CXCR7 Participates in Chemotaxis and Homing Mediated By CXCL12

Hematopoiesis is tightly orchestrated by a precise quiescence/cycling equilibrium that is required for the continuous controlled production of differentiated blood cells. Chemokines and their receptors play an essential role in maintaining the hematopoietic cells pool within cell niches. The CXCL12/CXCR4 axis has been identified as the central axis for migration, adhesion and homing of hematopoietic cells and for leukemic cells. Another CXCL12-binding receptor has recently, been identified, CXCR7, however the contribution of this receptor to CXCL12 - mediated effects in hematopoietic cells, is still controversial, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. We recently demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic (ALL) cells and that CXCR7 contributed to T-ALL cell chemotaxis induced by CXCL12 potentializing CXCR4 response. The mouse model with reduced expression of Arhgap21^+/- exhibits a 71% downregulation of CXCR7 protein in membrane of T cells, probably due to lower recruitment by β-arrestin since GPCRs recycling to the membrane needs association with this protein. Moreover, this model showed reduction in chemotaxis, adhesion and homing of hematopoietic stem and progenitor cells suggesting that Arhgap21 may be a strong candidate for CXCL12/CXCR4 axis regulation. Thus, in the present study we aimed to investigate the involvement of CXCR7 in chemotaxis induced by CXCL12 and homing using U937 myeloid leukemic cell line in NOD/SCID mice and T-lymphocytes in Arhgap21^+/- mice.

The subcellular location of CXCR4 and CXCR7 by confocal microscopy evidenced both receptors in the membrane and cytoplasm of U937 cells. After CXCL12 induction, the blocked of CXCR4 by AMD3100 and/or the inhibition of CXCR7 by transduction with lentivirus mediated shRNA resulted in significant changes in U937 cells chemotactic response analyzed by transwell assay (U937 treated with AMD3100, p=0.0019; U937 shCXCR7, p<0.001;U937 shCXCR7 treated with AMD3100, p=0.0056; Mann-Whitney test) and homing assay to spleen of NOD/SCID mouse after 16 hours of transplant (U937 treated with AMD3100, p=0.0070; U937 shCXCR7, p=0.044 6; U937 shCXCR7 treated with AMD3100, p=0.0243, Student's t test). Bone marrow analysis of Arhgap21^+/- mice showed a reduction of T lymphocytes (CD4⁺ andCD8⁺; p=0.02, Student's t test), erythroblasts (Ter119⁺, p=0.01) and myeloid cells (Gr1⁺ Mac⁺, p=0.02), but there was no difference however in B lymphocytes (B220⁺). Indeed, peripheral blood showed a significant reduction in CD8⁺ cells (p=0.04) but similar amounts of CD4⁺ cells. Thymus did not show difference in CD4⁺ and CD8⁺ populations. Herein, we show by transwell assay a lower response of T lymphocytes to CXCL12 (p=0.0050, Mann-Whitney test), corroborating to the reduced CXCR7 expression observed. No difference in CXCR4 expression was observed in any of the cell types.

Taken together, our results suggest that CXCR7 is involved in chemotaxis and homing of T-lymphocytes and myeloid leukemic cells, potentalizing the CXCR4 response. The reduction of T-lymphocytes in bone marrow of Arhgap21^+/- mice, may be related to a reduced homing due to the reduction of CXCR7 in the membrane of these cells, which leads to a lower attraction induced by CXCL12 ligant to bone marrow. Moreover, an inhibition of both receptors in myeloid leukemic cells could have a better effect in reducing their homing than the blocking of a single receptor.