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.

alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1).

The beta2 family of integrins, CD11a, CD11b, CD11c, and alphad, are expressed on most leukocytes. We show that the newest member of this family, alphad, is expressed on human eosinophils in peripheral blood, and surface expression can be upregulated within minutes by phorbol ester or calcium ionophore A23187. Culture of eosinophils with interleukin 5 (IL-5) leads to a two- to fourfold increase in alphad levels by 3-7 d without a change in alpha4 integrin expression. Eosinophils isolated from late phase bronchoalveolar lavage fluids express alphad at levels similar to that seen after 3 d of IL-5 culture. Regarding alphadbeta2 ligands, in both freshly isolated and IL-5-cultured eosinophils, as well as alphadbeta2-transfected Chinese hamster ovary cells, alphadbeta2 can function as a ligand for vascular cell adhesion molecule 1 (VCAM-1). This conclusion is based on the ability of monoclonal antibodies to alphad, beta2, or VCAM-1 to block cell attachment in static adhesion assays. In experiments with eosinophils, the relative contribution of alphadbeta2 integrin- mediated adhesion is enhanced after IL-5 culture. These experiments demonstrate that alphadbeta2 is an alternative ligand for VCAM-1, and this integrin may play a role in eosinophil adhesion to VCAM-1 in states of chronic inflammation.

Synovial distribution of alpha d/CD18, a novel leukointegrin. Comparison with other integrins and their ligands.

OBJECTIVE: To define the synovial distribution of the novel leukointegrin alpha d/CD18, and compare this with other members of the beta 2-integrin family of adhesion molecules, and their counter-receptors. METHODS: Monoclonal antibodies to the CD3, CD14, CD29, CD68, beta 2-integrin, and immunoglobulin supergene families were used to immunohistologically define the distribution of these molecules in synovial tissue samples from normal subjects and osteoarthritis (OA) and rheumatoid arthritis (RA) patients. RESULTS: The normal synovial lining cell layer (SLC) expresses CD68, vascular cell adhesion molecule 1, beta 1-integrin (CD29), the beta 2-integrins CD11b/CD18 (alpha m/beta 2, Mac-1), and alpha d/CD18, whereas CD11a/CD18 (alpha L/beta 2, lymphocyte function-associated antigen 1) and CD11c/CD18 (alpha x/beta 2, gp150/95) expression is generally absent. In RA synovitis, expression of beta 2-integrins in the SLC increases in proportion to the degree of hyperplasia. The ratio of cells in the SLC which express CD11c/CD18 increases substantially, approaching that of CD11b/CD18 and alpha d/CD18, while there is minimal increase in CD11a/CD18 expression. In the sublining areas of the tissues, aggregates and diffuse infiltrates of CD3/CD11a/ICAM-3+ lymphocytes are interspersed among CD68/CD14/CD11b/alpha d+ macrophages. A number of aggregates demonstrate intense alpha d staining of the lymphocytes. The synovial endothelium variably expresses intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule 1 (VCAM-1), with minimal evidence of ICAM-3 expression. CONCLUSION: The leukointegrin alpha d/CD18 is expressed constitutively by synovial macrophages and macrophage-like lining cells. In rheumatoid synovitis, the intense coexpression of this integrin and its known counter-receptor, ICAM-3, in the inflammatory infiltrates, suggests a potential role for this adhesion pathway in cellular interactions occurring the synovium.

A novel leukointegrin, alpha d beta 2, binds preferentially to ICAM-3.

The leukocyte-restricted beta 2 (CD18) integrins mediate cell adhesion in a variety of events essential for normal immune function. Despite extensive research in this field, only three members of this integrin subfamily have been described: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). We have identified a cDNA encoding a fourth alpha chain, alpha d, that associates with CD18. The alpha d subunit is more closely related to CD11b and CD11c than to CD11a. This integrin is expressed at moderate levels on myelomonocytic cell lines and subsets of peripheral blood leukocytes, and more strongly on tissue-compartmentalized cells such as foam cells, specialized macrophages found in aortic fatty streaks that may develop into atherosclerotic lesions. The alpha d/CD18 molecule exhibits preferential recognition of ICAM-3 over ICAM-1.

A novel canine leukointegrin, alpha d beta 2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood.

The beta 2 or leukointegrin family is comprised of three structurally related leukocyte surface heterodimers: LFA-1 (CD11a/CD18), Mac-1/Mo-1 (CD11b/CD18), and p150,95 (CD11c/CD18). In this work, we describe a novel canine beta 2 (CD18)-associated leukointegrin, designated alpha d. Expression of alpha d in tissues was prominent in macrophages in splenic red pulp, lymph node medullary regions, and bone marrow. In peripheral blood, alpha d expression was limited to a minor subpopulation of CD8+ T cells, which included small lymphocytes and large granular lymphocytes. A minor subpopulation of either CD8+ or CD4-CD8- splenic red pulp lymphocytes also expressed alpha d. Immunoprecipitation of alpha d from canine splenocytes revealed a heterodimer of 155 kDa and 95 kDa. Prior clearance of splenocyte extracts with an anti-CD18 mAb resulted in complete removal of alpha d. In addition, prior clearance of canine splenocyte extracts with anti-CD11a, anti-CD11b, or anti-CD11c mAb failed to clear alpha d. These immunoclearance data indicated that canine alpha d was antigenically distinct from the three known CD11 molecules, and occurred as an alpha d beta 2 heterodimer. Amino acid sequencing of canine alpha d affinity isolated from spleen further suggested that canine alpha d beta 2 probably represented a fourth member of the canine leukointegrin family via its homology to a subsequently discovered, novel human leukointegrin, alpha d beta 2, which further supported the uniqueness of the canine protein. The discovery of canine alpha d, and the demonstration of its highly restricted cell and tissue distribution, support a re-evaluation of leukointegrin-dependent inflammatory and immunologic interactions that involve cells now known to express alpha d.

Direct interaction with primed CD4+ CD45R0+ memory T lymphocytes induces expression of endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of vascular endothelial cells.

The process of recruitment of leukocytes at sites of inflammation involves direct cell-to-cell interactions between leukocytes and vascular endothelial cells (EC) mediated by various adhesion receptors on leukocytes and their inducible endothelial ligands. In this study we have examined the induction on EC of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) upon their interaction with subpopulations of human T cells. When co-cultured with EC both resting CD4+ T and CD8+ T cells caused a modest increase in the expression of endothelial ICAM-1. Moreover, resting CD4+ but not CD8+ T cells induced expression of ELAM-1 and VCAM-1 on a small fraction of unstimulated EC. Prior activation with phorbol 12-myristate 13-acetate (PMA) significantly increased the ability of T cells to up-regulate endothelial ICAM-1 and also induced the expression of both ELAM-1 and VCAM-1. PMA-primed CD4+ T cells induced both VCAM-1 and ELAM-1 on EC more efficiently than CD8+ T cells. Furthermore, the ability to induce the expression of ELAM-1 and VCAM-1 was confined to the CD4+ CD45R0+ memory/primed subpopulation of T cells. This induction of various endothelial adhesion ligands could also be mediated by antigen-primed CD4+ T cell lines. The CD4+ T cell-mediated induction of adhesion ligands required direct intercellular contact with EC because neither cultures of EC and PMA-primed CD4+ T cells separated by a microporous membrane insert nor the conditioned medium of PMA-primed T cells induced expression of ELAM-1 and VCAM-1 on EC. Cyclosporin A significantly inhibited the activation of T cells with PMA but had no effect on the ability of PMA-primed T cells to up-regulate endothelial CAM. Thus, CD4+CD45R0+ T cells via as yet unknown mechanism can significantly enhance the expression of each of the three endothelial adhesion ligands and, thereby, may facilitate the process of recruitment of additional leukocytes to exacerbate inflammation.