CCL25 and CCL28 promote alpha4 beta7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.     

The role of alpha and beta chains in ligand recognition by beta 7 integrins

Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the alpha(E) A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the alpha(E) MIDAS metal ion by E-cadherin Glu(31) and a novel projection of Phe(298) into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the alpha(E) A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the beta(7) MIDAS motif (D140A) abolished alpha(E)beta(7) binding to E-cadherin and alpha(4)beta(7)-mediated adhesion to MAdCAM-1, and alpha(4) chain mutations that abrogated binding of alpha(4)beta(1) to vascular cell adhesion molecule-1 and fibronectin similarly reduced alpha(4)beta(7) interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin alpha or beta chain, common structural features of both subunits are required for recognition of dissimilar ligands.     

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 chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.     

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

Adenosine suppresses alpha(4)beta(7) integrin-mediated adhesion of T lymphocytes to colon adenocarcinoma cells

The interaction of T lymphocytes with tumor cells, a key step in the antitumor immune response, is suppressed by adenosine, a nucleoside produced at increased levels within the hypoxic tumor environment. We have explored the mechanism by which adenosine interferes with the lymphocyte:tumor cell interaction. The adhesion of anti-CD3-stimulated T cells to syngeneic MCA-38 mouse colon adenocarcinoma cells did not involve LFA-1 (alpha(L)beta(2)) or VLA-5 (alpha(5)beta(1)). However, antibodies against either lymphocyte alpha(4) or beta(7) (but not beta(1)) integrin subunits, or against VCAM-1 on the tumor cells, significantly suppressed adhesion, showing that the recognition of MCA-38 cells by T cells is strongly dependent upon the association of alpha(4)beta(7) on the effector cells with VCAM-1 on the tumor targets. This association is modulated by adenosine: The ability of adenosine to suppress T cell adhesion to MCA-38 cells was lost if alpha(4)beta(7) was functionally blocked with anti-alpha(4) antibodies (i) prior to or (ii) during the adhesion assay or if (iii) alpha(+)(4) cells were depleted from the T lymphocyte population. The binding of T cells to fibronectin through alpha(4)beta(1) was not suppressed by adenosine. We conclude that adenosine partially inhibits the interaction of T lymphocytes with tumor cells by blocking the function of integrin alpha(4)beta(7).     

Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The alpha 4 beta 1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta-turn structure (residues 181-190) of the third N-terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin-alpha 5 beta 1 interaction. These findings suggest that the predicted beta-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.     

Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.     

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.     

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin

The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.     

Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY.

Most mononuclear leukocytes and cell lines express the integrin alpha 4 beta 1 (VLA-4) heterodimer. In this study we have used Northern blotting and immunoprecipitation experiments to demonstrate that a B lymphoblastoid cell line (JY) expressed the integrin beta 7 subunit in association with alpha 4. These alpha 4 beta 7-positive JY cells bound poorly or not at all to VLA-4 ligands (soluble form of vascular cell adhesion molecule 1 (sVCAM-1) and the CS1 region of fibronectin). In contrast, a beta 1-positive variant of JY cells (selected to express a mixture of alpha 4 beta 1 and alpha 4 beta 7) bound avidly to VLA-4 ligands, and this binding was completely inhibitable by anti-alpha 4 and anti-beta 1 monoclonal antibodies. Thus, beta 1 expression appears to be a critically important component of VLA-4-mediated binding to its ligands. After either JY or JY-beta 1 cells were stimulated for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, the majority of adhesion to VCAM or fibronectin remained alpha 4- and beta 1-dependent, but a low amount of adhesion to sVCAM-1 or fibronectin became alpha 4-dependent, beta 1-independent, thus suggesting a role for alpha 4 beta 7. In summary, we have found (i) that alpha 4 beta 7 makes little or no contribution to fibronectin or VCAM-1 binding on unstimulated JY cells, (ii) that alpha 4 beta 7 perhaps makes a minor contribution to ligand binding on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated cells, and (iii) that alpha 4 beta 1 is the functionally dominant VCAM-1 and fibronectin receptor even when expressed in relatively low amounts compared to alpha 4 beta 7.     

The CC' and DE loops in Ig domains 1 and 2 of MAdCAM-1 play different roles in MAdCAM-1 binding to low- and high-affinity integrin alpha4beta7

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4β7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4β7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4β7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4β7. The Asp-42 in the CC' loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4β7. The other CC' loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4β7 and α4β7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4β7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4β7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4β7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4β7. Moreover, DE loop residues were important for stabilizing the low-affinity α4β7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC' and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4β7 and suggest that the inactive α4β7 and α4β7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.     

The fourth blade within the beta-propeller is involved specifically in C3bi recognition by integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha M beta 2 are critical to phagocytosis of opsonized particles in host defense against foreign pathogens and certain malignant cells. Previous studies have mapped critical residues for C3bi binding to the I-domains of the alpha M and the beta2 subunits. However, the role of the alpha M beta-propeller in ligand binding remains less well defined, and the functional residues are still unknown. In the present study, we studied the function of the alpha M beta-propeller in specific ligand recognition by alpha M beta 2 using a number of different approaches, and we report four major findings. 1) Substitution of five individual segments (Asp398-Ala402, Leu412-Leu419, Tyr426-Met434, Phe435-Glu443, and Ser444-Thr451) within the W4 blade of the beta-propeller with their homologous counterparts in integrin alpha2 abrogated C3bi binding, whereas substitution of eight other segments outside this blade had no effect. 2) These five mutants defective in C3bi binding supported strong alpha M beta 2-mediated and cation-dependent cell adhesion to fibrinogen, suggesting that the conformations of these five defective mutants were intact. 3) Polyclonal antibodies recognizing sequences within the W4 blade significantly blocked C3bi binding by wild-type alpha M beta 2. 4) A synthetic peptide corresponding to Gln424-Gly440 within W4 interacted directly with C3bi. In conclusion, our data demonstrate that the W4 blade (residues Asp398 to Thr451) is involved specifically in C3bi but not fibrinogen binding to alpha M beta 2. Altogether, our study supports a model in which three separate domains of alpha M beta 2 (the alpha MI-domain, the alpha M beta-propeller, and the beta 2I-domain) function together and contribute to the formation of the C3bi-binding site.     

Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the alpha 4 beta 7-MAdCAM-1 interaction.

We studied interactions in shear flow of cells bearing integrins alpha4beta1 or alpha4beta7 with VCAM-1 and MAdCAM-1 substrates in different divalent cations. Interestingly, Ca(2+) was essential for tethering in flow and rolling interactions through both alpha4 integrins. Mg(2+) promoted firm adhesion of alpha4beta7-expressing cells on MAdCAM-1 but with much lower tethering efficiency in shear flow. The k(off) degrees of 1.28 s(-1) and resistance of the receptor-ligand bond to force (estimated as a bond interaction distance or sigma) for transient tethers on MAdCAM-1 were similar to values for E- and P-selectins. By contrast to results in Ca(2+) or Ca(2+) + Mg(2+), in Mg(2+) the alpha4beta7-MAdCAM-1 k(off) degrees decreased 20-fold to 0.046 s(-1), and the bond was weaker, providing an explanation for the finding of firm adhesion under these conditions. Shear enhanced tethering to MAdCAM-1, thereby contributing to the stability of rolling. Comparisons to selectins demonstrate that the kinetic and mechanical properties of the alpha4beta7 integrin are well suited to its intermediate position in adhesion cascades, in which it bridges rapid rolling through selectins to firm adhesion through beta2 integrins.     

Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin alpha4beta1

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.     

Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.     

Critical amino acid residues of the alpha4 subunit for alpha4beta7 integrin function

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the alpha and beta subunit of the alpha4 integrins, alpha4beta1 and alpha4beta7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human alpha4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for alpha4beta7 integrins. We present evidence that LDV-1 mutants (D489N) behave like alpha4 wt cells, but LDV-3 mutants (D811N) are impaired in alpha4beta7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on alpha4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced alpha4beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4beta7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for alpha4beta7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects alpha4beta7 heterodimer stability.     

Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1alpha (SDF-1alpha). Whereas the overlap between HIV-1 and SDF-1alpha functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1alpha functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. For this study, by employing alanine-scanning mutagenesis, (125)I-SDF-1alpha competition binding, Ca(2+) mobilization, and cell-cell fusion assays, we found that the mutation of many CXCR4 TM residues, including Tyr(45), His(79), Asp(97), Pro(163), Trp(252), Tyr(255), Asp(262), Glu(288), His(294), and Asn(298), could selectively decrease HIV-1-mediated cell fusion but not the binding activity of SDF-1alpha. Phe(87) and Phe(292), which were involved in SDF-1alpha binding, did not play a significant role in the coreceptor activity of CXCR4, further demonstrating the disconnection between physiological and pathological activities of CXCR4 TM domains. Our data also show that four mutations of the second extracellular loop, D182A, D187A, F189A, and P191A, could reduce HIV-1 entry without impairing either ligand binding or signaling. Taken together, our first detailed characterization of the different functional roles of CXCR4 TM domains may suggest a mechanistic basis for the discovery of new selective anti-HIV agents.     

CD14 is a ligand for the integrin alpha4beta1

Cell adhesion mediated by the integrin alpha4beta1 plays a key role in many biological processes reflecting both the number and functional significance of alpha4beta1 ligands. The lipopolysaccharide (LPS) receptor, CD14, is a GPI-linked cell surface glycoprotein with a wide range of reported functions and associations, some of which overlap with that of alpha4beta1. This overlap led us to test the specific hypothesis that alpha4beta1 and CD14 interact directly. Jurkat T cells (alpha4beta1(+)) were found to adhere to a recombinant CD14-Fc protein via alpha4beta1, whilst K562 cells (alpha4beta1(-)) did not. However, stable reexpression of the alpha4-subunit conferred this ability. The adhesion of both cell types to CD14 displayed activation state-dependent binding very similar to the interaction of alpha4beta1 with its prototypic ligand, VCAM-1. In solid-phase assays, CD14-Fc bound to affinity-purified alpha4beta1 in a dose-dependent manner that was induced by activating anti-beta1 mAbs. Finally, in related experiments, JY cells (alpha4beta7(+)) were also found to attach to CD14-Fc in an alpha4-dependent manner. In summary, CD14 is a novel ligand for alpha4beta1, exhibiting similar activation-state dependent binding characteristics as other alpha4beta1 ligands. The biological relevance of this interaction will be the subject of further studies.