CAE⁺ and Gr-1⁺ cells (neutrophils) but not F4/80⁺ or M-CSF receptor (c-fms)⁺ cells (monocyte/macrophages) developed in vitro from PU.1 deficient progenitors when cultured in SCF, IL-3, and IL-6. (A) Wright-Giemsa-stained cytospins of representative multilineage colonies from control and PU.1 null individuals revealed a conspicuous absence of macrophages in PU.1 deficient colonies. However, neutrophils and megakaryocytes were evident in both panels (1,150×). (B) The neutrophil enzyme CAE, demonstrated by a pink reaction product, was evident in PU.1 deficient and control polymorphonuclear cells. Note the larger, nonstaining macrophage in the control individual (1,150×). (C) Both PU.1 deficient and control polymorphonuclear cells expressed the cell surface marker Gr-1 which was demonstrated by immunoperoxidase staining. Detection was with diaminobenzidine (DAB) which yields an orange-brown reaction product (1,150×). (D) M-CSF receptor immunostaining revealed no M-CSF receptor-positive cells in PU.1 deficient colonies. Cell debris was present that stained nonspecifically with DAB in PU.1 null cultures (1,150×). (E) Immunocytochemical staining for the macrophage marker F4/80 revealed no positive cells in PU.1 deficient colonies, whereas many orange-staining F4/80⁺ cells were found in control colonies (1,150×).