Inhibition of the Neutrophil NADPH Oxidase by Adenosine Is Associated with Increased Movement of Flavocytochrome b Between Subcellular Fractions

Inflammation, Vol. 27, No. 1 February 2003 ( 2003)

0360-3997/ 03/ 0200-0045/ 0 2003 Plenum Publishing Corporation

45

Inhibition of the Neutrophil NADPH Oxidase byAdenosine Is Associated with Increased Movement ofFlavocytochrome b Between Subcellular Fractions

Steve D. Swain,1 Daniel W. Siemsen,1 Laura K. Nelson,1 Karen M. Sipes,1

Angela J. Hanson,1 and Mark T. Quinn1,2

Abstract—Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by theNADPH oxidase in f MLF-stimulated neutrophils. Although much is known about the pharama-cology and signal transduction of this effect, it is not known how adenosine affects assembly andlocalization of the NADPH oxidase components within the neutrophil. We report here that adeno-sine pretreatment of f MLF-stimulated neutrophils results in decreased plasma membrane/ secretorygranule content of the flavocytochrome b components (p22phox and gp91phox) of the NADPH oxi-dase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upreg-ulation of secretory and specific granule surface markers, confirming that degranulation was notimpaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in f MLF-stimulated neutrophils. These datasuggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocy-tosis and/ or redistribution of flavocytochrome b between various subcellular compartments.

KEY WORDS: neutrophil, adenosine, flavocytochrome b, granule, superoxide anion

INTRODUCTION

Neutrophils play an essential role in the body’s defenseagainst potentially pathogenic bacteria and fungi [re-viewed in (1, 2)]. These cells possess a complexarray of antimicrobial mechanisms, including proteolyticenzymes, peptides and proteins with direct bactericidalactivity, and the production of reactive oxygen species(ROS) (3). While the goal of this process is to destroy thepathogen, neutrophil-generated enzymes and ROS canalso damage host tissues near the site of inflammation

1 Department of Veterinary Molecular Biology, Montana State Univer-sity, Bozeman, MT 59717.

2 To whom correspondence should be addressed at the Montana StateUniversity, Department of Veterinary Molecular Biology, Bozeman,MT 59717. Telephone: (406) 994-5721; fax: (406) 994-4303, E-mail:[email protected].

and have been implicated in damage to host tissue invarious inflammatory conditions (4, 5).

The major source of ROS in neutrophils is theNADPH oxidase, a multi-component enzyme complexfound in many cell types, but highly enriched in neu-trophils [reviewed in (6, 7)]. The key redox compo-nent of the NADPH oxidase is flavocytochrome b, aheterodimer comprised of two integral membrane pro-teins (gp91phox and p22phox) (8). In resting cells, flavo-cytochrome b is localized primarily in the membranesof secretory and specific granules, but it relocates tothe plasma or phagosomal membranes during activation,presumably via degranulation events (6). The cytoso-lic components of the oxidase system include p40phox,p47phox, p67phox, and the GTPase Rac (7, 9). With appro-priate activating stimuli, these proteins translocate tothe membrane, where they associate with the membranecomponents and initiate O−

2 production (10).

Swain, Siemsen, Nelson, Sipes, Hanson, and Quinn46

Although quite a bit is known about the path-ways leading to NADPH oxidase activation, very lit-tle is known about the mechanisms involved in shut-ting off this system. One potentially useful approachto address this issue is to examine how endogenousinhibitory agents down-regulate NADPH oxidase activ-ity, and determine whether they affect the assemblyand localization of the oxidase complex within the neu-trophil. One such inhibitor is the endogenous purinenucleoside adenosine.

Adenosine is produced during ATP metabolism,and its rate of production is sharply increased duringmetabolic stress and ischemia (11). Adenosine has beencalled a “retaliatory metabolite” because it has potentlocal effects that act to protect tissue from cardiovas-cular, metabolic, and immunological stress (11). Theanti-inflammatory actions of adenosine on neutrophilsare well known. These include inhibition of adherence(12), actin polymerization (13), priming (14, 15), andROS production (16). Adenosine effects on neutrophildegranulation are more controversial, with some reportsof inhibition (17, 18) and other reports of no effect onthis process (19).

The role of various neutrophil signaling pathways inresponse to adenosine is not well understood. It is clearthat promotion of chemotaxis by low adenosine con-centrations is mediated via the A1 receptor, while anti-inflammatory actions of higher adenosine concentrationsare mediated via A2 and A3 receptors (16, 19). Less isknown about which second messengers mediate adeno-sine effects on ROS generation in neutrophils, althoughit has been proposed that adenosine may act to uncou-ple f MLF receptors from other signal transduction steps,including NADPH oxidase activation (20, 21). Althoughadenosine can increase cellular cAMP and diminish thelate phase of stimulant-induced increases in free Ca2+

concentration, it is not clear whether either of these isimplicated in inhibition of neutrophil ROS production(19, 22). Adenosine has also been implicated in the acti-vation of a specific protein phosphatase, which may pos-sibly alter the p47phox phosphorylation state (23).

Currently, the downstream effects of adenosine onNADPH oxidase assembly and localization in activatedneutrophils are not well known. One proposal is thatadenosine acts to uncouple f MLF receptors from othersignal transduction steps (20, 21). Another possibility,which has not been examined, is that adenosine mayaffect the assembly or localization of the NADPH oxi-dase components themselves. To begin to address thisissue, we examined the effects of adenosine on local-

ization of the NADPH oxidase within the neutrophiltogether with its effect on extracellular ROS produc-tion in f MLF-stimulated neutrophils. We show here thatadenosine pretreatment of human neutrophils, followedby f MLF activation, results in decreased amounts offlavocytochrome b in the plasma membrane/ secretorygranule component of fractionated neutrophils. Further-more, the decrease in flavocytochrome b correlateswith a decrease in extracellular ROS production bythese cells. The reduced membrane/ secretory granulecytochrome b content appears to be independent ofeffects on degranulation, as judged by conventionalmarkers of granule recruitment. However, adenosinetreatment does result in increased movement of cell-surface flavocytochrome b into heavy membrane frac-tions of stimulated neutrophils. These data suggest thatadenosine-mediated effects on neutrophil ROS produc-tion are due, in part, to effects on movement of flavocy-tochrome b between cellular compartments.

MATERIALS AND METHODS

Reagents

Antibodies CD35/ M0710, CD10/ F0826, andCD18/ M0782 were from DAKO Corporation (Carpinte-ria, CA) and polyclonal antihuman lactoferrin (L3262)was from Sigma Chemical Company (St. Louis, MO).Monoclonal antibodies recognizing cytoplasmic epitopesof p22phox (44.1) and gp91phox (54.1) and an extracellularepitope of gp91phox (7D5) were as previously described(24, 25). Dihydrorhodamine (DHR) and methylumbel-liferyl phosphate (MUP) were purchased from Molecu-lar Probes (Eugene, OR). Hanks buffered salt solution(HBSS) supplemented with 10 mM HEPES was pur-chased from GIBCO BRL (Grand Island, NY). Superox-ide dismutase (SOD) was purchased from Calbiochem (LaJolla, CA). All other reagents, including adenosine, N-formyl-methionine-leucine-phenylalanine ( f MLF), lumi-nol, and isoluminol were purchased from Sigma. Relaxbuffer consisted of 10 mM PIPES, 100 mM KCl, 3 mMNaCl, 3.5 mM MgCl2, 1.25 mM EGTA, pH 7.2, with 1mM ATP. All solutions to which cells were exposed wereprepared using endotoxin-free H2O.

Neutrophil Isolation

Blood was obtained from healthy human volunteersusing venipuncture into containers with either EDTA

Adenosine Alters Oxidase 47

(vacutainer tubes) or ACD (unit collection bags) as anti-coagulant. Neutrophils were isolated from blood usingdextran sedimentation of erythrocytes followed by cen-trifugation on Histopaque density media (1.077 g/ ml)and hypotonic lysis of residual erythrocytes, as previ-ously described (26). This procedure yielded neutrophilsof approximately 90% purity (based on flow cytometricanalysis) and viability of ≥98% (based on trypan blueexclusion).

Measurement of Neutrophil ROS Production

Neutrophil extracellular O−

2 production was mea-sured using SOD-inhibitable reduction of cytochrome c,as previously described (27). Briefly, neutrophils (2.5 ×106/ ml) in HBSS containing 1 mM CaCl2 and 0.3 mMcytochrome c were placed in 96-well microtiter platesand warmed to 378C for 5 min. Selected wells were thentreated with adenosine (final concentration of 10−7 or10−6 M), the plate was incubated for 3 min at 378C, andthe absorbance at 550 nm using a ThermoMax micro-titer plate reader (Molecular Devices, Sunnyvale, CA).f MLF (10−6 M final concentration) was then added tothe appropriate wells, and the absorbance at 550 nm wasmeasured at 3, 5, 10 and 15 min post-stimulation. Therate of O−

2 production was determined from the averageabsorbance of triplicate wells, minus the absorbance ofidentical wells containing SOD (1000 units/ ml), usingthe extinction coefficient of 21.1 × 103 M−1 cm−1 forcytochrome c.

A kinetic assessment of total neutrophil ROS pro-duction was made using luminol-enhanced chemilumi-nescence (28, 29). Briefly, neutrophils (2.5 × 106/ ml) inHBSS containing 1 mM CaCl2 supplemented with 150mM luminol were placed in white FluroNunc 96-wellmicrotiter plates and warmed to 378C. Adenosine (finalconcentration of 10−7 and 10−6 M) was added to the des-ignated wells, and incubation continued for 3 min priorto addition of f MLF (10−6 M) to the appropriate wells.Immediately after addition of f MLF, luminescence mea-surements were made on each well at 10-sec intervalsfor a total period of 15 min at 378C, using a FluoroskanAscent FL (Labsystems, Helsinki, Finland). Several vari-ations of this technique were used to examine extra-cellular versus intracellular ROS production. One varia-tion involved the use of SOD (1000 U/ ml) and catalase(10,000 U/ ml) to scavenge extracellular ROS, such thatmeasured chemiluminescence reflected only intracellularROS production (29). In another variation, isoluminolwas substituted for luminol. Isoluminol does not enter

cells; therefore, luminescence detected with this methodrepresents only extracellular ROS production (30). Afinal variation of this technique involved the use of lumi-nol without any scavengers, and a shorter measurementinterval (0.5 sec). In this technique, f MLF was addedto the wells using an injector to better examine the ini-tial kinetics of the f MLF-induced ROS production andthe effects that adenosine had on that response. In eachexperiment, all measurements were made in triplicate foreach treatment regimen.

As an alternative method, intracellular ROS pro-duction by f MLF-stimulated neutrophils was measuredusing the fluorescent probe DHR. Isolated neutrophilswere loaded with 200 ng/ ml DHR in the dark at 248C for10 min. After washing, loaded neutrophils were warmedto 378C and treated with appropriate combinations ofadenosine and f MLF, as described above. At designatedintervals (5, 10, 15 min), the cellular fluorescence ofDHR was measured by flow cytometry using a FACS-Calibur (Becton Dickinson, San Jose, CA).

Neutrophil Subcellular Fractionation

Aliquots of 108 neutrophils in 39 ml of HBSS con-taining 1 mM CaCl2 were prewarmed to 378C, adenosine(10−7 and 10−6 M final concentration) was added to theappropriate tubes, and incubation at 378C continued for5 min before f MLF (10−6 M) treatment. At either 2 or 6min after stimulation, the reaction was quenched by theaddition of 50 ml of ice cold HBSS containing 1 mMdiisopropylfluorophosphate (DFP). The cells were thencentrifuged at 950 g for 10 min at 48C, resuspended in2.0 ml relax buffer, and homogenized by N2 cavitation,as described previously (31). Nuclei, unbroken cells anddebris were removed from the cavitate by centrifugationat 500 g for five min at 48C. Neutrophil subcellular frac-tions were obtained by centrifugation on a two-step Per-coll gradient (1.050 g/ ml and 1.120 g/ ml) for 15 min at48C at 50,000 g (32). The gradients were fractionated in24 fractions, each of which was analyzed for subcellularmarkers, as described below.

The membrane/ secretory granule marker alkalinephosphatase was assayed using the fluorescent substrateMUP. Ten ml aliquots of each fraction were mixed with10 mM dithethanolamine buffer (pH 9.5) containing 140mM NaCl, 0.5 mM MgCl2, and 300 mM MUP. After15 min incubation at room temperature, fluorescence at360 nm excitation and 450 nm emission was measuredusing the Fluoroskan FL plate reader. The specific gran-ule marker lactoferrin was assayed using a single anti-


body ELISA, as previously described (33), except thata rabbit anti-human lactoferrin antibody was used asthe primary antibody. Myeloperoxidase, the azurophilgranule marker, was measured using perioxide-depen-dent oxidation of pyrogallol. Briefly, 50 ml aliquots ofeach fraction were suspended in 10 ml of MES buffer,pH 6.0, with 0.2% Triton X-100. Absorbance at 415 nmwas read with a ThermoMax plate reader to measure anybackground light scattering, pyrogallol (0.35 mg/ ml) andH2O2 (0.09%) were added, and, after 10 min at roomtemperature, absorbance at 415 nm was measured again.Background absorbance was then subtracted from each10-min reading.

Flow Cytometry

Changes in cell-surface markers of neutrophilrecruitment were assessed using flow cytometry. Isolatedneutrophils were incubated in HBSS containing 1 mMCa2+ at 378C for 3–5 min, and selected samples weretreated with adenosine (at 10−7 or 10−6 M) for 5 min.The appropriate samples were stimulated with 10−6 Mf MLF at 378C for 5 min, then quenched by the additionof 20 volumes of ice cold HBSS. After centrifugationat 500 g for 5 min at 48C, cells were resuspended incold HBSS containing 1% BSA and the appropriate pri-mary antibody. After incubation for 1 hour at 48C, thecells were washed with 20 volumes of cold buffer andincubated with the appropriate fluorescein-labeled sec-ondary antibody for 45 min at 48C. Finally, the cells werewashed, resuspended in a minimal volume of buffer, andanalyzed using a FACSCalibur flow cytometer.

Biotinylation of Cell-Surface Proteins

Biotinylation of cell surface neutrophil proteins wasperformed using EZ-Link Sulfo-NHS-LC-Biotin (PierceChemical Co., Rockford, IL), according to the manufac-turer’s instructions. Briefly, freshly isolated neutrophilswere resuspended in DPBS (pH 80) at 1 × 107/ ml. Thebiotinylation reagent was added from a 10× stock toa final concentration of 0.5 mg/ ml, and the cells werethen gently rocked at room temperature for 20 min. Thebiotinylated cells were washed twice in DPBS pH 8.0and once in HBSS. The cells were then stimulated withf MLF and fractionated on Percoll gradients exactly asdescribed above. Biotinylated proteins were harvestedfrom the three major membrane/ granule bands as fol-lows. The three fractions containing the highest contentof each marker (alkaline phosphatase, lactoferrin, and

myeloperoxidase) were pooled within each gradient andthen diluted by adding an equal volume of cold relaxbuffer. Percoll was removed from the membrane mate-rial by centrifugation at 155,000 g for 30 min at 48C.The granules/ membranes localized on top of the Percollpellet were aspirated in a total volume of 300 ml, andprotease inhibitors were added (Sigma mammalian pro-tease inhibitor cocktail at 1:100 dilution). These sampleswere then mixed with 300 ml of 10 mM HEPES (pH 7.4)containing 2% Triton X-100 and incubated on ice for 10min with occasional mild vortexing. To capture biotiny-lated proteins, 0.4 ml of each sample was mixed with60 ml of a 50% slurry of washed avidin beas (NeutrA-vidin, Pierce) and rotated at 48C for 80 min. The beadswere washed four times in HEPES buffer containing 1%Triton X-100 and boiled in 200 ml SDS-PAGE samplebuffer. Preliminary experiments verified that gp91phox

was the only NADPH flavocytochrome b protein biotiny-lated in intact neutrophils; therefore the isolated biotiny-lated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trans-ferred to nitrocellulose, and probed with an anti-gp91phox

antibody.

Alkaline Phosphatase Assay

For an additional assessment of secretory granulerecruitment, cell-surface alkaline phosphatase activitywas measured on neutrophils treated with various combi-nations of adenosine and f MLF, as previously described(34). Briefly, neutrophils were exposed to the appropri-ate adenosine/ f MLF treatment regimen for 5 min, andthen transferred to a cuvette containing 1.5 ml of 10 mMdiethanolamine (pH 9.5) with 150 mM NaCl, 0.5 mMMgCl2, and 0.2 mM MUP. Alkaline phosphatase activitywas measured over 5 min, the cells were then lysed todetermine total alkaline phosphatase activity, and secre-tory granule recruitment was assessed as the percentageof total alkaline phosphatase activity present on the cellsurface.

Immunoblotting

The three percoll gradient fractions within eachtreatment with the highest alkaline phosphatase activ-ity were pooled, and the Percoll was removed by cen-trifugation. The protein concentration of these sampleswas measured using the BCA method (Pierce), andequal amounts of protein from each sample were sep-arated by SDS-PAGE on polyacrylamide gradient gels


and transferred to nitrocellulose membrane as describedpreviously (31). Western blots were probed with pre-viously characterized monoclonal anti-p22phox antibod-ies (25, 35), followed by chemiluminescent detection(Pierce). The blots were analyzed using spot densitom-etry using an AlphaImager IS-1000 digital imaging sys-tem (Alpha Innotech, San Leandro, CA), and density val-ues were normalized between blots by setting the val-ues for p22phox band density of the f MLF-treated sampleequal to 100, and the control sample equal to 0.

RESULTS

Effects of Adenosine on f MLF-Induced NeutrophilROS Production

Although there have been multiple studies demon-strating that adenosine strongly inhibits neutrophil ROSproduction when cells are stimulated by f MLF, butnot PMA, differences in techniques make comparisonsbetween these reports problematic. Therefore, we firstexamined the oxidative burst of f MLF-stimulated neu-trophils, with and without adenosine inhibition, usingthree independent techniques. The first technique, utiliz-ing SOD-inhibitable reduction of cytochrome c, specifi-cally measured O−

2 released to the exterior of the cell.The advantage of this method is that it is quantifiable,which facilitates comparisons. The disadvantages of thismethod are that it does not measure intracellular ROSproduction, and it cannot show kinetic downturns inROS production, since reduced cytochrome c remains inthat state for the duration of the assay period. Our resultswith this method show that adenosine at 10−7 and 10−6

M inhibits f MLF-induced neutrophil ROS production by38% and 60%, respectively (Fig. 1), which is similar towhat has previously been reported (16). Virtually all ofthis ROS production occurred within the initial 5 min-utes after f MLF stimulation. Since the absorbance at 550nm increased very little after this point, subsequent ROSproduction must be either halted or predominately intra-cellular in nature.

Intracellular ROS production can also be measuredusing flow cytometry with the fluorescent probe DHR.Although our results with DHR showed that adenosineat 10−7 and 10−6 M also inhibited intracellular f MLF-induced ROS production (date not shown), the poorsignal-to-noise ratio of this technique made it inappro-priate for further experiments.

The third technique we used, luminol- or isolumi-

Fig. 1. Inhibition of neutrophil O−

2 production by adenosine: cytochromec-based assay. Neutrophils were pretreated with buffer (R), 10−7 Madenosine (I), or 10−6 M adenosine (˚) for 3 min at 378C. The cellswere then activated with 10−6 M f MLF, and absorbance at 550 nm wasmeasured at the indicated intervals. Replicate wells containing 1000U/ ml SOD were treated identically, and the absorbance of those wellswas subtracted from the treated wells to determine SOD-inhibitable O−

2production as described under Materials and Methods. Control cells(O) were incubated for the indicated times without activating agents.Values are means ±SEM, n c 3 replicates. The data shown representone experiment taken from four independent experiments.

nol-enhanced chemiluminescence, was chosen to pro-vide information regarding the relative kinetics of theintracellular versus extracellular neutrophil ROS produc-tion. This method, depending on the selection of lumi-nescent probe and scavenging enzymes, can discriminatebetween intra- and extracellular ROS production. Frommeasurements in a chemiluminescent plate reader, wewere able to obtain detailed kinetic information aboutadenosine inhibition of the f MLF induced neutrophilROS production. In neutrophils from most (∼90%) indi-viduals, f MLF produced a biphasic oxidative burst (Fig.2A). The addition of SOD and catalase to scavengeextracellular ROS when luminol (which measures bothextra- and intracellular reactions) was used eliminatedthe first peak of ROS production (Fig. 2A). Conversely,when isoluminol, which cannot enter cells and there-fore measures only extracellular ROS production (30),was used, only the first peak of ROS production wasdetected (Fig. 2B). These observations both confirm ourtests shown in Fig. 1, and corroborate published evidencethat f MLF-induced oxidative burst has an initial phaselasting 3 min, which is almost exclusively extracellularin nature, and is followed by a phase of predominantlyintracellular ROS production, which occurs during theperiod of 2 to 15 min after application of f MLF (36).Adenosine inhibition of f MLF-induced ROS production


Fig. 2. Analysis of intra- and extracellular ROS production in f MLF-stimulated neutrophil using a chemiluminescence-based assay. Neu-trophils were treated with 10−6 M f MLF at 378C, and chemilumines-cence was recorded at 10-second intervals for 14 min. Panel A: Thedetection buffer contained 150 mM luminol to measure total ROS pro-duction (solid line) or 150 mM luminol and 10,000 U/ ml catalase and100 U/ ml SOD to measure only intracellular ROS production (dottedline). Panel B: The detection buffer contained 150 mM isoluminol tomeasure only extracellular ROS production. The data shown representone experiment taken from 20 independent experiments.

occurred with both the extracellular component (Fig. 3A)and the intracellular component (Fig. 3B), although theinhibition was marginally stronger with the intracellularcomponent. When the kinetics of the extracellular por-tion of the f MLF-induced oxidative burst were exam-ined using a shorter measurement interval, it was appar-ent that adenosine inhibition of this response had a briefdelay. As seen in Fig. 4, the rate of ROS production inboth f MLF-stimulated and adenosine-pretreated/ f MLF-stimulated neutrophils was identical over the initial 5 secof the response (after an initial 5 sec lag in both groups),after which the rate of ROS production in adenosine-pre-treated cells sharply diminished.

Fig. 3. Inhibition of f MLF-induced ROS production by adenosine:chemiluminescence-based assay. Panel A: Total ROS production wasdetermined as described in Fig. 2A (without added catalase or SOD).Panel B: Measurements of intracellular ROS production as in Fig. 2Awith added catalase and SOD. Values are mean ±SEM, n c 3. Repre-sentative of 15 independent experiments.

Subcellular Localization of NADPH OxidaseComponents

Post-nuclear cavitates of unstimulated, f MLF stim-ulated, and adenosine pretreated/ f MLF stimulated neu-trophils were fractionated on two-step Percoll gradi-ents, and the subsequent fractions analyzed for stan-dard granule or plasma membrane markers (Fig. 5). Asdemonstrated previously by many groups, we show amajor band at higher density (1.138 g/ ml), which con-tains most of the cellular myeloperoxidase (MPO), anazurophil granule marker; a band at 1.102 g/ ml densitycontaining most of the cellular lactoferrin, a specific granule marker; and a smaller band at 1.027 g/ ml densitycontaining most of the cellular alkaline phosphatase, a


Fig. 4. Early kinetics of adenosine inhibition of f MLF-induced ROSproduction. Neutrophils were pretreated with buffer or 10−6 M adeno-sine for 3 min at 378C, followed by injection of 10−6M f MLF directlyinto the sample wells. ROS production was measured at 0.5 secondintervals for 120 sec. The data shown represent one experiment takenfrom 3 indpendent experiments.

marker for both plasma membranes and secretory gran-ules. Although flow cytometric analysis revealed thatrelease of lactoferrin occurred in response to f MLF treat-ment (see below), there were no consistent differences ineither the content of lactoferrin containing granules, ortheir position on the gradient after separation betweencontrol, stimulated, and adenosine pretreated cells. Thisdata suggests that the amount of lactoferrin released bythis treatment was small compared to overall cellularreserves. Although azurophil granules, unlike specificand secretory granules, do not contain NADPH oxidasecomponents, our data suggest that azurophil granules (orat least MPO activity) are affected by f MLF treatmentand adenosine pretreatment. MPO activity was typicallylower in the azurophil granules of untreated cells, ascompared to f MLF and adenosine/ f MLF-treated cells,although this is not apparent from the normalized datain Fig. 5. This suggests, as has been previously reported,that intracellular activity of MPO can be increased bytreatment with soluble activators (37). Because we choseto focus on the extracellular production of ROS dur-ing the first 3 min of the f MLF-induced oxidativeburst,we wished to determine the relative concentrationof NADPH oxidase components in the plasma membranefraction and whether adenosine pretreatment affectedtheir relative levels. As seen in Fig. 6A, the amount offlavocytochrome b [as determined by p22phox (shown)and gp91phox (not shown) staining] was increased in theplasma membrane/ secretory granule fraction of f MLF-stimulated neutrophils. In contrast, cells pretreated with

Fig. 5. Subcellular fractionation of adenosine/ f MLF-treated neu-trophils. Neutrophils were pretreated with buffer or 10−6 M adenosinefor 3 min at 378C, followed by activation with 10−6M f MLF for 2 min.Control cells were incubated for the indicated times without activat-ing agents. Following treatment, the cells were chilled with ice coldbuffer, resuspended in 48C pipes buffer, disrupted by N2 cavitation,and the post-nuclear supernatant was fractionated on two-step Percolldensity gradients. Each fraction was anlyzed for total alkaline phos-phatase, lactoferrin, and myeloperoxidase. The distributions of alka-line phosphatase (O), lactoferrin (I), and myeloperoxidase (R) on thegradients are shown. The data shown represent one experiment takenfrom eight independent experiments.

adenosine for 3 min prior to f MLF stimulation and frac-tionation showed a significantly decreased amount offlavocytochrome b associated with this fraction, and the


Fig. 6. Decreased p22phox in the plasma membrane/ secretory gran-ule fraction of adenosine/f MLF-treated neutrophils: correlation withreduced ROS production. Panel A: Neutrophils were pretreated withbuffer (lane 2), 10−7 M adenosine (lane 3), or 10−6 M adenosine (lane4) for 3 min at 378C, followed by activation with 10−6 M f MLF for 2min. Control cells were incubated for the indicated times without acti-vating agents (lane 1). Following treatment, the samples were fraction-ated on Percoll density gradients, as described under Fig. 5. The threegradient fractions containing the highest alkaline phosphatase activ-ity were pooled, and Percoll was removed by centrifugation. Equalamounts of protein from each sample (50 mg/ lane) were analyzedby SDS-PAGE (7–18% polyacrylamide gradient gels) and immuno-blotting with anti-p22phox monoclonal antibody. Panel B: Densomet-ric scans of Western blots (as in Fig. 6A) from three independentexperiments were normalized (0 c mean density of the p22phox bandfrom control neutrophils, 100 c mean density of f MLF-stimulated neu-trophils) and averaged. Values are mean normalized density ±SEM,n c 3. Panel C: ROS production was measured as in Fig. 3 for thesame preparations as described under Panel A. Values are the mean±SEM of the total integrated chemiluminescence over 3 min, n c 3.+P ≤ 0.05) and ∗(P ≤ 0.001) indicate a statistically significant differ-ence compared to f MLF-treated cells.

decrease in plasma membrane/ secretory granule-associ-ated flavocytochrome b was dose-dependent with respectto adenosine (Fig. 6A). Furthermore, this decrease inflavocytochrome b correlated with the decrease in extra-cellular ROS production during the first 3 min after stim-ulation (Fig. 6B, 6C). We were not able to consistentlydetect the major cytosolic-originating components of theNADPH oxidase, p47phox and p67phox, in the plasmamembrane/ secretory granule fraction. This is possiblydue to proteolysis, and Western blot analysis of the samesamples typically showed, for example, a proteolyzedform of p47phox, but not the full-length form (data notshown). Unfortunately, if the cells were treated with DFPprior to adenosine and f MLF treatments, the adenosineeffects on ROS production were inconsistent, and some-times much less than what was seen in non DFP-treatedcells. As a result we chose to apply DFP to the cells onlyduring the cold quenching step after the adenosine andf MLF treatments. While this treatment appeared to besufficient to prevent proteolysis of the flavocytochromeb subunits during fractionation, the cytosolic phox pro-teins were apparently quite labile under those conditions.

Degranulation of Specific and Secretory Granules

Recruitment of secretory or specific granules wasdetermined by analyzing changes in the cell-surfaceexpression of standard markers of the respective gran-ules. Secretory granules are believed to be the sole intra-cellular source of complement receptor 1 (CD35) (38)and the cell surface aminopeptidase CD10 (39) in neu-trophils. Both of these proteins were rapidly upregulatedon the neutrophil surface after treatment with f MLF, andpretreatment with adenosine did not inhibit the upregula-tion of either of these markers (Fig. 7A, 7B). To confirmthese data, we also used a fluorometric substrate assayto measure the cell-surface alkaline phosphatase activ-ity on untreated, f MLF-treated, and adenosine/ f MLF-treated neutrophils. This assay showed that unstimu-lated neutrophils had ∼55% of the total cellular alka-line phosphatase activity on the cell surface. Treatmentwith f MLF increased this value to 80%, and pretreat-ment with adenosine prior to f MLF stimulation did notsignificantly affect this response (78%), thus confirmingour flow cytometric analyses.

Lactoferrin has been considered a marker for neu-trophil specific granules. Although it is not a transmem-brane protein, it is detected on the cell surface of neu-trophils after specific granule degranulation, generally


Fig. 7. Flow cytometric analysis of neutrophil cell-surface markers in adenosine/ f MLF-treated neutrophils. Neutrophils were pretreated with buffer( f MLF), 10−7 M adenosine, or 10−6 M adenosine (as indicated) for 3 min at 378C, followed by activation with 10−6 M f MLF. Control cells wereincubated for the indicated times without activating agents. After 3 min, cells were chilled, washed, stained for the indicated antigen, and analyzedby flow cytometry. The data are expressed as relative mean fluorescence intensity compared to the mean fluorescence intensity of f MLF stimulatedneutrophils (arbitrarily set as 100%) ± SEM, of 3 pooled experiments, with n c 3 replicates in each experiment. Upper left: Staining for cell-surfaceCD35 (complement receptor 1), a secretory granule marker. Upper right: Staining for cell-surface CD10 (CALLA), a secretory granule marker.Lower left: Staining for cell-surface lactoferrin, a specific granule marker. Lower right: Staining for cell-surface CD11b/ CD18 (aMb2), which iscontained in both secretory granules and specific graules. +(P ≤ 0.05), #(P < 0.01), and *(P ≤ 0.001) indicate a statistically significant differencecompared to control cells.

in proportion to the degree of specific granule recruit-ment (33, 40). As seen in Fig. 7C, cell-surface lactofer-rin also increased after f MLF stimulation, and adeno-sine pretreatment had no effect on this response. Finally,the b2 integrin (aMb2 or CD11b/ CD18) is often usedas a marker for specific granule mobilization, althoughit is also present in secretory granules (41). As withall the markers we measured, aMb2 was upregulated inresponse to f MLF, and adenosine pretreatment had noeffect on this response (Fig. 7D). Therefore, it is clearfrom these experiments that adenosine pretreatment hadno effect on f MLF-induced recruitment of specific andsecretory granules and that changes in flavocytochrome

b levels were due to adenosine-specific signaling path-ways other than effects on granule mobilization.

Changes in Cell-Surface Flavocytochrome b

When human neutrophils are stimulated with 1 mMf MLF, there is a rapid (∼1 min) increase in cell-surfaceflavocytochrome b (42), and we confirmed this usingflow cytometry with monoclonal antibody 7D5 (Fig. 8).In cells pretreated with 1 mM adenosine and followed by1 mM f MLF, flavocytochrome b was also upregulated;however, there was consistently a lower level of cell-sur-face flavocytochrome b after adenosine treatment (Fig.


Fig. 8. Flow cytometric analysis of neutrophil cell-surface flavocy-tochrome. Neutrophils were pretreated with buffer or 10−6 M adeno-sine for 3 min at 378C, followed by activation with 10−6 M f MLF.At the indicated time points, the cells were rapidly chilled, washed,and stained with anti-flavocytochrome b antibody 7D5, as described.Mean fluorescence was determined using a FACSCalibur flow cytome-ter. The results are pooled from four separate experiments, expressedas the percent increase in mean fluorescence (±SEM) compared tountreated cells in each group.

8). In both conditions, the levels of cell-surface flavo-cytochrome b remained elevated for the duration of thef MLF-induced oxidative burst. These data suggest thatthe decrease in plasma membrane/ secretory granule flav-ocytochrome b compartment isolated on Percoll gradi-ents is predominately due to losses from the secretorygranule compartment.

When cell-surface proteins were conjugated withbiotin prior to treatment and fractionation, biotiny-lated gp91phox was abundant in the uppermost (plasmamembrane/ secretory granule) Percoll gradient band (Fig.9A); however the amount of gp91phox present in theplasma membrane/ secretory granule fraction obtainedfrom adenosine-treated samples (lane 7) was signifi-cantly (p < 0.001) less than that of control (lane 1)or f MLF-treated (lane 4) samples (relative band den-sities for f MLF and adenosine + f MLF were 113.3 ±7.2% and 63.0 ± 10.2% of control), which is consis-tent with our flow cytometric studies described above. Itwas also apparent that the intermediate Percoll gradientband (specific granules) of all three samples containedsmall amounts of biotinylated gp91phox, although therewere no consistent differences in the relative intensitiesof gp91phox staining between the three treatment groups(lanes 2, 5, 8). Interestingly, biotinylated gp91phox wasalso present in the lowest (azurophil granule) band ofthe Percoll gradient, but apparently only in samples that

Fig. 9. Analysis of flavocytochrome b in granule fractions isolatedfrom biotinylated neutrophils. Neutrophils were biotinylated, pre-treated with buffer or adenosine for 3 min, and then activated with 10−6

M f MLF at 378C. The cells were then disrupted by N2 cavitation andfractionated on two-step Percoll density gradients as described underFig. 5. Biotinylated proteins localized in granule fractions were iso-lated with avidin beads and immunoblotted, as described under Materi-als and Methods. Panel A: Biotinylated proteins retrieved from plasmamembrane/ secretory granule fractions (lanes 1, 4, 7), specific granulefractions (lanes 2, 5, 8), and azurophil granule fractions (lanes 3, 6, 9)of cells treated with buffer (lanes 1–3), 10−6 M f MLF (lanes 4–6), and10−6 M adenosine followed by f MLF (lanes 7–9) were immunoblottedfor gp91phox. Panel B: Biotinylated proteins retrieved from azurophilgranule fractions of neutrophils treated with buffer (lane 1), f MLF(lane 2), and adenosine + f MLF (lane 3). Greater sample loads (2× )and development times were used for these samples compared to thosein Panel A. Silver staining of replicate gels was used to confirm equalloading of samples (not shown). Representative blots are shown from3 separate experiments.

had been treated with adenosine prior to f MLF stimu-lation (lane 9). Because of the very faint nature of thisband, we doubled the amount of sample loaded onto thegel and the development time of the resultant blot. It wasapparent from these experiments that in each treatmentgroup, the azurophil granule fraction actually did containsome biotinylated gp91phox (Fig. 9B); however, samplesfrom adenosine-treated neutrophils always showed muchhigher levels of biotinylated gp91phox than untreated or


f MLF-treated neutrophils. Indeed, densitometric anal-ysis of the gp91phox blots showed that samples fromadenosine-treated cells contained significantly (p < 0.05)more gp91phox than untreated controls (175.0 ± 14.1%;n c 3). In comparison, f MLF-treated cells were slightly(123.7 ± 16.0%; n c 3) but not significantly higher thanuntreated controls.

DISCUSSION

Because of the potentially damaging effects ofexcessive ROS production, activation of the neutrophilNADPH oxidase is tightly regulated on multiple lev-els. At the level of protein–protein interactions, sev-eral events are necessary for activation of the oxidase.These include phosphorylation of the cytosolic phox pro-teins, which may regulate further interactions among thecytosolic phox proteins and small GTPases, followed byassociation of this complex of cytosolic proteins withthe membrane-bound flavocytochrome b (10). Only afterthese associations are made is the oxidase fully activeand able to produce O−

2 . Furthermore, for O−

2 productionto continue over time, there must be a stable associa-tion of the cytosolic components of the oxidase with themembrane components. This may be achieved by contin-uous recruitment of new cytosolic proteins to membranesites that contain flavocytochrome b (43, 44).

Beyond assembly of the oxidase components at theprotein level, NADPH oxidase activation also involvesmovement of the membrane components between dif-ferent subcellular compartments (45–47). This in turnmay determine the sites and magnitude of O−

2 produc-tion within the neutrophil. We show here that treat-ment of neutrophils with adenosine has a significanteffect on the relative amount of flavocytochrome b in theplasma membrane/ secretory granule fraction of f MLF-treated cells, and that this change correlates with thedecrease in extracellular ROS production during theinitial phase of the f MLF-induced oxidative burst. Inunstimulated neutrophils, the majority of the flavocy-tochrome b is normally contained within the specificgranule and secretory granule components of the cell(48). Upon stimulation with f MLF (and to a greaterextent with PMA) flavocytochrome moves from intra-cellular granules (especially the specific granules) tothe plasma membrane of the neutrophil (46, 48). Thisevent is usually considered to be a result of degran-ultion, i.e., granules within the neutrophil fuse withthe plasma membrane and the proteins in their mem-

branes (e.g., flavocytochrome b) are then incorporatedinto the plasma membrane (49). It is firmly establishedthat stimulation of neutrophils with various agents cancause recruitment of both secretory and specific gran-ules to the plasma membrane, with a resultant up-reg-ulation of the cell-surface expression of many proteins,including integrins, receptors for cytokines, complementcomponents, chemotactic peptides, and possibly flavo-cytochrome b itself (42, 48, 50). As described above,adenosine has been reported to either inhibit or have noeffects on neutrophil degranulation in response to sol-uble activators. Therefore, to determine whether adeno-sine effects on degranulation and upregulation of cell-surface proteins could account for the observed differ-ences in plasma membrane cytochrome b, we comparedcell-surface markers of secretory and specific granulemobilization on neutrophils treated with identical regi-mens to those analyzed for oxidative burst and sub-cel-lular fractionation. We found that adenosine pretreatmenthad no significant effect on the recruitment of secretoryor specific granules to the cell surface of f MLF-stim-ulated neutrophils. Therefore, it is not likely that theobserved decrease in membrane flavocytochrome b wasdue to adenosine limiting its recruitment from intracel-lular stores.

Since a decreased level of flavocytochrome b inthe plasma membrane/ secretory granule fraction did notappear to be due to decreased trafficking to this com-partment via modulation of degranulation by adeno-sine, we considered whether adenosine acted to promoteremoval of the flavocytochrome b from this compart-ment. Indeed, our results suggest that adenosine treat-ment of neutrophils does promote increased traffickingof flavocytochrome b between cellular compartmentsafter f MLF stimulation. When neutrophil surface pro-teins are biotinylated, small amounts of biotinylatedgp91phox appear in the specific granule fraction, as wellas in the azurophil granule fraction. Whether the flavo-cytochrome b actually leaves the plasma membrane anddirectly joins specific granules and/ or azurophil gran-ules is unknown, but is not likely. It is more likelythat membrane vesicles containing flavocytochrome bnormally undergo cycles of endocytic recycling, as hasbeen shown for other neutrophil membrane proteins (51,52). During the course of this process, flavocytochromeb-containing membranes may become associated withother components involved with intracellular trafficking(e.g., Rab proteins, cytoskeletal proteins, etc.). These andother changes would result in altered densities of themembrane vesicles and subsequent changes in migration


of the vesicles on Percoll gradients once the cells werefractionated. While the Percoll gradient system used hereis well established for the separation of membrane, spe-cific granules, and azurophil granules (32), these frac-tions are likely not homogenous, as the fractionationprinciple is simply by vesicle density. We show herethat adenosine treatment results in significantly increasedmovement of flavocytochrome b from the plasma mem-brane into some form of higher density membranes, andthis coincides with the accelerated shut down of theextracellular production of ROS by the neutrophil. Acomparison between results obtained from the examina-tion of total amounts of flavocytochrome b protein in amembrane compartment of fractionated neutrophils, andthe movement of biotinylated proteins between mem-brane compartments can be problematic. By necessity,neutrophil cell-surface proteins are biotinylated on rest-ing cells; therefore biotinylated gp91phox found in heavygranule compartments would only be derived from thatportion of flavocytochrome b located at the cell surfaceduring labeling. Flavocytochrome b that originated ininternal granules, which then participated in intracellu-lar trafficking during the oxidative burst, would not bebiotinylated. Thus, it is likely that the amount of biotiny-lated gp91phox found in the heavy granule fraction repre-sents a fraction of that which is being internalized. Thisconclusion is supported by the downregulation in cellsurface flavocytochrome b during the later portion of thef MLF-induced oxidative burst (as determined by flowcytometry).

The f MLF-induced oxidative burst of neutrophilsis normally self-limiting, and the entire burst is usu-ally limited to 5–10 min, while the extracellular por-tion of the burst is complete in 3–5 min. In previ-ous studies, Cronstein et al. (20) reported that occu-pancy of A2 adenosine receptors promoted associa-tion of f MLF-bound receptors with the cytoskeleton,thereby contributing to termination of the response. Ourkinetic results suggest that adenosine may accelerate thisshut-off event via an additional mechanism. Accordingto this scenario, recruitment of flavocytochrome b tothe plasma membrane through degranulation occurs inf MLF-stimulated neutrophils whether or not adenosineis present. Within seconds, however, adenosine-treatedcells begin to reduce the amount of flavocytochrome bin the plasma membrane/ secretory granule compartment,probably through endocytosis and membrane traffick-ing. These flavocytochrome b-containing vesicles prob-ably acquire higher densities as their constituents arechanged, so that they begin to co-isolate with specific

granules and azurophil granules. During this process,the NADPH oxidase is presumably inactivated, and theflavocytochrome b is either degraded or recycled backinto a storage compartment such as specific granules.The exact trafficking pathway of endocytosed flavocy-tochrome b and the possible site of inactivation willrequire more detailed experimentation than what wepresent in this study. In any case, it appears that thispathway is normally active in resting neutrophils, butadenosine increases the trafficking of flavocytochromeb through this pathway.

Recently, there has been a reevaluation of the func-tion and behavior of neutrophil granules. While onceviewed as containers of enzymes and proteins withstrictly defined structures and roles, it is becoming appar-ent that these organelles are more varied and dynamicthan previously thought. This is particularly true withrespect to the secretory granule. Morphological and his-tochemical studies at the electron microscope level haveshown this compartment consists of a dynamic set oftubules, as opposed to simply a group of discreet vesicles(53, 54). When neutrophils are stimulated with f MLF,these tubules can undergo rapid reorganization and formconnections with the extracellular environment (53, 54).Furthermore, these structures appear to be important assites of ROS production in stimulated neutrophils (55,56). Unfortunately, biochemical analysis of these com-partments in neutrophils has been limited, due to insuf-ficient techniques for their isolation from other subcel-lular compartments. Although the technique of free-flowelectrophoresis can potentially be used to isolate secre-tory vesicles from vesicles originating from the plasmamembrane, this method is costly and not widely avail-able (57). More commonly, investigators utilize the stan-dard method of subcellular fractionation on Percoll gra-dients (32, 48). By this method, plasma membrane vesi-cles are not physically separated from secretory granulevesicles, although measurement of latent alkaline phos-phatase is believed to give a relative indication of theproportion of the two components, at least in samplesfrom unstimulated cells (58). In our studies, adenosinecaused a decrease in the amount of flavocytochromeb (both p22phox and gp91phox) present in this plasmamembrane/ secretory granule fraction at both 2 min post-f MLF stimulation (when ROS production was extracel-lular) and at 6 min post-f MLF stimulation (when ROSproduction was intracellular). While these data reinforcethe fact that plasma membrane and secretory granulecomponents are not separated with this technique, theyalso lend credence to the observations that this cellular


compartment is the site of ROS production, both intra-cellular and extracellular, because of the correlation withflavocytochrome b concentration and ROS productionrates. Previous work suggests that there is dynamic recy-cling between the plasma membrane and intracellularparts of this component (59). This type of dynamic extra-and intracellular membrane remodeling would certainlyprovide a basis for the observations reported here.

Acknowledgments—The authors would like thank Dr. MichioNakamura (Institute of Tropical Medicine, Nagasaki Univer-sity, Nagasaki, Japan) for the kind gift of monoclonal antibody7D5. This work was supported in part by USDA-NRICGP 99-03600, USDA-NRICGP 99-03508, USDA-NRICGP 00-02262,NIH RO1 HL66575, and the Montana State University Agri-cultural Experimental Station. This is manuscript 2001-43from the Montana Agricultural Experiment Station, MontanaState University-Bozeman.

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Inhibition of the Neutrophil NADPH Oxidase by Adenosine Is Associated with Increased Movement of Flavocytochrome b Between Subcellular Fractions