Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.

We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.

A novel neutrophil-activating factor produced by human mononuclear phagocytes.

The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.

Cytochemical localization of catalase in leaf microbodies (peroxisomes).

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.

Cytochemical localization of peroxidatic activity of catalase in rat hepatic microbodies (peroxisomes).

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H(2)O(2), and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H(2)O(2) appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H(2)O(2) by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H(2)O(2), completely abolished microbody staining in the absence of H(2)O(2). Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.

Inhibition of biogenic amine uptake by hydrogen peroxide: a mechanism for toxic effects of 6-hydroxydopamine.

Hydrogen peroxide, dialuric acid, or 6-hydroxydopamine inhibited the uptake of dopamine, norepinephrine, and serotonin into rat brain synaptosomal preparations. The addition of catalase protected all systems, but catalase was only partially protective for 6-hydroxydopamine acting upon catecholamine uptake. The data show that 6-hydroxydopamine generates hydrogen peroxide and that hydrogen peroxide can damage the biogenic amine uptake systems. Part of the damage caused by the 6-hydroxydopamine that accumulates in the catecholamine nerve terminals in vivo may be attributed to the hydrogen peroxide.

Correction of metabolic deficiencies in the leukocytes of patients with chronic granulomatous disease.

Polymorphonuclear leukocytes from patients with chronic granulomatous disease (CGD) exhibit metabolic and bactericidal deficiencies that may be the result of inadequate production of H(2)O(2). A hydrogen peroxide-generating system was, therefore, inserted into CGD leukocytes. This was accomplished by allowing the cells to phagocytize latex spherules coated with glucose oxidase. This produced an amelioration in the known metabolic deficiencies of these cells during phagocytosis: (a) intracellular (catalatic) formate oxidation dependent upon hydrogen peroxide production was enhanced fourfold; and (b) hexose monophosphate shunt activity, which other workers have shown to be at least partially dependent upon the availability of H(2)O(2), was markedly stimulated. These data strengthen the evidence that the fundamental metabolic lesion in CGD cells during phagocytosis is indeed deficient production of hydrogen peroxide, probably, as previously shown, due to diminished oxidase for reduced nicotinamide adenine dinucleotide.

In vivo formation of H2O2 in red cells during exposure to hyperoxia.

Chow-fed and tocopherol-deficient mice were given aminotriazole (AT), exposed to 100% O(2) at 60 pounds per square inch absolute for 1 hr (OHP), and red blood cells were assayed for catalase activity and lipid peroxide levels. A decrease of catalase activity (CA) in the presence of AT can be taken as evidence of excess formation or accumulation of H(2)O(2). No differences of CA were observed among chow-fed mice, with or without AT and/or OHP. Tocopherol-deficient mice with AT had lower CA (0.174+/-0.040) than chow-fed mice with AT (0.225+/-0.028) P < 0.01. Tocopherol-deficient mice with AT exposed to OHP had even lower CA, 0.137+/-0.024, P < 0.01.The data are consistent with the hypothesis that H(2)O(2) is formed or accumulated in excess in red cells of tocopherol-deficient mice, an effect that is enhanced in the presence of hyperoxia. They imply that tocopherol plays a role in the detoxification of H(2)O(2).

Myeloperoxidase-mediated iodination by granulocytes. Intracellular site of operation and some regulating factors.

The intracellular site of operation of the myeloperoxidase-H(2)O(2)-halide antibacterial system of granulocytes has been determined by utilizing measurements of the fixation of iodide to trichloracetic acid (TCA) precipitates of subcellular fractions, including intact phagocytic vesicles. Na(125)I was added to suspensions of guinea pig granulocytes in Krebs-Ringer phosphate buffer, and they were then permitted to phagocytize different particles. Phagocytic vesicles were formed by allowing cells to ingest a paraffin oil emulsion (POE) and collected by flotation on sucrose after homogenization. Measurement of (125)I bound to TCA precipitates of the different fractions and the homogenates disclosed that the lysosome-rich fraction obtained by centrifugation from control (nonphagocytizing) cells accounted for a mean 93.1% of the total cellular activity. With phagocytosis of POE, TCA-precipitable iodination increased two- to sevenfold, and the lysosomal contribution fell to a mean 36.9% of the total. The appearance of activity within phagocytic vesicles accounted for almost the entire increase seen with phagocytosis (a mean 75.7%), and iodide was bound within these structures with high specific activity. More iodide was taken up by cells than fixed, regardless of iodide concentration, and was distributed widely throughout the cell rather than selectively trapped within the vesicles. The amount of iodide taken up and fixed varied considerably with the phagocytic particle employed. Yeast particles were found to stimulate iodination to a far greater degree than the ingestion of POE or latex. Such observations are consistent with the concept that the ingested particle is a major recipient of the iodination process. Measurements of metabolic activities related to the formation and utilization of peroxide by cells phagocytizing different particles were made and correlated with iodination. The findings suggest that mechanisms must exist within granulocytes to collect or perhaps even synthesize H(2)O(2) within phagocytic vesicles to serve as substrate for myeloperoxidase. The simultaneous stimulation of other metabolic pathways for peroxide disposal and its release into the medium by phagocytizing cells is consistent with the high diffusability of this important bactericidal substance.

Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine.

We have examined whether the toxic effects of homocysteine on cultured endothelial cells could result from the formation and action of hydrogen peroxide. In initial experiments with a cell-free system, micromolar amounts of copper were found to catalyze an oxygen-dependent oxidation of homocysteine. The molar ratio of homocysteine oxidized to oxygen consumed was approximately 4.0, which suggests that oxygen was reduced to water. The addition of catalase, however, decreased oxygen consumption by nearly one-half, which suggests that H2O2 was formed during the reaction. Confirming this hypothesis, H2O2 formation was detected using the horseradish peroxidase-dependent oxidation of fluorescent scopoletin. Ceruloplasmin was also found to catalyze oxidation of homocysteine and generation of H2O2 in molar amounts equivalent to copper sulfate. Finally, homocysteine oxidation was catalyzed by normal human serum in a concentration-dependent manner. Using cultured human and bovine endothelial cells, we found that homocysteine plus copper could lyse the cells in a dose-dependent manner, an effect that was completely prevented by catalase. Homocystine plus copper was not toxic to the cells. Specific injury to endothelial cells was seen only after 4 h of incubation with homocysteine plus copper. Confirming the biochemical studies, ceruloplasmin was also found to be equivalent to Cu++ in its ability to cause injury to endothelial cells in the presence of homocysteine. Since elevated levels of homocysteine have been implicated in premature development of atherosclerosis, these findings may be relevant to the mechanism of some types of chronic vascular injury.

Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity.

A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.

Respiration and glucose oxidation in human and guinea pig leukocytes: comparative studies.

A comparison has been made of the metabolic shifts in human and guinea pig leukocytes when they phagocytize. Respiration of guinea pig polymorphonuclear leukocytes (PMN) and the increment during phagocytosis were each about 2(1/2)-fold that of human PMN. This was also true of the direct oxidation of glucose-6-P (hexose monophosphate shunt). Enzymes potentially responsible for these phenomena have been compared in each species. Cyanide-insensitive NADH oxidase and NADPH oxidase were measured and only the formed exhibited adequate activity to account for the respiratory stimulus durintg phagocytosis. The hydrogen peroxide formed by this enzyme stimulates the hexose monophosphate shunt by oxidizing glutathione which upon reduction by an NADPH-linked glutathione reductase provides NADP to drive the hexose monophosphate shunt. Other linkages between respiratory stimulation and that of the hexose monophosphate shunt also pertain in the guinea pig.

Erythropoietic protoporphyria: lipid peroxidation and red cell membrane damage associated with photohemolysis.

The mechanism by which long wavelength ultraviolet light hemolyzes red cells obtained from patients with erythropoietic protoporphyria (EPP) was investigated. Previous studies had suggested that irradiation of these red cells with wavelengths of light capable of eliciting dermatological manifestations led to oxygen-dependent colloid osmotic hemolysis through the formation of peroxides. In the present report, lipid peroxidation during in vitro irradiation of EPP red cells with long ultraviolet light was demonstrated by: (a) the formation of 2-thiobarbituric acid reactants; (b) the presence of conjugated diene bonds in red cell lipid; and (c) the selective loss of unsaturated fatty acids proportional to the number of carbon-carbon double bonds in each. Irradiation of EPP red cells was also shown to result in the formation of hydrogen peroxide.Before photohemolysis there was a decline in cell membrane sulfhydryl groups and a loss in activity of the cell membrane enzyme acetylcholinesterase. These parameters provide further evidence suggesting that the cell membrane is a primary site of the photohemolytic effect of long ultraviolet light in EPP red cells. Further evaluation of the radiation-induced inactivation of EPP red cell acetylcholinesterase was performed by radiating mixtures containing bovine erythrocyte acetylcholinesterase and protoporphyrin IX. These studies revealed that the rate of decline in enzyme activity is accelerated by the addition of linoleic acid, an unsaturated fatty acid, but not by palmitic acid, a saturated fatty acid. Partial protection against both photohemolysis and acetylcholinesterase decline is provided by alpha-to-copherol. This lipid antioxidant loses its activity during the irradiation of EPP red cells suggesting that it is utilized in this process.

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles.

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.

Phorbol myristate acetate induced oxidation of 2',7'-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease.

The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2-, H2O2, OH-) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2',7'-dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X-linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.

Endocytic and secretory repertoire of the lipid-loaded macrophage.

We have studied the effects of intracellular lipid storage on macrophage function. Mouse peritoneal macrophages were lipid loaded by three regimens modeling loading through the scavenger receptor [acetylated low density lipoprotein (Ac-LDL) cells], by extracellular matrix-bound LDL [low density lipoprotein complexed with dextran sulfate (DS-LDL) cells], and by conditions of reduced cholesterol acceptors in the medium [low serum, oleic acid, Ac-LDL (LS/OI) cells]. Significantly increased cholesterol levels in all three regimens were measured by cholesterol determination and Oil Red O staining of fixed cells. Lipid-laden cells were equal to control macrophages in binding and ingesting immunoglobulin-coated sheep erythrocytes, reflecting Fc-mediated endocytosis. The lipid-laden cells were compared to control cells for secretory functions of macrophages that could be important in the atherosclerotic artery. They were still capable of producing all secretory products examined, but the quantities of H2O2 and arachidonic acid metabolites were reduced in some cases, and fibrinolytic activity appeared to be increased. Western blot analysis showed a five-fold increase in the release of tumor necrosis factor by DS-LDL-loaded cells. We suggest that the location of intracellular lipid pools as well as the type of lipids (and/or lipid complexes) ingested may determine the extent of functional changes.

The calcium ionophore ionomycin can prime, but not activate, the reactive oxygen generating system in differentiated HL-60 cells.

Both the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and the calcium ionophore ionomycin induced a metabolic response in normal neutrophils. However, the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, was required to detect any release of hydrogen peroxide induced by ionomycin. In differentiated HL-60 cells, only FMLP stimulation was associated with any notable metabolic activation. The response to FMLP proceeds with a rate and time course similar to that seen in normal cells. The use of ionomycin as a stimulating agent did not result in any detectable activation of the system that generates reactive oxygen metabolites, even if azide was present in the measuring system. Raising the concentration of cytoplasmic free Ca2+ is therefore not sufficient to activate the system responsible for the generation of reactive oxygen metabolites in HL-60 cells. However, preincubation with ionomycin primed HL-60 cells to an increased response during stimulation with the chemotactic peptide FMLP and the phorbol ester PMA. Since HL-60 cells lack specific granules but have an intact ligand-receptor coupling mechanism, a role for the subcellular granule is proposed, in the generation of reactive oxygen species in normal granulocytes, and analysis of the data presented leads to two conclusions: 1) FMLP, which acts through cells surface receptors, causes the cells to produce oxygen radicals, which to a large extent are released from the cells, a process that is not dependent on the specific granule content of the cells, whereas 2) ionomycin, which bypasses cell-surface receptors, is also capable of stimulating an oxygen-radical formation that is granule dependent and retained inside the cells. Furthermore, the results suggest that an increase in intracellular Ca2+ is not sufficient to initiate activation of the plasma membrane-bound system that generates reactive oxygen metabolites, but the results support a role for Ca2+ in the priming event.

Tumor necrosis factor-alpha induces increased hydrogen peroxide production and Fc receptor expression, but not increased Ia antigen expression by peritoneal macrophages.

It has recently been shown that tumor necrosis factor (TNF) induces increased Ia antigen expression on a malignant murine macrophage cell line, and that TNF is synergistic with gamma interferon (IFN) in inducing Ia expression. This finding raises the possibility that TNF serves as a non-interferon macrophage activating factor in vivo. Since it is known that TNF has different effects on malignant and benign cells, we chose to evaluate the effects of recombinant TNF on primary cultures of murine peritoneal macrophages (MP). Neither human nor murine TNF increased the proportion of MP which expressed Ia antigen, and TNF actually partially prevented the IFN-induced increase in Ia. However, culture with TNF activated MP for increased hydrogen peroxide production in response to phorbol myristate acetate (PMA) and antagonized the IFN-induced decrease in the proportion of the MP bearing receptors for the Fc region of IgG2b. TNF and IFN were additive in increasing peroxide production. Both human and murine TNF had the same effects on MP, and MP from C3H/HeN (endotoxin sensitive) and C3H/HeJ (endotoxin resistant) mice responded comparably to TNF and IFN. Our results support the hypothesis that TNF has some macrophage-activating activity, but its effects are selective and not identical with those of IFN.

Human granulocyte-macrophage colony stimulating factor: an effective direct activator of human polymorphonuclear neutrophilic granulocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to modulate different granulocyte functions. In the present study we investigated the effect of purified and recombinant human GM-CSF, particularly on the oxidative metabolism of isolated human granulocytes. In addition, ultrastructural changes upon stimulation were evaluated. For detection of granulocyte activation the following assay systems were used: 1) lucigenin-dependent chemiluminescence (CL), 2) superoxide-dismutase (SOD) inhibitable cytochrome C-reduction (superoxide), 3) horseradish peroxidase-mediated oxidation of phenol red (hydrogen peroxide), 4) release of myeloperoxidase, 5) ultrastructural detection of hydrogen peroxide-production, and 6) scanning and transmission electron microscopy (SEM and TEM, respectively). A significant CL response was seen upon stimulation with recombinant human GM-CSF at concentrations ranging from 1 to 10(3) U/ml. The CL response started within 5-10 min with a maximum at 60-90 min and lasted more than 3 h. Thereafter granulocytes were completely deactivated to restimulation with the same mediator and with Tumor Necrosis Factor, but responded to other triggers of the oxidative burst, whereas the response to f-met-leu-phe was significantly increased. The CL signal was completely blocked by an antiserum to GM-CSF. Moreover, the response was significantly inhibited by SOD and D-Mannitol, suggesting the involvement of distinct reactive oxygen species (ROS) in generating the CL response. Significant amounts of superoxide were detected within 180 min after stimulation with GM-CSF, whereas release of hydrogen peroxide and peroxidase were only minimal as shown by functional and ultrastructural assays. Activation of granulocytes could be visualized by SEM and TEM. GM-CSF stimulated cells showed an increased adherence to the substratum developing polarized filopodia and an increased number of intracellular vesicles within 30 min after addition of the stimulus. The results clearly demonstrate that GM-CSF directly stimulates granulocytes and, particularly, their oxidative metabolism. Therefore, GM-CSF which is probably released by epidermal cells appears to be a candidate for neutrophil activation in the skin, and thereby may play a crucial role in inflammatory skin diseases.

Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes.

Microorganisms ingested by PMNs are exposed to a variety of antimicrobial systems. Together they comprise a formidable armamentarium, and few organisms survive. The predominant antimicrobial system would be expected to vary with the species, the availability of oxygen and the type of microorganism ingested. There is considerable evidence that the MPO-mediated antimicrobial system plays an important role in the destruction of certain microorganisms in most species; chicken heterophils, however, do not contain MPO,40 and some microorganisms are resistant to this system due to the nature of their cell wall material.146 Further, microbial catalase may offer some protection. The granulocytes of some species (e.g., rabbit, chicken) are rich in cationic proteins and these agents may play a particularly important role in these cells. Granular cationic proteins are less plentiful in human cells.111 Organisms vary in their susceptibility to lysozyme and this enzyme is absent from bovine leukocytes.113 It is probable that the total microbicidal potential of the leukocyte is in excess of its needs under most circumstances. This "overkill" capacity is a reflection of both the level of activity of individual systems and their variety. Particular organisms are susceptible to more than one antimicrobial system and thus may be effectively handled by back-up systems when one is absent. Thus, an organism normally killed by the peroxidase system may be handled less efficiently but adequately when MPO is absent by other oxygen-dependent antimicrobial systems. When a defect in oxidative metabolixm is present as in CGD, both MPO-catalyzed and nonenzymatic oxygen-dependent systems are absent. The ingested organism can, in some instances, supply the needed product of oxidative metabolism (i.e., H2O2); in other instances, oxygen-independent antimicrobial systems are adequate to prevent microbial growth. However, in yet other instances, the organisms survive and multiply and severe infection results.

Perspectives on hydrogen peroxide and drug-induced hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency.

G-6-PD-deficiency is a genetic disorder of erythrocytes in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidized glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidase. Although a variety of free-radical species may be produced during the interaction of xenobiotic agents with erythrocytes and hemoglobin, the inability to destroy peroxides seems to be the hallmark of the disease. Colloid osmotic hemolysis is seldom observed in this disorder and it is possible that hydroxyl radicals derived from peroxide damage both lipid and protein constituents of the plasma membrane so that its intrinsic mechanical properties are altered. Erythrocytes with damaged membranes become less deformable and may be subjected to mechanical entrapment in the microcirculation. Ultimate recognition of damaged cell and sequestration by phagocytes leads to anemia.