ARTHRITIS & RHEUMATISM
Vol. 39, No. 12, December 1996, pp 1951-1960
0 1996, American College of Rheurnatology
REVIEW
MOLECULAR THERAPEUTICS
Methotrexate and its Mechanism of Action
BRUCE N. CRONSTEIN
Since its reintroduction to the practice of rheu-
matology in the mid-l980s, methotrexate (MTX) has
become the most widely used second-line agent for the
treatment of rheumatoid arthritis (RA). To date, MTX
has been used to treat patients who have RA or other
inflammatory diseases without a clear understanding of
either its mechanism of action or the molecular basis for
its toxicity. A thorough understanding of the biochemi-
cal mechanism(s) of action of MTX as well as the events
that lead to the toxic effects of MTX may result in the
development of new, more effective and less toxic agents
for the treatment of RA and other inflammatory dis-
eases. In this review, the effects of MTX treatment on
those factors thought to be most important in the
pathogenesis of chronic inflammatory arthritis will be
discussed and the evidence regarding the molecular
mechanism of MTX will be examined.
MTX is a cytotoxic agent
The first report on the efficacy of MTX in the
treatment of RA appeared in 1951 (1). MTX had
recently been introduced as a cytotoxic agent for the
treatment of malignancies, and it was thought that the
potent inhibition of purine and pyrimidine synthesis
mediated by MTX might lead to similar suppression of a
malignant immune response. When reintroduced to
clinical rheumatology in the 1980s, it was thought to act
as a chemotherapeutic agent for inhibiting the replica-
Supported by grants from the USPHS (AR-AI-41911, AR-
11949, and HL-19721), the GCRC (MOl-RR-00096), and the Kaplan
Cancer Center (CA-16087).
Bruce N. Cronstein, MD: New York University Medical
Center, New York.
Address reprint requests to Bruce N. Cronstein, MD, Profes-
sor of Medicine and Pathology, Division of Rheumatology, New York
University Medical Center, 550 First Avenue, New York, NY 1UO16.
Submitted for publication June 18, 1996; accepted in revised
form August 21, 1996.
1951
tion of activated lymphocytes and other cells responsible
for tissue injury in RA-similar to its role in the
treatment of cancer. Clearly, the side effects of low-dose
MTX therapy, stomatitis and marrow suppression, are
due to inhibition of cell replication; however, there is no
evidence that MTX specifically depletes specific “auto-
immune” cells.
In clinical use, the development of marrow sup-
pression is seen most often in patients who are relatively
folate-deficient at the start of therapy (2,3), and its
occurrence is usually a signal to either reduce the dosage
of MTX or discontinue the drug. Prevention of marrow
suppression, stomatitis, and other more common toxi-
cities of MTX without diminution of the therapeutic
effect in RA patients by folk acid or folinic acid
supplements (4-6) would suggest that inhibition of
cellular proliferation (or complete suppression of other
folate-dependent cellular reactions) by MTX is not
critical to its therapeutic effect (7).
MTX modulates humoral immunity
One of the hallmarks of RA is the presence of
serum antibodies directed against the Fc portion of IgG
rheumatoid factor (RF), although its role in the patho-
genesis of RA is debatable. Several groups of investiga-
tors have reported that MTX treatment does not affect
circulating levels of IgM-RF (8-10). In contrast, Alarcdn
and colleagues (11) used a sensitive enzyme-linked
immunosorbent assay to study the concentrations of IgM
and IgA RFs in patients involved in a blinded study of
the efficacy of MTX in the treatment of RA; those
authors observed a significant drop in R F levels in
patients who improved. When the data were analyzed,
there was a strong statistical association between MTX
treatment and a reduction in R F levels and a less
impressive association between improvement in re-
sponse to MTX and reduction in R F levels. Other
1952
investigators (12) have reported that R F levels de-
creased when inflammation was controlled by either
MTX or gold salts.
Olsen and coworkers (13) reported that treat-
ment of RA patients with MTX is associated with
diminished RF production by lymphocytes cultured ex
vivo, although similar effects on R F production were
reported for gold salts as well. Moreover, MTX treat-
ment of cultured peripheral blood mononuclear cells
(PBMC) has been shown to result in diminished RF
production in vitro (14,15). In some of these studies, the
investigators were able to reverse the effects of MTX on
RF production by treating the cells with folinic acid.
Since reductions in the R F level may accompany
successful therapy with other second-line agents (e.g.,
gold salts and penicillamine) and since the role of R F in
the pathogenesis of RA is unclear, it is difficult to
conclude that the effects of MTX therapy on R F levels
in vivo or on RF production in vitro are causally related to
the beneficial effects of this drug in the therapy of RA.
MTX modulates cellular immunity
The central role of cellular immune reactions and
T cells in the development and pathogenesis of RA has
been generally accepted for a number of years. The
strong HLA-DR-associated risk of developing RA and
the presence of large numbers of T cells in the affected
synovium support the contention that T cells are impor-
tant determining elements in the pathogenesis of joint
inflammation and destruction in RA. Thus, one poten-
tial explanation for the therapeutic effects of MTX in
RA is diminution in the number or reactivity of T cells
involved in the pathogenesis of inflammation (16). How-
ever, there is little evidence to support the hypothesis
that MTX affects either the number or function of T
cells involved in the pathogenesis of RA. Moreover,
recent controlled studies of monoclonal antibodies di-
rected against CD4+ T cells have provided little evi-
dence that the elimination or modulation of T cells
significantly alters the course of RA (17,18).
Although a reduction in T cell number or func-
tion might be the predicted result of MTX treatment,
Weinblatt and colleagues (19) reported that, in fact,
long-term therapy with MTX leads to an increase in the
percentage of CD3 and CD4 cells in the peripheral
blood. In contrast, Wascher et a1 (20) reported that
short-term therapy (12 weeks) diminishes the number of
circulating T and B cells. One caveat in interpreting
these studies is that the peripheral blood T cell number,
subsets, or function may not accurately reflect synovial T
CRONSTEIN
cell responses. In general, though, there are few data to
support the contention that modulation of T cell num-
bers or functions by MTX is responsible for the thera-
peutic effects of MTX.
MTX diminishes phlogiston production and responses
A large and increasing number of soluble agents
have been reported to play a role in the development of
synovial inflammation. These factors include lipid deriv-
atives (prostaglandins, leukotrienes, platelet-activating
factor), complement activation products, cytokines, and
chemokines. Although MTX has not been reported to
affect the generation of complement activation products
or prostaglandins, it may affect either the generation of
or response to leukotrienes, cytokines, and chemokines.
Leukotrienes, most notably leukotriene B4
(LTB,), are potent lipid-derived stimuli for leukocytes.
The effect of MTX therapy on the generation of leuko-
trienes remains somewhat controversial. Sperling and
coworkers (21,22) have reported that neutrophils from
RA patients or normal controls treated with MTX
produce less LTB, than do cells from individuals not
treated with MTX (21,22). Similarly, Leroux and co-
workers (23) reported that MTX treatment (a single
dose) inhibited the generation of 5- and 12-lipoxygenase
products. In contrast, Hawkes and colleagues (24) ob-
served that MTX administered in vivo did not inhibit the
production of lipoxygenase products by neutrophils
stimulated ex vivo. Methodologic differences may ac-
count for the differing results. Nonetheless, the resulting
diminution in LTB, synthesis and synovial fluid concen-
tration is probably not responsible for the therapeutic
effects of MTX, since the magnitude of the decrement in
synovial fluid LTB, induced by substitution of dietary
lipid with fish oil is similar to that induced by MTX
therapy, but does not lead to as great an improvement in
arthritis (25-27).
Cytokines are low molecular weight proteins that
are secreted by various immune and inflammatory cells.
Based on studies in animal models of RA and, more
recently, in RA patients, it is increasingly clear that
cytokines play a critical role in the development of the
clinical and laboratory manifestations of RA. Although
it is currently thought that tumor necrosis factor a (TNFa)
plays the central role in the cytokine cascades responsi-
ble for synovial inflammation in RA, interleukin-1 (IL-1)
and IL-6, among others, are responsible for many of the
manifestations of RA (28). The effects of MTX therapy
on both the response to and the production of these
MECHANISM OF ACTION OF MTX
cytokines have been studied extensively in animal mod-
els, in RA patients, and in their isolated cells.
IL-1 was among the first cytokines to be discov-
ered. Because of the availability of reagents with which
I L 1 production or the response to IL-1 can be mea-
sured, the levels of this cytokine in both the circulation
and the synovial fluid of animals and patients with RA
has been studied in great detail. In two studies, it was
shown that MTX treatment of animals with inflamma-
tory arthritis decreased the production of IL-1 in vivo as
well as ex vivo (29,30). Subsequent studies produced
more ambiguous results; MTX treatment of animals or
patients or their cells did not inhibit IL-1 production, but
significantly diminished the ability of their cells to
respond to IL-1, an effect which could be overcome in
some studies by treatment with folinic acid (31-33).
One potential explanation for the effects of MTX
treatment on cellular responses to IL-1 was reported by
Brody and colleagues (34) who observed that, when
studied in vitro, MTX markedly inhibited the binding of
IL-1p to its receptor on peripheral blood cells, an effect
that could be overcome by an excess of IL-1p. Although
those authors' findings may explain the in vitro effects of
MTX, it is difficult to understand how reversible inhibi-
tion of IL-1 binding can be relevant to the mechanism of
action of a drug which is administered on a weekly basis
and which is present as free drug in biologic fluids for
only a few hours after the dose is administered (35).
Later studies reported that MTX treatment of patients
leads to diminished I G 1 concentrations in the synovial
fluid of patients who achieved a response to the drug;
however, no reduction in serum IL-1 concentrations was
noted (36,37). These observations suggested that a local
effect of MTX treatment on IL-1 production might
contribute to the therapeutic effects of low-dose MTX.
Although none of these studies is definitive, they are all
consistent with the hypothesis that interference with
either IL-1 production or the effects of IL-1 is related to
the reduction in symptomatic synovial inflammation
observed in patients with RA who are being treated with
MTX. With the possible exception of the study by Brody
et a1 (34), none of the studies summarized here have
determined how MTX decreases IL-1 secretion or the
response to IL-1.
Recently, investigators have concentrated their
attention on the central role of TNFa in the pathogen-
esis of RA. This focus has led to the development of
potentially useful therapeutic agents (monoclonal anti-
TNFa antibodies and soluble TNF receptors). Adminis-
tration of anti-TNFa antibodies to patients with RA has
resulted in dramatic, albeit temporary, improvements
1953
(for review, see ref. 28). In general, in vitro studies of
MTX using either animal or human cells have shown
that there is little effect of MTX on TNFa production,
although liposomal preparations of MTX dramatically
inhibit TNFa production in vitro, most likely because of
improved uptake of liposomal MTX by target cells
(38-41). Studies in the adjuvant arthritis model of RA
have demonstrated that MTX treatment leads to a
profound decrease in the synovial fluid TNFa concen-
tration (42), and treatment with liposomal MTX results
in diminished TNFa production ex vivo in this model
(39). No such study in humans has yet been reported.
The studies reported to date do not demonstrate any
significant effect of MTX therapy on TNFa concentra-
tions in the peripheral blood of patients with RA
(43,44). As with IL-1 (see above), there may be a
disparity between the effects of MTX on the synovial
production of TNFa and total circulating levels of
TNFa, and the utility of measuring circulating TNFa
concentrations as an indicator of response to therapy
remains unclear. Thus, although MTX therapy leads to a
decrease in TNFa production locally, the role of this
reduction in the mechanism of action of MTX is not
settled.
A variety of other pro- and antiinflammatory
proteins have been measured in the serum of RA
patients treated with MTX. Among these, the one
cytokine that is most consistently diminished in the
serum is IL-6, a cytokine most tightly linked to produc-
tion of acute-phase reactants by the liver. Serum con-
centrations of IL-6 were found to decline during MTX
therapy in 4 studies (44-47) but did not change in
another (48). Although MTX therapy leads to an in-
crease in the cellular production of IL-1 receptor antag-
onist and soluble TNF receptors (43), these changes are
not reflected by alterations in the circulating levels of
these antiinflammatory proteins. The chemokine IL-8 is
a potent chemoattractant for neutrophils and an angio-
genic factor produced by mononuclear cells, among
others; in patients with RA, MTX therapy was shown to
inhibit the spontaneous production of IL-8 by PBMC
but not synoviocytes (49,50). As with the other cytokines
studied, the mechanistic role of IL-8 inhibition by MTX
therapy in the antiinflammatory effects of MTX remains
uncertain.
The studies summarized above suggest that at
least one of the mechanisms by which MTX inhibits
inflammation in RA is by modulating the production of
cytokines in the synovium. Although the production of
some of these factors was clearly inhibited by MTX
treatment in vitro or ex vivo, other agents also affected
1954
putrescin
M e t h o;t e x a t e
Methionine
D H F -**TH F- N 5-C H ,-TH F 1 Methylation of
Homocysteine
Adenosine
Figure 1. Inhibition of transmethylation reactions and polyamine formation by methotrexate.
DHF = dihydrofolate; THF = tetrahydrofolate; N"-CH,-THF = N"-methyl-tetrahydrofolate;
ATP = adenosine triphosphate; THF = tetrahydrofolate; SAM = S-adenosyl-rnethionine; SAH =
S-adenosyl-hornocysteine.
cytokine release (e.g., corticosteroids), indicating that
diminution of cytokine release at sites of inflammation is
not a specific property of MTX. Finally, none of the
studies cited addressed the question of the molecular
mechanism by which MTX treatment modulates cyto-
kine release, although a number of hypotheses have
been suggested.
The molecular mechanism(s) of MTX
One potential explanation for the effects of MTX
therapy on cellular and humoral immunity and cytokine
secretion is that MTX, via its effects on the purine and
pyrimidine synthesis required for cell division, is cyto-
toxic for the cells that generate cytokines or incite other
cells to generate cytokines. As discussed above, there is
no evidence that a specific subset of immune or other
cells is depleted in patients undergoing MTX therapy,
although the cytotoxic effects of MTX probably play a
role in the toxicity of MTX (stomatitis, cytopenias).
Alternatively, MTX treatment may induce a biochemical
change in the cells of the inflamed synovium that leads
to a decrease in the secretion of soluble mediators of
inflammation.
After absorption from the gut (or after systemic
administration), MTX is present in the circulation as
either the native compound or its active metabolite
7-hydroxy-MTX for a relatively short period of time.
Both of these compounds are taken up by cells and
converted intracellularly to their polyglutamates, which
are both active and quite long-lived. It is likely that the
accumulation of MTX polyglutamates is responsible for
both the therapeutic effects and the toxic effects of the
drug (35). The therapeutic effects of low-dose MTX in
CRONSTEIN
P oly am in e s
(S permine, sp ermidine)
SAM
p h o sp h olipid s ,
proteins, R N A ,
DNA, etc
the treatment of RA have been ascribed to two distinct,
although not mutually exclusive, biochemical mecha-
nisms, as follows.
The biochemical mechanism of action of MTX:
two hypotheses
MTX inhibits methylation reactions. MTX and
its polyglutamates inhibit a number of intracellular
folate-dependent reactions, among which is the methyl-
ation of homocysteine to methionine. The conversion of
homocysteine to methionine is required for the genera-
tion of S-adenosyl-methionine ( S A M ) , which acts as the
proximal methyl donor to RNA, DNA, amino acids,
proteins, and phospholipids, as well as in the synthesis of
polyamines such as spermidine and spermine (Figure 1).
Methylation of all of these biologically active molecules
is required for cellular survival and function, particularly
in the immune system. After donation of its methyl
group, SAM is converted to S-adenosyl-homocysteine
( S A H ) which, in turn, is metabolized to adenosine and
homocysteine, which can be recycled to form SAM
(Figure 1).
During the late 1970s and early 1980s, investiga-
tions into the pathogenesis of severe combined immu-
nodeficiency associated with adenosine deaminase
(ADA) deficiency demonstrated that SAH accumulates
in the ADA-deficient cells and that the accumulation of
SAH leads, via competitive inhibition of SAM-
dependent methyl-transfer reactions, to marked inhibi-
tion of transmethylation reactions (51-54). This obser-
vation led to the hypothesis that the inhibition of
transmethylation reactions was an important factor in
the development of severe combined immunodeficiency.
MECHANISM OF ACTION OF MTX
In othcr studics, investigators induced a marked increase
in intracellular SAH concentration by incubating cells
with large cxcesses of adcnosine and homocystcine in
the prcsence of an ADA inhibitor in order to inhibit
transmethylation rcactions; they found that this treat-
ment markedly inhibited lymphocyte and mononuclear
cell function (55-57). The immunologic cffects of inhib-
iting transmethylation reactions were thought to be of
potcntial use in the treatment of RA or othcr forms of
inflammatory disease in which cellular and humoral
immunity play a role.
Evidencc that polyamincs, the synthesis of which
is dependent upon SAM, might be important in the
pathogenesis of KA was provided in later studies that
reported an increase in polyaminc levels in urinc, syno-
vial fluid, synovial tissuc, and PBMC from paticnts with
KA (58-61). Indeed, Fleschcr and coworkcrs (60,61)
reported that the defect in IL-2 secretion observed whcn
peripheral blood T cells from patients with RA were
cultured ex vivo could be rcversed by inhibitors of
polyamines. Thosc investigators also suggcsted that the
ammonia, H,O,, or other toxic substances generated
from the oxidation of polyamines by mononuclear cells
(but not lymphocytes) in these cultures was responsiblc
for the inhibition of IL-2 synthesis. The association of
polyamine accumulation with this specific defect in
immune regulation has only bcen documented in KA.
Although similar defects in IL-2 production have bcen
documented for the lymphocytcs of patients with sys-
temic lupus erythcmatosus (see ref. 62) and Sjogrcn’s
syndrome (see ref. 63), the role of polyamines in the
pathogenesis of these alterations has not been explored.
Intracellular polyaminc concentrations and IL-2 re-
sponses in inflammatory cells, tissucs, or fluids of pa-
tients with other forms of inflammatory arthritis that
respond to MTX have also not been studied.
In 1990, Neshcr and Moore (64) noted that M‘IX
might also inhibit transmethylation reactions, and first
tested the hypothcsis that inhibition of these transmeth-
ylation reactions was responsiblc for thc beneficial ef-
fects of MTX treatment in RA. In their initial studies,
they found that MTX inhibited the production of
IgM-RF by cultured PBMC. Trcatrnent of the cells with
folinic acid reversed the cffect of MTX treatment on thc
measured function. Morc telling, howevcr, was the ca-
pacity of methionine or spermidine (a polyamine whose
synthesis is dependent upon SAM) to revcrse thc effects
of MTX on mononuclear cell function (64). In a subse-
quent study, this same laboratory rcported further in
vitro evidence that MTX inhibits cellular, in this case
monocytc, function via inhibition of transmethylation
1955
reactions. Again, SAM and folinic acid (but not spcrmi-
dine) rcversed the cffect of MTX on monocytc function
(65). Although the concentrations of MTX that were
required to inhibit cellular function in these two studies
wcrc high, the results rcportcd are consistent with the
hypothesis that inhibition of transmethylation reactions
contributcs to the antiinflammatory cffects of MTX.
A further test of the hypothesis that inhibition of
transmethylation reactions is responsible for the antiin-
flainmatory effects of MTX would be to determine
whether other inhibitors of transmethylation reactions
are also antiinflammatory. Because of the interest in
transmethylation reactions that followed from thc stud-
ies of the mechanism of immunodeficiency in childrcn
lacking ADA, a potent inhibitor of S A H hydrolase that
thereby inhibited transmethylation reactions,
3-deazaadenosine, was developed. In in vitro and in vivo
studies, this compound was found to be an active
antiinflammatory agcnt (66-71). Moreover, the drug was
studied in clinical trials of paticnts with RA, although its
efficacy has never been reported (72). Intercstingly,
although most of its effects were initially ascribed to its
capacity to inhibit transmethylation reactions, some
antiinflammatory cffects of 3-deazaadenosine wcre later
shown to be independent of its ability to inhibit trans-
methylation rcactions (73-75). Thus, the evidence to
date suggcsts that the inhibition of transmethylation
reactions by MTX may inhibit the cellular synthesis of
polyamines, which accumulate in the synovium of pa-
tients with RA and which may contribute to joint
destruction in this disease.
MTX promotes adenosine release. As discussed
above, MTX is taken up by cells and convcrted to
long-livcd polyglutamatcs (76). Rcsults of studies per-
formed by Nlcgra et a1 (77) and, subsequently, Baggott
and coworkcrs (78) demonstrated that MTX polygluta-
matcs were as potent, if not more potent, inhibitors of a
varicty of folate-dependent enzymcs as thc native mol-
ecule. Moreovcr, the enzyme inhibited most effectively
by MTX polyglutamatcs was 5-aminoimidazole-4-
carboxarnide ribonucleotide (AICAR) transformylase
(77,78). If the synthesis of AICAR is less inhibited than
its metabolism, as predicted by the rclative scnsitivitics
of the enzymatic steps to inhibition by MTX polygluta-
mates, then the inhibition of AICAR transformylase by
MTX polyglutamates would result in intracellular accu-
mulation of AICAR (Figure 2). Since AICARibosidc
directly inhibits ADA and AICAR inhibits AMP deami-
nasc, the intracellular accumulation of AICAR could
lead to the respective release of adenosine or AMP
(which is convcrted by 5’-nucleotidase to adenosine)
1956
INTRACELLULAR
---+.l
/
/
/ AIC#R
METHOTREXATEdy
FAICAh
1
,,,q=+..
O0
1 .
l n o s i n e m Adenosine,
1
I
1
Uric Acic)
into the extracellular space (78,79). That MTX treat-
ment may lead to the accumulation of AICAR was
supported by the finding that humans treated with high
doses of MTX excrete increased concentrations of amino-
imidazole carboxamide in their urine (80).
The observations described above suggested the
hypothesis that adenosine, which is present in increased
concentrations in the extracellular space, mediates at
least some of the antiinflammatory effects of low-dose
MTX therapy. This hypothesis was first tested in a series
of in vitro experiments. Treatment of cultured human
fibroblasts and endothelial cells with MTX led to in-
creased adenosine release but only after prolonged
incubations, as might be predicted if the increase in
adenosine release resulted from intracellular accumula-
tion of AICAR (81). Moreover, MTX-induced adeno-
sine release was enhanced when the MTX-treated cells
were subject to exposure to a stressful agent, stimulated
neutrophils. The adenosine that was released from the
MTX-treated cells was both necessary and sufficient to
diminish the adhesion of stimulated neutrophils, a sur-
rogate antiinflammatory effect (81).
Subsequent studies on the effects of MTX treat-
ment in a murine model of acute inflammation revealed
that treatment of mice with pharmacologically relevant
doses of MTX at weekly intervals leads to intracellular
AICAR accumulation and increased concentrations of
adenosine in inflammatory exudates. Moreover, the
effects of low-dose, intermittent MTX treatment on
CRONSTEIN
EXTRACELLULAR
ATP
11
ADP
tl
AMP
tl
b
~’N1
T
AMP
* ADENOSINE
*Uric Acid
leukocyte accumulation in this model of inflammation
were due to the excess adenosine released into the
inflammatory exudate, since elimination of extracellular
adenosine by the addition of ADA to the exudate
reversed the effects of MTX treatment (82). Similar
findings in a different animal model were reported by
Asako and colleagues (83,84).
As with inhibition of transmethylation reactions,
a further test of the hypothesis that adenosine mediates
the antiinflammatory effects of MTX would be the
demonstration that other agents that induce adenosine
release are antiinflammatory as well. Both adenosine
kinase inhibitors (85-88) and sulfasalazine (89), an
antiinflammatory agent that directly inhibits AICAR
transformylase (90), were also antiinflammatory by an
adenosine-dependent mechanism in in vitro and in vivo
models of inflammation. This provides further evidence
that the antiinflammatory effects of MTX are mediated,
at least in part, by adenosine. More support for the
hypothesis that MTX enhances adenosine release in
humans was provided by Bernini et a1 (91), who reported
that high-dose MTX increases adenosine concentrations
in the cerebrospinal fluid of children treated for child-
hood leukemia. Thus, MTX treatment does lead to local
increases in adenosine concentration in both animals
and humans, and the evidence is consistent with the
notion that agents that promote adenosine release by
alternative mechanisms are also antiinflammatory in in
vitro and animal models. Further proof of the hypothesis
MECHANISM OF ACTION OF MTX
that agents that induce adenosine release are antiinflam-
matory will require trials in patients with RA or other
inflammatory diseases.
The antiinflammatory and immunologic effects
of adenosine result from the interaction of adenosine
with specific receptors on the cell surface. These aden-
osine receptors, of which 4 subtypes have been char-
acterized at the pharmacologic and molecular levels
(Al, A, AJ, are all members of the family of 7
transmembrane-spanning receptors related to the ad-
renergic receptors. Via interaction with an A,, receptor
on stimulated neutrophils, adenosine inhibits stimulated
release of toxic oxygen metabolites, adhesion to a variety
of different cell types, and neutrophil-mediated injury
both in vitro and in vivo (92-96). Adenosine also inhibits
lymphocyte proliferation to mitogens and induces sup-
pressor phenotype and function (97-102). Moreover,
adenosine, acting at one or another of its receptors on
monocyte/macrophages, inhibits the production of
TNFa, IL-6, and IL-8 (88,103-105) and, by a less clear
mechanism, increases the secretion of the potent antiin-
flammatory cytokine IL-10 (106). One study demon-
strated that synovial cell synthesis of collagenase, but not
tissue inhibitor of metalloprotease or stromelysin, is
specifically inhibited in patients with RA who have been
treated with MTX (107), and this highly selective effect
of MTX therapy can be accounted for by an adenosine
receptor-mediated effect as well (108). The antiinflam-
matory effects of MTX treatment observed in a murine
model appeared to be mediated primarily by A, adeno-
sine receptors, since an A, receptor antagonist com-
pletely reversed the antiinflammatory effects of MTX
(82). The role of A, receptors in the antiinflammatory
effects of adenosine has not yet been explored in animal
models of inflammation.
Whether or not adenosine mediates the thera-
peutic effects of MTX, it is likely that some of the toxic
effects of MTX may result from engagement of adeno-
sine receptors. One possible example of adenosine-
mediated MTX toxicity is accelerated rheumatoid nodu-
losis, an increase in the number of rheumatoid nodules
that occurs in MTX-treated patients with nodular RA,
often despite good therapeutic responses. Merrill and
coworkers studied an in vitro model of rheumatoid
nodule formation and found that MTX increases aden-
osine concentrations in supernatants of cultured mono-
nuclear cells (Merrill J, Schreibman D, Cronstein BN:
unpublished data) and enhances giant cell formation (a
surrogate for nodule formation in this in vitro model) by
a mechanism that is reversed by a specific A, adenosine
receptor antagonist and is mimicked by a specific aden-
1957
osine Al receptor agonist (109). Thus, enhanced aden-
osine release in extraarticular tissues may promote nod-
ule formation in susceptible patients.
Central nervous system (CNS) toxicity (head-
aches, light-headedness, and cognitive dysfunction) is
not an uncommon problem for patients with rheumatic
diseases treated with MTX. Similar toxic reactions are
seen in children treated with higher doses of MTX for
leukemia. Bernini and colleagues (91) showed that the
severe CNS toxicity observed in MTX-treated leukemia
patients was associated with dramatic elevations in cere-
brospinal fluid adenosine concentrations and was com-
pletely reversed by administration of an adenosine re-
ceptor antagonist (aminophylline). These observations
suggest that the well-established CNS effects of adeno-
sine are responsible for the CNS toxicity experienced by
some patients.
Conclusion
The introduction of MTX for the treatment of
inflammatory diseases has transformed the way in which
RA is treated. Indeed, the standard of comparison for
trials of new second-line agents is MTX. Although new
developments in the area of cytokine antagonists or gene
therapy may lead to the development of potent, specific
antiinflammatory drugs, the recognition that MTX may
act by two complementary biochemical mechanisms
should also permit the development of new agents that
are safer and more effective for the treatment of RA.
ACKNOWLEDGMENT
The author wishes to thank Dr. Susan M. Goodman for
reviewing the manuscript.
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