Effect of theophylline and specific phosphodiesterase IV inhibition on proliferation and apoptosis of progenitor cells in bronchial asthma

Abstract

1. Theophylline possesses anti-inflammatory activities in asthma. We examined whether theophylline and agents that modulate cyclic AMP can determine the survival and proliferation of progenitor cells. 2. Progenitor cells from the blood of normal and asthmatic subjects were cultured for 14 days in methylcellulose with GM-CSF, stem cell factor, IL-3 and IL-5. Apoptosis was measured by flow cytometry of propidium-iodide-stained cells. 3. A greater number of colonies with a higher proportion of cells of eosinophil lineage from asthmatics compared to normal subjects were grown. Theophylline (at 5 and 20 micro g ml(-1)) significantly inhibited colony formation and increased apoptotic cells in asthmatics compared to control. Salbutamol (0.1, 1, 10 micro M), dibutyryl-cAMP (0.1, 1 mM) and rolipram (0.1, 1 mM), a phosphodiesterase IV inhibitor, also dose-dependently decreased colony numbers and increased apoptosis of progenitor cells from asthmatics. 4. There was no significant effect of theophylline, db-cAMP, salbutamol or rolipram on colony formation or the survival of progenitor cells from normal subjects. AMP did not affect the colony formation and apoptosis. Expression of Bcl-2 protein on progenitor cells of asthma was downregulated by theophylline, salbutamol, db-cAMP and rolipram. 5. Theophylline and rolipram decreased colony formation committed to the eosinophil lineage, together with an increase in apoptosis through an inhibition of Bcl-2 expression effects that may occur through cAMP. The anti-inflammatory properties of theophylline include an inhibition of circulating progenitor cells.

Figure 1

Effects of theophylline, salbutamol, db-cAMP, rolipram and AMP on the colony formation of progenitor cells cultured for 14 days from normal subjects (n=7, in each group) and patients with mild intermittent asthma (n=8, in each group). Data shown as mean±s.e.m. ^*P<0.05, ^**P<0.01 compared with unstimulated controls in patients with asthma.

Figure 2

Detection of apoptosis of progenitor cells induced by theophylline plus growth factors (GM-CSF, IL-3, SCF and IL-5) using PI staining by flow cytometric analysis. Progenitor cells from normal subjects (a) or patients with asthma (b) were treated with various concentrations of theophylline, collected after 72-h culture period and stained with PI to detect DNA fragmentation by flow cytometry. Percentages indicate the percentage of apoptotic cell nuclei/total cell nuclei.

Figure 3

Effect of theophylline, salbutamol, db-cAMP, rolipram and AMP on the apoptosis of progenitor cells from normal subjects (n=7, in each group) and patients with mild intermittent asthma (n=8, in each group). The progenitor cells were incubated for 72 h in the presence of SCF, GM-CSF, IL-3 and IL-5. Data are shown as mean±s.e.m. ^*P<0.05, ^*ast;P<0.01 compared to controls in patients with asthma.

Figure 4

Direct immunofluorescence flow cytometry was performed to detect intracellular expression of Bcl-2 on CD34+ cells. Cells from normal subjects and asthmatic patients were incubated with or without 5 μg ml⁻¹ theophylline for 24 h. The graph shows data expressed in the form of histogram depicting specific Bcl-2 staining with (dashed line) or without (solid line) theophylline treatment for a normal subject (a) and for an asthmatic (b).

Figure 5

Direct immunofluorescence flow cytometry was performed to detect intracellular expression of Bcl-2 on CD34+ cells. The mean fluorescence intensity (MFI) of Bcl-2 expression in cells treated with (solid bars) or without (hatched bars) 5 μg ml⁻¹ theophylline (a) or 0.1 mM db-cAMP (b) or 0.1 mM rolipram (c) or 1 μM salbutamol (d) for 24 h in normal subjects (normal, n=7) or patients with mild intermittent asthma (asthma, n=8). ^*P<0.05 compared with the progenitor cells from normal subject in the absence of treatment. ^**P<0.05 compared with the progenitor cells in the absence of treatment. Data are means±s.e.m.