Gi Protein Signals Are Required for BM Homing of Hemopoietic Progenitor Cells, and Cooperate with Alpha4-Intergrin and Endothelial Selectins.

Several examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of Gi protein coupled receptor signaling and as such, it blocks hemopoietic progenitor cell (HPC) migration in vitro. However, contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on BM homing of HPC. We found that prolonged incubation of BM cells or fetal liver cells with PTX inhibited their BM homing in irradiated or non-irradiated recipients by >75%. A concomitant increase in circulating CFU-C in blood provided added assurance that the data were not due to non-specific toxicity. This inhibition of BM homing was of functional consequence, since PTX-treated cells provided impaired radioprotection and markedly decreased short-term engraftment. Detailed studies showed that inhibition of in vitro migration and ribosylation of Gi proteins were complete only after extended incubation with PTX, whereas short-term incubation for 1–2 h, as used in previous studies, was insufficient. In addition, the incubation of BM cells with SCF may have exaggerated the negative effect of PTX on the inhibition on BM homing. We next sought to test the basis of the residual 25% homing of PTX treated cells. As PTX did not directly inhibit hemopoietic cell adhesion, we tested homing of PTX-incubated and control BM cells in genetic models deficient in certain adhesion molecules on either the hemopoietic cells or the hemopoietic microenvironment. BM homing of PTX-treated α4integrin-/- BM cells transplanted into wild-type (WT) mice, or PTX-treated WT cells transplanted into EPselectin-/- recipients inhibited BM homing significantly stronger than either modality alone, to >90% and >95%, respectively. Of note, untreated WT cells have normal homing in EPselectin-/- mice. These data demonstrate cooperativity between Gi blockade and α4integrin or endothelial selectin blockade in inhibiting BM homing of progenitor cells. Absence of β2integrin or L-selectin in the presence or absence of PTX did not inhibit BM homing. In summary, these studies show that Gi protein signals are required for BM homing of progenitor cells. PTX is a very strong inhibitor of BM homing, suggesting that it may inhibit convergent signals from more than one mediator. We further show that the residual BM homing of migration-impaired BM cells is due to participation of α4 integrin on the transplanted cells, or to endothelial selectins on the host side.