Involvement of calpain in the process of Jurkat T cell chemotaxis

Abstract

Massive T cell infiltration into the central nervous system is a hallmark of multiple sclerosis (MS) and its rodent model experimental autoimmune encephalomyelitis (EAE), resulting in the induction of many of the pathophysiological events that lead to neuroinflammation and neurodegeneration. Thus, blocking T cell migration into the central nervous system may reduce disease severity in MS and EAE. One potential target for reducing T cell migration is inhibition of the Ca 2+ -activated neutral protease calpain. Previous studies in other cell types have demonstrated that migration is reduced by incubation of cells with calpain inhibitors. Thus, we hypothesize that calpain inhibition will reduce migration of T cells in response to and toward the chemokine CCL2. To test this hypothesis, the intracellular free Ca 2+ levels in Jurkat E6-1 T cells was first measured by the fura-2 assay to assess whether the intracellular ion environment would support calpain activation. The intracellular free Ca 2+ levels were found to increase in response to CCL2. The cells were next treated with the calpain inhibitor calpeptin in a multiwelled Boyden chamber with CCL2 used as the chemoattractant. These studies demonstrate that inhibition of calpain with its inhibitor calpeptin produces a dose-dependent inhibition of chemotaxis. Calpain activity, as measured by live cell imaging, was also increased in response to CCL2, providing further evidence of its involvement in the process of chemotaxis and migration. These studies provide evidence for the involvement of calpain in the mechanisms of chemotaxis and warrants further exploration in MS patient and EAE animal samples. Keywords calpain; chemotaxis; EAE/MS; T cellsT cell involvement in the disease process of multiple sclerosis (MS) (Hickey, 1991;McFarland and Martin, 2007) and its animal model experimental autoimmune encephalomyelitis (EAE) are well established (Waksman and Adams, 1962;Swanborg, 2001). T cell infiltration into the central nervous system (CNS) is greatly increased in the plaques of MS patients (Hickey, 1991) as well as of EAE animals immediately before the onset of pathological symptoms and during clinical course of the disease (Waksman and Adams, 1962). Increased activity and expression of the Ca 2+ -activated protease calpain has been demonstrated in inflammatory cells (Shields et al., 1998(Shields et al., , 1999 during the disease state in EAE animals. Moreover, inhibition of *Correspondence to: Naren L. Banik, PhD, Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 309 CSB, PO Box 250606, Charleston, SC 29425. E-mail: E-mail: baniknl@musc.edu. calpain has been demonstrated to decrease the T cell infiltration during acute EAE (Hassen et al., 2006). Calpain has signaling functions and is involved in a multitude of cellular processes including apoptosis , T cell activation (Penna et al., 1999;Schaecher et al., 2004), cell cycle (Janossy et al., 2004), and cell migration (Huttenlocher et al., 1997;Franco and Huttenl...

Calpain 2 Controls Turnover of LFA-1 Adhesions on Migrating T Lymphocytes

“…Our previous study and others indicate that calpain has a positive role in terms of promoting LFA-1- and β1- mediated integrin adhesion [21], [24], [25], [26], [27]. However in a recent report, no LFA-1 related adhesion defects were observed in T cells from mice where the calpain 4 subunit shared by calpain 1 and calpain 2 was conditionally deleted [23].…”

“…It is known that calpain is involved in the migration of slow moving cells such as fibroblasts, but whether calpain makes a contribution to the migration of immune cells has been uncertain with reports of a suppressive role in myeloid cells [22] and a recent study in T lymphocytes indicating a null effect [23]. However other studies in T lymphocytes show calpain to make a positive contribution to adhesion and migration [24], [25], [26], [27]. It is possible that conflicting results are due to off-target effects of calpain inhibitors.…”

Acute-Phase Protein α1-Antitrypsin Inhibits Neutrophil Calpain I and Induces Random Migration

“…Thus, according to our current knowledge, constitutively active calpain I expressed in human neutrophils most likely contributes to their spherical phenotype. On the other hand, the random migration of neutrophils induced by calpain inhibition is associated with their decreased directional migration toward chemotactic stimuli (24,(36)(37)(38). This latter finding and the fact that chemotaxis of activated neutrophils is inhibited by AAT (9) prompted us to investigate the putative ability of AAT to inactivate calpain I activity.…”

“…Remarkably, calpain I inhibition occurred irrespectively of the transient increase in the intracellular Ca 2+ level in response to AAT treatment. Importantly, other calpain inhibitors have also been found to induce levels of intracellular Ca 2+ (38,39 activation of Rac1/Cdc42. However, Rac1 activity has been shown to promote random cell motility (42,43) concentration.…”

Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression

“…T lymphocyte activation after TCR/CD3 complex engagement increases calpain expression (5). In turn, calpain activity is involved in T lymphocyte migration (6), secretion of IL-2 and cell surface expression of IL-2R subunit a (CD25) (7), secretion of IFN-g and Th1 commitment (8), and secretion of IL-17 and Th17 commitment (9).…”