INVOLVEMENT OF AN NAD(P)H OXIDASE-LIKE ENZYME IN SUPEROXIDE ANION ANDHYDROGEN-PEROXIDE GENERATION BY RAT TYPE-II CELLS

Background - Although alveolar macrophages are considered to be the pr imary cellular mediators of host defence in the lung, there is increas ing evidence that type II cells may also play an active role in host d efence. A study was undertaken to investigate whether type II cells ge nerate O-2(-.) and H2O2 via an NADPH oxidase-like system and whether e xposure of the type II cells to soluble or particulate stimuli known t o activate NADPH oxidase in macrophages also leads to increased produc tion of H2O2. Methods - Rat type II cells and alveolar macrophages wer e exposed to 10, 100, or 1000 nM phorbol-12-myristate-13-acetate (PMA) and the production of O-2(-.) and H2O2 was determined by chemilumines cence. Thirty minutes before stimulation with 1 mu M PMA type II cells were also exposed to the same concentrations of a protein C (PKC) ant agonist GF109203x, non-selective protein kinase inhibitor staurosporin e (1, 10, or 100 nM), or the NADPH oxidase inhibitor diphenyliodonium chloride (DPI) (1, 10, 100, or 1000 mu M). The effects of arachidonic acid, zymosan and Staphylococcus aureus on H2O2 production were determ ined. Cell membrane fractions from type II cells and macrophages were assayed for NADPH oxidase activity. Results - After exposure to 1 mu M PMA, O-2(-.) and H2O2 generation increased 6.3-fold and 9.0-fold, res pectively, in type II cells and 2.4-fold and 5.2-fold, respectively, i n macrophages. In contrast to the macrophages, the increase in O-2(-.) and H2O2 generation by type II cells was completely prevented by 1 mM KCN. Preexposure to GF109203x, staurosporine, or DPI completely preve nted the rise in O-2(-.) and H2O2 generation. Mean (SD) NADPH oxidase activity of 138 (38) nmol O-2(-.)/min/mg protein was found in membrane fraction I of the type II cells, and 102 (31) nmol O-2(-.)/min/mg pro tein in fraction II. Macrophages showed higher NADPH oxidase activity in membrane fraction II. In type II cells exposure to arachidonic acid led to a significant 5.3-fold increase in H2O2 generation, exposure t o zymosan increased H2O2 generation 46-fold, and exposure to S aureus 25-fold with a maximum 30-50 minutes after addition of the bacteria. C onclusions - Type II cells generate O-2(-.) and H2O2 via a PKC-mediate d activation of an NAD(P)H oxidase-like membrane bound enzyme. Arachid onic acid, zymosan, and bacteria also give rise to increased H2O2 prod uction. Type II cells might thus play an active role in host defence.