LIGATION OF SELECTIN-L AND INTEGRIN CD11B CD18 (MAC-1) INDUCES RELEASE OF GELATINASE-B (MMP-9) FROM HUMAN NEUTROPHILS/

Objective and Design: To examine whether ligation of the adhesive rece ptors - selectin L and Mac-1 on the neutrophil surface could induce ge latinase B exocytosis. Materials: Neutrophils were isolated from fresh heparinized blood of human donors by Gradisol G centrifugation and hy potonic lysis of erythrocytes. Methods: Integrin CD11b/CD18 and select in L mediated adhesive interaction of human neutrophils were mimicked by binding antibodies to these receptors on the surface of isolated le ukocytes. Neutrophils (5 x 10(6)/ml) were incubated with antibodies ag ainst selectin L (40 mu g/ml) and CD18 or CD11b (10 mu g/ml). The secr etion of gelatinase was examined by determination of enzyme activity a nd gelatin substrate zymography of cell supernatants. Results: Ligatio n of selectin L, CD18 and CD11b integrin subunits by monoclonal antibo dies induced a rapid release of 24.6 +/- 1.8% (p < 0.005), 24.0 +/- 2. 9% (p < 0.001) and 22.7 +/- 2.0% (p < 0.005) of total neutrophil gelat inase, respectively as compared with 11.1 +/- 1.6% in the control. The se values were equivalent to N-formyl-methionylleucyl-phenylalanine (f MLP)-stimulated secretion of gelatinase. Under these experimental cond itions there was no significant beta-glucuronidase release from azurop hilic granules. Gelatinase exocytosis elicited by selectin L and CD18 ligation was inhibited by 82.7 +/- 10.1% and 49.3 +/- 5.9%, respective ly after preincubation of the neutrophils with 10 mu M herbimycin A. C onclusions: Ligation of selectin L and integrin CD11b/CD18 provides st imulatory signals to neutrophils which induce secretion of gelatinase B that may facilitate their transmigration into sites of inflammation.