HISTOCHEMICAL-DEMONSTRATION OF HYDROGEN-PEROXIDE PRODUCTION BY LEUKOCYTES IN FIXED-FROZEN TISSUE-SECTIONS OF INFLAMMATORY LESIONS

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by i ncubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 mu g/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O 2 was required to produce the chromogenic DAB product. Granulocytes (P MNs and eosinophils) were the main types of cells stained by the DAB r eaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell de ath and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presenc e of peroxidases to produce the colored reaction product. Within our t issue sections, such peroxidases were evidently present in excess, bec ause addition of low concentrations of H2O2 significantly increased th e reaction product. Although some of the H2O2 produced by the granuloc ytes may have been derived from the dismutation of superoxide (O-2(-)) the NADPH oxidase pathway for O-2(-) formation did not seem to be inv olved: NADPH oxidase, a rather labile enzyme, should not be active aft er mild fixation, and diphenyleneiodonium (100 mu M), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactiv e nitrogen intermediates were also not involved, because N-G-monomethy l-L-arginine and N-G-nitro-L-arginine methyl ester, inhibitors of nitr ic oxide synthetase, did not appreciably inhibit the reaction. We conc lude that stable, non-flavine-requiring oxidases, possibly cyclooxygen ases or lipoxygenases, produced the H2O2 measured histochemically by o ur DAB reaction. These studies were made on tissue sections of acute d ermal inflammatory lesions produced in rabbits by the topical applicat ion of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chl oride. Both intact PMNs and disintegrating PMNs in the base of the cru st produced H2O2. Despite the production of H2O2 and the presence of p eroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well prote cted by the antioxidants present in this self-limiting inflammatory re action.