Release Of Reactive Oxygen Metabolites Research Articles

RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming

Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar “primed” phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF) using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1). However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF) on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05). These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation.

In vitro evaluation of the inflammatory activity of ultra-high molecular weight polyethylene

To understand the inflammatory potential of oxidised ultra high molecular weight polyethylene (ox-UHMWPE) compared with the virgin one (UHMWPE), we analysed in vitro the predisposition of their interaction with plasma proteins and cells involved in the inflammatory response. The adsorption on the surface of the two materials of adhesion proteins (Fibronectin and Albumin), and pro-inflammatory proteins (IgG and IgA) have been studied. Moreover, we have evaluated the materials effect on complement activation and on macrophages and monocytes-neutrophils behaviour. The two UHMWPE chemical forms adsorbed all the proteins studied; the only difference was in complement activation. Enzyme immunoassay results evidenced higher levels of factor Bb and iC3b in plasma after the contact with the oxidised form. Physico-chemical properties of the oxidised UHMWPE affected the attachment of the cells as demonstrated by macrophages adhesion experiments. UHMWPE favoured only a limited peritoneal macrophages (PMs) spreading (round-shaped cells); the cell spreading and presence of microvilli on the cell membranes was evident in the case of the oxidised form, suggesting the activation of these cells on this chemical form. Ox-UHMWPE evidenced a statistically significative increase in chemiluminescence values respect to human unstimulated peripheral blood mononuclear cells, an index of increased cell release of reactive oxygen metabolites. In conclusion, UHMWPE oxidative degradation with its chemical modification induces monocytes-neutrophils chemiluminescence activation and PMs morphology changes correlated with macrophage activation, data consistent with the complement activation results obtained in this study; such modifications, along with changes in mechanical properties, are related to implant failure.

Apparent role of traveling metabolic waves in oxidant release by living neutrophils.

Cell metabolism self-organizes into two types of dissipative structures: chemical oscillations and traveling metabolic waves. In the present study we test the hypothesis that traveling NAD(P)H waves within neutrophils are associated spatially and temporally with the release of reactive oxygen metabolites (ROMs). Using high-speed optical microscopy and taking advantage of the autofluorescence of NAD(P)H, we have observed the propagation of NAD(P)H waves within cells. When NAD(P)H waves reach the lamellipodium of morphologically polarized neutrophils, a diffusing plume of superoxide is released as evidenced by the conversion of hydroethidine in the extracellular environment to ethidium bromide. Parallel results were obtained by using high-speed emission microspectrophotometry. These experiments indicate that the spatial and temporal properties of NAD(P)H waves are transformed into ROM pulses in the extracellular environment. Propagating NAD(P)H waves allow neutrophils to specifically deliver substrate to the lamellipodium at high concentrations, thus facilitating the local and periodic release of ROMs in the direction of cell movement and/or a target.

Interaction of bioactive glasses with peritoneal macrophages and monocytes in vitro

Macrophage activation was analyzed following exposure to pure, crystalline alpha-quartz powders, two bioactive gel-glass powders of different compositions, and a melt-derived glass, 45S5 Bioglass. The release of reactive oxygen metabolites (chemiluminescence test), modifications of cell morphology, the amount of tumor necrosis factor alpha (TNFalpha) secreted, and the amount of TNFalpha mRNA expression were evaluated. The 45S5 Bioglass powders elicited the highest chemiluminescence response while the two solgel glasses had a lower response with less of an oxidative burst difference between them. Particulate bioactive glasses are actively ingested by mouse peritoneal macrophages, and only the 58S solgel glass had a moderate toxic effect on the macrophages. Macrophage cell morphology showed increased size and cell spreading, consistent with the high level of cytokine secretion induced by 45S5 Bioglass. The 45S5 Bioglass powders led to an increased release of TNFalpha and expression of TNFalpha mRNA relative to unstimulated and control treated monocytes. Bioactive glasses (and particularly 45S5 Bioglass) that in vivo induce rapid bone growth appear to activate an autocrine-like process in which the response evoked by the material (for example monocyte and macrophage activation with cytokine production) enhances subsequent interactions with cells in contact with the material.

Immunoglobulin enhances the bioactive-glass-induced chemiluminescence response of human polymorphonuclear leukocytes.

Bioactive glasses are bone substitutes that chemically bind to bone. Implanted materials always elicit a response from surrounding tissues and thereby can activate inflammatory cells, with subsequent release of biomaterial and tissue-damaging agents. Bioactive glasses can activate polymorphonuclear leukocytes (PMNL) and induce a release of reactive oxygen metabolites (ROM). Adsorption of proteins on the surface of the implanted material may influence the subsequent inflammatory cell response. The effect of Sandoglobulin(R) (SG) and albumin on the ROM release by PMNLs induced by a bioactive glass was studied by a chemiluminescence (CL) assay. An enhanced effect for SG and inhibitory effect for albumin on the CL response of PMNLs was observable. The CL response of the PMNLs was dependent on the incubation time of the glass in solution.

IgG Immune Complex Binding to and Activation of Liver Cells

Background/Aims: The aim was to study IgG immune complex (IC) binding to isolated hepatocytes, Kupffer cells (KCs) and sinusoidal endothelial cells (SECs). Further, we wished to analyze the capacity of IgG ICs to induce release of reactive oxygen metabolites by the IC-binding liver cells. Methods: ICs were formed between ¹²⁵I-tyramine-cellobiose-labelled dinitrophenyl-conjugated human serum albumin (¹²⁵I-TC-DNP₁₀HSA) and polyclonal rabbit IgG antibodies. Binding of ICs to different rat liver cells in suspension was studied at 4°C. Production of reactive oxygen metabolites was measured by luminol-enhanced chemiluminescence at 37°C. Results: IgG mediated binding of ¹²⁵I-TC-DNP₁₀HSA to both KCs and SECs, but not to hepatocytes. The binding showed saturation kinetics and was blocked by an excess of unlabelled IgG-ICs. IgG-ICs activated KCs, but not SECs, to a chemiluminescence response. Conclusions: Both KCs and SECs bind IgG-ICs in vitro, probably via Fc receptor interaction. IgG-ICs activate KCs to produce reactive oxygen metabolites. The binding of IgG-ICs to isolated hepatocytes is small.

Inhibition of neutrophil apoptosis after elective surgery

Neutrophils play a crucial role in host defense against infections, but their inappropriate infiltration and activation within tissues can cause host tissue damage through release of reactive oxygen metabolites, metalloproteinases, and proinflammatory cytokines. The termination of a neutrophil-mediated inflammatory response is effected through programmed cell death or apoptosis. Delayed neutrophil apoptosis is associated with proinflammatory diseases, such as the systemic inflammatory response syndrome. Surgery induces a profound inflammatory response; therefore, neutrophil apoptosis of patients undergoing elective surgery was investigated. Nonseptic patients undergoing elective orthopedic surgery while under epidural anesthesia had neutrophils and platelet-poor isolated from whole venous blood harvested at 4 time points: pre-epidural, 45 minutes postepidural but before surgical intervention, 1 hour postsurgical incision, and 24 hours postsurgery. Neutrophil apoptosis was quantified at 1, 12, and 24 hours in culture by immunofluorescence flow cytometry of annexin V and propidium iodide staining and confirmed by TUNEL (terminal deoxynucleotidyl transferase nick end labeling) assay for DNA strand breaks. Serum cytokines were quantified by specific enzyme-linked immunosorbent assay. Spontaneous neutrophil apoptosis after elective surgery was significantly (P < .001) inhibited with an effect evident within an hour of surgical incision and persisting at 24 hours postsurgery. The addition of patients' 24 hour postoperative plasma to healthy neutrophils markedly (P < .01) reduced neutrophil apoptosis, whereas plasma taken an hour after surgical incision was ineffective. Interleukin (IL)-6 was notably increased (1395 +/- 196 pg/mL, P < .01) 24 hours postsurgery and at this postoperative concentration inhibited (P < .01) apoptosis of normal neutrophils. Levels of other inflammatory mediators (IL-1 beta, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, soluble Fas, soluble Fas ligand) were unaltered. The anti-inflammatory cytokine IL-10 was only slightly increased 24 hours postsurgery (8.32 +/- 2.99 pg/mL); however, the addition of recombinant human IL-10 (10 ng/mL) counteracted (P < .05) inhibition of neutrophil apoptosis induced by IL-6 and post-surgery plasma. These results identify marked inhibition of neutrophil apoptosis after elective surgery and suggest that the inhibition of neutrophil apoptosis in the postoperative period is, at least in part, a result of soluble circulating factors. The marked imbalance favoring proinflammatory over anti-inflammatory cytokine release in the immediate postoperative period mediates the overwhelmingly antiapoptotic net capacity of postsurgery plasma.

Effects of hypoxia-reoxygenation on isolated liver nonparenchymal cells

To clarify the relationship between Kupffer cells (KC) and hepatocytes in hepatic ischemia-reperfusion injury, isolated liver nonparenchymal cells (NPC), including a large proportion of KC, from C3H/HeN and C3H/HeJ mice were utilized in an in vitro hypoxia-reoxygenation system which enabled precise control of the oxygen concentration in the circumferential air during incubation. The viability of NPC, concentrations of cytokines and superoxides released from NPC, and the effects of antioxidants on hypoxiareoxygenation were investigated. The results were: (1) The deterioration of NPC was slow under hypoxia, but a significant decrease in their viability was observed with reoxygenation. These results were virtually identical in C3H/HeN and C3H/HeJ mice. (2) The concentration of tumor necrosis factor (TNF)-α from NPC of C3H/HeJ was much lower than that from NPC of C3H/HeN, and production of O2− was lower in the NPC of C3H/HeJ than in C3H/HeN, but changes in the viability of NPCs which were reoxygenated following various intervals of hypoxia were almost identical in both types of mice. (3) Viability after reoxygenation was improved by the addition of catalase or superoxide dismutase (SOD), and further improved with the combined use of catalase and SOD. These results suggested that the changes in KC function after reoxygenation decreased the viability of the cells, due predominantly to the release of reactive oxygen metabolites, not to the release of TNF-α.

Multiple Emulsion-Mediated Enhancement of the Therapeutic Effect of Tetrandrine against Silicosis

Abstract Using a water-in-oil-in-water multiple emulsion system developed for pulmonary drug targeting, the effectiveness of tetrandrine as an antifibrotic agent and the therapeutic advantage of a tetrandrine emulsion over drug in solution for the treatment of silicosis were investigated in rats. Previously we have shown that the action of tetrandrine is attributed to its ability to inhibit the release of reactive oxygen metabolites and inflammatory cytokines by alveolar macrophages, and that targeted delivery of tetrandrine to alveolar macrophages using a multiple emulsion system minimizes drug toxicity, maintaines the drug's pharmacological activity, and enhances tetrandrine distribution in the lungs while reducing systemic drug distribution. The purpose of the present study is to provide in vivo evidence of emulsion-mediated enhancement of drug action in the lungs against silica-induced lung injury using a rat model. The antifibrotic action of tetrandrine was evaluated by examinations of lung histology...

Escherichia coli-induced activation of neutrophil NADPH-oxidase: lipopolysaccharide and formylated peptides act synergistically to induce release of reactive oxygen metabolites

The prevailing view of neutrophil NADPH-oxidase activation during interaction with bacteria is that the production of toxic oxygen metabolites should be directed into the phagosome containing the engulfed prey. However, in this report we show that a common Escherichia coli strain, HB101, may induce a release of neutrophil oxygen metabolites to the extracellular milieu. This phenomenon is dependent on three factors: (i) the mobilization (upregulation) of neutrophil secretory vesicles prior to interaction with the bacteria, (ii) soluble bacterial factors binding to the formylmethionyl-leucyl-phenylalanine receptor and tentatively identified as formylated peptides, and (iii) a bacterium-associated priming factor identified as lipopolysaccharide.

Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear and polymorphonuclear phagocytes*

Isolated human mononuclear and polymorphonuclear phagocytes were stimulated in the presence and absence of serotonin (5-HT), the major secretory product of activated platelets, and the release of reactive oxygen metabolites during the respiratory burst was assessed by luminol-enhanced chemiluminescence. In the presence of 5-HT, a dose-dependent suppression of the chemiluminescence signal occurred, irrespective of the stimulus used to elicit the respiratory burst. A similar suppression of luminol-enhanced chemiluminescence was also seen in a radical generating cell free system. 5-HT was found to be oxidized by the reactive oxygen species released by stimulated phagocytes. This oxidation is prevented in the presence of other antioxidants. The major 5-HT oxidation product was isolated by gel chromatography and identified by mass-spectrometry as a 5-HT dimer, probably 5,5'-dihydroxy-4,4'-bitryptamine. It is concluded that the 5-HT released from activated thrombocytes at sites of inflammation and endothelial cell damage acts as a true scavenger of reactive oxygen species generated during the respiratory burst of stimulated phagocytes and may thus modulate various aspects of cell-mediated defence reactions.

Evidence for environmentally derived immunomodulation in mussels from the Venice Lagoon

Mussels, Mytilus galloprovincialis, were sampled at three times of year, from various sites within the Venice Lagoon and a reference Platform site in the north Adriatic. The immune response of the mussels was assessed using a range of assays which included total and differential cell counts, phagocytosis, degradative enzyme levels and release of reactive oxygen metabolites. Chlorinated hydrocarbons, including lindane, DDT and PCBs, together with trace metal levels were measured in digestive gland tissues from the mussels. The measurements of immune response and the contaminant levels showed seasonal fluctuations. However, the results did demonstrate significant differences in a number of immunocompetence assays which in some instances showed some correlation with the levels of contaminants measured in the tissues. In particular, total and differential cell counts, phagocytosis and release of reactive oxygen metabolites showed a degree of correlation with contaminant burdens although other factors were clearly involved. The results confirm the requirement for using a range of assays for monitoring pollution effects.

Effects of fluoranthene on the immunocompetence of the common marine mussel, Mytilus edulis

The effects of a model polycyclic aromatic hydrocarbon on the immunocompetence of the marine mussel, Mytilus edulis, were demonstrated in laboratory-controlled experiments. Haemolymph from mussels, exposed for 7 days to three concentrations of fluoranthene, was extracted and assays carried out to investigate total and differential haemocyte numbers, peroxidase and phenol oxidase activity, production of reactive oxygen metabolites and release of lysosomal enzymes. Fluoranthene exposure at 200 and 400 μg l −1 resulted in an increase in the total number of circulating haemocytes but did not affect proportions of eosinophilic to basophilic blood cells. The release of reactive oxygen metabolites was also significantly stimulated. The percentage of haemocytes exhibiting peroxidase and phenol oxidase activity increased significantly at the highest contaminant concentration. The release during phagocytosis of chymotrypsin-like enzyme from the haemocytes was inhibited at all fluoranthene levels. The observed effects on immunocompetence of the mussel, on exposure to environmentally realistic levels of contaminant, demonstrate the necessity for further work in order to establish a relationship between pollutants and disease susceptibility. The variability in effects on different aspects of the immune response emphasises the need for a multi-assay approach to pollution monitoring.

Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.

Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.

Enhanced basal and stimulated PMN chemiluminescence activity in children with atopic dermatitis: stimulatory role of colonizing staphylococci?

In adults, intense staphylococcal skin colonization and hyperactivity of polymorphonuclear leukocyte (PMN) oxidative metabolism are characteristic features of atopic dermatitis. Precise data on childhood atopic dermatitis are lacking. In a prospective study we analysed the PMN chemiluminescence activity with special reference to staphylococcal stimuli in 19 children (mean age 6.2 years) with mild to moderate atopic dermatitis. Staphylococcus aureus was isolated from 17/19 (90%) of children with atopic dermatitis and 13/45 (29%) of healthy age-matched controls (p less than 0.001). The mean (SEM) chemiluminescence activity of unstimulated atopic dermatitis-PMN was 0.34 (0.009) (controls: 0.092 (0.003) x 10(6) cpm/10(6) PMN/min (p less than 0.02). Staphylococcal antigens (S. aureus, S. epidermidis, S. haemolyticus) induced a 1.9-3.1-fold higher peak chemiluminescence response in children with atopic dermatitis than in controls (p less than 0.05). The time interval until peak chemiluminescence activity was considerably shorter for all stimuli in atopic dermatitis. We conclude that PMN of children with atopic dermatitis are "primed", showing enhanced release of reactive oxygen metabolites even in the "resting" state, and are easily stimulated by staphylococcal antigens present on the skin of patients with atopic dermatitis from early childhood on. We speculate that PMN hyperreactivity may contribute to chronic skin damage in atopic dermatitis.

Phagocytosis by lipopolysaccharide-primed human neutrophils is associated with increased extracellular release of reactive oxygen metabolites.

The effect of priming human neutrophils with lipopolysaccharide was investigated regarding the respiratory burst activity generated during phagocytosis of IgG- or C3b-opsonized yeast particles. LPS pretreatment significantly enhanced the respiratory burst activity, measured as luminol-amplified chemiluminescence, of both types of opsonized particles. In control cells most of the activity was produced intracellularly, probably in the phagosomes. In the primed cells, however, extracellular release of reactive oxygen metabolites was significantly increased during Fc- and CR3-mediated phagocytosis (P less than 0.01 and P less than 0.002, respectively). The release was most pronounced when using C3b-opsonized particles. Potent oxygen metabolites acting together with lysosomal enzymes are of importance in inflammatory-induced tissue damage. An increased extracellular release of reactive oxygen species by phagocytizing primed neutrophils can therefore lead to greater damage to the surrounding tissues.

Pathogenetic Significance of Reactive Oxygen Species in Diffuse Fibrosing Alveolitis

Excessive release of reactive oxygen metabolites (ROM) from lung inflammatory cells has been claimed to be of major pathogenetic significance in diffuse fibrosing alveolitis. In the present study, the content of oxidized methionine residues [Met(O)] as a percentage of total methionine (Met) in BAL-derived proteins was used to assess the biologic effect of ROM. In addition, procollagen-III-peptide was measured in BAL fluid as a marker of fibroblast activation. We investigated bronchoalveolar lavage (BAL) samples from seven control patients without evidence of interstitial lung disease and from 42 patients with fibrosing alveolitis caused by idiopathic pulmonary fibrosis (IPF), n = 20, or by collagen vascular disease (CVD), n = 22. Met(O) was elevated in the patients with IPF or CVD compared with that in the control subjects (8.86 +/- 1.26 and 8.13 +/- 1.44% versus 3.36 +/- 0.49%, p less than 0.01 and p less than 0.05, respectively; mean +/- SEM). A positive correlation was found between percentage of neutrophils in BAL and Met(O) in both groups separately and combined (IPF, r = 0.84; p less than 0.001; CVD, r = 0.44; p less than 0.05; IPF and CVD, r = 0.60; p less than 0.001), whereas an inverse relationship existed between Met(O) and the percentage of alveolar macrophages in BAL (IPF, r = -0.59; p less than 0.01; CVD, r = -0.24; NS; IPF and CVD, r = -0.41; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell chemiluminescence. A novel aspect of T-cell membrane activation studied with a Jurkat tumour cell line.

The binding of mitogenic lectins phytohaemagglutinin (PHA), concanavalin A (Con A) and/or of monoclonal antibodies to different receptors such as antigen receptor complex or CD2 on human T cells generates increases in the concentrations of inositol triphosphate (IP3) and cytoplasmic free calcium. This T lymphocyte requires the delivery of two signals; the first can be provided by specific monoclonal antibodies or by mitogenic lectins, and the second by a phorbol ester, phorbol myristate acetate (PMA). In other cells such as macrophages, the rise of intracellular calcium via the generation of IP3 and stimulation of protein kinase C can activate the phospholipase A2, a calcium-dependent enzyme. This enzyme initiates the release of reactive oxygen intermediates and metabolites of arachidonic acid. In order to know whether this other metabolic pathway can be generated in T cells, we tested the capacity of different T-cell lines and clones to produce superoxide anion after stimulation by the above-mentioned activating agents. In this paper, we demonstrate that treatment of the Jurkat human cell line with Con A, PHA, and PMA results in a significant release of reactive oxygen metabolites. Of the various T-cell lines and clones tested, only Jurkat exhibited an oxidative burst. Moreover, none of the antibodies tested (anti-CD3, anti-CD2, and anti-CD28) and known to activate T cells, and none of the immune complexes was able to mediate such an effect. The existence of an oxidative metabolism in at least one T-cell line suggests that T-cell activation may in some instances use another metabolic pathway.

Gamma-interferon restores listericidal activity and concurrently enhances release of reactive oxygen metabolites in dexamethasone-treated human monocytes.

By preventing macrophages from destroying phagocytized microorganisms, glucocorticoids lower natural resistance of the host against many pathogens. gamma-Interferon has an opposite effect and restores activity of dexamethasone-treated mononuclear phagocytes against some but not all microorganisms. In the present studies we show that dexamethasone impairs activity of human blood monocytes kept in culture for 36 h against Listeria monocytogenes, without impairing H2O2 or O2- secretion. Likewise dexamethasone does not interfere with activation of systems generating oxygen metabolites by lymphokines. Thus gamma-interferon increases three- to fivefold the capacity of dexamethasone-treated monocytes to secrete O2- or H2O2 upon stimulation by opsonized zymosan, live bacteria, or phorbol myristate acetate. Concurrently gamma-interferon restores listericidal activity of dexamethasone-treated monocytes. After gradual activation by exposure to gamma-interferon for progressive time periods, listericidal activity becomes tightly correlated (r = 0.922-0.994) with the amount of H2O2 or O2- secreted by dexamethasone-treated monocytes. Activation of oxidative systems by the lymphokine is, however, not correlated with the restoration of activity against Aspergillus spores, lost during dexamethasone treatment, which does not depend on antimicrobial oxygen metabolites. Taken together, these observations lend to the hypothesis that glucocorticoids impair nonoxidative defense mechanisms of "resting" macrophages, while gamma-interferon restores macrophage function impaired by glucocorticoids by activating alternate killing systems, which are at least partly of oxidative nature.

Generation of reactive oxygen metabolites by phagocytosing endothelial cells

We have studied the ability of particulate stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human endothelial cells. Basal release of superoxide (O 2 −) and hydrogen peroxide from undisturbed monolayers was very low (108 pmol (O 2 − and 75 pmol H202 in 3 h from dishes of 3 × 10 5 cells). Addition of 1-μm diameter polystyrene microspheres, which were phagocytosed by the cells progressively, caused a dramatic increase in release of both metabolites; by 3 h, a 13.5- and 6.6-fold increase over controls was observed respectively ( P < 0.001). Addition of formaldehyde-fixed human platelets or chylomicron-size lipid particles also increased production of reactive oxygen species. Similar rises in H 2O 2 and (O 2 − production were induced by treatment with 10 7 M phorbol myristate acetate. Pretreatment of endothelial cells with neuraminidase, heparinase or heparitinase to alter their glycocalyx composition substantially enhanced the effect of microspheres on H 2O 2 and (O 2 − generation. We conclude that the interactions of particles, including platelets and lipids, with endothelial cells leads to the generation of significant pericellular levels of reactive oxygen species. These metabolites can oxidise a wide variety of nearby molecules, leading to cell damage and altered uptake characteristics for lipoproteins containing peroxidised lipids. These effects are exacerbated when endothelial cell glycocalyx composition is disrupted.