Chemoattractant-induced release of elastase by tumor necrosis factor-primed human neutrophils: auto-regulation by endogenous adenosine.
OBJECTIVE
In the present work, we studied the role of cell-derived adenosine in both the physiologic regulation and pharmacologic control of the exocytosis of azurophilic granules of neutrophils exposed to tumor necrosis factor alpha (TNF) and stimulated with some chemoattractants.


METHODS
Human neutrophils were pre-incubated in the absence or presence of TNF. Thereafter, the appropriate chemoattractant was added to the cells. After incubation, the cell-free supernatant was collected for testing 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 comparisons test.


RESULTS
Neutrophil incubation with 10 ng/ml TNF or 0.1 micromol/l N-formyl-met-leu-phe (fMLP) failed to release elastase activity (NE) (NE in absence of 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 amounts of TNF, released elastase in response to 0.1 micromol/l fMLP in a dose-dependent manner (NE in presence of 10 ng/ml TNF and 0.1 micromol/l fMLP: 133.7 +/- 24.0 nmoles/h). As compared with fMLP, C5a had lower activity (NE in presence of 10 ng/ml TNF and 0.1 micromol/l C5a: 66.4 +/- 25.1 nmoles/h), whereas interleukin-8, platelet activating factor and leukotriene B4 were ineffective. The secretory response of TNF-primed neutrophils to fMLP was inhibited by adenosine in a dose-dependent manner (IC50 = 5.18 +/- 7.1 micromol/l). The addition of adenosine deaminase (ADA) to TNF-primed neutrophils resulted in increased secretory response to fMLP (NE in absence and presence of 0.25 U/ml ADA: 71.5 +/- 11.0 and 107.3 +/- 18.6 respectively, P = 0.060). Moreover, two inhibitors of phosphodiesterase type IV (RO 20-1724 and nimesulide) reduced the elastase release only in the absence of ADA (RO 20-1724: percent 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 ADA = 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 +/- 97.5 and 47.3 +/- 53.3 respectively; nimesulide: percent cAMP increment in absence or presence of ADA = 177.7 +/- 19.0 and 19.5 +/- 29.3 respectively).


CONCLUSIONS
Endogenous adenosine down-regulates the cell secretory response and is instrumental in uncovering the susceptibility of azurophilic granule exocytosis to control by inhibitors of phosphodiesterase type IV.
Ottonello
L
L
Semeiotica e Metodologia Medica 2, Dipartimento di Medicina Interna, Genova, Italy. otto@csita.unige.it
Amelotti
M
M
Barbera
P
P
Dapino
P
P
Mancini
M
M
Tortolina
G
G
Dallegri
F
F
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000241:Adenosine / Q000502:physiology*; D000243:Adenosine Deaminase / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002630:Chemotactic Factors / Q000494:pharmacology*; D015936:Complement C5a / Q000494:pharmacology; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002630:Chemotactic Factors; D003470:Culture Media; D007202:Indicators and Reagents; D010726:Phosphodiesterase Inhibitors; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D010196:Pancreatic Elastase; D000243:Adenosine Deaminase; D000241:Adenosine; C012655:nimesulide