Chemoattractant-induced release of elastase by tumor necrosis factor-primed human neutrophils: Auto-regulation by endogenous adenosine

Objective and Design: In the present work, we studied the role of cell-deri ved adenosine in both the physiologic regulation and pharmacologic control of the exocytosis of azurophilic granules of neutrophils exposed to tumor n ecrosis factor alpha (TNF) and stimulated with some chemoattractants. Material and Methods: Human neutrophils were pre-incubated in the absence o r presence of TNF. Thereafter, the appropriate chemoattractant was added to the cells. After incubation, the cell-free supernatant was collected for t esting elastase activity and intracellular cAMP levels. Results, expressed as mean +/-1 SD, were evaluated by unpaired two-tailed Student's t-test and by analysis of variance followed by Student-Newman-Keuls multiple comparis ons test. Results: Neutrophil incubation with 10 ng/ml TNF or 0.1 mu mol/l N-formyl-m et-leu-phe (fMLP) failed to release elastase activity (NE) (NE in absence o f stimulus: 23.1 +/- 5.7 nmol/h, TNF-induced NE: 26.4 +/- 14.4 nmol/h: fMLP -induced NE: 27.0 +/- 9.9 nmol/h). Neutrophils, pre-exposed to various amou nts of TNF, released elastase in response to 0.1 mu mol/l fMLP in a dose-de pendent manner (NE in presence of 10 ng/ml TNF and 0.1 pmol/l fMLP: 133.7 /- 24.0 nmoles/h). As compared with fMLP, C5a had lower activity (NE in pre sence of 10 ng/ml TNF and 0.1 mu mol/l C5a: 66.4 +/- 25.1 nmoles/h), wherea s interleukin-8, platelet activating factor and leukotriene B-4 were ineffe ctive. The secretory response of TNF-primed neutrophils to fMLP was inhibit ed by adenosine in a dose-dependent manner (IC50 = 5.18 +/- 7.1 mu mol/l). The addition of adenosine deaminase (ADA) to TNF-primed neutrophils resulte d in increased secretory response to fMLP (NE in absence and presence of 0. 25 U/mlADA: 71.5 +/- 11.0 and 107.3 +/- 18.6 respectively, P = 0.060). More over, two inhibitors of phosphodiesterase type IV (RO 20-1724 and nimesulid e) reduced the elastase release only in the absence of ADA (RO 20-1724: per cent inhibition in absence or presence of ADA = 20.2 +/- 15.0 and 4.4 +/- 5 .1 respectively; nimesulide: percent inhibition in absence or presence of A DA = 22.2 +/- 19.6 and 0.8 +/- 3.0 respectively). Similarly, RO 20-1724 and nimesulide increased intracellular cAMP levels only in absence of ADA (RO 20-1724: percent cAMP increment in absence or presence of ADA = 215.4 +/- 9 7.5 and 47.3 +/- 53.3 respectively; nimesulide: percent cAMP increment in a bsence or presence of ADA = 177.7 +/- 19.0 and 19.5 +/- 29.3 respectively). Conclusions: Endogenous adenosine down-regulates the cell secretory respons e and is instrumental in uncovering the susceptibility of azurophilic granu le exocytosis to control by inhibitors of phosphodiesterase type IV.