Immunohistology of murine BM identifies megakaryocytes as producers of the neutrophil chemoattractants KC and MIP-2. (A) Three KC-positive megakaryocytes (arrowheads) surround a KC-expressing BM vessel in naive BM (representative image of 4 independent experiments with 3 mice per treatment group). The lower image was stained with an IgG2a isotype control mAb. Megakaryocytes are indicated by arrowheads. (B) Expression of KC and MIP-2 on megakaryocytes and associated vessels 2 hours after injection of mice with either control PBS or G-CSF (representative image of 4 independent experiments with 3 mice per treatment group). Arrowheads indicate megakaryocytes. (C) Quantification of loss of KC and MIP-2 expression by image analysis from megakaryocytes after G-CSF or PBS treatment of mice as in panel B (AU = arbitrary units, n = 16-20 megakaryocytes from 4 independent experiments for each condition, the horizontal bar indicates the mean value). (D) Fold increase in Cxcr2 chemokine mRNAs in murine BM 2 hours after systemic G-CSF treatment as detected by qRT-PCR. Data are means ± standard deviation (SD) of a total of 6 animals measured separately in 3 independent experiments each time against 2 PBS-treated control mice. (E) Production of KC and MIP-2 by primary purified BM mouse megakaryocytes over 4 hours ± G-CSF (mean ± SD, 3 separate experiments per condition). (F) Production of IL-8 by the human megakaryocyte cell line MEG-01 over a 24-hour period (mean ± SD, 3 separate experiments per condition). Also shown is the lack of effect of coincubation of megakaryocytes with G-CSF at 20 and 50 μg/mL.