The antiinflammatory mechanism of methotrexate Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation

Full text

(1)

The antiinflammatory mechanism of

methotrexate. Increased adenosine release at

inflamed sites diminishes leukocyte

accumulation in an in vivo model of

inflammation.

B N Cronstein, … , D Naime, E Ostad

J Clin Invest.

1993;

92(6)

:2675-2682.

https://doi.org/10.1172/JCI116884

.

Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in

low concentrations. We examined the hypothesis that the antiphlogistic effects of

methotrexate result from its capacity to promote intracellular accumulation of

5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury,

increases local adenosine release. We now present the first evidence to establish this

mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice

were injected intraperitoneally with either methotrexate or saline for 3-4 wk during induction

of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte

AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air

pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The

methotrexate-mediated reduction in leukocyte accumulation was partially reversed by

injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a

specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but

not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine.

Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of

animals treated with either saline or the potent antiinflammatory steroid dexamethasone.

These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the

antiphlogistic action of which is due to increased adenosine release at inflamed sites.

Research Article

(2)

The

Antiinflammatory Mechanism of Methotrexate

Increased Adenosine Release at Inflamed Sites Diminishes Leukocyte Accumulation inan InVivoModel of Inflammation

Bruce N. Cronstein, Dwight Naime, and Edward Ostad

DepartmentofMedicine, Division ofRheumatology, New York University Medical Center, New York 10016

Abstract

Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined thehypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of5-aminoimidazole4-carboxamide ribonucleotide (AICAR)

that,underconditionsof cellinjury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for34wkduring induction of air pouches. Pharmacologically relevant doses of methotrex-ate increasedsplenocyte AICAR content, raised adenosine

con-centrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in

in-flamed air pouches. Themethotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of

adenosine deaminase (ADA) into the air pouch, completely reversed by aspecific adenosineA2 receptor antagonist, 3,7-di-methyl-l-propargylxanthine (DMPX),but notaffectedby an adenosine

Al

receptor antagonist, 8-cyclopentyl-dipropylxan-thine. NeitherADAnorDMPXaffected leukocyte accumula-tion in theinflamedpouchesof animalstreated with either sa-line or the potent antiinflammatory steroid dexamethasone. Theseresults indicate that methotrexate isanonsteroidal

an-tiinflammatoryagent, theantiphlogisticactionofwhich is due toincreased adenosinereleaseatinflamed sites.(J. Clin. In-vest. 1993.92:2675-2682.) Keywords:leukocyte-

adenosine-purine*inflammation* methotrexate

Introduction

Methotrexateisafolate

antagonist

first developed forthe treat-mentof

malignancies

andnow

widely

usedin thetreatmentof rheumatoid arthritis (1). Unlike its use in the treatment of

malignancies

(pulses of 20-250

mg/kg),

methotrexate is

ad-ministeredweeklyinlowdoses(0.1-0.3 mg/kg)totreat

rheu-This materialwaspresented in abstract formattheannualmeetingof the American Federation for ClinicalResearch/Am.Soc. for Clinical Investigation/Am. Association ofPhysicians in Wash. DC, 2 May 1993.

AddresscorrespondencetoDr.Bruce N.Cronstein,Dept. of Medi-cine, Division of Rheumatology, New York University Medical Center, 550 First Avenue,NewYork,NY 10016.

Receivedfor publication6July1993 andinrevisedform26July 1993.

matoid arthritis and other inflammatory diseases (1). Al-though theoriginalrationale for the use of methotrexate in the treatmentofrheumatoid arthritis was "immunosuppression," themolecular mechanism by which methotrexate suppresses

inflammationis not well understood. It is unlikely that, in the dosesgiven,methotrexate diminishesproliferationof immune cells by inhibiting de novo purine and pyrimidine synthesis

sinceleukopeniaand mucosalulcerations,phenomena best

ex-plainedby theantiproliferative effects ofmethotrexate, are

con-sidered evidence ofdrugtoxicityand indications to decrease or stoptherapy. Otherproposed mechanisms includeadecrease inneutrophil (but not macrophage) leukotriene synthesis (2) andinhibition of transmethylation reactionsby inhibiting the

formationof S-adenosyl-methionine,amethyl donor required

forproteinandlipid methylation(3).

We have recently proposed an alternative biochemical

mechanism of action ofmethotrexate; methotrexate promotes therelease ofthe antiinflammatory autocoid adenosineat

in-flamed sites (4). Previous studies havesuggestedthat metho-trexateaccumulates within cells and, both directly and

indi-rectly, inhibits 5-aminoimidazole-4-carboxamide ribonucleo-tide

(AICAR)'

transformylase, resulting in the intracellular accumulation ofAICAR (Fig. 1; references 5-9). Increased

intracellular concentrations ofAICAR promote, byacomplex mechanism, theincreasedreleaseofthe potent

antiinflamma-tory autocoid adenosine ( 10, 11). Results ofinvitro studies

supportthis

hypothesis

(4);lowconcentrations (< 10nM) of

methotrexateorhigher concentrations ofthecell-soluble,

non-phosphorylated precursor of AICAR, AICARibonucleoside (acadesine), promote adenosine release from fibroblastsand

endothelial cells. Theincrease in extracellularadenosine

con-centration

diminished,

viaoccupancy of

specific

cell surface

receptors,the

capacity

ofstimulated

neutrophils

toadhereto themethotrexate-treated endothelialcells and

fibroblasts,

inan in vitro model ofan

inflammatory

interaction. Asako et al. (1.2)haveconfirmedthat methotrexate suppresses inflamma-tion by

increasing

adenosinerelease

using

the hamstercheek pouchmodelofacuteinflammationbut

high

concentrations of

topically applied

methotrexate(1

MM)

wereused intheir

study.

We report here thefirst evidence frominvivostudiesthat demonstrates that low-dose weekly methotrexate treatment causesintracellularaccumulation of

AICAR,

increased adeno-sine releaseatsitesof

inflammation,

andadenosine-dependent inhibitionof inflammation. Moreover,wehaveconfirmedthat inmethotrexate-treated mice adenosine diminishes inflamma-tion viaoccupancyof adenosine A2receptors. These data

pro-vide the first in vivo demonstration ofa novel biochemical mechanism ofactionformethotrexate.

1.Abbreviations usedin thispaper:AICAR, 5-aminoimidazole-4-car-boxamideribonucleotide; DMPX,3,7-dimethyl-1-propargylxanthine.

J.Clin.Invest.

©TheAmericanSocietyfor Clinical

Investigation,

Inc.

0021-9738/93/12/2675/08 $2.00

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DeNovo Purine Biosynthesis

A

I.

Transmethylation

Reactions

-GAR WF-FGAR-l

I

MTXu AICAR ATP

DHF8u FAICAR ADP SAM

I

II Methionine

KIMP: AMP THF

I

II II

I

Hoc

ysteine

Inosine Adenosine SAH

-Hypocanthine

Sat

I ExtracellularSpace

Uric Acid

Figure1.Proposed molecular mechanism of action of methotrexate. Shown herearethemajorsteps inpurine synthesisanddegradation. Abbreviations: GAR,

fl-glycinamide

ribonucleotide;FGAR, a-N-for-mylglycinamide ribonucleotide; MTXSU, methotrexate polygluta-mate;DHFSIU, dihydrofolate polyglutamate; AICAR, 5-aminoimida-zole-4-carboxamide ribonucleotide;FAICAR,formyl-AICAR;IMP, inosinicacid;THF,tetrahydrofolate(reduced); SAM, S-adenosyl-methionine;SAH,S-adenosylhomocysteine.

Methods

Materials. 3,7-Dimethyl-l-propargylxanthine and 8-cyclopentyl-di-propylxanthine were obtained from Res. Biochem. Inc. (Natick, MA). Injectable methotrexate (US Pharmacopeia)waspurchased from

Le-derle Laboratories Division of American Cyanamid (Wayne, NJ). Adenosine deaminase (Type IV, calf intestinal), carrageenan, and dexamethasone wereobtainedfromSigmaChemical Co. (St. Louis, MO). All other reagents were the highest grade that could be obtained. Induction ofairpouchesandcarrageenan-induced inflammation. To induceanair pouch, mice (6-wk-old, female Balb/c mice, Taconic Farms Inc.,Germantown, NY) were injected subcutaneously with 3 cc ofair (on the back) three times per wk. After 3-4wk,theair pouchwas

injectedwith 1 mlofa2%(wt/vol) suspension of carrageenan and mice were returned to their cages, where they were allowed to run free for4h.Attheend of4h, the animalswerekilled, 2 cm3 of normal salinewasinjected into the pouch and the contents of the pouchwere

aspirated,diluted 1:2with normal saline, and samples were taken for cellcount.Smearsof the undiluted fluidwereprepared andanaliquot

wasstained (Wright-Giemsa), revealing that >95% of the exudate cellswere PMNs. In someexperimentstheair pouchwasdissected from the subcutaneous tissue, fixed in formalin, processed for histo-logic sections, and stained (hematoxylin and eosin and Giemsa stains) usingstandard methods ( 13). These studies werereviewed and ap-proved by the Institutional Animal Care and Use Committee of New YorkUniversity Medical Center.

Treatment ofmice with methotrexate. Mice were treated with weekly intraperitoneal injections ( 1 ml) of methotrexate (U.S.P., 0.05-0.5 mg/kg)orpyrogen-free(U.S.P.) normal saline (0.9%) for 3 or4

wk. Therewere noapparent adverse effects of either treatment that

could bedetected by visual inspection of the animals and there were no apparent differences between the animals treated with saline and those treatedwith methotrexate.

Preparationofadenosinedeaminase. Adenosine deaminase(50,l,

4000U/ml)wasdialyzedagainstPBSovernight (4°C) before dilution andinjectioninto the air pouch.Forsomeexperiments, dialyzed adeno-sinedeaminasewasincubated with deoxycoformycin(1 MM) for 30 minat roomtemperaturebefore dilution ( 1:2600) in PBS containing carrageenan( 14).

Injectionofadenosine deaminase andadenosine receptor

antago-nists. In someexperiments adenosine deaminase, previously inacti-vatedadenosine deaminase (see above), and adenosine receptor antag-onistswereadded to the carrageenan suspension to an appropriate final concentration. The final volume of the carrageenan suspension (with

inhibitors)

didnotdiffer from that in control mice

(1

ml).

Histologic analysis ofsections ofairpouches. Slides of stained (Giemsa) sections ofmouseairpouchwereexaminedmicroscopically usingaLeitz researchmicroscopetowhichwasattacheda high-resolu-tion videocamera.Videoimageswereprojected directlyonto a screen

foranalysisbyuseof JAVA software(JandelSci.,CorteMadera,CA)

runon aZenith 386 computer. Allimagesweredigitized directlyand enhanced forcontrastandbrightness usingPHOTOSTYLER software (Aldus, Inc.,Seattle, WA).

QuantitationofAICAR.In someexperimentsthespleenswere har-vested and the cellswereisolatedbyscraping throughgauze. The cells were washed and resuspendedat100X 106/mlin PBS. The cellswere

thenlysed and theproteinsweredenaturedbyaddition of 1 vol of trichloroaceticacid (10% vol/vol). The trichloroacetic acidwas

ex-tracted withfreon-octylamineand the supernatantswerecollected and stored at -80'C before analysis. Nucleotides were quantitated by HPLC by amodification of the methodofChenetal. (15). Briefly, nucleotideswereinjectedonto aPartisil-10SAX column(Whatman Inc.,Clifton,NJ), isocratic elution with 0.007 MNH4H2PO4,pH4.0, followedbyalinear gradientto0.25 MNH4H2PO4, pH 5,formedover

30 minat aflowrateof 2 ml/min. Absorbancewasmonitoredat250 and 260nmandconcentrationwascalculatedbycomparisonto

stan-dards.Preliminary studies showed that 85% of addedAICARibotide was recoveredusing this technique.

Adenosine determination. Aliquots of pouch exudates were added

to asimilar volume oftrichloroaceticacid ( 10%vol/vol),followedby extraction of theorganic acid,asdescribed above. The adenosine

con-centration ofthe supernatantswasdetermined by reverse-phase HPLC,

as wehavepreviouslydescribed( 14).Briefly, sampleswereappliedto a

CI8gBondapack

column (Waters Chromatography Div., Milford, MA) and eluted witha0-40%-linear gradient(formedover60min)of 0.01 M ammoniumphosphate (pH 5.5) andmethanol, with a 1.5 ml/minflowrate.Adenosinewasidentified by retention time and the characteristic UV ratio of absorbanceat250/260,and the

concentra-tionwascalculatedbycomparisontostandards. Insomeexperiments theadenosine peakwasdigested bytreatmentwith adenosine

deami-nase(0.15 IU/ml, 30 min at 37°C)toconfirm that the peakso identi-fied containedonly adenosine (16). Preliminarystudies demonstrated that 90% of added adenosinewasrecoveredusingthistechnique.

Digitizationofchromatograms. Chromatograms were digitized us-ingaHewlett-Packard(Palo Alto, CA) Scanjet apparatus and the

re-sultingimageswereenhancedforcontrastandbrightnessusing PHO-TOSTYLER softwarerunon aZenith 386 personal computer.

Statisticalanalysis. The data wereanalyzed by the appropriate levelof ANOVAperformedby EXCEL 4.0software (Microsoft,Inc., Bothell,WA).

Results

Low-dose weeklymethotrexate markedly inhibits leukocyte ac-cumulation inthe airpouch in response to carrageenan. Low-dose weekly methotrexate isa potent form of antiinflamma-torytherapy in patients suffering fromrheumatoid arthritis. To

confirm that the murine airpouch model of inflammation was a reasonable model in which to study the antiinflammatory

effects ofmethotrexate, we determined the effect of various dosesoflow-doseweekly methotrexate (administered

intraperi-toneally) on accumulation ofleukocytes in the murine air

pouch after injection ofcarrageenan. Methotrexate

dimin-ished, inadose-dependentmanner, the number of leukocytes that accumulated in carrageenan-treated air pouches by as

muchas60% (IC50=0.08 mg/kg perwk,P < 0.0002 vs. saline-treatedanimals; Fig.2). Moreover, the doses of methotrexate

requiredtoachieveamaximalantiinflammatoryeffect in this

animalmodel aresimilarto those required for the treatment of

rheumatoid arthritis(the equivalent of 10-15 mg/wk in a

(4)

metho-6- Figure 2. Weekly injec-tionof low-dose metho-trexateis antiinflamma-tory in the airpouch model. Methotrexate wasgivento the mice

(2 \by intraperitoneal

injec-tionattheindicated dosesfor 3 to4wk

dur-2 inginduction of the air

.05 .1 5

pouch.Theair pouch

[Methotrexatel (mg\kg\wk) wasinjected with

carra-geenan(2% wt/vol), exudateswere harvested4hlater, and the cells were counted. Each point represents themean(±SEM)of cell counts from three mice. Analysis of variance demonstrates that the exudate cell count varied significantly with the dose of methotrexate (P<0.0002).

trexate-mediated inhibition ofleukocyte accumulation was

similarevenwheninflammation wasinducedupto 6 dafter the lastdoseof methotrexate (datanotshown).

Low-dose weekly methotrexate treatment increases intra-cellular concentrations ofAICAR. We have proposed that the antiinflammatory actions ofmethotrexate result, bothdirectly

andindirectly, from theinhibition ofAICARtransformylase

(4).If thismechanism iscorrect,specific inhibitionofAICAR

transformylaseshould result inhigher intracellular

concentra-tions ofAICAR. Wedirectlytested thevalidity of this

hypothe-sis byexamining AICAR concentrations in splenocytes from

saline- andmethotrexate-treated mice (0.5 mg/kg by weekly

intraperitoneal injection for4 wk) by HPLC. We found that

splenocytes from micetreatedwithmethotrexatecontained

sig-nificantlymoreAICARthan those treatedwith saline (Table I,

Fig. 3). These results are consistent with the hypothesis that

low-dosemethotrexatetreatmentleads tofunctionalinhibition

ofAICARtransformylase.

Low-dose weekly methotrexate treatment increases

adeno-sineconcentrations in

inflammatory

exudates. We have previ-ouslyshown thattreatmentofcells inculture witheither meth-otrexate orAICARibonucleoside (acadesine),a

nonphosphor-ylated, cell-soluble precursorofAICAR, promotes release of

adenosine into thesupernate and thatadenosinerelease was

TableLMethotrexate (0.5mg/kgperwk) Treatment IncreasesIntracellular AICAR and Extracellular Adenosine

Exudate adenosine AICARconcentration concentration Condition (pmol/106splenocytes±SEM) (1M, ±SEM)

n=6 n=16

Control 26.5±10 0.57±0.09

Methotrexate

(0.5 mg/kg per wk) 72.4±16* 1. 1 1±0.1 9t

Miceweretreatedwith aweeklyintraperitoneal injectionof sterile salineormethotrexate for4wkduringwhich timeanairpouchwas

inducedonthebacks of thesemice,asdescribed. After4wkthe air poucheswereinjectedwith carrageenan(2%wt/vol),thesplenocyte lysates andinflammatoryexudateswerecollected andanalyzed by HPLC,asdescribed. *P<0.02vs.control, Student'st test. *P

<0.008vs.control,Student'sttest.

A.

B.

5

Time

(min)

Figure3. Intracellular concentration of AICARishigherin

spleno-cytesfrom animalstreatedwith methotrexate(0.5 mg/kgperwk).

Miceweretreated with methotrexate for4wkduring induction of

the airpouch.Afterthe animalswerekilled,and-the airpouch

exu-datewasharvested,thespleenswerecollected,and the cellswere

col-lected. The cellnumberwasadjusted, the cellswerelysed, and the

AICAR-concentration analyzed by reverse-phase, ion exchange HPLC and detectedatOD260.Shown isarepresentative

chromato-gramof six ofsplenocyteAICAR from(A)acontrol(22.6 pmol/106

splenocytes)and(B)amethotrexate-treatedmouse(87.3) pmol/106

splenocytes).

enhanced under conditions of "stress" (4). To determine whether low-dose weekly methotrexate treatment also

pro-motesadenosinereleaseinvivowequantitated the adenosine

concentration in inflammatory exudates taken from air pouches in saline- and methotrexate-treated (0.5 mg/kgper

wk) mice. We found that methotrexatetreatmentledtoa

signif-icantly higher adenosine concentration in the pouch exudate (Table I, Fig. 4). Thus, low-dose, intermittent methotrexate

therapypromotesadenosine releaseataninflamed site.

Adenosine mediates the antiinflammatory effect

ofmetho-trexateinthe airpouch. Todetermine whether the methotrex-ate-induced increase in adenosine concentration observed in pouch fluid exudateswasrelatedtothe antiinflammatory ef-fects ofmethotrexate,westudied the effect of adenosine

deami-nase onleukocyte accumulationinmethotrexate-treatedmice. Adenosine deaminase irreversibly deaminates extracellular adenosine to its inactive metabolite, inosine, and thereby renders it inactiveat adenosine receptors. Adenosine

deami-nase(0.15 IU/ml)did notsignificantly affect the number of

leukocytesrecovered from pouchesof saline-treated animals, but partially reversed the antiinflammatory effect of metho-trexate treatment(Fig. 5). Histologic examination oftheair

pouch tissuerevealed that,similarto its effectson leukocyte

(5)

A.

Animals

Control ADA DMPX MTX MTX+ MTX+

ADA DMPX

18 9 9 18 9 8

I5 20 25

B.

Time

(Minutes)

Figure 4. Theconcentrationofadenosine ishigherinexudates of

mice treated with methotrexate (0.5 mg/kgperwk). Micewere

treated with methotrexate for4 wkduringinductionof the airpouch. Aftertheanimalswerekilled theairpouchexudatewasharvested

and soluble adenosinewasextracted aftertreatmentof the exudates

with10%trichloroacetic acid.Theadenosineconcentration of

exu-dateextractswasthenanalyzedbyreverse-phase HPLC,asdescribed,

and detectedat

OD260.

Shown isarepresentative chromatogramof

16 of pouchexudateadenosinefrom(A)acontrol(0.39,uM)and

(B)amethotrexate-treatedmouse(1.3

,M).

filtration into thepouch tissue (38±2 vs. 106±14 cells/ 160X

field, methotrexatevs.control,P<0.01),andadenosine deam-inase reversed the antiinflammatory effect of methotrexate (88±3 cells/ 160x field,P<0.01 vs.methotrexatealone)

with-outaffecting leukocyte infiltrationincontrol mice(91±6 cells/ 160X field, P=NSvs.control,Fig. 6). Adenosine

deaminase-mediated reversal of the antiinflammatory effect of

methotrex-atetreatmentwasspecific since adenosine deaminase didnot

reverse the antiinflammatory effects of dexamethasone ( 1.5

mg/kg, injected intraperitoneally 1 h beforeinjection of the pouch with carrageenan, Fig. 7). Moreover, conversion of adenosinetoinactive metaboliteswasresponsible for reversal

of the antiinflammatory effect since adenosine deaminase whichwasinactivated by prior incubation with its tight-bind-ing, irreversible inhibitor deoxycoformycin (1 MM), did not affect the antiinflammatory capacity of methotrexate treat-ment(datanotshown). We conclude from these experiments that the increase inextracellular adenosineinthe methotrex-ate-treatedanimals isresponsible,atleastinpart,for the antiin-flammatory effects of methotrexate.

Theantiinflammatory effect of adenosineis mediatedvia

adenosineA2receptors.Thereareatleasttwomajor subtypes of adenosinereceptor,

A,

andA2, that canbedifferentiated, in

part,onthebasis ofagonist and antagonist specificity ( 17, 18). Since extracellular adenosineappearedtomediatethe antiin-flammatory effects of methotrexate, wesought to determine whether the antiinflammatory actions of adenosinewere me-diatedbyoccupancyofaspecific adenosinereceptor.We

there-Figure5. Adenosine deaminase (ADA,0.15 lU/ml)and DMPX

(mg/kg)reversetheantiinflammatoryeffects of methotrexate treat-ment(0.5 mg/kgperwk). Miceweretreated with saline(control)

ormethotrexate for 3to 4 wk beforeinflammationwasinduced in

the airpouch. Shownarethemeans(±SEM)of the numberof cells

that accumulatedinthepouchexudates. Methotrexatesignificantly

inhibited the accumulation ofleukocytesinthepouchexudate

(4.0±0.4vs. 1.5±0.1 X 106cells/pouch,controlvs.methotrexate,P

<3X 10-6). NeitherADA(4.8±0.5X 106cells/pouch)norDMPX

(4.8±0.4X 106cells/pouch)significantlyaffectedthenumber of cells

that accumulatedinthe controlairpouches,but both ADA(2.3±0.8

X 106cells/pouch) andDMPX(3.8±0.5X 106cells/pouch)

signifi-cantlyreversed theantiinflammatoryeffectof methotrexate(P

<0.006 and P<0.001 vs.methotrexatealone,respectively).

fore injected receptor-specific adenosinereceptorantagonists into the airpouch with the inflammatory stimulus. The adeno-sine

A,

receptor antagonist 8-cyclopentyl-dipropylxanthine (0.2 mg/kg) didnotaffect leukocyte accumulationintheair pouch in either control animals ormethotrexate-treated

ani-mals (Fig. 8). Because of its poorsolubility in aqueous

me-dium, higher concentrations of 8-cyclopentyl-dipropylxan-thine could not be utilized forstudy. In contrast, a specific

adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargyl-xanthine (DMPX), completely reversed the antiinflammatory effect of methotrexatetreatment(IC50= 0.2 mg/kg, P<0.01;

Fig. 9) without affecting accumulation of leukocytes in either control animals (Fig. 5) or dexamethasone-treated animals

(Fig. 7). We conclude from these experiments that the in-creased adenosine found at inflamed sites in methotrexate-treated animalsmediates theantiinflammatory effects of meth-otrexatebyengaging adenosine A2receptors.

Discussion

The results of the experiments reported here provide the first in vivo demonstration ofamolecular mechanism for the

antiphlo-gistic actions of methotrexate. Methotrexate, either acting di-rectlyorbypromoting the intracellular accumulation of

dihy-drofolatepolyglutamate, increases intracellularcontentof Al-CAR. The increase in intracellular AICAR concentration is associated with(and probably leads to)anincrease in

extracel-lular adenosine ininflammatory exudates. The increase in lo-cal adenosine concentrations at sites of inflammation sup-pressesinflammationviaoccupancyof adenosine A2receptors

oninflammatoryorconnective tissuecells.

Adenosine

5

OD260

4

3 I0

V-x

44

0

i'

(6)

I-A~~~~~~VC_

_~~~~~~~~~~~~~~~~4

ws

'owsL

I'-

!~~~~~~~~~~~~I

a

a''

t~~~~~~~~~~~~~~~~~o

'#E-1.'*i

Figure 6. Adenosine deaminase (ADA, 0.15 IU/ml)reversestheantiinflammatoryeffects of methotrexatetreatment(0.5 mg/kgperwk).Mice weretreated with saline (control) or methotrexate for 3wkbefore inflammationwasinduced in the airpouch. The air pouchesweredissected

outof theanimals,andfixed andprepared by standardhistopathological techniquesforphotomicroscopy.Thephotographic imageswere

digi-tizeddirectly using JAVA software and theimagesshownwereadjusted onlyforbrightnessandcontrast.Shownarerepresentativefields(of10 examined) fromonesection fromoneoftwoanimals studied under eachcondition.

Theobservation that low-dose weekly methotrexate ther-apy promotes the intracellular accumulation of AICAR in

splenocytes indicatesthat the"folate antagonism" oflow-dose weekly methotrexate is highlyspecific. Viainhibition of

dihy-drofolate reductase, high concentrations ofmethotrexate di-minishthe cellular contentofthe methyl donors required for

synthesis of purines and pyrimidines(6). In addition to the

synthesis of formyl-AICAR from AICAR (Fig. 1),reduced

fo-lateis required forthesynthesis ofa-N-formylglycinamide

ribo-nucleotide from

3-glycinamide

ribonucleotide, precursors of

AICAR. Thus, under the conditions studied, if

methotrexate

inhibited folate-dependent reactions nonspecifically, then we would have expected either no change or a decrease in cellular AICAR content. We found the opposite, a net increase in

cellu-lar AICARcontent, an observation thatindicates that

treat-mentwithlowconcentrations ofmethotrexateleadsto

selec-tive inhibition of AICAR

transformylase

without

inhibiting

the

enzymatic

steps

required

forthe

production

of AICAR. The

selective effect oflowconcentrations of methotrexateon pur-ine biosynthesis mostlikely follows from themetabolism of methotrexatetoits

polyglutamated derivatives

(for

reviewsee

reference 6).

Polyglutamated

methotrexate

directly

inhibits several steps in the

synthesis

and metabolism of purines and

pyrimidines (5, 7-9). In

particular, polyglutamated

metho-trexateis apotent direct inhibitor ofAICAR

transformylase

(7). Moreover, inhibition of

dihydrofolate

reductase

by

meth-otrexate (and methotrexate

polyglutamate)

leadstothe intra-cellular accumulation of

dihydrofolate polyglutamate,

a

known and potentinhibitor of AICAR

transformylase

(7-9).

(7)

polygluta-8

-C

4)

-o0

4

2-Control Dex Dex Dex

(1.5mg\kg) +ADA +DMPX

Figure 7. Neither adenosine deaminase(ADA,0.15lU/ml)nor

DMPX(mg/kg)reversetheantiinflammatoryeffects of

dexametha-sone treatment(1.5mg/kg).Airpoucheswereinducedonmice for 3wk. 1hbeforeinjection ofcarrageenaninto the airpouch,themice receivedanintraperitonealinjectionof dexamethasone(1.5mg/kg)

orsaline. The exudateswereharvested 4h afterinjectionof

carra-geenanand the cell numberwasquantitated.Dexamethasone signifi-cantly diminished the number of cells that accumulated in theair pouch and neitherADAnorDMPXsignificantlyaltered the number of cellsthataccumulated in the airpouchof animals treated with dexamethasone.

mates(7)arerequiredtoinhibit AICARtransformylase, it is morelikelythatdihydrofolate polyglutamates areresponsible

for the intracellular accumulation of AICAR. Nonetheless, treatment with methotrexate may leadtoinhibition ofAICAR

transformylase (andaccumulationofAICAR) bytwodifferent butcomplementarymechanisms.

Previousstudieshave demonstratedthatintracellular

accu-mulation of AICAR increases adenosine release from some, butnotall,cell types(11).Barankiewiczetal. have shownthat treatmentofB-lymphoblastswithhighconcentrationsof Al-CARibonucleoside diminishes adenosine uptakeand utiliza-tion,resultinginincreased releaseof adenosine into the

extra-cellular space,

particularly

underconditionsof ATP

degrada-tion(11,19).In contrast to T

lymphoblasts,

which release little

3

0

-2

0

Control DPCPX MTX MTX+

DPCPX

Figure 8. 8-Cyclopentyl-dipropylxanthine (DPCPX, 0.2 mg/kg) does

notreversetheantiinflammatory effect ofmethotrexate (0.5 mg/kg perwk). Miceweretreatedwith methotrexate for3 to 4 wkbefore

inflammationwasinduced in the air pouch. Shownare themeans

(±SEM) of thenumberofcells that accumulated in the pouch

exu-dates fromsix miceinthepresenceof the indicatedconcentrations

ofDPCPX.

0

0

-- 0 4)

5-0

.05 .1 .5

IDMPXI (mg\pouch)

Figure9. DMPX(mg/kg)reversestheantiinflammatoryeffect of methotrexate(0.5 mg/kgperwk). Miceweretreatedwith

metho-trexatefor 3to4wk beforeinflammationwasinduced in the air pouch. Shownarethemeans(±SEM) of the number of cells that

ac-cumulated in thepouch exudates from three mice in thepresenceof theindicated concentrations of DMPX. Analysis of variance

demon-stratesthat the numberof cells in the pouch exudate varies signifi-cantly with thedoseof DMPX (P<0.01).

adenosine under any condition, B lymphoblasts possess in-creased AMP-S'-nucleotidase activity (adenosine formation) and relatively littleadenosine kinase or adenosinedeaminase activity (adenosineutilization[ 1]). Thus, Barankiewiczetal. postulated that, since AICARibonucleoside does not affect adenosine production or transport, intracellular accumula-tions of AICAR must inhibit adenosine kinase or adenosine deaminaseactivityinordertopromotetheincreasein extracel-lularadenosine observed (11, 19). AICARibonucleoside may also lead to anincreaseinadenosine releaseatsites of "stress," such asreperfusion after ischemic insult totheheart, andthe increased extracellular adenosine that accumulates in ischemic tissueprotectstheaffected tissue from leukocyte-mediated in-jury (10). Ourdata suggest that treatment invivo with low-dose methotrexate similarly increases intracellular AICAR contentand,moreimportantly, promotes adenosine releaseat inflamed sites.

Methotrexateinduced increasedadenosine concentrations ininflammatory exudates and was a potent antiinflammatory agentinthe air pouch model. To prove that the effects of meth-otrexate on purine metabolism and inflammation were causally related, we used two different approaches: elimination ofextracellular adenosine by adenosine deaminase and antago-nism of adenosine at its receptors with a specific antagonist (DMPX). Bothof these experimental maneuvers reversed the

antiinflammatory effect of methotrexate but did not reverse the antiinflammatory effect of dexamethasone in this same model. Dexamethasone is a potent agonist at glucocorticoid receptors thatdiminishes leukocyte accumulation at inflamma-tory sitesbyamechanism that is not related to purine metabo-lism(forreview see reference 20). Thus, our observation that bothspecific eliminationandantagonism of adenosine reverse theantiinflammatoryeffectsofmethotrexateis strong evidence that adenosine mediates the antiphlogistic effect of metho-trexate.

Wehavepreviously observed that methotrexate treatment, in vitro, promotes an increase in adenosine release at the ex-pense ofhypoxanthine and inosine release (4). In this study we were unable to detect inosine in most samples and the HPLC

(8)

othercompoundspresent inthesecomplexbiologic fluids.

Nev-ertheless, the adenosine concentration present in inflamma-tory exudates ofmethotrexate-treated animals ( 1. 1 ,uM) is more thansufficientto account for thediminished inflamma-tionobserved; maximal inhibition ofstimulated neutrophil ad-hesion and generation of superoxide anion and H202 is achieved with adenosine concentrations greater than or equal to 1 juM ( 14, 21). Indeed, the concentration of adenosine foundin exudatesfromcontrolanimalswas less than the

con-centrationofadenosine foundintransudatesfrom "stressed"

isolated rabbithearts (during hypoxia, 1225±300 nM;

refer-ence22). Although the adenosine concentration measured in the inflammatory exudate probably reflects the metabolic

changes in methotrexate-treated animals and is sufficient to

inhibittheproduction of toxicoxygenmetabolitesby the cells present inthe inflammatoryexudate, it islikely that the in-crease in extracellular adenosine responsible for diminished inflammation isthatwhichoccursinthesurrounding tissues,a lessreadily accessible site forsampling.

There are atleast twomajorsubclassesof adenosine recep-torthat can bedistinguished on pharmacologic grounds,

A,

and A2 ( 17, 18). Adenosine

A,

receptors arerelatively high-af-finityreceptorsthat arelinked to pertussis toxin-inhibitedG

proteins (23-33). Adenosine

A,

receptors have been demon-strated on neutrophils and macrophages (but notperipheral

blood mononuclear cells) where theymediate,whenoccupied, enhanced chemotaxis and phagocytosis of

immunoglobulin-coated particles (34-38). AdenosineA2 receptors are low-af-finityreceptors linked toGassignal transduction proteinsin many cell types.AdenosineA2 receptors arepresent on

neutro-phils,monocytes,lymphocytes,andbasophils and,when

occu-pied, generally suppress the

inflammatory

or immune func-tions ofthese cells

(for

review see references 39-41).

Using

relatively selective antagonistswe found that the

antiinflam-matory effects of adenosine in methotrexate-treated animals

weremediatedbyoccupancyof adenosineA2 receptors,results

that wereidentical tothoseobtained byAsakoetal.(12).In contrast,Schrieretal.(42)observed,

utilizing

receptor-specific agonists,that occupancyof adenosine

A,

receptorsrather than

A2 receptors isantiinflammatoryin a rat modelof inflamma-tion.Thediscrepancymay be due tospecies differencesin

ago-nistsensitivityoradenosinereceptorexpression. Alternatively, the apparent difference in receptor

specificity

for the

antiin-flammatory

effects of adenosine results fromadifference inthe

distribution, lipid

solubility,

orother

pharmacologic properties

oftheadenosine

receptor-specific agonists

studied.

Wefirst suggestedthatadenosinemightbe anendogenous

antiinflammatory

agent whenwe observed thatadenosine

in-hibitsthegeneration of toxicoxygenmetabolites by stimulated neutrophils ( 14). In subsequent studieswe have shownthat

adenosine,

both addedexogenouslyorreleasedendogenously, diminishesendothelial cell

injury

mediated by stimulated

neu-trophils (43). The

cytoprotective

effects of adenosine result

from inhibition ofthegeneration of toxicoxygen metabolites

andinhibition ofthestimulatedadhesionofneutrophilstothe

endothelium (43).Inthe modelunderstudy,the apparent ef-fect of adenosine wastodiminish extravasation of leukocytes into an

inflammatory

exudate. There may be an additional beneficial effectof methotrexate therapy for the

synovial

tis-suesof

patients

treatedwith

methotrexate;

theconcentration of adenosinepresent in the

inflammatory

pouchexudatesismore than sufficient to inhibit generation of toxic oxygen

metabo-lites by stimulated leukocytes. Thus, treatment with metho-trexatemay bothdiminish the number ofleukocytes that accu-mulate in aninflammatoryexudate and inhibitthe destructive capacity of thoseleukocytesthatdo arrive at theinflamed site. Inthe model studied here,inflammation was acute and was characterized by the accumulation of a neutrophilic infiltrate in both the air pouch and the surrounding tissues. Although it

islikelythat theadenosine released is acting directlyon neutro-phil adenosine receptors, it is also possible that adenosine in-hibits the generation of cytokines or chemoattractants required for accumulation of the inflammatory exudate. Indeed,

adeno-sine,

probably actingat an A2 receptor, inhibitssynthesis of

cytokines (TNFa) and other inflammatory proteins (comple-ment C2) by macrophages (44, 45). Moreover, adenosine,

act-ing at its receptor, inhibits lymphocyte proliferation and

in-duces suppressoractivityin culturedlymphocytes(39).Thus, theantiinflammatory effects ofmethotrexate(actingvia

adeno-sine)aremoregeneral thanthosestudiedinthis model ofacute

inflammation.Indeed,itis likelythat theeffectsof methotrex-ate, acting via adenosine, onlymphocyteormonocyte function play a greater roleindiminishingthechronicinflammation of rheumatoid arthritisthan the effects on acute inflammation observed inthis model.

We have demonstrated a novelbiochemicalmechanism of

action of methotrexate. Low-doseweekly methotrexate ther-apy leads tointracellular accumulation of AICAR, which pro-motesincreasedadenosinerelease (and/ordiminished

adeno-sine uptake)atsitesofinflammation. This increasein extracel-lular adenosine diminishes both the accumulation and

function of leukocytesininflamed sites.Thesefindingssuggest several novel approaches to the development ofnew agents that inhibitinflammation by increasing adenosinerelease: de-velopmentofdirectinhibitors ofAICAR transformylase, inhib-itorsofadenosine deaminaseandadenosinekinase,and

adeno-sine uptake inhibitors.

Acknowledgments

Wewould liketo thankCampbell Bunce for his excellenttechnical assistance with this project. Wewouldalsoliketothank Dr. Gerald Weissmann for his discussions and advice and for reviewing this

manu-script.

This workwasmadepossible bygrantsfrom the Arthritis

Founda-tion,New Yorkchapter, The AmericanHeartAssociation,NewYork affiliate,and theNational Institutes of Health (AR-1 1949, HL-19721 ) and with assistance from the General Clinical Research Center (MOIRR00096) and the Kaplan Comprehensive Cancer Center (CA16087).

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