Fig. 5.
Fig. 5. IL-5R expression by developing primary human eosinophils. / (A) The effect of cytokine treatment on IL-5Rα isoform mRNA expression by human cord blood CD34+ cell-derived eosinophil progenitors. RT-PCR using primers specific for the TM-transcript, both TM and SOL transcripts, and β actin mRNA was performed on RNA extracted from cord blood CD34+ cells cultured in IL-3+IL-5 for 0, 3, 5, 7, or 14 days. Results are representative of 5 separate experiments. (B) Interleukin 5Rα surface expression is detected after 7 days of culture of CD34+progenitors in IL-3 and IL-5. Flow cytometry plot shows surface staining with α16 (an IgG1 mouse monoclonal antibody to human IL-5Rα chain) or isotype control IgG1 antibody. CD34+cord blood-derived progenitors were stained at the time of isolation (day 0) and after 7 days of culture in IL-3 and IL-5. The plot shown is typical of 10 independent experiments. (C) CD34+ umbilical cord blood progenitors acquire biologic responsiveness to IL-5 by day 7 of culture in IL-3 and IL-5. Chemokinetic and polarization responses of cord blood-derived CD34+ cells cultured in IL-3 and IL-5 for 4, 7, and 14 days. Cells added to the top well were exposed to medium alone (open bars) or IL-5 at 0.1 nM (hatched bars) or 1 nM (black bars). Chemokinetic index is the number of cells in the bottom well at 1 hour with stimulus divided by that with medium alone. Polarization in response to medium alone or 0.1 nM and 1 nM IL-5 was assessed by change in forward scatter (FSC) signal on flow cytometry (mature peripheral blood eosinophils showed a mean change in forward scatter of 120 units to 1 nM IL-5, n = 3, data not shown). The figure shows a typical response at days 7 and 14. No response to IL-5 was seen at day 3 to 4 in 7 independent experiments. Responsiveness of cells cultured for 7 days to IL-5 was variable and may reflect different rates of eosinophil development in different cultures; however, similar responses to those shown were seen from cells cultured for 7 days in IL-3 and IL-5 in 4 independent experiments.

IL-5R expression by developing primary human eosinophils.

(A) The effect of cytokine treatment on IL-5Rα isoform mRNA expression by human cord blood CD34+ cell-derived eosinophil progenitors. RT-PCR using primers specific for the TM-transcript, both TM and SOL transcripts, and β actin mRNA was performed on RNA extracted from cord blood CD34+ cells cultured in IL-3+IL-5 for 0, 3, 5, 7, or 14 days. Results are representative of 5 separate experiments. (B) Interleukin 5Rα surface expression is detected after 7 days of culture of CD34+progenitors in IL-3 and IL-5. Flow cytometry plot shows surface staining with α16 (an IgG1 mouse monoclonal antibody to human IL-5Rα chain) or isotype control IgG1 antibody. CD34+cord blood-derived progenitors were stained at the time of isolation (day 0) and after 7 days of culture in IL-3 and IL-5. The plot shown is typical of 10 independent experiments. (C) CD34+ umbilical cord blood progenitors acquire biologic responsiveness to IL-5 by day 7 of culture in IL-3 and IL-5. Chemokinetic and polarization responses of cord blood-derived CD34+ cells cultured in IL-3 and IL-5 for 4, 7, and 14 days. Cells added to the top well were exposed to medium alone (open bars) or IL-5 at 0.1 nM (hatched bars) or 1 nM (black bars). Chemokinetic index is the number of cells in the bottom well at 1 hour with stimulus divided by that with medium alone. Polarization in response to medium alone or 0.1 nM and 1 nM IL-5 was assessed by change in forward scatter (FSC) signal on flow cytometry (mature peripheral blood eosinophils showed a mean change in forward scatter of 120 units to 1 nM IL-5, n = 3, data not shown). The figure shows a typical response at days 7 and 14. No response to IL-5 was seen at day 3 to 4 in 7 independent experiments. Responsiveness of cells cultured for 7 days to IL-5 was variable and may reflect different rates of eosinophil development in different cultures; however, similar responses to those shown were seen from cells cultured for 7 days in IL-3 and IL-5 in 4 independent experiments.

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