Primitive erythroblasts interact with fetal liver macrophage cells in vitro. (A) Immunohistochemical analysis of 2 erythroblast islands reconstituted with E14.5 blood and E14.5 liver macrophages. (Left panel) ϵγ-globin–positive primitive erythroid cells (red) attached to F4/80-positive macrophage cells (blue). (Right panel) The same erythroblast islands with nuclei stained by HO. Small bright nuclei of the primitive erythroblasts are seen as well as larger fainter macrophage nuclei (m). An enucleated primitive erythrocyte is indicated by an arrowhead. (B) Primitive blood cells preferentially bind F4/80-positive macrophages compared with F4/80-negative adherent cells. The percentage of F4/80-positive macrophages (left panel) and F4/80-negative adherent cells (right panel) with 0, 1, 2, 3, or 4 or more ϵγ-globin–positive cells attached is plotted. These data represent the mean plus or minus SEM of 3 independent experiments. (C) The presence of α4-integrin blocking antibody during the reconstitution of erythroblast islands resulted in both a significant decrease in the percentage of macrophages in erythroblast islands, defined as containing 4 or more erythroblasts (4+, red bars) and a significant increase in the percentage of macrophages lacking any attached primitive blood cells (0, black bars) compared with isotype controls (P < .02). These data represent the mean plus or minus SEM of 3 independent experiments. (D) The addition of α4-integrin blocking antibody also caused a decrease in the average number of primitive erythroid cells per reconstituted erythroblast island (defined as 4 or more primitive erythroid cells per macrophage) compared with isotype controls (P ≤ .005). The results of 4 independent experiments are shown.