Institute of Immunology, Laboratory for Rheumatology Research, and the Center for Rheumatic Diseases, The National Hospital, Oslo, Norway and
1 Department of Immunology and Rheumatology, Hôpital Edourd Herriot and Immunovirology Laboratory, UMR CNRS 5537, Faculté de Médicine Laennec, Lyon, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Three different synovial tissue (ST) samples were obtained from one patient with erosive RA. The characterization of Th1 and Th2 responses was performed by interferon- and interleukin-4 measurements and by expression of the chemokine receptors CCR5 and CCR3. Measurements of secreted and surface immunoglobulin determined the types of B cells.
Results. The first ST sample yielded 31 CD4+ T cell clones which showed an unusual Th2 dominant pattern in the inflamed synovium. The Th2 response was associated with predominantly synovial IgG B cells, and a predominantly Th1 profile in the peripheral blood. In contrast, ST samples obtained 2 and 2.5 yr later displayed first a Th0 and thereafter a Th1 profile, and the synovial B cell response was predominantly of IgM type. The T cell lines from the Th1/Th0 tissues expressed the Th1 marker CCR5 but not CCR3, while the T cells from the Th2 tissue expressed the Th2 marker CCR3 and no CCR5.
Conclusion. These results demonstrate that a predominantly Th2 response can be associated with active erosive RA. However, the Th2 profile was not permanent and changed into a Th0 and thereafter a Th1 profile.
KEY WORDS: Th1/Th2, Th subsets, B cells, Rheumatoid arthritis.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the existence of polarized Th1 and Th2 populations has been confirmed, these represent the extremes in a spectrum where intermediate stages are also revealed [13]. Within this spectrum, differentiated T cells producing a mixture of Th1 and Th2 cytokines, such as IFN and IL-10, have been identified [14, 15]. Furthermore, alternative regulatory populations exist that are associated with but distinct from the Th2 cells. These subgroups are termed Th3 which produces transforming growth factor-ß (TGFß) but little or no IL-4 or IL-10 [16] and Tr1 which produces high levels of IL-10, low levels of IL-2 and no IL-4 [17].
Th1 and Th2 cells are known to antagonize each other in a variety of ways [18, 19], and several chronic inflammatory autoimmune diseases such as multiple sclerosis (MS), diabetes and rheumatoid arthritis (RA) have been described as Th1 dominant diseases [20], while atopy and allergy are Th2 dominant diseases [21]. In the case of RA, the synovium is highly infiltrated by T lymphocytes that are predominantly of the Th1 type [22]. The Th1 cells are thought to contribute to the inflammation by inducing high levels of the pro-inflammatory cytokines TNF, IL-1, IL-6 and IL-17 in the synovium [23, 24], which leads to cartilage destruction and bone erosions [25, 26]. IL-4 mRNA is almost absent in RA synovium and IL-4-producing Th2 clones can rarely be detected [22, 27]. We have previously analysed the Th1/Th2 profile in the synovium of 14 RA patients. One of these patients did not show the Th1/Th0 profile normally associated with RA, but in contrast displayed an unusual Th2 profile in the synovium. It was important to see if this Th2 profile in the inflamed synovium was a fixed response. Therefore, this unusual patient was investigated during a kinetic study in which three tissue samples were analysed over a period of 2.5 yr.
![]() |
Patient and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The first synovial tissue (STI) for immunological examinations was obtained from a synovectomy of the right elbow on 1 October 1996. The surgeon found massive synovitis. On admission on 25 September 1996 the erythrocyte sedimentation rate (ESR) was 18 mm, C-reactive protein (CRP) was 31 mg/l, TBC was 323 x 109/l and white blood cell count (WBC) was 8.2 x 109/l. At the time of the operation she was on medication with NSAID and anti-allergic drugs.
The second tissue sample (STII) was from a bursectomy on 5 November 1998. A fairly large bursa with synovitis had developed under the metatarso-phalangeal (MTP) II joint where the metatarsal part of the joint had been removed in 1981. The following day the ESR was 42 mm, CRP was 115 mg/l, TBC was 451 x 109/l, WBC was 10.1 x 109/l, and with 74% neutrophils. The medication was the same as on 1 October 1996.
The third time tissue was obtained (STIII) was from a synovectomy on 14 February 1999 of the PIP III of the right hand. During the operation bulging synovitis was observed and removed. The following day the ESR was 36 mm, CRP was 79 mg/l, TBC was 433 x 109/l, WBC was 11.4 x 109/l and with 72% neutrophils. Medication was the same as administered on the previous operation.
In addition, the patient had a history of multiple allergies. At the age of 25 yr she reacted by itching on receiving morphine for pain in connection with an appendectomy and she has also reacted to trimethoprim/sulphamethoxazole and xylometazoline hydrochloride. Some time after 1970 she also developed a strong reactivity to wasp, bee and insect bites. She did not have any severe allergic reactions prior to any of the operations when we received ST samples.
Preparation of MNC
A buffy coat (from normal blood donors at the blood bank, Ullevål Hospital) was layered on Isopaque-Ficoll separation media (Lymphoprep, Nycomed, Oslo, Norway) and centrifuged at 320 g for 25 min. The cells were washed three times with Hanks balanced salts (HBSS) and resuspended at a concentration of 1 x 106/ml in RPMI-1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% pooled human serum. This medium was used for the culturing of T cells throughout the study. ST were cut into small pieces and digested with 4 mg/ml collagenase (Sigma, MO, USA) in PBS Dulbecco's (Gibco, Grand Island, NY, USA) for 1.5 h at 37°C. After centrifugation at 320 g for 10 min, the cells were washed three times with HBSS and cultured in RPMI overnight to remove adherent cells. Tissue debris was removed and the supernatant laid on Isopaque-Ficoll separation media and separated and washed as described above.
T cell lines, clones and B cell lines
The protocol for stimulation of lymphocytes from ST and the subsequent T cell cloning and the number of viable clones from each patient are given in Table 1. MNC (1.5 x 106) from ST were initially stimulated with 2.5 µg/ml PHA (phytohaemagglutinin, Sigma BioSciences, MO, USA) or 10 µg/ml bacillus Calmette-Guérin (BCG) (the culture fluid was a kind gift from H. Wiker, Institute of Immunology and Rheumatology, Oslo, Norway). After 3 days, and subsequently every fourth day, the cell lines were fed with 20 U/ml recombinant IL-2 (Boehringer Mannheim). Cloning was performed on day 14 by limiting dilution (0.31 cells/well) in 20 µl wells (Terasaki; Nunc, Roskilde, Denmark), using 2 x 104 irradiated allogenic peripheral blood mononuclear cells (PBMNC), 2.5 µg/ml PHA and 20 U/ml IL-2. Growing T cell clones were expanded after 8 days to 96-well plates (Nunc) in fresh complete medium containing 20 U/ml IL-2, 2.5 µg/ml PHA and 1 x 106 irradiated allogenic PBMC and later to 24-well plates (Falcon, Becton Dickinson, New Jersey, USA). T cell clones were fed twice weekly, and restimulated every 12 days with PHA, irradiated allogenic PBMC and IL-2. To obtain B cells lines, ST lymphocytes (5 x 106/ml) were mixed with an equal volume of 1% sheep red blood cells (SRBC) and centrifuged at 200 g for 7 min. The pellets were resuspended and underlaid with 10 ml of lymphoprep and centrifuged at 320 g. The B cell fraction was washed twice in PBS and resuspended in 5 ml of EpsteinBarr virus (EBV)-containing supernatant and incubated for 1 h before the addition of 15 ml of complete medium.
|
Stimulation of T cell lines and clones
T cell lines and clones were stimulated with anti-CD3 (HIT3a, Pharmingen, San Diego, CA, USA). Anti-CD3 diluted in PBS (0.25 µg/ml) was coated on 24-well plates (Falcon) at 37°C for 3 h, and after incubation the wells were washed twice in PBS. Cloned cells (1 x 106 cells/ml in medium) were added to each well at 1.5 x 106 cells/well. As a control, cloned cells were added to wells that had been coated with PBS only. The clones had been stimulated with PHA 12 days prior to the assay. After 24 h incubation, the cells and supernatants were collected and centrifuged at 320 g for 10 min, and stored at -20°C until cytokine quantification could be performed. In parallel, T cell clones were set up in 96-well plates (Nunc) in triplicate, with 50 µl/well at the same concentration. After 24 h incubation the cells were pulsed with 0.5 mCi of tritiated thymidine (Amersham, Amersham, UK) for 18 h after incubation with anti-CD3. The incorporation of tritiated thymidine was measured by a beta counter. The clonality of the T cell clones was assured by cloning down to 0.3 cells/well and the low cloning frequency (Table 1).
Phenotyping of T cells
Cells were phenotyped using a standard immunofluorescent technique [29], and analysed with a FACscan (Becton-Dickinson). The monoclonal antibodies used for staining were: anti-TCR/ß (FITC-T10B9.1A-31), anti-TCR
/ß (PE-B1.1), anti-CD4 (PE-RPA-T4), anti-CD8 (FITC-HIT8a), anti-CD28 (PE-CD28.2), anti-CD27 (PE-M-T271), anti-CD57 (FITC-NK-1), anti-CD38 (PE-HIT2), anti-CCR5 (PE-2D7) (all Pharmingen, San Diego, CA, USA), anti-CD45RB (FITC-PD7/26) (DAKO, Denmark). Anti-CCR3 (7B11) was kindly provided by Dr Walter Newman and Dr Paul Ponath (LeukoSite, Inc., Cambridge, MA, USA) and developed with anti-mouse IgG Fc (pan IgG Fc PE) a kind gift from Dr K. Thomson.
Cytokine measurements
IFN (Schering-Plough Research Institute, Kenilworth, NJ, USA), IL-4 and IL-10 (RD System, Abington, UK) levels were measured by a two-site sandwich enzyme-linked immunosorbent assay (ELISA) using pairs of anti-IFN
, anti-IL-4 and anti-IL-10 monoclonal antibodies. Mouse A35 anti-human IFN
, MAB604 anti-IL-4 and MAB217 anti-IL-10 monoclonal antibodies were used for coating and biotinylated mouse B27 anti-human IFN, BAF204 anti-IL-4 and BAF217 anti-IL-10 monoclonal antibodies were used for detection. After subsequent incubation with a streptavidin peroxidase complex, and development with orthophenylenediamine (OPD), the optical densities were measured at 492 nm.
The same ELISA technique was used to measure IgG, IgM and IgE antibodies. Goat anti-human IgG (I-2136), goat anti-human IgM (I-2386) and goat anti-human IgE (I-0632) were used for coating, and alkaline phosphatase-labelled anti-human IgG (A-3312), anti-human IgM (A-3437) and anti-human IgE (A-3525) (all Sigma) were used for detection. After development with p-nitrophenyl phosphate (pNPP), the optical densities were measured at 405 nm.
Proliferation assay
Clones were tested in a proliferation assay 10 days after the last addition of feeder cells. Cloned T cells (2 x 104/well) were stimulated with 2.5 µg/ml PHA in the presence of irradiated autologous peripheral blood (PB) MNC (7 x 104/well). After 4 days of incubation, 0.5 mCi of tritiated thymidine was added, and the cells were harvested 18 h later. The incorporation of tritiated thymidine was measured by a beta counter.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Thus, in contrast to the Th1 predominance normally found in RA synovium, this patient showed an unusual predominance of Th2 as well as Tc2 cells in the first sample obtained from the inflamed RA synovium.
Th2 clones express similar membrane markers regardless of various cytokine profiles
Because Th2 clones are not normally associated with the inflamed synovium it was important to characterize these cells further. The groups of Th2 clones which secreted various combinations of Th2 cytokines were screened for CD28 expression, the memory markers CD27 and CD45RB, the marker of germinal centre T cells CD57, and also analysed for their proliferative capacity (Tables 4 and 5
). In contrast to the normal situation where the synovium has increased frequency of CD28-negative cells, all the Th2 clones expressed the co-stimulatory molecule CD28. The IL-4-producing clones consisted of the most differentiated phenotypes, all having low expression of CD45RB and only one clone expressed some CD27. In the group of clones that produced IL-4 and IL-10, 88% expressed markers of a highly differentiated memory phenotype, and in the IL-10-producing group the percentage was 78%. CD57 was also expressed in one IL-4-producing clone and in one IL-10-producing clone. This shows that there were no marked differences in the expression of markers between the various Th2-producing clones. Clones in these different groups were further tested in a proliferation assay (Table 5
), but no differences were observed in their proliferative capacity against PHA.
|
|
A Th1 phenotype in PB confers a selective attraction of Th2 cells into the joint
Since it has been reported that RA patients have increased production of IL-4 in PB [30] we investigated if the Th2 profile in the synovium was associated with an increased production of IFN or IL-4 in the PB. Lymphocytes were isolated from PB on the same day as the first ST sample was obtained, and cultured for 3 weeks before testing the production of cytokines and expression of CCR3/CCR5 of the T cells (Fig. 1B
). T cells from PB produced high levels of IFN
and only small amounts of IL-4, and showed a Th1 dominant profile with CCR5 expression and almost no expression of the Th2 marker CCR3. In contrast, the ST T cells were negative for CCR5 but expressed CCR3 (Fig. 1A
). This indicates that the Th2 cells were selectively attracted to the joint at that time.
|
Various Th profiles can be associated with rheumatoid inflammation
Two new ST samples were obtained, 2 (STII) and almost 2.5 yr (STIII) later to determine if a Th2 pattern still dominated the inflamed synovium. The second ST sample (STII) was obtained from a bursa with synovitis. Lymphocytes were isolated, and after 2 weeks in culture they produced both IL-4 and IFN (Fig. 2A
), and expressed high levels of CCR5 and small amounts of CCR3, and were characterized as a Th0 pattern. Lymphocytes isolated from PB at this time were cultured for the same period (Fig. 2B
) and the T cells showed the same cytokine profile and CCR3/CCR5 expression as in the ST T cells, which indicates no selective attraction of particular Th cell subsets. The third ST (STIII) sample was an active synovitis obtained by synovectomy almost 6 months after the STII sample. T cells from this tissue produced high levels of IFN
and small levels of IL-4 and expressed only CCR5 (Fig. 3A
). The T cells isolated from PB at this time showed the same Th1 profile with production of IFN
and expression of CCR5 (Fig. 3B
). This demonstrates that the Th1/Th2 profile in RA synovium is not fixed but can change over time.
|
|
A Th2 to Th0/Th1 shift was associated with a shift in B cell isotypes
To investigate if the various Th profiles observed in the synovium at the various time points were associated with particular B cell responses, EBV-transformed B cell lines were made from STI and STIII. After 3 weeks in culture, the B cells were stained for membrane IgE, IgG and IgM and production of IgG, IgM and IgE antibodies was measured by ELISA (Fig. 4A and Table 6
). In the B cell population from STI, 58% stained positive for IgG, 15% for IgM and about 4% were positive for IgE. However, no IgE antibody could be detected in the supernatant but the ratio between IgG and IgM antibodies was similar to that of the membrane expression. This shows that the Th2 response was accompanied by a B cell response predominantly of IgG type. In contrast, the B cell line from STIII with a Th1 profile (Fig. 4B
and Table 6
) consisted predominantly of B cells of IgM types. No membrane IgG could be detected, but small levels of IgG antibody could be detected in the supernatant. This shows that the shift in Th profile from Th2 to Th1 was accompanied by a change in the B cell isotypes from predominantly IgG to predominantly IgM (Table 7
).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the first ST sample with a Th2 profile, 31 CD4+ clones producing various combinations of the cytokines IL-4 and/or IL-10 identified the Th2 profile. In the clones that produced IL-10 either alone or in combination with IL-4, the IL-10 concentration was low compared with the concentration of IL-4. This suggests that none of these clones was of the regulatory Tr1 type, which secretes high amounts of IL-10 and low amounts of IL-4. Tr1 cells have been shown to have low proliferative capacity and the three groups of clones producing either IL-4, IL-4 + IL-10 or only IL-10 were therefore tested in a proliferation assay. However, no difference was observed between the groups, which further indicates that the IL-10-producing population could not be of the Tr1 type. Both Tr1 and Th3 have been shown to suppress Th1 and Th2 responses. Only one single clone from STI was clearly of a Th3 type. This indicates that there were typical regulatory T cell subsets in the Th2 dominated tissue, but their effect may have been insufficient to control the disease. The importance of both of these Th2 and Th3 populations in suppression of the inflammation has been observed in experimental arthritis where combination treatment with IL-4 and IL-10 suppressed the disease [35].
In our study, the clones were developed using PHA, IL-2 and irradiated allogenic PBMC. By this technique, T cell clones were developed three times from the first tissue, and all showed the same Th2 profile, while T cell clones developed from other patients repeatedly showed a usual Th1/Th0 phenotype [15]. PHA is a strong mitogen and should be able to activate all kinds of T cells. If overgrowth of individual T cell clones occurs during such PHA activation, variations in the Th cell populations in individual tissues would not be observed, as was the case here. Moreover, by the use of this method during the activation of PB T cells, the cytokine profile of Th cells is quite different and most T cells secrete a mixture of IFN and IL-4, resembling the naive T cell population. In addition, the technique has been tested previously [22, 36] and has been shown to allow growth of virtually all different T cells and should not introduce any bias in the T cell repertoire. We have also observed another patient where we only obtained two CD4+ T cell clones from a ST sample. However, both of these clones were clearly of the Th2 type [22], and this patient may show similarities with the patient analysed in this paper.
A pathogenic role of Th2 cells has been reported in immunodeficient individuals [37], and in animals with no immunodeficiencies [38]. These observations indicate that Th2 cells generated under different conditions or from animals with different genetic backgrounds can also differ in some of their properties despite their common production of Th2 cytokines. These studies have identified mechanisms by which Th2 cells can give rise to pathogenic effects. In addition, in models of arthritis such as collagen induced arthritis (CIA) mice, the addition of IL-4 at the onset resulted in a markedly enhanced inflammatory mass and induced increased tissue fibrosis [39]. However, the cartilage destruction was markedly reduced and bone erosions were virtually absent. This indicates that IL-4 can induce both inflammatory and anti-inflammatory effects in the joint, and as such could explain how a Th2 response might be associated with an inflamed synovitis. Th2 cells are also more potent at providing B cell help than Th1 cells. In the RA synovium, a Th2 dominance could lead to increased production of autoantibodies such as rheumatoid factor which might perpetuate the inflammation [40].
The patient was diagnosed as seronegative erosive RA in 1979 and at this time point the patient also had some allergic reactions. In subsequent years the patient developed several additional allergic reactions. The patient was typed as HLA-DRB1*0404, and combined with a severe erosive disease this patient most probably resembles a classical RA. However, allergy is a typical Th2 disease, and is not frequently found in patients with chronic diseases involving Th1 cells because of the cross-regulatory properties of Th1 and Th2 cells. As such the combination of allergy and RA in this patient demonstrates an unusual feature and a particular case.
The mechanisms responsible for Th2 development are not completely understood, but co-stimulatory signals such as interactions between CD28/CTLA-4 and their ligands on APC CD80/86 have been shown to be important for Th2 development [41, 42]. In the most frequent RA conditions, CD4+ T cells both in PB and ST have an increased frequency of CD28-negative cells [43]. In contrast, all the Th2 clones analysed in this study expressed CD28. This indicates that the primary activation of these cells is markedly different from the activation of CD4+ T cells during normal Th1 RA conditions, and may be a result of allergic reactions in the patient. However, the Th2 profile was not stable and varied between different time points. In a bursa with synovitis (STII) obtained 2 yr later, T cells produced both IL-4 and IFN. The same profile was present in PB, although the IFN
level was higher compared with the ST, which indicates a higher Th2/Th1 ratio in the ST as compared with the PB. Moreover, in another ST sample (STIII) obtained 4 months after STII, the Th profile in the synovium was dominated by Th1 cells. In addition, we have previously shown that the Th2 clones from STI were not stable during culture and could switch into Th1 types with high production of IFN
[34]. In contrast, all Th1 clones obtained from other patients remained stable. This indicates that the Th2 cells dominating the synovium at that time point were not committed to their phenotype, and as such did not reflect a fixed Th2 response.
The various Th cells infiltrating the synovium were not only different regarding cytokine production, but also in their expression of chemokine receptors, which enable them to migrate to particular inflamed areas [44]. The T cell population producing Th2 cytokines expressed CCR3 and no CCR5, while the T cell population from the two other tissue samples with predominantly Th0/Th1 types expressed CCR5 and no CCR3. As such, the T cells in the first tissue sample (STI) not only produced Th2 cytokines but also expressed Th2-associated markers. No clinical differences were observed at the different time points with regard to allergic reactions or medication that could have influenced the type of Th response. Since the ST samples were obtained from different sites we cannot completely exclude that the STI from the right elbow might be atypical and that other joints would show a different profile. However, being a systemic disease with affection of several joints this is not likely.
Th1 and Th2 cells differ in their capacity to give B cell help and isotype switching. Th2 cells can induce isotype switching to IgE and IgG4 [45]. Even though the isotype switch to IgG2a and IgG3 in mice is IFN and Th1 dependent, there is no real evidence that the production of such complement fixing and opsonizing antibodies in humans is Th1 and IFN
dependent [1, 6]. B cells from the tissue with Th2 dominance (STI) were mostly of IgG type, and in 4% of the B cells membrane IgE could be detected but the level of secreted IgE antibodies was under the detection limit. In contrast, the B cell population from the tissue with Th1 dominance (STIII) consisted predominantly of IgM types and the presence of IgG could only be detected at the secreted antibody level. As such, the different B cell isotypes present in the two ST samples correlate with the change in Th cell subsets.
This report shows the interesting observation that ST from an erosive arthritis was dominated by Th2 cells, which is rarely seen in the local RA inflammation. However, the Th1/Th2 balance changed over time in this RA patient and gradually became dominated by Th0 cells and ultimately Th1. Even if the observation includes only one patient, it brings up the possibility that the Th1 cytokine profile in the local RA inflammation is not necessarily fixed. Because of the ongoing joint inflammation despite a Th2 profile in our case, it is unclear if the Th2 cells were responsible for joint degradation or had anti-inflammatory properties. However, the identification of mechanisms that induce changes in the cytokine balance in RA synovium could be important with regard to treatment.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|