ZAP-70 enhances migration of malignant B lymphocytes toward CCL21 by inducing CCR7 expression via IgM-ERK1/2 activation

Abstract

IntroductionZAP-70 protein is a 70-kDa member of the Syk family of protein tyrosine kinases that was first identified as a crucial element for proximal signaling from the T-cell receptor. 1 Similar to Syk protein after the B-cell receptor (BCR) stimulation, ZAP-70 is recruited to the phosphorylated immunoreceptor tyrosine activation motifs of the -and CD3-chains present in the T-cell receptor, where it subsequently becomes phosphorylated and initiates several signaling cascades. 2 Expression of ZAP-70 protein was considered to be restricted to T lymphocytes and natural killer cells. However, it has also been found expressed in normal B-cell precursors and in some subsets of activated B cells. [3][4][5][6] Among B cell-derived malignancies, ZAP-70 is mainly expressed in chronic lymphocytic leukemia (CLL; 37%-57% of cases), 7-9 B-acute lymphoblastic leukemia (B-ALL; 56%-59% of cases) 4,10 and Burkitt lymphoma (8%-31% of cases). 11,12 An increased ZAP-70 expression in CLL has been associated with particular adverse biologic features, such as the presence of unmutated IgHV genes 7 or high CD38 expression, 13 and correlates with a poor clinical outcome. [7][8][9] In the same vein, ZAP-70 expression in B-ALL correlates with a short survival as well. 10 Although ZAP-70 expression in B-cell malignancies has an adverse prognostic influence, its role in the biology of the tumoral B cell is not fully defined. In this regard, the expression of ZAP-70 protein in CLL cells has been related to an enhanced BCR signaling. [13][14][15][16][17] In addition, increased ZAP-70 expression has been associated with increased migration capabilities of CLL cells toward different chemokines, such as CXCL12, CCL19, and CCL21, [18][19][20] and with increased signaling and survival on CXCL12 treatment. 18,21 However, whether these increased migrative capabilities are a direct effect of ZAP-70 expression or a mere reflection of the distinct biology features of ZAP-70-expressing cells needs to be further investigated.To ascertain the direct implication of ZAP-70 in B-cell signaling and migration, we analyzed the phenotypic effects of ectopic ZAP-70 expression in a B-cell system and studied the expression of adhesion molecules and chemokine receptors in CLL primary cells with high or low ZAP-70 expression within the same patient. Herein, we report that IgM, but not IgD, stimulation mobilizes and activates ZAP-70, which in turn enhances BCR-induced ERK1/2 and Akt phosphorylation and delays IgM and CD79b internalization. Moreover, we show that ZAP-70 induces the expression of the chemokine receptor CCR7 via ERK1/2 activation, thus directly enhancing the capacity of signaling and migration of the ZAP-70-expressing B cells toward CCL21. Finally, we show that CLL cells with higher ZAP-70 expression within the same patient have a different expression profile of adhesion molecules and chemokine receptors and enhanced migration capacity toward CCL21. Methods Cell lines and primary cellsThe Burkitt lymphoma B-cell lines Raji and Ramos were obtained from ...

The role of ZAP70 kinase in acute lymphoblastic leukemia infiltration into the central nervous system

“…38,39 ALL cells upregulate chemokine receptors, such as CXCR4, CCR3, CCR4 and CCR7, 25,40,41 and ZAP70 was shown to enhance the migration of lymphocytes towards chemokines, e.g., CXCL12, CCL21 or CCL19. 21,23,24 Herein we show that CCR7/CXCR4 expression was reduced in 697-shZAP70 cells, which impaired their migration toward CCL19/CXCL12. Similarly, enforced ZAP70 expression in 697 cells increased the expression of CCR7/CXCR4 and subsequent migration toward their ligand (Figure 2).…”

“…This observation differs from the situation in B-CLL, where cells with lower ZAP70 expression showed a reduced BM homing. 21,22 In JURKAT T-ALL cells, downregulation of ZAP70 did not prevent CNS infiltration; however, it was associated with a reduction in CNS blasts recovered from xenografts ( Figure 1D). Downregulating ZAP70 in JURKAT cells did not prolong xenograft survival.…”

“…21,24 Chemokine receptors CCR7 and CXCR4 in turn have been associated with an increased capability of T-ALL cells to enter the CNS. 25,26 We hypothesized that ZAP70 mediated upregulation of CCR7 and CXCR4 may enhance the homing and the survival of CNS-prone BCP-ALL cells in the CNS niche.…”

“…21 5 × 10 5 cells were added to the top chamber of a transwell culture insert (Corning) and allowed to migrate toward media containing 2 μg/ml CCL19 or 0.1 μg/ml CXCL12 (ImmunoTools) for 16 h. The cell number in the lower chamber was determined.…”

“…ZAP70 has been shown to enhance the migration of malignant B cells to the bone marrow (BM) by upregulating adhesion molecules and chemokine receptors (CCRs), 21,22 and is also required for C-X-C motif chemokine ligand 12 (CXCL12)-mediated T-cell transendothelial migration. 23,24 In T-cell acute lymphoblastic leukemia (T-ALL), the chemokine receptors CCR7 and CXCR4 have been associated with an increased capability of T-ALL cells to enter the CNS, mainly in cell line models.…”