Abstract
Signal regulatory protein-α (SIRPA) is an immunoglobulin superfamily transmembrane protein with intracellular docking sites for two Src homology domain containing tyrosine phosphatases, and most abundantly expressed in neurons and myeloid cells. SIRPA is a critical immune inhibitory receptor on macrophages. CD47 is a ligand for SIRPA, and CD47 interaction with SIRPA serves as a ‘self-recognition’ that prevents phagocytosis of the cells expressing CD47. Recently, we (Nature immunology 2007) identified polymorphism in murine Sirpa as a critical modulator of engraftment in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation, and non-obese diabetic (NOD) mouse Sirpa polymorphism has far greater reactivity with human CD47 than that of the respective alleles of other strains resulting in effective engraftment of human hematopoietic cells. In addition, as well as the mouse, human SIRPA is highly polymorphic in the IgV domain. Then, we examined whether polymorphism in SIRPA induced the difference for suppressive effect of human macrophage on hematopoiesis. First, we examined sequence alignment of SIRPA IgV domain (exon 3 of SIRPA) of healthy control 18 people. We identified two major variants; 6 people with variant1 (V1) and 12 people with variant2 (V2). The SIRPA amino acid sequences of V1 and V2 are different in 13 residues, and its residues in orthologous position between species that is polymorphic between NOD and other strains as well as between V2 and V1. Next, we examined that whether there is a difference in the effect on hematopoiesis between V1 macrophage and V2 macrophage by long term culture-initiating cells (LTC-IC) assay on MS-5 stromal cells. For macrophage preparation, peripheral mononuclear cells from healthy donors were purified by positive selection using MACS CD14 Micro Beads (Miltenyi Biotec), and they were incubated with M-CSF for 3 days. For LTC-IC assay, 5×102 differentiated macrophages were seeded onto established MS-5 stroma in 96-well tissue culture plates. The next day, human hematopoietic CD34+ cells were seeded at doses of 102 to 5×103 cells per well and cultured for 4 weeks. At the end of the culture, cells were detached and plated into methylcellulose progenitor assays. Macrophage had a suppressive effect on the number of LTC-IC in all cultures. There was tendency that V1 macrophage had a greater suppression compared to V2 macrophage, however the differences between V1 and V2 were marginal (p=0.03). These data suggests that human macrophages have suppressive effect on hematopoiesis, and human SIRPA polymorphism modulates macrophage-mediated suppression of hematopoiesis in allogenic model, likewise in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation. Moreover, SIRPA polymorphism might be related to graft failure in the allogenic hematopoietic stem cell transplantation.
