Abstract
Mononuclear phagocytes (MNPs) are critical in health to maintain tissue homeostasis and in disease as major effectors of innate immunity. In the adult, MNPs develop from bone marrow (BM) progenitors that differentiate to monocytes, tissue macrophages (Mϕs), and specialized cells (dendritic cells, microglia and osteoclasts). Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of MNPs. GAB2, a member of the GAB family of scaffolding proteins (GAB1-3), modulates and amplifies signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and Shp2 phosphatase. Knockdown studies in the 32D myeloid cell line from our lab showed that GAB2 is required for CSF-1 induced mitogenesis and activation of Akt, a PI3K effector. To test the hypothesis that the GAB2-PI3K axis is important for MNP development in vivo, we examined Mϕ development in GAB2 +/+ and −/− mice (gift of Josef Penninger). GAB2 is upregulated 14-fold during CSF-1-induced differentiation of primary BM cells from GAB2+/+ mice. A significant difference is detected in the steady state percentage of F4/80+ BM cells (F4/80 is a mature Mϕ marker): 17.5 ± 1.6 (GAB2+/+, n=8) vs. 11.4 ± 1.6 (GAB2–/−, n=6) (p=0.025, 2-sided t-test). Using the CFU-C progenitor assay with CSF-1 as the only growth factor, primary BM cells from GAB2 −/− mice show a striking 7-fold reduction in colony numbers compared to those from GAB2 +/+ mice (p=0.004) and the colonies were much smaller. Thus GAB2 is essential for optimal CSF-1-dependent Mϕ colony formation. We then used CD31 and Ly6C and flow cytometry to follow the kinetics of Mϕ formation during BM differentiation. These markers monitor sequential stages of Mϕ development: CD31highLy6C– -> CD31+Ly6C+ -> CD31-Ly6Chigh (Eur. J. Immunol.24:2279). As early as 2 days after differentiation induction, GAB2−/− BM cells show a 2-fold reduction in the CD31+Ly6C+ subset (p=6×10−6) and a 6-fold increase in the CD31-Ly6Chigh subset (p=1×10−4), indicating that in the absence of GAB2, CSF-1 promotes a smaller increase in myeloid progenitors and an earlier appearance of more mature cells. To assess proliferation in the progenitor population, day 2 BM cells were labeled with CFSE. Consistent with decreased cell division during early stages of Mϕ development in the absence of GAB2, we observed a 66% reduction in CFSE intensity in GAB2+/+ compared to −/− cells after 3 days in culture. A 2-fold reduction in proliferation by the MTS assay is similarly observed during late Mϕ development (days 5-7) (p=10−4). No difference in viability or expression of CSF-1R or CD11b is found in day 7 Mϕs from GAB2+/+ and −/− mice, excluding increased cell death or arrested differentiation as causes. To investigate the role of GAB2-PI3K, we transduced BM cells with viruses expressing WT-GAB2, 3YF-GAB2 (defective in PI3K binding), both in MSCV-IRES-GFP, or empty MSCV. WT- and 3YF-GAB2 expression in GAB2−/− cells increase the numbers of CFU-Cs by 5- and 2-fold respectively and by 8- and 2.4-fold in GFP+ colonies ≥ 500 μ. Conversely, 3YF-GAB2 exerts a dominant-negative effect on GAB2+/+ cells (a decrease of 30% and 76% in unsorted cells and GFP+ colonies ≥ 500 μ respectively). Therefore PI3K recruitment by GAB2 is required for CSF-1-induced Mϕ colony formation but other GAB2 effector pathways are also important. Our findings support the conclusion that GAB2 is crucial for CSF-1 mediated Mϕ development in the BM, by regulating monocyte/Mϕ progenitor expansion and Mϕ proliferation, in part through PI3K.
Glycosaminoglycans in rat mucosal mast cells