ALBERT

Notes: Summary Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and nonparticulate (phorbol myristate acetate) stimuli. This membraned activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn++, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.

Notes: Summary The PMN is exquisitely designed to combat invading micro-organisms. The relationship between structure and function is nowhere more evident than in this cell type. The elaborate biochemical machinery which the PMNs possess for killing ingested micro-organisms works as a highly integrated system, with each step occurring in sequence and at a particular site. In the past decade or so it has become apparent that the Klebanoff system (myeloperoxidase-halide-H2O2) and possibly other active O2 species as well, play an important role in the bactericidal activity of PMNs. Application of cytochemical techniques for oxidative enzymes and for end-products of oxidative reactions has localized the sites within the phagocytosing or stimulated PMN at which these various components of the cidal systems are active and generated. In this fashion, biochemical data have been not only confirmed, but in several instances, the cytochemical approach has led the way in extending our knowledge and thinking regarding PMN metabolism and cidal functions. In our laboratory we have studied the bactericidal machinery of PMNs by cytochemical means. We have established, at the ultrastructural level, that the myeloperoxidase-containing azurophil granules fuse with the phagosome membrane and empty their contents into the phagosome (Baehneret al., 1969). We have shown that H2O2 is generated within the phagosome (Briggset al., 1975b). This established that the myeloperoxidase-H2O2 system could work within the phagosome, since both of these components are present following phagocytosis. We determined that H2O2 could be detected on the cell surface and within the phagosome following phagocytic stimulation of NADH oxidase activity (Briggset al., 1975a). The cell surface localization of H2O2 was an important finding since the phagosome membrane is derived from the plasmalemma. Thus internalization of the plasmalemma, with components capable of generating H2O2, can explain the presence of H2O2 within the phagosome. We have also shown that when PMNs are treated with non-particulate stimuli of the respiratory burst, similar results are found, that is, H2O2 is present on the cell surface and within vesicles, which are presumed to be of surface origin (Badweyet al., 1980). We have shown that D-amino acid oxidase, another enzyme capable of generating H2O2 is cytochemically demonstrable and that it can utilize cell wall components of ingested bacteria as substrates for enzyme activity (Robinsonet al., 1978). The PMNs from CGD patients do not kill certain bacteria. This inability to kill bacteria is related to the low levels of H2O2 produced during phagocytosis. Using the cerium reactioon we determined that PMN from CGD patients produce little cytochemically detectable H2O2 and that what little is present is restricted to the phagosome (Briggset al., 1977). Some PMNs contain other oxidases which are capable of generating H2O2 and O 2 − from O2 consumed during phagocytosis. The guinea-pig PMN (but not human) has an unusual aldehyde oxidase. Cytochemically the aldehyde oxidase activity is restricted to the phagosome (Robinsonet al., 1979). We have also developed a method for localization of sites of O 2 − production following stimulation. In phorbol myristate acetate-stimulated PMNs, reaction product for O 2 − is present within surface-derived vesicles, and in some cases, on the cell surface. Cytochemical detection of enzymes and products of enzymatic activity (H2O2 and O 2 − ) associated with stimulation of the respiratory burst in PMN has thus provided further evidence for the importance of active oxygen species in phagocytosis. Furthermore, the site-specific information obtained from cytochemistry has provided an important link in understanding the structure-function interplay associated with phagocytosis in PMNs. It should be realized, however, that the cytochemical methods we have utilized detect in most instances the end product of an enzymatic reaction (for example, H2O2) and not the site of the enzyme itself. This is important, for instance in the case of H2O2, because this entity appears to begenerated on the surface of the plasmalemma or on the luminal surface of the phagosomal membrane. However, the enzyme responsible may well be situated on the cytoplasmic side of these membranes, and the generation of the H2O2 may involve an electron shuttle across the membrane. Such a mechanism may involve cytochrome and quinone compounds as carriers (Segal & Jones, 1979; Millardet al., 1979). Experiments are now being designed to localize the sites of the enzymesper se by immunocytochemistry. This approach should help resolve these important questions.

Notes: We have compared the subcellular sites of H2O2 and presumably also superoxide-(O2-) production, and certain aspects of metabolic responses (O2 consumption, O2- production) of stimulated neutrophils from human blood and those elicited into guinea pig peritonea. Stimulation was accomplished with either opsonized zymosan or phorbol-12-myristate-13-acetate (PMA). Striking quantitative differences were observed between these cell types with regard to the increased respiration and O2- production observed during stimulation. These differences were most apparent when opsonized zymosan served as the stimulating agent. They were minimized when the soluble stimulating agent, PMA, was used. With either stimulus, the subcellular sites of H2O2 production were the same for both types of neutrophils, i.e., the plasmalemma and phagosomal membranes. No H2O2 production could be detected cytochemically in the absence of stimulation.Treatment of both unstimulated human blood and elicited guinea pig peritoneal neutrophils with the nonpenetrating, covalently linking reagent, p-diazobenzenesulfonic acid, failed to diminish O2- production upon subsequent stimulation, in contrast to a previous report. These data are discussed in terms of the possible cytological arrangements of the respiratory enzyme(s), and the different modes of stimulation of neutrophil metabolism by various agents. Ancillary data on elicited mouse peritoneal neutrophils are presented.

Notes: Maximal rates of superoxide (O-2) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O-2 as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14-18 nmol O-2/min/107 cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O-2 upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H2O2 by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H2O2 staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H2O2 staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.