Role of Mitochondrial NCX on CXCL12-Induced Chemotaxis in A20 B Lymphocytes

Abstract

Lymphocyte chemotaxis plays important roles in immunological reactions. It has been reported that mitochondria redistribute at the uropod during T lymphocyte migration triggered by a chemokine ligand (CXCL12). However, how mitochondria are involved in lymphocyte chemotaxis has been unclear. We studied roles of mitochondria Ca2+ handling proteins, Na-Ca exchange (NCXm) and Ca2+ uniporter, on CXCL12-induced chemotaxis in A20 B lymphocytes. CXCL12 (100 ng/ml) increased transwell migration of A20 cells from 4.6 ± 0.5 % (non-stimulated cells) to 12.6 ± 0.6 % (P < 0.05). This increase was dose-dependently inhibited by CGP-37157 (an inhibitor of NCXm), but not affected by Ru360 (an inhibitor of mitochondrial Ca2+ uniporter). Knock-down of a gene of NCXm (NCLX) by siRNA reduced NCLX protein expression to 52 ± 9 % and inhibited transwell migration similarly to CGP-37157. In the 8 hrs observation of cell migration under microscope, mean displacement of NCLX siRNA cells without CXCL12 was larger (21.4 ± 1.5 μm) than control siRNA cells (13.5 ± 2.2 μm, P < 0.05), and applying CXCL12 did not increase mean displacement and percentage of directional migration in NCLX siRNA cells. Intracellular Ca2+ measured by fura-2 was higher in NCLX siRNA cells (fura-2 ratio 0.49 ± 0.01) than the control siRNA cells (0.45 ± 0.01, P < 0.05) in the absence of CXCL12. After 2 hrs CXCL12 stimulation, the intracellular Ca2+ increased in control siRNA but not in NCLX siRNA cells. Treating A20 cells with BAPTA-AM (an intracellular Ca2+ chelator) suppressed the cell migration. Our results suggest that NCLX-mediated Ca2+ signaling is associated with CXCL12-induced chemotaxis in B lymphocytes.