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Clin Exp Immunol. Jun 1999; 116(3): 534–541.
PMCID: PMC1905307

Beneficial effect of the inosine monophosphate dehydrogenase inhibitor mycophenolate mofetil on survival and severity of glomerulonephritis in systemic lupus erythematosus (SLE)-prone MRLlpr/lpr mice

Abstract

The aim of the present study was to evaluate the therapeutic effect of mycophenolate mofetil (MMF) on the course of disease in SLE-prone MRLlpr/lpr mice. Three-months-old mice displaying clinical symptoms of glomerulonephritis were given MMF (100 mg/kg per day) orally via the drinking water. Control mice received i.p. injections of cyclophosphamide (CYC) (1.8 mg/mouse per week) or saline. Survival, albuminuria and haematuria, immunoglobulin levels and anti-dsDNA antibodies in serum, frequencies of immunoglobulin-producing B lymphocytes and glomerular deposits of immunoglobulin and C3 were analysed. The results showed that MMF treatment significantly prolonged survival and reduced the occurrence of albuminuria and haematuria in MRLlpr/lpr mice. In addition, the number of immunoglobulin-producing B cells and serum levels of IgG and IgG anti-dsDNA antibodies were reduced after MMF and CYC treatment. MMF treatment significantly reduced the extent of deposition of C3 in glomeruli. We conclude that the reduced severity of glomerulonephritis following treatment of lupus-prone mice with MMF was as efficacious as that of CYC. These results warrant clinical trials of MMF in SLE patients with glomerulonephritis.

Keywords: mycophenolate mofetil, glomerulonephritis, MRLlpr mice, systemic lupus erythematosus, cyclophosphamide

INTRODUCTION

The MRLlpr/lpr mouse strain spontaneously develops an autoimmune disease resembling human SLE. The disease is characterized by immune complex-mediated glomerulonephritis, enlargement of spleen and lymph nodes, production of various autoantibodies such as anti-DNA antibodies and rheumatoid factors (RF) [1]. These mice also have impaired T cell functions, as evidenced by a low proliferative response to antigens and mitogens in vitro and decreased DTH in vivo [24]. A lymphoproliferation (lpr) gene recessively expressed in the MRLlpr/lpr mice leads to deficiency in Fas-mediated apoptosis of lymphocytes [5,6].

MRLlpr/lpr mice were used in this study to examine the effects of the immunomodulating substance mycophenolate mofetil (MMF) on the progression of the SLE-like disease. MMF is a prodrug converted in the blood after gastrointestinal absorption to the active compound mycophenolic acid (MPA). MPA reversibly and non-competitively inhibits the eukaryotic enzyme inosine monophosphate dehydrogenase (IMPDH) [7], which is involved in the de novo pathway of guanosine synthesis [7]. Lymphocytes, and to a lesser extent monocytes, are dependent on the de novo guanosine synthesis. MMF treatment thus specifically inhibits T and B cell proliferation and production of antibodies. In contrast to lymphocytes, most other cell types can utilize the salvage pathway for guanosine synthesis and are thus not affected by the MMF treatment [7]. Furthermore, glycosylation of proteins, specifically the transfer of fucose and mannose to glycoproteins, is inhibited by MMF. Lymphocyte attachment to endothelial cells and extravasation are often mediated by glycoproteins such as adhesion molecules, thus MMF treatment leads to decreased recruitment of lymphocytes and monocytes to sites of chronic inflammation [7].

Autoimmune diseases in experimental animal studies which have shown improvement after MMF treatment include spontaneous diabetes in Bio-Breeding rats [8] and uveoretinitis (EAU) in Lewis rats [9]. In addition, MMF has been utilized in the treatment of psoriasis [10] and rheumatoid arthritis [11]. Recent published case reports have revealed beneficial effects of MMF in immune complex-mediated bullous pemphigoid [12] and pemphigus vulgaris [13] as well as in systemic vasculitis and IgA nephritis [14]. Interestingly, a recently published abstract described a beneficial effect of MMF in some cyclophosphamide (CYC)-resistant proliferative lupus nephritis patients [15]. However, no controlled clinical trails on the effects of MMF in systemic autoimmune rheumatic diseases have yet been published.

In this study the effect of MMF on established lupus disease in MRLlpr/lpr mice was compared with that of CYC, the drug of choice in treatment of murine [16] and human [17,18] SLE with glomerulonephritis. Our results suggest that MMF is at least as efficient in controlling the SLE disease as CYC, an alkylating agent with considerably lower specificity and thus higher risk of adverse effects.

MATERIALS AND METHODS

Mice

MRLlpr/lpr mice, originally purchased from Bomholtgård (Ry, Denmark) were bred in the animal facility of the Department of Rheumatology and Clinical Immunology in Göteborg. Male and female mice aged 5–12 weeks were housed 3–10 animals per cage under standard conditions of temperature and light and were fed standard laboratory chow ad libitum.

Treatment of mice

In earlier studies MMF has been administered daily in a suspension through gastric gavage only. The solubility of MMF in tap water is approx. 0.2 mg/ml, but the solubility was increased to > 2 mg/ml by buffering the solution with PBS dissolved in distilled water 1:4 and lowering the pH to 3.6 with HCl. In a preliminary study we found that the mice accepted drinking water containing 0.4 mg/ml of MMF. A following dose titration experiment comparing 1.0 and 2.0 mg/ml revealed an optimal dose of 1.0 mg MMF per ml drinking water (see below). By daily control of the volume of water consumed by the mice in each cage we calculated that 1.0 mg/ml of MMF corresponds to a dose of approx. 100 mg/kg per day. The water consumption was rather stable throughout the experiments and did not significantly differ between the treatment groups.

In each experiment three different treatment regimens were employed: (i) control mice were continuously given acidified drinking water; (ii) one test group was given MMF (Roche Pharmaceutical, Basel, Switzerland) dissolved in the drinking water at various concentrations (0.4, 1.0 and 2.0 mg/ml); (iii) a second test group was given CYC (Asta Medica AG, Frankfurt am Main, Germany) injected intraperitoneally at a dose of 1.8 mg/mouse per week, a dose previously used for treatment of lupus-prone mice [16,19].

Clinical parameters

All the mice were weighed weekly. After collection of sera, mice were killed by cervical dislocation and cervical lymph nodes and spleens removed and weighed. The degree of albuminuria and haematuria in freshly voided urine was monitored weekly with standard dipsticks (Redia test; Boehringer, Mannheim, Germany). Mice were considered to have developed significant proteinuria or haematuria when the readings showed either 3 once, or 2 on at least two separate occasions.

Serological parameters

Collection of blood

Blood was taken from mice prior to sacrifice. The sera were individually stored at −20°C until use.

Antibodies to double-stranded DNA

Serum IgG and IgM antibody levels specific for double-stranded (ds)DNA were measured by ELISA. To avoid contamination with single-stranded (ss)DNA, calf thymus DNA (Sigma, St Louis, MO) was dissolved in 0.1 m NaCH3COO including 1 mm ZnCl2 pH 5.5 and treated with S1 nuclease followed by extensive dialysis against PBS. Methylated bovine serum albumin (mBSA; 10 μg/ml) was used to precoat wells followed by coating with 50 μg/ml of S1 nuclease pre treated native calf thymus DNA. All the sera were incubated in serial dilutions. To measure the levels and class specificity of anti-dsDNA antibodies bound to the solid phase, affinity-purified and alkaline phosphatase-labelled F(ab′)2 fragments of goat anti-mouse IgG or IgM (Southern Biotechnology Inc., Birmingham, AL) diluted 1:1000 in PBS were used followed by addition of p-nitrophenyl phosphate in 10% diethanolamine buffer pH 9.8. The absorbance was measured in a Titertek Multiscan photometer (Flow Labs, McLean, VA) at 405 nm. All optical density (OD) values were converted to antigen-specific arbitrary units (AU) with calibration curves based on the OD obtained from serial dilutions of a reference pool of sera from 4-month-old female MRLlpr/lpr mice.

Determination of IgG1, IgG2a, IgG3 and IgM levels in serum

The single radial immunodiffusion technique [20] was used for determination of IgG1, IgG2a, IgG3 and IgM levels in sera as previously described.

Histopathological and cellular parameters

Tissue collection, and single cell preparation

Kidneys from treated mice were rapidly frozen for immunohistochemical studies (see below). Spleens were mashed and passed through a nylon wool sieve to give a single-cell suspension. The cells were centrifuged at 515 g for 5 min and the pelleted cells were resuspended in Tris-buffered 0.83% NH4Cl to lyse erythrocytes. After washing in PBS the total number of cells was calculated and the cells were used for FACS analysis and ELISPOT assays (see below).

Immunofluorescence analysis

Glomerular deposits of immunoglobulin and complement factor C3 were visualized by direct immunofluorescence on cryostat sections of kidney specimens using F(ab′)2 fragments of FITC-conjugated goat anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) and anti-mouse C3 antibodies (Cappel Labs, Cochraneville, PA). The intensity of staining in mesangium and peripheral loop areas was scored accordingly from 0 (background staining) to 3 (maximal staining) [19,21]. Background staining was defined as staining of kidney sections from young, healthy MRL+/+ mice. All specimens were coded and read by two independent observers. Statistical comparison between the groups was made after scoring as follows: 0–1 (none or mild disease) and 2–3 (severe disease).

Flow cytometry for analysis of cell phenotypes

Isolated spleen cells were stained with FITC-labelled antibodies to CD8 (Becton Dickinson, San Jose, CA) and immunoglobulin (Dakopatts) and with PE-conjugated antibodies to CD4 (Becton Dickinson). The frequency of cells expressing the given phenotype was analysed on a FACStar (Becton Dickinson) and expressed as the percentage of gated mononuclear cells. The total number of immunoglobulin-expressing B cells, CD4- and CD8-expressing cells per spleen was also calculated.

ELISPOT

The ELISPOT technique [22] was used for enumeration of IgG- and IgM-secreting cells. Briefly, shallow wells (Costar, Cambridge, MA) were coated with 200 μl of PBS containing 10 μg/ml of affinity-purified F(ab′)2 fragments of goat anti-mouse IgG and IgM (Southern Biotechnology). After overnight incubation at 4°C and blocking with 5% fetal calf serum (FCS), 100 μl of Iscove's culture medium containing 107, 106 or 105 freshly isolated spleen cells per ml were added to each well. Incubation was carried out for 3.5 h at 37°C in 5% CO2 and 95% humidity. The plates were subsequently rinsed and each well was incubated stepwise with 200 μl of alkaline phosphatase-conjugated F(ab′)2 fragments of goat anti-mouse IgG or IgM (Southern Biotechnology) diluted 1:750 in PBS–Tween 20 and finally with enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate (Sigma). After 1 h the enzyme–substrate reaction was stopped by wash in tap water and each well was examined for the appearance of dark blue spots. All cell samples were run in duplicate in the ELISPOT assay. The numbers of immunoglobulin-secreting cells were expressed as the frequency of spot-forming cells per 106 mononuclear cells (SFC/106 MNC) or per 106 B cells.

Statistical analysis

The Mann–Whitney and Fisher's exact tests were used as statistical methods. Results are presented as mean ± s.d. of the mean. P ≤ 0.05 was considered statistically significant.

RESULTS

Dose titration experiments

In a pilot experiment we treated 6-week-old female MRLlpr/lpr mice with MMF (40 mg/kg per day) administered in acidified drinking water. The MMF-treated mice displayed somewhat reduced albuminuria and haematuria frequency compared with control mice. Survival was not prolonged with this low dose of MMF compared with controls (data not shown).

To titrate the optimal dose of MMF, 8-week-old female mice (n = 7–8) were given 100 or 200 mg/kg per day of MMF and compared with both control mice as well as with mice treated with 1.8 mg CYC per week. Mice treated with 100 mg/kg per day of MMF in the drinking water displayed diminished frequency of albuminuria and haematuria, decreased serum levels of immunoglobulin and anti-dsDNA antibodies as well as prolonged survival compared with control mice. Similar results were obtained in CYC-treated mice. In contrast, the 200 mg/kg per day dose of MMF was toxic, since the mice displayed considerable weight loss and increased frequency of albuminuria and haematuria in comparison with the 100 mg/kg per day group (results not shown). Based on these results we regard 100 mg/kg per day of MMF administered in the drinking water as the optimal dose. Thus, in all the following experiments mice were treated with 100 mg/kg per day of MMF or 1.8 mg/mouse per week of CYC and were compared with untreated controls.

Effects of MMF on clinical course of lupus

Survival

Twelve-week-old female MRLlpr/lpr mice, with symptoms of glomerulonephritis (as demonstrated by high frequency of albuminuria and haematuria), were randomly selected for inclusion in a survival experiment comparing MMF and CYC treatments (MMF n = 14, CYC n = 10 and controls n = 15). As shown in Fig. 1a, treatment with MMF significantly prolonged survival of diseased mice compared with controls. This outcome was similar to that seen with CYC.

Fig. 1Fig. 1
(See previous page) (a) Survival (%) of 12-week-old female MRLlpr/lpr mice treated with either 100 mg/kg per day of mycophenolate mofetil (MMF) in drinking water ([filled triangle]) (n = 14), 1.8 mg/mouse per week of i.p. injections with cyclophosphamide (CYC) ...

Kidney disease

In Fig. 1b,c, the cumulative occurrence of albuminuria and haematuria in mice treated with MMF (n = 14), CYC (n = 10) or in controls (n = 15) is shown. Pretreatment values show relatively high frequency of albuminuria and haematuria, clearly demonstrating that the mice already suffered from glomerulonephritis. MMF treatment reduced both albuminuria and haematuria during the first 4 weeks of treatment, whereas CYC only had an effect on albuminuria. Statistically significant reduction of both albuminuria and haematuria was found after 5–6 weeks of treatment with MMF compared with the control mice. Five months after initiation of treatment, albumin and erythrocyte levels in the urine were still diminished in MMF-treated mice compared with controls. In contrast, CYC-treated mice displayed no significant long-term reduction of albuminuria or haematuria.

Frozen kidney sections from female 19-week-old mice treated with MMF or CYC for 12 weeks were stained with FITC-labelled antibodies against immunoglobulins and complement (C3). As shown in Fig. 2, all control mice displayed heavy deposits of immunoglobulin and C3 in glomeruli. In contrast, approx. 50% of MMF-treated mice showed little or no immunoglobulin and C3 deposits in glomeruli. The majority of mice treated with CYC displayed heavy deposits of immunoglobulin and C3.

Fig. 2
Seven-week-old female MRLlpr/lpr mice were treated with either 100 mg/kg per day of mycophenolate mofetil (MMF) (n = 9), 1.8 mg/mouse per week of cyclophosphamide (CYC) (n = 6) or left untreated (n = 9) for 12 weeks. At sacrifice three controls and one ...

Lymphoid proliferation, immunoglobulin and anti-DNA antibody production

Five-week-old premorbid male MRLlpr/lpr mice were treated with either MMF or CYC for 11 weeks. All the mice were then killed and lymphoid organs were weighed, spleen MNC counted and analysed by ELISPOT. Both treatment regimens reduced spleen and cervical lymph node weights compared with controls despite unaltered total body weight (Table 1) and the number of MNC in spleens was reduced in both MMF- and CYC-treated mice. The overall frequencies of IgM- and IgG-producing MNC were similar in treated and control mice with the exception of a lowered frequency of IgM SFC in CYC-treated mice (Table 1). However, the reduced total number of MNC in spleens of MMF- and CYC-treated mice implied a significantly diminished number of IgG- and IgM-producing cells per spleen in these two groups in comparison with controls.

Table 1
Effect on organ weights, spleen mononuclear cell (MNC) numbers and IgM- and IgG-spot forming cells (SFC) in male mice treated with 100 mg/kg per day of mycophenolate mofetil (MMF) administered in drinking water, intraperitoneally 1.8 mg/mouse per week ...

In a second experiment 7-week-old female MRLlpr/lpr mice were treated with either MMF or CYC for 12 weeks and then killed. It should be noted that three control animals and one CYC-treated mouse died (having developed albuminuria and haematuria and having displayed weight loss) before the termination of the experiment and thus were excluded from further analyses. The cervical lymph node weight was slightly reduced after CYC treatment and there was a reduction in spleen cell numbers and spleen weight in MMF- and CYC-treated animals compared with controls (Table 2). MMF- and CYC-treated mice displayed reduced polyclonal B cell activation and autoantibody production in serum. Thus, both MMF- and CYC-treated animals showed significantly lowered IgG2a and IgG3 serum levels. IgG1 levels were significantly reduced only after CYC treatment, whereas IgM levels did not differ between the groups (Table 3). In addition, serum levels of IgG anti-dsDNA antibodies were significantly reduced after both MMF and CYC treatment compared with the controls. The IgM anti-dsDNA antibody levels were not significantly altered after either treatment (Table 3).

Table 2
Effect of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) on body weight, lymphoid organ weight, spleen mononuclear cell number (MNC) number and frequency of IgM- and IgG-producing cells (SFC/106 MNC) in 19-week-old female MRLlpr/lpr mice after ...
Table 3
Effect of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) on serum levels of immunoglobulin and anti-dsDNA antibodies in 19-week-old female MRLlpr/lpr mice treated from 7 weeks of age

Spleen cell populations

Freshly isolated spleen cells from female MRLlpr/lpr mice treated with either MMF or CYC were subjected to FACS analysis. Table 4 shows that in the MMF-treated group the frequency of immunoglobulin-expressing B cells was significantly reduced compared with control mice, whereas T cell frequencies (CD4+ and CD8+ cells) were not significantly altered. Notably, the total number of B cells per spleen was reduced in MMF-treated mice whereas the number of splenic T cells was not significantly reduced compared with controls. A reduction of frequency of both IgG- and IgM-secreting cells per million spleen cells accompanied the slightly lowered total number of spleen cells (Table 2). The frequency of IgG-secreting B cells was somewhat decreased (Table 4). Thus, the MMF suppression of B cell activity seems to be due to both reduction of the number of B cells and the inhibition of their immunoglobulin production.

Table 4
Effect of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) on frequency of B and T cells and SFC/106 B cells in spleen from 19-week-old female MRLlpr/lpr mice treated from 7 weeks of age

DISCUSSION

In this study we have shown that MMF is an effective drug for treatment of nephritis in lupus-prone MRLlpr/lpr mice. Importantly, the therapeutic efficacy of MMF is comparable to that of CYC, the current drug used for treatment of human [17,18] and murine SLE [23]. Earlier it has been demonstrated that MMF-treated premorbid NZB/W lupus-prone mice displayed prolonged survival and limited renal damage [24]. The use of already diseased MRLlpr/lpr mice in our study clearly demonstrates the therapeutic effect of MMF on established lupus nephritis as opposed to the mainly prophylactic effect discovered in the NZB/W model [24,25]. Recently, van Bruggen et al. showed that MMF-treated MRLlpr/lpr mice develop less severe glomerulonephritis, and reduced incidence of albuminuria compared with vehicle-treated control mice, corroborating our findings [26]. However, in contrast to our results no clear-cut immunomodulating effects were found in their study.

Several immunomodulating drugs have been tested for their effect on the clinical course of SLE in MRLlpr/lpr mice. CYC was established in 1984 as an effective inhibitor of lymphadenopathy, arthritis and nephritis in MRLlpr/lpr mice [23], and 2 years later it was shown that Linomide (LS-2616) also exhibited disease-retarding properties comparable to that of CYC in these mice [19]. Conflicting results have been obtained after treatment of MRLlpr/lpr mice with cyclosporin A (CsA). While one study showed no demonstrable effect of CsA [27], another revealed amelioration of immunopathology and prolonged survival [28]. The newer immunomodulators, such as FK506 [29] and leflunomide [30], when tested in MRLlpr/lpr mice, have revealed disease-retarding effects. However, among all long-term treatment studies published on immunomodulating drug effects on lupus disease in MRLlpr/lpr mice, our present results are the first to demonstrate equivalent therapeutic efficacy of MMF on disease outcome compared with CYC.

Intermittent pulse CYC therapy in combination with corticosteriods has been long established as the optimal treatment modality in SLE nephritis [31]. In several clinical studies this treatment has been proved superior to corticosteroids alone or per oral CYC administration [17,18]. The use of CYC is limited by its extensive side-effects, including severe infections [32] due to bone marrow suppression, the risk of development of malignancies and azospermi, as well as ovarian toxicity. The latter is a serious problem since the majority of lupus nephritis sufferers are women of childbearing age. Intravenous CYC therapy for > 15 months resulted in sustained amenorrhoea in 39% of patients [33]. An important challenge is thus to find alternative and well tolerated treatment modalities offering the efficacy of CYC treatment but avoiding the severe side-effects.

MMF is a relatively lymphocyte-specific immunomodulating drug displaying fewer and less severe side-effects compared with CYC, and is potentially useful in the treatment of SLE. The active metabolite of MMF, MPA, inhibits IMPDH, leading to depletion of guanosine triphosphate (GTP) pools in T and B lymphocytes in vitro, thereby reducing lymphoproliferation [7]. The lymphocyte-specific anti-proliferative property of MMF is also valid in vivo, as demonstrated by strongly inhibited DNA synthesis in lymph nodes, compared with intermediate inhibition in spleens and no inhibition in testis in mice given 100 mg/kg per day of MMF orally. Furthermore, MPA (25 or 50 mg/kg per day orally) has been shown to inhibit antigen-specific antibody formation significantly in mice [34]. These findings indicate that the dose of MMF used in our study should be enough to deplete GTP sufficiently to reduce the number of B cells and to decrease antibody formation.

The main cause of death in MRLlpr/lpr mice is renal failure, and the significantly prolonged survival we found during MMF treatment is probably due to inhibition of glomerulonephritis. Interestingly, albuminuria and haematuria were significantly reduced by MMF treatment. Furthermore, the observation that MMF treatment decreased the occurrence of albuminuria and haematuria after 2 weeks of treatment in already diseased animal indicates the therapeutic capacity of MMF to influence ongoing renal disease. It has previously been shown that CYC treatment of lupus mice protects against the development of albuminuria [16]. Since 50% of the mice in the CYC group already displayed albuminuria at the start of treatment, we were unable to corroborate these previous findings.

In addition, MMF-treated animals had significantly lower levels of C3 depositions in glomeruli compared with control mice, and one third of these mice displayed only trace amounts of C3 deposits. Since three of the control mice died of renal failure before we were able to ascertain the level of immunoglobulin deposits, and since the number of mice in the CYC group was small, we are unable to draw conclusions regarding the level of immunoglobulin deposition.

In accordance with our results, treatment with CYC has previously been shown to reduce adenopathy and splenomegaly in MRLlpr/lpr mice [23]. Our present study demonstrates that MMF-treated mice display less inhibition of the lymphoproliferative disease, but at least as pronounced amelioration of glomerulonephritis when compared with CYC. Indeed, earlier studies have shown that the lymphoproliferative disease can be disassociated from autoantibody production and glomerulonephritis [35].

The mechanism whereby MMF ameliorates glomerulonephritis is not fully understood. Clearly, a reduction of the number of B lymphocytes (a consequence of the decreased proliferation discussed above) and the concomitant reduction in the production of pathogenic IgG anti-dsDNA antibodies are important factors in amelioration of the disease. In addition, inhibition of the glycosylation of proteins involved in leucocyte adhesion to endothelial cells influencing the extravasation of lymphocytes and monocytes to target organs could be important. It has been shown that in vitro exposure of T lymphocytes and/or endothelial cells to MPA strongly decreases cell adhesion, and this effect is mediated by reduction of expression of VLA-4, the ligand for VCAM-1, on lymphocytes [36]. In a recently published paper it was shown that MMF treatment totally abolished lymphocyte and macrophage infiltration to rat kidney allografts [37]. A consequence of reduced MNC influx into glomerulonephritic kidneys could be the limitation of local production of disease-promoting cytokines. In order to test this hypothesis renal immunohistochemical studies are underway in our laboratory.

In conclusion, we have shown that the immunomodulating drug MMF significantly reduces the progression of already established glomerulonephritis and prolongs survival in MRLlpr/lpr mice. The ameliorating effect of MMF was as efficacious as that of CYC. In addition, due to the relatively selective inhibition of B and T cell proliferation of MMF, we propose that fewer serious side-effects might occur with MMF than with CYC. Thus, MMF is potentially an attractive alternative drug for treating SLE patients with glomerulonephritis.

Acknowledgments

We thank Mrs Ing-Marie Nilsson for excellent technical assistance and Professor Andrzej Tarkowski and Vincent Collins for critically reading the manuscript. This study was supported by grants from the Börje Dahlin foundation, the Göteborg Medical Society, Swedish Association against Rheumatism, the King Gustav V's 80 years foundation, the Anna-Greta Crafoord foundation, the Medical Faculty of University of Göteborg (LUA), Roche AB Pharmaceuticals and the Swedish Medical Research Council.

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