Modulation of Responsiveness of Human CD34+ Cells to CXL12/SDF-1 through Desensitization of CXCR4 by CCR5 Chemokines: Possible Implications for Mobilization during Inflammation.

Abstract In addition to G-CSF, agents like chemokines and the CXCR4 antagonist AMD3100 mobilize hematopoietic stem/progenitor cells (HSPCs). Unlike G-CSF-induced mobilization that has slow kinetics; chemokine- and AMD3100-mediated mobilization is rapid and generally occurs within a few hours. Although a number of pathways have been implicated in mobilization, disruption of the CXCL12/SDF-1-CXCR4 axis is considered central to mobilization. In addition to the above agents, HSPCs are also mobilized during infection. We were interested in unraveling possible mechanism(s) underlying mobilization of HSPCs from bone marrow into the circulation during inflammation. Endotoxin, a component of the cell wall of gram negative bacteria, plays a role in mobilization of HSPCs (Broxmeyer HE and Williams DE Crit Rev Oncol Hematol , 1988,173 ; Velders GA et al Blood , 2004, 340). Endotoxin directly affects chemotactic response of neutrophils by signaling through TLR4 and modulating G-receptor kinases activities (Fan J and Malik AB Nature , 2003, 315). Towards our goal, we studied agents that might modulate CXCL12 -mediated responses (chemotaxis and adhesion) of CD34+ cells. Using purified CD34+ cells from human cord blood (CB) we found that unlike neutrophils, endotoxin did not affect chemotaxis of CD34+CB cells towards SDF-1, thus ruling out a direct effect of endotoxin on this event. HSPCs are highly selective in their chemotaxis response and only known to respond to CXCL12 (Wright DE et al J Exp Med , 2002, 1145). However, they express low levels of receptors for other chemokines, including CCR5. During inflammation, levels of many chemokines, including CCR5 responsive chemokines, are elevated. We reasoned that during inflammation, production of inflammatory chemokines leads to activation of their receptors on HSPCs cell surface, and possibly these chemokine receptors could cross-talk with CXCR4, the receptor for CXCL12 and desensitize CXCR4. Since disruption of CXCL12/CXCR4 axis leads to mobilization, desensitization of CXCR4 could result in mobilization of HSPCs into the circulation. We evaluated CCR5 responsive chemokines, CCL3, CCL4 and CCL5 for modulation of responses of CD34+CB cells to CXCL12. We first studied the chemotactic response of CD34+CB cells to CXCL12 alone and in the presence of various concentrations of CCL4, CCL4 and CCL5 using traditional transwell chambers. CCL4 had the most pronounced effect and decreased the chemotactic response of CD34+CB cells towards CXCL12. However, none of the CCR5 ligands induced chemotaxis. Inhibition of CXCL12 mediated chemotaxis by CCL4 was dose-dependent and maximal inhibition was observed in the presence of 1000 ng/ml of CCL4. CCL4 also reduced CXCL12 stimulated adhesion of CD34+CB cells to fibronectin. Expression of CXCR4 on cell surface was not altered in the presence of CCL4, but both calcium flux and Erk1/Erk2 phosphorylation in response to SDF-1 was reduced in the presence of CCL4. Interestingly, we found that although only a small proportion of CD34+CB cells express CCR5 on the surface, greater than 90% of CD34+ cells have a large pool of CCR5 inside the cell. Current studies are underway to determine whether expression of CCR5 on CD34+CB cells can be modulated by inflammatory cytokine(s)/chemokine(s). Our study demonstrates cross-talk from CCR5 ligands can down-modulate intracellular responses of CXCL-12 in CD34+ cells. This may provide a possible mechanism that underlies mobilization of HSPCs during inflammation.