Life Sciences, Vol. 49, pp. 1879-1886 Printed in the U.S.A.

Pergamon Press

ENZYMATIC DEGRADATION OF TUMOR NECROSIS FACTOR BY ACTIVATED HUMAN NEUTROPHII•: ROLE OF ELASTASE Jo~lle Nortier 1, Peter Vandenabeele2, Etienne Noel3, Yves Bosseloir 1, Michel Goldman 1,4 and Monique Deschodt-Lanckman1 1 Laborat0ire Pluridisciplinaire de Recherche Exp~rimentale Biom(:dicale, Facult~ de M&iecine, Universit~ Libre de Bruxeiles, Campus Erasme, 1070 Brussels 2 Laboratorium voor Moleculaire Biologie, Rijksuniversiteit of Gent, 9000 Ghent 3 Laboratoire de Chimie Biologique and 4 Service d'Immunologie, H6pital Erasme, 1070 Brussels, Belgium (Received in final form October ii, 1991)

Summary Although the role of tumor necrosis factor-alpha ('INF) as mediator of inflammation is now well established, its interactions with polymorphonuclear neutrophils (PMN) are not fully understood. Therefore, we investigated the possible hydrolytic action on TNF of intra-lysosomal enzymes released by activated PMN in the extracellular medium. We first incubated 125I radiolabeled TNF in vitro with activated PMN and by HPLC analysis, we observed a degradation process completely blocked by the previous addition of alpha 1Antitrypsin (AT) to the incubation medium. By comparing several degradative patterns of TNF obtained with purified leukocyte proteases and supornatant of activated PMN, we identified elastase as the major enzyme involved in this catabolic process of "INF. In a second part, we determined the bioactivity of the cleavage fragments of recombinant human TNF (rhTNF) by a cytotoxicity assay. None of the fragments was found biologically active. Our results suggest that, at inflammatory sites, an enzymatic degradation of TNF may occur in the poricellular area of activated PMN. This new catabolic pathway leading to inactivation of TNF might be regarded as an effective local negative feed-back process limiting the potentially toxic effects of this cytokine. Beside its role in the pathophysiology of septic shock and tumor-associated cachexia, tumor necrosis factor-alpha (TNF) is also involved in local inflammatory responses (I). This latter effect is at least in part related to the action of 'INF' on polymorphonuclear neutrophils 0PMN). Indeed, several functions of mature granulocytes have been shown to be activated by TNF, such as respiratory burst (2), chemotaxis (3), antibody-dependent cellular cytotoxicity (4), phagocytic activity

Correspondence to : Jo~lle NORTIER, MD - Laboratoire Pluridisciplinaire de Recherche Exp~rimentale Biom6,dicale, Facult~ de M6decine, Universit~ Libre de Bruxelles, Campus Erasme, B~t. G2- niv4, mute de Lennik, 808, 1070 Brussels - Belgium. 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

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and degranulation leading to the release of proteolytic enzymes in the perieellular area (5). In the present study, we analyzed the impact of the PMN degranulation process on 'INF. The in vitro observation of a proteolytic digestion of 125I-TNF incubated with activated PMN led to investigation of the enzymes involved in this pheno- menon, paying particular attention to elastase and cathepsin G. These two neutral proteases contained in azurophilic granules (6) are known to degrade protein components of the extracellular matrix such as elastin, several types of collagen (7) and fibronectin (8). Finally, we examined the biological activity of the fragments of recombinant human TNF (rhTNF) resulting from enzymatic hydrolysis. Exnerimental vroeedure Materials. 125I labeled TNF (specific radioactivity approx. 600 Ci/mmoi) was purchased from Amersham International (Amersham, Bucks, U.K.). rhTNF was a generous gift of Dr. W. Fiers (Laboratorium voor Moleculaire Biologic-Gent, Belgium). Both purified leukocyte elastase and cathepsin G (specific activity of 60 and 52 units/rag protein respectively) as well as alphalAntitrypsin (AT) and phorboi 12-myristate 13-acetate (PMA) were obtained from Sigma (Sigma Chemical Company, St. Louis, MO, USA). Neutrophils preparation and activation. Venous blood from healthy human volunteers was collected in 10 ml heparinized syringes. PMN suspensions were prepared according to Verheugt et al. (9) and were finally diluted in Dulbecco's modified Eagle's medium (DMEM, GIBCO Laboratories, Grand Island, NY, USA) without Phenol Red at approx. 2 x 106 celis/mi. Stimulation of the neutrophils was performed at 37"C by adding PMA at a f'mal concentration of 200 ng/ml. Supernatant of stimulated PMN was collected at different time intervals and elastase release was determined by an automatic immunoassay using the Kit Elastase-PMN Imac from Merck. TNF incubation and HPLC fractionation of the cleavage product~. 125I-TNF (20-25,000 cpm per assay) was incubated during different time intervals at 370C in the presence of PMN (activated or not by PMA), or in the presence of supernatant of PMA-activated PMN, of purified leukocyte elastase or cathepsin G (both at 10 pg/ml), in a final volume of 200 ft. Reaction was stopped by adding 20/A trifluoroacetic acid (TFA) and samples were applied to a Waters C4 column (Waters Associate Milford, MA, USA). Elution was carried out at room temperature at a 1.0 ml/min flow rate with a 0-60 % (v/v) linear acetonitrile gradient in 0.08 % (v/v) TFA over 60 min. Fractions of 1.0 ml were collected and their radioactivity was determined in a gammaradiation counter. In experiments using rhTNF (1 mg/ml), elution was monitored at 280 nm and 1.0 ml fractions were tested for bioactivity. Biological activitv of the cleavage fragments of ~'hTNF. TNF bioactivity was determined by a cytotoxicity assay on actinomycin-D-treated WEHI-164 clone 13 cells (10), the number of surviving cells being evaluated by the use of MTI" (M-2128, Sigma) (11), as previously described (12). Results

PMN activation by PMA. PMN were stimulated with PMA and elastase concentration was measured in the supernatant. This treatment resulted after 240 minutes in a six-fold increase in secreted elastase as compared to unstimulated PMN (165 ng/ml versus 24 ng/ml).

Vol. 49, No. 25, 1991

TNF Degradation by Leukocyte Enzymes

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Enzymatic degradation of tumor necrosis factor by activated human neutrophils: role of elastase.

Although the role of tumor necrosis factor-alpha (TNF) as mediator of inflammation is now well established, its interactions with polymorphonuclear ne...
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