Proc. Nail. Acad. Sci. USAVol. 88, pp. 2441-2445, March 1991Physiology/Pharmacology

Methotrexate inhibits neutrophil function by stimulating adenosinerelease from connective tissue cells

(inflammation/endothelium/fibroblast/adenosine receptor/purine receptor)

BRUCE N. CRONSTEIN*t, MARK A. EBERLE*, HARRY E. GRUBERf, AND RICHARD I. LEVIN**Department of Medicine, Divisions of Rheumatology and Cardiology, New York University Medical Center, 550 First Avenue, New York, NY 10016; andtGensia Pharmaceuticals, Inc., 11025 Roselle Street, San Diego, CA 92121-1207

Communicated by Robert M. Berne, December 17, 1990 (received for review October 1, 1990)

ABSTRACT Although commonly used to control a variety ofinflammatory diseases, the mechn of action of a low dose ofmethotrexate remains a mystery. Methotrexate accumulates in-tracellularly where it may interfere with purine metabolism.Therefore, we determined whether a 48-hr pretreatment withmethotrexate affected adenosine release from [14Cladenine-labeled human fibroblasts and umbilical vein endothelial cells.Methotrexate n y increased aden e release by fibro-blasts from 4 ± 1% to 31 + 6% of total purinereleased (EC50,1 nM) and by endothelial cells from 24 ± 4% to 42 ± 7%.Methotrexate-enhanced adenosine release from fibroblasts wasfurther increased to 51 ± 4% (EC5u, 6 nM) and from endothelialcells was increased to 58 ± 5% of total purine released byexposure to stimulated (fMet-Leu-Phe at 0.1 ,uM) neutrophils.The effect of methotrexate on adenosine release was not due tocytotoxicity since cells treated with maximal concentrations ofmethotrexate took up [14C~adenine and released 14C-labeled pu-rine (a measure of cell injury) in a manner identical to controlcells. Methotrexate treatment of fibroblasts dramatically inhib-ited adherence to fibroblasts by both mulated neutrophils(IC50, 9 nM) and stimulated neutrophils (IC5*, 13 nM). Meth-otrexate treatment inhibited neutrophil adherence by enhingadenosine release from fibroblasts since digestion of extracellularadenosine by added adenosine deaminase completely abrogatedthe effect ofmethotrexate on neutrophil adherence without, itself,affecting adherence. One hypothesis that explains the effect ofmethotrexate on adenosine release is that, by inhibiton of 5-ami-noimidazole-4-carboxamideribonnceotide (AICAR) transformy-lase, methotrexate induces the accumulation of AICAR, thenucleoside precursor of which (5- _ a ixaderibonucleoside referred to hereafter as acade) has previouslybeen shown to cause adenosine release from ischmic cardiactissue. We found that acadesine also promotes adennreleasefrom and inhibits neutrophil adherence to connective tissue cells.The observation that the andinflammatory actions of methotrex-ate are due to the capacity of methotrexate to induce adenosinerelease may form the basis for the development of an additionalclass of antiinflammatory drugs.

First reported to be useful in the treatment of rheumatoidarthritis (1), methotrexate is now widely used to treat avariety of inflammatory diseases, most notably rheumatoidarthritis (for review, see ref. 2). The mechanism by whichmethotrexate modulates inflammation remains, however, amystery. The antineoplastic (antiproliferative) effects ofmethotrexate are due to inhibition of dihydrofolate reductasewith resulting inhibition of purine and pyrimidine synthesis.However, folate depletion probably does not account for thetherapeutic effects of methotrexate in inflammatory disease.(i) At the doses of methotrexate administered, leukopenia

due to inhibition of DNA synthesis, does not occur (2), afinding not consistent with the hypothesis that methotrexateis antiinflammatory due to inhibition by methotrexate ofdihydrofolate reductase. (ii) In two (3, 4) of three (5) pub-lished trials neither folate supplementation nor administra-tion of reduced folate (folinic acid) reversed the therapeuticeffects of this agent (although both agents reduced toxicity),direct evidence against inhibition of dihydrofolate reductase.Recent observations have suggested a different mechanism

to explain the antiinflammatory characteristics of methotrex-ate. Methotrexate and its polyglutamated analogues are verypotent inhibitors of 5-aminoimidazole-4-carboxamide ribo-nucleotide (AICAR) transformylase (6-8), an enzyme re-quired for de novo purine synthesis. In a study of caninemyocardial injury Gruber et al. (9) found that administrationof 5-aminoimidazole-4-carboxamide ribonucleoside(acadesine), the nucleoside precursor of AICAR, increasesadenosine release from, diminishes neutrophil accumulationin, and increases collateral flow into ischemic myocardium.Thus, methotrexate, by inhibiting AICAR transformylase,may increase the intracellar concentration of its substrate,AICAR, which would lead, in turn, to increased release ofadenosine, a potent antiinflammatory autocoid, at sites ofinflammation.We report that methotrexate, at pharmacologically rele-

vant doses, induces adenosine release from human dermalfibroblasts and umbilical vein endothelial cells. The increaseis most marked in the presence of neutrophils stimulated withthe chemoattractant fMet-Leu-Phe (0.1 uM). In turn, thereleased adenosine inhibited neutrophil adhesion. Acadesinealtered, in a manner similar to methotrexate, both adenosinerelease and neutrophil adherence.

MATERIALS AND METHODSMaterials. Tissue culture media [Dulbecco's modified Ea-

gle's medium (DMEM) and medium 199] were obtained fromGIBCO. [14C]Adenine was purchased from NEN/DuPontand DEAE-cellulose thin layer chromatography plates wereobtained from Eastman Kodak. The scintillant Filtron-X wassupplied by National Diagnostics (Manville, NJ). Lym-phoprep (Hypaque/Ficoll) was obtained from Nyegaard(Oslo). Trioctylamine was purchased from Aldrich Chemical(Orangeburg, NY) and Freon-113 was obtained from Math-eson. Methotrexate, fMet-Leu-Phe, AICAR, and all otherreagents were obtained from Sigma. All reagents were of thehighest quality available.

Endothelial Cell Cultures. Endothelial cells were culturedand grown as described by Jaffe et al. (10). Briefly, segmentsof freshly obtained human umbilical veins were treated with

Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide ribonu-cleotide; ANOVA, analysis of variance; PMN, polymorphonuclearleukocyte.tTo whom reprint requests should be addressed.

2441

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

1

2442 Physiology/Pharmacology: Cronstein et al.

collagenase (0.1%), and the endothelial cells were collectedand grown to confluence in gelatin-coated flasks containingmedium 199/20% (vol/vol) fetal bovine serum at 370C in a 5%C02/95% air atmosphere. The endothelial cells were thenpassed as necessary and grown to confluence in gelatin-coated 96-well tissue culture plates in medium 199/20% fetalbovine serum. All cells were used in the third passage.Human Dermal Fibroblasts. Normal human fibroblasts

(GM08389) were obtained from the National Institute ofGeneral Medical Sciences Human Genetic Mutant Cell Re-pository (Camden, NJ) and cell line HM was a generous giftof Frank Martiniuk (New York Univ. Medical Center, NewYork). The cells were grown to confluence in DMEM/20ofetal bovine serum and passed as necessary. All cells wereused during passages 5-15. Nearly identical results wereobtained when either cell line was used.

Incubation of Cell Cultures with Methotrexate. Fibroblastsor preconfluent cultures of endothelial cells were washedthree times with medium and then incubated for 48 hr at 370Cin a 5% C02/95% air atmosphere in fresh medium containingmethotrexate at various concentrations. At the end of theincubation, cells were washed three times with fresh medium.When examined microscopically, there was no difference incellular morphology between wells treated with methotrexateand those treated with medium alone.

Isolation of Neutrophils. Human neutrophils were isolatedfrom whole blood after centrifugation through Hypaque/Ficoll gradients, sedimentation through dextran, 6% (wt/vol), and hypotonic lysis of erythrocytes. This procedureallowed study of populations that were 98 ± 2% neutrophilswith few contaminating erythrocytes or platelets. Neutro-phils were suspended in a Hepes-buffered saline solutionconsisting of 150mM Na+, 5 mM K+, 1.3 mM Ca2+, 1.2 mMMg2+, 155 mM Cl-, and 10 mM Hepes (pH 7.45) (11).

Labeling of Connective Tissue Cells with [14C]Adenine.After washing the cell cultures with fresh medium, cells wereincubated in Hepes-buffered saline containing [14C]adenine(25 ,Ci/ml; 1 Ci = 37 GBq) in a final volume of 250 1.d per wellfor 3 hr at 37°C in a 5% C02/95% air atmosphere. At the endof this incubation wells were again washed three times withfresh medium before use in the experiments.

Assay for 14C-Labeled Purine Release. To study the effectsof preincubation with methotrexate or incubation withacadesine on 14C-labeled purine release, endothelial cells orfibroblasts were incubated at 37°C in a 5% C02/95% airatmosphere in the presence or absence of 1.25 x 106 neu-trophils per ml with or without fMet-Leu-Phe (0.1 ,uM) andacadesine in a final volume of 200 ,l. This concentration ofneutrophils is 12.5% of that which we have found (12) toinjure endothelial cells. Because in preliminary experimentspreincubation of connective tissue cells with acadesine mark-edly reduced [14C]adenine uptake, acadesine was addedduring final incubations. In some experiments adenosinedeaminase (0.125 international unit/ml), which had beendialyzed for 3-4 hr at 4°C against phosphate-buffered saline,was added to tissue culture wells. At the end of the incuba-tion, samples of supernatant medium were collected, treatedwith 10o (vol/vol) trichloroacetic acid, and extracted with amixture of Freon/trioctylamine, 31:9 (vol/vol), before cen-trifugation at 10,000 x g. The aqueous layer was thencollected and frozen (-20°C) until assayed for purine con-tent. In some experiments the remaining supernatant mediumwas removed, the remaining cells were lysed by overnightincubation with water, and the lysates were collected forquantitation of radioactivity. All experimental conditionswere performed in duplicate with <5% variation betweenreplicates. In preliminary experiments we found that additionof the chemoattractant fMet-Leu-Phe (0.1 ,gM) in the absenceof neutrophils did not affect adenosine release from connec-tive tissue cells regardless of whether or not they were treated

with methotrexate (100 ,M) or acadesine (100 AtM, data notshown).

Separation and Quantitation of 14C-Labeled Purines. A50-Al portion of each sample was spotted onto DEAE-cellulose thin layer chromatography sheets. Separation wasthen carried out by chromatography in water/isobutanol/methanol/ammonium hydroxide in a ratio of 30:10:1:10 (vol/vol). After drying, the labeled purines and their carriercompounds (AMP, hypoxanthine, inosine, and adenosine,each at 500 mg/dl) were visualized under ultraviolet, cut out,and placed in scintillation vials. Radioactivity was quanti-tated in a Packard scintillation counter to an error of <0.2%(13).

Neutrophil Adherence to Endothelial Cell or FibroblastMonolayers. After removal of medium for quantitation ofpurines, the monolayers and adherent neutrophils were fixedby addition of formaldehyde to 3.7% (vol/vol). Monolayersand their adherent neutrophils were then washed three timesto remove nonadherent neutrophils and then stained withWeigert's hematoxylin. Adherent neutrophils were easilydifferentiated from underlying fibroblasts and endothelialcells on the basis of size and nuclear-staining characteristics(12). The number of neutrophils in three x 100 fields per wellwas quantified and the mean was calculated. Counts wereperformed on two replicate wells, which differed by <5%.

Statistical Analysis. All results represent the mean (±SEM), unless otherwise stated. The significance of the effectsof agents and neutrophils and their interactions on adenosinerelease from and neutrophil adhesion to connective tissuecells was determined by the appropriate level of analysis ofvariance (ANOVA).

RESULTSTreatment of fibroblasts with methotrexate caused a dose-dependent increase in release of adenosine from 4 ± 1% to amaximum of 31 ± 6% of the total purine released (Fig. 1).Methotrexate was a surprisingly potent promoter of adeno-sine release with an EC50 of 1 nM. When fibroblasts weretreated with methotrexate and then incubated with neutro-phils, there was a nearly identical dose-dependent increase inrelease of adenosine from 5 ± 2% to 23 ± 5% of total purinereleased (Fig. 1, P < 0.01). However, treatment of fibroblastswith methotrexate followed by incubation with neutrophilsstimulated with fMet-Leu-Phe (0.1 ,uM) markedly enhanced

6*MEDIUM, 60- *PMNs

Z vPMNs+fMLP I

, 201 /w

O

40.0

.01 1 100o 10o,000IMETHOTREXATEI (nM)

FIG. 1. Normal human fibroblasts were incubated with meth-otrexate at the indicated concentrations for 48 hr, washed, andlabeled with [14C]adenine. After washing, the fibroblasts were incu-bated in the presence of medium alone, neutrophils, or stimulated(fMet-Leu-Phe at 0.1 ~M) neutrophils. After 2 hr the supernatant wascollected and anlalyzed by thin layer chromatography. Data are themean (± SEM) offourexperiments performed in duplicate. Two-wayANOVA indicates that the percentage of purine released as adeno-sine varies with dose of methotrexate (P < 0.0001) and with thepresence of stimulated neutrophils (P < 0.003). PMN, polymorpho-nuclear leukocyte; fMLP, fMet-Leu-Phe.

Proc. Natl. Acad. Sci. USA 88 (1991)

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

1

Proc. Natl. Acad. Sci. USA 88 (1991) 2443

adenosine release from 4 ± 1% to 51 ± 4% of total purinereleased (Fig. 1). The quantity of adenosine detected corre-sponds to -400 nM, a concentration well within the effectivephysiologic range of activity for adenosine (14). Althoughstimulated neutrophils induced a greater shift in purine re-lease from methotrexate-treated fibroblasts, there was nosignificant change in the concentration of methotrexate re-quired to shift purine release (EC50, 6 nM). As shown (Fig. 1),neither unstimulated nor stimulated neutrophils altered basaladenosine release from fibroblasts. Moreover, methotrexateor neutrophils or their combination did not affect total purinerelease from fibroblasts (7 ± 2, 7 ± 1, 7 ± 1%, respectively,of total purine pool released vs. 7 ± 1% from control cells).As compared to fibroblasts, endothelial cells, under con-

trol conditions, released a greater percentage of their purineas adenosine (24 ± 4% vs. 4 ± 1%, P < 0.01, n = 4). Whenendothelial cells were treated with methotrexate (100 ALM),there was an increase in the percentage ofadenosine releasedto 42 ± 7% of total purine released (P < 0.01, n = 4). As withfibroblasts, unstimulated neutrophils did not affect the per-centage of purine released as adenosine from control ormethotrexate-treated endothelial cells (20 ± 4% and 39 ± 8%,respectively, n = 4). Stimulated neutrophils also did notaffect adenosine release (18 ± 3% of total purine released, n= 4) from endothelial cells but increased adenosine releasefrom methotrexate-treated cells to 58 ± 5% of total purinereleased (P < 0.003 vs. control, n = 4).To examine the hypothesis that inhibition ofAICAR trans-

formylase by methotrexate is responsible for the release ofadenosine, we determined whether acadesine, the nucleosideprecursor of AICAR, also increases adenosine release.Acadesine (100 ,uM) induced fibroblasts to release a greaterpercentage of purine as adenosine (from 3 ± 1% to 19 ± 5%of total purine released, P < 0.01, n = 4; Fig. 2). However,acadesine was far less potent than methotrexate. As withmethotrexate, stimulated but not unstimulated neutrophilsalso enhanced the effect of acadesine (100 ,uM) on adenosinerelease (53 ± 7% and 22 ± 13% of total purine released,respectively, P < 0.001, n = 4; Fig. 2).Acadesine (100 ,uM) treatment increased the percentage of

purine released as adenosine from endothelial cells from 24 ±4% to 39 ± 6% (n = 4, P < 0.01). Stimulated neutrophilsfurther increased the percentage of purine released as aden-osine to 62 ± 9o of total purine released (P < 0.001, n = 4).To determine whether methotrexate or acadesine was toxic

to endothelial cells or fibroblasts, we compared both uptakeand release of purine by treated cells. Cells treated withmethotrexate (100 ,uM) took up as much [14C]adenine ascontrol cells (101 ± 7% of control uptake, n = 4) and did not

z EMEDIUMg 60 *PMNsz vPMNs+fMLP

40-

"a 40l20

- 20 > = g

z

0 ,0M .01 1 100

[ACADESINEI (pM)

FIG. 2. After labeling with [14C]adenine, normal human fibro-blasts were incubated with acadesine at the indicated concentrationsin the presence or absence of neutrophils or stimulated neutrophils(fMet-Leu-Phe at 0.1 jM). After 2 hr the supernatant was collectedand analyzed by thin layer chromatography. Data are the mean (±SEM) of two to four experiments performed in duplicate. fMLP,fMet-Leu-Phe.

release any greater percentage of the labeled purine poolduring these experiments (7 ± 1 vs. 7 ± 2% of total labelreleased from control and methotrexate-treated cells, n = 4).Similarly, fibroblasts treated with acadesine (100 ,uM) orexposed to stimulated neutrophils plus methotrexate (100jM) also released no more of their labeled purine pool thancontrol cells (7 ± 1 and 7 ± 1% of total label released).Moreover, no change in cell morphology was detectedwhether cells were treated with methotrexate, acadesine,stimulated neutrophils, or their combination. These resultsindicate that the increase in adenosine release from meth-otrexate-treated fibroblasts was not due to toxicity of meth-otrexate for fibroblasts. Similar results were obtained usingendothdlial cells (data not shown).We next determined whether the release of adenosine from

connective tissue cells treated with methotrexate was rele-vant to the antiinflammatory activity of methotrexate. Wehave previously demonstrated that adenosine, presumablyacting at adenosine A2 receptors on neutrophils, inhibitsneutrophil adherence to endothelial cells (12). Therefore, wedetermined whether adherence by unstimulated and stimu-lated neutrophils to connective tissue cells was affected bytreatment of the connective tissue cells with methotrexate.Treatment ofconnective tissue cells with methotrexate mark-edly inhibited adherence ofboth unstimulated and stimulatedneutrophils to fibroblasts (EC50, 9 nM and 13 nM, -respec-tively, P < 0.001; Fig. 3). Similarly, acadesine also inhibitedunstimulated and stimulated neutrophil adherence to fibro-blasts (EC50, 13 ,uM and 18 ,uM, respectively, P < 0.001; Fig.4) at concentrations similar to those required for promotionof adenosine release. Methotrexate and acadesine inhibitedneutrophil adherence to endothelial cells in a similar fashion(data not shown).To determine whether the diminished adherence of neu-

trophils was related to the increase in adenosine release fromconnective tissue cells, we determined whether addition ofadenosine deaminase, which metabolizes adenosine to inos-ine, reverses the effect of methotrexate treatment on neu-trophil adherence. Adenosine deaminase alone did not affectadherence ofeither unstimulated or stimulated neutrophils toeither fibroblasts or endothelial cells (Figs. 5 and 6). Incontrast, and as described above, treatment of connectivetissue cells with methotrexate (100 uM) markedly inhibitedneutrophil adherence to connective tissue cells and thisinhibition was completely abolished by the addition of aden-osine deaminase. Similarly, adenosine deaminase completelyreversed the effect of acadesine on adherence to endothelial

700

600 O*STIMULATED PMNZ \ *UNSTIMULATED PMNz 500

=

300I\xk

200- F

100.01 1 100 10,000

[METHOTREXATE] (nM)

FIG. 3. Normal human fibroblasts were treated with methotrex-ate as indicated for 48 hr and then washed extensively. The fibro-blasts were then incubated with neutrophils in the presence (stimu-lated) or absence of fMet-Leu-Phe (0.1 jiM) for 2 hr. After fixationand washing the monolayers were stained and the neutrophils werecounted in three fields per well. Data are the mean (+ SEM) of fourexperiments performed in duplicate. Two-way ANOVA demon-strates that neutrophil adherence varied significantly with the dose ofmethotrexate (P < 0.0001) and with stimulation (P < 0.0001).

Physiology/Pharmacology: Cronstein et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

1

2444 Physiology/Pharmacology: Cronstein et al.

800 -

z2 600-

zw1= 400-I

200

*PMN

*STIMULATED PMN

.01 1[ACADESINE1 1pM)

100

FIG. 4. Normal human fibroblasts were incubated in the presenceof acadesine at the indicated concentrations with stimulated (fMet-Leu-Phe at 0.1 ,uM) or unstimulated neutrophils. After fixation andwashing the monolayers were stained and the neutrophils werecounted in three fields per well. Data are the mean (± SEM) of fourexperiments performed in duplicate. Two-way ANOVA demon-strates that neutrophil adherence varied significantly with the dose ofacadesine (P < 0.01) and with stimulation (P < 0.001).

cells (Fig. 7). Nearly identical results were found withfibroblasts (data not shown).

DISCUSSIONThe results of the experiments reported herein demonstratean antiinflammatory action of methotrexate: increased aden-osine release. Treatment of both fibroblasts and endothelialcells with methotrexate at pharmacologically relevant dosesincreases adenosine release from these cells, an effect that iseven more marked in the presence of stimulated neutrophils.The adenosine released from methotrexate-treated connec-tive tissue cells, in turn, inhibits adhesion of neutrophils toconnective tissue cells, a critical initial step for infiltration orinjury by neutrophils of connective tissue cells. These ob-servations suggest that this is a mechanism by which meth-otrexate diminishes inflammation in vivo.We have shown herein that the concentration of adenosine

released from methotrexate-treated cells that remains extra-cellar (equivalent to a final concentration of 400-500 nM)inhibits neutrophil function but the antiinflammatory effectsof extracellular adenosine are not confined to neutrophilfunction. Previous studies have demonstrated that adenosineoccupies adenosine A2 receptors on monocyte-macrophages(15-19) and lymphocytes (20-24), cells that play a major rolein the pathogenesis of chronic inflammation. In general,occupancy of adenosine receptors on monocytes and lym-

700

500

300 -

ECONTROL PMNOSTIMULATED PMN

IiIF~

+ADA +ADA+METHOTREXATE

FIG. 5. Normal human fibroblasts were incubated with meth-otrexate (100 ,uM) for 48 hr, washed extensively, and then incubatedfor 2 hr with unstimulated or stimulated (fMet-Leu-Phe at 0.1 ,uM)neutrophils in the presence or absence of adenosine deaminase(ADA, 0.125 international unit/ml). After fixation and washing themonolayers were stained and the neutrophils were counted in threefields per well. Data are the mean (± SEM) of three experimentsperformed in duplicate. Two way ANOVA demonstrates that neu-trophil adherence varied significantly with the presence of meth-otrexate (P < 0.01) and that adenosine deaminase induced a signif-icant increase in adherence of both stimulated and unstimulatedneutrophils to methotrexate-treated fibroblasts (P < 0.05).

O STIMULATED* UNSTIMULATED

700-

zSO0.

500-zw

o300-

100+ADA +ADA

METHOTREXATE (10O0pM)

FIG. 6. Preconfluent monolayers of human umbilical vein endo-thelial cells were incubated in the presence or absence of methotrex-ate (100 ,uM) for 48 hr and washed extensively. The monolayers werethen incubated with neutrophils in the presence (stimulated) andabsence (unstimulated) of fMet-Leu-Phe (0.1 AM) and adenosinedeaminase (ADA, 0.125 international unit/ml) for 2 hr. After fixationand washing the monolayers were stained and the neutrophils werecounted in three fields per well. Data are the mean (± SEM) of threeexperiments performed in duplicate. Two-way ANOVA demon-strates that neutrophil adherence varied significantly with the pres-ence of methotrexate (P < 0.01) and that adenosine deaminaseinduced a significant change in adherence of both stimulated andunstimulated neutrophils to methotrexate-treated endothelial cells (P< 0.05).

phocytes inhibits their ability to induce tissue damage. It hasbeen demonstrated (12, 14, 25-30) that adenosine occupiesspecific A2 receptors on the surface of neutrophils to inhibitthe generation oftoxic oxygen metabolites such as 0°-, H202,and adherence to endothelium. Thus, for example, increasedrelease of adenosine from synovial cells could dampen boththe acute and chronic inflammation present in the joints ofpatients with rheumatoid arthritis.Although the functional effects of adenosine are not re-

stricted to a single type of inflammatory cell, we wouldpredict that the effects of the adenosine released from meth-otrexate-treated cells would be restricted to the areas mostdirectly infiltrated by inflammatory cells. Adenosine is veryshort-lived in whole blood where it is rapidly taken up byerythrocytes or metabolized by adenosine deaminase (31).Moreover, at sites of tissue necrosis intracellular enzymessuch as adenosine deaminase are released that can metabo-lize adenosine to the functionally inactive purine ribosideinosine.

O STIMULATED* UNSTIMULATED

700-z

zI- 500-

w

<300-

100 l+ADA +ADA

ACADESINE (1 OOpM)

FIG. 7. Normal human endothelial cells were incubated for 2 hrwith unstimulated or stimulated (fMet-Leu-Phe at 0.1 ,tM) neutro-phils in the presence or absence of acadesine (100 ,uM) and adenosinedeaminase (ADA, 0.125 international unit/ml). After fixation andwashing, the monolayers were stained and the neutrophils werecounted in three fields per well. Data are the mean (± SEM) of threeexperiments performed in duplicate. Two-way ANOVA demon-strates that neutrophil adherence varied significantly with the pres-ence of acadesine (P < 0.01) and that adenosine deaminase induceda significant change in adherence ofboth stimulated and unstimulatedneutrophils to acadesine-treated endothelial cells (P < 0.03).

Proc. Natl. Acad. Sci. USA 88 (1991)

z

z

Ul.,rCi

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

1

Proc. Natl. Acad. Sci. USA 88 (1991) 2445

The molecular mechanism by which methotrexate andacadesine promote adenosine release from connective tissuecells remains unknown; however, our data suggests onepossible pathway by which methotrexate may induce in-creased extracellular adenosine concentrations. Methotrex-ate and its polyglutamated derivatives are potent inhibitors ofAICAR transformylase (8). Inhibition ofAICAR transformy-lase could cause accumulation of its substrate, AICAR andacadesine, a compound previously shown to promote aden-osine release by an unknown mechanism (9). Our data areconsistent with this hypothesis. Thus, the parallel effects ofacadesine and methotrexate on adenosine release and neu-trophil adherence suggest that the effect of methotrexate onadenosine release is due to inhibition by methotrexate ofAICAR transformylase with accumulation of AICAR.An alternative pathway by which methotrexate could mod-

ulate inflammatory cell interactions is suggested by studies ofNesher and Moore (32), who found that methionine reversesthe effects of methotrexate on in vitro immunoglobulin pro-duction and hypothesized that uptake of methionine leads toregeneration of S-adenosylmethionine, a methyl donor thatmay be depleted in methotrexate-treated cells due to inhibi-tion of dihydrofolate reductase. Our results do not excludethe hypothesis of Nesher and Moore (32) but suggest analternative interpretation of their studies. In methotrexate-treated cells exogenous methionine may degrade to homo-cysteine that could recondense with adenosine thereby"trapping" excess adenosine intracellularly as S-adenosyl-homocysteine. If increased adenosine release contributes tothe antiinflammatory activity of methotrexate, then intracel-lular "trapping" of adenosine would reverse the effects ofmethotrexate treatment. Alternatively, methotrexate mayinhibit the function of various cell types by different mech-anisms.Our results show that, in contrast to untreated connective

tissue cells, cells treated with either methotrexate oracadesine release more adenosine after exposure to stimu-lated neutrophils. The mechanism by which stimulated neu-trophils enhance adenosine release only from cells treatedwith methotrexate or acadesine is unknown. However, it iswell known that intracellular stores of reduced gluthathioneprotect connective tissues from oxidant injury. Stimulatedneutrophils release a variety of toxic oxygen metabolites thatrequire detoxification and, ultimately, ATP turnover to re-generate reduced glutathione. Moreover, ATP is used toreestablish membrane ion gradients in connective tissue cellsafter exposure to the toxic products of neutrophils. There-fore, it is possible that neutrophils enhance adenosine releasefrom connective tissue cells treated with methotrexate oracadesine because such treatment might diminish reutiliza-tion of adenosine generated during adenine nucleotide turn-over.Whereas our studies do not rule out a direct effect of

methotrexate on neutrophil function, our results do indicatean antiinflammatory mechanism by which methotrexate mayameliorate rheumatoid arthritis; methotrexate increasesadenosine release from connective tissue cells, specificallyconnective tissue cells under stress. Since the effects ofadenosine are confined to those areas where the adenosine isreleased and because of the extremely rapid metabolism ofadenosine in tissues and in the blood, the potential toxicity ofexcess adenosine release is reduced. Thus, the demonstra-tion that agents capable of stimulating adenosine release atinflamed sites are antiinflammatory could lead to the devel-opment of an additional class of antiinflammatory drugs.

We thank Melissa Dworkin and Phoebe Recht for their outstandingtechnical assistance and Dr. Paula Marchetta for her contributions.We also thank Drs. Rochelle Hirschhorn and Gerald Weissmann fortheir helpful discussions and suggestions during the performance of

these experiments and Dr. Weissmann for reviewing this manuscript.B.N.C. is the recipient of a Clinical Investigator Award from theNational Institutes of Health (AR-01490) and is the Irene DugganArthritis Investigator of the Arthritis Foundation. This research wasperformed with the support of grants from the American HeartAssociation (New York City Affiliate), Lederle Laboratories, GensiaPharmaceuticals, Inc., the Evans Foundation, the National Institutesof Health (HL19721 and AR11949 to Dr. Gerald Weissmann), and agrant to R.I.L. from the Aaron Diamond Foundation under theClinical Scientist Program.

1. Gubner, R., August, S. & Ginsberg, V. (1951) Am. J. Med. Sci.221, 176-182.

2. Furst, D. E. & Kremer, J. M. (1988) Arthritis Rheum. 31,305-314.

3. Morgan, S. L., Baggott, J. E., Vaughn, W. H., Young, P. K.,Austin, J. V., Krumdieck, C. L. & Alarcon, G. S. (1990)Arthritis Rheum. 33, 9-18.

4. Tishler, M., Caspi, D., Fishel, B. & Yaron, M. (1988) ArthritisRheum. 31, 906-908.

5. Hanrahan, P. S. & Russell, A. S. (1988) J. Rheumatol. 15,1078-1080.

6. Allegra, C. J., Drake, J. C., Jolivet, J. & Chabner, B. A. (1985)Proc. Natl. Acad. Sci. USA 82, 4881-4885.

7. Allegra, C. J., Hoang, K., Yeh, G. C., Drake, J. C. & Baram,J. (1987) J. Biol Chem. 262, 13520-13526.

8. Baggott, J. E., Vaughn, W. H. & Hudson, B. B. (1986) Bio-chem. J. 236, 193-200.

9. Gruber, H. E., Hoffer, M. E., McAllister, D. R., Laikind,P. K., Lane, T. A., Schmid-Schoenbein, G. W. & Engler,R. L. (1989) Circulation 80, 1400-1411.

10. Jaffe, E. A., Nachman, R. L., Becker, C. G. & Minick, C. R.(1973) J. Clin. Invest. 52, 2745-2756.

11. Boyum, A. (1968) Scand. J. Clin. Lab. Med. Suppl. 21, 77-89.12. Cronstein, B. N., Levin, R. I., Belanoff, J., Weissmann, G. &

Hirschhorn, R. (1986) J. Clin. Invest. 78, 760-770.13. Henderson, J. F., Fraser, J. H. & McCoy, E. E. (1974) Clin.

Biochem. 7, 339-358.14. Cronstein, B. N., Rosenstein, E. D., Kramer, S. B., Weiss-

mann, G. & Hirschhorn, R. (1985) J. Immunol. 135, 1366-1371.15. Lappin, D. & Whaley, K. (1984) Clin. Exp. Immunol. 57,

454-460.16. Riches, D. W. H., Watkins, J. L., Henson, P. M. & Stan-

worth, D. R. (1985) J. Leukocyte Biol. 37, 545-557.17. Eppell, B. A., Newell, A. M. & Brown, E. J. (1989) J. Immu-

nol. 143, 4141-4145.18. Elliott, K. R. F., Stevenson, H. C., Miller, P. J. & Leonard,

E. J. (1986) Biochem. Biophys. Res. Commun. 138, 1376-1382.19. Leonard, E. J., Shenai, A. & Skeel, A. (1987) Inflammation 11,

229-240.20. Marone, G., Vigorita, S., Triggiani, M. & Condorelli, M. (1986)

Adv. Exp. Med. Biol. 195, 7-14.21. Bonnafous, J.-C., Dornand, J., Favero, J. & Mani, J.-C. (1981)

J. Recept. Res. 2, 347-366.22. Kammer, G. M., Birch, R. E. & Polmar, S. H. (1983) J.

Immunol. 130, 1706-1712.23. Mandler, R., Birch, R. E., Polmar, S. H., Kammer, G. M. &

Rudolph, S. A. (1982) Proc. Natl. Acad. Sci. USA 79, 7542-7546.

24. Kammer, G. M. & Rudolph, '~.A. (1984) J. Immunol. 133,3298-3302.

25. Cronstein, B. N., Kramer, S. B., Weissmann, G. & Hirsch-horn, R. (1983) J. Exp. Med. 158, 1160-1177.

26. de la Harpe, J. & Nathan, C. F. (1989) J. Immunol. 143,596-602.

27. Cronstein, B. N., Kubersky, S. M., Weissmann, G. & Hirsch-horn, R. (1987) Clin. Immunol. Immmunopathol. 42, 76-85.

28. Schrier, D. J. & Imre, K. M. (1986) J. Immunol. 137, 3284-3289.

29. Nielson, C. P. & Vestal, R. E. (1989) Br. J. Pharmacol. 97,882-888.

30. Roberts, P. A., Newby, A. C., Hallett, M. B. & Campbell,A. K. (1985) Biochem. J. 227, 669-674.

31. Moser, G. H., Schrader, J. H. & Deussen, A. (1989) Am. J.Physiol. 256, C799-C806.

32. Nesher, G. & Moore, T. M. (1990) Arthritis Rheum. 33, 954-959.

Physiology/Pharmacology: Cronstein et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

1