cells (DCs) regulate immune responses, they exhibit functional heterogeneity depending on their anatomical location. We examined the functional properties of intestinal DCs after oral administration of cholera toxin (CT), the most potent mucosal adjuvant. Two CD11c+ DC subsets were identified both in Peyer's patches and mesenteric lymph nodes (MLN) based on the expression of CD8alpha (CD8+ and CD8- DCs, respectively). A third subset of CD11c+CD8int was found exclusively in MLN. Feeding mice with CT induced a rapid and transient mobilization of a new CD11c+CD8- DC subset near the intestinal epithelium. This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8int DCs in MLN. MLN DCs from CT-treated mice were more potent activators of naive T cells than DCs from control mice and induced a Th2 response. This increase in immunostimulating properties was accounted for by CD8int and CD8- DCs, whereas CD8+ DCs remained insensitive to CT treatment. Consistently, the CD8int and CD8- subsets expressed higher levels of costimulatory molecules than CD8+ and corresponding control DCs. Adoptive transfer experiments showed that these two DC subsets, unlike CD8+ DCs, were able to present Ags orally coadministered with CT in an immunostimulating manner. The ability of CT to mobilize immature DCs in the intestinal epithelium and to promote their emigration and differentiation in draining lymph nodes may explain the exceptional adjuvant properties of this toxin on mucosal immune responses.     

Identification of ITGA4/ITGB7 and ITGAE/ITGB7 expressing subsets of decidual dendritic-like cells within distinct microdomains of the pregnant mouse uterus

Several leukocyte populations have been described within the pregnant mouse uterus, some of which express the integrin beta 7 (ITGB7). Here we demonstrate that the majority of the ITGB7(+) decidual leukocytes belong to the dendritic cell (DC) lineage. By multiparameter flow cytometric analysis we demonstrated the existence of three distinct DC subsets, characterized by differential expression of ITGA4/ITGB7 (formerly alpha4beta7-integrin) and ITGAE/ITGB7 (formerly alphaEbeta7-integrin). Importantly, the predominant DC subsets reside in distinct microdomains of the Day 9 pregnant mouse uterus. ITGAX(+) ITGAM(med) ITGA4/ITGB7(+) ITGAE(-) (formerly CD11c(+) CD11b(med) alpha4beta7(+) alphaE(-)) cells represent the majority of DCs in the vascular zone (VZ), whereas ITGAX(+) ITGAM(-) ITGAE/ITGB7(+) (formerly CD11c(+) CD11b(-) alphaEbeta7(+)) DCs are mainly located in the lower central decidua basalis (cDB) and the underlying myometrium. A population of ITGAX(+) ITGAM(low) DCs lacking ITGB7 are restricted to the cDB. Confocal microscopy studies show direct contact of VZ DCs with uterine natural killer (uNK) cells, suggesting a functional relationship between both cell populations. Collectively, our data identify three phenotypically distinct DC subsets residing in distinct microdomains of the uterus. The differential expression of ITGA4/ITGB7 and ITGAE/ITGB7 suggests distinct functional roles of the different DC subsets during early pregnancy.     

TNF-alpha-dependent and -independent maturation of dendritic cells and recruited CD11c(int)CD11b+ Cells during oral Salmonella infection

Maturation of dendritic cells (DC) is crucial for their ability to induce adaptive immunity. Although several mediators of DC maturation have been found, their contributions to DC maturation during infection are poorly understood. In this study we show that murine conventional (CD11c(high)) DC up-regulate costimulatory molecules in a subset-specific manner after oral Salmonella infection. Although both CD8alpha+ and CD8alpha- subsets increase CD86 expression, CD40 was preferentially up-regulated on CD8alpha+ DC, and CD80 was preferentially increased on CD8alpha- DC. In addition, high levels of CD80 and CD86 were found on CD11c(int)CD11b+ cells that accumulated in infected organs. Costimulatory molecules were simultaneously induced on CD11c(high) and CD11c(int)CD11b+ cells in Peyer's patches, mesenteric lymph nodes and spleen 5 days after infection despite different kinetics of peak bacterial burden in these organs. Up-regulation of costimulatory molecules occurred on all DC within the respective subset. Moreover, <1% of CD11c-expressing cells associated with Salmonella expressing enhanced GFP in vivo. Thus, DC maturation did not depend on bacterial uptake. Rather, infection-induced up-regulation of CD80, CD86, and CD40 on CD11c-expressing cells of mesenteric lymph nodes was dependent on TNFR type I (TNFRI) signaling. Although indirect up-regulation of costimulatory molecules on DC and CD11c(int)CD11b+ cells was TNFRI dependent, cells directly associated with Salmonella were able to mature independently of TNFRI signaling. Thus, Salmonella-induced TNF-alpha is an important mediator of indirect DC maturation during infection, whereas a TNF-alpha-independent maturation pathway contributes to direct maturation of bacteria-associated DC.     

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

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

E-cadherin is a ligand for integrin alpha2beta1.

E-cadherin is a 120-kDa transmembrane glycoprotein expressed mainly on the surface of epithelial cells. The best characterised function of E-cadherin is homotypic, calcium-dependent cell-cell adhesion; however, the observation that E-cadherin is also capable of interacting with the alphaEbeta7 integrin to mediate leukocyte cell-cell adhesion [Nature 372 (1994) 190] suggests that it also participates in heterotypic interactions. To investigate the possibility that E-cadherin may interact with integrins expressed on non-leukocytic cells, cell adhesion and solid-phase receptor-ligand binding experiments were performed using a pentameric E-cadherin construct designed to detect low affinity, high avidity interactions. HT1080 human fibrosarcoma cells specifically adhered to pentameric E-cadherin, and this adhesion was inhibited by anti-functional monoclonal antibodies directed against the integrin alpha2 and beta1 subunits, but not by a series of antibodies recognising other subunits. This suggested that the E-cadherin receptor was alpha2beta1, a previously characterised collagen/laminin receptor. Pentameric E-cadherin, but not monomeric E-cadherin, specifically bound, in a divalent cation-dependent manner, to both purified alpha2beta1 and to a recombinant form of the A-domain of the alpha2 subunit, which has been shown to be a major ligand-binding site within this and other integrins. These findings demonstrate that E-cadherin can interact with alpha2beta1 and suggest that heterotypic interactions between E-cadherin and integrins may be more common than originally thought.     

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.     

L-selectin, alpha 4 beta 1, and alpha 4 beta 7 integrins participate in CD4+ T cell recruitment to chronically inflamed small intestine

CD4+ T cells are essential for development and perpetuation of Crohn's disease, a chronic immune-mediated condition that affects primarily the small intestine. Using novel models of Crohn's disease-like ileitis (i.e., SAMP1/YitFc and CD4+ T cell transfer models), we have begun to understand the adhesive pathways that mediate lymphocyte trafficking to the chronically inflamed small bowel. Expansion of the CD4/beta7+ population and increased mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression were observed within the intestinal lamina propria with disease progression. However, Ab blockade of the beta7 integrin, the alpha4beta7 heterodimer, MAdCAM-1, or L-selectin did not attenuate inflammation. Blockade of two pathways (L-selectin and MAdCAM-1 or alpha4 integrins) was required to improve ileitis. Further analyses showed that 55 +/- 7% of the mesenteric lymph node alpha4beta7+CD4 expressed L-selectin. These L-selectin+ T cells were the main producers of TNF-alpha and the predominant ileitis-inducing subpopulation. Mechanistically, combined blockade of L-selectin and MAdCAM-1 depleted the intestinal lamina propria of CD4+ T cells that aberrantly coexpressed alpha4beta7 and alpha4beta1 integrins, markedly decreasing local production of TNF-alpha and IFN-gamma. Thus, pathogenic CD4+ T cells not only use the physiologic alpha4beta7/MAdCAM-1 pathway, but alternatively engage alpha4beta1 and L-selectin to recirculate to the chronically inflamed small intestine.     

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.     

Phospholipase Cgamma binds alpha1beta1 integrin and modulates alpha1beta1 integrin-specific adhesion.

Integrin adhesion receptors have been implicated in bidirectional signal transduction. The dynamic regulation of integrin affinity and avidity as well as post-ligand effects involved in outside-in signaling depends on the interaction of integrins with cytoskeletal and signaling proteins. In this study, we attempted to identify cytoplasmic binding partners of alpha(1)beta(1) integrin. We were able to show that cell adhesion to alpha(1)beta(1)-specific substrates results in the association of phospholipase Cgamma (PLCgamma) with the alpha(1)beta(1) integrin independent of PLCgamma tyrosine phosphorylation. Using peptide-binding assays, the membrane proximal sequences within the alpha(1)beta(1) integrin subunits were identified as binding sites for PLCgamma. In particular, the conserved sequence of beta(1) subunit binds the enzyme very efficiently. Because purified PLCgamma also binds the integrin peptides, binding seems to be direct. Inhibition of PLC by leads to reduced cell adhesion on alpha(1)beta(1)-specific substrates. Cells lacking the conserved domain of the alpha(1) subunit fail to respond to the PLC inhibition, indicating that this domain is necessary for PLC-dependent adhesion modulation of alpha(1)beta(1) integrin.     

Adaptor protein-2 exhibits alpha 1 beta 1 or alpha 6 beta 1 integrin-dependent redistribution in rhabdomyosarcoma cells

Downregulation of several signaling pathways, such as those stimulated by growth factor receptors, occurs by internalization of signaling receptors through clathrin-coated pits. The first step in internalization or endocytosis is interaction with AP-2, which results in coated pit formation by assembly of clathrin to AP-2. Changes in endocytosis are reflected in the distribution of AP-2 molecules at the cell surface. Integrins are receptors which mediate attachment to the extracellular matrix and also stimulate numerous intracellular signaling pathways; however, it is not known how signaling through integrins is terminated or downregulated. Endocytosis through clathrin-coated pits offers an attractive mechanism for this. This work explores the relationship between AP-2 and beta(1) integrins. RD cells grown for 24 h on collagen or laminin exhibit a redistribution of AP-2 to the cell periphery relative to those grown on fibronectin or polylysine. The total AP-2 protein levels in the cells are unaffected. Blocking alpha(1)beta(1) integrin ligand binding on collagen prevents this redistribution fully. On laminin where alpha(1)beta(1) and alpha(6)beta(1) integrins are engaged, both receptors must be simultaneously blocked to prevent AP-2 redistribution, confirming that the redistribution depends on the specific engagement of the receptors. Immunofluorescence reveals that the majority of alpha(1)beta(1) integrins colocalize with alpha(6)beta(1) integrins in linear structures identified as focal adhesions. A separate fraction of alpha(1)beta(1) integrins colocalize with AP-2 in coated pits. Interestingly, alpha(6)beta(1) integrins are not located in coated pits, demonstrating that integrin colocalization with AP-2 is not necessary to induce redistribution of AP-2.     

Structural requirements of MLD-containing disintegrins for functional interaction with alpha 4 beta 1 and alpha 9 beta1 integrins

Three non-RGD-containing disintegrins, VLO5, EO5, and EC3, belong to the heterodimeric family of these snake venom-derived proteins. They are potent inhibitors of certain leukocyte integrins such as alpha4beta1, alpha4beta7, and alpha9beta1, and act through the MLD motif present in one of their subunits. However, the selectivity of these disintegrins to interact with integrins is related to the amino acid composition of the integrin-binding loop in the MLD-containing subunit. The most important amino acid is that preceding the MLD motif. In vitro experiments in adhesion and ELISA assays revealed that the TMLD-containing disintegrins, VLO5 and EO5, appeared to be very potent inhibitors of human alpha4beta1 and alpha9beta1 and less effective in inhibition of the alpha4beta7 integrin. The reverse effect was observed for the AMLD-containing disintegrin, EC3. The data with native disintegrins were confirmed by experiments with synthetic peptides displaying TMLD and AMLD motifs. The MLD-containing disintegrins showed differential activities to inhibit human and murine alpha4beta1 integrin. EC3 was a weaker inhibitor of human integrin, whereas VLO5 and EO5 less actively inhibited murine alpha4beta1. These data describe a useful set of potent and selective integrin antagonists and suggest conformational requirements of human and mouse integrins for interaction with ligands.     

Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.     

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells

We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer's patch. CD11b(+)/CD8alpha(-) (myeloid) DCs are localized in the subepithelial dome, CD11b(-)/CD8alpha(+) (lymphoid) DCs in the interfollicular regions, and CD11b(-)/CD8alpha(-) (double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand(-) trimer, or Staphylococcus aureus and IFN-gamma. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-gamma production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.