Functional and structural characterization of integrins and their ligands ICAM-2, vitronectin and fibronectin in leukocytes

Sammanfattning: FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF INTEGRINS AND THEIR LIGANDS ICAM-2, VITRONECTIN AND FIBRONECTIN IN LEUKOCYTES Rosalba Salcedo Amaya Doctoral dissertation from the Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden Transient adhesion of leukocytes to cells and extracellular matrices is a crucial process in leukocyte physiology. Cell surface adhesion molecules such as integrins and members of the immunoglobulin (Ig) superfamily are involved. beta2 integrins are major contributors of cell-cell adhesion by recognition of Ig- related intercellular adhesion molecules ICAM-I, ICAM-2 and ICAM-3, whereas ,beta1, beta 3 and other integrins mediate cell adhesion to extracellular matrix components such as collagens, vitronectin (Vn) and fibronectin (Fn). In spite of major advances during the last decade, the molecular basis of leukocyte adhesion is not fully understood. This thesis aimed to the identification and characterization of adhesion molecules of Iymphoid cells and polymorphonuclear leukocytes (PMNs) in different experimental conditions. The results predominantly describe integrins and their ligands ICAM-2, Vn and Fn. Similarly to phorbol ester treatment, expression of the Epstein Barr virus (EBV)-encoded latent membrane protein (LMP)-I gene in a murine pre-B cell line, A/J-95, induced homotypic adhesion. By using blocking monoclonal antibodies, the , beta2 integrin CDlla/CD18 (LFA-I) was found to mediate intercellular adhesion in both cases, without a quantitative increase in the cell surface expression of this receptor. By inducing activation of the integrin, both phorbol ester and LMP-I appear to have CDllalCD18 as a target. Although expressed at the cell surface, ICAM-l did not participate in the intercellular adhesion, suggesting participation of ICAM-2. The cellular distribution of human ICAM-2 was studied by using a new mAb. This antibody, generated against a recombinat ICAM-2 protein, reacted mainly with Iymphoblastoid B cells, B Iymphoma cells and vascular endothelial cells and immunoprecipitated from the latter, a cell surface glycoprotein with apparent molecular weight of 55 kDa. To localize binding sites within ICAM-2, adherence of NAD-20 EBV-transformed Iymphoblastoid cells to three immobilized synthetic peptides corresponding to both Ig-like domains was tested. One peptide, corresponding to the beginning of the second domain, mediated adhesion of the cells through CDlla/CD18 and, to a minor extent, the beta1 integrin CD49d/CD29 (VLA-4). In contrast to other EBV-transformed Iymphoblastoid cell lines, which grow in cell clusters, IB-4 cells grow as a monolayer. To define the molecular basis of this phenotypic difference, blocking mAbs to adhesion molecules and purified serum proteins were used. IB-4 cells were found to adhere to the tissue culture vessel through vitronectin, provided by the fetal bovine serum, by using aVbeta3 integrin. aVbeta3 also mediated IB-4 cell adhesion to human serum Vn and to purified Vn and Fn. This constitutive adherence was not enhanced by phorbol ester and was inhibited by RGD-containing peptides. Searching for endogenous and secreted adhesion molecules of leukocytes, blood PMN fibronectin was studied. In resting cells, fibronectin was detected intracellularly in the specific granules as demonstrated by immunostainning, image digital analysis, and fluorescence and electron microscopy. Immunochemical studies with a battery of mAbs demonstrated that PMN Fn was similar to plasma Fn and lacked the ED-A spliced region. Stimulation of PMNs with phorbol ester or chemoattractants rapidly induced secretion of intact Fn which was proteolytically cleaved by cell surface-bound elastase and, to a minor extent, cathepsin G. Proteolytic fragments of fibronectin are known to posses biological properties not inherent to intact fibronectin. Endogenous PMN Fn and/or its fragments might mediate leukocyte adhesion and stimulate PMN activities such as migration, chemotaxis, phagocytosis, degranulation and oxidative metabolism. These studies contribute to a better definition of the molecular basis of leukocyte adhesion to cells and matrices, and exemplifies the multitude of adhesive mechanisms used by these highly motile cells. Key words: Leukocytes, Adhesion molecules, Integrins, ICAM-2, Vitronectin, Fibronectin.