Cell migration in the immune system: the evolving inter-related roles of adhesion molecules and proteinases.

Leukocyte extravasation into perivascular tissue during inflammation and lymphocyte homing to lymphoid organs involve transient adhesion to the vessel endothelium, followed by transmigration through the endothelial cell (EC) layer and establishment of residency at the tissue site for a period of time. In these processes, leukocytes undergo multiple attachments to, and detachments from, the vessel-lining endothelial cells, prior to transendothelial cell migration. Transmigrating leukocytes must traverse a subendothelial basement membrane en route to perivascular tissues and utilize enzymes known as matrix metalloproteinases to make selective clips in the extracellular matrix components of the basement membrane. This review will focus on the evidence for a link between adhesion of leukocytes to endothelial cells, the induction of matrix metalloproteinases mediated by engagement of adhesion receptors on leukocytes, and the ability to utilize these matrix metalloproteinases to facilitate leukocyte invasion of tissues. Leukocytes with invasive phenotypes express high levels of MMPs, and expression of MMPs enhances the migratory and invasive properties of these cells. Furthermore, MMPs may be used by lymphocytes to proteolytically cleave molecules such as adhesion receptors and membrane bound cytokines, increasing their efficiency in the immune response. Engagement of leukocyte adhesion receptors may modulate adhesive (modulation of integrin affinities and expression), synthetic (proteinase induction and activation), and surface organization (clustering of proteolytic complexes) behaviors of invasive leukocytes. Elucidation of these pathways will lead to better understanding of controlling mechanisms in order to develop rational therapeutic approaches in the areas of inflammation and autoimmunity.

Distinct roles for matrix metalloproteinase-2 and alpha4 integrin in autoimmune T cell extravasation and residency in brain parenchyma during experimental autoimmune encephalomyelitis.

Expression of alpha4 integrin by auto-reactive T cells is critical for their ability to induce EAE, an autoimmune disease of the central nervous system in mice, used as a model to study human multiple sclerosis. Having previously identified one role for alpha4 integrin in adhesion-mediated induction of matrix metalloproteinase-2 (MMP-2), an enzyme that degrades the subendothelial basement membrane matrix, we investigated independent roles for MMP-2 and alpha4 integrin during EAE. The data suggest that expression of alpha4 integrin by auto-reactive T cells is important not only in mediating MMP-2 induction to facilitate entry into the CNS, but also plays a role in maintaining residency within the CNS.

PECAM-1 (CD31) expression modulates bleeding time in vivo.

PECAM-1 is a 130-kd member of the Ig superfamily present on endothelial cells, platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. Its expression begins early in development and persists through adulthood. PECAM-1 functions as an adhesion and signaling molecule between adjacent endothelial cells and between endothelial cells and circulating blood elements. Antibodies directed against PECAM-1 have been shown to affect angiogenesis, endothelial cell migration, and polymorphonuclear leukocyte transmigration. Furthermore, its dimerization is associated with the modulation of integrin affinity. Antibody inhibition studies suggest that PECAM-1 plays a role in modulating thrombosis; however, recent in vitro aggregation studies performed on platelets harvested from PECAM-1-deficient mice revealed no abnormalities. In this report we demonstrate prolonged in vivo bleeding times in PECAM-1-deficient mice. This abnormality was not corrected when wild-type hematopoietic precursors were engrafted into marrow-ablated PECAM-1-deficient mice. Furthermore, normal bleeding times were observed when marrow-ablated wild-type mice were engrafted with hematopoietic precursors harvested from PECAM-1-deficient mice. These studies are consistent with a role for PECAM-1 in modulating thrombosis in the vasculature, which is potentially mediated by endothelial cell PECAM-1 expression.

The interrelationship of alpha4 integrin and matrix metalloproteinase-2 in the pathogenesis of experimental autoimmune encephalomyelitis.

Previous studies have suggested that surface expression of alpha4 integrin by autoreactive T-cell clones is necessary for the clones to induce experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. To provide direct evidence for this phenomenon, we have transfected alpha4 integrin into C19alpha4-LO, a myelin basic protein-reactive T-cell clone that does not express alpha4 integrin and does not induce EAE when adoptively transferred into a susceptible mouse strain. Transfection of alpha4 integrin converted this clone to an alpha4 integrin-expressing clone that induced EAE. We then examined potential mechanisms by which alpha4 integrin may facilitate the disease process. C19 T-cell clones adhered equally to a monolayer of microvascular endothelial cells, regardless of level of alpha4 integrin expression. However, in contrast to T-cell clones that do not express alpha4 integrin, T-cell clones that express alpha4 integrin (endogenously or by transfection) transmigrated through an endothelial cell layer and subendothelial matrix at an enhanced rate and adhered to recombinant vascular cell adhesion molecule-1 (rVCAM-1) and the CS1 fragment of fibronectin, and after adhesion to these ligands, a matrix-degrading metalloproteinase (MMP-2) was induced and activated. The clones were also shown to constitutively express the membrane-type matrix metalloproteinase (MT1-MMP), an enzyme that activates MMP-2. GM6001 and UK-221,316, inhibitors of metalloproteinases, reduced alpha4 integrin-mediated transmigration and EAE induction by C19 T-cell clones. In addition, we studied a second EAE-inducing T-cell clone, MM4, which constitutively expresses alpha4 integrin and MMP-2. Engagement of alpha4 integrin on the MM4 clone up-regulated the expression and activation of MMP-2, without changing the expression of MT1-MMP. MMP inhibitors also reduced transmigration of and EAE induction by the MM4 T-cell clone. These studies demonstrate directly that expression of alpha4 integrin by autoreactive T-cell clones is required for adoptive transfer of EAE in this model. We also define a role for alpha4 integrin in the disease process in mediating the induction and coordinate activation of a matrix metalloproteinase (MMP-2), which facilitates T-cell transmigration.

T cell adhesion to endothelial cells and extracellular matrix is modulated upon transendothelial cell migration.

During inflammation, T cells transmigrate from the bloodstream into perivascular tissues. As T cells transmigrate, they undergo a series of attachments to and detachments from the endothelium and then extravasate into the extracellular matrix (ECM). T cell migration into the ECM involves a number of mechanisms that influence cell-ECM interactions. The modulation of integrin expression and affinity are two such mechanisms in which cells can alter their ability to interact with other cells and ECM. We show in vitro that transmigrated T cells exhibit down-regulation of very late activation antigen-4 and leukocyte function-associated antigen-1 integrin surface expression and a decrease in binding to recombinant vascular cell adhesion molecule-1 and recombinant intercellular adhesion molecule-1. Also, transmigrated T cells displayed an increase in binding to collagens I and IV and fibronectin. Further, brain sections of experimental autoimmune encephalomyelitis mice demonstrated that as T cells migrated farther into the tissue, very late activation antigen-4 expression was lost while CD4 expression remained unchanged. The significance of these findings in the modulation of the inflammatory response is discussed.

The roles of adhesion molecules and proteinases in lymphocyte transendothelial migration.

T cell extravasation into perivascular tissue during inflammation involves transmigration through the endothelial cell (EC) layer and basement membrane. We have demonstrated that matrix metalloxproteinase-2 (MMP-2) is induced in T cells upon adhesion to endothelial cells and that the induction of MMP-2 is mediated by binding of T cell VLA-4 to VCAM-1. Cloned murine Th1 cells antigenic to myelin basic protein, either expressing VLA-4 on their cell surface and causing experimental autoimmune encephalomyelitis (EAE) or not expressing VLA-4 and not causing EAE, were used. VLA-4 positive (+) T cells that adhered to VCAM-1 positive (+) endothelial cells exhibited an induction in MMP-2 mRNA, protein, and activity, whereas MMP-2 was not induced in the T cells that adhered to the VCAM-1 negative (-) endothelial cells or VLA-4 negative (-) T cells that adhered to VCAM-1+ endothelial cells. Incubating T cells with rVCAM-1-coated dishes showed that VLA-4+ T cells adhered to the molecule and that adhesion to rVCAM-1 was sufficient to induce MMP-2. VLA-4+ T cells that had transmigrated through a VCAM-1+ endothelial cell monolayer exhibited MMP-2 activity. TIMP-2 was shown to reduce T cell transmigration in vitro. Transmigrated T cells exhibited downregulation of VLA-4 and LFA-1 integrin surface expression and decreased binding to rVCAM-1 and rICAM-1 and increased binding to collagens I and IV, fibronectin, and laminin. Brain sections of mice demonstrated that as T cells migrated farther into the tissue, VLA-4 expression was lost, although CD4 expression remained unchanged. These results demonstrate that binding to VCAM-1 on endothelial cells induces MMP-2 in T cells, which, in turn, may facilitate T cell migration into perivascular tissue. The significance of these findings in the modulation of the inflammatory response is discussed.

Interferon-gamma inhibits proliferation, differentiation, and creatine kinase activity of cultured human muscle cells. II. A possible role in myositis.

OBJECTIVE: To investigate the effects of human interferon-gamma (IFN-gamma) on cultured human skeletal muscle cells. METHODS: Muscle cell cultures were treated with various concentrations of recombinant human IFN-gamma, and muscle cell proliferation, creatine kinase synthesis and muscle cell cytotoxicity were analyzed. RESULTS: Treatment of muscle cell cultures with IFN-gamma resulted in significant inhibition of myoblasts proliferation, growth, and fusion into multinucleated myotubes. IFN-gamma inhibited creatine kinase synthesis if applied before, but not after, the myoblasts begin to differentiate into myotubes. The effect of IFN-gamma was dose dependent and observed at a concentration of IFN-gamma as low as 10 U/ml. Despite these cytostatic effects, IFN-gamma was not cytotoxic to cultured muscle cells even with very high (10,000 U/ml) IFN-gamma doses. CONCLUSION: IFN-gamma inhibits muscle cell proliferation and differentiation in vitro. These findings suggest that IFN-gamma, a T cell lymphokine, may inhibit muscle regeneration and the repair of injured muscle fibers in myositis.