PMA-stimulated Superoxide Production Research Articles

Olfactomedin 4 contributes to hydrogen peroxide-induced NADPH oxidase activation and apoptosis in mouse neutrophils.

Neutrophils increase production of reactive oxygen species, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, to destroy invading microorganisms under pathological conditions. Conversely, oxidative stress conditions, such as the presence of H2O2, induce neutrophil apoptosis, which helps to remove neutrophils after inflammation. However, the detailed molecular mechanisms that are involved in the latter process have not been elucidated. In this study, we investigated the potential role of olfactomedin 4 (Olfm4) in H2O2-induced superoxide production and apoptosis in mouse neutrophils. We have demonstrated that Olfm4 is not required for maximal-dosage PMA- and Escherichia coli bacteria-induced superoxide production, but Olfm4 contributes to suboptimal-dosage PMA- and H2O2-induced superoxide production. Using an NADPH oxidase inhibitor and gp91phox-deficient mouse neutrophils, we found that NAPDH oxidase was required for PMA-stimulated superoxide production and that Olfm4 mediated H2O2-induced superoxide production through NADPH oxidase, in mouse neutrophils. We have shown that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-induced apoptosis compared with neutrophils from wild-type mice. We also demonstrated that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-stimulated mitochondrial damage and membrane permeability, and as well as reduced caspase-3 and caspase-9 activity, compared with neutrophils from wild-type mice. Moreover, the cytoplasmic translocation of the proapoptotic mitochondrial proteins Omi/HtrA2 and Smac/DIABLO in response to H2O2 was reduced in neutrophils from Olfm4-deficient mice compared with neutrophils from wild-type mice. Our study demonstrates that Olfm4 contributes to H2O2-induced NADPH oxidase activation and apoptosis in mouse neutrophils. Olfactomedin 4 might prove to be a potential target for future studies on inflammatory neutrophil biology and for inflammatory disease treatment.

Modulation of ovine neutrophil function and apoptosis by standardized extracts of Echinacea angustifolia, Butea frondosa and Curcuma longa

Impaired neutrophil function has been associated with increased infectious diseases in ruminants. Attachment of neutrophils to endothelium and superoxide production is critical features of their immune activity. Once the infection is cleared, programmed cell death ensures the rapid resolution of inflammation. To develop new natural therapeutics for ruminants, standard extracts of Echinacea angustifolia (Polinacea™), Butea frondosa and Curcuma longa (Curcuvet™) were first evaluated on ovine neutrophil functions. Curcuvet™ strongly reduced PMA-stimulated adhesion and superoxide production. Polinacea™ and B. frondosa extract also reduced these functions, but with less efficacy than Curcuvet™. We analyzed the effect of extracts on spontaneous apoptosis and gene expression in neutrophils aged in vitro for up to 22 h. IL8 is critical for neutrophil recruitment and the immune response; Bcl2-related proteins, Bcl2A1 and Bax, are key regulators of neutrophil fate. Spontaneous apoptosis strongly increased in ovine neutrophils cultured for 22 h (T22), accompanied by an upregulation of IL8 and a decreased Bcl2A1:Bax ratio. Curcuvet™ stimulated spontaneous apoptosis and inhibited IL8 and Bcl2A1 gene expression at T22, whereas Polinacea™ and B. frondosa extract inhibited spontaneous apoptosis and stimulated IL8 expression at T22. These results suggest that Curcuvet™ has antiinflammatory activity, whereas Polinacea™ and B. frondosa have an immunomodulatory action on sheep neutrophils.

Bone Marrow-derived Cells Require a Functional Glucose 6-Phosphate Transporter for Normal Myeloid Functions

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose 6-phosphate transporter (Glc-6-PT). Glc-6-PT activity has been shown to be critical in the liver and kidney where a deficiency disrupts glucose homeostasis. GSD-Ib patients also have defects in the neutrophil respiratory burst, chemotaxis, and calcium flux. They also manifest neutropenia, but whether Glc-6-PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To address this, we transferred bone marrow from Glc-6-PT-deficient (Glc-6-PT(-/-)) mice to wild-type mice to generate chimeric mice (BM-Glc-6-PT(-/-)). As a control, we also transferred bone marrow between wild-type mice (BM-Glc-6-PT(+/+)). While BM-Glc-6-PT(+/+) mice have normal myeloid functions, BM-Glc-6-PT(-/-) mice manifest myeloid abnormalities characteristic of Glc-6-PT(-/-) mice. Both have impairments in their neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in granulocyte colony-stimulating factor and chemokine KC levels. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is decreased in both BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice along with depressed peritoneal neutrophil accumulation. The neutrophil recruitment defect was less severe in BM-Glc-6-PT(-/-) mice than in Glc-6-PT(-/-) mice. These findings demonstrate that Glc-6-PT expression in bone marrow and neutrophils is required for normal myeloid functions and that non-marrow Glc-6-PT activity also influences some myeloid functions.

Taurine chloramine inhibits PMA-stimulated superoxide production in human neutrophils perhaps by inhibiting phosphorylation and translocation of p47 phox

Neutrophils produce microbicidal oxidants to destroy the invading pathogens using nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a membrane-associated enzyme complex that generates superoxide anion (O 2 −). Upon stimulation, the cytosolic components of NADPH oxidase, p47 phox and p67 phox and the small GTPase Rac move to phagosomal and plasma membranes where they become associated with the membrane components of NADPH oxidase, gp91 phox and p22 phox and express enzyme activity. We previously showed that taurine chloramine (Tau-Cl) inhibits O 2 − production in mouse peritoneal neutrophils (Kim, 1996). In the present study, we investigated the mechanisms underlying Tau-Cl-derived inhibition on O 2 − production using a human myeloid leukemia cell line, PLB-985 cell, which has been differentiated into neutrophil-like cell. Tau-Cl inhibited the phorbol myristate acetate (PMA)-elicited O 2 − production as previously observed in murine peritoneal neutrophils. Translocation of p47 phox , p67 phox and Rac was increased in response to PMA, and Tau-Cl inhibited the PMA-stimulated translocation of p47 phox and p67 phox to plasma membrane without affecting the translocation of Rac. In addition, Tau-Cl inhibited the PMA-derived phosphorylation of p47 phox , a requirement for the translocation of cytosolic NADPH oxidase component to the plasma membrane. These results suggest that Tau-Cl inhibits PMA-elicited O 2 − production in PLB-985 granulocytes by inhibiting phosphorylation of p47 phox and translocation of p47 phox and p67 phox , eventually blocking the assembly of NADPH oxidase complex.

Immune‐Modulating Effects of Melatonin, N‐Acetylserotonin, and N‐Acetyldopamine

Melatonin and N-acetylserotonin (NAS) have antioxidant properties. In the present study, we examined whether melatonin, NAS, and N-acetyldopamine (NAD) have a modulatory effect on tumor necrosis factor-alpha (TNF-alpha) synthesis and superoxide production. Differentiated THP-1-derived human monocytes were coincubated with Escherichia coli lipopolysaccharide (LPS) and rising concentrations of melatonin, NAS, or NAD. After 24 h, TNF-alpha was measured in cell supernatants. In addition, the production of superoxide by HL-60-derived human neutrophils upon stimulation with 4-beta-phorbol 12-beta-myristate 13-alpha-acetate (PMA) or N-formyl methionyl-leucyl-phenylalanine (fMLP) and increasing concentrations of melatonin, NAS, or NAD was determined. Incubation of THP-1-derived monocytes with increasing concentrations of melatonin, NAS, or NAD resulted in a marked decrease in LPS-stimulated TNF-alpha production, which was dose-dependent and on the order of 96-98%. Incubation of HL-60-derived neutrophils with increasing concentrations of melatonin, NAS, or NAD resulted in a modest decrease in PMA-stimulated superoxide production, which was dose-dependent. At the 100 microM dose, melatonin, NAS, or NAD resulted in a 14 +/- 4%, 30 +/- 1%, and 29 +/- 1% decrease in PMA-stimulated superoxide production, respectively. Coincubation of HL-60 cells with melatonin, NAS, or NAD also resulted in a modest dose-dependent decrease in fMLP-stimulated superoxide production. At the 100 microM dose, melatonin, NAS, or NAD resulted in a 13 +/- 1%, 14 +/- 1%, and 14 +/- 1% decrease in superoxide production, respectively. Our results indicate that the inhibitory effect of melatonin, NAS, or NAD on LPS-induced TNF-alpha production is robust and dose-dependent. These compounds are equally effective in attenuating the generation of oxidant radicals, although to a lesser degree.

The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the production of superoxide by human monocytes, but not neutrophils, activated in vitro.

To evaluate the regulatory properties of the essential oil of Melaleuca alternifolia (tea tree oil) on the production of oxygen derived reactive species by human peripheral blood leukocytes activated in vitro. The ability of tea tree oil to reduce superoxide production by neutrophils and monocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) was examined. The water-soluble fraction of tea tree oil had no significant effect on agonist-stimulated superoxide production by neutrophils, but significantly and dose-dependently suppressed agonist-stimulated superoxide production by monocytes. This suppression was not due to cell death. Chemical analysis identified the water-soluble components to be terpinen-4-ol, alpha-terpineol and 1,8-cineole. When examined individually, terpinen-4-ol significantly suppressed fMLP- and LPS- but not PMA-stimulated superoxide production; alpha-terpineol significantly suppressed fMLP-, LPS- and PMA-stimulated superoxide production; 1,8-cineole was without effect. Tea tree oil components suppress the production of superoxide by monocytes, but not neutrophils, suggesting the potential for selective regulation of cell types by these components during inflammation.

Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate.

The inhibitory effects of five tea polyphenols, namely theaflavin (TF1), theaflavin-3-gallate (TF2), theaflavin-3,3'-digallate (TF3), (-)-epigallocatechin-3-gallate (EGCG), and gallic acid, and propyl gallate (PG) on xanthine oxidase (XO) were investigated. These six antioxidant compounds reduce oxidative stress. Theaflavins and EGCG inhibit XO to produce uric acid and also act as scanvengers of superoxide. TF3 acts as a competitive inhibitor and is the most potent inhibitor of XO among these compounds. Tea polyphenols and PG all have potent inhibitory effects (>50%) on PMA-stimulated superoxide production at 20 approximately 50 microM in HL-60 cells. Gallic acid (GA) showed no inhibition under the same conditions. At 10 microM, only EGCG, TF3, and PG showed significant inhibition with potency of PG > EGCG > TF3. The superoxide scavenging abilities of these six compunds are as follows: EGCG > TF2 > TF1 > GA > TF3 > PG. PG was the most potent inhibitor of PMA-stimulated H(2)O(2) production in HL-60 cells. The order of H(2)O(2) scavenging ability was TF2 > TF3 > TF1 > EGCG > PG > GA. Therefore, the antioxidative activity of tea polyphenols and PG is due not only to their ability to scavenge superoxides but also to their ability to block XO and related oxidative signal transducers.

The effect of molecular weight and beta-1,6-linkages on priming of macrophage function in mice by (1,3)-beta-D-glucan.

1,3-beta-D-glucans (glucans) are structural elements in the cell walls of yeast and fungi with immunomodulatory properties, mediated through their ability to activate macrophages. This study assessed the activation of cells of the peritoneal cavity between 3 and 90 days after i.p. injection of particulate yeast glucan differing in molecular weight (MW) and degree of (1,6)-linkages. Female QS mice, 7-9 weeks of age, were injected, i.p., with varying doses of low (< 5 x 10(5)), medium (1-2 x 10(6)) or high (> 3 x 10(6)) MW glucans, all with low (< 5%) beta-(1,6)-linkages, or high MW (> 3 x 10(6)) glucan with high 1,6-linkages (> 20%). All glucans induced a transient increase in the proportion of neutrophils and eosinophils and a reduction in mast cell numbers in the peritoneal cavity. Peritoneal macrophages showed an altered morphology, increased intracellular acid phosphatase, increased LPS-stimulated NO production and increased PMA-stimulated superoxide production. There were no significant changes in serum lysozyme levels. Most macrophage activities returned to control levels by 28 days post injection of 1, 3-beta-D-glucan. There was a trend for higher MW or (1,6)-linked, (1, 3)-beta-D-glucans to be more stimulatory. It was concluded that particulate yeast (1,3)-beta-D-glucan is an effective stimulator of immune function, the efficiency of which may be influenced by the MW and degree of (1,6)-linkages.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet-activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10(-10) and 10(-9) M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (> or = 10(-7) M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10(-9) M) caused secretory granule degranulation, moderate doses (> or = 10(-7) M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10(-5) M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations > or = 10(-7) M also caused up-regulation of the adhesion molecules Mac-l and L-selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10(-6) M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10(-5) M) caused an increased rate of PMA-stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA-induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up-regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus.

Priming of human monocyte superoxide production and arachidonic acid metabolism by adherence to collagen- and basement membrane-coated surfaces.

Monocytes (m phi s) come into intimate contact with basement membranes and extracellular matrix proteins as they extravasate from the blood to the interstitium or to sites of tissue injury. We examined the in vitro effects of m phi adherence to an endothelial cell-derived basement membrane or to purified extracellular matrix proteins on phorbol myristate acetate (PMA)-stimulated superoxide production and prostanoid secretion. Elutriation-purified human peripheral blood m phi s were adhered to tissue culture wells that were precoated with the following purified proteins: bovine serum albumin (BSA), collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), or laminin (LM). To model the provisional matrix at sites of tissue injury, m phi s were also adhered to wells coated with either denatured collagen type I or gelatin (GEL) or coated with basement membrane (BM) derived from endothelial cell monolayers. The m phi s were adhered to the protein-coated surfaces for 1 h at 37 degrees C in serum-free medium and washed to remove nonadherent cells, and the number of adherent m phi s was measured. Monolayers of m phi s were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA-stimulated superoxide production by adherent m phi s was determined after 1 and 18 h of adherence to the protein-coated surfaces. PMA-stimulated release of two prostanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) was measured after 18 h of m phi adherence to the surfaces. Following 18 h of adherence, PMA-stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented of primed by m phi s adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by m phi s adherent to COL IV, FN, or LM. The results suggest that adherence to basement membranes, collagen type I-containing surfaces in the interstitium, or denatured collagen at sites of tissue injury primes m phi respiratory burst and arachidonate metabolism to inflammatory agonists. Induction of priming events by substrate-specific adherence may be an important factor regulating host defense functions of m phi s in the extracellular matrix.

Signal transducing properties of the N-formyl peptide receptor expressed in undifferentiated HL60 cells.

Differentiated HL60 cells respond to challenge with ligand by mobilizing intracellular second messengers, resulting in superoxide production, degranulation, and actin polymerization with subsequent chemotaxis and phagocytosis. The functional capabilities of undifferentiated HL60 cells have not been similarly characterized due to the absence of the cell surface receptors required to initiate these processes. To investigate these properties, undifferentiated HL60 cells were transfected with one of the better characterized neutrophil chemotactic receptors, the N-formyl peptide receptor (FPR). Expression of the recombinant FPR gene product in FPR-transfected HL60 cells and the absence of the endogenous FPR in vector-transfected HL60 cells was demonstrated by Northern blot and flow cytometric analyses. FPR-transfected HL60 cells retained their ability to undergo granulocytic differentiation with dibutyryl cAMP, as determined by FMLP- and PMA-stimulated superoxide production. Furthermore, incubation of FPR-transfected HL60 cells for 5 days in the presence of FMLP resulted in limited differentiation as evidenced by the expression of functional C5a receptors. Binding studies of FPR-transfected HL60 cells demonstrated the presence of two binding affinities with dissociation constants of 0.6 and 33 nM, similar to dibutyryl cAMP differentiated HL60 cells and human neutrophils but contrasting the single high affinity state of the FPR expressed in mouse L cell fibroblasts. FPR-transfected HL60 cells displayed FMLP-dependent calcium mobilization with an EC50 of 3 nM and actin polymerization with an EC50 of approximately 10 nM. Actin polymerization was not observed in FPR-transfected L cell fibroblasts or undifferentiated vector-transfected HL60 cells. Both calcium mobilization and actin polymerization were sensitive to treatment with pertussis toxin, indicating the requirement for a Gi-like protein. Stimulation of either undifferentiated or differentiated HL60 cells with ATP resulted in pertussis toxin-insensitive calcium mobilization but was ineffective in producing actin polymerization. The results described herein show for the first time that undifferentiated HL60 cells can respond to chemoattractant receptor stimulation with many of the properties of the mature neutrophil. Transfected HL60 cells will provide an excellent system to study the characteristics of chemotactic receptors as well as the functional properties of myeloid cells.

The relationship between sugar metabolism, transport and superoxide radical production in rat peritoneal macrophages

Dexamethasone inhibits sugar-dependent phorbol myristate acetate (PMA)-stimulated superoxide production and 2-deoxy-spd-glucose (2-dGlc) transport in rat peritoneal macrophages (Rist, R.J., Jones, G.E. and Naftalin, R.J.(1991) Biochem. J. 278, 119–128; Rist, R.J. and Naftalin, R.J. (1991) Biochem J. 278, 129–135). Here it is shown that with glucose as a substrate, dexamethasone (0.1 μM) acts as a non-competitive inhibitor of PMA-induced superoxide production; decreasing the maximal rate of superoxide production ( P < 0.001) without altering the K m . In contrast, with 2-dGlc as a substrate, dexamethasone shows competitive inhibition of PMA-stimulated superoxide production; increasing the K m of superoxide production, ( P < 0.001) without altering the V max . The maximal rate of PMA-stimulated superoxide production with glucose as substrate is 10–12-fold in excess of the maximal rate with 2-dGlc as substrate. Diphenylene iodonium (DPI) is a non-competitive inhibitor of PMA-stimulated glucose-dependent superoxide production in macrophages, ( K i = 1–5 μM ) and significantly reduces the activity of the PMA-in-duced hexose monophosphate shunt, (HMPS) ( P < 0.01). However, DPI (1 μM) has no significant effect on the PMA-induced increase in 2-dglc uptake, suggesting that the stimulus for HMPS activity and superoxide production is separate from the stimulus for hexose transport. A model is described which explains the observed differences in hexose transport and glucose- and 2-dGlc-dependent superoxide production in terms of the differences in metabolism of the two sugars. Accumulation of free 2-dGlc within the cytosol leads to saturation of hexokinase and hence, the effects of PMA and dexamethasone, which alter the coupling between hexokinase and the transporter, are only observed at low concentrations of 2-dGlc, where it is accumulated to sub-saturating levels. Since glucose is completely metabolized within the cell, PMA and dexamethasone increase and decrease, respectively, net uptake of sugar and superoxide production at all glucose concentrations.

Direct observation of hexokinase translocation in stimulated macrophages.

1. Fluorescence imaging of antibodies was used to show that phorbol 12-myristate 13-acetate (PMA) induces a 4-fold increase in the amount of hexokinase relative to the control in the cortical shell of rat peritoneal macrophage cytosol adjacent to the plasma membrane, and a corresponding depletion in the amount of hexokinase in the central core of the cytosol. However, there was no significant PMA-dependent change in the distribution of glucose-6-phosphate dehydrogenase. 2. Cytochalasin D, an inhibitor of actin microfilament polymerization, prevented the PMA-induced hexokinase translocation and also reduced the PMA-dependent increases in 2-deoxy-D-glucose transport and glucose-dependent PMA-stimulated superoxide production. 3. PMA caused a contraction of the width of the cortical F-actin zone. Cytochalasin D caused some dispersal of F-actin within the cell, increasing the density of F-actin within the central cytosolic core and causing aggregation of the F-actin within the cortex. These data are consistent with the view that PMA induces attachment of hexokinase to microfilaments within the cortical zone adjacent to the cell membrane of macrophages, and cytochalasin D prevents this attachment. This is the first direct demonstration of the translocation of hexokinase to the plasma membrane in activated cells, and supports the view that enhanced hexokinase activity in the cortical region of the cytosol is an important early component of the macrophage activation process.

Aerosolized Lipopolysaccharide Increases Pulmonary Clearance of99mTc-DTPA in Rabbits

Bacterial endotoxins alter the permeability of endothelium, but little is known of their effect on epithelium. We exposed specific pathogen-free rabbits to aerosolized Pseudomonas aeruginosa LPS or saline and performed serial measurements of RL, Cdyn, BP, WBC count and differential, and platelet counts. Pulmonary 99mTc-DTPA half-life was measured 4, 6, or 8 h after exposure. The animals were sacrificed and BAL performed. Background and PMA-stimulated superoxide production was measured from individual AM using electrooptical determination of reduction of NBT. Lung tissue was processed for light microscopy and ratio of wet to dry weight. 99mTc-DTPA half-life was significantly shorter in LPS-exposed animals at 6 h (p < 0.05) and 8 h (p < 0.001). There were no differences in Cdyn, RL, BP, WBC, differential, platelet, or BAL cell count at any time between groups. No histologic changes or differences in lung wet to dry weight ratios were found. PMA-stimulated AM superoxide production was significantly increased (p < 0.01) in LPS-exposed animals. This effect was time dependent and could be duplicated in AM from control animals following a 4-h incubation with LPS, lavage fluid from LPS-exposed animals, or recombinant murine TNF. These results demonstrate that aerosolized Pseudomonas LPS increases pulmonary epithelial permeability and primes AM superoxide production.

Selective up-regulation of macrophage function in granulocyte-macrophage colony-stimulating factor transgenic mice.

Peritoneal and pleural cells from mice transgenic for GM-CSF were studied with regard to their phenotype and functional capacity, and compared with cells from normal littermates. Transgenic mice showed markedly elevated peritoneal and pleural cell counts compared with littermates, and a significantly higher proportion of cells in the transgenic populations were macrophage in phenotype. Transgenic macrophages were larger than the littermate cells, showing abundant foamy cytoplasm and enhanced spreading on plastic. Analysis by flow cytometry showed a more than sixfold increased expression of the macrophage activation markers MAC-2 and MAC-3, but not other markers, on transgenic macrophages. Superoxide production was measured in whole cell populations, both in their basal state and in response to particulate (zymosan) and soluble (PMA) stimuli. Both basal and stimulated superoxide production were markedly elevated in transgenic mice of 12 wk of age, with the largest differences seen in response to PMA. In younger mice, however, only PMA-stimulated superoxide production was significantly greater in transgenic macrophages than in littermate cells and levels of superoxide were generally lower than those seen in 12-wk-old mice. These findings suggest that the enhanced functional capacity of transgenic cells is a maturation-dependent event. In contrast to these findings, drug-dependent cytotoxicity assays performed on cells from 12-wk-old mice revealed no significant differences in killing capacity between the two mouse strains. Taken together these data indicate a selective rather than uniform functional up-regulation in transgenic macrophages compared with their littermates, with a time scale suggestive of a maturational rather than activation process. These findings may provide an indication of the functional macrophage phenotype resulting from long term exposure to GM-CSF in vivo, and help to explain the macrophage-associated pathology seen in GM-CSF-transgenic mice.

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression.

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.

Inhibition by linoleic acid hydroperoxide of alveolar macrophage superoxide production: Effects upon mitochondrial and plasma membrane potentials

Linoleic acid hydroperoxide (LOOH) is a naturally occurring product of lipid peroxidation. Incubation of rat alveolar macrophages with LOOH produced alterations of membrane properties and function at concentrations of LOOH as low as 0.1 μ m. These included phorbol myristate acetate (PMA)-stimulated superoxide production, mitochondrial membrane potential, and plasma membrane potentials. These effects were clearly separated from gross loss of structural integrity as measured by lactate dehydrogenase release, in terms of both time of incubation and concentration of LOOH. PMA-stimulated superoxide production measured 15 min after addition of 10 μ m LOOH was inhibited ≈50%; however, addition of this concentration of the hydroperoxide after PMA stimulation was without effect. Superoxide production was also measured in a cell-free system produced by incubation of alveolar macrophages with sodium dodecyl sulfate. Prior incubation of alveolar macrophages with LOOH, H 2O 2, or t-butyl hydroperoxide, under conditions that significantly inhibited superoxide production by the intact cells, did not produce inhibition of the NADPH-dependent superoxide generating system in the cell-free preparation. These results suggest that the effect of LOOH was upon signal transduction involved in the stimulation of superoxide production rather than on the NADPH oxidase itself. Measurements of membrane potential changes were made using the lipophilic ions, 3,3′-dipentyloxacarbocyanine (DiOC 5(3)) and bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V). On the basis of their charge, DiOC 5(3) fluorescence primarily reports mitochondrial potential and oxonol V absorbance reports plasma membrane potential. With 10 μ m LOOH, depolarization of the plasma and mitochondrial membranes appeared to occur within seconds. As prior depolarization depresses superoxide production, these hydroperoxide-induced changes in membrane potential may be responsible for decreased PMA-stimulated superoxide production.

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli.

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.

Effect of cyclooxygenase inhibitors and protease inhibitors on phorbol-induced stimulation of oxygen consumption and superoxide production by rat pulmonary macrophages

Oxygen consumption and Superoxide anion production by pulmonary macrophages are both increased by phorbol myristate acetate (PMA), but the two processes have been separated using protease inhibitors and cyclooxygenase inhibitors. Pretreatment with the protease inhibitors (1-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK) and N-α-p- tosyl- L - lysine chloromethyl ketone (TLCK), as well as with the cyclooxygenase inhibitors acetylsalicylic acid (ASA) and ibuprofen (IBU), inhibited the stimulation of Superoxide production and oxygen consumption by phorbol myristate acetate. However, whereas the order of potency for inhibition of stimulation of Superoxide production was TPCK > TLCK > IBU > ASA, the order of potency for inhibition of stimulation of oxygen consumption was ASA > IBU > TPCK = TLCK. Although all four agents were effective inhibitors of PMA-stimulated Superoxide production and oxygen consumption when added before PMA, in contrast to the cyclooxygenase inhibitors, TPCK was unable to inhibit oxygen consumption by more than 70–80% regardless of the concentration used, although Superoxide generation could be inhibited completely. When added after PMA, ASA did not suppress either oxygen consumption or Superoxide production and ibuprofen was only one-half as effective as an inhibitor. TPCK and TLCK, when added after PMA, accelerated the return to basal rates of both oxygen consumption and Superoxide production. None of the four agents had any effect on basal Superoxide production or oxygen consumption at the concentrations used. The data support the interpretation that both prostagiandin biosynthesis and protease activity may be associated with the activation of the superoxide-generating system of pulmonary macrophages. The consumption of molecular oxygen following stimulation of the cells with phorbol myristate acetate is not due solely to the generation of Superoxide, however, since each process is inhibited with different potency by the same group of inhibitors. There appears to be a component of oxygen consumption which results from the activation of cyclooxygenase and, unlike Superoxide production, cannot be completely inhibited by treatment with protease inhibitors.