Endogenous thrombospondin-1 and proteases in the regulation of lymphocyte adhesion and motility

Abstract: The human immune system, which protects the body from invading pathogens, largely depends on the proper function of lymphocytes, which are highly motile and constantly recirculate the blood and lymph. Adhesive and motile capability is often amplified or uncontrolled during chronic inflammatory conditions such as autoimmune diseases. This thesis comprises four studies of T lymphocyte motility and adhesion aiming to elucidate the regulative role of endogenous secretion of enzymes and the matricellular protein thrombospondin-1 (TSP-1). We initially investigated the expression of matrix metalloproteinases (MMPs) in seven leukemia T cell lines and found a strict correlation between secretion of MMP-9, its natural inhibitor tissue inhibitor of MMPs (TIMP-1) and ability to infiltrate a threedimensional (3D) gel of extracellular (ECM) components. However, cell migration to two-dimensional (2D) ECM-components was not correlated to MMP-9/TIMP-1 expression. The role for MMP-9 and TIMP-1 in motility was unclear since an inhibitor of MMP-9 activity rather enhanced infiltrative capacity over 24h. We conclude that MMP-9 and TIMP-1 play a role for spontaneous T lymphocyte motility in 3D-matrices, which is a functional property separated from ability to migrate to 2D ECMcomponents. In our following papers (Paper II-IV), we found that T cell contact with collagen type I or beta1- and beta2-integrin ligands induced cell surface expression of TSP-1 and the TSP-1 receptor low density lipoprotein receptor-related protein (LRP)/CD91. Interaction of TSP-1 with its cell surface receptors calreticulin (CRT), LRP and integrin associated protein (IAP)/CD47 was promoting motility of T cells in 3D collagen through CD47. T cell surface TSP-1 also induced polarized spreading on fibronectin and ICAM-1 via binding to and signaling through CD47. T cell lines without endogenous expression of TSP-1 showed no spontaneous infiltration of collagen type I but became motile in the presence of exogenous TSP-1. We further found constitutive cell surface association of functional granzyme B on activated T cells, which continuously cleaved TSP-1, reduced infiltration of collagen type I and maintained non-polarized spreading on fibronectin and ICAM-1. This process depended on internalization of TSP-1 fragments via LRP. In summary, we have included TSP-1 and its receptors CRT, LRP and CD47 in a model for regulation of T cell motility and adhesion, stating that TSP-1 drives infiltrative capacity and polarized spreading of T cells through receptor communication in cis within the same plasma membrane, generated by cross-linking of its receptors CRT, LRP and CD47. Cleavage of TSP-1 by granzyme B and possibly other enzymes, followed by internalization of fragments via LRP, reduces motility and results in nonpolarized spreading.