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.

Fig. 1

Changes in intracellular free [Ca²⁺] in response to various ionic stimulus, chemokine exposure, and protease inhibition. Changes in intracellular free [Ca²⁺] were measured with the ratio-metric intracellular fluorescent dye fura-2 loaded into Jurkat E6-1 T cells and read on a fluorescent plate reader after exposure to experimental compounds. A: Intracellular free [Ca²⁺] rises to a steady state in response to increased extracellular [Ca²⁺]. B: Ionomycin, a calcium ionophore, effectively transports Ca²⁺ across the plasma membrane and creates an intracellular free [Ca²⁺] spike in the cell. C: The chemokine CCL2 causes an increase in intracellular free [Ca²⁺] at an optimum concentration of 10 ng/mL. D: The calpain inhibitor calpeptin causes an increase of intracellular free [Ca²⁺] in a concentration-dependent manner. E: The calpain inhibitor PD150606 causes an increase of intracellular free [Ca²⁺] in a concentration-dependent manner. Each time point represents the mean ± SEM (n = 6–8/time point).

Fig. 2

Effect of the calpain inhibitor calpeptin on CCL2-induced chemokinesis and chemotaxis in Jurkat E6-1 T cells. Cells were loaded with the fluorescent dye calcein AM to aid in cell tracking and then loaded into a 96-well Boyden Chamber with various treatments to assess migration status. A: CCL2 (50 ng/mL) was placed both above and below the chamber’s filter membrane to study the chemokinetic response or was only placed below the filter to study the chemotactic response. Mean ± SEM (n = 8). *P < 0.05 vs. unstimulated; †P < 0.05 vs. chemokinesis. B: Calpain inhibition with any of the concentrations of calpeptin in the absence of CCL2 did not significantly alter the levels of migration. C: Inhibition of calpain with calpeptin did not significantly alter the migration of Jurkat T cells in response to chemokinetic stimulation (50 ng/mL CCL2 placed above and below the filter membrane). D: Calpeptin caused a concentration-dependent inhibition of migration of cells toward CCL2 (50 ng/mL). Mean ± SEM (n = 8). *P < 0.05 vs. vehicle (set as 100% chemotaxis).

Fig. 3

Effect of the calpain inhibitor calpeptin on CCL2-induced chemotaxis in human PBMCs. PBMCs were loaded with the fluorescent dye calcein AM to aid in cell tracking and then loaded into a 96-well Boyden chamber with various treatments to assess migration status. Calpeptin caused a concentration-dependent inhibition of migration of cells toward CCL2 (100 ng/mL). Mean ± SEM (n = 3). *P < 0.05 vs. control (set as 100% chemotaxis).

Fig. 4

Effect of chemoattractant concentration on calpain inhibitor-induced reduction in chemotaxis: a response distinct from the inhibition by cytochalasin D. Chemoattractant was loaded in the lower wells of a 96-well Boyden chamber in increasing concentrations while Jurkat T cells were loaded in the upper chamber. A: The chemotaxis of Jurkat T cells in the increasing chemoattractant gradient was compared with chemotaxis in the same gradient when an IC₅₀ concentration of calpeptin was added to the cells in the upper wells. Chemotaxis of Jurkat T cells was highest at an optimum CCL2 concentration of 50 ng/mL. Calpeptin-induced reduction in chemotaxis was observed only up to 50 ng/mL CCL2 and the effect was lost at higher CCL2 concentration. Mean ± SEM (n = 8). *P < 0.05 between groups for media in upper chamber vs. calpain inhibitor; †P < 0.05 migration inhibition compared with control. B: The chemotaxis of Jurkat T cells in the increasing chemoattractant gradient was compared with chemotaxis in the same gradient when the actin polymerization inhibitor cytochalasin D was added to the cells in the upper wells. Cytochalasin D groups were not significantly different from control; however, it could significantly attenuate the CCL2-induced chemotaxis. Mean ± SEM (n = 8). *P < 0.05 between groups for media in upper chamber vs. cytochalasin D. C: The chemotaxis of Jurkat T cells in the increasing chemoattractant gradient was compared with chemotaxis in the same gradient when cytochalasin D and calpeptin was added to the upper wells. Combined treatment group displayed the same trend as cytochalasin D treatment alone. Mean ± SEM (n = 4). *P < 0.05 between groups for media in upper chamber vs. calpeptin and cytochalasin D.

Fig. 5

Increased intracellular calpain activity in response to chemokine CCL2. Increased calpain activity was imaged in live Jurkat E6-1 T cells based on the increasing fluorescence from the calpain-specific substrate CMAC, t-BOC-Leu-Met after exposure to the chemokine CCL2 (50 ng/mL) or the calpain inhibitor calpeptin (100 μM). A: Representative micrographs from a 15-min time lapse exposure measuring fluorescence of 50 μM CMAC at 370 nm. B: Representative images of spot density selections of random cells, repeating for every time point. C: The average spot density of 10 individual cells over the time lapse series for each exposure group. The mean of 10 cells was also included on each of the graphs. D: The change in average fluorescence density for the mean of each experimental group. Mean ± SEM (n = 10). *P < 0.05 between the experimental groups and basal dF/dt; †P < 0.05 between the CCL2 group and CCL2 + calpeptin group. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]