TUMOR-NECROSIS-FACTOR INCREASES THE ELASTOLYTIC POTENTIAL OF ADHERENTNEUTROPHILS - A ROLE FOR HYPOCHLOROUS ACID

Neutrophils adhered to biologic surfaces exhibit proteolytic cleavage of surface proteins even in the presence of proteinase inhibitors. Suc h proteolysis is restricted to the pericellular space and appears to r equire the dual action of proteinases and reactive oxygen species. The present study was designed to investigate the mechanism by which tumo r necrosis factor-alpha (TNF) stimulates neutrophil proteolysis. Tissu e culture wells were coated with insoluble H-3-labeled elastin substra te. Human blood neutrophils (0.5 to 2.0 x 10(6) cells/ml/well) were in cubated in the coated wells for 4 to 18 h at 37-degrees-C in the prese nce of varying concentrations of serum or purified alpha1-antitrypsin (A1AT). TNF (0 to 1,000 U/ml) was also present in the incubations. Ela stin degradation was determined as soluble H-3-elastin fragments relea sed into the supernatants. As previously reported, cells (no TNF) exhi bited spontaneous elastolysis even in the presence of 1% serum or 4 mu M A1AT. Compared with cells incubated alone (no TNF), TNF increased el astolysis 3-fold in the 4-h incubations and 83% in 18-h incubations. T NF also significantly increased proteolysis when neutrophils were conc urrently treated with phorbol myristate acetate or N-formylmethionylle ucyl-phenylalanine. Since TNF is known to prime neutrophils for hypoch lorous acid (HOCl) release, the present study hypothesized that the en hancement of proteolysis by TNF was related to increased release of HO Cl. First, TNF caused a 4-fold increase in HOCl release by neutrophils adhered to elastin surfaces. Second, the effect of methionine on elas tolysis by adherent neutrophils was studied. These studies showed that trapping of HOCl by methionine led to a marked decrease in elastin br eakdown, an effect that was accentuated in TNF-treated cells. In summa ry, these studies show that TNF augments the proteolytic activity of n eutrophils cultured on biologic surfaces in the presence of proteinase inhibitors. TNF-mediated increases in proteolysis are caused by incre ases in HOCl and possibly by other site-directed effects in the perice llular space.