Theophylline accelerates human granulocyte apoptosis not via phosphodiesterase inhibition.

Theophylline, in addition to its bronchodilator effect, is reported to have an antiinflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airway.In bronchial asthma, such inflammatory cytokines as GM-CSF and IL-5 are upregulated and have been proposed to cause granulocyte infiltration (neutrophils and eosinophils) in the airway by inhibition of granulocyte apoptosis.We examined the abilities of theophylline to counteract the prolongation of human granulocyte survival caused by cytokines.Theophylline was shown to shorten granulocyte survival in a dose-dependent manner.Upon incubation with a therapeutical concentration of theophylline ( 0.1 mM; 18 g/ml), percentages of GM-CSF (10 ng/ml)-induced delayed apoptosis increased from 18 Ϯ 2% to 38 Ϯ 3% ( P Ͻ 0.02) in neutrophils and from 21 Ϯ 2% to 35 Ϯ 2% ( P Ͻ 0.02; 24-h incubation) in eosinophils.The percentage of IL-5 (5 ng/ml)-induced delayed eosinophil apoptosis also increased from 22 Ϯ 4% to 33 Ϯ 2% ( P Ͻ 0.05).In contrast, cyclic AMP (cAMP)-increasing agents (3-isobutylmethylxanthine, dibutyryl cAMP, and rolipram) inhibited granulocyte apoptosis in the control and anti-Fas antibody-treated cells.In eosinophils, the expression of bcl -2 protein decreased after incubation with theophylline.These findings suggest that theophylline accelerates granulocyte apoptosis, which may play an essential role in inflammation, and controls granulocyte longevity regardless of the elevation of intracellular cAMP levels.( J. Clin.Invest.1997.100:1677-1684.)Key words: cell death • Fas antigen • neutrophils • eosinophils • bcl -2 protein MethodsReagents.Percoll and dextran T500 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden).Theophylline, dibutyryl-adenosine 3 Ј ,5 Ј monophosphate, 3-isobutylmethylxanthine (IBMX, 1 a phosphodiesterase inhibitor), DMSO, and Hepes were purchased from Sigma Chemical Co.(St.Louis, MO).A specific phosphodiesterase IV inhibitor (Rolipram; ME3167) was a gift from Meiji Seika Kaisha (Kawasaki, Japan).Recombinant human GM-CSF (rhGM-CSF) was supplied by Kirin Brewery (Tokyo, Japan) and human IL-5 was purchased from Genzyme Corp. (Cambridge, MA).IgM anti-Fas antibody (CH11), FITC-labeled anti-Fas monoclonal antibody, and FITC-labeled anti-IgG1 mAb were purchased from MBL (Nagoya, Japan).FITC-labeled antibcl -2 (clone 124) was from Dako Japan (Kyoto, Japan).Anti-CD16 mAb-coated immunologic magnetic beads and the magnetic cell sorter system were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany).