type i ifn protects against antigen-induced arthritis

Type I IFN protects against antigen-induced arthritis

Fei Ying1, Jaya Prakash Chalise2, Sudeep Chenna Narendra2

and Mattias Magnusson2

1 Affiliated Hospital of Guiyang Medical College, Department of Microbiology and Immunology,

Guiyang, Guizhou, P. R. China2 Clinical and Experimental Medicine, Autoimmunity and Immune Regulation, Linkoping

University, Linkoping, Sweden

Autoimmune diseases including rheumatoid arthritis (RA) involve immune reactions

against specific antigens. The type I IFN system is suspected to promote autoimmunity in

systemic lupus erythematosus, but may also dampen immune reactions in e.g. inflam-

matory bowel disease. This prompted us to investigate the role of type I IFN in antigen-

induced arthritis (AIA). The importance of type I IFN in methylated (m) BSA-induced

arthritis was studied by using mice deficient for the type I IFN receptor (IFNAR) and by

administration of the IFN-a activator viral double-stranded (ds) RNA or recombinant IFN-a

at antigen sensitization. In IFNAR knock-out mice, arthritis severity was significantly

higher than in WT mice. Administration of dsRNA at antigen sensitization protected WT

but not IFNAR KO mice from arthritis. Also, addition of recombinant IFN-a during the

immunization, but not the induction phase of arthritis, almost abolished arthritis.

Protection mediated by IFN-a was accompanied by delayed and decreased antigen-specific

proliferative responses, including impaired lymph node recall responses after intra-

articular antigenic challenge. In conclusion, we demonstrate that type I IFN can prevent

joint inflammation by downregulating antigen-specific cellular immunity.

Keywords: Arthritis . Tolerance . Type I IFN

Introduction

The cause of autoimmunity remains unknown, but a better

understanding of what determines whether encounter with an

antigen results in immunological attack or tolerance should

provide strategies for deviating an existing autoimmune

response. The type I IFNs, initially discovered for their direct

anti-viral activity [1], are pluripotent cytokines with bearing also

on adaptive immune responses [2], especially humoral immunity

[3]. The rapid onset of type I IFN production in response to viral

infection has suggested type I IFNs as potential instigators of

viral-induced autoimmunity [4], although the link is only

circumstantial. Interestingly, a number of auto-antibody related

diseases, in particular systemic lupus erythematosus are char-

acterized by a type I IFN signature, i.e. elevated levels of type I

IFNs and products regulated by type I IFNs [5]. This may

represent pro-inflammatory properties of type I IFN on adaptive,

humoral immune responses, which may contribute to autoimmu-

nity [6]. However, the effects of type I IFNs on antigen-specific

immunity cannot be clearly categorized as either pro- or anti-

inflammatory. In vaccine studies, e.g. both an enhancing [7] and

a clear dampening effect [8] of type I IFN signalling on the

antigen-specific immune response has been reported. Similarly,

in experimental models of autoimmunity, type I IFN may either

aggravate [9, 10] or mitigate [11–13] inflammation. The under-

lying mechanism(s) explaining these apparent contradictory

findings remain to be determined. In arthritis, viral infections

are known to exacerbate or precipitate inflammation [14], and

viral interferogenic double-stranded (ds)RNA and IFN-a can be

found at the site of inflammation in rheumatoid arthritis (RA)Correspondence: Dr. Mattias Magnussone-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2011. 41: 1687–1695 DOI 10.1002/eji.201040956 Immunomodulation 1687

patients [15]. RA is in part characterized by antigen-specific-

driven autoimmunity including autoantigens such as IgG and

citrinullated circular peptides. To study the role of viral nucleic

acids and type I IFN on the development of antigen-specific

immunity leading to arthritis, we used the methylated bovine

serum albumin (mBSA)-induced arthritis model. This model

resembles human RA in that it is characterized by joint swelling

including infiltration of macrophages and neutrophils to the

synovium, followed by pannus formation and cartilage degrada-

tion [16] and has the advantage of allowing the controlled

comparison of arthritic and healthy joints in the same individual.

In this study, we report that the absence of type I IFN-signalling

clearly aggravates antigen-induced arthritis (AIA) and that type I

IFN-inducing stimuli or recombinant IFN-a can prevent the

development of AIA.

Results

dsRNA protects against AIA

Viral infections are suspected to contribute to arthritis. A pro-

inflammatory component of most viruses is their dsRNA [17]. To

evaluate the impact of dsRNA on the development of AIA, dsRNA

was administered by immunization with mBSA as described in

Materials and methods. Two weeks later, mice were challenged

with an intra-articular injection of mBSA. As shown in Fig. 1, the

group of mice receiving dsRNA had a lower frequency and

developed significantly milder arthritic symptoms than control

mice receiving mBSA plus PBS. A representative image of a joint

from a mouse treated with dsRNA, showing lack of infiltrating

cells to the synovium, is depicted in Fig. 1C (upper left). For

comparison, a representative image of an untreated control

mouse with infiltrated cells scattered all over the synovial tissue is

shown (Fig. 1C, upper right). Thus, administration of dsRNA

along with the antigen mBSA at immunization clearly prevented

the development of arthritis upon intra-articular challenge with

mBSA.

Protection by dsRNA is mediated via type I IFN

Many of the proinflammatory effects of dsRNA are mediated via

type I IFNs We therefore evaluated whether the protective effect

of dsRNA on AIA was also dependent on type I IFNs. To this end,

WT mice and mice unable to signal via the type I IFNs receptor

(IFNAR KO) were immunized with mBSA with or without dsRNA.

As shown in Fig. 1, only WT mice were protected from arthritis by

dsRNA. In contrast, IFNAR KO mice developed severe arthritis

regardless of administration of dsRNA. This shows that the

protective effect of dsRNA on arthritis is mediated via type I IFNs.

Moreover, these data also revealed that the absence of type I IFN

signalling resulted in more severe arthritis. In WT mice and in

IFNAR KO mice the arthritic frequencies are comparable in that

80–100% of animals develop arthritis upon mBSA challenge

(Fig. 1A). In contrast, mice unable to signal via the type I IFN

receptor developed significantly more severe arthritis than WT

mice (Fig. 1B), indicating that endogenous type I IFN signalling

may have a mitigating effect on arthritis development. Repre-

sentative images of arthritis in IFNAR KO mice, treated or not

with dsRNA, are depicted in Fig. 1C (lower panel).

Administration of recombinant IFN-a protects againstAIA

The inability of dsRNA to protect from arthritis in IFNAR KO mice

shows that type I IFN signalling is a prerequisite for this effect.

We next evaluated whether signalling by a type I IFN alone is

sufficient to protect against AIA. To this end, recombinant IFN-awas administrated before and at the time of immunization with

mBSA, followed by intra-articular challenge with mBSA alone

2 wk later, as described in Materials and methods. As depicted in

Fig. 2, IFN-a clearly protects from development of arthritis. This

is evident from a clearly lower frequency and severity of arthritis

in WT mice treated with IFN-a as compared with WT mice treated

with PBS. As expected, only WT mice, but not IFNAR KO mice

were protected against arthritis by IFN-a treatment (Fig. 2A

and B), which confirms receptor specificity of the response.

To test whether a similar dose of IFN-a would have the same

effect if administered at the time of arthritis induction, mice were

immunized with mBSA at days 1 and 7, and at the day of intra-

articular injection (day 21) treated with 1000 U IFN-a. This had

no effect on arthritis development, because mice treated with

IFN-a at day 21 developed arthritis to the same extent as PBS-

treated animals (control). The arthritis frequency (per cent

animals developing arthritis) and severity (mean arthritic

score7SEM) was 83% and 1.6770.42, respectively in the control

group versus 100% and 1.6770.33 in the IFN-a-treated group,

n 5 6, ns). Thus, activation of type I IFN signalling must occur

prior to intra-articular challenge in order to be protective.

Type I IFN downmodulates mBSA-specific cellularresponses

Arthritis development upon intra-articular injection of mBSA in

pre-immunized animals is a result of developing mBSA-specific

IgG and activation of mBSA-specific T cells [18]. To determine

whether type I IFN-mediated protection was accompanied by

altered antibody responses, the levels of anti-mBSA IgG in serum

were determined at day 13, day 20 and day 28 following the first

immunization with mBSA. As depicted in Fig. 3, the addition of

IFN-a or lack of endogenous type I IFN signalling did not alter the

generation of the anti-mBSA antibody response.

Arthritis induced by mBSA is also dependent on antigen-

specific T cells [18]. The amount of activated, antigen-specific

T cells can be assessed by quantifying the proliferative response

to antigenic re-challenge of leukocytes ex vivo. To determine

whether IFN-a could regulate the generation of antigen-specific


Eur. J. Immunol. 2011. 41: 1687–1695Fei Ying et al.1688

proliferation, leukocytes from immunized animals were

re-stimulated in vitro with mBSA. Immunization in the presence

of IFN-a decreased the proliferative response in lymph node cells

isolated at termination (day 28) of AIA from WT (Fig. 4A), but

not from IFNAR KO mice (Fig. 4B), which indicates receptor

specificity. The response was dose dependent with a maximum

proliferation at 50mg/mL mBSA (Fig. 4A and B). This concen-

tration and a concentration mBSA that resulted in a lower

response (12.5mg/mL mBSA) were used to determine the effect

of IFN-a signalling on the kinetics of the anti-mBSA proliferative

response. Spleen and lymph node cells were isolated on 0, 13, 20

and 28 days after the first mBSA immunization of AIA from WT,

IFNAR KO and WT mice immunized in the presence of IFN-a.

Immunization in the presence of IFN-a had three distinct

effects on the kinetics of the mBSA-induced response. First,

immunization in the presence of IFN-a resulted in a slower onset

of the response, manifested by significantly lower proliferation at

day 13 in both lymph node (Fig. 4C) and spleen (Fig. 4D) cells.

Second, IFN-a reduced the maximum proliferative response by

half in splenocytes (Fig. 4D), and the inhibition was even more

pronounced in lymph node cells (Fig. 4C). Third, IFN-a impaired

the lymph node recall response after the intra-articular challenge

on day 21 (Fig. 4C, day 28). No significant differences were

observed for Con A-stimulated responses between leukocytes

from mice treated with IFN-a or control cells at any time point

(Fig. 4A and B inserts, and data not shown).

control (IFNAR ko)

+ dsRNA (wt)

+ dsRNA (IFNAR ko)

control (wt)

0.1 mm 0.1 mm

0.1 mm0.1 mm

A

C

B

Figure 1. Effect of dsRNA and type I IFN signalling in mBSA-induced arthritis. Arthritis was induced in WT and mice lacking the type I IFN receptor(IFNAR KO) as described in Materials and methods. (A) Percent and (B) severity of arthritis in WT and IFNAR KO mice treated with dsRNA or PBS.Arthritis was scored 0–3, depending on the degree of inflammation as described in Materials and methods. (C) Representative images of jointsections from WT and IFNAR KO mice treated with dsRNA or PBS (control). n 5 6. �, po0.05, ��, po0.01, (Mann–Whitney). The induction of arthritis inthe presence of dsRNA was repeated once and the induction of arthritis in WT and IFNAR KO mice in the absence of dsRNA was repeated in fourindependent experiments with similar results.


Eur. J. Immunol. 2011. 41: 1687–1695 Immunomodulation 1689

Intriguingly, lack of endogenous type I IFN signalling resulted

in reduced maximum proliferation in lymph node cells (Fig. 4C,

day 13) and impaired lymph node recall responses after the intra-

articular challenge day 21 (Fig. 4C, day 28). In spleen cells, the

anti-mBSA-response did not differ between WT and IFNAR KO

mice at any time point (Fig. 4D).

A dose-dependent response was also observed throughout the

kinetic study in that the mBSA-response observed day 13–28 at

50 mg/mL mBSA (Fig. 4C and D) was higher than that observed

for 12.5mg/mL mBSA (data not shown).

Discussion

Joint manifestations including RA may worsen or develop during

viral infections [14]. Our earlier results have shown that dsRNA,

a proinflammatory nucleic acid expressed by most viruses [17]

induces local inflammation if present in the joint [19] and that

such RNAs can be found in synovial fluid from RA patients [15].

In order to evaluate the importance of dsRNA for the develop-

ment of AIA we administered dsRNA or PBS as a control along

with the antigen in the immunizations of mBSA-induced arthritis.

+ IFN-alpha (wt) control (wt)

control (IFNAR ko)+ IFN-alpha (IFNAR ko)

0.1 mm

0.1 mm 0.1 mm

0.1 mm

A B

C

Figure 2. Effect of recombinant IFN-a in mBSA-induced arthritis. Arthritis was induced in the presence or absence of recombinant IFN-a in WT andIFNAR KO as described in Materials and methods. (A) Percent and (B) severity of arthritis in WT and IFNAR KO mice treated with IFN-a or PBS. (C)Representative images of joint sections from WT and IFNAR KO mice treated with IFN-a or PBS (control). nZ5. �, po0.05, �� po0.01, (Mann–Whitney).The induction of arthritis in the presence of recombinant IFN-a was repeated in four independent experiments with similar results.



To isolate the effect of dsRNA from that of other microbial

constituents, we immunized mice with mBSA in the presence of

incomplete Freund’s Adjuvant (i.e. without mycobacterial

products), which upon intra-articular injection of mBSA produces

a comparable, but transient acute joint inflammation as

compared with immunization in the presence of complete

Freund’s Adjuvant [20]. Surprisingly, dsRNA lowered the

frequency and severity of arthritis, indicating that dsRNAs have

anti-inflammatory properties. In fact, it has been showed that

repeated i.p. administration of dsRNA inhibits joint swelling by

decreasing the amount of inflammatory tissue in the effector

phase of arthritis, as demonstrated in collagen-antibody induced

arthritis [21] and in the K/BxN serum transfer model [21, 22].

This inhibition [21, 22], and many of the immune effects of

dsRNA are mediated via type I IFNs, cytokines directly activated

by dsRNA upon viral infection and mandatory for the innate anti-

viral immune response [23].

To test whether the protective effect of dsRNA was mediated

by type I IFNs, we used mice lacking the type I IFN receptor

(IFNAR KO mice). These mice lack the ability to respond to

IFN-a/b [24], and produce minimal amounts of type I IFNs in

response to dsRNA or herpes simplex virus as compared with WT

mice [19], probably due to the lack of positive feed-back [25].

Administration of dsRNA to WT and IFNAR KO mice showed that

the ability of dsRNA to inhibit development of mBSA-induced

arthritis was totally dependent on type I IFN signalling. A role of

type I IFN signalling has also been reported in NADPH-mediated

suppression of arthritis in rats [13], indicating that this pathway

may be operable not only in mice. Further underscoring the effect

of type I IFN signalling was the finding that WT mice, also

without addition of dsRNA, developed significantly milder

arthritis than receptor knock out mice (Fig. 1A and B), which has

also been observed in the K/BxN serum transfer arthritis model,

indicating that arthritis development is regulated by type I IFN

Figure 3. Effect of type I IFN on the humoral response against mBSA.At indicated time points after the first mBSA-immunization, bloodsamples were collected from the tail vein of WT mice (�), WT miceimmunized in the presence of IFN-a (& ) and IFNAR KO mice (m). Thelevels of total anti-mBSA IgG were determined by ELISA as described inMaterials and methods and expressed as the mean absorbance(405 nm)7SEM of each treatment group and time point, nZ20. Dataare the mean of four independent experiments.

Figure 4. Effect of type I IFN on mBSA-induced proliferation ex vivo. At day 28, total lymph nodes from (A) immunized WT, and (B) IFNAR KO micetreated or not with IFN-a were isolated and re-stimulated with 0–100 mg/mL mBSA or 1.25 mg/mL Con A (inserts). At days 0, 13, 20 and 28 after thefirst mBSA immunization, (C) total lymph nodes and (D) spleen cells from WT, IFNAR KO and WT mice immunized in the presence of IFN-a wereisolated and re-stimulated with medium or 50 mg/mL mBSA. After 60 h incubation, in the presence of tritiated thymidine during the last 12 h,proliferation (cpm) was assessed in a b-counter. In (C and D), the cpm-value from mock-stimulation (medium) for each individual has beensubtracted. Values are expressed as cpm7SEM, nZ5. �1po0.05 at 0–100 mg/mL mBSA, �2po0.05, WT versus IFNAR KO, ��3po0.01, WT versus WT1IFN,�4po0.05, WT versus WT1IFN and WT versus IFNAR KO, ��5 po0.01, WT versus WT1IFN, (Mann–Whitney). Data are representative of threeindependent experiments.



signalling. However, immunization per se did not elicit detectable

levels of IFN-a in serum (data not shown). This indicates that also

low-intensity signalling via the type I IFN receptor affects arthritis

development. Similarly, mice deficient for IFN-b develop more

severe [26], and treatment with IFN-b–expressing fibroblasts

ameliorates, collagen-induced arthritis [27], but it remains to be

determined whether activation of IFN-a signalling in the sensiti-

zation phase also can protect against this form of AIA.

The anti-inflammatory property of type I IFN signalling was

further confirmed by including recombinant IFN-a in the immu-

nizations. The protective effect required that IFN-a was present in

the sensitization phase, because administration of IFN-a only in

the arthritis induction phase (day 21) had no ameliorating effect.

In contrast, administration of type I IFNs in antibody-mediated

models of the effector phase of arthritis shows an ameliorating

effect [21, 22]. For example, 5� 104 U IFN-a administered at the

time of CAIA induction and thereafter 104 U IFN-a every other

day mitigated joint swelling [21]. Thus, type I IFN may down-

regulate arthritis by several mechanisms. If continously admi-

nistered it may mitigate antibody-induced joint inflammation. In

this paper, we show that administration of 103 U IFN-a only at the

antigen sensitization, but not in the induction phase (day 21)

protects against arthritis development upon intra-articular chal-

lenge with the same antigen. This is a hitherto unknown property

of IFN-a.

Still, almost 20 years ago IFN-a was reported to improve joint

scores in RA patients [28]. However, the clinical picture is

complicated by controversial results [29] including the develop-

ment of RA during IFN-a therapy of viral infections [30]. As

outlined below, in experimental arthritis, the opposing effects

could possibly be understood by distinguishing the innate and

adaptive immune effects of IFN-a.

In apparent contrast to the direct pro-inflammatory effects

exerted by dsRNA and IFN-a if they are present in the joint [19],

the present data show that IFN-a is clearly anti-inflammatory in

AIA. The pro-arthritogenic properties of dsRNA and IFN-a, which

is dependent on type I IFN signalling [19], are established 3 days

after intra-articular injection, and thereafter subsides within a

week [31]. The innate immune system is furthermore sufficient to

establish dsRNA-induced arthritis because SCID mice, which are

devoid of functional T and B cells (i.e. adaptive immune system),

are also susceptible to this form of arthritis [31]. Thus, the

contradictory effects type I IFN signalling can possibly be

explained by the different effects that type I IFN have on arms of

the innate, and adaptive immune system. Indeed, as outlined

below, type I IFN signalling can downmodulate adaptive, cellular

immune responses.

Severe arthritis in mBSA-induced arthritis can be accom-

panied by marked titers of mBSA-specific IgG [18]. Although

mice unable to signal through the type I IFN receptor developed

more severe arthritis (Figs. 1 and 2), the levels of anti-mBSA IgG

at days 13–28 after the initial immunization did not differ from

WT mice. In fact, reduced arthritis severity in the absence of

clearly lower levels of total anti-mBSA IgG has also been observed

by Rontzsch et al. [32]. In line with this, protection from arthritis

induced by exogenously added IFN-a was not associated with a

decreased antibody response (Fig. 3). It remains to be deter-

mined whether type I IFN-mediated amelioration of mBSA-

induced arthritis can be reflected in the levels of different isotypes

of anti-mBSA antibodies.

Antigen-specific T cells are of crucial importance in the

establishment of mBSA-induced arthritis [18]. Type I IFNs can

impair the ability of DC, possibly by altering their phenotype and

survival, to induce naıve Th-cell proliferation [33] and this may

explain the delayed onset of antigen-specific proliferation in

spleen and lymph node cells from IFN-a–treated animals. In

control cells, the mBSA proliferative response showed a peak at

13 days after the first immunization (Fig. 4C and D). Later, the

response clearly declined, but after the intra-articular injection of

mBSA at day 21, the anti-mBSA response increased in lymph

node (Fig. 4C), but not in spleen (Fig. 4D), cells. Isolated lymph

nodes include the popliteal and inguinal lymph nodes that drain

the joint. Thus, intra-articularly injected mBSA may accumulate

in the draining lymph nodes, resulting in potent re-activation of

mBSA-specific lymphocytes. This likely explains why the recall

response at day 28 was observed in lymph node but not spleen

cells. Interestingly, this recall response in lymph node cells was

inhibited by IFN-a (Fig. 4C). Thus, type I IFN downregulates

cellular immunity against mBSA, which may explain why

IFN-a–treated mice were either protected or developed signifi-

cantly less severe arthritis. In this regard, it is interesting to note

that type I IFN signalling, apart from its direct anti-proliferative

effects [34, 35], can promote the development of regulatory

T cells, with a suppressive function on proliferating T cells. This

was demonstrated by administration of high doses of inter-

ferogenic CpG-DNA to WT and IFNAR KO mice. In WT, but not in

mice lacking type I IFN signalling, this resulted in an indoleamine

dioxygenase dependent accumulation of regulatory T cells [36].

Also, depletion of T cells with a regulatory phenotype has been

shown to aggravate experimental arthritis [37]. It remains to be

determined whether IFN-a treatment in mBSA-induced arthritis

results in the activation of regulatory T cells and if these can

contribute to the downmodulation of arthritis. This is the focus of

our future studies. Another possible T-cell effect contributing to

the protective properties of IFN-a is the inhibition of pro-arthri-

togenic Th17 [38] cell development by IFN-a [39].

The effect on proliferation of adding IFN-a at the time of

immunization was not seen in IFNAR KO mice (Fig. 4B), indi-

cating receptor specificity. The absence of a functional type I IFN

receptor did not affect the proliferative anti-mBSA response in

spleen cells (Fig. 4D) but resulted in lower lymph node prolif-

eration and an impaired lymph node recall response at day 28

(Fig. 4C). Intriguingly, cells from IFNAR KO mice and cells from

mice treated with IFN-a were both unable to mount a lymph node

recall response. Total lack of type I IFN signalling may impair

antigen-presenting cells from IFNAR KO mice to stimulate T-cell

proliferation [40], likely by affecting MHC class II expression

[41]. Thus, the apparent contradictory effects on the lymph node

recall response of (i) IFNAR deficiency and (ii) adding IFN-a to

WT mice can possibly be explained by the enhancing effects



of IFN-a signalling on (i) MHC class II expression and (ii) the

inhibitory effects of IFN-a on proliferating T cells [34–36], its

ability to expand regulatory T cells [36] and to inhibit [39] pro-

arthritogenic Th17 cells [38].

Thus, the more severe arthritis observed in type I IFN receptor

knock-out mice as opposed to WT mice, cannot be explained by

higher antigen-specific proliferation in IFNAR KO mice. This

raises the possibility that the mechanisms contributing to the

protective effect of administration of IFN-a, and the ameliorating

effect of a functional type I IFN receptor on arthritis, differ. When

IFN-a is administered in the sensitization phase, IFN-a–mediated

protection against arthritis is reflected in diminished antigen-

specific proliferation (Fig. 4). Similarly, co-delivery of IFN-a to an

HCV-DNA vaccine [8], and treatment with IFN-a before immu-

nization with Mycobacterium tuberculosis reduces antigen-specific

proliferation [42], indicating that the presence of IFN-a at the

time of antigen sensitization may downregulate subsequent

proliferation induced by antigenic re-challenge. In contrast, the

amelioration of arthritis in WT, as opposed to IFNAR KO mice

(Figs. 1 and 2), was not accompanied by decreased cellular

(Fig. 4) or humoral (Fig. 3) immunity. The fact that type I IFN

signalling mitigated arthritis, despite high levels of anti-mBSA

IgG, is supported by the fact that endogenous type I IFN signal-

ling also reduces joint swelling in a model of the effector phase of

arthritis, where arthritis is induced by arthritogenic antibodies

[22]. Thus, low-intensity type I IFN signalling could prevent

severe mBSA-induced arthritis by mechanisms active in the

effector phase without affecting antigen-specific responses. In

contrast, administration of IFN-a in the sensitization phase

downregulates antigen-specific proliferation (Fig. 4) and subse-

quently prevents development of arthritis (Fig. 2), but is without

effect when anti-mBSA-immunity is already established.

As mentioned above, the anti-inflammatory property of type I

IFN presented here in AIA stands in sharp contrast to the short-

lasting pro-inflammatory properties of IFN-a directly administered

to the joint [19]. IFN-a, apart from activating the proinflammatory

transcription factor STAT1, may also activate STAT3, which inhi-

bits transcriptional activation by STAT1, by prevention of STAT1-

DNA homodimer formation [43]. This mechanism is proposed to

protect from tissue damage resulting from prolonged immune

responses, while still allowing for a rapid and effective anti-viral

response. Thus, the STAT1/STAT3 ratio resulting from activation

by IFN-a may determine whether activation by IFN-a will result in

arthritis or inhibition of joint inflammation. Another way by which

IFN-a may ameliorate arthritis is via its modulating effects on

tumor necrosis factor a (TNF-a). TNF-a is a cytokine with an

important pathogenic role in RA including the AIA [44] employed

here. In fact, type I IFNs can directly inhibit induction of TNF-a by

activating the Twist molecule, which upon binding to TNF-aregulatory DNA elements prevents synthesis of TNF-a [45].

In conclusion, the present study shows that activation of type I

IFN signalling at the time of antigen-sensitization prevents

subsequent antigen-triggered joint inflammation. This finding

will be of importance to develop therapeutics based on immune

tolerance rather than immune suppression.

Materials and methods

Animals and induction of arthritis

Mice deficient for subunit 1 of the type I IFN receptor (A129) and

wild type (129) congenes were a kind gift from Maries van den

Broek, Zurich University [24]. Mice were housed in the animal

facility of the Department of Rheumatology and Inflammation

Research, Goteborg University, and at the animal facility of

Linkoping University, Sweden. Mice were kept under standard

conditions of temperature and light, and fed laboratory chow and

water ad libitum. The study was approved by the Ethical

Committees of Goteborg University (No. 176-2008) and Linkoping

University (No. 72-2009). Arthritis was induced by intra-articular

injection of 30mg mBSA at day 21 in the left knee (and an equal

volume (20mL of PBS) in the right knee as control) of pre-

immunized animals. Animals were pre-immunized day 0 and day 7

with 200mL antigen emulsion containing 200 and 100mg mBSA,

respectively. The antigen was prepared by diluting mBSA in PBS

and then emulsified 1:1 in Freund’s incomplete Adjuvant (Sigma-

Aldrich). DsRNA was administered as follows: On day 0, 200mg

and on day 7 100mg of the dsRNA analogue poly I:C (Sigma-

Aldrich) was added to the antigen emulsion by including it in the

mBSA–PBS mixture before addition of Freund’s incomplete

adjuvant. Recombinant murine IFN-a (PBL, Interferon Source)

was administrated in the same manner (1000 U on days 0 and 7)

and by intra-peritoneal injection on day�1 and day 6 or at day 21.

Evaluation of arthritis

Histologic examination of joints was performed after routine

fixation, decalcification and paraffin embedding. Sections were cut

and stained with H&E. All the slides were coded and evaluated

blindly. Arthritis was evaluated with regard to synovial lining

hyperplasia and cellular infiltration in synovial tissue. Intra-

articular injection of PBS never resulted in detectable inflamma-

tion. The frequency of arthritis among individuals in different

experimental groups was calculated and the severity of synovitis of

each specimen was judged on an arbitrary scale from 0 to 3. No

signs of inflammation (0); mild inflammation with proliferation of

the synovial lining layer (1). Grades 2 and 3 represent different

degrees of inflammation characterized by influx of inflammatory

cells scattered throughout the synovial tissue.

Determination of anti-mBSA IgG

Microtiter plates were coated overnight with 10mg/mL mBSA

diluted in 50 mM carbonate/bicarbonate buffer. After blocking two

hours at RT with 2% w/v casein, serum samples diluted 1:500 in

2% casein were added. After sample incubation at RT for 2 h with

gentle agitation, plates were consecutively incubated 1 h at

RT with 1mg/mL biotinylated goat-anti mouse IgG (Jackson



Immunoresearch) and horseradish peroxidase-conjugated Extra-

vidin (0.5mg/mL, Sigma-Aldrich) and washed three times in PBS-

0.5% Tween 20 between each step. All reagents were diluted in 2%

casein. Plates were later developed by the addition of substrate for

horseradish peroxidase (0.1 mg/mL TMB (Sigma-Aldrich) accord-

ing to the manufacturer’s instructions). After 2–5 min incubation at

RT with gentle shaking, the reaction was stopped by the addition

of 100mL 1 M H2SO4. The signal was quantified by reading at

450 nm on a Spectra Max Plus spectrophotometer.

Lymphocyte proliferation

At days 0, 13, 20 and 28 of the mBSA-induced arthritis, lymph node

and spleen cells were isolated aseptically as follows. Lymph node

cells (pooled body lymph nodes) and splenocytes were isolated by

passing the organ through a 70mm cell strainer. Splenic erythrocytes

were lyzed by Red Blood Cell Lysis buffer (Sigma) according to the

manufacturer’s instructions. After wash, the resulting cell suspensions

were resuspended in Iscove’s complete medium (10% FCS, 50mM

2-ME, 4mM L-glutamine, and 20mg/mL gentamicin) and stimulated

in 96-well cell culture plates (1� 106 cells per mL) with 0–100mg/

mL mBSA, medium only or 1.25mg/mL Con A (Sigma-Aldrich) in a

total volume of 200mL. 3 H–Thymidine was added after 48h of

culturing, and the cells were harvested on glass-fiber filter (AB

Ninolab, Upplands Vasby, Sweden) after 10–12h and the amount of

incorporated 3H–Thymidine was counted in a beta counter.

Statistical analysis

Differences between groups were compared using the

Mann-Whitney sum of ranks test (Using Prism 5.04, GraphPad

Software). A p-value less than 0.05 was considered significant.

Acknowledgements: The authors express their gratitude to the late

Berit Ericsson for preparing joints for analysis and thank Malin

Erlandsson and Margareta Rosenqvist (Rheumatology and

Inflammation Research, University of Gothenburg, Sweden) for

excellent technical assistance. The study was supported by grants

from Gustav V 80-years foundation, the Ake Wiberg foundation,

Magnus Bergvall foundation, the Swedish Association against

Rheumatism (Reumatikerforbundet) and Svenska Lakaresallskapet.

Conflect of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: AIA: antigen-induced arthritis � IFNAR: IFN-a/b-receptor

� mBSA: methylated BSA � RA: rheumatoid arthritis

Full correspondence: Dr. Mattias Magnusson, Autoimmunity and

Immune Regulation, Clinical and Experimental Medicine, Linkoping

University, 581 83 Linkoping, Sweden

Fax: 146-13-13-22-57

e-mail: [email protected]

Received: 16/8/2010

Revised: 17/2/2011

Accepted: 22/3/2011

Accepted article onine: 6/4/2011



type i ifn protects against antigen-induced arthritis

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