REVIEWS

Mechanism of action of methotrexate

in rheumatoid arthritis, and the search
for biomarkers
Philip M. Brown, Arthur G. Pratt and John D. Isaacs
Abstract | The treatment and outcomes of patients with rheumatoid arthritis (RA) have been
transformed over the past two decades. Low disease activity and remission are now frequently
achieved, and this success is largely the result of the evolution of treatment paradigms and the
introduction of new therapeutic agents. Despite the rapid pace of change, the most commonly
used drug in RA remains methotrexate, which is considered the anchor drug for this condition.
In this Review, we describe the known pharmacokinetic properties and putative mechanisms of
action of methotrexate. Consideration of the pharmacodynamic perspective could inform the
development of biomarkers of responsiveness to methotrexate, enabling therapy to be targeted
to specific groups of patients. Such biomarkers could revolutionize the management of RA.

Institute of Cellular Medicine,
Faculty of Medical Sciences,
Framlington Place, Newcastle
upon Tyne, NE2 4HH, UK.
Correspondence to P.M.B.
philip.brown@newcastle.ac.uk
doi:10.1038/nrrheum.2016.175
Published online 27 Oct 2016
The past 25 years have seen dramatic changes in the out-
comes of patients with rheumatoid arthritis (RA). The
advent of effective biologic disease-modifying therapies
(DMARDs) has made it possible to arrest progression
of the disease and prevent associated joint damage and
disability1. However, despite the wealth of new agents,
methotrexate remains the main starting therapy and
anchor drug for the treatment of RA.
In the 1940s, rational drug design led to the develop-
ment of methotrexate, an antifolate agent that inhibits the
enzyme dihydrofolate reductase (DHFR). Methotrexate
was initially administered at high doses (often as a single
dose of up to 1,000 mg) as a treatment for leukaemia,
but was subsequently found to be effective at much lower
doses (15–25 mg per week) in patients with inflamma-
tory arthritides2. The first documented use of metho-
trexate for the treatment of RA was in 1951 (REF. 3), but
widespread use only occurred following its licensing for
this indication in the 1980s4,5. Results from a number of
studies have confirmed the efficacy of methotrexate for
the treatment of RA; the authors of a Cochrane Review
calculated that achieving a 50% improvement in disease
parameters required a pooled number-needed-to‑treat of
seven patients6. Several trials have directly compared the
efficacy of methotrexate with that of biologic therapies in
early, DMARD-naive RA7–21, without identifying consist-
ent superiority of any particular first-generation biologic.
Considerations of the relative effectiveness, costs and
toxic effects of these therapies suggest that conventional
DMARDs, including methotrexate, should still form the
initial basis of treatment strategies in early RA.
Despite its widespread use and known clinical effi-
cacy, the mechanisms by which low-dose methotrexate
exerts its therapeutic effect in RA remain incompletely
understood. By contrast, the pathways involved in
metho­trexate transport and metabolism in cancer ther-
apy have been mapped in detail22. At the high drug doses
used in oncological settings, methotrexate antagonizes
the synthesis of purines (and, therefore, DNA), result-
ing in cell-cycle arrest during S phase. Apoptosis ensues,
which accounts for the cytotoxic effect of methotrexate
on rapidly dividing malignant cells, and also explains its
frequent adverse effects of pancytopenia and mucositis22.
These adverse effects can be reversed by supplementa-
tion with calcium or sodium folinate (also known as
folinic acid, leucovorin, or 5‑formyltetrahydrofolate)22.
High doses of folate are needed to rescue the toxic effects
of methotrexate in oncology patients. However, at the
low doses of methotrexate used to treat patients with RA,
no loss of therapeutic benefit occurs with the concomi-
tant administration of 5 mg folate (to minimize toxicity)23.
These observations suggest that the antirheumatic effect
of methotrexate exploits alternative mechanisms to its
anticancer effect.
Methotrexate benefits from low cost, an extensive
safety record and a weekly treatment regimen, making it
an attractive option in early RA24. However, these advan-
tages are somewhat offset by its slow onset of action, and
the fact that not all patients who are treated with metho-
trexate achieve an adequate clinical response. A review of
the results of clinical trials involving methotrexate demon-
strated that although ~40% of patients with early arthritis

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Key points with renal impairment 29,35,37,44. Following low-dose

• Methotrexate shows good efficacy in a proportion of patients: 40% of treated
patients with rheumatoid arthritis achieve an ACR50 response
• The mechanism of action of methotrexate has not fully been defined, however
potentiation of adenosine signalling carries the most robust data
• Pharmacokinetic parameters, particularly intracellular methotrexate
polyglutamation, show some association with disease activity, although they cannot
yet be used to predict treatment response
• An exploration of the expression and polymorphisms of genes encoding molecules
linked to proposed mechanisms of methotrexate action is underway to identify
methotrexate-responsive signatures
• At present, no robust markers or predictive models exist for methotrexate
responsiveness in RA
achieve a good response (defined as meeting American
College of Rheumatology ACR50 criteria) with metho-
trexate monotherapy, no robust biomarkers are yet avail-
able to identify this subset of patients25. Consequently, the
determination of responder status requires a lengthy trial
of methotrexate treatment, which creates a potential win-
dow for joint damage to accrue. Biomarkers that predict
methotrexate response are, therefore, urgently required.
Pharmacokinetics of methotrexate
Methotrexate (4‑amino‑10‑methylfolic acid) is a mod-
ified form of folate that was designed to have greatly
increased binding affinity for DHFR compared with
the parent molecule. The UK licensing information for
methotrexate in patients with RA recommends oral, sub-
cutaneous or intramuscular administration as a single
weekly dose of 15–20 mg, although in clinical practice
doses of up to 25 mg can be used. In pharmacokinetic
assessments, compared with the subcutaneous form,
oral methotrexate has demonstrated significant inter-
patient and interdose variability in bioavailability26–30.
Reported bioavailability ranges from <30% to >70%,
with an apparent plateau for single oral doses >15 mg
per week31, although the upper limit of bioavailability
can be increased by splitting the dose32. In a study involv-
ing 143 patients who alternately received both intramus-
cular and oral methotrexate, therapeutic benefits and
improvements in adverse events were observed with the
parenteral formulation33.
Oral methotrexate is primarily absorbed from the
small intestine by the proton-coupled folate transporter,
which is active at pH 5.5 and transports reduced folates
and methotrexate, but shows virtually no activity at
neutral pH34. Around 50% of circulating methotrexate
is bound to plasma proteins35. Excretion of methotrexate
is primarily renal, occurring through both glomerular
filtration and active tubular secretion36,37. A small pro-
portion is subject to first-pass metabolism in the liver,
producing 7‑OH-methotrexate36, which has therapeutic
efficacy in cancer38 and intracellular activity in putative
anti-inflammatory pathways in vitro39–41. Approximately
10% of excretion is biliary, and some enterohepatic recy-
cling also occurs (primarily of 7‑OH‑methotrexate)42,43.
The plasma half-life of low-dose methotrexate varies
from 4.5 h to 10.0 h, and can be prolonged in patients
administration, methotrexate typically reaches peak
plasma concentrations after 1–2 h, and by 24 h has
almost completely disappeared from the circulation45.
Cellular uptake of methotrexate is mediated by folate
transporter 1 (FOLT, also known as reduced folate car-
rier protein (RFC1), encoded by SLC19A1), with some
contribution from the α, β and γ folate receptors34 (FIG. 1).
Methotrexate is exported from cells by the ATP-binding
cassette proteins (ABCC1–ABCC5 and ABCG1), which
have biological activity for a number of chemotherapeu-
tic agents34. In common with naturally occurring folates,
a proportion of intracellular methotrexate undergoes
polyglutamation (the serial addition of glutamate res-
idues)46,47. This process is catalysed by folylpolygluta-
mate synthetase (FPGS), and produces methotrexate
derivatives with various polyglutamate chain lengths.
Methotrexate derivatives with chains longer than three
glutamate residues are not substrates for the ABCC
exporter proteins, so polyglutamation — balanced by the
reverse process, deglutamation (catalysed by lysosomal
γ‑glutamyl hydrolase (GGH)) generates a steady-state
level of intracellular methotrexate48. As well as causing
retention of intracellular methotrexate, polyglutamation
also alters the cellular effects of methotrexate, enabling
it to interact with other elements of the purine-synthesis
pathways49,50 (FIG. 1).
Mechanism of action
In patients receiving chemotherapy, methotrexate is
administered in short courses at very high doses, pro-
ducing measurable cytotoxic effects. These time courses
and doses can be readily studied in vitro, and the effects
of methotrexate on depletion of thymidine and purine
residues and cell-cycle arrest at S1 are well established51.
Determination of the mechanism of action of low-dose
methotrexate administered for a long time in the treat-
ment of RA is more challenging. An appreciable time lag
occurs in vivo before a clinical benefit is seen. In addi-
tion, the accumulation of intracellular polyglutamated
methotrexate is a gradual process. Although these fac-
tors limit the applicability of in vitro studies, a number of
mechanisms that are potentially involved in determining
the efficacy of methotrexate in RA are currently under
investigation (FIG. 2).
Folate antagonism
The depletion of metabolite levels observed with the use
of methotrexate in patients receiving cancer treatment
could also be relevant to the replication or survival of
lymphocytes or pathogenic cell types in RA (FIG. 3). The
role of methotrexate-induced depletion of purine and
pyrimidine pools in T‑lymphocyte activity has been
investigated in vitro52,53. In primary human T lympho-
cytes, treatment with low-dose methotrexate reduced
levels of ATP and GTP, and increased synthesis of UTP,
compared with untreated control cells54. The effects of
these changes were a reduction in proliferation and an
increase in apoptosis in mitogenically stimulated cells,
but not in resting cells. Methotrexate-induced apoptosis
was also demonstrated ex vivo in activated T cells from

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Folate Of note, the generation of adenosine seems to be driven

receptors RFC1 ABCC1–ABCC5
FPGS
Methotrexate MethotrexateGlu(1–7)
GGH
DHFR TYMS ATIC
Figure 1 | Pharmacodynamics of methotrexate in RA. Cellular methotrexate
uptake is primarily governed by folate transporter 1 (FOLT, also known as RFC1) with
limited contribution from folate receptors. Cellular effluxNature Reviews | Rheumatology
of methotrexate is governed
by ATP-binding cassette (ABCC)-family transporters. Within the cell, methotrexate is
serially polyglutamated in a reversible reaction governed by folylpolyglutamate
synthetase (FPGS) and γ‑glutamyl hydrolase (GGH). Polyglutamation of methotrexate
(producing methotrexateGlu(1–7)) alters its spectrum of activity, increasing inhibition of
dihydrofolate reductase (DHFR), thymidylate synthetase (TYMS), and 5‑aminoimidazole-
4‑carboxamide ribonucleotide transformylase (ATIC). Polyglutamation also reduces
efflux from the cell via ABCC transporters.
six patients with RA55. Addition of purines and pyrimi-
dines (particularly thymidine) to cell cultures abrogated
the induction of apoptosis55,56.
Evidence from clinical practice does not support the
purine–pyrimidine-antagonism hypothesis, however.
Concomitant administration of folate has been investi-
gated as a way to mitigate the toxic and adverse effects
associated with methotrexate therapy; folate admin-
istered up to six times per week reduces the incidence
of treatment-related adverse effects and liver-function
abnormalities23. However, folate co‑therapy does not
result in a loss of clinical benefit, suggesting that a reduc-
tion in folate-dependent purine–pyrimidine synthesis
is not a major factor underpinning the clinical effect of
methotrexate in RA.
Adenosine signalling
Adenosine is an important anti-inflammatory mediator,
with wide-ranging actions across a variety of immune-
cell subtypes57. The potential role of adenosine in the
activity of methotrexate was suspected because of the
observation of an inhibitory effect of polyglutamated
methotrexate on bifunctional purine biosynthesis protein
PURH (also known as 5‑aminoimidazole-4‑carbox­amide
ribonucleotide (AICAR) formyltransferase (ATIC))49.
ATIC is involved in purine metabolism, and its inhibition
by methotrexate results in increases in both intracellular
AICAR levels and extracellular adenosine levels49 (FIG. 4).
largely by ATP or ADP release and extracellular cleavage,
although the main effector molecule seems to be aden-
osine rather than ATP. The accumulation of AICAR
has been demonstrated to occur with both high-dose
metho­trexate in cancer therapy and low-dose metho­
trexate in the treatment of psoriasis, through detection
of the AICAR metabolite aminoimidazole carboxamide
in urine58,59.
Adenosine has an extremely short half-life (meas-
ured in seconds) because of its degradation by adeno-
sine deaminase60. Adenosine acts as a paracrine signalling
agent via four distinct purinergic G‑protein-coupled
receptors: adenosine receptor A1 (ADORA1), adeno-
sine receptor A2a (ADORA2A), adenosine receptor A2b
(ADORA2B) and adenosine receptor A3 (ADORA3)61,62.
The anti-inflammatory effects of adenosine are largely
thought to be associated with signalling via ADORA2A63.
In RA, adenosine receptors are overexpressed on immune
cells, probably because of the high levels of TNF64–66. Such
overexpression might ‘prime’ patients with RA to respond
to any potentiation of adenosine signalling, such as that
induced by methotrexate therapy57. ADORA3, which is
the most abundantly expressed adenosine receptor in
RA, also potentially has a role in this process67. Signalling
via ADORA3 downregulates production of TNF and
nuclear factor κ‑light-chain-enhancer of activated B cells
(NF‑κB)68, and a specific agonist of ADORA3 has shown
promise in patients with RA who have high ADORA3
expression69. The four adenosine-receptor subtypes are
all expressed by synovial cells in RA, and expression of
ADORA2A and ADORA2B is higher in patients receiving
methotrexate therapy than in untreated patients67.
Studies conducted both in vitro and in vivo have
provided data that support the adenosine-signalling
hypothesis. In vitro, treatment with short courses of low-
dose methotrexate led to reduced adherence of neutro-
phils to fibroblasts70. This effect was associated with the
release of adenosine from fibroblasts, and abrogated by
the addition of adenosine deaminase. In experiments
involving the mouse air-pouch model of inflammation,
weekly intraperitoneal injections of methotrexate led to
reduced leukocyte accumulation within inflammatory
exudates, compared with saline injections71. This change
was partially reversed by injection of adenosine deam-
inase into the air pouch, and was completely reversed
by injection of an ADORA2A antagonist, whereas an
ADORA1 antagonist had no effect71. Similar results were
obtained in experiments involving induction of inflam-
mation in rat mesenteric venules72. Methotrexate respon-
siveness in the mouse air-pouch model demonstrated
strain specificity, and was linked to a single gene locus
that seemed to confer methotrexate responsiveness via
adenosine signalling73.
The role of adenosine in the action of methotrexate
has also been explored in gene-knockout mouse models.
In a mouse model of peritonitis, treatment with meth-
otrexate increased adenosine levels in inflammatory
exudates in all strains of mice tested, compared with
pretreatment levels74. In wild-type mice and Adora3-
knockout mice, but not in Adora2a-knockout mice,

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unresponsive to methotrexate had significantly lower

Folate antagonism
Adhesion molecules Adenosine signalling
Cytokines Methotrexate Methyl
donors
Eicosanoids and MMPs Reactive oxygen species
Figure 2 | Potential mechanisms of action of low-dose
methotrexate in rheumatoid arthritis. Several
mechanisms have been proposed to explain the observed
Nature Reviews | Rheumatology
clinical effects of methotrexate in rheumatoid arthritis,
including antagonism of folate-dependent processes,
stimulation of adenosine signalling, inhibition of
methyl-donor production, generation of reactive oxygen
species, downregulation of adhesion-molecule expression,
modification of cytokine profiles and downregulation of
eicosanoids and matrix metalloproteinases (MMPs).
methotrexate treatment also reduced leukocyte accu-
mulation and TNF levels, suggesting that Adora2a is the
principal anti-inflammatory adenosine receptor in this
model74. Furthermore, in the air-pouch model of inflam-
mation, the response to methotrexate was not seen in
5ʹ‑nucleotidase (CD73)-deficient mice, suggesting that
the majority of extracellular adenosine is generated by
cleavage of released adenine nucleotides, rather than
by direct release of adenosine75. In a study of human
microvascular endothelial cells, a specific inhibitor of
CD73 abolished the increase in extracellular adenosine
level that resulted from treatment with methotrexate76. In
a study involving 17 patients with RA, levels of synovial
cyclic AMP were inversely correlated with disease activity
scores, erythrocyte sedimentation rates and IL‑18 levels77.
In a case report published in 2010, infusions of ATP led
to improvements in disease activity, fatigue, pain and
physical functioning (versus levels achieved with metho­
trexate alone) in a patient with RA78, but other studies of
this approach are lacking.
Adenosine might be one of the main mediators of
downregulation of the activation and proliferation of
T lymphocytes. Regulatory T cells (Treg cells) that express
forkhead box protein P3 (FOXP3) are thought to be an
important immunoregulatory cell subset involved in
reducing type 17 T helper cell (TH17) activity. FOXP3+
Treg cells are potentially dysregulated in autoimmune
diseases such as multiple sclerosis79, systemic lupus
erythematosus80 and RA81,82. These cells express CD39
(ectonucleoside triphosphate diphosphohydrolase 1),
which cleaves ATP and ADP to AMP, and CD73, which
cleaves AMP to release adenosine. The beneficial effects
of low-dose methotrexate in RA might, therefore, be
mediated by potentiating the generation of adenosine by
FOXP3+ Treg cells, creating an immunotolerant environ-
ment. In support of this hypothesis, the results of a pro-
spective study showed that patients with RA who were
pretreatment expression of CD39 on Treg cells than did
methotrexate-responsive patients83.
A number of chemical agents in widespread use pos-
sess anti-adenosine activity, including caffeine. Clinical
observations suggest that patients with RA who have
high caffeine intakes have impaired methotrexate
responsiveness84. Animal models have also shown a
modest suppressive effect of caffeine and theophyl-
line (another adenosine antagonist) on the response to
metho­trexate85. However, the results of a large observa-
tional study that stratified patients with RA by levels of
caffeine intake found no significant effect of this agent
on the effectiveness of methotrexate86. Notably, levels
of adenosine deaminase in synovial fluid and serum
are higher in patients with RA than in patients with
osteo­arthritis (OA)87, and serum levels of adenosine
deaminase might also correlate with RA disease activity
scores88.
Conflicting evidence also exists with regard to other
aspects of the adenosine signalling hypothesis. In mice
with antigen-induced arthritis, methotrexate efficacy
was not affected by treatment with adenosine-receptor
antagonists, although the methotrexate doses used in
this study were 10 times higher than those used in clin-
ical practice89. By contrast, another study in the same
mouse model showed that adenosine signalling via
ADORA2B drove bone destruction and abrogated the
beneficial effect of methotrexate on bone resorption90.
In an ex vivo study, RA synovial fluid demonstrated
antiapoptotic properties when added to cultured neutro-
phils91. This activity correlated positively with the syno-
vial fluid adenosine concentration, and was reversed by
addition of adenosine deaminase, suggesting that adeno-
sine promotes the survival of neutrophils within joints91.
In a study involving seven patients with RA, serum levels
of adenosine did not change during or after methotrex-
ate therapy (although, given the very short half-life of
adenosine, these measurements might not have accu-
rately reflected local tissue concentrations)92. In addi-
tion, no change was seen in the AICAR concentration
within erythrocytes up to 1 week after administration
of the first dose of methotrexate; however, this timescale
might be too short to observe the intracellular accumula-
tion of methotrexate polyglutamates92. In a clinical study
involving 10 patients with inflammatory bowel disease,
methotrexate injection had no significant short-term
effect on serum or rectal adenosine concentrations93.
Interference with methyl donation
Many cellular metabolic functions involve the addition of
methyl groups to biomolecules, including DNA and pro-
teins. A number of folate derivatives are capable of acting
as methyl donors, and are involved in the regeneration of
methionine from homocysteine. Methionine is a substrate
for conversion to S‑adenosylmethionine, the main methyl
donor within the cell. These processes are dependent on
DHFR, which is inhibited by methotrexate (FIG. 5).
The polyamine pathway, which includes the synthesis
of spermine and spermidine, is one such methyl-donation-
dependent metabolic pathways. Levels of polyamines

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Pentose DHFR
Ribose 5P phosphate
pathway

DHF THF
GAR

Methotrexate/
methotrexateGlu(1–7) 5-CH3-THF 5,10-CH2-THF
FGAR

MTHFR

AICAR UMP

ATIC TYMS

FAICAR
AMP

IMP DNA synthesis dTMP

GMP

Figure 3 | Potential role of methotrexate in depletion of the nucleotide pool. Methotrexate and its polyglutamated
Nature Reviews | Rheumatology
derivatives inhibit the enzymes dihydrofolate reductase (DHFR) and thymidylate synthetase (TYMS), reducing de novo
synthesis of thymidine residues. Methotrexate polyglutamates can inhibit 5‑aminoimidazole-4‑carboxamide
ribonucleotide (AICAR) transformylase (ATIC), reducing synthesis of adenosine and guanidine precursors from the
pentose-phosphate pathway. The net result is reduced DNA synthesis and subsequent cytostasis. DHF, dihydrofolate,
dTMP, deoxythymidine monophosphate; FAICAR, 5‑formamidoimidazole-4‑carboxamide ribotide; FGAR, N2-formyl-
N1-(5‑phospho-D‑ribosyl)glycinamide; GAR, N1-(5‑phospho-D‑ribosyl)glycinamide; IMP, inosine monophosphate;
MTHFR, methylenetetrahydrofolate reductase; ribose 5P, ribose 5‑phosphate; THF, tetrahydrofolate; UMP, uracil
monophosphate.

in synovial fluid are higher in patients with RA than in
those with OA94. In experiments conducted both in vitro
and in vivo, treatment with methotrexate reduced levels
of polyamines and rheumatoid factor, compared with
either pretreatment levels or levels in untreated cells; this
inhibition was antagonized by addition of spermidine,
spermine or S-adenosylmethionine95–97. Overexpression
of the polyamine-synthesizing enzyme ornithine decar-
boxylase also antagonizes the effect of methotrexate on
T lymphocytes98. These results suggest that downregu-
lation of polyamine synthesis via inhibition of methyl-
donor generation could contribute to the mechanism
of action of methotrexate. However, although the trans-
methylation inhibitor 3‑deaza-adenosine has demon-
strated antagonism of transmethylation in vivo, it has
not shown any therapeutic benefit in RA99,100.
Patients with RA have global DNA hypomethylation,
compared with healthy controls, and DNA methylation is
upregulated by methotrexate therapy101. Hypomethylation
of DNA in synovial fibroblasts might conceivably
result from depletion of S‑adenosylmethionine due to
high turnover of polyamines102. However, the role of
genome-wide hypomethylation in the pathology of RA
is unknown, and no data are available to demonstrate that
site-specific DNA methylation is altered by methotrexate,
nor whether such an alteration could be involved in the
therapeutic efficacy of this drug in RA.
Reactive oxygen species
Methotrexate therapy results in an increase in the
level of apoptosis in transformed T cells, the extent of
which is dependent on the dose and duration of treat-
ment103,104. This effect is associated with the generation
of reactive oxygen species (ROS), and is ameliorated by
the addition of antioxidants. Antagonism of polyamines
might also have a role in the generation of ROS, as poly­
amines have ‘oxygen-scavenging’ properties. An alter-
native mechanism for ROS generation by methotrexate
is thought to involve tetrahydrobiopterin, which is an
important cofactor for a number of enzymes, and a
ligand of endothelial nitric oxide synthase (eNOS)105,106.
Tetrahydrobiopterin can be generated de novo from
GTP107, and one of the intermediates in this process
(neopterin) is associated with RA108,109 and with levels
of IL‑2 receptor109. A recycling pathway also exists to
convert oxidized dihydrobiopterin back to tetrahydro­
biopterin. This process is regulated by DHFR, and
antagonism of this pathway by methotrexate might
result in reduced levels of tetrahydrobiopterin (FIG. 6).
In mice, both tetrahydrobiopterin and dihydrobiop-
terin can act as ligands for eNOS105. Tetrahydrobiopterin
drives production of nitric oxide, whereas dihydrobiop-
terin uncouples eNOS, leading to superoxide generation.
In genetically engineered mice with constitutively low lev-
els of tetrahydrobiopterin, treatment with methotrexate

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Extracellular Paracrine signalling antigen found on T lymphocytes, which acts as a homing

ATP ADP AMP Adenosine
CD73
ADOR
ATP ADP AMP Adenosine
AMPDA ADA
MethotrexateGlu(1–7) IMP Inosine
↑ AICAR
ATIC
Anti-inflammatory effects
Intracellular FAICAR
Figure 4 | Proposed mechanism by which methotrexate increases adenosine
signalling. Methotrexate (in particular polyglutamated methotrexate)
Nature Reviewsantagonizes the
| Rheumatology
activity of 5‑aminoimidazole-4‑carboxamide ribonucleotide (AICAR) transformylase
(ATIC), resulting in accumulation of AICAR, which antagonizes the activity of adenosine
deaminase (ADA) and AMP deaminase (AMPDA), increasing concentrations of AMP and
adenosine, which are externalized by the cell. AMP in the extracellular environment is
cleaved to adenosine by the ecto‑5ʹ‑nucleotidase CD73, which is potentiated by AICAR.
Extracellular adenosine signals in an autocrine and paracrine fashion via adenosine
receptors (ADOR). FAICAR, 5‑formamidoimidazole-4‑carboxamide ribotide.
reduces tetrahydrobiopterin levels, resulting in increased
superoxide generation compared with placebo treatment.
In wild-type mice, treatment with methotrexate increases
levels of dihydrobiopterin without affecting tetrahydro-
biopterin levels or eNOS activity.
Methotrexate-induced, ROS-driven apoptosis of
T lymphocytes has only been demonstrated using a
transformed cell line (Jurkat cells) in vitro. In these
cells, rates of apoptosis — and ROS levels — increased
with methotrexate exposure (compared with untreated
cells)110. These effects were associated with increased
c‑Jun N‑terminal kinase (JNK) signalling, and were
reversed by the addition of tetrahydrobiopterin. Unlike
transformed T cells, fibroblast-like synoviocytes showed
adenosine-dependent JNK activation that was unaf-
fected by tetrahydrobiopterin supplementation111. In
fibroblast-like synoviocytes, IL‑6 stimulates prolifera-
tion and ROS production, both of which are inhibited
by treatment with methotrexate112.
Adhesion-molecule expression
Adhesion-molecule expression is an area of growing
research interest in a number of chronic inflammatory
conditions. For example, the cutaneous lymphocyte
signal for the skin in psoriasis, is downregulated by
low-dose methotrexate treatment113. Measurements of
the expression of adhesion molecules might provide
response biomarkers in RA therapy. For example, low-
dose methotrexate could mediate its effect on T lympho-
cytes not by induction of apoptosis, but by reduction of
the expression of adhesion molecules and the activation
and trafficking of cells into joints114.
In synovial biopsies taken from patients with RA
16 weeks after initiation of methotrexate therapy, expres-
sion of the adhesion molecules E‑selectin and vascular
cell adhesion protein 1 (VCAM1) were reduced versus
pretreatment levels115. Levels of IL‑1 and TNF, as well
as the numbers of inflammatory cells in the joint, were
also lower than before treatment115. Methotrexate might,
therefore, reduce trafficking of inflammatory cells to
the joint by reducing the expression of adhesion mol-
ecules. Methotrexate treatment of antigen-stimulated
peripheral blood mononuclear cells caused an adeno-
sine-independent reduction in expression of cutane-
ous lymphocyte antigen, and an adenosine-dependent,
folate-dependent reduction in intercellular adhesion
molecule 1 (ICAM1)114. ICAM1 is involved in the migra-
tion of leukocytes, suggesting that methotrexate treat-
ment has a role in restricting immune-cell trafficking
to the joint. Serum levels of a number of soluble adhe-
sion molecules (sICAM1, sVCAM1 and sE‑selectin) are
also elevated in patients with RA relative to those with
OA, and are downregulated by treatment with metho-
trexate116. Reductions in levels of sE‑selectin correlate
with improvements in clinical markers of RA severity117.
Furthermore, stimulation of ADORA2A expression or
adenosine signalling in cell lines in vitro can lead to
reductions in levels of E‑selectin, VCAM1 and ICAM1
(REFS 118,119). Alteration in adhesion-molecule expression
could be a downstream manifestation of methotrexate-
induced potentiation of adenosine signalling118,119.
Reduction of adhesion-molecule expression by
methotrexate might interfere with cell–cell contact that
is required for inflammation. Results from a study of
the bidirectional interaction between synovial fibro-
blasts and peripheral blood T lymphocytes suggested
that fibroblast-derived IL‑15 drives expression of TNF,
INFγ, IL‑17, CD25 and CD69 from T lymphocytes, in
a process that is dependent on cell contact120. This, in
turn, drives expression of ICAM1 and IL‑15 by synovial
fibroblasts, creating an inflammatory feedback loop.
Methotrexate antagonizes this crosstalk and reduces
adhesion of T lymphocytes to fibroblasts in culture120.
Expression of cell-surface Fcγ receptors (FcγRs),
which are involved in immune-complex signalling,
might also be involved in the activity of methotrexate. In
a study conducted both ex vivo and in vitro, methotrexate
treatment of patients with RA resulted in reduced expres-
sion of FcγRI and FcγRIIa on monocytes compared with
pretreatment levels, whereas levels of FcγRIIIa were not
significantly altered121. In patients with early RA, base-
line FcγRIIIa expression on CD14+ monocytes negatively
correlated with the level of response to methotrexate
therapy122.

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Eicosanoids and metalloproteinases

Methotrexate/ DHFR
methotrexateGlu(1–7)
DHF THF
MS
Homocysteine 5-CH3-THF 5,10-CH2-THF
MTHFR
SAH Methionine
SAM 10-CH3-THF
Methyl donor
Figure 5 | Role of dyhydrofolate reductase (DHFR) in the generation of methyl
donors. S‑Adenosylmethionine (SAM) is the main cellular methyl donor in
humans. Synthesis of SAM is dependent on methionine, which
Nature is generated
Reviews from
| Rheumatology
homocysteine, with 5‑methyl-tetrahydrofolate (5‑CH3-THF) as a cofactor. 5‑CH3-THF
production is dependent on tetrahydrofolate (THF) production. Methotrexate acts
to antagonize DHFR, and thereby reduces the availability of THF and metabolites
for subsequent methyl-donor production. Some evidence also suggests that
methotrexate polyglutamates directly antagonize synthesis by methionine
synthetase (MS). DHF, dihydrofolate; MTHFR, methylenetetrahydrofolate reductase;
SAH, S‑adenosylhomocysteine.
Modification of cytokine profiles
The extent to which methotrexate modifies RA pathobi-
ology via a direct influence on cytokine production by
immune cells is unclear. Evidence suggests that even a
single dose of methotrexate can inhibit the production of
proinflammatory cytokines123,124. After ex vivo activation,
T cells isolated from methotrexate-treated patients with
RA have a reduced capacity to produce the cytokines char-
acteristic of type 1 and type 2 T helper cells (namely IFNγ,
IL‑4 and IL‑13), as well as reduced production of TNF and
granulocyte macrophage colony-stimulating factor, com-
pared with T cells from methotrexate-naive patients125.
Methotrexate treatment reduces the proportion of TNF-
positive CD4+ T cells in peripheral blood of patients with
RA, and increases the proportion of IL‑10‑positive T cells,
relative to pretreatment levels126. Co‑culture of synovial
fibroblasts and T cells from patients with RA induces
production of proinflammatory cytokines including IL‑6,
IFNγ and IL‑17; this effect is attenuated if the T cells are
taken from methotrexate-treated patients, a difference
that has been attributed to disruption of cell adhesion
rather than cytokine dysregulation per se120. Although a
general inhibitory effect of methotrexate on NF‑κB sig-
nalling has been proposed127, information is lacking on
the direct molecular mechanisms by which cytokine net-
works might be modified by methotrexate, and the extent
to which they underpin (rather than merely accompany)
its therapeutic efficacy.
Treatment of RA ultimately results in abrogation of the
inflammatory response in joints, and the effects of metho­
trexate on eicosanoids and metalloproteinases (MMPs)
as end-mediators of joint damage have also been studied.
Prostaglandin E2 (PGE2) production by prostaglandin
G/H synthase 2 (PTGS2, also known as cyclooxygenase‑2)
was lower in whole blood from methotrexate-treated
patients with RA than in blood from healthy controls129.
In cultured synovial cells from patients with RA, meth-
otrexate treatment caused a dose-dependent reduction
in PGE2 production, without affecting PTGS1 or PTGS2
mRNA expression128,129. In a rabbit model of antigen-
induced arthritis, intra-articular levels of PGE2 and
thromboxane B2 were lower in methotrexate-treated ani-
mals than in saline-treated controls130. In a study of eight
metho­trexate-naive patients with RA, ex vivo production
of leukotriene B4 was lower in samples of neutrophils or
plasma taken 24 h after methotrexate administration than
in pretreatment samples131. In patients with early RA, pre-
treatment serum concentrations of MMP1, MMP3, MMP9
and MMP13 fell significantly after 6 months of treatment
with methotrexate132–134. Levels of tissue inhibitor of met-
alloproteinases 1 (TIMP1) can either fall133 or rise134 with
methotrexate treatment, which leads to reductions in both
the ratio of MMP to TIMP, and MMP activity132.
Methotrexate-induced changes lead to reductions in
inflammation and in the degradation of cartilage. These
changes contribute to the clinical response to methotrex-
ate, but whether they represent a specific mechanism of
action or a common final pathway is unclear. For example,
changes in PGE2 production and TIMP1 levels could rep-
resent downstream effects of the influence of methotrex-
ate on production of IL‑1 and IL‑6, respectively. Some of
these changes are also observed following treatment with
NSAIDs, and the methotrexate-specific pathways have yet
to be delineated.
Integrating the proposed mechanisms
At present, the hypothesis with the most-robust evidence
suggests that methotrexate mediates its effect in RA via
potentiation of adenosine signalling. However, given the
difficulty of directly assaying adenosine, and the lack of
robust in vitro models of low-dose methotrexate therapy,
the role of adenosine is yet to be definitively confirmed.
The potentially variable influence of adenosine (and,
therefore, of methotrexate) on different cell types should
also be determined.
Evidence also exists for methotrexate-dependent
pathways that involve generation of ROS, synthesis of
methyl donors and maintenance of nucleotide pools,
and methotrexate conceivably has pleiotropic thera-
peutic effects on various immune cells and mediators,
resulting in an overall dampening of the inflammatory
response. Furthermore, some proposed mechanisms
of methotrexate activity, including adhesion-molecule
regulation and alteration of cytokine networks, could
represent indirect effects rather than primary responses.
Improving our understanding of pivotal methotrexate
pharmacodynamics should facilitate tailoring of therapy
in early RA and implementation of a stratified approach

NATURE REVIEWS | RHEUMATOLOGY VOLUME 12 | DECEMBER 2016 | 737

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Extracellular Neopterin Pterin Tetrahydrobiopterin

Dihydroneopterin Dihydrobiopterin

Lactoyltetra-
hydrobiopterin Dihydrobiopterin

Pyruvoyl Sepiapterin
tetrahydropterin reductase DHFR
Pyruvoyl reductase
GTP cyclo- tetrahydropterin
hydrolase Dihydro- synthetase Pyruvoyl
GTP neopterin tetra- Tetrahydrobiopterin Methotrexate
triphosphate hydropterin

Sepiapterin Sepiapterin DHPR

reductase reductase
Quinonoid
Intracellular Siapterin dihydrobiopterin

Figure 6: | Metabolism of pterins. Tetrahydrobiopterin can be generated from GTP through a series of enzymatic steps.
Tetrahydrobiopterin is a co-factor in a number of cellular reactions, and is oxidized to quinonoid dihydrobiopterin, which
can be recycled by dihydropteridine reductase (DHPR), and to dihydrobiopterin, which is Nature
recycledReviews | Rheumatology
by dihydrofolate
reductase (DHFR). The latter enzyme is antagonized by methotrexate).

to treatment. These developments could synergize with
early aggressive therapy and treat‑to‑target strategies to
improve patient outcomes.
Biomarkers of response to methotrexate
With the advent of expensive biologic therapies that
are highly effective only in subpopulations of patients,
consideration must be given to the rational use of these
medications. Similarly, the ability to identify in advance
the ~40% of patients who will have a good response
to methotrexate therapy would be hugely beneficial24.
In this regard, the problem of methotrexate resistance in
cancer therapy has led to the characterization of meta­
bolic pathways that control methotrexate levels, and
to the identification of several perturbations in these
pathways that can result in treatment resistance.
In the study of RA, data enabling prediction of the
response to methotrexate are lacking. Polyglutamation
seems to be integral to the maintenance of steady-state
methotrexate concentrations and to methotrexate
activity, so polyglutamate levels might correlate with
treatment effectiveness. Current studies of inflamma-
tory arthropathy have identified accumulation of only
methotrexateGlu(1–5) (1–5 glutamate chain lengths) in
erythrocytes, in contrast to the 1–7 chain lengths found
in leukaemia cells. This difference might be related to
differing drug kinetics across different cell types or
the lower methotrexate doses used in RA than oncol-
ogy. However, although the results of some studies of
erythro­cytes in RA and JIA suggest that levels of polyglu-
tamated methotrexate (particularly levels of long-chain
polyglutamates) are associated with the response to treat-
ment47,135–138, this association has not been observed in all
studies139,140. Another potential benefit of high levels of
methotrexate polyglutamates is a low risk of develop-
ing antibodies against the anti-TNF agent infliximab141.
This observation is of particular interest in patients
with RA who are resistant to conventional DMARDs, as
these patients often require switching to anti-TNF bio-
logic therapies such as infliximab. Increasing evidence
suggests that the development of anti-drug antibodies
is a driver of loss of treatment efficacy; amelioration of
this risk, therefore, is likely to help preserve treatment
response142.
Parameters such as the dosage, timing and the route of
methotrexate administration143, but also the presence
of single-nucleotide polymorphisms (SNPs) in folate-
pathway genes SLC19A1 and GGH144, might influence
methotrexate polyglutamate accumulation. Notably,
modelling studies suggest that the time required to
achieve steady-state methotrexate polyglutamate con-
centrations in erythrocytes is in excess of 1 year, which
is considerably longer than the typical 4–6 month trial
of treatment in clinical practice145. Consequently, meas-
urement of erythrocyte methotrexate polyglutamate
levels currently has no practical utility in predicting the
response to this treatment, as steady-state levels will not
have been reached by the end of the treatment trial.
Methotrexate polyglutamation occurs more rapidly
in T lymphocytes than in erythrocytes (2 weeks ver-
sus 49 weeks, respectively, to reach a steady state), and
average polyglutamate chain length is higher in T lym-
phocytes145. However, no studies have yet determined a
correlation between lymphocyte methotrexateGlu(1–7) levels
and therapeutic response. Erythrocyte folate levels have
also been investigated for determination of methotrexate

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responsiveness; in one study, a low baseline folate level
was associated with a poor response to methotrexate136,
and low levels of polyglutamated folate (in patients on
established methotrexate therapy) are associated with low
disease activity138.
The effects of methotrexate on folate and purine
metabolism have been investigated by gene-expression
profiling. Expression of genes encoding folate-metabo-
lizing enzymes and transporters is upregulated in periph-
eral blood cells from methotrexate-naive patients with
RA, compared with those in peripheral blood cells from
healthy controls and methotrexate-treated patients146.
However, well-powered studies that correlate baseline
gene expression with long-term patient outcomes during
methotrexate treatment are lacking.
At the genomic level, HLA‑DRB1 ‘shared epitope’
alleles have been associated with a lack of response to
methotrexate monotherapy147. In addition, a number of
SNPs have been investigated for the prediction of metho­
trexate treatment response. These studies have generally
been small, with varied definitions of methotrexate
response, leading to identification of a variety of dif-
ferent and sometimes contradictory correlates. SNPs in
methotrexate transport and metabolism genes (SLC19A1,
FOLR1, FPGS, GGH and ABCB1) and purine-synthesis
genes (ATIC, AMPD1, ADA and ADORA2A) have
been investigated in methotrexate-treated patients with
RA148,149. The C3435T (rs1045642) SNP in ABCB1 was
significantly associated with the risk of poor response to
methotrexate, and a possible significant (P = 0.05) inter-
action of SNPs in GGH and ABCB1 was suggested by
multifactor dimensionality reduction (a nonparamet-
ric alternative to traditional statistical methods, such as
logistic regression; multifactor dimensionality reduction
is used to identify interactions among discrete varia-
bles that influence a binary outcome)148. Given the large
number of post hoc analyses conducted in this study148,
however, it is unclear whether multiple-test correction
is appropriate for this novel analysis method. Univariate
analysis of SNPs in the ADA and ADORA2A genes cor-
related with methotrexate response, but the association
was lost after multiple-test correction149. In regression
analy­sis, SNPs in FPGS, TYMS and DHFR were associ-
ated with methotrexate response and accounted for 9.6%
of the variance in the change in disease activity149.
SNPs in genes associated with adenosine release
(AMPD1, ATIC, ITPA, MTR and MTRR) were studied
in 205 methotrexate-treated patients with newly diag-
nosed RA150. Specific alleles in AMPD1, ATIC and ITPA
were significantly associated with the likelihood of a
good response (defined as a disease activity score ≤2.4).
The presence of these SNPs, along with a further SNP
in MTHFD1, were combined with clinical parameters
of baseline disease activity, sex, smoking status and the
presence of rheumatoid factor to produce a predictive
metric for methotrexate response151. The scoring system
for this metric ranged from 0 to 11.5; scores ≤3.5 had
a true-positive response rate of 95% and scores ≥6 had a
true-negative predictive rate of 86%151. On validation with
a sample of 75 patients with RA, a negative predictive
value of 81% and positive predictive value of 47% were
achieved, when comparing pretreatment metric values
with clinical responses at 6 months. However, despite
the promising negative predictive value reported for the
metric, only 75% of the cohort had a predictive score at
baseline (that is, ≤3.5 or ≥6) and of those only 26 were
predicted to be non-responders from a final total of 50
clinical non-responders152. This result suggests, therefore,
that at best this metric might be able to identify only a
subpopulation of non-responders at baseline. This model
also requires further validation in larger cohorts.
Large numbers of SNPs in methotrexate-pathway
genes have now been investigated. A multicentre SNP
analysis has identified several gene–gene interactions
associated with methotrexate responsiveness153. A gen-
otype containing specific SNPs in the ATIC, SLC19A1
and ITPA genes was associated with poor response to
methotrexate in two patient cohorts153. In a third cohort,
age, sex and anti-citrullinated protein antibody (ACPA)
status were also required to enable prediction of respon-
siveness; the genotype was associated with methotrexate
response only in ACPA-positive older men153. A different
study revealed potential associations between metho-
trexate responsiveness and the presence of SNPs in GGH,
as well as in ATIC and SLC19A1 genes154.
Methotrexate response in early RA is also associated
with a number of genetic variants in CHST11, which
encodes carbohydrate sulfotransferase 11155. Genome-
wide association studies (GWAS) identified potential
risk loci for poor methotrexate response, including
confirmation of previously identified associations with
DHFR, FPGS and TYMS genes156. To date, SNPs in only
two genes have been subjected to meta-analyses. The
results showed that the 80G>A SNP (rs1051266) in
SLC19A1 has a significant association with methotrexate
efficacy157. By contrast, meta-analysis demonstrated that
two MTHFR SNPs have no significant association with
methotrexate efficacy158.
The lack of consensus from studies of SNPs is per-
haps unsurprising, given the relatively small populations
genotyped and the pleotropic nature of RA. The perfor-
mance of GWAS with larger populations should expand
the list of ‘genes of interest’ with novel loci that have not
previously been considered. Furthermore, pooling of
SNP analyses to maximize sample sizes could help to
clarify the role of these genes in response prediction.
Conclusions and future directions
Despite a long history of clinical application and demon-
strable efficacy in RA, the precise mechanism by which
low-dose methotrexate provides therapeutic benefit
remains incompletely understood. The adenosine-signal-
ling hypothesis has received considerable support from
studies in animal models, some of which are corrobo-
rated by the results of human studies, but this hypoth-
esis cannot yet explain all the benefits of methotrexate.
Several other competing hypotheses also exist, and deter-
mining the relevance of each is difficult. Methotrexate
has a slow onset of action and intermittent dosing reg-
imen in vivo, which are challenges for in vitro model-
ling. Furthermore, methotrexate might have pleiotropic
effects that differ in dominance between individuals.

NATURE REVIEWS | RHEUMATOLOGY VOLUME 12 | DECEMBER 2016 | 739

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Perhaps the two most pertinent clinical questions
relating to methotrexate in the treatment of RA are
where does this drug fit into the therapeutic para-
digm, and how can we predict which patients will
respond best to this therapy? The answer to the latter
question will probably guide responses to the former.
Thus, in early RA, methotrexate monotherapy could
be reserved for ‘good responders’, with other patients
receiving alternative DMARDs or potentially escalating
directly to biologic therapy. Experience in oncology
suggests that a mechanistic understanding of metho­
trexate might facilitate such individualized therapy.
Evidence suggests that accumulation of methotrexate
polyglutamates is a useful pharmacokinetic measure
that might correlate with methotrexate response. In
addition, SNP and GWAS analyses are beginning to
identify possible ‘responder genotypes’ based on mark-
ers in genes encoding methotrexate transporter and
target proteins. In the future, sufficiently large genomic
studies should identify the most important elements
of methotrexate pharmacodynamics in RA. Current
predictive models for methotrexate efficacy are cum-
bersome and limited to the research setting, but with
further refinement they could provide true clinical util-
ity, enabling a tailored approach to DMARD therapy
in RA.

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Acknowledgements
The authors’ work is supported by the National Institute for
Health Research (NIHR) Newcastle Biomedical Research
Centre based at Newcastle Hospitals National Health Service
(NHS) Foundation Trust and Newcastle University, UK. The
views expressed are those of the authors and not necessarily
those of the NHS, the NIHR or the Department of Health.
Author contributions
P.M.B. researched the data for the article, and wrote the
manuscript. All authors contributed substantially to discus-
sions of the article content and to review or editing of the
manuscript before submission.
Competing interests statement
The authors declare no competing interests.

742 | DECEMBER 2016 | VOLUME 12 www.nature.com/nrrheum

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