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Emily O'Koren, Rose Mathew, Daniel Saban; Searching for phenotypic differences between microglia and monocyte-derived macrophages in the neuroretina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):871.
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© ARVO (1962-2015); The Authors (2016-present)
It is now established that monocyte-derived macrophages seen in neuroinflammation and normal tissue resident microglia have distinct developmental origins (bone marrow and yolk sac, respectively). Lack of discriminatory markers for these two populations has thwarted efforts to understand their respective contributions in pathological states. The current study sought to identify differentially expressed cell surface markers of monocyte-derived macrophages versus microglia in the neuroretina.
C57BL/6 mice were lethally irradiated and reconstituted with GFP+ bone marrow cells in order to discriminate microglia (GFP-) from monocyte-derived macrophages (GFP+). One cohort of host mice was given lead helmet shielding against irradiation-induced neuroretinal injury, whereas the other cohort was left unshielded (n=4 to 5 mice/group). Non-irradiated/naïve mice (n=5) were also included. 4 months post-irradiation, flow cytometric analysis of neuroretinal immune cells was performed. Discrimination of intravascular cells was enabled by infusion of CD45/BV650 Ab five minutes prior to euthanasia. Discrimination of extravascular cells was enabled via additional ex vivo staining with CD45/APCCy7 Ab. Samples were additionally stained for CD11b, Ly6C, Ly6G, F4/80, CD64, I-A/I-E.
Microglia and monocyte-derived macrophages in neuroretina were identified as BV650/CD45- APCCy7/CD45+ CD11b+ Ly6C- Ly6G- F4/80+ CD64+. Greater than 60% of these cells were GFP+ in unshielded mice, whereas GFP+ cells were completely undetectable in shielded counterparts. GFP+ cells from unshielded mice were analyzed for mean fluorescence intensity (MFI) levels of CD45, CD11b, F4/80, CD64, I-A/E expression, and compared to GFP- cells from unshielded, shielded, and naïve mice. While the MFI of CD45 and I-A/E were not different (p>0.05), the MFI of CD11b was slightly higher (p=0.002) and CD64 was slightly lower (p=0.02) in GFP+ cells. Strikingly, MFI of F4/80 showed a 4-fold increase in GFP+ cells (p=0.001).
Our data suggest that the two populations express certain markers at different levels. Surprisingly, CD45 was not differentially expressed, which may suggest that differentiation of monocytes into macrophages in the neuroretina is associated with down-regulation of CD45. Most striking difference was in F4/80, possibly suggesting that it can help distinguish the 2 populations in neuroinflammation.
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