Effect of TNF- on gelatinolytic activities expressed by PB and BM CD34⁺ cells and on migration of clonogenic progenitors. (A) Time dependence of gelatinolytic activity and the effect of TNF-. The zymographic analysis of gelatinases secreted by CD34⁺ cells from PB and BM was performed in the absence (control lanes) and presence of TNF- (final concentration, 1 ng/mL). Cell-free supernates were withdrawn after incubating the cells at 37°C and 5% CO₂ in serum-free IMDM at the times (2, 16, and 24 hours) indicated. Data are representative of three independent experiments using CD34⁺ cells from both steady-state BM and G-CSF–mobilized PB. (B) RT-PCR analysis of transcripts expressed by PB and BM CD34⁺ cells in the absence (c) or presence of 1 ng/mL TNF-. mRNA was isolated from cell pellets obtained after incubating the cells at 37°C and 5% CO₂ in serum-free IMDM for 24 hours. GAPDH was used as the mRNA internal control to ensure equivalence of loading. (C) Dose-dependence of the stimulatory effect of TNF- on gelatinolytic activity of PB CD34⁺ cells. Zymographic analysis of media conditioned by PB CD34⁺ cells in the presence of various concentrations of TNF-. The cells were incubated in serum-free IMDM for 16 hours in the absence (c lane) and in the presence of 0.1, 1.0, and 20 ng/mL TNF-. (D) Migration of PB CD34⁺ cells and clonogenic progenitor cells in response to various concentrations of TNF- (0, 1.0, and 20.0 ng/mL). Bar graphs represent the mean and standard deviations of duplicate experiments. There was a significant increase in the percentage migration of CD34⁺ cells in the presence of TNF- relative to the control (P = .016 for 1 ng/mL TNF- and P = .001 for 20 ng/mL TNF-). For the clonogenic assay, cells suspended in equal volumes of media obtained from the lower compartments of the Boyden chambers were plated. The graph shows the mean number of CFU-GM, BFU-E, CFU-GEMM, and CFU-MK progenitors scored from quadruplicate plates. Except for the CFU-MK, the numbers of colony-forming progenitors in the presence of 1 and 20 ng/mL TNF- are significantly different (P ≤ .005) compared with the control.