Engagement of adenosine receptors inhibits hydrogen peroxide (H2O2-) release by activated human neutrophils

Adenosine and its analogs, acting at specific cell surface receptors, inhibit generation of superoxide anion by neutrophils. Since it has been suggested that hydrogen peroxide (H2O2) release may not be contingent upon superoxide anion release, we studied the effects of 2-chloroadenosine, a potent adenosine receptor agonist, on the formation of H2O2 by neutrophils exposed to various stimuli: n-formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A, phorbol myristate acetate (PMA), serum-treated zymosan particles (STZ), and immune complexes. 2-Chloroadenosine (0.01-10 microM) inhibited formation of H2O2 by neutrophils exposed to FMLP, concanavalin A, and STZ particles. As we have found with O2- generation, 2-chloroadenosine failed to inhibit H2O2 release by neutrophils stimulated by either phorbol myristate acetate or immune complexes. The data show that whereas adenosine and its analogs inhibit neutrophil release of H2O2 and superoxide anion in response to most ligands, they fail to inhibit activation of neutrophils by immune complexes. Nor do they inhibit neutrophil activation by PMA, an agent which bypasses cell surface receptors by direct activation of protein kinase C. Surprisingly, we found that adenosine deaminase activity was adsorbed onto zymosan particles during opsonization and enhanced release of H2O2 by neutrophils exposed to STZ. These studies with yeast cell walls suggest that if microorganisms adsorb adenosine deaminase from serum, then the intracellular microbicidal activity of neutrophils is enhanced.     

Effect of adenosine and its stabile analogue 2-chloroadenosine on cerebrocortical microcirculation and NAD/NADH redox state

In the present study, the effects of topically applied adenosine (ADO) and its stabile analogue 2-chloroadenosine (CADO) on cerebrocortical microcirculation and NAD/NADH redox state (oxidized/reduced nicotinamide adenine dinucleotide) were investigated. Vascular volume (CVV), mean transit time of blood flow (t _m), blood flow (CBF), and NADH fluorescence of the cat brain cortex were measured through a cranial window with a microscope fluororeflectometer. The reference values of CVV,t _m, and CBF, measured in the artificial cerebrospinal fluid (mock CSF) which superfused brain cortex, were regarded as 100%. Adenosine and 2-chloroadenosine, in the concentration range of 10^–6–10^–3 M, resulted in concentration-dependent increases in CBF and NAD reduction. 10^–5 M adenosine and 2-chloroadenosine increased CBF by 49.6±5.6% and 80.4±10.3%, respectively. At a pharmacologically high concentration (10^–3 M), ADO increased CBF by 164.6±13.5%, CADO by 333±44%. At the same time, 10^–3 M ADO and CADO shifted the cortical NAD/NADH redox state by 7.9±0.4% and 12.4±0.7%, respectively toward a more reduced state. Our results, concerning the vasodilator potency of adenosine and 2-chloroadenosine, accord with available data in the literature. However, the pronounced NAD reduction obtained with these adenosine nucleosides suggests that, besides an action on vascular adenosine receptors, some other changes, such as increased substrate mobilization and possibly cAMP production, may contribute to the vasodilator effect of adenosine and 2-chloroadenosine.     

[3H]Adenosine binding to rat mast cells — pharmacologic and functional characterization

Rat serosal mast cell adenosine receptors were characterized by [³H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [³H]adenosine binding to sonicated mast cell membrane preparations at 0°C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400±1600 high affinity [³H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97±3.0 nM. Competition studies reveal that adenosine>2-chloroadenosine>NECA>l-PIA>d-PIA in competing for [³H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, andl-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting anR _a cell surface adenosine receptor subtype; these same analogs potentiate mast cellb-hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [³H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states.     

Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: Adenosine dependent and independent mechanisms

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil aggregation (homotypic cell-cell adhesion) and do so without affecting expression of CD11b/CD18. Since the first step in acute inflammation is a critical interaction between neutrophils and the vascular endothelium (heterotypic cell-cell adhesion), we determined whether NSAIDs diminish the adherence of neutrophils to the endothelium. At antiinflammatory concentrations (0.5–5 mM) sodium salicylate, an NSAID that does not inhibit prostaglandin synthesis, inhibited stimulated but not unstimulated neutrophil adherence to endothelial cells (IC₅₀ < 1 mM,P < 0.00001). Salicylates have previously been shown to inhibit oxidative phosphorylation and, predictably, sodium salicylate inhibited oxidative phosphorylation, as evidenced by depletion of ATP stores (875±75 pmol/10⁶ PMN, [2.92±0.25 mM]) in stimulated (FMLP, 0.1M) but not resting neutrophils treated with antiinflammatory doses of sodium salicylate (EC₅₀=1 mM,P < 0.00001). Indomethacin and piroxicam (10 and 30M) only minimally decreased ATP concentrations in stimulated and resting neutrophils. ATP is metabolized to adenosine, and we have previously demonstrated that both endogenously released (180–200 nM) and exogenous adenosine (IC₅₀=250 nM) inhibit stimulated neutrophil adherence to endothelial cells. To determine whether the increased metabolism of ATP and the resultant increase in adenosine release were responsible for inhibition of neutrophil adhesion to endothelium, we determined whether addition of adenosine deaminase (ADA, 0.125 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite, inosine, affected inhibition of neutrophil adhesion to endothelium by stimulated neutrophils. ADA significantly reversed inhibition of neutrophil adherence to endothelium by sodium salicylate (0.5–5 mM,P < 0.00001). This suggests that sodium salicylate inhibits neutrophil adherence by increasing adenosine release. Whereas indomethacin and piroxicam (10–50M) also inhibited stimulated neutrophil adherence to endothelial cells, ADA did not affect their inhibition of adherence. These studies demonstrate a heretofore unexpected antiinflammatory mechanism for salicylates: salicylates increase ATP hydrolysis and thereby enhance release of adenosine. Moreover, these data are consistent with the hypothesis that NSAIDs differ from one another with respect to their mechanisms of action.     

Unique characteristics of superoxide production by human eosinophils in eosinophilic states

Eosinophils from patients with peripheral blood eosinophilia and human neutrophils from normal subjects and patients with neutrophilia produced superoxide anion (O₂ ^–) in vitro at similar rates in the absence of stimulation and exhibited comparably increased rates of O₂ ^– production during the initial 1 h of incubation with opsonized zymosan. In the presence of opsonized zymosan, the rate of O₂ ^– production by eosinophils was constantly high for 3 h, whereas the rate of production by neutrophils fell by more than 65% after 1 h. Consequently, the amount of superoxide produced by phagocytizing leukocytes was twofold higher for eosinophils than for neutrophils at 3 h. O₂ ^– production by cell-free sonicates of zymosan-stimulated eosinophils and neutrophils exhibited the same preference for NADPH over NADH. One mM sodium azide significantly decreased the generation of O₂ ^– by phagocytizing eosinophils, but lacked an effect on neutrophils. The prolonged release of O₂ ^– by eosinophils engaged in phagocytosis may contribute both to their unique microbicidal profile and to the capacity of eosinophils to injure host tissues in some eosinophilic syndromes.     

Characterization of adenosine receptors in bone. Studies on the effect of adenosine analogues on cyclic AMP formation and bone resorption in cultured mouse calvaria

The effect of different adenosine analogues on cyclic AMP (cAMP) formation and bone resorption in cultured mouse calvarial bones was investigated. 5'-N-ethylcarboxamidoadenosine (NECA), R-N6-phenylisopropyl-adenosine (PIA), N6-cyclohexyl-adenosine (CHA) and 2-chloroadenosine all stimulated cyclic AMP formation with a threshold close to 1 mumol l-1); NECA was the most potent agonist. Theophylline (10, 100 mumol l-1) inhibited the cAMP accumulation induced by NECA and 2-chloroadenosine (30 and 300 mumol l-1), dose dependently. There was no inhibition of cAMP formation by PIA and CHA in forskolin-treated bone tissue. SQ 22, 536 and 2',5'-dideoxyadenosine (100 mumol l-1) both inhibited rolipram-stimulated cAMP formation. Cyclic AMP accumulation in isolated osteoblast-like cells from neonatal mouse calvarial bones was stimulated by NECA (10 and 100 mumol l-1) and 2-chloroadenosine (100 mumol l-1). 2-chloroadenosine (10 and 30 mumol l-1), but not NECA, PIA nor CHA, caused a dose-dependent stimulation of 45Ca release in both 48- and 120-h culture. The effect of 2-chloroadenosine on 45Ca release could not be antagonized by theophylline. Neither NECA, PIA, CHA nor 2-chloroadenosine could affect PTH-stimulated 45Ca release in short term cultures (6, 24 h). By contrast, stimulation of cAMP formation by forskolin or dibutyryl cAMP caused a rapid (6 h) inhibition of PTH-stimulated bone resorption. The results demonstrate functional A2 and P-site receptors in mouse calvaria and osteoblast-like cells, but no A1-receptor was detected. These adenosine receptors regulate cAMP, but are not intimately linked to bone resorption. The calcium mobilization induced by 2-chloroadenosine appears to be unrelated to adenosine receptors.     

Effect of Mycotoxins on Some Activities of Isolated Human Neutrophils

Activities of mycotoxins—citrinin, ochratoxin B, rubratoxin B, and zearalenol β—that affected superoxide anion (O₂^?) production were studied on human neutrophils with regard to hypochlorous acid (HOCl) generation, nitric oxide (NO) formation, and chemotaxis of isolated cells. At the doses of 10^{? 8}, 10^{? 6}, 10^{? 4}, or 10^{? 2} mg/mL, in stimulated cells the mycotoxins inhibited HOCl production proportionally to that of O₂^? because HOCl production is directly dependent on O₂^? generation. But unlike the others, zearalenol β decreased HOCl production much more than one of O₂^?, indicating an effect also on myeloperoxidase secretion. All mycotoxins inhibited NO generation but only at the dose of 10^{? 2} mg/mL. Formyl-met-leu-phe–induced chemotaxis of neutrophils was deeply affected as well, as all mycotoxins at all doses used decreased it. All considered effects were not dose-dependent, likely connected to high variability of individual sensitivity to these compounds and because they already are present in blood introduced with foods. These activities of neutrophils confirm and extend the danger for immune systems by mycotoxins also in very small quantities, since the smaller concentrations we used are easily reachable in blood.     

Mechanisms of the Macrolide-Induced Inhibition of Superoxide Generation by Neutrophils

The effect of macrolides on the superoxide (O₂ ^?) production by neutrophils was studied. Resting neutrophils become primed by lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), and primed neutrophils generate O₂ ^? in response to fMLP or adhesion, respectively. Both LPS-primed fMLP-stimulated O₂ ^? generation by macrolide-treated neutrophils and adhesion-stimulated O₂ ^? generation by macrolide-treated fMLP-primed neutrophils were inhibited. Macrolide inhibition of O₂ ^? generation was dependent on serum or pH. Serum could be substituted by NaHCO₃. The intensity of inhibition was azithromycin = roxithromycin > clarithromycin > erythromycin, in that order. Non-antimicrobial derivatives of erythromycin, that is, EM703 and EM900, inhibited O₂ ^? generation at pH 7.4. NH₄Cl abolished the activity of azithromycin (AZ) only when added to neutrophils with AZ but not after incubation with AZ, suggesting that NH₄Cl prevented the influx of AZ. AZ did not affect the expression of alkaline phosphatase, CD11b, and cytochrome b₅₅₈ in both resting and LPS-primed neutrophils. These results suggested that macrolides did not affect granule mobilization but inhibited O₂ ^? generation selectively.     

Dissociation of phagocytosis,metabolic stimulation and lysosomal enzyme release in human leukocytes

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O ₂ ^– ) generation by, and lysosomal enzyme release from normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O ₂ ^– generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O ₂ ^– when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O ₂ ^– production of these cells. In all cases a time and dose-dependent stimulation was found of both the O ₂ ^– generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O ₂ ^– generation. When PMN's were preincubated with 5×10^–4 M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16–35%. Hydrocortisone also inhibited O ₂ ^– generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O ₂ ^– production).Financially supported by a travel grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Contribution of prostaglandins in hypoxia-induced vasodilatation in isolated rabbit hearts. Relation to adenosine and KATP channels

The mechanism of hypoxia-induced coronary vasodilatation was studied in isolated, saline-perfused rabbit hearts under constant flow conditions. Reduction in the perfusion solution PO₂ (from 520±6 to 103±9 mm Hg) under control conditions halved the coronary resistance and was accompanied by a significant release of the prostaglandin (PG) 6-keto-PGF₁ (from 1.8±0.3 to a maximum of 4.4±0.9 pmol min^–1 g^–1). The cyclooxygenase inhibitor, diclofenac (1 M), blocked the release of PGI₂ and reduced hypoxia-induced vasodilatation (from 47±8% to 25±5%, P<0.05). The relative contribution of adenosine, prostaglandins, and adenosine triphosphate (ATP)-sensitive K⁺ channel (K_ATP channel) activation in hypoxia-induced vasodilatation was assessed by comparing the differential change (control response minus response after treatment) in coronary perfusion pressure (CPP) during infusion of 8-phenyltheophylline (8-PT), diclofenac, and glibenclamide, respectively. The differential change in CPP with 8-PT and diclofenac given together (–48 ±7%) was found to be equivalent to the sum of their respective effects (–24±7 and –19±4%, respectively). Glibenclamide (0.3 M) reduced significantly hypoxia-induced vasodilatation (differential change in CPP of –27±6%) as well as the dilator response to 10 M adenosine and to the stable PGI₂-analogue, iloprost. Forskolin-induced coronary vasodilatation in arrested hearts was slightly, but significantly, reduced by glibenclamide. Our results suggest that both cyclooxygenase products and adenosine, acting independently and concomitantly, contribute to the dilator response of coronary resistance vessels to hypoxia, in part through the activation of K_ATP channels. K_ATP channel activation by prostacyclin and adenosine may involve both cyclic adenosine monophosphate-dependent and independent pathways.     

Estimation of endogenous adenosine activity at adenosine receptors in guinea-pig ileum using a new pharmacological method

Aim: Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. Methods: Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. Results: Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10⁻⁶ m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). Conclusion: Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose–response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.     

Characterization of arachidonic acid metabolism,superoxide production,and bacterial killing by bovine alveolar neutrophils elicited with leukotriene B4 and zymo-san-activated plasma

Leukotriene B₄ (LTB₄) and zymosan-activated plasma (ZAP) were each instilled into the lungs of steers to elicit alveolar neutrophils for subsequent functional analysis. Prior to instillation of either agent, bronchoalveolar lavage cell populations consisted of 95.8 ± 0.4% macrophages (mean ±SEM). Four hours after instillation of LTB₄ or ZAP, the lavage cell populations consisted of 75.0 ± 8.8% and 90.7 ± 0.7% neutrophils, respectively. Alveolar neutrophils elicited with LTB₄ and stimulated with the calcium ionophore A23187 released diminished amounts of LTB₄ and increased amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) as compared to circulating neutrophils. Release of superoxide anion was decreased for LTB₄-elicited alveolar neutrophils as compared to circulating cells, while bacterial killing was unchanged. ZAP-elicited alveolar neutrophils released diminished amounts of LTB₄ when stimulated with A23187 as compared to circulating neutrophils. There were no differences observed in 5-HETE levels between the two cell populations. In addition, release of superoxide anion was diminished among ZAP-elicited alveolar cells, while bacterial killing was unchanged. Incubation of circulating neutrophils with LTB₄ did not influence the release of arachidonate metabolites, superoxide anion, or bacterial killing. However, incubation of circulating neutrophils with ZAP, followed by A23187 resulted in a reduction in the release of LTB₄, as compared to control cells. Prior exposure to ZAP did not influence the release of superoxide anion or bacterial killing by the circulating neutrophils.     

Adenosine reduces GABAergic IPSC frequency via presynaptic A1 receptors in hypothalamic paraventricular neurons projecting to rostral ventrolateral medulla

Adenosine is an inhibitory modulator of neuronal transmission, including GABAergic transmission in the hypothalamus. It is known that the local GABAergic inputs tonically inhibit the hypothalamic paraventricular neurons projecting to the rostral ventrolateral medulla (RVLM; PVN-RVLM neurons) which regulate sympathetic outflow. In this study, we examined the effects of adenosine on GABAergic synaptic transmission in the PVN-RVLM neurons using whole cell patch-clamp combined with the retrograde labeling technique. Adenosine (100 μM) reversibly decreased the frequency of miniature IPSCs (from 3.41 ± 0.75 to 2.19 ± 0.49 Hz) in a concentration-dependent manner (IC₅₀ = 1.0 μM) without affecting the amplitude and the decay time constant of miniature IPSCs. Adenosine increased the paired-pulse ratio of evoked IPSCs from 1.19 ± 0.05 to 2.28 ± 0.09 (P < 0.001). The effects of adenosine was mimicked by a selective A₁ receptor agonist (CHA, 10 μM), and blocked by a selective A₁ receptor antagonist (DPCPX, 2 μM), but not by a selective A₂ receptor antagonist (DMPX, 10 μM). In conclusion, the results showed that adenosine inhibits synaptic GABA release via presynaptic A₁ receptors in the PVN-RVLM neurons, indicating a potential of adenosine A₁ receptors in regulating sympathetic tone in normal and disease states.     

Neutrophils may directly synthesize both H2O2 and O2 − since surface stimuli induce their release in stimulus-specific ratios

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O₂ ^– and H₂O₂, the latter being presumed to arise by spontaneous dismutation of the former. If H₂O₂ were indeed derived exclusively from released O₂ ^– according to the equation 2O₂ ^– + 2H⁺ H₂O₂ + O₂, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H₂O₂ should not form when cytochromec is present to scavenge O₂ ^– before it can dismutate. Although H₂O₂ cannot be measured directly in the presence of cytochromec because it is consumed in reoxidizing reduced cytochromec, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochromec. We found that the relative amounts of extracellular H₂O₂ and O₂ ^– that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochromec from H₂O₂ oxidation when some stimuli were used but not with others. For example, the ratio of O₂ ^– to H₂O₂ produced by neutrophils exposed to PMA was about 2:1, the expected result if H₂O₂ were derived from O₂ ^–. However when cytochalasin B was added to the cells before the stimulus, the yield of H₂O₂ was reduced but not the yield of O₂ ^–. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O₂ ^– and H₂O₂. Coating the plastic with IgG doubled cytochromec reduction without effecting H₂O₂. In contrast, coating with albumin reduced H₂O₂ without effecting cytochromec reduction. Soluble IgG aggregates induced production of mostly O₂ ^– whereas immune complexes resulted in release of both metabolites.FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O₂ ^– than H₂O₂. The addition of catalase to the cytochromec solution improved the yield of reduced cytochromec when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H₂O₂ release is not a linear function of the amount of O₂ ^– generated and that either a variable fraction of O₂ ^– spontaneously dismutates to H₂O₂ or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H₂O₂ as well as O₂ ^–. If the latter were true, the pathologic consequences of neutrophil activation would vary depending on whether O₂ ^– was the primary product (chemotactic activation) or whether H₂O₂ was released as well (immune complex stimulation).     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Effect of fibronectin on the generation of superoxide anion by neutrophils in alimentary toxicoinfections and experimental salmonella endotoxemia

Production of O₂ ⁻ by neutrophils and the effect of fibronectin on it are studied in patients with alimentary toxicoinfections and in rats with salmonella endotoxemia. Fibronectin is found to affect the generation of superoxide anion by neutrophils, the nature of its effect depending on the number of neutrophils and background free radical activity. Fibronectin stimulates the suppressed and lowers the enhanced generation of O₂ ⁻ by neutrophils. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 628–631, June, 1996     

Alloantibody bipolar bridging; A new mechanism of cell surface activation

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found: (1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (FcγR) were specific for IgG₂ subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG₁, IgG₂ and F(ab′)₂ anti-DNP-DNP₂₅ BSA); and (3) that specific alloantibodies of IgG₂ subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O₂^?) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG₁ nor F(ab′)₂ portion were able to trigger O₂^? release.This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.     

Specific effects on human neutrophils of antibodies to a membrane protein constituent of neutrophil receptors for chemotactic formyl-methionyl peptides

IgG and Fab were prepared from goat antisera to MP-2, the quantitatively predominant membrane protein constituent of human neutrophil receptors for chemotactic formyl-methionyl peptides. Only 10%–25% of the f-Met-Leu-Phe combining sites of MP-2 purified from neutrophil membranes that had been solubilized in Nonidet P40 exhibited binding constants similar in magnitude to those of the receptors in intact neutrophils, while the remainder of the sites retained a mean of 2% of the affinity of native receptors. Purified MP-2 elicited IgG antibodies predominantly to framework determinants, rather than the combining site, of the f-Met-Leu-Phe receptors. IgG antibodies, but not Fab, evoked the release of significant quantities of β-glucuronidase and lysozyme from neutrophils. Saturating concentrations of Fab bound to a mean of 65,000 determinants per neutrophil, as assessed with ¹²⁵I-Fab, but failed to stimulate neutrophil chemotaxis or chemokinesis, and inhibited by 15% or less the binding of [³H]f-Met-Leu-Phe to intact neutrophils. Fab of anti-MP-2 inhibited neutrophil chemotactic responses to f-Met-Leu-Phe by up to 80%, without influencing the responses to equally chemotactic concentrations of fragments of C5 and of leukotriene B₄. Preincubation of neutrophils for 2–30 min at 37° with concentrations of f-Met-Leu-Phe which suppressed significantly the number of receptors available to [³H]f-Met-Leu-Phe, increased the number of receptors detected by ¹²⁵I-Fab of anti-MP-2, while neither fragments of C5 nor leukotriene B₄ altered the number of receptors determined by either assay. Antibodies to non-combining site determinants of chemotactic peptide receptors provide a novel immunospecific probe for studies of the regulation of neutrophil chemotaxis.     

Effect of deuterium oxide on neutrophil oxidative metabolism,phagocytosis, and lysosomal enzyme release

We have previously shown that deuterium oxide (D₂O) enhances the oxidation of methionine, a myeloperoxidase (MPO)-mediated reaction, by human neutrophils during phagocytosis. However, D₂O has no effect on the oxidation of methionine by the purified MPO-H₂O₂-Cl^– system. To explain this observation, we studied the effect of D₂O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D₂O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D₂O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D₂O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H₂O₂ production by neutrophils in the presence of latex particles or PMA were also stimulated by D₂O. In contrast, D₂O inhibited the ingestion of latex particles. D₂O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D₂O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D₂O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.