ORIGINAL ARTICLE

Methotrexate attenuates the Th17/IL-17 levels in peripheral bloodmononuclear cells from healthy individuals and RA patients

Yanshan Li • Lindi Jiang • Si Zhang •

Lianhua Yin • Lili Ma • Dongyi He •

Jie Shen

Received: 26 November 2010 / Accepted: 18 February 2011 / Published online: 21 June 2011

� Springer-Verlag 2011

Abstract To investigate whether the inhibition of Th17/

interleukin (IL)-17 contributes to the beneficial effects of

methotrexate (MTX) in the treatment of rheumatoid

arthritis (RA). Peripheral blood mononuclear cells

(PBMCs) from healthy donors and RA patients were col-

lected. The cells were stimulated with monoclonal anti-

bodies to CD3 and CD28 in the absence or presence of

MTX. After coincubation, IL-17 production was detected

at both the mRNA and protein levels, and the percentage of

cells positive for both CD4 and IL-17 in PBMCs was

analyzed by flow cytometry. PBMCs of healthy donors and

RA patients were stimulated with CD3 and CD28 mono-

clonal antibodies to produce high levels of IL-17. The

augmentation of IL-17 at the mRNA and protein levels was

significantly inhibited when PBMC cultures were prein-

cubated with MTX. Compared with PBMCs of healthy

donors, PBMCs of RA patients produced higher levels of

IL-17, and this increase in IL-17 levels was more inhibited

by MTX pretreatment. MTX inhibited IL-17 at the mRNA

level in a dose-dependent manner, but not at the protein

level, in both PBMCs of healthy donors and RA patients.

MTX did not affect the percentage of CD4- and IL-17-

positive cells in PBMCs. MTX dose dependently sup-

pressed the production of IL-17 at the mRNA level by

PBMCs from healthy donors and RA patients. Suppression

of IL-17 by MTX may contribute to its potent anti-

inflammatory role in RA therapy.

Keywords IL-17 � Methotrexate � Arthritis � Rheumatoid

Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune and

inflammatory disease. It is characterized by chronic and

erosive synovitis that mainly involves the peripheral joints

[1]. Although the cause is unclear, immune dysregulation is

believed to be crucial in the pathogenesis of RA, and

various cytokines that act as mediators of inflammation and

joint destruction are thought to be involved. Methotrexate

(MTX), a first-line drug in the treatment of RA, has been

shown to improve the tenderness and swelling of involved

joints and to exhibit more efficacy and better tolerability

than most other DMARDs [2, 3]. However, an under-

standing of the mechanism of action of MTX for attenu-

ating the disease process in RA remains elusive [4, 5].

MTX was conventionally considered to affect intracellular

purine levels, pyrimidine metabolism, and DNA synthesis

by the inhibition of dihydrofolate reductase (DHFR) and

some folate-dependent enzymes [6, 7]. Anti-inflammatory

effects of MTX have been reported in various aspects of an

inflammatory response such as the inhibition of cycloox-

ygenase 2 (COX-2) [8], the increase in adenosine release

[9], the production of reactive oxygen species [10], and the

Y. Li � L. Jiang (&) � L. Ma

Department of Rheumatology, Zhongshan Hospital,

Fudan University, No 180, Road Fenglin,

200032 Shanghai, China

e-mail: zsh-rheum@hotmail.com

S. Zhang

Department of Biochemistry and Molecular Biology,

Shanghai Medical College, Fudan University, Shanghai, China

L. Yin

Department of Physiology and Pathophysiology,

Shanghai Medical College, Fudan University, Shanghai, China

D. He � J. Shen

Department of Rheumatology, Guanghua Hospital,

Shanghai, China

123

Rheumatol Int (2012) 32:2415–2422

DOI 10.1007/s00296-011-1867-1

downregulation of the expression of adhesion molecules

[11]. Recently, some studies have focused on the effects of

MTX on cytokines and found that MTX inhibited inter-

leukin (IL)-6, IL-4, IL-13, interferon (IFN)-c, tumor

necrosis factor (TNF)-a, and granulocyte macrophage

colony-stimulating factor (GM-CSF) induced by T-cell

activation in the whole blood of healthy, juvenile RA

(JRA), and RA patients [12, 13].

IL-17, which is predominantly produced by a specific

subset of CD4 T-helper cells called Th17 cells, is a crucial

inflammatory cytokine for inducing and perpetuating

chronic inflammation, cartilage damage, and bone erosion

[14–17]. Th17 response dysfunction leads to overwhelming

IL-17 production by CD4 cells and other sources, which

may be associated with chronic inflammation. Many lines

of evidence have shown that IL-17 is present at the sites of

inflammatory arthritis and amplifies the inflammation

induced by synergistic interactions with other cytokines,

supporting the use of IL-17 as a new therapeutic target in

RA [18–21].

In this study, we investigated the effects of MTX

treatment on IL-17 at the mRNA and protein levels in

peripheral blood mononuclear cells (PBMCs) from healthy

individuals and RA patients and on the percentage change

of cells positive for both CD4 and IL-17.

Materials and methods

Subjects

Eleven RA patients (2 males and 9 females; mean age:

54 years, range: 40–67 years) and 25 healthy donors (9

males and 16 females; mean age: 29 years, range:

19–67 years) were enrolled in the study. All RA patients

fulfilled the 1987 revised criteria of the American College

of Rheumatology for RA. The blood samples were

obtained after the subjects included in the study consented.

All experiments using human subjects were performed in

accordance with the Declaration of Helsinki and approved

by the Institutional Review Board, Fudan University.

Antibodies and reagents

MTX was purchased from Unterach (Austria). Purified

anti-CD3 monoclonal antibody (mAb), purified anti-CD28

mAb, and fluorescein isothiocyanate (FITC) anti-human

CD4 were purchased from eBioscience (USA). The anti-

human IL-17-phycoerythrin (PE) mAb was purchased from

R&D Systems (USA), and Histopaque�-1077 was pur-

chased from Sigma–Aldrich (USA). The IL-17 enzyme-

linked immunosorbent assay (ELISA) kit was obtained

from Raybiotech (USA), and the PrimeScriptTM RT

reagent Kit and Ex TaqTM enzyme were purchased from

Takara (Japan). The Fix and Perm reagents and TRIzol�

reagent were obtained from Invitrogen (USA). Complete

RPMI-1640 medium contained 0.1% 2-mercaptoethanol

(ME), 100 U/mL penicillin, and 100 g/mL streptomycin

was from Usen (China). Fetal calf serum (FCS) was from

HyClone Laboratories (USA).

Cell preparation and alternative treatments

PBMCs were isolated from the heparinized blood of

healthy donors and patients with RA using Ficoll-Hypaque

density gradient centrifugation. The cells were finally

adjusted to a final concentration of 4 9 106/mL in com-

plete RPMI-1640 medium with 10% heat-inactivated FCS.

PBMCs were plated onto a flat-bottomed 24-well plate at

2 mL/well in triplicate and incubated in the absence (for

the non-drug group) or the presence of various concentra-

tions of MTX (0.1, 1.0, 5.0, and 25.0 lg/mL) for the MTX

group for 1 h. Subsequently, the cells were incubated with

anti-CD3 (0.2 lg/mL) and anti-CD28 (1.0 lg/mL) at 37�C

in an atmosphere of 5% CO2.

Extraction of total RNA and reverse transcriptase-

polymerase chain reaction

PBMCs were first cultured with various concentrations of

MTX for 1 h and then incubated with anti-CD3 (0.2 lg/

mL) and anti-CD28 (1.0 lg/mL) for 20 h. Total RNA was

extracted using TRIzol reagent according to the manufac-

turer’s instructions. The cDNA was prepared using the

PrimeScriptTM reverse transcriptase (RT) reagent kit.

Message RNA levels were quantified by polymerase chain

reaction (PCR) using the GeneAmpR PCR System 9700

(ABI, USA). Amplification reactions were performed with

primers specific for IL-17 (forward, 50ATC TCC ACC

GCA ATG AGG AC30; backward, 50 GTG GAC AAT

CGG GGT GAC AC30; resulting in an amplification of

232-bp-long fragment) and for glyceraldehyde phosphate

dehydrogenase (GAPDH), which was used as an endoge-

nous reference gene for relative quantification (forward,

50GGT GAA GGT CGG AGT CAA CG30; backward,

50CAA AGT TGT CAT GGA TGA CC30; resulting in the

amplification of a 496-bp-long fragment).

ELISA

The supernatants were harvested and assayed for IL-17

according to the manufacturer’s protocol for the corre-

sponding ELISA kit. The detection range of IL-17 was

10–1,000 pg/mL.

2416 Rheumatol Int (2012) 32:2415–2422

123

Flow cytometry

PBMCs were diluted with RPMI-1640 to a concentration of

4 9 106/mL, incubated in the absence or presence of

varying concentrations of MTX for 1 h, and stimulated

with 0.2 lg/mL anti-CD3 mAb, 1.0 lg/mL anti-CD28

mAb, and 1.5 lg/mL monensin for 5 h. Cells were stained

with FITC-conjugated anti-human CD4, followed by fixa-

tion, permeabilization, and staining with anti-human IL-17-

PE mAb. Flow cytometric analysis was performed within

24–48 h by BD FACSCalibur (BD, USA).

Statistical analysis

Descriptive characteristics are presented as mean (SD).

Comparisons of the drug group with the non-drug group or

the control group were tested with an independent 2-sam-

ple t-test or paired samples t-test. Linear associations were

assessed by Pearson’s correlation. Statistical analyses were

performed using SPSS statistical software (v. 18). P values

of \0.05 were considered significant.

Results

Stimulation with anti-CD3 and anti-CD28 mAbs

increased IL-17 levels in PBMCs

PBMCs isolated from healthy donors and RA patients were

stimulated with anti-CD3 and anti-CD28 mAbs. Compared

to PBMCs stimulated with vehicle, the IL-17/GAPDH

mRNA ratios of PBMCs from healthy donors [0.71 (0.17)

vs. 0.34 (0.14), P \ 0.01] or from RA patients [0.78 (0.14)

vs. 0.54 (0.15), P = 0.02] were significantly increased

after stimulation for 20 h (Fig. 1a). After stimulation for

48 h, IL-17 protein levels of PBMCs from healthy donors

[271.83 (145.11) pg/mL vs. 11.48 (7.12) pg/mL, P \ 0.01]

and RA patients [436.16 (205.08) pg/mL vs. 15.36 (6.57)

pg/mL, P \ 0.01] were significantly increased, compared

to stimulation with vehicle control (Fig. 1b). Additionally,

the basal levels of IL-17 mRNA of RA patients were higher

than those of healthy donors [0.54 (0.15) vs. 0.34 (0.14),

P = 0.02], but no differences were found between these 2

groups after stimulation with anti-CD3 and anti-CD28

mAbs.

MTX treatment dose dependently suppressed

the augmentation of IL-17 mRNA levels in PBMCs

stimulated with anti-CD3 and anti-CD28

In order to evaluate the effect of MTX on the expression of

IL-17 mRNA, PBMCs were pretreated with 4 different

concentrations (0.1, 1.0, 5.0, and 25.0 lg/mL) of MTX for

1 h and then stimulated with anti-CD3 and anti-CD28

mAbs for 20 h. In the healthy donor group, the ratios of IL-

17/GAPDH for the 4 concentrations of MTX were 0.65

(0.20), 0.55 (0.19), 0.51 (0.18), and 0.49 (0.17), respec-

tively. In comparison with the non-drug group [0.71

(0.17)], P values were 0.51, 0.06, 0.02, and 0.02, respec-

tively (Fig. 2a).

A similar inhibitory effect was observed in the PBMCs

of RA patients. The IL-17 mRNA level in the groups

receiving the lower doses of MTX (0.1 and 1.0 lg/mL) had

no statistically significant differences when compared with

the non-drug group [0.67 (0.14) vs. 0.78 (0.14), P = 0.21;

and 0.56 (0.19) vs. 0.78 (0.14), P = 0.06, respectively].

The IL-17 mRNA levels in the groups receiving the higher

doses of MTX (5.0 and 25.0 lg/mL) had statistically sig-

nificant differences when compared with the non-drug

group [0.54 (0.17) vs. 0.78 (0.14), P = 0.03; and 0.45

(0.12) vs. 0.78 (0.14), P = 0.01, respectively; Fig. 2b].

The relationship between the inhibitory effects of MTX

and the varying dosages was analyzed. For the PBMCs

Fig. 1 Anti-CD3 and anti-CD28 mAbs treatment elevated the level

of IL-17 produced at the mRNA and protein levels by PBMCs from

RA patients and healthy donors. Freshly isolated PBMCs from RA

patients and healthy donors were stimulated with anti-CD3 (0.2 lg/

mL) and anti-CD28 (1.0 lg/mL) for 20 h [a IL-17 mRNA detected by

RT–PCR, n = 6–11] or 48 h (b secreted IL-17 analyzed by ELISA,

n = 11–21). The control was incubated with a NaCl-balanced

solution

Rheumatol Int (2012) 32:2415–2422 2417

123

from the healthy donors and the patients with RA, the

treatment concentrations of MTX, which were logarith-

mically normalized as lg MTX, were negatively correlated

with the ratio of IL-17/GAPDH (R2 = 0.94, P = 0.03; and

R2 = 0.96, P = 0.02, respectively; Fig. 2c, d).

MTX treatment suppressed the anti-CD3 and anti-

CD28 stimulation of IL-17 protein levels in PBMCs

In order to investigate the effect of MTX treatment on IL-

17 secretion in culture solution, PBMCs were pretreated

with 4 different concentrations (0.1, 1.0, 5.0, and 25.0 lg/

mL) of MTX for 1 h and then stimulated with anti-CD3

and anti-CD28 mAbs for 48 h. The IL-17 protein levels of

the supernatant that were measured by ELISA were 120.77

(67.44), 116.90 (51.63), 93.39 (42.57), and 109.79 (44.92)

pg/mL, respectively, in the PBMCs of healthy donors. Each

of these values was lower than that measured in the

PBMCs of the non-drug group [271.83 (145.11) pg/mL,

P \ 0.01 for all comparisons; Fig. 3a]. IL-17 protein levels

in the PBMCs of RA patients treated with the 4 concen-

trations of MTX were 133.49 (36.96), 115.61 (26.58),

93.38 (27.79), and 100.19 (29.08) pg/mL, respectively.

Each of these values showed statistically significant dif-

ferences when compared with that measured in the PBMCs

of the non-drug group [436.16 (205.08) pg/mL, P \ 0.01

for all comparisons; Fig. 3a].

MTX suppressed the production of IL-17 in PBMCs,

regardless of whether the PBMCs were from healthy

donors or RA patients. Although the secreted IL-17 levels

of the PBMCs from the non-drug group of the RA patients

were higher than those of the healthy donors, there were no

statistically significant differences between the RA patients

and the healthy donors after MTX treatment (P [ 0.05 for

all comparisons; Fig. 3a). The percentage change (the

value change between the non-drug group and the post-

treatment group divided by the value of the non-drug

group) of IL-17 in the culture supernatants of the PBMCs

was analyzed. The percentage decrease of the IL-17 levels

from the RA patients in the 0.1 and 5.0 lg/mL MTX

groups was more significant than that of the healthy donors

[0.73 (0.07) vs. 0.52 (0.24), P = 0.03; and 0.81 (0.05) vs.

0.60 (0.19), P = 0.01, respectively; Fig. 3b].

MTX treatment did not change the percentage of CD4-

and IL-17-positive cells from PBMCs stimulated

with anti-CD3 and anti-CD28

The percentage of CD4- and IL-17-positive cells was

increased after stimulation with anti-CD3 and anti-CD28 in

the PBMCs of healthy donors. After treatment with MTX

(1.0 and 5.0 lg/mL), the percentage of CD4- and IL-17-

positive cells decreased from 0.94 (0.65) to 0.43 (0.52) and

0.64 (0.61), with P [ 0.05, respectively (Fig. 4).

Fig. 2 MTX treatment

suppressed the augmentation of

IL-17 mRNA levels in PBMCs.

PBMCs were isolated from

healthy donors (a n = 9–11),

and RA patients (b n = 5–6)

were treated with different

concentrations of MTX for 1 h

and then were stimulated with

anti-CD3 and anti-CD28 mAbs.

After 20 h, IL-17 mRNA

product and GAPDH product of

PBMCs were measured by RT–

PCR. There was a linear

regression of IL-17 mRNA

levels by PBMCs of healthy

donors (c) and of RA patients

(d) and the concentrations of

MTX. The concentration of

MTX was logarithmically

normalized by the base of 10

and is presented as lg MTX

2418 Rheumatol Int (2012) 32:2415–2422

123

The correlation between clinical data and MTX effects

on IL-17

The clinical data of the enrolled RA patients [i.e., age, sex,

duration of disease, erythrocyte sedimentation rate (ESR),

C-reactive protein (CRP) levels, rheumatoid factor (RF)

levels, anti-cyclic citrullinated peptide (anti-CCP) levels,

glucose-6-phosphate isomerase (GPI) levels, joint pain, and

joint swollen count] are listed in Table 1. The relationship

between the change of IL-17 in the supernatants of the

PBMCs between the non-drug group and the MTX treat-

ment groups and the clinical serologic markers was ana-

lyzed. The change in the values of IL-17 treated with

0.1 lg/mL MTX and 5.0 lg/mL MTX in vitro was posi-

tively correlated with serum RF levels (R2 = 0.62,

P = 0.04; and R2 = 0.64, P = 0.03, respectively, Fig. 5).

Correlations could not be found for any other index,

including ESR, CRP, anti-CCP, GPI, joint pain, or joint

swollen count.

Discussion

Our data identified that pretreatment with MTX inhibits the

production of IL-17 in vitro in PBMCs of healthy indi-

viduals and RA patients. Consistent with other reports [14],

IL-17 production by PBMCs from patients with RA was

increased highly when stimulated with mAbs to CD3 and

CD28. The addition of MTX into the cell cultures inhibited

the IL-17 production by PBMCs at both the mRNA and

protein levels. The inhibitory effect of MTX on IL-17

mRNA showed a dose-dependent pattern in our study.

IL-17 is an important inflammatory cytokine that is

involved in the pathogenesis of RA. Several studies have

shown IL-17 overexpression in PBMCs and fibroblast-like

synoviocytes or synovial fluids [14, 15, 18, 19]. The col-

lagen-induced arthritis (CIA) model of mice indicated that

IL-17 contributed to joint inflammation. This is supported

by the finding that IL-17-deficient mice from the CIA

Fig. 3 MTX treatment suppressed the secretion of IL-17 in PBMCs.

a IL-17 levels showed the same trend of change, regardless of

whether the PBMCs were from healthy donors (n = 19–21) or from

RA patients (n = 8–11) when the cells were treated with different

concentrations of MTX under being stimulated with anti-CD3 and

anti-CD28 mAbs for 48 h. b Compared to PBMCs of healthy donors,

the level of IL-17 in PBMCs of RA patients was markedly inhibited

by MTX. The figure shows that the percentage change of IL-17 levels

(the change value between the non-drug group and the post-treatment

group divided by the value of the non-drug group) in PBMCs from

healthy donors and RA patients

Fig. 4 The percentage of CD4- and IL-17-positive cells in PBMCs of

healthy donors (n = 7–9) after treatment with MTX. a a PBMCs

were stimulated with anti-CD3 and anti-CD28 for 5 h. a b PBMCs

were pretreated with 1.0 lg/mL MTX for 1 h and then stimulated

with anti-CD3 and anti-CD28 for 5 h. a c PBMCs were pretreated

with 5.0 lg/mL MTX for 1 h and then stimulated with anti-CD3 and

anti-CD28 for 5 h. b The percentage of CD4- and IL-17-positive cells

showed no statistically significant differences between the non-drug

group and each of the 2 different concentrations of MTX groups

Rheumatol Int (2012) 32:2415–2422 2419

123

model showed little inflammation [22]. Importantly, the

therapeutic role of IL-17 antagonism was confirmed in CIA

mice where joint inflammation and cartilage destruction

were further ameliorated by neutralizing or inhibiting

Th17/IL-17 [20, 23]. The inhibitory effect of MTX on the

production of IL-17 may be a potential mechanism for its

therapeutic effect.

MTX is one of the commonly used DMARDs, and its

mode of action is not fully established [4, 5]. A potential

mechanism for the remission of joint inflammation may be

attributable to diminished cytokine production. In a study

using whole blood of JRA patients [12], MTX pretreatment

inhibited the production of IL-6 that was induced by

lipopolysaccharide (LPS), but had no effect on the pro-

duction of TNF-a. Gerards et al. examined the whole blood

of RA patients and found that MTX dose dependently

inhibited the levels of IL-4, IL-13, IFN-c, TNF-a, and GM-

CSF that were induced by antibodies to CD3 and CD28. In

postmyocarditis rats, MTX also reduced the plasma levels

of TNF-a and IL-6 [24]. In this study, we observed in vitro

that MTX inhibited IL-17 production at the mRNA and

protein levels in PBMCs that had been stimulated with

antibodies to CD3 and CD28. The analysis of the rela-

tionship between the dose and the efficacy of MTX showed

that MTX suppressed IL-17 mRNA in a dose-dependent

manner. The same association was not found at the protein

level. This finding may be because we only detected the

extracellular protein of IL-17 in PBMCs. We did not find

that MTX significantly decreased the percentage of CD4-

and IL-17-positive cells. This could be because the mAbs

to CD3 and CD28 upregulated IL-17 by increasing cellular

activity rather than by increasing cellular proliferation [14]

or by stimulating other T-cell types to produce IL-17 [25].

IL-17 levels in PBMCs from RA patients were higher than

those of healthy donors. MTX has a stronger ability to

suppress IL-17 in RA PBMCs than in PBMCs from healthy

individuals. This phenomenon indicated that PBMCs from

RA patients are easily activated for the production of IL-17

and are significantly decreased by MTX treatment. The

diminution of IL-17 may be one of the mechanisms of

MTX treatment in RA. Previous studies [26, 27] have

reported that there was no significant difference of serum

IL-17 levels between predrug treatment and postdrug

treatment. This may be caused by the complex interactions

of drugs when MTX is used in combination with other

drugs in vivo. In our study, we tried to confirm IL-17

inhibition by MTX with less interfering factors.

Previous studies [28–30] have reported that RF levels

may be one of the predictive factors for MTX response of

RA patients. In our study, interestingly, we found that the

effect of MTX on suppressing IL-17 in vitro was positively

Table 1 Clinical data of RA

patients in the study

ESR erythrocyte sedimentation

rate, CRP C-reactive protein,

RF Rheumatoid factor, Anti-CCP anti-cyclic citrullinated

peptide antibodies, GPIGlucose-6-phosphate isomerase

Patient factors Mean M (P25–P75) No. of trials missing

Number 11 / /

M/F 2/9 / /

Ages, year 54.1 63.0 (40.0–67.0) 0

Disease duration, months 78.9 54.0 (8.8–120.0) 1

ESR, mm/h 53.1 50 (21.0–83.0) 0

CRP, mg/L 35.2 14.4 (9.4–49.4) 1

RF, IU/mL 222.8 180.0 (73.0–273.0) 0

Anti-CCP, RU/mL 323.1 200 (35.9–200.0) 0

GPI, ng/mL 2.1 1.4 (0.5–3.8) 2

Joint pain count 13.7 12.0 (6.8–22.3) 1

Joint swollen count 8.4 7.0 (3.0–9.3) 1

Fig. 5 Linear correlations of the value change of IL-17 (between

non-drug and post-treatment of MTX) of RA PBMCs in vitro after

treatment with MTX and the level of serum RF in RA patients:

a PBMCs of RA patients pretreated with 0.1 lg/mL MTX; b PBMCs

of RA patients pretreated with 1.0 lg/mL MTX; c PBMCs of RA

patients pretreated with 5.0 lg/mL MTX; d PBMCs of RA patients

pretreated with 25.0 lg/mL MTX. *P \ 0.05

2420 Rheumatol Int (2012) 32:2415–2422

123

related with the level of serum RF (Fig. 5). A high titer of

RF indicates that B cells are in an activated state, and the

cells have highly efficient antigen-presenting function [31,

32]. PBMCs from RA patients with high RF are activated

to a sensitized state by a RF-related immune environment.

This state is easily affected by internal and external factors

such as MTX on IL-17.

Taken together, our results showed that MTX can sup-

press IL-17 production and inhibit IL-17 mRNA expression

in a dose-dependent manner. This may be a potential

mechanism of MTX in improving RA signs and symptoms.

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