The point mutation of tyrosine 759 of the IL-6 family cytokine receptor gp130 synergizes with HTLV-1 pX in promoting rheumatoid arthritis-like arthritis
Katsuhiko Ishihara1,3,
Shin-ichiro Sawa3,
Hideto Ikushima3,
Seiichi Hirota4,
Toru Atsumi2,
Daisuke Kamimura3,
Sung-Joo Park2,
Masaaki Murakami3,
Yukihiko Kitamura4,
Yoichiro Iwakura5 and
Toshio Hirano1,2,3
1 Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan 2 Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan 3 Department of Molecular Oncology (C7), Osaka University Graduate School of Medicine, Suita, Osaka, Japan 4 Department of Pathology (C2), Osaka University Graduate School of Medicine, Suita, Osaka, Japan 5 Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Correspondence to: T. Hirano, Department of Molecular Oncology (C7), Osaka University Graduate School of Medicine, 2-2 Yamada-oka Suita, Osaka 565-0871, Japan. E-mail: hirano{at}molonc.med.osaka-u.ac.jp
Transmitting editor: H. Karasuyama
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Abstract
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Rheumatoid arthritis (RA) is a polygenic autoimmune disease. The autoimmunity develops from synergistic actions of genetic and environmental factors. We generated a double-mutant mouse by crossing two murine models of RA, a gp130 mutant knock-in mouse (gp130F759/F759) and an HTLV-1 pX transgenic mouse (pX-Tg), in a C57BL/6 background, which is resistant to arthritis. The mice spontaneously developed severe arthritis with a much earlier onset than the gp130F759/F759 mice and with a much higher incidence than did the pX-Tg mice. The symptoms of gp130F759/F759 mice, including lymphoadenopathy, splenomegaly, hyper-
-globulinemia, autoantibody production, increases in memory/activated T cells and granulocytes in the peripheral lymphoid organs, and a decrease in the class II MHCbright CD11c+ population, were augmented in the double mutants. Marked reductions in incidence, severity and immunological abnormalities were seen in the triple mutant, IL-6//gp130F759/F759/pX-Tg, indicating that the arthritis in the double mutant is IL-6 dependent. gp130F759/F759/pX-Tg is a unique mouse model for RA.
Keywords: autoimmune disease, knock-in mouse, rheumatoid arthritis, Tax
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Introduction
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Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the progressive chronic inflammation of multiple joints, which leads to their destruction, thus disabling the patients. The incidence of RA is
1% worldwide, but its etiology is not yet known. Several characteristics of RA, such as hyper-
-globulinemia, autoantibody production, genetic linkage with the HLA-DR locus, and infiltration of T cells and plasma cells into the synovium, have suggested that immunological disorders play crucial roles in the pathogenesis of this disease (1). RA is a polygenic disease, and is caused by immunological disorders that develop from the synergistic actions of genetic and environmental factors, such as bacterial or viral infections. Clinical and experimental studies have revealed the involvement of inflammatory cytokines, such as tumor necrosis factor (TNF)-
, IL-1, and IL-6 in the pathophysiology of RA (2). For example, TNF-
promotes the growth of synovial cells and blocking the TNF-
signal is an effective therapy (3).
IL-6 is a pleiotropic cytokine that regulates multiple biological functions, such as the development of the nervous and hematopoietic systems, acute-phase responses, inflammation, and immune responses (4). A causative role for IL-6 in autoimmune disease was first recognized because of the observation that the autoimmune symptoms of patients with cardiac myxoma, such as hyper-
-globulinemia and autoantibody production, disappeared with the surgical removal of the tumor which produced IL-6 (5). Furthermore, a high concentration of IL-6 was detected in the synovial fluid and serum of RA patients (6). Since then, clinical studies have provided more evidence supporting the involvement of IL-6 in the pathogenesis of autoimmune diseases (4,7). Studies using IL-6 knock-out mice revealed that IL-6 is involved in the severity and progress of experimentally induced arthritis (810). However, it was not known whether an abnormality in IL-6 family cytokine/gp130 receptor signaling was involved in spontaneous autoimmune diseases until we showed that a point mutation of the IL-6 family cytokine receptor, gp130, spontaneously caused RA-like autoimmune disease (11).
The IL-6 receptor consists of two molecules, the IL-6 receptor
chain and gp130, which is shared among the receptors for the IL-6 family cytokines. The ligand binding of gp130 activates two major signal-transduction pathways, the STAT3-mediated signal and the SHP-2/Gab/MAPK signal, in a manner dependent on the YXXQ motif and tyrosine (Y) 759 of gp130 respectively (12,13).
To clarify the roles played by the SHP-2- and STAT3-mediated signal-transduction pathways in vivo, we generated a series of knock-in mouse lines in which the gp130-mediated STAT3 or SHP-2 signals are selectively disrupted, by mutating the tyrosine residues of all the YXXQ motifs or Y759 to phenylalanine (gp130FXXQ/FXXQ and gp130F759/F759 mice respectively). Our analysis of these mice indicated that the SHP-2-mediated or Y759-dependent signals negatively regulate the biological responses elicited by the STAT3-mediated signals in vivo, and that the balance of positive and negative signals generated through gp130 is skewed or shifted to positive STAT3 signaling in gp130F759/F759 mice (14). Importantly, the gp130F759/F759 mice spontaneously develop an RA-like autoimmune disease at
1 year of age (11). gp130F759/F759 mice show severe immunological abnormalities, including autoantibody production, increased memory/activated T cells, impaired thymic negative selection and peripheral clonal deletion. Most importantly, the development of the RA-like disease is totally dependent on mature lymphocytes.
HTLV-1 is the causative agent of adult T cell leukemia. HTLV-1 encodes a transcriptional trans-activator, Tax, in the env-pX region, that trans-activates transcription from the cognate viral promoter (15,16). Tax also activates many cellular genes for cytokines, cytokine receptors and immediate early transcriptional factors (1720). HTLV-I is also associated with several chronic inflammatory diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis and HTLV-I-associated arthropathy (21). The HTLV-1 transgenic mouse is an animal model of arthritis that is triggered by viral infection (22). This mouse develops a form of arthritis resembling human RA, accompanied by increased gene expression of inflammatory cytokines including IL-6, MHC molecules in the joints and resistance of T cells to Fas-mediated apoptosis (23). The susceptibility to arthritis is dependent on the genetic background of the mice; BALB/c mice are susceptible, but C57BL/6 mice are resistant to arthritis (24).
Here, we report clear synergism between the point mutation of Y759 of gp130 and the HTLV-1 pX gene in the C57BL/6 genetic background, in which the HTLV-1 pX gene alone cannot induce RA-like joint disease. The double-mutant mouse gp130F759/F759/pX-Tg developed severe arthritis much earlier than the gp130F759/F759 mouse. Lympho-hematopoietic abnormalities of the gp130F759/F759 mice, such as splenomegaly, lymphoadenopathy, hyper-
-globulinemia, autoantibody production, and increased granulocytes and memory/activated T cells, were augmented in the gp130F759/F759/pX-Tg mice. These abnormalities, as well as the onset, incidence and severity of the arthritis, were ameliorated in IL-6-deficient gp130F759/F759/pX-Tg mice. The gp130F759/F759/pX-Tg double-mutant mouse is a unique mouse model for the polygenic autoimmune disease RA.
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Methods
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Mice
gp130F759/F759 knock-in mice that had been backcrossed to C57BL/6 5 times or more were mated with HTLV-1 env-pX-transgenic mice (pX-Tg) that had been backcrossed 17 times to C57BL/6 (B6). The litters of the F1 generation were intercrossed to obtain gp130F759/F759/pX-Tg double mutants. Thus the double mutants were thought as backcrossed 6 times to B6 (N6). In all the analyses including the studies of clinical course, we used the wild-type and single-mutant mice derived from the same littermates to generate the double- or triple-mutant mice.
IL-6-deficient mice were kindly given by Dr Kopf (25) and backcrossed 8 times to B6. They were then crossed with gp130F759/F759/pX-Tg+/ to generate IL-6/gp130F759/F759/pX-Tg+/ triple mutants, which were equivalent to 7 times backcrossed to B6 (N7). All these mice were kept at the Institute of Experimental Animal Sciences at Osaka University Medical School.
Analyses of arthritis
Clinical assessment of arthritis
Mice were inspected every week for up to 24 weeks and assessed for signs of arthritis: redness, swelling and restriction of mobility. The severity of the arthritis (arthritis score) was based on the restriction of mobility, and swelling of the wrist and ankle joints, which were each examined bilaterally. The severity of the arthritis was graded on a scale of 04 as follows: 0 (no change), 1 (minimal change), 2 (mild change), 3 (moderate change) and 4 (severe change). The arthritis score shown is a sum of the scores assessed bilaterally to give a possible maximum of 16 points for each mouse. The incidence was defined as the percentage of mice with a score
2 points.
Radiologic and histologic analyses of arthritis
Radiologic and histologic data from B6 gp130F759/F759/pX-Tg and 129/B6. gp130F759/F759 mice were obtained at 611 and 18 months of age respectively. X-ray photographs of the bones were taken using a Softex CMB-2 (Tokyo, Japan) and Fuji Film FR. For the histologic examination, joints were fixed in 10% formalin/neutral phosphate buffer, decalcified in 10% EDTA-4Na and embedded in paraffin. Sections were stained with hematoxylin & eosin.
Immunohistochemistry
Synovium of the knee joint was excised and frozen in OCT compound using liquid nitrogen. Serial sections were cut at a thickness of 5 µm and fixed with acetone. After inactivation of the endogenous peroxidase with 0.01% H2O2 in PBS, the sections were blocked with 5% goat serum in PBS and then incubated with rat or hamster mAb, or rabbit antibody. Bound rat mAb was detected with horseradish peroxidase (HRP)-labeled goat anti-rat IgG (Histofine; Nichirei, Japan) and visualized with 3,3'-diaminobenzidine (Dako, Kyoto, Japan ). Bound hamster mAb and rabbit antibodies were detected using a biotinylated mouse anti-hamster mAb mixture (PharMingen, San Diego, CA) and biotinylated goat anti-rabbit IgG (Zymed, South San Francisco, CA) respectively. For class II MHC staining, a biotinylated mouse anti-class II MHC mAb (25-9-17; PharMingen) was used. Bound biotinylated antibodies were visualized with ABC for HRP (Vector, Burlingame, CA) and 3,3'-diaminobenzidine or ABC for alkaline phosphatase (Vector) and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. For nuclear counterstaining, hematoxylin or methyl green was used in single- or dual-staining experiments respectively. The antibodies used were: rat anti-mouse IL-6 (MP5-20F; PharMingen), rat anti-CD4 (RM4-5; PharMingen), anti-CD11b (M1/70) and anti-granulocytes (Gr-1, RB6-8C5), hamster anti-CD3 mAb (48-2B; Santa-Cruz, CA), and rabbit anti-phospho-STAT3 (Tyr705) (Cell Signaling, Beverly. MA). Anti-CD11b and Gr-1 were purified from the culture supernatant of hybridomas in our laboratory.
Assay for serum antibodies and IL-6
Serum IgG concentrations were measured with an ELISA system as described previously (26). For the concentrations of serum autoantibodies and IL-6, commercially available ELISA systems for rheumatoid factor of the IgG class (Shibayagi, Shibukawa, Japan), anti-dsDNA antibody (Shibayagi) and IL-6 (Biosource, Camarillo, CA) were used.
Flow cytometry analysis
A single-cell suspension prepared from the spleen or lymph nodes by teasing the tissue with slide glass was stained with a combination of labeled mAb, and analyzed with a FACSCalibur and CellQuest software, as described previously (27). The mAb used were: FITC-conjugated Gr-1 (a marker for granulocytes), IM7 (anti-CD44; PharMingen), PC61 (CD25; PharMingen), AM/3 (IgM) and 25-9-17 (class II MHC, I-Ab; PharMingen), phycoerythrin (PE)-conjugated MEL14 (CD62L; Caltag, Burlingame, CA), H1.2F3 (CD69; PharMingen) and HL3 (CD11c; PharMingen), CyChrome-conjugated 53-6.7 (CD8; PharMingen) and RM4-5 (CD4; PharMingen), PECy5-conjugated Gr-1 (Southern Biotechnology Associates, Birmingham, Alabama), TriColor-conjugated RA3-6B2 (CD45R; Caltag), allophycocyanin-conjugated M1/70 (CD11b; PharMingen), CTCD4 (CD4; Caltag) and CTCD8 (CD8; Caltag), biotinylated CS/70 (IgD), and CyChromestreptavidin (PharMingen). mAb Gr-1, AM/3 and CS/70 were purified from culture fluid supernatant and labeled with FITC or biotin by standard procedure.
Statistical analysis
Arthritis score, cell numbers of spleen and inguinal lymph nodes, ELISA assays, and frequencies of flow cytometry analyses of cell populations were compared by the MannWhitney U-test.
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Results
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Synergy between the Y759F mutation of gp130 and the HTLV-1 pX gene in the development of RA-like arthritis
pX-Tg mice in the C57BL/6 background (B6.pX-Tg mice) are resistant to arthritis (24). We generated double-mutant mice by crossing gp130F759/F759 with B6.pX-Tg mice to determine the effects of pX gene expression with the gp130F759/F759 mutation. The gp130F759/F759/pX-Tg double mutants developed arthritis as young as 5 weeks old (Fig. 1). A 50% incidence of arthritis was observed at 11 weeks of age in B6.gp130F759/F759/pX-Tg mice and within the 24-week observation period the incidence reached 70100%. The incidences of male and female B6.gp130F759/F759/pX-Tg mice at 24 weeks were 86 and 56% respectively. Only 7% of the B6.pX-Tg mice developed arthritis, with onset at
16 weeks of age, consistent with the previous report that the C57BL/6 genetic background is resistant to the development of pX-dependent arthritis (24). Although the earliest symptom appeared in the B6.g130F759/F759 mice at
10 weeks of age, 50% incidence was observed at
38 weeks of age (data not shown), indicating that the progress of arthritis in the gp130F759/F759/pX-Tg double mutants was much faster than in the single mutants. The average severity score of the gp130F759/F759/pX-Tg double mutants gradually increased to 6 points (male 7 points and female 5 points), while that of the g130F759/F759 mice was <2 points within the 24-week observation period. These results indicate that the gp130 Y759F mutation and pX gene synergize in the development and progress of arthritis. This synergistic effect was also observed in the double mutants generated by mating with g130F759/F759 mice backcrossed 8 times to B6, which generated the mutants with 9 times backcrossed to B6.

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Fig. 1. Facilitated development and accelerated progress of arthritis in gp130F759/F759/pX-Tg double-mutant mice. Double-mutant mice of the following genotypes were generated in the C57BL/6 background (N6) by crossing. gp130WT/WT/pX-Tg/ (black diamonds, n = 16; male 12, female 4), gp130F759/F759/pX-Tg/ (triangles, n = 31; male 14, female 17), gp130WT/WT/pX-Tg+/ (white circles, n = 15; male 6, female 9) and gp130F759/F759/pX-Tg+/ (black circles, n = 16; male 7, female 9). The severity of arthritis of each mouse was assessed weekly using the criteria described in Methods. Mice with scores 2 points were defined as arthritic. The incidence and average severity scores are indicated in the left and right graphs respectively.
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The earliest symptom of this arthritis was restriction of the movement of the wrist or ankle joints, which progressed to ankylosis with severe deformity of the wrists (not shown) and ankles (Fig. 2, top center). In radiologic analyses, the ankle joints of the B6.gp130F759/F759/pX-Tg mice at 24 weeks of age (Fig 2, middle center) had erosions and deformity of bones, and ankylosis. Histological analysis of the gp130F759/F759/pX-Tg mice at 24 weeks revealed hyperplasia of synovial fibroblasts, infiltration of neutrophils, narrowing of the joint spaces and ankylosis of the bone (Fig. 2, bottom center). These histological changes are similar to those observed in the arthritis of gp130F759/F759 mice on the 129/B6 background at the age of 18 months (Fig. 2, right panels). Presence of the erosive arthritis and lack of nephropathy support this model to be RA-like rather than systemic lupus erythematosus-like, although both rheumatoid factor and anti-dsDNA antibody were detected in the sera.

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Fig. 2. Arthritis of gp130F759/F759/pX-Tg+/. Macroscopic (top), radiologic (middle) and histologic (bottom) analyses of B6.gp130WT/WT/pX-Tg/ (left; 6 months old), B6.gp130F759/F759/pX-Tg+/ (center; 6 months old) and 129/B6.gp130F759/F759 (right; 18 months old) mice are shown. The histological analysis is of the synovium in the frontal portion of the ankles.
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To clarify the localization, interaction and activation status of the cells involved in this arthritis, we performed an immunohistochemical analysis of the synovium. Staining with anti-CD11b (Fig. 3C) and anti-Gr-1 (Fig. 3B) mAb revealed that cells expressing CD11b or Gr-1 were mainly located in the layers of the synovial lining cells, indicating these were type A synovial cells. Co-localization of CD11b and Gr-1 was observed in the superficial lining cells, while the independent localizations of CD11b+ or Gr-1+ cells were observed in the sublining area, representing macrophages and granulocytes. There were areas containing scattered CD4+ cells that overlapped with regions dominated by CD11b+ cells in the sublining (Fig. 3C and E). Dual staining showed co-localization of CD3+ cells with CD4+ cells, confirming that most of the CD4+ cells were T cells (not shown). Very few CD8+ cells were observed (not shown). The area containing CD4+ cells was closely located around or overlapping the area containing class II MHC+ cells (Fig. 3E), suggesting this to be a location where certain immune responses take place. Class II MHC+ cells overlapped with a portion of the CD11b+ cells (Fig. 3C). Since CD11c+ cells were rarely observed in the synovial sections (not shown), class II MHC+CD11b+ cells could be the main cell population presenting antigens in the synovium.

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Fig. 3. Immunohistochemical analysis of the arthritic joints of gp130F759/F759/pX-Tg+/ mice. Cryosections of the synovium of knee joints from a gp130F759/F759/pX-Tg+/ mouse at 6 months old were stained with (A) hematoxylin & eosin, (B) anti-Gr-1 (brown), (C) anti-CD11b (brown; white arrow heads) and class II MHC (dark blue; black arrow heads), (D) anti-IL-6 (brown; arrow heads) and anti-TNF (dark blue), (E) anti-CD4 (brown: a white arrow head) and anti-class II MHC (dark blue; a black arrow head), and (F) IL-6-producing cells (left; brown) and the nuclear localization of Y705 phosphorylated STAT3 (right; brown) in serial sections.
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We then examined the production of inflammatory cytokines. An ELISA of serum IL-6 levels in gp130F759/F759/pX-Tg mice aged 45 months old (101 ± 38 pg/ml; average ± SEM of n = 9) showed a 31 or 53% increase compared with pX-Tg (78 ± 32 pg/ml; n = 7) or gp130F759/F759 (66 ± 36 pg/ml; n = 7) mice respectively. Although these differences are not significant, it suggests that local IL-6 production is augmented in gp130F759/F759/pX-Tg mice, probably due to the action of Tax, as reported previously (18). Immunohistochemically, local IL-6 production was observed at the superficial lining layers extending to the sublining areas in the arthritic synovium (Fig. 3D and F, left). The stronger staining at the sublining area and the shape of the cells indicate that synovial fibroblasts were the major IL-6 producers (Fig. 3F, left). IL-1ß (not shown) and TNF-
(Fig. 3D) were faintly detected, but the staining was much weaker than that of IL-6, suggesting that IL-6 is the major inflammatory cytokine in this arthritis. To detect IL-6-mediated signaling, we examined the localization of phospho-STAT3. Some cells that were located in the area of IL-6 production also showed nuclear localization of phospho-STAT3 (Fig. 3F, right), suggesting that these cells were receiving aberrant gp130 signals from IL-6 produced at the site.
Synergy between the gp130 Y759F mutation and HTLV-1 pX for the generation of arthritis was dependent on IL-6.
Among the IL-6 family cytokine members that use gp130 as a signal-transducing receptor subunit, IL-6 plays a major role in the regulation of immune responses (28). Furthermore, the immunohistochemical analyses in the present study demonstrated that IL-6 was produced at the synovium in the arthritic joints and activated STAT3-mediated signaling. Thus, we examined the roles of IL-6 in the pathogenesis of the severe arthritis in gp130F759/F759/pX-Tg double mutants by generating IL-6//gp130F759/F759/pX-Tg triple mutants.
As shown in Fig. 4(A), arthritis in the gp130F759/F759/pX-Tg double mutants started to appear in mice at 7 weeks old, whereas that of the IL-6/gp130F759/F759/pX-Tg triple mutants started in mice at 16 weeks old, 9 weeks later. The incidence of arthritis in the IL-6/gp130F759/F759/pX-Tg triple mutants then gradually increased, but at 24 weeks, when the incidence of arthritis in the gp130F759/F759/pX-Tg double mutants had reached 100%, that in the IL-6/gp130F759/F759/pX-Tg triple mutants showed a plateau at 50%. Furthermore, the average severity score of the IL-6/gp130F759/F759/pX-Tg triple mutants was much lower than that of the gp130F759/F759/pX-Tg double mutants (2 versus 6). These data indicated that IL-6 plays pivotal roles in determining the time of onset, final incidence and severity of the arthritis in the double mutants.


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Fig. 4. IL-6 dependency of the arthritis in gp130F759/F759/pX-Tg+/ mice. (A) Clinical course of arthritis in gp130F759/F759/pX-Tg+/ (white circles, n = 10; male 6, female 4) and IL-6//gp130F759/F759/pX-Tg+/ (black circles, n = 8; male 4, female 4) mice was examined. (B, top) Histologic analyses of the knee joint from 24-week-old IL-6+/+/gp130WT/WT/pX-Tg/ (left), IL-6+/+/gp130F759/F759/pX-Tg+/ (center) and IL-6//gp130F759/F759/pX-Tg+/ (right) mice. (B, bottom) Radiologic analyses of ankle joints arranged in the same order as in (B, top).
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Histological examination revealed that hyperplasia of the synovial fibroblasts, infiltration of neutrophils and bone destruction were much ameliorated in the IL-6/gp130F759/F759/pX-Tg triple mutants (Fig. 4B, top). Radiologic analysis showed that the osteoporotic changes, erosive changes of the toes and reactive calcification of the tarsal bones seen in the gp130F759/F759/pX-Tg mice were not observed in the IL-6/gp130F759/F759/pX-Tg triple mutants (Fig. 4B, bottom).
Immunological abnormalities caused by the synergy between the gp130Y759F mutation and HTLV-1 pX were dependent on IL-6
To clarify the role of IL-6 in the arthritis of gp130F759/F759/pX-Tg double-mutant mice, various parameters of the lympho-hematopoietic systems of each mouse were examined and compared. The average age of the mice in the groups analyzed was 32 weeks (range 2440 weeks) and the genotypes and numbers of mice used were: gp130WT/WT/pX-Tg/ (n = 6), gp130F759/F759/pX-Tg/ (n = 5), gp130WT/WT/pX-Tg+/ (n = 5), gp130F759/F759/pX-Tg+/ (n = 8) and IL-6/gp130F759/F759/pX-Tg+/ (n = 5). The average severity score of gp130F759/F759/pX-Tg (11.6 ± 1.2) was much higher than that of the arthritic mice gp130F759/F759 (1.4 ± 0.9) and that of IL-6/gp130F759/F759/pX-Tg was significantly decreased (4.0 ± 1.6) (Fig. 5A).

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Fig. 5. IL-6 dependency of various parameters examined in the analysis of gp130F759/F759/pX-Tg+/. The arthritis score (A), total cell number in the spleen (B) and bilateral inguinal lymph nodes (C), and concentrations of serum IgG (D), rheumatoid factor (E) and anti-dsDNA antibody (F) in mice averaging 32 weeks old (range 2436) of the following genotypes are shown. The numbers on the horizontal axis indicate: 1, gp130WT/WT/pX-Tg/ (n = 6); 2, gp130F759/F759/pX-Tg/ (n = 5); 3, gp130WT/WT/pX-Tg+/ (n = 5); 4, gp130F759/F759/pX-Tg+/ (n = 8); and 5, IL-6//gp130F759/F759/pX-Tg+/ (n = 5). The circles and horizontal bars indicate the values for each mouse and the average respectively. *P < 0.05; **P < 0.01
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Similar to the severity score, splenic cell number (Fig. 5B) and the titer of rheumatoid factor (Fig. 5E) of gp130F759/F759/pX-Tg were significantly higher than those of wild-type or single mutants. The cell numbers of inguinal lymph node (Fig. 5C), and the concentrations of serum IgG (Fig. 5D) and anti-dsDNA antibody (Fig. 5F) showed increased values in the gp130F759/F759 mouse group both in the presence or absence of pX expression, indicating these changes are dependent on the gp130F759/F759 mutation. These parameters, except for the inguinal lymph node cell numbers, normalized in the absence of IL-6, indicating significant IL-6 dependency.
Then we performed flow cytometry analyses to investigate the IL-6-dependent changes in the immune-competent cell populations of the inguinal lymph nodes (Fig. 6 and Table 1). Around 7 months old, only the reduction of naive CD4 T cells (CD62L+CD44) and slight increases of activated CD4 T cells (CD62L+CD44+ or CD69+) were observed in gp130F759/F759 mice (Fig. 6A). In contrast, marked decreases of naive CD4 and CD8 T cells, and increases of memory/activated CD4+ T cells (CD62L+CD44+, CD62LCD44+ and CD69+ cells), activated CD8+ T cells (CD62L+CD44+ and CD69+ cells) and granulocytes (CD11b+Gr-1+) were observed in gp130F759/F759/pX-Tg, whereas these changes were almost normal in IL-6/gp130F759/F759/pX-Tg+/ (Fig. 6A and B, and Table 1), suggesting that these changes are IL-6 dependent, and intimately related to autoimmunity and the severity of arthritis.


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Fig. 6. IL-6-dependent changes of immune-competent cell populations in the inguinal lymph nodes of gp130F759/F759/pX-Tg+/. The inguinal lymph node cells of each genotype at 7 months old were stained with the combination of mAb and analyzed with a flow cytometer as described in Methods. (A) Analyses of T cell subsets. (B) Analyses of myeloid cells. Numbers in the quadrants of dot-plots for CD4/CD8 and Gr-1/CD11b indicate the frequencies in total inguinal lymph node cells. Numbers in the quadrants of dot-plots for CD44/CD62L and CD25/CD69 indicate the frequencies in CD4+ or CD8+ inguinal lymph node cells. Numbers in the regions of dot-plots for CD11c/CIIMHC indicate the frequency of CD11c+ cells in the inguinal lymph node cells. In the histogram, the frequencies of CIIMHCbright cells in the CD11c+ cells are indicated. Representative data from the analyses summarized in Table 1 are shown.
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Since T cell activation is mainly regulated by professional antigen-presenting cells (29), we examined the dendritic cells in the lymph nodes of arthritic mice (Fig. 6B and Table 1). The average frequencies of CD11c+ cells in the lymph nodes of wild-type and gp130F759/F759/pX-Tg mice were 5 and 6% respectively, indicating that this double mutation did not greatly affect the development of dendritic cells. However, the expression levels of class II MHC (CIIMHC) molecules were drastically changed in the gp130F759/F759/pX-Tg dendritic cells. Among CD11c+ cells, mature dendritic cells can be distinguished by the bright expression of class II MHC. In wild-type control mice, the CIIMHCbright population represented 35% of the CD11c+ cells, whereas in gp130F759/F759/pX-Tg mice it was only 4%. Furthermore, of the IL-6/gp130F759/F759/pX-Tg CD11c+ cells, the CIIMHCbright population recovered to 29%, indicating that the decreased frequency of the CIIMHCbright population was dependent on IL-6/gp130 signaling. A similar reduction of the CIIMHCbright population was also observed in the CD11c+ cells of gp130F759/F759/pX-Tg mice at 16 weeks of age, before the increase of memory or activated T cells was beginning to be obvious. Since this change of T cells follows a decreased frequency of CIIMHCbrightCD11c+ cells among lymph node cells, it may affect the regulation of T cell responses.
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Discussion
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We recently reported that a point mutation of gp130, a signal-transducing receptor subunit common to IL-6 family cytokines, causes RA-like autoimmune arthritis. This model, the gp130F759/F759 mouse, is unique and important because it provided the first experimental evidence that a point mutation of a cytokine receptor could cause autoimmune disease. Furthermore, the autoimmune disease spontaneously developed as a result of deregulated IL-6/gp130 signaling. In the gp130F759/F759 mouse, a point mutation of gp130 Y759F selectively disrupts the SHP-2-mediated gp130 signals, which results in the loss of ERK activation and in the prolonged activation of STAT3. Such unbalanced signaling is likely to be involved in triggering the breakdown of self-tolerance.
In this paper, we showed a clear synergy between the gp130 Y759F mutation and the HTLV-1 pX gene in causing RA-like arthritis, i.e. a genetic factor (the point mutation of gp130) and an environmental factor (the overexpression of pX, which mimics HTLV-1 infection) synergize, and facilitate the onset and progress of arthritis. It was surprising that the combination of a genetic factor for arthritis with late onset and slow progression, and a virus gene expression that evokes arthritis only rarely in a B6 genetic background, resulted in arthritis that had much earlier onset and faster progress than was seen in either single mutant. In the gp130F759/F759/pX-Tg mouse, abnormalities previously observed in gp130F759/F759 mice were augmented. These include splenomegaly, lymphoadenopathy, hyper-
-globulinemia, autoantibody production, increases in granulocytes and memory/activated T cells, and decreases in naive T cells and class II MHCbrightCD11c+ cells. The clinical course of the triple mutant, IL-6/gp130F759/F759/pX-Tg, was markedly ameliorated, indicating that the acceleration of arthritis in gp130F759/F759/pX-Tg is largely dependent on IL-6. Furthermore, almost all of the abnormalities seen in the gp130F759/F759/pX-Tg mice were found to be dependent on IL-6, suggesting that these abnormalities are intimately related to the onset or progress of the disease.
Although we did not clarify the molecular mechanisms for the acceleration of the arthritis, we can speculate that the mutations in gp130 and the pX gene play distinct roles in this process. Since the latest analysis of arthritis in 8 times backcrossed B6.gp130F759/F759 mice revealed that the incidence reached 100% at 54 weeks (unpublished observation), the major effect of the pX gene seems to be to accelerate the progression of the disease. The increased cytokine gene expression in pX-Tg reported previously supports this notion. In particular, IL-6 gene expression is known to be enhanced by pX and the synergy we observed was dependent on IL-6; therefore, it is likely that a pX-induced up-regulation of the IL-6 gene is one of the mechanisms. Since the major effect of pX expression is mediated by NF-
B activation, a role for pX could be as a substitute for TNF-
or IL-1 in virus-mediated effects.
On the other hand, because B6.pX-Tg rarely develops arthritis, the role of Y759F-mutated gp130 seems to be to trigger the disease in the pX-Tg mutant of an arthritis-resistant strain. Although IL-6 gene targeting in gp130F759/F759/pX-Tg could not completely block the development of arthritis, the incidence reached a plateau at 50%, suggesting the involvement of other IL-6 family cytokines that utilize gp130.
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Acknowledgements
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This work is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, and the Osaka Foundation for the Promotion of Clinical Immunology. We would like to thank Drs Y. Saeki and S. Ohshima for valuable advice and suggestions. We thank Ms R. Okuda for mouse maintenance, and Ms R. Sato for making cryo-sections and performing excellent technical assistance. We also thank Ms R. Masuda and A. Kubota for secretarial assistance.
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Abbreviations
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CIIclass II
HRPhorseradish peroxidase
PEphycoerythrin
RArheumatoid arthritis
Tgtransgenic
TNFtumor necrosis factor
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References
|
---|
- Firestein, G. S. 2003. Evolving concepts of rheumatoid arthritis. Nature 423:356.[CrossRef][ISI][Medline]
- Feldmann, M., Brennan, F. M. and Maini, R. N. 1996. Role of cytokines in rheumatoid arthritis. Annu. Rev. Immunol. 14:397.[CrossRef][ISI][Medline]
- Feldmann, M. and Maini, R. N. 2001. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu. Rev. Immunol. 19:163.[CrossRef][ISI][Medline]
- Hirano, T. 1998. Interleukin 6 and its receptor: ten years later. Int. Rev. Immunol. 16:249.[Medline]
- Hirano, T., Taga, T., Yasukawa, K., Nakajima, K., Nakano, N., Takatsuki, F., Shimizu, M., Murashima, A., Tsunasawa, S., Sakiyama, F., et al. 1987. Human B-cell differentiation factor defined by an anti-peptide antibody and its possible role in autoantibody production. Proc. Natl Acad. Sci. USA 84:228.[Abstract]
- Hirano, T., Matsuda, T., Turner, M., Miyasaka, N., Buchan, G., Tang, B., Sato, K., Shimizu, M., Maini, R., Feldmann, M., et al. 1988. Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Immunol. 18:1797.[ISI][Medline]
- Ishihara, K. and Hirano, T. 2002. IL-6 in autoimmune disease and chronic inflammatory proliferative disease. Cytokine Growth Factor Rev. 13:357.[CrossRef][ISI][Medline]
- Alonzi, T., Fattori, E., Lazzaro, D., Costa, P., Probert, L., Kollias, G., De Benedetti, F., Poli, V. and Ciliberto, G. 1998. Interleukin 6 is required for the development of collagen-induced arthritis. J. Exp. Med. 187:461.[Abstract/Free Full Text]
- Ohshima, S., Saeki, Y., Mima, T., Sasai, M., Nishioka, K., Nomura, S., Kopf, M., Katada, Y., Tanaka, T., Suemura, M. and Kishimoto, T. 1998. Interleukin 6 plays a key role in the development of antigen-induced arthritis. Proc. Natl Acad. Sci. USA 95:8222.[Abstract/Free Full Text]
- Sasai, M., Saeki, Y., Ohshima, S., Nishioka, K., Mima, T., Tanaka, T., Katada, Y., Yoshizaki, K., Suemura, M. and Kishimoto, T. 1999. Delayed onset and reduced severity of collagen-induced arthritis in interleukin-6-deficient mice. Arthritis Rheum. 42:1635.[CrossRef][ISI][Medline]
- Atsumi, T., Ishihara, K., Kamimura, D., Ikushima, H., Ohtani, T., Hirota, S., Kobayashi, H., Park, S. J., Saeki, Y., Kitamura, Y. and Hirano, T. 2002. A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis. J. Exp. Med. 196:979.[Abstract/Free Full Text]
- Hirano, T., Ishihara, K. and Hibi, M. 2000. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19:2548.[CrossRef][ISI][Medline]
- Kamimura, D., Ishihara, K. and Hirano, T. 2003. IL-6 signal transduction and its physiological roles: the signal orchestration model. Rev. Physiol. Biochem. Pharmacol. 149:1.
- Ohtani, T., Ishihara, K., Atsumi, T., Nishida, K., Kaneko, Y., Miyata, T., Itoh, S., Narimatsu, M., Maeda, H., Fukada, T., Itoh, M., Okano, H., Hibi, M. and Hirano, T. 2000. Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Immunity 12:95.[ISI][Medline]
- Sodroski, J. G., Rosen, C. A. and Haseltine, W. A. 1984. Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science 225:381.[ISI][Medline]
- Fujisawa, J., Seiki, M., Sato, M. and Yoshida, M. 1986. A transcriptional enhancer sequence of HTLV-I is responsible for trans-activation mediated by p40 chi HTLV-I. EMBO J. 5:713.[Abstract]
- Maruyama, M., Shibuya, H., Harada, H., Hatakeyama, M., Seiki, M., Fujita, T., Inoue, J., Yoshida, M. and Taniguchi, T. 1987. Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-1-encoded p40x and T3/Ti complex triggering. Cell 48:343.[ISI][Medline]
- Muraoka, O., Kaisho, T., Tanabe, M. and Hirano, T. 1993. Transcriptional activation of the interleukin-6 gene by HTLV-1 p40tax through an NF-kappa B-like binding site. Immunol. Lett. 37:159.[CrossRef][ISI][Medline]
- Inoue, J., Seiki, M., Taniguchi, T., Tsuru, S. and Yoshida, M. 1986. Induction of interleukin 2 receptor gene expression by p40x encoded by human T-cell leukemia virus type 1. EMBO J. 5:2883.[Abstract]
- Fujii, M., Niki, T., Mori, T., Matsuda, T., Matsui, M., Nomura, N. and Seiki, M. 1991. HTLV-1 Tax induces expression of various immediate early serum responsive genes. Oncogene 6:1023.[ISI][Medline]
- Kitajima, I., Yamamoto, K., Sato, K., Nakajima, Y., Nakajima, T., Maruyama, I., Osame, M. and Nishioka, K. 1991. Detection of human T cell lymphotropic virus type I proviral DNA and its gene expression in synovial cells in chronic inflammatory arthropathy. J. Clin. Invest. 88:1315.[ISI][Medline]
- Iwakura, Y., Tosu, M., Yoshida, E., Takiguchi, M., Sato, K., Kitajima, I., Nishioka, K., Yamamoto, K., Takeda, T., Hatanaka, M., et al. 1991. Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-I. Science 253:1026.[ISI][Medline]
- Kishi, S., Saijyo, S., Arai, M., Karasawa, S., Ueda, S., Kannagi, M., Iwakura, Y., Fujii, M. and Yonehara, S. 1997. Resistance to fas-mediated apoptosis of peripheral T cells in human T lymphocyte virus type I (HTLV-I) transgenic mice with autoimmune arthropathy. J. Exp. Med. 186:57.[Abstract/Free Full Text]
- Iwakura, Y., Itagaki, K., Ishitsuka, C., Yamasaki, Y., Matsuzawa, A., Yonehara, S., Karasawa, S., Ueda, S. and Saijo, S. 1998. The development of autoimmune inflammatory arthropathy in mice transgenic for the human T cell leukemia virus type-1 env-pX region is not dependent on H-2 haplotypes and modified by the expression levels of Fas antigen. J. Immunol. 161:6592.[Abstract/Free Full Text]
- Kopf, M., Ramsay, A., Brombacher, F., Baumann, H., Freer, G., Galanos, C., Gutierrez-Ramos, J. C. and Kohler, G. 1995. Pleiotropic defects of IL-6-deficient mice including early hematopoiesis, T and B cell function, and acute phase responses. Ann. NY Acad. Sci. 762:308.[Medline]
- Itoh, M., Ishihara, K., Hiroi, T., Lee, B. O., Maeda, H., Iijima, H., Yanagita, M., Kiyono, H. and Hirano, T. 1998. Deletion of bone marrow stromal cell antigen-1 (CD157) gene impaired systemic thymus independent-2 antigen-induced IgG3 and mucosal TD antigen-elicited IgA responses. J. Immunol. 161:3974.[Abstract/Free Full Text]
- Ishihara, K., Kobune, Y., Okuyama, Y., Itoh, M., Lee, B. O., Muraoka, O. and Hirano, T. 1996. Stage-specific expression of mouse BST-1/BP-3 on the early B and T cell progenitors prior to gene rearrangement of antigen receptor. Int. Immunol. 8:1395.[Abstract]
- Betz, U. A., Bloch, W., van den Broek, M., Yoshida, K., Taga, T., Kishimoto, T., Addicks, K., Rajewsky, K. and Muller, W. 1998. Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects. J. Exp. Med. 188:1955.[Abstract/Free Full Text]
- Mellman, I. and Steinman, R. M. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255.[ISI][Medline]