Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils

Wan-chun Sun Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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James N. Moore Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602
Departments of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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David J. Hurley Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602
Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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Michel L. Vandenplas Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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Thomas F. Murray Department of Pharmacology, School of Medicine, Creighton University, Omaha, NE 68178

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Abstract

Objective—To assess the anti-inflammatory effects of an adenosine analogue on lipopolysaccharide (LPS)-stimulated equine neutrophils.

Sample Population—Neutrophils obtained from 10 healthy horses.

Procedures—An adenosine analogue (5′-N-ethylcarboxamidoadenosine [NECA]) was tested for its ability to inhibit production of reactive oxygen species (ROS) in LPS-stimulated equine neutrophils. Selective adenosine receptor antagonists were used to identify the receptor subtype responsible for effects. To assess the mechanism of action of NECA, cAMP concentrations were measured, and effects of dibutyryl cAMP (a stable analogue of cAMP) and rolipram (a type 4 phosphodiesterase inhibitor) were investigated.

Results—NECA elicited concentration-dependent inhibition of ROS production that was inhibited by ZM241385, a selective adenosine A2A receptor antagonist; this effect of NECA was not affected by the adenosine A2B receptor antagonist MRS1706. Also, ZM241385 blocked NECA-induced increases in cAMP concentrations, whereas MRS1706 did not alter this effect of NECA. Rolipram potentiated NECA-induced inhibition of ROS production, and dibutyryl cAMP also inhibited ROS production.

Conclusions and Clinical Relevance—Activation of adenosine A2A receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A2A receptor agonists may be developed as suitable anti-inflammatory drugs in horses.

Abstract

Objective—To assess the anti-inflammatory effects of an adenosine analogue on lipopolysaccharide (LPS)-stimulated equine neutrophils.

Sample Population—Neutrophils obtained from 10 healthy horses.

Procedures—An adenosine analogue (5′-N-ethylcarboxamidoadenosine [NECA]) was tested for its ability to inhibit production of reactive oxygen species (ROS) in LPS-stimulated equine neutrophils. Selective adenosine receptor antagonists were used to identify the receptor subtype responsible for effects. To assess the mechanism of action of NECA, cAMP concentrations were measured, and effects of dibutyryl cAMP (a stable analogue of cAMP) and rolipram (a type 4 phosphodiesterase inhibitor) were investigated.

Results—NECA elicited concentration-dependent inhibition of ROS production that was inhibited by ZM241385, a selective adenosine A2A receptor antagonist; this effect of NECA was not affected by the adenosine A2B receptor antagonist MRS1706. Also, ZM241385 blocked NECA-induced increases in cAMP concentrations, whereas MRS1706 did not alter this effect of NECA. Rolipram potentiated NECA-induced inhibition of ROS production, and dibutyryl cAMP also inhibited ROS production.

Conclusions and Clinical Relevance—Activation of adenosine A2A receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A2A receptor agonists may be developed as suitable anti-inflammatory drugs in horses.

Initial events in inflammation include activation of polymorphonuclear leukocytes (neutrophils), adherence of neutrophils to vascular endothelium, and migration of neutrophils into tissues to engulf and kill invading microorganisms.1 A part of this last process is the production of ROS via NADPH-dependent oxidase. In situations characterized by the release of endotoxins from gram-negative bacteria, there may be widespread activation of neutrophils, release of enzymes and ROS, and substantial tissue damage.2-4 These processes have been implicated in the development of the systemic inflammatory response syndrome and multiple organ failure.5

Analysis of results of several studies indicates that adenosine, a ubiquitous purine nucleoside released during ATP metabolism, has anti-inflammatory effects and may be an important natural dampening mechanism for the inflammatory response. Furthermore, there are indications that adenosine may be useful in the treatment of conditions characterized by excessive stimulation of the inflammatory system. Because adenosine has an extremely short life span in the circulation, more stable analogues of adenosine are required for therapeutic use. In the extracelluar environment, adenosine may bind to 4 G-protein–coupled receptors (A1, A2A, A2B, and A3), the expression of which varies considerably among tissues. Because these receptor subtypes differ in their downstream signaling pathways, physiologic responses elicited by adenosine vary considerably.6,7 Concurrent stimulation of several adenosine receptor subtypes may result in anti-inflammatory effects as well as deleterious effects on cardiovascular function. Analysis of the results of studies8-10 performed in several species suggests that selective stimulation of adenosine A2A receptors can reduce the production of ROS by activated neutrophils. Consequently, there is considerable interest in pharmacologic interventions that selectively stimulate adenosine A2A receptors and result in anti-inflammatory effects.

Endotoxemia is an important component of many diseases in horses, most notably those with high mortality rates. In adult horses, these diseases include intestinal inflammation or ischemia and bacterial infections that involve the pleural or peritoneal cavities.5 In neonatal foals, endotoxemia is most commonly associated with failure of passive transfer of colostral components and subsequent development of septicemia.11 As determined in several clinical studies5,11-14 in which the limulus amoebocyte lysate assay was used to detect endotoxin in the circulation, approximately 30% to 40% of horses examined at university hospitals because of clinical signs of colic and 40% to 50% of neonatal foals examined because of suspected septicemia have endotoxins in their circulation. Because of the impact of endotoxemia in these animals and the fact that anti-inflammatory treatment currently is limited to administration of nonsteroidal anti-inflammatory drugs, new therapeutic options are needed to control the deleterious effects of the systemic inflammatory response to bacteria and bacterial components. Therefore, objectives of the study reported here were to determine whether incubation of equine neutrophils with endotoxin stimulates the production of ROS, to compare the effects of stimulation of adenosine receptors on this response, and to evaluate the subtype of adenosine receptor and signal transduction pathway responsible for effects attributable to stimulation of adenosine receptors.

Materials and Methods

Sample population—Blood samples for all experiments reported here were obtained from 10 healthy adult horses owned and managed by the University of Georgia Equestrian Team. Horses were housed indoors during the day and maintained on pasture at night. Use of these horses was approved by the Institutional Animal Care and Use Committee of the University of Georgia.

Collection and preparation of samples—Blood samples were obtained from the jugular vein of these horses into syringes that contained EDTA as an anticoagulant, and RBCs were allowed to settle for approximately 20 minutes. Leukocyte-rich plasma was expressed from each syringe through a needle bent to an angle of approximately 135°. Leukocyte-rich plasma was diluted with an equal volume of Dulbecco PBS solution without calcium or magnesium, layered onto a solution of polysucrose and sodium diatrizoate,a and centrifuged at 400 × g for 30 minutes at 20°C. The RBCs that contaminated the resulting granulocyte pellet were lysed with distilled water, and tonicity was then restored by the addition of 2X PBS solution. When necessary, this step was repeated until the granulocyte pellet was visually free of contaminating RBCs.

Granulocytes (typically > 95% neutrophils, as determined by use of differential staining of cytocentrifuged preparations) were washed 3 times with PBS solution and then suspended at a final concentration of 3 × 107 cells/mL in RPMI 1640 medium containing 10% fetal bovine serum,b 2mM L-glutamine, 2mM sodium pyruvate, and 50 μg of gentamicin/mL. In preliminary experiments, it was determined that equine neutrophils produced ROS immediately after being isolated. However, incubation of the isolated neutrophils at 37°C in an atmosphere of 5% carbon dioxide for 90 minutes reduced endogenous ROS production to values comparable to those measured for wells that lacked neutrophils. Thus a 90-minute incubation period was used in the experiments described here. After this incubation period, neutrophils were counted, and viability was determined to be ≥ 98% by use of exclusion of trypan blue dye. Neutrophils were diluted immediately before use to achieve a final concentration of 3 × 106 cells/mL by the addition of the aforementioned medium.

Experimental procedures—Inflammatory responses are rapidly amplified in horses and are involved in the pathogenesis of many diseases. Therefore, we performed a series of experiments to evaluate the anti-inflammatory potential that activation of adenosine receptors, specifically the A2A receptor, may have in modulating cellular responses induced by LPS.

Activation of adenosine A2A receptors is coupled to Gprotein–mediated responses that lead to the accumulation of cAMP in a variety of cell types.10 Therefore, we evaluated whether NECA would increase cAMP concentrations in neutrophils from horses and whether the A2A-receptor antagonist, ZM241385, would shift the NECA concentration-response curve to the right as expected for a competitive receptor antagonist. To evaluate the effect of NECA on cAMP concentrations and whether this was mediated via adenosine A2B receptors, we tested the specific A2B receptor antagonist MRS1706 for its ability to block NECA-induced cAMP production (Figure 1).

Figure 1—
Figure 1—

Schematic summarizing the effects of interactions between adenosine and its receptor subtypes (A1, A2A, A2B, and A3) on the production of ROS as well as specific adenosine-receptor antagonists used in the study reported here. TLR4 = Toll-like receptor 4. PKC = Protein kinase C. NF-κB = Nuclear factor KB. MAPK = Mitogen-activated protein kinase. - = Inhibitory effect.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Inhibition of ROS production and generation of cAMP induced by adenosine analogues may be dependent on activation of adenylyl cyclase. Therefore, we tested the effect of a type 4 phosphodiesterase inhibitor (rolipram, which prevents degradation of intracellular cAMP) and the addition of a nonhydrolyzable form of cAMP (ie, db-cAMP) on LPS-induced production of ROS by equine neutrophils.

Measurement of in vitro generation of ROS—Generation of ROS by neutrophils (100 μL/well) was monitored in 96-well flat-bottom plates. Wells contained NECA,c adenosine deaminase,d Escherichia coli O55: B5 LPS,e ZM241385,f MRS1706,g MRS1220,h 4-(3-[cyclopentyloxy]-4-methoxyphenyl)-2-pyrrolidinone (ie, rolipram),i db-cAMP,j or a combination of these substances; PMAk was used as a positive control stimulus in all experiments. To detect ROS, 10 μL of a nonfluorescent dyel was added to each well (final concentration of dye in each well, 10μM). The dye oxidizes to form green-fluorescent rhodamine 123 in response to oxygen radicals (principally H2O2) produced by neutrophils. Plates were incubated in a humidified atmosphere of 5% carbon dioxide at 37°C for 2 hours, after which fluorescence was measured by use of a fluorescent plate readerm with a 485-nm excitation filter and a 538-nm emission filter. Values were reported as the number of AFUs. All reagents were diluted with RPMI 1640 medium that lacked phenol red but contained 10% fetal bovine serum, 2mM L-glutamine, 2mM sodium pyruvate, and 50 μg of gentamicin/mL.

To permit comparison of data among experiments, fluorescence in each group of wells was adjusted to that obtained for the unstimulated cells, which reflected endogenous production of ROS activity. To evaluate the degree of inhibition of LPS-induced responses, the following equation was used:

article image

where AFU LPS is the value for cells incubated with LPS alone, AFU treatment is the value for cells incubated with LPS and the particular treatment being evaluated, and AFU cells is the basal value for unstimulated cells.

To provide a quality-control index, a parallel set of wells was included in each experiment; in those parallel wells, ROS production was determined for unstimulated cells incubated with polymyxin B.n When ROS production by those cells was < 90% of ROS production of cells incubated without polymyxin B, we concluded that there had been LPS contamination at some point during the experiment, and results for those experiments were excluded from the study.

Measurement of total cellular cAMP concentrations—Neutrophils (2 × 106 cells/ mL) were suspended in RPMI 1640 medium that contained 1 U of adenosine deaminase/ mL and 50μM rolipram. Aliquots (160 μL) of cell suspensions were placed into duplicate wells of 96-well plates. Various concentrations of the adenosine-receptor agonist NECA were added in 20 μL of RPMI 1640 medium with or without the A2A-receptor antagonist ZM241385 or the A2B-receptor antagonist MRS1706. After incubation in an atmosphere of 5% carbon dioxide at 37°C for 20 minutes, reactions were terminated by adding 20 μL of stop solution provided in a commercially available kit° to each well. Concentrations of cAMP were then determined in duplicate by use of the protocol provided by the manufacturer of the kit.

Data analysis—All experiments were repeated 3 times by use of neutrophils isolated from various horses. Concentration-response data were analyzed by use of nonlinear regression analysisp and were fit by use of the following logistic equation:

article image

where Y is the response; top and bottom are the maximum and minimum plateaus of the concentration response curve, respectively; × is the adenosine analogue concentration, and n is the Hill slope. The EC50 values were expressed as mean ± 95% confidence intervals. Pairwise comparison of best fit values for log EC50, top, and bottom were performed by use of the F test to identify significant differences between concentrationresponse curves generated with and without each of the receptor antagonists. Values were considered significant at P < 0.05.

Results

LPS stimulation of ROS production—In the experiments reported here, LPS-induced production of ROS was used as a model for the interaction between leukocytes and the microbial products that drive inflammatory responses in endotoxemic horses. In another study15 conducted by our laboratory group, we developed and used the interaction between peripheral blood neutrophils and E coli LPS to generate predictable and concentration-dependent responses in vitro. To define a suitable concentration of LPS to use in these experiments, we measured ROS production by equine neutrophils incubated with a wide range of concentrations of E coli LPS. The EC50 for LPS-induced ROS production was 8.6 ng/mL (range, 5.6 to 13.0 ng/mL), and the lowest concentration tested that induced maximal ROS production was 100 ng of LPS/mL (Figure 2). The concentration of 100 ng of LPS/mL was used in subsequent experiments.

Figure 2—
Figure 2—

Mean ± SEM values for LPS-induced production of ROS by equine neutrophils. Values represent results for 6 replicates; similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Effects of NECA on LPS-induced ROS production— Incubation with NECA inhibited LPS-induced production of ROS by equine neutrophils in a concentration-dependent manner (Figure 3). This effect of NECA was reduced in a concentration-dependent manner by the selective A2Areceptor antagonist ZM241385; responses of cells to 10, 100, and 1,000nM ZM241385 differed significantly from responses of cells incubated without the receptor antagonist. Analysis of a Schild plot of these data revealed the KB value of ZM241385 was 1.2nM (Figure 4).

Figure 3—
Figure 3—

Mean ± SEM effects of various concentrations of ZM241385 (A), MRS1706 (B), and MRS1220 (C) on NECA-mediated inhibition of LPS-induced production of ROS by equine neutrophils. The LPS was used at a concentration of 100 ng/mL. Values represent results for 6 replicates; similar results were obtained in 3 additional experiments. *†Values differ significantly (*P < 0.05; †P < 0.001) from values for 0nM.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Figure 4—
Figure 4—

Schild plot of the effect of various concentrations of ZM241385 on NECA-mediated inhibition of LPS-induced production of ROS by equine neutrophils. In this plot, dose ratio (DR) is calculated from EC50 values determined for NECA with and without the addition of ZM241385 and the negative logarithm of the dissociation constant, pA2 (pA2 = 8.916).

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

To determine the selectivity for inhibition of LPS-induced ROS production by NECA, parallel experiments were performed by use of the adenosine A2B-receptor antagonist MRS1706 and the adenosine A3-receptor antagonist MRS1220. The IC50 value for NECA was not significantly affected by concentrations of MRS1706 as high as 1,000nM, which suggested that the effects of NECA were not attributable to activation of the adenosine A2B receptor subtype (Figure 3). Similarly, the inhibitory effect of NECA on LPS-induced ROS production was not altered by 10nM MRS1220. However, the effect of NECA was significantly reduced by 100 and 1,000nM MRS1220.

Effect of NECA on accumulation of cAMP—Incubation of neutrophils with a range of concentrations of NECA resulted in a concentration-dependent increase in cAMP accumulation with an EC50 value of 1.6μM (range, 9 to 2.4μM). Incubation of neutrophils with increasing concentrations of ZM241385 increased the EC50 value for NECA-induced cAMP accumulation (Figure 5). Analysis of a Schild plot of these data yielded a KB value of 0.7nM for ZM241385 (Figure 6). Responses of cells to 10, 100, and 1,000nM ZM241385 differed significantly from responses of cells incubated without the receptor antagonist. In contrast, incubation of neutrophils with the A2B-receptor antagonist MRS1706 (100nM) did not change the EC50 value for NECA-induced cAMP accumulation.

Figure 5—
Figure 5—

Mean ± SEM effects of various concentrations of ZM241385 (A) and MRS1706 (B) on NECA-induced changes in cAMP concentrations in equine neutrophils. Values represent results for 3 replicates; similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Figure 6—
Figure 6—

Schild plot of the effect of various concentrations of ZM241385 on NECA-induced changes in cAMP concentrations in equine neutrophils. pA2 = 9.182. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Effect of cAMP on LPS-induced ROS production—Incubation of neutrophils with rolipram inhibited LPS-induced ROS production in a concentration-dependent manner, with an IC50 value of 111.3nM (range, 52.0 to 238.3nM; Figure 7). The IC50 values for NECAinduced inhibition of LPS-induced ROS production after incubation with 0, 10, 30, and 100nM rolipram were 64.3nM (range, 48.2 to 85.7nM), 36.4nM (range, 28.8 to 45.9nM), 24.9nM (range, 17.1 to 36.4nM), and 18.4nM (range, 12.1 to 28.1nM), respectively. Incubation with 30 or 100nM rolipram significantly decreased the IC50 value for NECA-induced inhibition of LPS-induced ROS production, and the effect of the combination of rolipram and NECA was more evident as the concentration of rolipram increased. Incubation with db-cAMP inhibited LPS-induced ROS production in a concentration-dependent manner, with an IC50 value of 10.2μM (range, 6.3 to 16.6μM; Figure 8).

Figure 7—
Figure 7—

Mean ± SEM effects of various concentrations of rolipram (A) and the combined effect of various concentrations of rolipram and NECA (B) on LPS-induced ROS production by equine neutrophils. The LPS was used at a concentration of 100 ng/mL. Values represent results for 6 replicates; similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Figure 8—
Figure 8—

Mean ± SEM effects of various concentrations of db-cAMP on LPS-induced ROS production by equine neutrophils. The LPS was used at a concentration of 100 ng/mL. Values represent results for 3 replicates; similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.649

Incubation of equine neutrophils with 100μM NECA almost completely inhibited LPS-induced production of ROS (Table 1). In contrast, NECA had minimal effects on non–receptor-mediated activation of protein kinase C by PMA, inhibiting only approximately 20% of ROS production at the highest concentration of NECA. Similarly, whereas db-cAMP almost completely inhibited LPS-induced ROS production, it had a small effect on PMA-induced ROS production. Similar results, although less robust, were obtained with rolipram.

Table 1—

Mean ± SEM values for effects of various agents that increased the intracellular cAMP concentration on LPS- and PMA-induced ROS activity in equine neutrophils obtained from 4 horses.

AgentInhibition of LPS-induced* ROS production (%)Inhibition of PMA—induced ROS production (%)
NECA (μM)
10094.8 ± 3.4 20.4 ± 4.7
1086.0 ± 6.1 5.2 ± 5.1
179.5 ± 9.2  
db-cAMP(μM)
10099.9 ± 2.4 18.3 ± 8.5
3083.4 ± 14.1 9.2 ± 5.4
1046.3 ± 13.0 4.3 ± 2.8
Rolipram (μM)
5067.4 ± 19.1 7.5 ± 4.8
551.3 ± 16.1 1.5 ± 2.1

The LPS was used at a concentration of 100 ng/mL.

The PMA was used at a concentration of 0.1 μM.

Significantly (P < 0.001) different from value obtained with LPS alone; NECA, db-cAMP, or rolipram did not significantly (P < 0.05) alter PMA-induced ROS production.

Discussion

Adenosine exerts its biological effects by interacting with specific G-protein–coupled receptors.16 Of the 4 adenosine receptor subtypes, A1 and A3 receptors are coupled to inhibitory G-protein receptors, whereas A2A and A2B receptors are coupled to stimulatory G-protein receptors. Therefore, activation of the latter 2 receptors activates adenylyl cyclase and causes intracellular accumulation of cAMP.9 Analysis of results of several studies9,10,17,18 indicates that adenosine inhibits an array of leukocyte functions via activation of A2A receptors, thereby causing anti-inflammatory effects. Analysis of results of the study reported here indicated that NECA, a stable analogue of adenosine, inhibits LPS-induced production of ROS by equine neutrophils and does so through activation of adenosine A2A receptors. This conclusion is based on our finding that the effects of NECA were competitively inhibited by coincubation with the selective adenosine A2A-receptor antagonist ZM241385 and that Schild analysis of the data for coincubation with ZM241385 yielded a KB value of 1.2nM. The latter finding is consistent with the KD value reported from radioligand binding experiments performed on HEK293 cells expressing equine A2A receptors.19 Furthermore, the inhibition of LPS-induced production of ROS by NECA detected in the study reported here was not affected by coincubation with the selective adenosine A2B-receptor antagonist MRS1706 or by incubation with concentrations of MRS1220 that selectively bind adenosine A3 receptors. Although the effect of NECA was reduced by higher concentrations of MRS1220, those concentrations have not been reported to distinguish between adenosine A2A and A3 receptor subtypes.10 Collectively, the findings of the study reported here indicated that NECA-mediated inhibition of LPS-induced ROS production by equine neutrophils occurs via activation of adenosine A2A receptors, and these findings are consistent with results of a study10 in which the oxidative activity of human neutrophils was inhibited by stimulation of adenosine A2A receptors.

An ample body of evidence indicates that agents that increase intracellular concentrations of cAMP also modulate neutrophil function.8,10,20 Analysis of results of the study reported here revealed that incubation of equine neutrophils with NECA caused an accumulation of cAMP, and this response was attributable to stimulation of adenosine A2A receptors because this effect of NECA was antagonized by ZM241385 with a KB value of 0.7nM. The KB values for incubation with ZM241385 derived from the antagonism of the inhibitory effect of NECA on ROS production and stimulatory effect of NECA on cAMP production were consistent with the affinity of ZM241385 for adenosine A2A receptors. In contrast, 100nM MRS1706 did not affect cAMP accumulation in response to NECA, which indicated that this effect of NECA did not involve adenosine A2B receptors.

To further investigate the role of cAMP in LPS-induced ROS production by equine neutrophils, we evaluated the effects of rolipram, which is a selective type 4 phosphodiesterase inhibitor that increases intracellular concentrations of cAMP by preventing the breakdown of cAMP to 5′AMP.21 In the study reported here, rolipram partially inhibited LPS-induced ROS production in a concentration-dependent manner (Figure 6). When neutrophils were incubated with a combination of NECA and rolipram, there was complete inhibition of LPS-induced ROS production. Furthermore, the IC50 value of NECA decreased as the concentration of rolipram increased, which suggested that inhibition of ROS production by NECA was potentiated by the inhibition of cAMP degradation. These results are consistent with cAMP mediation of this response to NECA in equine neutrophils.

Disagreement exists over increases in intracellular cAMP concentrations associated with adenosine A2Areceptor agonists and their effects on neutrophil function.22 Although results of several studies8–10,18 support a role for cAMP in the synergistic effects of adenosine A2A-receptor agonists and type 4 phosphodiesterase inhibitors, results of 1 study23 on oxidative activity of neutrophils induced by N-formyl-methionyl-leucylphenylalanine failed to identify a synergistic effect of NECA and type 4 phosphodiesterase inhibitors. Because of these conflicting findings, we also determined the effects of incubation of equine neutrophils with a stable cAMP analogue (ie, db-cAMP). In these experiments, db-cAMP inhibited LPS-induced ROS production in a concentration-dependent manner (Figure 8). This provided additional support for the role of cAMP in the anti-inflammatory effects of agents that stimulate receptors, such as the adenosine A2A receptors, that are coupled to stimulatory receptors on G-proteins and stimulation of adenylyl cyclase. In the study reported here, agents that increase cAMP concentrations (ie, NECA, db-cAMP, and rolipram) also had a degree of selectivity in their ability to modulate intracellular signaling pathways that control ROS production in equine neutrophils because they were more potent in regulating LPS-induced ROS production than in regulating PMA-induced ROS production (Table 1). Because ROS production induced via activation of toll-like receptor 4 or protein kinase C may involve similar downstream effectors (eg, nuclear factor KB and mitogen-activated protein kinases), these pathways may share signal transduction elements that are regulated by cAMP and its effectors, albeit to varying degrees.

Results of the study reported here also are consistent with findings of 2 other studies24,25 in which investigators determined that adenosine exerted anti-inflammatory effects in equine chondrocytes by interacting with adenosine A2A receptors. In one of those studies,24 adenosine and NECA inhibited production of nitric oxide induced by LPS or recombinant human interleukin-1α, whereas in the second study,25 adenosine and the adenosine A2A-receptor agonist (N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl] adenosine; ie, DPMA) or the phosphodiesterase inhibitor rolipram reduced chondrocyte production of nitric oxide induced by LPS. Analysis of these results indicates that adenosine and adenosine-receptor agonists play anti-inflammatory roles in equine chondrocytes via activation of adenosine A2A receptors in a cAMP-dependent manner.

It is worthwhile to mention that results reported here regarding LPS-induced production of ROS differ from those of another study26 with equine neutrophils in which LPS alone failed to induce ROS production. This difference may have been attributable to differences in the methods used to isolate and handle the cells or in the techniques used to monitor ROS production. In preliminary studies, we determined that cells incubated with LPS shortly after isolation by use of a solution of polysucrose and sodium diatrizoate did not consistently produce additional ROS, whereas LPS-induced ROS production was obvious when the neutrophils were initially incubated in an atmosphere of 5% carbon dioxide at 37°C for 90 minutes. We presume that this improved response to LPS was attributable to a change in the activation status of the cells that may have resulted from interactions with the solution of polysucrose and sodium diatrizoate. Although analysis of these findings suggests that the neutrophils in the study reported here were not quiescent at the time of their interaction with LPS, the objective of the study was to evaluate the ability of stimulation of adenosine A2A receptors to modify responses of neutrophils to proinflammatory stimuli, such as LPS, and then to understand the mechanisms by which the modified response was effected.

Analysis of results of the study reported here indicated that stimulation of adenosine A2A receptors inhibits LPS-induced ROS production in equine neutrophils by increasing intracellular concentrations of cAMP. Analysis of these findings suggests that stable adenosine-receptor agonists that function via activation of A2A receptors may be useful as anti-inflammatory drugs in horses.

ABBREVIATIONS

ROS

Reactive oxygen species

NADPH

Reduced form of nicotinamide adenine dinucleotide phosphate

LPS

Lipopolysaccharide

NECA

5′-N-ethylcarboxamidoadenosine

db-cAMP

Dibutyryl cAMP

PMA

Phorbol myristate acetate

AFU

Arbitrary fluorescent unit

EC50

Concentration that produces 50% of the maximum plateau effect

KB

Dissociation constant

IC50

Concentration that produces 50% of maximum inhibition

a.

Histopaque 1077, Sigma-Aldrich Corp, St Louis, Mo.

b.

Fetal bovine serum, Hyclone, Logan, Utah.

c.

NECA, Tocris Bioscience, Ellisville, Mo.

d.

Adenosine deaminase, Roche, Indianapolis, Ind.

e.

Escherichia coli O55:B5 LPS, List Biologics, Campbell, Calif.

f.

ZM241385, Tocris Bioscience, Ellisville, Mo.

g.

MRS1706, Tocris Bioscience, Ellisville, Mo.

h.

MRS1220, Tocris Bioscience, Ellisville, Mo.

i.

Rolipram, Sigma-Aldrich Corp, St Louis, Mo.

j.

db-cAMP, Sigma-Aldrich Corp, St Louis, Mo.

k.

PMA, Sigma-Aldrich Corp, St Louis, Mo.

l.

DHR123, Invitrogen Molecular Probes, Carlsbad, Calif.

m.

Fluoroskan Ascent FL, Thermo Labsystems, GMI Inc, Albertville, Minn.

n.

Polymyxin B, Bedford Labs, Bedford, Ohio.

o.

cAMP Biotrak EIA kit, Amersham Biosciences, Piscataway, NJ.

p.

Prism software, version 4.0, Graph Pad Software Inc, San Diego, Calif.

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