Inflammatory arthritis and dermatitis in thymectomized, CD25+ cell-depleted adult mice

A. Loughry, S. Fairchild2, N. Athanasou1, J. Edwards1 and F. C. Hall2

Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, 1 Nuffield Orthopaedic Centre, Oxford and 2 Department of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK.

Correspondence to: F. C. Hall, Box 157, Department of Clinical Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK. E-mail: fch22{at}medschl.cam.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
Objective. To investigate the effect of CD25+ or CTLA-4+ cell depletion on the natural history of collagen-induced and spontaneous arthritis in male DBA1/J mice.

Methods. Male DBA/1J mice were treated with anti-CD25 depleting antibody (PC61) or isotype control (GL113), or with anti-CTLA-4 depleting antibody (4F10) at various time-points peri- and post-immunization with bovine collagen type II, emulsified in adjuvant. In order to develop a model system in which long-term depletion of CD25+ regulatory T cells can be achieved prior to immunization, adult male DBA/1J mice were thymectomized prior to administration of either PC61 or GL113. An ELISA demonstrated that PC61 and GL113 antibodies were undetectable by 21 days after administration and FACS analysis confirmed the long-term depletion of CD25+ cells in peripheral blood.

Results. In the thymectomized mice treated with PC61, the CD25+ population was depleted and a spontaneous arthritis developed (P = 0.03). In the non-thymectomized mice, administration of CTLA-4-depleting antibody prior to immunization exacerbated arthritis in mice immunized with bovine collagen type II emulsified in incomplete Freund's adjuvant (P<0.01). However, no significant difference in the natural history of arthritis was evident in mice treated with CD25-depleting antibody (PC61) compared with control antibody (GL113).

Conclusions. Two separate models implicate CD25+ CTLA-4+ constitutive cells in suppression of arthritis in susceptible DBA/1 males: exacerbation of collagen-induced arthritis following CTLA-4 depletion at the start of induction and spontaneous arthritis in the thymectomy/CD25+ depletion model.

KEY WORDS: Inflammatory arthritis, Dermatitis, CD25, Thymectomy, Mice


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
Chronic inflammatory arthritis affects over 1% of the population world-wide, most commonly as rheumatoid arthritis (RA). In contrast, acute polyarthralgia or polyarthritis has a higher lifetime incidence, occurring transiently in most individuals during infections. This implies that regulatory mechanisms serve to limit collateral tissue damage and to promote resolution of the inflammatory response. This study examined the effect on murine models of arthritis of depletion of cells expressing markers associated with a regulatory T-cell population.

Collagen-induced arthritis (CIA) in susceptible murine strains is a well-characterized model of inflammatory arthritis. In common with RA, susceptibility to disease is oligogenic and includes the MHC class II locus [1]. CD4+ T cells are implicated in the initiation of disease, although B cells, CD8+ T cells and cells of the monocyte/macrophage lineage are also involved [2–4]. In contrast with RA, CIA is clearly initiated by exposure to antigen, usually heterologous collagen type II (CII), emulsified in adjuvant, and the disease is more frequent in males, with an incidence of >90% in males of the DBA/1 strain of mice. CIA was therefore adopted as a model in which to assess the effect of removing regulatory immune cell subsets.

Evidence for regulatory activity by subsets of T cells has emerged from the study of transplantation immunology [5, 6], autoimmunity [7–9] and excessive response to commensal organisms [10]. It appears that a number of distinct CD4+ T-cell populations have regulatory activity. Roncaralo and colleagues described the Tr1 CD4+ T-cell subset that produces IL-10 [11]. Meanwhile, Weiner and colleagues, working in experimental allergic encephalitis, described the Th3 CD4+ cell, which produces TGF-ß. The majority of data emerging in the last 5 yr on regulatory T cells, however, relates to a minority population of CD4+ T cells that express CD25 (IL-2R{alpha}) and CTLA-4 constitutively [8, 12]. Mice which undergo neonatal thymectomy (between days 3 and 5) fail to develop this population, which normally constitutes 5–10% of the peripheral CD4+ T-cell compartment, and they develop a variety of organ-specific or systemic autoimmune diseases [13]. Depletion of CD25+ or CTLA-4+ cells in adult mice has also resulted in the development of chronic inflammatory diseases, including colitis [14], insulin-dependent diabetes [15] and experimental allergic encephalitis [16] in susceptible strains. Despite widespread acceptance of the presence of regulatory activity, definition of the ‘natural’ regulatory T-cell lineage by surface markers has been problematic since CD25, CTLA-4 and other surface markers, which can be used to enrich regulatory subsets, are also expressed on other populations. Recently, expression of the transcription factor FoxP3 has been shown to determine regulatory activity [17]. This tool has facilitated the study of a range of phenotypically defined subsets with regulatory activity. A currently prevalent model proposes that a population of ‘natural’ regulatory T cells mature in the thymus, as a result of high-affinity interactions between the T-cell receptor and MHC class II/self peptides. The regulatory activity of these CD4+CD25+CTLA-4+ FoxP3+ T cells is contact-dependent. In the periphery, however, further regulatory subsets can be ‘induced’ by TGF-ß secreted by the ‘natural’ regulatory T cells [18]. In this process, CD4+CD25FoxP3 T cells are induced to express FoxP3 and CD25 and these cells acquire regulatory activity. Although FoxP3 is the most specific marker for regulatory activity, it cannot be used to separate live cell populations, and less specific cell surface markers are still required to enrich or deplete regulatory subsets.

In this study, either CD25+ or CTLA-4+ cells were depleted from DBA/1 male mice using depletion regimens either peri- or post-immunization with bovine CII emulsified in adjuvant. In order to clarify the effect of selective and prolonged depletion of constitutive CD25+ cells, adult mice were thymectomized prior to CD25 depletion. These mice developed spontaneous inflammatory arthritis and dermatitis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
Mice
Male DBA1/j mice were obtained from Harlan. C57Bl/6 mice were obtained from the Oxford Biomedical Services Unit. C57Bl/10 mice were purchased from Jackson Bar Harbor. All mice were caged in a conventional facility. All protocols involving live animals were subjected to ethical review as part of the application process for a Home Office Project Licence. A range of welfare score sheets and action plans was developed to monitor and minimize the impact of the study on the animals.

Antibodies
Monoclonal antibodies administered to mice were: anti-CD25-blocking and -depleting rat IgG1 antibody, clone PC61, rat IgG1 anti-ßGAL isotype control, clone GL113, anti-CTLA-4 depleting hamster IgG antibody clone 4F10. FACS analysis was performed using FITC (FITC)-, phycoerythrin (PE)- or PerCP-conjugated anti-CD4 antibody clone LT4- (Pharmingen), PE- or APC-conjugated anti-CD25 antibody (PC61; Pharmingen), FITC-conjugated anti-CD69 antibody (Pharmingen) and FITC-conjugated anti-CD62L (Pharmingen). Purified anti-CD3 antibody (Pharmingen) was used to stimulate T cells in proliferation assays in vitro. The following antibodies were used for the anti-CII specific IgG subtype enzyme-linked immunosorbent assay (ELISA): murine IgG1 standard clone MOPC-21 (Pharmingen), murine IgG2a standard clone G155–178, alkaline phosphatase (ALK)-conjugated anti-murine IgG1 clone X56 (Pharmingen), ALK-conjugated anti-murine IgG2a clone R19-15 (Pharmingen). For the rat IgG1 ELISA, either GL113 or PC61, purified from hybridoma supernatant, was used as rat IgG1 standard. Biotinylated anti-rat IgG1, clone RG11/39.4 (Pharmingen) was used as a detection antibody.

B-cell hybridomas PC61, GL113 and 4F10 were grown in the cellular compartment of a CL1000 flasks (Integra) in RPMI medium supplemented with 10% low-immunoglobulin serum (Sigma), penicillin, streptomycin and L-glutamine. RPMI supplemented only with penicillin, streptomycin and L-glutamine was added to the acellular compartment of the cell farm. The supernatant from the cellular compartment of the cell farm was harvested twice weekly, according to the manufacturer's instructions and stored at –20°C. Antibody was purified from the supernatant by removing cell debris by centrifugation (200 g for 30 min) and filtration (0.22 µm) followed by two rounds of precipitation with saturated ammonium sulphate (12 h at 4°C). The precipitated antibody was resuspended in the minimum volume of endotoxin-free water and dialysed against sterile phosphate-buffered saline (PBS). The purified antibody concentration and size were assessed by measuring absorbance at 280 nm and by sodium dodecyl sulphate gel electrophoresis.

Histology
Tissues were harvested post-mortem and preserved in PBS/1% paraformaldehyde for a minimum of 48 h. Tissues containing bone were decalcified in 5% nitric acid for 24–48 h. The tissue was dehydrated and fat was extracted by a series of automated exchanges: 70% ethanol, 90% ethanol, 100% ethanol (x3), xylene (x3), paraffin (x3). The specimen was then embedded in wax, cooled to –20°C and sectioned at 5-µm intervals. Sections were stained with haematoxylin and eosin, and with toluidine blue.

Arthritis induction and monitoring
Adult male DBA/1j mice (aged approximately 2 months) were immunized with 100 µg bovine CII (MD Biosciences) emulsified in complete Freund's adjuvant (CFA) intradermally at the base of the tail. Arthritis onset and severity were assessed by performing an arthritis score three times weekly. Each paw was assigned a score between 0 and 4: 0 = no arthritis, 1 = mild involvement in a single area (single interphalangeal joint, mid-foot or wrist/ankle); 2 = moderate/severe involvement in a single area (as above) or mild involvement in 2–3 areas; 3 = severe involvement in two areas or moderate involvement in several areas; 4 = severe involvement in all areas. This method of assessment was identical for both CIA and spontaneously occurring arthritis. In CIA, the day to arthritis onset was defined as the number of days between immunization with CII and the occurrence of arthritis with a severity score ≥2.

Thymectomy
Mice aged approximately 5 weeks were anaesthetized using intraperitoneal fentanyl/fluanisone/midazolam. Thymectomy was performed using a surgical approach from the ventral neck area. A small incision in the paratracheal fascia enabled the insertion of a tapered glass pipette into the thoracic apex and aspiration of the thymic lobes. Skin closure was achieved with the tissue adhesive VetbondTM (3M Animal Care).

ELISAs
ELISAs were performed to detect the presence and isotype of anti-bovine CII antibodies, anti-murine CII antibodies and the presence of rat IgG1 [isotype of PC61 (anti-CD25)] and GL113 (anti-ßGAL isotype control). For the anti-CII assays, ELISA-grade bovine or murine CII (Chondrex) was diluted to 5 µg/ml in coating buffer (1 M sodium bicarbonate mixed with 1 M disodium carbonate at a ratio of ~2.5:1 (v/v) and adjusted to pH 9.6). Eighty wells of 96-well Maxisorp (Nunc) plates were coated with CII at 4°C overnight and a standard curve of either mouse IgG1 or mouse IgG2a was plated in duplicate in the remaining 16 wells. The plate was washed with PBS/0.1% Tween, blocked with PBS/1% bovine serum albumin/0.02% sodium azide overnight at 4°C, and dilutions of mouse serum (1:100, 1:300, 1: 900) were added to the CII-coated wells. After incubation at 4°C overnight, the plates were washed five times with PBS/0.1% Tween and second-layer antibody (either ALK-anti-IgG1 or ALK-IgG2a, 1:1000 in PBS/1% bovine serum albumin/0.02% azide) was added, either at 4°C overnight or room temperature for 2 h. After three washes with PBS/0.1% Tween, 100 µl freshly prepared p-nitrophenyl phosphate substrate was added to each well. After 10–20 min, the reaction was stopped by the addition of 25 µl 3 M sodium hydroxide and the emission at 405 nm was quantified using a µQuant plate-reader (Biotek Instruments). The graphical relationship between optical density at 405 nm and IgG concentration was fitted using µQuant software and the concentrations of IgG in each well were calculated from the linear part of the curve.

The ELISA for rat IgG1 was performed using a similar protocol. Plates were coated with serial dilutions of the test sera and a standard curve of purified rat IgG1 (PC61) was plated in duplicate. After overnight incubation, washing and blocking of plates, biotinylated anti-rat IgG1 2 µg/ml was added to the wells for 2 h at room temperature. After washing, 100 µl streptavidin-conjugated ALK (Pharmingen) 1:1000 was added to each well for between 30 min and 1 h. The substrate and readout were performed as described above.

Study design and statistical analysis
Mice were assigned to study groups using computer-generated random numbers. Each cage was randomized separately, ensuring that all study groups were equally represented in each cage.

The outcome measures for the arthritis induction experiments were: incidence, onset speed (number of days elapsing between immunization and development of arthritis with a score ≥2), peak arthritis severity score, area under the curve (AUC) for arthritis severity score plotted against time. The AUC was calculated using the trapezium rule [19]. The AUC data sets were assessed for normality using the Anderson–Darling normality test (Minitab). Significance testing of normally distributed data sets was performed using Student's t-test. For non-parametric data, the Mann–Witney test was applied (Minitab).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
CD25 is induced on activated CD4+ T cells during arthritis induction
CD25 is an activation marker on T cells, in addition to being a constitutive marker on a subgroup of CD4+ T cells. In order to assess the changing nature of CD4+CD25+ T cells post-immunization, peripheral blood was sampled at baseline and at day 13 post-immunization with 100 µg bovine CII/CFA in ten 2-month-old DBA/1 males. Peripheral blood mononuclear cells were stained with PE-anti-CD4, APC-anti-CD25 and either FITC-anti-CD69 (an early activation marker) or FITC-conjugated anti-CD62L (marker of naive or resting cells; facilitates recirculation through lymph nodes via high endothelial venules). Figure 1 demonstrates that the percentage of CD25+ cells within the CD4+ T-cell subset is significantly elevated at day 13 post-immunization (mean ± S.E.M.): 9.3 ± 0.3% compared with 8.0 ± 0.4% at baseline (P<0.01; one-tailed paired t-test). Since mice have approximately 3.5 x 106 total lymphocytes and approximately 1 x 106 CD4+ lymphocytes per millilitre of blood (data not shown), this equates to 8.0 x 104 CD4+CD25+ lymphocytes/ml at baseline, rising to 9.3 x 104/ml at day 13. The percentage of CD4+CD25+ T cells expressing the activation marker CD69 had risen from 1.1 ± 0.2 to 3.9 ± 0.6% (P<0.001), whereas the percentage of CD4+CD25+ T cells expressing CD62L (L-selectin) had decreased from 70.7 ± 1.0 to 60.9 ± 4.0% (P<0.05). Therefore, in the 13 days post-immunization, the absolute number of CD4+CD25+CD69+ T cells in peripheral blood rose approximately four-fold from 8 x 102/ml to 3.6 x 103/ml; cells of this phenotype probably represent the activated effector population. In contrast, the absolute number of CD4+CD25+CD62L+ T cells remained constant at approximately 5.6 x 104/ml; these cells probably represent the regulatory T cell population.



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FIG. 1. The changing proportions of CD4+CD25+ subsets in adult DBA/1 male mice pre- and 13 days post-immunization with 100 µg bCII/CFA. (a) The proportion of CD4+ T cells that express CD25 is significantly elevated post-immunization (P<0.01). (b) The proportion of CD4+CD25+ cells that express the activation marker CD69 is elevated; this equates to a rise in absolute CD4+CD25+CD69+ lymphocyte numbers of 8 x 102 to 3.6 x 103. (c) The proportion of CD4+CD25+CD62L+ (phenotype more specific for regulatory population) are depressed post-immunization but the absolute cell numbers (5.6 x 104) are unchanged.

 

    Depletion of CD25+ cells at or after immunization with CII/CFA does not exacerbate CIA
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
The optimal dose of anti-CD25-blocking and -depleting antibody, PC61, was determined by administering 2, 1, 0.5, 0.25 or 0.125 mg intraperitoneally to 2-month-old male DBA/1 mice. Two administrations separated by a 1-week interval were given to two mice for each dose. The minimum dose of CD25-depleting antibody (PC61) that reduced CD4+CD25 medium and CD4+CD25hi T cells each to ≤0.05% of total CD4+ T cells was 1 mg (data not shown). This dose was used in subsequent studies.

Two-month-old male DBA/1j mice were immunized intradermally at the base of the tail with either 100 µg bovine CII emulsified in CFA or PBS emulsified with CFA. Mice were treated with three 1 mg intraperitoneal doses of monoclonal antibody, either CD25-depleting antibody, (PC61) or the isotype control, GL113. PC61 can potentially block and deplete both CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells and it is clear that the subset of CD25+CD69+CD62L effector cells increases post-immunization with CII/CFA (see previous section). In an attempt to deplete predominantly the CD4+CD25+ regulatory T cells, two antibody administration regimens were adopted: either early (days –1, +6 and +13) or late (days +14, +21 and +28) relative to the day of immunization (designated day 0). There were five mice in each of eight treatment groups, which ran concurrently. Arthritis severity scores were performed approximately three times weekly and outcome measures were the day of onset, peak arthritis severity score over the duration of the experiment and the AUC calculated from a plot of arthritis severity score against time.

Figure 2a displays the AUC for each individual mouse in the four groups immunized with CII/CFA. There are no significant differences between the groups that received the CD25-depleting antibody (PC61) and the isotype control (GL113), irrespective of whether the antibody was administered early or late, as described above. Mild arthritis was evident in some unimmunized mice but the severity was minimal compared with the immunized group (data not shown). For the CII/CFA-immunized groups (early and late groups combined), the speed of onset of arthritis was 41 days post-immunization in the PC61-treated group and 34 days in the GL113-treated group. The peak arthritis severity scores were 7.3 and 9.4 respectively in the mice that received PC61 or GL113. There was no significant difference between these groups but the trend was for arthritis to occur later and with reduced severity in the groups treated with PC61.



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FIG. 2. (a) AUC (derived from plots of arthritis severity score against time) for DBA/1 male mice immunized with 100 µg bCII/CFA and treated with three doses at weekly intervals of 1 mg intraperitoneally of PC61 (anti-CD25 antibody) or GL113 (rat IgG1 isotype control). Early antibody administration was on days –1, +6 and +13 relative to immunization whereas late administration was on days +14, +21 and +28. Each filled circle represents an individual mouse. There was no significant difference in AUC between the groups. (b) AUC and anti-bCII IgG serum concentrations of mice treated with anti-CTLA-4 antibody (4F10) or no antibody. Mice were immunized with either 100 µg bCII/CFA or 100 µg bCII/IFA. Closed circles indicated AUCs for individual mice. In the group of mice immunized with bCII/IFA, administration of 4F10 was associated with reduced severity of arthritis. The table displays the mean ± S.E.M. AUC for each group and the concentrations (µg/ml) of bCII-specific IgG1 and IgG2a antibodies in the serum 5 weeks post-immunization. Baseline IgG1 ≤0.5 ± 0.4 µg/ml and baseline IgG2a ≤1.9 ± 1.7 µg/ml (data not shown). There was no significant difference in antibody concentrations between the CTLA-4-depleting antibody (4F10)-treated or control groups.

 

    Depletion of CTLA-4+ cells prior to immunization with CII/incomplete Freund's adjuvant exacerbates CIA
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
Since the subset of CD4+ T cells that constitutively express CD25 also includes cells that constitutively express CTLA-4, the anti-CTLA-4 monoclonal antibody 4F10 was used to deplete CTLA-4+ cells from mice during arthritis induction. Initially, 1 mg 4F10 was administered on days –3 and +4 relative to immunization with bovine CII emulsified in adjuvant on day 0. A control group received no antibody. The experiment was performed in parallel for mice immunized with 100 µg bovine CII emulsified in either CFA or incomplete Freund's adjuvant (IFA). There were 10 adult male DBA/1j mice per group. Serum from the mice was stored at day 0, prior to immunization and again at day +35. The concentration of serum anti-bovine CII IgG1 and IgG2a was measured in these samples by ELISA.

Figure 2b displays the AUC (derived from arthritis severity plotted against day of assessment) for each of the four groups. Data from all four groups approximated to a normal distribution and paired Student's t-tests were performed independently for the mice immunized using CFA or IFA. There was no significant difference in the CII/CFA-immunized group receiving 4F10 compared with no antibody. However, 4F10 administration reduced the AUC significantly in the group immunized with CII/IFA (P<0.01). The table accompanying Figure 2b displays the mean (±S.E.M.) AUC for each group and the mean (±S.E.M.) serum bovine CII-specific IgG1 and IgG2a concentrations 5 weeks post-immunization for each group. The administration of 4F10 was not associated with a significant difference in specific IgG concentrations. Baseline anti-bovine CII concentrations were low: IgG1, 0.2 ± 0.1 µg/ml; IgG2a, 0.7 ± 0.4 µg/ml.

A second CTLA-4+ depletion experiment was subsequently conducted, in which DBA/1 males received 1 mg CTLA-4-depleting antibody (4F10) at day +1 and day +7 relative to the day of immunization (day 0). Six groups were studied in parallel: three immunization regimens, each with or without 4F10 administration. The immunization regimens were 100 µg bovine CII /IFA or 50 µg bovine CII/IFA; each was administered at day 0 by intradermal injection at the base of the tail. Figure 3 displays the AUC for each group. There were no significant differences between groups that received 4F10 compared with no antibody treatment, either for the AUC or for the titre of anti-bovine CII IgG (data not shown).



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FIG. 3. AUCs of mice treated with anti-CTLA-4 antibody (4F10) or no antibody. Mice were immunized with either 100 µg bCII/IFA or 50 µg bCII/IFA. There was no significant difference in AUC or antibody concentrations between the 4F10-treated or control groups.

 

    Thymectomy and depletion of CD25+ cells in adult DBA/1j mice results in prolonged depletion of CD4+CD25hi and CD4+CD25med T cells
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
In the models above, selective depletion of either CD25+ or CTLA-4+ regulatory cells relied on the timing of antibody administration. The only regimen that appeared to deplete regulatory activity was the administration of 4F10 at days –3 and +4 relative to immunization. Even this regimen was problematic, since residual CTLA-4-depleting antibody (4F10) may have depleted emerging CTLA-4+ effector T cells and, once the antibody had cleared, the regulatory T cell subset could be replenished. In order to selectively deplete the naturally occurring regulatory T cells, an adult thymectomy/depletion model was developed in male DBA/1 mice. Adult mice aged 6–8 weeks underwent thymectomy. These mice received 1 mg intraperitoneally either of CD25-depleting antibody (PC61) or the isotype control (GL113) at 2 and 3 weeks post-thymectomy. Presence of exogenous antibody can exert a number of immunomodulatory effects, both by depletion of CD25+ cells (PC61) or by binding to Fc receptors (either PC61 or GL113). In order to establish the point at which the exogenous antibody had been cleared, concentrations of rat IgG1 (either PC61 or GL113) were measured in sera collected at baseline and on days 2, 9 and 20 after antibody administration. Figure 4a displays these results; serum concentrations of the administered antibody were undetectable by ELISA at 20 days after administration.



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FIG. 4. (a) Concentration of rat IgG1 in the serum of mice, estimated by ELISA, at baseline and 2, 9 and 20 days after the final administration of 1 mg rat IgG1 intraperitoneally, either CD25-depleting antibody (PC61) or control antibody (GL113). No rat IgG1 is detectable at day 20. (b–d) FACS dot-plots, gated on the lymphocyte population, which display the effect of PC61 on the percentage of CD4+ lymphocytes that express CD25. CD25neg, CD25lo, CD25med and CD25hi subsets of CD4+ lymphocytes have been defined according to the expression level of CD25 (b). (c) Administration of PC61 removes both CD25med and CD25hi subsets. In contrast, (d) shows that control antibody, GL113, does not deplete these subsets (c and d display peripheral blood CD4+CD25+ subsets 2 days following the final administration of antibody). The only discernible change with isotype control is a shift from CD25neg to CD25lo expression; this is also evident in the PC61-treated mice.

 
Peripheral blood mononuclear cells harvested on days 2, 9, 20, 36 and 44 after antibody administration were stained with PerCP-conjugated anti-CD4 and APC-conjugated CD25. Figure 4b–d depicts four groups of CD4+ lymphocytes, which were defined by the level of CD25 expression as CD4+CD25, CD4+CD25lo, CD4+CD25med and CD4+CD25hi subsets. Treatment with CD25-depleting antibody (PC61) ablated the CD4+CD25med and CD4+CD25hi subsets without significantly altering the frequency of CD4+CD25 and CD4+CD25lo subsets or the absolute number of cells in these subsets. Treatment with the control antibody (GL113) did not significantly alter the size of the CD4+CD25hi or CD4+CD25med T-cell subsets. However, a similar shift from CD4+CD25 to CD4+CD25lo expression was evident in both CD25-depleting antibody (PC61) and GL113-treated groups. Figure 5 indicates the duration of CD4+CD25med and CD4+CD25hi subset depletion. A subset of CD4+CD25med T cells reappeared progressively from 20 days after CD25-depleting antibody (PC61) administration but CD4+CD25hi T cells remained depleted during this period.



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FIG. 5. This displays the time-course of depletion of the CD25med and CD25hi subsets of CD4+ lymphocytes. A subset of CD25med T cells reappears from approximately 20 days after PC61 administration. The CD4+ CD25hi subset remains depleted beyond day 36 but reconstitution appears to be occurring by day 44.

 

    Thymectomy and depletion of CD25+ cells in adult DBA/1j mice results in spontaneous occurrence of arthritis and dermatitis
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
Adult DBA/1 males aged 6–8 weeks underwent thymectomy and then received either CD25-depleting antibody (PC61) or control antibody (GL113) (12 mice/group), according to the protocol described above. Depletion of CD4+CD25+ cells in the PC61-treated group and clearance of the administered rat IgG1 antibody was confirmed as described in the previous section. A low-grade arthritis (arthritis severity score 1–3) was evident in approximately 50% of mice from each treatment group during the first month following antibody administration (mice aged 3–4 months). Between 5 and 7 weeks following the second administration of antibody (PC61 or GL113) an arthritis severity score between 3 and 7 occurred in nine of the 12 mice treated with CD25-depleting antibody (PC61), compared with three of the 12 mice treated with control antibody (GL113). The AUCs for the individual mice during the period between antibody administration and the end of the experiment are shown in Figure 6a. The AUC for the TxGL113 group were not normally distributed and a non-parametric significance test was applied to these data. The differences in scores between the groups treated with CD25-depleting antibody (PC61) and control antibody (GL113) were significant (P = 0.03; Mann–Whitney two-tailed test). The pattern of arthritis in the TxPC61 group was distinct from that observed by our group in CIA in DBA/1 males. In TxPC61 mice, arthritis predominated in the hindpaw proximal interphalangeal joints and involvement of forepaws and the ankle joint were less frequently involved, in comparison with CIA (Fig. 6b). Figure 7 compares the histological appearance of interphalangeal joints from a control DBA/1 male, a thymectomized, CD25-depleted DBA/1 male and a thymectomized, isotype control (GL113) treated DBA/1 male. Pannus formation, destruction of articular cartilage and bone erosion were all evident in the affected joint from the thymectomized, CD25-depleted male.



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FIG. 6. (a) AUC scores for individual mice undergoing either the TxPC61 protocol (thymectomy followed by depletion of CD25+ cells with the anti-CD25 antibody, PC61) or the control protocol (thymectomy followed by administration of isotype control antibody, GL113). Although low-grade arthritis was evident in approximately 50% of mice from each group, a more severe arthritis occurred in the TxPC61 protocol mice (P<0.03; Mann–Witney non-parametric test). (b) Comparison of the pattern of joint involvement in mice undergoing the TxPC61 (CD25-depleting antibody) and TxGL113 (control antibody) protocols with DBA/1 males subjected to the standard CIA protocol (100 µg bovine CII emulsified in CFA administered intradermally at the base of the tail) and unimmunized littermates. The graph shows the percentage of hindpaws and forepaws in each group that show involvement at the distal interphalangeal joints [(Hind) HDIP or (Fore) FDIP), mid-foot (HM or FM), ankle (AJ) or wrist (WJ)]. The number of paws scored in each group were as follows: 24 hind- and 24 forepaws in each of the TxPC61 and TxGL113 groups and 20 hind- and 20 forepaws in the CIA group and 10 hind- and forepaws in the unimmunized controls. The score merely documents involvement of the given joint/region and provides no information on the severity of joint involvement.

 


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FIG. 7. (a) Arthritis in proximal interphalangeal joint (arrow) in the hindpaw of a mouse that underwent the TxPC61 protocol. (b) Histology of normal interphalangeal joint from control (unthymectomized) DBA/1 mouse (haematoxylin and eosin) (c) Histology of normal interphalangeal joint from DBA/1 mouse that underwent the control protocol (haematoxylin and eosin). (d) Histology of interphalangeal joint from DBA/1 mouse that underwent the thymectomy and CD25-depletion protocol (haematoxylin and eosin). Inflammatory pannus with associated cartilage and bone erosion is evident.

 
In addition to spontaneous arthritis, dermatitis occurred in eight of 12 thymectomized, CD25-depleted mice compared with two of 12 control antibody (GL113)-treated mice. Macroscopically, the eruption consisted of crops of ulcerated lesions 2–3 mm in diameter. These occurred particularly over the back, abdomen and scrotum (Fig. 8a). Histologically, the lesions were characterized by full-thickness ulceration of the epidermis and the presence of a diffuse, focally heavy neutrophil infiltrate. There was no evidence of vasculitis (Fig. 8c). No parasitic infection was evident in the mouse colony. Furthermore, each cage of 10 mice included both CD25-depleted (PC61)- and control (GL113)-treated animals. In each cage, the PC61-treated animals proved more susceptible to the development of dermatitis.




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FIG. 8. (a) An ulcerative depilating rash on a DBA/1 mouse that underwent thymectomy and CD25-depletion protocol. (b) A normal murine skin from the lower back (haematoxylin and eosin). (c) Section through rash over lower back of a DBA/1 mouse that had undergone thymectomy and CD25 depletion (haematoxylin and eosin). A dense inflammatory infiltrate underlies the ulcerated area. (d) High-power view of inflammatory infiltrate.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 
CD25 is expressed both by regulatory and by activated effector CD4+ T cells. The increased expression of CD25 by activated CD4+ T cells in mice 13 days post-immunization with CII/CFA has been demonstrated in this study. The increased percentage of CD4+ T cells expressing CD25 was shown to be a result of increased activated CD4+CD25+CD69+ T cells. Interestingly, the absolute number of CD4+CD25+CD62L+ T cells in the peripheral blood did not change in the 2 weeks post-immunization. This phenotype is probably more specific for regulatory cells than CD4 and CD25 expression alone, since several laboratories have demonstrated that the CD4+CD25+CD62L+ subset has higher regulatory potency than the unfractionated CD4+CD25+ subset [20, 21].

The expression of CD25 on effector cells post-immunization hampers attempts to selectively deplete CD4+CD25+ regulatory T cells during an immune response. In this study, there was no exacerbation of CIA in (unthymectomized) mice, which underwent depletion of CD25+ cells. PC61 was administered between days –1 and +28, relative to immunization with CII and adjuvant at day 0. Since PC61 is detectable in the peripheral blood for approximately 2 weeks, the CD25+ cells depleted with these regimens would include CD4+ T cells with both constitutive and activation-induced CD25 expression. This would be consistent with the observation that the PC61-treated group had a trend towards less severe arthritis. Morgan and colleagues reported that depletion of CD25+ cells with PC61 exacerbated CIA in DBA/1 males [22]. This group used several PC61 regimens over a similar time course to those used in this study and, in these, no exacerbation of arthritis was evident. However, when PC61 was administered on days –28, –24, –21 and –14 relative to immunization, arthritis exacerbation occurred. This early PC61 administration probably permitted the selective depletion of CD4+ T cells that constitutively expressed CD25, since the antibody would have been undetectable in the circulation at the time when effector CD4+ T cells expressed activation-induced CD25.

Depletion of CTLA-4+ cells by administration of 4F10 antibody on days –3 and +4 relative to immunization with CII emulsified in IFA exacerbated arthritis. Later depletion of CTLA-4+ (4F10 administration on days +1 and +7) had no discernible effect on arthritis severity. It is probable that both regulatory and effector subsets expressing CTLA-4 were depleted using this regimen. The exacerbation of arthritis with the earlier CTLA-4-depletion was evident in the mice immunized with CII/IFA but not CII/CFA. CFA contains mycobacterial fragments and is a more potent stimulant of the innate immune response. Although CD4+CD25+ T cells can regulate innate immune as well as adaptive effector responses [23], it is possible that a stimulus of sufficient potency can overcome the regulatory influence of CD4+CD25+ T cells. This has been demonstrated in vitro where optimal concentrations of plate-bound anti-CD3 and/or addition of exogenous IL-2 or anti-CD28 abrogates the suppressive effect of CD4+CD25+ T cells on CD4+CD25 effectors [24].

Adult thymectomy followed by administration of the anti-CD25 antibody, PC61 (TxD protocol) results in the prolonged depletion of CD25+ cells from the peripheral blood of DBA/1 mice. The demonstration that administered antibody (PC61 or GL113) was undetectable within 3 weeks post-administration confirmed that the prolonged CD4+CD25hi regulatory T-cell depletion was not dependent on continuing removal of CD25+ cells (including any emerging CD4+CD25+ effector T cells). Furthermore, this indicates that any non-specific immunomodulatory effects of rat IgG1 antibody would be insignificant from 2–3 weeks post-administration. Adult thymectomy followed by CD25+ cell depletion facilitates emergence of inflammatory arthritis and neutrophilic dermatitis in male DBA/1j mice. A minority of the adult thymectomized but CD25+-replete control mice also developed arthritis and dermatitis. However, the incidence and severity of inflammatory disease were both lower in the CD25+-replete mice and the low-grade inflammatory arthritis in this group is reminiscent of that described in unmanipulated elderly DBA/1 males [25]. There was no evidence of inflammatory disease in organs other than joints and skin of the DBA/1 mice studied (data not shown).

The re-emergence of a CD4+CD25+ population after adult thymectomy and CD25-depletion raises important questions concerning the origin and function of this subset. The replenishing CD4+CD25+ population expressed medium levels of CD25 and appeared between 20 and 30 days post-depletion. Since the depleting antibody was probably cleared from the circulation between 2 and 3 weeks post-administration, it is possible that the CD4+CD25med cells were generated earlier in the time-course but were depleted by persisting anti-CD25 antibody. Alternatively, it is possible that CD25-depletion was incomplete and that CD4+CD25+ T cells that were sequestered in lymphoid tissue during the depletion phase subsequently recirculate to the peripheral blood. However, if this occurred, recirculation of CD4+CD25hi T cells would also be expected. There is no sign of CD4+CD25hi replenishment until at least day 42 post-antibody administration. We have not tested the regulatory capacity of the replenishing CD4+CD25+ populations. It is possible that these cells represent effector T-cell populations involved in the autoimmune process. However, it has been demonstrated that CD4+CD25+ regulatory T cells can be induced in the periphery [18]. Since TGF-ß, from thymically derived natural regulatory T cells, is believed to drive this process, an alternative source of TGF-ß would be required. However, several cell types can secrete TGF-ß. These issues require clarification.

It is probable that depletion of the subset of CD4+ T cells that constitutively express CD25 is responsible for the emergence of spontaneous inflammatory disease, since this phenotypic subset contains the natural regulatory T-cell population. However, CD25 is constitutively expressed on other cell subsets, including a minority of CD8+ T cells and some B cells. We have not formally excluded the involvement of these other CD25-positive cell populations. The development of arthritis and dermatitis following the TxD protocol may reflect an exacerbation of an inherent tendency to inflammatory disease at these sites in the DBA/1 strain. This role for genetic predisposition in the expression of inflammatory disease would be consistent with the observation that neonatal thymectomy (and hence failure of emergence of the CD4+CD25+ T cell population) results in different patterns of spontaneous inflammatory disease in different mouse strains [13]. Preliminary data from thymectomy and CD25-depletion in other strains of mouse support this notion. Three out of three C57BL/10.Q mice developed arthritis compared with only one of three C57BL/6 mice (data not shown). Although these data suggest that susceptibility of mouse strains to arthritis following thymectomy and CD25-depletion correlates with susceptibility to CIA [26], the diseases are distinct. In addition to an increased predilection for arthritis in interphalangeal joints, mice with TxD-induced arthritis have no anti-mouse CII antibodies (data not shown).

In humans, CD4+CD25+CTLA-4+ T cells have also been shown to exert regulatory activity in vitro [27, 28]. Recent data suggest that the CD4+CD25hi subset is dysfunctional in patients with active rheumatoid arthritis and that a clinical response of these patients to anti-TNF-{alpha} therapy correlates with a normalization of CD4+CD25+ T-cell function [29]. This study and the work of Morgan and colleagues [22] indicate that CD4+CD25+ regulatory T cells can oppose arthritogenic immune responses, at least in certain arthritis-susceptible strains of mice. Further study is required to understand their mechanism of action in this context and their activity in both self-limiting and chronic synovitis in humans.


    Acknowledgments
 
We thank Awen Gallimore for provision of B cell hybridomas PC61, 4F10, GL113, Colin Hetherington for assistance in BMSU and Caroline Atkinson for preparing skin histology slides. AL and SF and FCH were supported by the Arthritis Research Campaign, UK, and NA and JE were supported by Research into Ageing, UK.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Depletion of CD25+ cells...
 Depletion of CTLA-4+ cells...
 Thymectomy and depletion of...
 Thymectomy and depletion of...
 Discussion
 References
 

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Submitted 2 August 2004; revised version accepted 12 October 2004.



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