Defective expression of Bcl-2 in the bone marrow of Kostmann patients. (A) Immunohistochemical detection of the antiapoptotic protein Bcl-2 was performed in bone marrow sections from patient 2 prior to and after the initiation of G-CSF treatment. Sections were also stained for the proapoptotic protein Bax prior to and after the initiation of treatment. Original magnification × 400. A markedly reduced expression of Bcl-2 was observed in myeloid cells in the pretreatment specimen, whereas Bcl-2-positive small T lymphocytes were readily seen in the same section. After initiation of G-CSF therapy, the expression of Bcl-2 in myeloid cells was normalized. No differences were seen in Bax expression in sections obtained before and during treatment with G-CSF. Similar data were obtained in all Kostmann patients included in the present study. As an external control, bone marrow sections from a healthy individual were stained for Bcl-2 and Bax. (B) Bone marrow cell suspensions from Kostmann patients and controls (mean ± standard deviation [SD]; n = 3) were stained for cell surface markers and intracellular Bcl-2 as described in “Patients, materials, and methods.” Patients were off G-CSF therapy for 60 hours prior to bone marrow aspiration. Data shown are the mean fluorescence intensities (MFIs) of Bcl-2-FITC in CD34^-CD13dimCD11bdim cells (▪), and CD3⁺ cells (□) (the latter data serve as an internal control for Bcl-2 staining procedures). The percentages of cells in the different stages of granulopoiesis were normal in all patients, with the exception of patient 4, who displayed an increase in the number of early precursor cells (data not shown).