Introduction
Rheumatoid arthritis (RA) is the most common autoimmune disease in humans, affecting 1% of the population in western countries. Histologically, RA is characterized by hyperplasia and infiltration of the synovial membrane with mononuclear cells, development of an aggressive tissue called pannus and secretion of proteases, which are responsible for the destruction of articular cartilage and adjacent bone. It is well established that macrophages and synovial fibroblasts are effector cells of joint destruction, and it is presumed that autoreactive CD4
+ T cells are involved in their activation [
1]. There is now a large body of evidence that, in rodents, regulatory T cells (T
reg) actively control the activation of autoreactive T cells and thus maintain immunological self-tolerance. Apart from adaptive T
reg cells, which can be induced by antigen-specific stimulation of conventional peripheral T cells under tolerogenic conditions (for review [
2]), there is no doubt that naturally occurring T
reg cells exist in healthy mice as well as in humans and rats, and these are characterized by constitutive expression of CD25 [
3‐
5]. Absence of these cells
in vivo results in a multi-organ autoimmune syndrome [
3,
6]. These CD4
+CD25
+ T
reg cells leave the thymus as committed 'professional' suppressor T cells [
7‐
9], proliferate in the periphery, and acquire an effector/memory-like phenotype [
10]. In unmanipulated mice, T
reg cells can also be found in the CD25
- compartment, based on the expression of the integrin α
Eβ
7 [
10,
11], possibly reflecting differences in developmental stages of these cells.
The exact role played by naturally occurring CD4
+CD25
+ T
reg cells in the pathogenesis of arthritis remains controversial. Arthritis is part of the autoimmune syndrome induced by transfer of CD25-depleted splenocytes into lymphopenic hosts [
3], and CD4
+CD25
+ cells are protective in collagen-induced arthritis [
12]. However, Bardos and coworkers [
13] ruled out a role for naturally occurring CD4
+CD25
+ T
reg cells in proteoglycan-induced arthritis.
To clarify this issue, we used the antigen-induced arthritis (AIA) model. AIA is a Tcell-dependent experimental arthritis that is induced by intra-articular injection of antigen (methylated bovine serum albumin [mBSA]) into knee joints of preimmunized mice [
14,
15]. This results in an acute inflammatory reaction, which is characterized by exudation of neutrophils and fibrin, which later proceeds to a chronic arthritis with synovial hyperplasia, infiltration of mononuclear cells, and cartilage and bone destruction – histopathological changes similar to those that occur in RA. Autoimmune responses against cartilage constituents such as collagen types I and II and proteoglycans are involved in rendering the disease chronic [
16,
17]. Beyond the 100% incidence of arthritis, another major advantage of the AIA model is that the time point of induction of arthritis is known, allowing manipulation of CD4
+CD25
+ T
reg cell number
in vivo at defined stages in the disease. Using depletion of CD25-expressing cells or transfer of CD4
+CD25
+ cells, in the present study we demonstrated that T
reg cells modulate the onset of AIA but are ineffective at later stages, calling into question their value as a new therapeutic approach to established chronic arthritis.
Methods
Animals, arthritis induction and assessment
For all animal experiments, female C57Bl/6 mice (Charles River, Sulzfeld, Germany; age range 6–10 weeks) were used. Animals were kept under standard conditions, fed a standard diet and given free access to water. All animal studies were approved by the government commission for animal protection.
At 21 and 14 days before arthritis induction, mice were subcutaneously injected with 100 μg mBSA (Sigma, Deisenhofen, Germany), emulsified in complete Freund's adjuvant (Sigma) supplemented to 2 mg/ml heat-killed Mycobacterium tuberculosis (strain H37RA; Becton Dickinson [BD], Heidelberg, Germany). Simultaneously, mice received 5 × 108 heat-inactivated Bordetella pertussis (Chiron-Behring, Marburg, Germany) intraperitoneally. Arthritis was induced by intra-articular injection of 100 μg mBSA in 25 μl phosphate-buffered saline (PBS) into the right knee joint cavity.
Arthritis severity was monitored by measurement of lateral joint diameter using a vernier caliper (Oditest, Kroeplin Längenmesstechnik, Schlüchtern, Germany). Histological severity of arthritis was scored in a blinded manner by two investigators (PKP and MG) in frontal knee joint sections, stained with haematoxylin and eosin and prepared as described previously [
14]. Briefly, at least four sections per knee joint were semiquantitatively examined on a 0–3 point scale for each of the following: extent of synovial hyperplasia, mononuclear infiltration, cartilage destruction and pannus formation.
Antibodies and reagents
The following antibodies were grown and purified from the culture supernatants in our laboratory: anti-CD25 (PC61), anti-CD3 (145 2C11), anti-CD4-FITC and FITC-labelled anti-CD4-F(ab) (GK1.5), anti-CD8 (TIB105), anti-CD28 (37.51) and anti-Mac-1 (M1/70). The following antibodies and secondary reagents were purchased from BD Pharmingen (Heidelberg, Germany): PE-Cy5-labelled anti-CD4 (H129.9), biotinylated anti-αEβ7 (M290), biotinylated anti-CD25 (7D4), allophycocyanine or FITC-conjugated anti-CD25 (PC61), streptavidin-allophycocyanine and streptavidin-PE, and matched antibody pairs for ELISPOT analysis of IFN-γ (R4-6A2 and biotinylated XMG1.2) and IL-4 (BVD4 1D11 and biotinylated BVD6-24G2) production.
In vivodepletion
Mice were injected with 0.5 mg purified anti-CD25 antibody (PC61) 4 and 2 days before intra-articular antigen injection. Polyclonal rat IgG, purified from normal rat serum, was used as control. The degree of depletion was determined by fluorescence-activated cell sorting, using a non-cross-reactive biotin-labelled anti-CD25, FITC-labelled anti-CD4 and streptavidin-conjugated allophycocyanine. Measurement was performed using FACSCalibur
® (BD) and data were analyzed using WinMDI
http://www.scripps.edu.
Preparation, pre-activation and transfer of regulatory T cells
Pooled spleen and lymph node cells from naive C57Bl/6 donors or, if indicated, from immunized mice were incubated with anti-CD4-FITC (clone GK1.5) and anti-CD25-biotin (clone 7D4; BD). CD4+ T cells were isolated using an anti-FITC-Multisort-Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) in accordance with the manufacturer's instructions. CD4+ T cells were sorted into CD25- and CD25+ cells using anti-biotin MicroBeads (Miltenyi Biotech). Purity was greater than 92% for CD4+CD25- and greater than 80% for CD4+CD25+ cells.
CD25-expressing and αEβ7-expressing subsets were sorted by FACS. Briefly, pooled spleen and lymph node cells from naive mice were stained with anti-CD25-FITC, anti-αEβ7-biotin and streptavidin-PE. The stained cells were enriched with anti-FITC and anti-PE MicroBeads, using the AutoMACS separation unit (Miltenyi Biotech). Thereafter, the cells were sorted into subsets according to their expression of CD25 or αEβ7 using a FACSDiVa cell sorter (BD). The purity was 90–95%, as determined by FACS.
For activation, cells were cultured for 24–72 hours in the presence of plate-bound anti-CD3 (3 μg/ml), anti-CD28 (10 μg/ml) and rhIL-2 (100 U/ml; Chiron, Ratingen, Germany) in RPMI 1640 containing 10% fetal calf serum (FCS; Gibco, Karlsruhe, Germany). Thereafter, cells were washed with PBS and transferred intravenously via lateral tail vein into mice at the time point of AIA induction or at later time points when indicated.
Delayed-type hypersensitivity reaction
Seven days after arthritis induction, mice were challenged by intradermal injection into their ears of 5 μg mBSA in 10 μl PBS. Ear thickness was measured before injection and 24 and 48 hours later using a vernier caliper (Kroeplin).
Proliferation assay and ELISPOT analysis
Single cell suspension from spleens and lymph nodes (inguinal, popliteal, axillary) were cultured at a density of 1 × 106/ml in RPMI 1640, containing 10% FCS, 2 mmol/l L-glutamine, 10 mmol/l Hepes, 1 mmol/l sodium pyruvate, 0.5 μmol/l 2-mercaptoethanol and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin; all from Gibco) in the presence of medium alone or 25 μg/ml mBSA for 72 hours in 96-well tissue culture plates (Greiner Bio One, Nürtingen, Germany). Cells were pulsed with 0.5 μCi [3H]thymidine (Amersham-Buchler, Braunschweig, Germany) for the last 18 hours of culture. Thereafter, cells were harvested onto 96-well glass fibre filters (Packard Bioscience, Groningen, The Netherlands), and [3H]thymidine incorporation was measured with a scintillation counter (Top-Count; Packard Bioscience).
For ELISPOT analysis, PVDF-membrane 96-well microplates (Millipore, Eschborn, Germany) were coated overnight at 4°C with the primary antibody diluted in sterile PBS. After washing, plates were blocked for 2 hours with RPMI 1640 containing 10% FCS. Thereafter 2 × 105 (IL-2 and IFN-γ) or 1 × 106 (IL-4) cells were cultured in duplicate wells for 24 (IL-2 and IFN-γ) or 48 hours (IL-4). After washing again plates were incubated overnight at 4°C with the secondary antibody diluted in PBS/1% BSA. Extravidin–alkaline phosphatase conjugate (1:30,000 in PBS/1% BSA) and BCIP/NBT solution (bromochloroindolyle phophate/nitroblue tetrazolium; both from Sigma) were used for spot development. The number of spots was quantified using a KS-ELISPOT-Reader (Carl Zeiss, Oberkochen, Germany).
Determination of serum IgG by ELISA
Microplates (96-well; Greiner Bio One) were coated with antigen (0.125 μg/ml mBSA), collagen type I (from rat tail tendon) and type II (10 μg/ml), and proteoglycans (10 μg/ml both from bovine cartilage) and left overnight, as described previously [
14]. After washing, plates were incubated with serially diluted serum samples and the amount of bound IgG was determined using anti-mouse IgG-peroxidase conjugate (ICN, Eschwege, Germany) and ortho-phenylendiamine (Sigma) as substrate. Extinction was measured at 492 nm against 620 nm with an ELISA reader (Tecan, Crailsheim, Germany).
Cell transfer for in vivohoming assay
For
in vivo homing assay, cells were sorted with a modified protocol and labelled with
111indium, as described elsewhere [
10]. Briefly, CD4
+ cells were enriched by negative selection. Enriched CD4
+ T cells were stained with FITC-conjugated anti-CD4-F(ab) and anti-CD25-allophycocyanine and sorted into CD4
+CD25
+ or CD4
+CD25
- cells by FACS (BD). Cells were labelled with
111In (Indiumoxin; Amersham-Buchler) for 20 min at room temperature; 1 × 10
6 labeled cells were injected intravenously, and 24 hours later mice were killed and the distribution of radioactivity in various organs and the rest of the body was measured in a γ-counter (Wallac Counter, Turku, Finnland).
Alternatively, a proportion of these cells was labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) by incubation with 5 μmol/l CFSE (Molecular Probes, Leiden, The Netherlands) in RPMI 1640 for 5 min at room temperature. After washing, 1 × 106 cells were injected intravenously. Twenty-four hours later single cell suspensions were prepared from the draining and nondraining peripheral and mesenteric lymph nodes, the spleen and the peripheral blood, and stained with anti-CD4 and analyzed by FACS. Dead cells were excluded using propidiumiodide.
Statistical analysis
Data are expressed as mean ± standard error of mean, unless otherwise indicated. Experimental groups were tested for statistically significant differences with the Mann–Whitney U-test using SPSS 10.0 (SPSS Inc, Chicago, IL, USA).
Discussion
Our findings provide clear evidence that CD4
+CD25
+ T
reg cells are critical for regulating the severity of AIA in mice. We showed this by manipulating the T
reg cell numbers using two different approaches: depletion of CD25-expressing cells and transfer of purified CD4
+CD25
+ T
reg cells. It is important to stress that we depleted CD25-expressing cells in the interval between immunization and AIA induction, because CD25-depletion before immunization profoundly increases the resulting humoral and cellular immune responses [
3,
12]. These data are consistent with studies conducted in collagen-induced arthritis; however, in these experiments CD25-expressing cells were depleted before immunization with collagen type II, and the resulting more severe arthritis could be interpreted as the result of stronger immunization state [
12]. With our experimental design, we were able to examine the effect of T
reg cells in ongoing joint inflammation directly. Because CD25 is also expressed on activated conventional T cells, it could be assumed that injection of an anti-CD25 antibody would deplete not only T
reg cells but also effector T cells, but the exacerbated AIA in CD25-depleted mice argues against such a depletion of effector T cells. Accordingly, in control experiments lymph node cells from CD25-depleted mice isolated at the time of induction of AIA were able to mount a similar anti-mBSA response
in vitro as compared with control mice (data not shown). Furthermore, CD4
+CD25
+ cells isolated from immunized donors can suppress development of AIA (Fig.
4b). Taken together, these data imply that the CD25
+ compartment in immunized mice largely consists of T
reg cells.
AIA induction in CD25-depleted mice resulted in a much more severe arthritis in the acute and chronic stages of disease. We recently showed, with the use of a depleting anti-CD4 antibody, that this acute stage of AIA is already under the control of T cells [
15]. Nevertheless, early AIA is dominated by cells of the innate immune system [
19], and the exacerbation of arthritis in CD25-depleted mice could be due to a lack of suppression of these cells by T
reg cells. In accordance with this view, CD4
+CD25
+ T
reg cells are able to suppress innate immune cells in a model of bacteria-induced colitis [
20].
In later stages exacerbated arthritis in CD25-depleted mice is accompanied by increased mBSA-specific proliferation and IgG production. This enhanced responsiveness emerged during arthritis development and is due to sustained T cell activation. Such prolonged T cell activation in the absence of CD4
+CD25
+ cells has also been described in other disease models [
21] and is probably the cause of the increased AIA severity. Moreover, the PC61 antibody used in our study has a half-life of approximately 3 weeks
in vivo (Sutmuller R, personal communication), which makes it possible that T
reg cell function is not only impaired by depletion but also by blockade of IL-2 binding to CD25 by the PC61 antibody. IL-2 or IL-2 signalling via CD25 has been shown to be critical to the regulatory action of T
reg cells [
22,
23]. Also, activation-induced cell death of pathogenic T cells, which is regulated by IL-2, could be impaired by withdrawal of IL-2 signalling and therefore contribute to the observed high levels of cellular immune responses in our study [
24].
The fact that depletion of CD4
+CD25
+ T
reg cells enhances the immune response against the foreign antigen mBSA clearly demonstrates that their suppressive effect is not strictly limited to autoreactive T cells. Taking into consideration that T
reg cells are also critically involved in the control of immune responses against pathogens [
25,
26], their physiological function is not just to prevent autoimmunity but also to control the extent of inflammatory reactions in order to prevent tissue damage to the host. Further support for the influence of CD4
+CD25
+ T
reg cells on arthritis development came from the transfer experiments. When transferred at the time of induction of AIA, CD4
+CD25
+ cells were able to ameliorate ongoing disease. Analysis of the recipients did not reveal a remarkable long-lasting suppression of systemic mBSA-specific immune reactions. Thus, prevention of AIA appears to be possible without inducing anergy or abrogating previously induced T-cell effector functions [
27]. In contrast to this, transferred CD4
+CD25
- cells significantly enhance cell-mediated and humoral immune responses.
Furthermore, the homing data presented here demonstrate that CD4
+CD25
+ cells can migrate into the arthritic knee joint. Functional T
reg cells have repeatedly been found within such effector sites and/or draining lymph nodes, for instance in tolerated allografts [
28], in Langerhans islets and pancreatic lymph nodes in inflammation-induced diabetes [
29], in chronically inflamed skin in a
Leishmania infection model [
30], and in the mucosa and mesenteric lymph nodes in inflammatory colitis in severe combined immunodeficient (SCID) mice [
31]. Interestingly, two recent papers [
32,
33] reported an accumulation of functional T
reg cells in the inflamed joints of patients with RA, juvenile arthritis and other rheumatic diseases.
It is most likely that the transferred CD4
+CD25
+ T
reg cells act in the draining lymph node as well as in the inflamed tissue. Within such a scenario, it could be possible that T
reg cells inhibit the activation of effector T cells and their subsequent migration to the joints. Such a mechanism was recently speculated in modulation of virally induced immunopathology by T cells [
26].
Huehn and colleagues [
11] recently demonstrated that CD4
+CD25
+ T
reg cells can be divided into subsets based on the expression of the integrin α
Eβ
7. Moreover, this marker identifies CD25
- T
reg cells [
34]. Both α
Eβ
7-expressing subsets had better capacity to reach the inflamed joint and to prevent arthritis in the AIA model, as compared with α
Eβ
7- T
reg cells [
10]. Thus, suppression at the site of inflammation is also an important part of the activity of T
reg cells. How this effect is mediated is unclear but an involvement of IL-10 or transforming growth factor-β is possible [
20,
35,
36].
If these hypotheses are correct, then they could explain why the transfer of T
reg cells after arthritis induction is not effective. On the one hand, transfer of T
reg cells 24 hours after intra-articular antigen challenge might be too late to inhibit activation of effector T cells and their migration to the joint. Indeed, T-cell activation is an early event in AIA because CD4
+ T cell depletion ameliorates the acute stage of the model [
15]. On the other hand, it could be possible that the suppressive function of regulatory T cells is switched off under the inflammatory conditions present in the inflamed tissue by factors such as IL-6 or glucocorticoid-induced tumor necrosis factor family-related gene (GITR) and GITR-ligand interactions, abrogating the suppressive effect of T
reg cells [
37]. With this in mind, it could be interesting to investigate whether the accumulated T
reg cells in patients with arthritis function properly
in vivo and whether these patients could really benefit from a therapeutic enhancement of T
reg function, as suggested by some enthusiastic investigators in this field.
In this regard, data on the curative effects of T
reg cells in experimental disease models are conflicting. To best of our knowledge, a curative effect of CD4
+CD25
+ T
reg cells has only been demonstrated in the colitis model induced by transfer of CD45RB
high T cells into SCID mice [
31,
38]. In contrast, other authors were unable to demonstrate such an inhibitory effect of T
reg cells on SCID colitis when they were transferred 1 week after administration of pathogenic CD45RB
high T cells [
39]. Because arthritis in the AIA model has a hyperacute onset, it could be assumed that the time window for an ameliorative effect of T
reg cell transfer ends very shortly after intra-articular injection of antigen. However, further studies on the role of T
reg cells in other arthritis models are clearly needed to clarify whether enhancement in T
reg cell function might be beneficial in experimental arthritis and perhaps in human disease.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
OF purified the anti-CD25 hybridoma and purified the monoclonal antibodies from the supernatant; planned and conducted all animal experiments, including ELISA and ELISPOT analysis; and drafted the manuscript. PKP and MG scored the histological changes in arthritic joints. KS, JH and AH conducted the migration experiments, as well as the αEβ7 transfer experiments. RB supervised the project and participated together with AS and AR in the design of the study and its coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.