The Role of C5a in the Mobilization of Hematopoietic Stem/Progenitor Cells

Abstract

The complement system, a vital component of the immune system, has been shown to play a role in hematopoietic stem/progenitor cell (HSPC) trafficking. C3a is known to be important in the retention of HSPC in the bone marrow (BM) as C3a-deficient mice are good mobilizers, and C5a is important in the mobilization of HSPC because C5a-deficient mice are poor mobilizers (Stem Cells 2007; 25: 3093). Further, granulocyte-colony stimulating factor (G-CSF) activates the complement system via the classical pathway, down-regulates stromal-derived factor (SDF)-1 in BM stromal cells and decreases expression of its receptor, CXCR4, in myeloid cells. In this work investigated the mechanism of C5a involvement in HSPC mobilization. Using RT-PCR and FACS we examined the expression of the C5a receptor (CD88) on mobilized peripheral blood (PB) and steady-state PB HSPC and mature white blood cells, and in vitro-expanded myeloid, erythroid, and megakaryocytic progenitor cells. We found thatCD88, like the G-CSF receptor, is not expressed on BM, PB or cord blood HSPC (CD34+ cells);during CD34+ cell differentiation the expression of CD88 increases in myelocytic and megakaryocytic progenitors; andthe percentage of monocytes and polymorphonuclear (PMN) cells expressing CD88 is significantly higher in mobilized than in steady-state PB.Examing the function of C5a (using flow cytometry) we found that, unlike C3a, C5a decreases CXCR4 expression in a dose-dependent manner in monocytes and PMN, but not in lymphocytes; and this effect was not seen when anti-C5a antibody was added. Interestingly, we found that G-CSF down-regulation of CXCR4 expression on PMN was partially restored by anti-C5a antibody, suggesting that the mobilizing effects of G-CSF are at least in part due to the action of C5a. Moreover, chemotaxis of PMN towards SDF-1 (chemotaxis assay) increased when these cells were stimulated with C3a but decreased with both C5a and G-CSF, reflecting the CXCR4 expression status of these cells after stimulation. We also examined the effect of C5a on matrix metalloproteinase (MMP) secretion in BM leukocytes and found that, like G-CSF, C5a increased MMP-9 and MMP-2 secretion into media (zymography). Since the SDF-1/CXCR4 axis plays an integral role in the retention of HSPC in the BM, we conclude that C5a promotes mobilization by disrupting this axis, as well as increasing MMP-9 and MMP-2 secretion by BM leukocytes, thereby allowing egress of HSPC into the PB.