CXCL-12 Induces Prolonged Erk-Phosphorylation in Zap-70 Positive B-CLL.

Introduction

Zap70 is superior to mutation status as a prognostic indicator in B-CLL, but whether it is causally implicated or just associated with progressive disease is unclear. Zap-70 transduces the TCR signal through the CD3 zeta-unit in T cells, but although it is important in preB development, it has no known function in mature B-cells. Zap-70 positive B-CLL show enhanced B cell receptor mediated Syk-phosphorylation upon surface IgM (sIgM) crosslinking, simulating antigen driven B cell proliferation. This may increase the proliferative drive in Zap-70+ B-CLL compared to Zap-70- leading to a higher risk of secondary chromosomal damage. Zap-70 was recently implied in Erk activation via CXCL-12/CXCR4 in T-cells and this is enhanced by cell adhesion through beta-1 integrins that may influence P-Erk phosphatases. Since both CXCL12/CXCR-4 and integrins influence B-CLL survival in vitro, we investigated the duration of downstream Erk activation in Zap-70 + versus Zap-70 B-CLL after CXCL-12 stimulation in the presence and absence of integrin binding by fibronectin.

Materials/methods

Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-gradient. Zap-70 was measured by flow cytometry (Upstate Ab), and PCR.

B-CLL cells from 6 patients were stimulated with 100 ng/ml CXCL-12 after preincubation in serum free RPMI for 2 hrs at 37°in fibronectin or BSA coated plates and in suspension culture. After CXCL-12 addition supernatant and adherent cells were separately lysed at 0, 2 and 10 minutes and studied by Western blot for total Erk, Akt, P-Erk and P-Akt. Parallel cultures were extended for 24 hours and apoptosis of supernatant versus adherent cells was performed by AnnexinV-PI staining and flow cytometry.

Results

Erk-phosphorylation was markedly stimulated by CXCL-12 after 2 minutes, both in supernatant and adherent fractions of fibronectin and BSA coated plates compared to suspension cultures. This stimulation decreased in Zap-70- B-CLL after 10 minutes but remained high in Zap-70+ B-CLL particularly in the fibronectin adherent fraction. These cells were also protected from spontaneous apoptosis at 24 hours when compared to supernatant Zap70+ and Zap-70- cells.

Conclusion

CXCL-12/CXCR4 ligation leads to prolonged Erk-phosphorylation in Zap-70+ but not in Zap70- B-CLL cells. Prolonged Erk-activation is required for nuclear translocation and effective signal transduction along this way. This effect is enhanced by concomitant fibronectin engagement of integrins, which may give Zap-70+ B-CLL cells a proliferative advantage upon homing in lymphoid tissues.