Calcium-dependent degradation of surfactant protein A by activated neutrophils due to serine proteases

The effects of activated leukocytes on surfactant function and composition were examined to better define the mechanisms by which acute inflammation c ontributes to respiratory distress syndrome (ARDS). Peripheral blood leukoc ytes from healthy volunteers were incubated in vitro with surfactant for a hours: (I) in the absence of activation; (2) following activation by additi on of phorbol myristate acetate (PMA); (3) following addition of PMA + FeCl 2/EDTA; (4) PMA + FeCl2; (5) PMA + EDTA; (6) PMA + FeCl2 + superoxide dismu tase (SOD) + catalase; (7) PMA + EDTA + serine or cysteine protease inhibit ors. Surfactant was then repurified by sucrose gradient centrifugation, and function, phospholipid and protein composition, and extend of lipid peroxi dation were assessed. PMA caused activation of leukocytes as detected by di chlorofluorescene assay. Lipid peroxidation (assessed by conjugated diene a ssay) was detected in all samples containing PMA. Abnormal function was not ed in surfactant exposed to activated cells with 1 mM EDTA, whereas activat ion alone, or with FeCl2, had no effect. SOD + catalase prevented lipid per oxidation, but did not prevent leukocyte-mediated dysfunction, which was as sociated with a marked reduction in surfactant protein A (SP;A), but no det ectable change in surfactant protein B (SP-B) oz phospholipid composition. Serine protease inhibitors prevented SP-A degradation and dysfunction, wher eas cysteine protease inhibitors had no protective effect. Addition of puri fied SP-A (5% w/w) to dysfunctional surfactant restored normal function, wh ile SP-B/C mixture (3%) did not. Activated leukocytes cause surfactant dysf unction in vitro by serine protease-mediated degradation of SP-A, which occ urs only in the presence of EDTA, and is prevented by addition of calcium. Although lipid peroxidation mediated by leukocyte release of free-radical p roducts was also detected, surfactant dysfunction appears to be unrelated t o this process.