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The Importance of IL-1 and TNF-, and the Noninvolvement of IL-6, in the Development of Monoclonal Antibody-Induced Arthritis

Biological Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF- was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 or TNF-, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1, TNF-, or MIP-1. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R^-/- mice and TNFR^-/- mice was not observed at all, but severe arthritis was developed in IL-6^-/- mice. These results suggest that IL-1 and TNF- play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Collagen-induced arthritis (CIA)² is commonly used as a rheumatoid arthritis (RA) model in the screening of antirheumatic drugs because it has various similarities to human RA (1, 2). In the CIA model, anti-type II collagen (CII) Ab plays an important role and is shown to induce arthritis in mice by passive transfer (2). An arthritis model using a mixture of four monoclonal anti-CII Abs was established (3, 4). This arthritis model can be prepared in constant yields using various strains of mice independent of MHC haplotypes (4), but, to date, the mechanism of arthritis development remains unclear.

In RA, not only anti-CII Ab but also inflammatory cytokines and chemokines are believed to be involved in the development of arthritis (5, 6, 7, 8, 9, 10, 11). Inflammatory cytokines such as IL-1, IL-6, and TNF- are shown to contribute to the development of arthritis in some arthritis models (8, 9, 10, 11). IL-1 is shown to have various physiological functions such as induction of inflammation, modification of immune response, and activation of osteoclasts (12, 13, 14, 15). IL-6 and TNF- are also involved in inflammation, differentiation and proliferation of T and B cells, and bone absorption (15, 16, 17, 18, 19). All these cytokines are regarded as targets for RA treatment. Therefore, an anti-cytokine therapy has been developed and its effectiveness is being clinically tested. Recently, anti-RA therapy by soluble TNFR and anti-TNF- Ab was reported to be effective (5, 20), but the effectiveness of other cytokines has not been discovered yet. Thus, the most important cytokine in RA is still unknown.

Many chemokines, including macrophage inflammatory protein (MIP)-1, MIP-2, and monocyte chemoattractant protein (MCP)-1, are estimated to be involved in the development of arthritis because of their increased expression levels in the synovial fluid of RA patients and in the arthritic foot of animal models (6, 7, 8, 11, 21). The estimated function of chemokines in arthritis development is the regulation of inflammation including infiltration of lymphocytes, monocytes, and neutrophils, among others (22, 23, 24, 25). The effectiveness of anti-MIP-1, MIP-2, and MCP-1 treatments on arthritis development has been reported in some arthritis models (26, 27), but their exact contributions have not been fully elucidated.

In this study, we characterized the anti-CII Ab-induced arthritis model and investigated the roles of cytokines and chemokines in the development of arthritis. The expression level of every cytokine (IL-1, IL-6, and TNF-) and chemokine (MIP-1, MIP-2, and MCP-1) investigated in this study was elevated in the arthritic hind paw, and cytokine and chemokine levels were highly correlated with the arthritis score. IL-1 and TNF- were found to be most important in arthritis development among these cytokines and chemokines, and the anti-MIP-1 Ab treatment was also effective in this model. Surprisingly, however, the involvement of IL-6 in the development of arthritis was refuted due to the noninduction of arthritis by rIL-6 or in IL-6^-/- mice and noninhibition of arthritis using anti-IL-6 neutralizing Ab. We also report the unique lack of T and B cell involvement of this arthritis model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Male BALB/cAnNCrj (BALB/c) mice were purchased from Charles River (Tokyo, Japan). Male IL-1R^-/- mice (28), IL-6^-/- mice (29), and p55 and p75 TNFR^-/- mice (30) were from The Jackson Laboratory (Bar Harbor, ME). C57BL/6J Jcl mice, C.B-17/lcr-scid Jcl (scid/scid) (31) mice and their corresponding control, C.B-17/lcr-+/+ Jcl (+/+ control), were from CLEA Japan (Tokyo, Japan). All mice were purchased at the age of 5–6 wk, housed at Sankyo Laboratories (Tokyo, Japan), and given a standard rodent chow diet and water ad libitum.

Reagents

Arthritogenic mAb mixture and LPS were purchased from Immuno-Biological Laboratories (Gunma, Japan). BSA, mouse IgG, goat IgG, protease inhibitor mixture, and indomethacin were obtained from Sigma-Aldrich (St. Louis, MO). Recombinant murine IL-1, IL-6, TNF-, MIP-1, MIP-2, and MCP-1, neutralizing Abs to murine IL-1, IL-6, MIP-1, MIP-2, and MCP-1, rat IgG1 and IgG2a isotype control Abs, and ELISA kits of murine IL-1, IL-6, and TNF- were from Genzyme (Minneapolis, MN). ELISA kits of murine MIP-1 and MIP-2 were from R&D Systems (Minneapolis, MN). Anti-murine TNF--neutralizing Ab and a murine MCP-1 ELISA kit were from BioSource International (Camarillo, CA). Rat IgG2b isotype control Ab was from BD PharMingen (San Diego, CA). TRIzol reagent was purchased from Life Technologies (Grand Island, NY). TaqMan Universal PCR Master Mix, TaqMan Rodent GAPDH Control Reagent, TaqMan Reverse Transcription Reagent, and TaqMan Probes were from PE Applied Biosystems (Foster City, CA). PCR primers were from Amersham Pharmacia Biotech (Tokyo, Japan). Protein assay dye reagent was purchased from Bio-Rad (Hercules, CA).

Induction of arthritis in mice

Arthritis was induced by the method of Terato and colleagues (3, 4) using an arthritogenic mAb mixture. Briefly, mice were injected i.v. with 2 mg of anti-CII Ab (2 mg/500 µl/body) from the tail vein, and 3 days later 50 µg of LPS (50 µg/100 µl/body) was injected i.p. As a control, 2 mg of mouse IgG dissolved in PBS (2 mg/500 µl/body) was injected in place of anti-CII Ab.

Measurement of cytokine concentration in arthritic hind paws

Hind footpads of sacrificed mice were cut at the borderline of fur growth and frozen in liquid N₂. The footpads were homogenized in ice-cold PBS supplemented with 0.5% protease inhibitor mixture, 10 mM EDTA, and 100 µM indomethacin using a Polytron homogenizer (KINEMATICA, Lucerne, Switzerland). The volume of PBS used for homogenization was adjusted to 75 mg of tissue per milliliter of PBS. The homogenate was centrifuged for 15 min at 1,870 x g, and the supernatants were centrifuged for 5 min at 13,230 x g. The supernatants were subjected to ELISA analysis. ELISA kits of murine IL-1, IL-6, TNF-, MIP-1, MIP-2, and MCP-1 were used according to the protocol of each ELISA kit. The concentration of total protein in the supernatants was measured according to the method of Biuret using a protein assay dye reagent calibrated against the concentration of BSA. The concentration of cytokines and chemokines was expressed in picograms per milligram of protein.

Induction of arthritis by anti-CII Ab and cytokine

Murine recombinant proteins (rIL-1, rIL-6, rTNF-, rMIP-1, rMIP-2, and rMCP-1) of 500 ng (solved in 50 µl PBS) per footpad were s.c. injected into the footpads of mice on day 3, with or without an i.v. injection of anti-CII Ab (2 mg/500 µl/body) on day 0. As a control, BSA was injected s.c. on day 3 with or without anti-CII Ab.

Treatment with neutralizing Abs to cytokines and chemokines

To evaluate the effects of neutralizing Abs against cytokines and chemokines on arthritis development, mice were preinjected with 500 µg/ml PBS per body of anti-IL-1 Ab, anti-IL-6 Ab, anti-TNF- Ab, anti-MIP-1 Ab, anti-MIP-2 Ab, or anti-MCP-1 Ab 1 h before anti-CII Ab injection. In the control mice, the same dose of rat IgG1 isotype Ab, rat IgG2a isotype Ab, rat IgG2b isotype Ab, or goat IgG was injected before arthritis induction.

Clinical assessment of arthritis

Mice were carefully examined daily post-LPS or post-cytokine injection for swelling of the hind paws as a sign of arthritis. The severity of arthritis was graded on a 0–3 scale as follows: 0, normal; 1, swelling of one digit; 2, swelling of more than two digits; 3, swelling of entire paw.

Histopathological assessment of arthritis

For histopathology, the hind legs were removed (by cutting between the knee and ankle), fixed in phosphate-buffered 10% formaldehyde, decalcified in 10% EDTA, and paraffin-embedded as previously described (32). Sections of hind paws were made (by slicing the footpads horizontally) and stained with H&E. Evaluation was done on synovial membranes, bone, and cartilage tissues of the tarsal joint. With regard to synovial membranes, scoring was done for the following events: edema, congestion and/or hemorrhage, infiltration of neutrophils, infiltration of lymphocytes, infiltration of blood plasma cells, infiltration of macrophage cells, proliferation of synovial cells, proliferation of papilla (villi), proliferation of fibroblast, proliferation of granulation tissues, presence of debris in the joint cavity, and increase of blood vessels. On bone and cartilage tissues, degeneration and/or death of chondrocytes, destruction of cartilage tissues, infiltration of neutrophils, destruction of bone tissues, increase in osteoclasts, and ostitis and/or periostitis were scored. The severity was graded as follows: -, normal; +, slight change; ++, mild change; +++, severe change. Scoring was also done on the interphalangeal joint and was used in the scoring of the tarsal joint for better accuracy.

Quantitative real-time PCR

Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems) to analyze the induction or suppression of cytokine and chemokine mRNAs by the development of arthritis, following the manufacturer’s instructions. Briefly, to obtain total RNA, arthritis was induced in 35 mice by injecting anti-CII Ab and LPS, and 10 rear footpads from five mice were collected daily from days 1 to 7. Total RNA was extracted daily from the pooled footpads using TRIzol reagent. For cDNA synthesis, 1 µg of total RNA was transcribed with TaqMan reverse transcription reagents using random hexamers. Primers and TaqMan probes were designed using the primer design software Primer Express (PE Applied Biosystems), except for GAPDH, which was available commercially. The primer and probe sequences were as follows: IL-1, forward primer (5'-AACCTGCTGGTGTGTGACGTTC), reverse primer (5'-CAGCACGAGGCTTTTTTGTTGT), and probe (5'-TTAGACAGCTGCACTACAGGCTCCGAGATG); IL-6, forward primer (5'-ACAACCACGGCCTTCCCTACTT), reverse primer (5'-CACGATTTCCCAGAGAACATGTG), and probe (5'-TTCACAGAGGATACCACTCCCAACAGACCT); MIP-2, forward primer (5'-ATCCAGAGCTTGAGTGTGACGC), reverse primer (5'-AAGGCAAACTTTTTGACCGCC), and probe (5'-CGCCCAGACAGAAGTCATAGCCACTCTCAA); MCP-1, forward primer (5'-CCACTCACCTGCTGCTACTCAT), reverse primer (5'-TGGTGATCCTCTTGTAGCTCTCC), and probe (5'-CACCAGCAAGATGATCCCAATGAGTAGGC). TaqMan probes were labeled at the 5' end with the reporter dye molecule FAM, except for GAPDH, which was labeled with VIC, and at the 3' end with the quencher dye molecule TAMRA. cDNA samples (100 ng each, except for GAPDH (10 ng)) were mixed with primers and TaqMan Universal PCR Master Mix in a total volume of 50 µl as described in the manufacturer’s directions (protocol 4304449; PE Applied Biosystems). The PCR was conducted using the following parameters: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. Quantitative real-time PCR was performed for IL-1, IL-6, MIP-2, and MCP-1 and normalized to the copies of GAPDH mRNA from the same sample. Acquired data were analyzed by Sequence Detector software version 1.63 (PE Applied Biosystems). All PCR assays were performed in duplicate and results are represented by the mean values.

Statistical analysis

Statistical significance was determined by parametric Dunnett’s test in the comparison of cytokine and chemokine concentration in hind paws, and by nonparametric Dunnett’s test in the comparison of arthritis score. The significance of the arthritis score was determined on day 7 post-arthritis induction. To elucidate the correlation of cytokine or chemokine concentration and arthritis score, Spearman’s rank order correlation coefficient was calculated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anti-CII Ab is essential for arthritis development

The combined injection of anti-CII Ab on day 0 and LPS on day 3 to BALB/c mice induced arthritis (Fig. 1A). The first sign of slight swelling was detected around day 4 and paw swelling with redness reached a maximum on days 6–7. The swelling continued until day 14 in most cases and decreased gradually after that. In contrast, mice injected with murine IgG in place of anti-CII Ab showed slight redness around the footpad after injecting LPS, but no swelling occurred. Histopathological analyses on hind footpads showed apparent infiltration of neutrophils and proliferation of fibroblasts around synovial membranes 7 days after arthritis induction (Fig. 1B). As shown in Table I, the severity of the above events was decreased on day 14, although degeneration and/or death of chondrocytes and destruction of cartilage tissues were observed. Ostitis, periostitis, increase in the number of osteoclasts, and destruction of bone tissues, which did not appear on day 7, were seen on day 14. The histology of mice injected with IgG and LPS was almost normal on day 7, although slight changes, such as infiltration of macrophage cells, were observed on day 14. Paw swelling, which was highest around days 6–10, gradually decreased, but the destruction of the tarsal joints as indicated in the histological examinations was greatest on day 21 (data not shown). Histological changes around the bone and cartilage occurred until past day 35, although infiltration of inflammatory cells abated considerably (data not shown). These results suggest that anti-CII Ab is essential for arthritis development in this animal model.

View larger version (66K): [in this window] [in a new window]   FIGURE 1. Time course of arthritis development induced by anti-CII Ab and LPS injection. A, Arthritis scores are shown. Mice were injected with IgG (2 mg per body; , Control (-)) or anti-CII Ab (2 mg per body; •, Control (+)) on day 0 and injected with LPS (50 µg per body) on day 3. The severity of arthritis was judged and scored as described in Materials and Methods, and the sum score of both hind paws was recorded. The data were expressed as means ± SEM of five mice in each group. B, Representative histologies of tarsal joint using H&E stain of normal (Norm.), IgG plus LPS-injected (C (-)), and anti-CII plus LPS-injected joints (C (+)) on days 7 and 14. No swelling and almost no change in histology were observed in the hind paws of C (-) mice.

Because involvement of inflammatory cytokines and chemokines has been suggested based on results from RA models as well as from clinical examinations, we measured the concentration of cytokines and chemokines in the hind paws of arthritis-induced mice (Fig. 2). After the induction of arthritis, hind footpads were collected daily from day 0 (the day of anti-CII Ab injection) to day 7. Levels of IL-1 in the footpads were low from days 0 to 3, increased from day 3, and reached a plateau on day 5 (Fig. 2A). However, the plasma level of IL-1 remained low from days 0 to 7, except for the period of 1–12 h post-LPS injection on day 3 (data not shown). The concentrations of IL-6, MIP-1, MIP-2, and MCP-1 in the footpads were also low before the development of arthritis, but rose from day 4 (Fig. 2, B and D–F). The maximum concentration of each of these factors was reached on slightly different days. Although the concentration of TNF- in hind paws was lower than the lower detection limit of ELISA from days 0 to 7, a transient elevation of the TNF- level was confirmed from days 3 to 4 (Fig. 2C). IL-1 and IL-6 were also elevated transiently after LPS injection and reached a peak at 4 h after LPS injection (Fig. 2, A and B), although the TNF- level reached a peak at 2 h after the injection of LPS (Fig. 2C).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Time course of cytokine and chemokine levels in hind paws of arthritis-induced mice. Hind paws of mice were obtained daily from days 0 (untreated) to 7 post-arthritis induction; they were then homogenized and centrifuged to obtain the supernatants. The concentrations of IL-1 (A), IL-6 (B), MIP-1 (D), MIP-2 (E), and MCP-1 (F) in the supernatants were measured by ELISA and are shown by •. A transient elevation of IL-1 (A, inset), IL-6 (B, inset), and TNF- (C) concentration was seen post-LPS injection, as shown by (x-axis shows hours after LPS injection (each graduation = 4 h); y-axis shows concentration of IL-1, IL-6, and TNF- (each graduation = 100, 200, or 20 pg/mg protein, respectively)). The data are expressed as the means ± SEM of five mice in each group. The value was significantly different from that of the control group on day 0 (*, p < 0.05; **, p < 0.01).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. High correlation between arthritis score and cytokine and chemokine levels. Hind paws were obtained from untreated and normal or arthritis-induced mice on days 6 and 7 after the scoring of arthritis severity, and the concentrations of cytokines and chemokines in hind paws were measured as described in Materials and Methods. The concentration of each cytokine and chemokine was plotted against the arthritis score. The r value represents Spearman’s rank-order correlation coefficient (normal, n = 8; score 0, n = 12; score 1, n = 6; score 2, n = 4; score 3, n = 14; , individual data; horizontal bars represent the mean of each group).

Development of arthritis by the combined injection of anti-CII Ab with rIL-1 or rTNF-, but not with rIL-6 or recombinant chemokines

Because the expression levels of cytokines and chemokines in the arthritic hind paws were high, cytokines and chemokines could be playing important roles in arthritis development. Therefore, we investigated whether the injection of recombinant cytokines and chemokines in the footpad could induce arthritis (Fig. 4). First, BSA, recombinant cytokines, or chemokines (500 ng/50 µl/footpad) were injected into the footpads of hind paws. These recombinant cytokines and chemokines were injected at a concentration >300 times that in the homogenate of arthritic footpad. Subcutaneous injection of BSA (data not shown), rIL-1, rIL-6, rTNF-, rMIP-1, rMIP-2, or rMCP-1 alone did not induce arthritis (Fig. 4). Then, BSA (data not shown), cytokines, or chemokines were injected into the footpad s.c. on day 3, after the i.v. injection of anti-CII Ab (2 mg/500 µl/body) on day 0. Mice injected with BSA (data not shown), rIL-6, rMIP-1, rMIP-2, or rMCP-1 did not induce arthritis, even if anti-CII Ab was injected beforehand (Fig. 4, B and D–F). However, interestingly, rIL-1 and rTNF- induced arthritis if anti-CII Ab was injected previously (Fig. 4, A and C). The swelling was considerable and continued for >14 days (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Induction of arthritis by s.c. injection of recombinant cytokine and chemokine with anti-CII Ab. Mice were injected with () or without () anti-CII Ab (2 mg/500 µl/body) i.v. on day 0 and injected s.c. with recombinant cytokine and chemokine (500 ng/50 µl/footpad) on day 3. The severity of arthritis was judged and scored as described in Materials and Methods, and the sum score of both hind paws was recorded. The data were expressed as means ± SEM of five mice in each group. **, The value was significantly different from that of the normal group (p < 0.01).

Inhibition of arthritis development by neutralizing Abs against IL-1, TNF-, and MIP-1, but not by anti-MCP-1 Ab and anti-IL-6 Ab

To evaluate the importance of cytokines and chemokines in the development of arthritis, neutralizing Abs against cytokines and chemokines were injected before the induction of arthritis (Fig. 5). Anti-TNF- Ab completely inhibited the onset of arthritis (p < 0.05, Fig. 5C), and anti-IL-1 Ab also inhibited the development almost completely (p < 0.05, Fig. 5A). Dosing anti-MIP-1 Ab also suppressed paw swelling significantly (p < 0.05, Fig. 5D), and anti-MIP-2 Ab reduced the arthritic severity score partially (NS), compared with the score of mice injected with isotype Ab. In contrast, Abs to IL-6 and MCP-1 did not inhibit the development of arthritis at all (Fig. 5, B and F). Each cytokine or chemokine turned out to contribute to arthritis development to various degrees, despite the high correlation of their expression with the arthritis score.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Inhibition of arthritis development by neutralizing Abs against IL-1, TNF-, and MIP-1, but not by Abs against IL-6 and MCP-1. Arthritis was induced in mice by the i.v. injection of anti-CII Ab (2 mg/500 µl/body) on day 0 and LPS (50 µg/100 µl/body) i.p. on day 3. One hour before this arthritis induction, mice were injected with anti-cytokine (anti-IL-1, anti-IL-6, anti-TNF-) or anti-chemokine (anti-MIP-1, anti-MIP-2, anti-MCP-1) neutralizing Abs (500 µg/ml/body) () i.v. As a control, an isotype Ab (•) corresponding to each cytokine and chemokine (rat IgG1, rat IgG2a, rat IgG2b, goat IgG) was injected. The data were expressed as means ± SEM of four or five mice in each group. *, Values are significantly different from the control (p < 0.05).

For further evaluation of the contribution of cytokines in the onset of arthritis, IL-1R^-/- mice, IL-6^-/- mice, TNFR^-/- mice, and their control background (wild-type (WT)) mice were injected with anti-CII Ab and LPS to induce arthritis (Fig. 6). Arthritis was developed in WT mice from day 5 and their arthritic score reached a plateau around day 7 (Fig. 6Aa). However, in IL-1R^-/- mice and TNFR^-/- mice, arthritis was not developed by day 7 (Fig. 6A, b and d). Furthermore, in these mice, arthritis was not developed for the next 7 days (data not shown). In contrast, IL-6^-/- mice developed arthritis at the incidence of 100% (data not shown), and the severity score was the same as that of WT mice (Fig. 6Ac). On day 7 post-induction of arthritis, mice were sacrificed and hind paws were sectioned for histopathological examination (Fig. 6B). As shown in Fig. 6Bc, inflammation and tissue injury, including infiltration of neutrophils and macrophages, proliferation of fibroblast, and presence of debris in cavity, could be seen in control and IL-6^-/- mice. In contrast, there were few histopathological changes in IL-1R^-/- mice or TNFR^-/- mice on day 7 (Fig. 6B, b and d). Table III shows the concentration of IL-1 in the hind paws of the above three types of deficient mice on day 7 post-arthritis induction. Although the level of IL-1 in hind footpads of arthritis-induced IL-1R^-/- and TNFR^-/- mice was as low as that of untreated normal mice, the concentration of IL-1 in the hind paw of IL-6^-/- mice was elevated to >20 pg/mg protein, which is close to the level of control mice. MIP-1 and MIP-2 levels were also elevated in control and IL-6^-/- mice but were low in IL-1R^-/- and TNFR^-/- mice (data not shown).

View larger version (55K): [in this window] [in a new window]   FIGURE 6. Inhibition of arthritis development in IL-1R-/- and TNFR-/- mice, but not in IL-6-/- mice. Arthritis was induced by injecting anti-CII Ab (2 mg/500 µl/body) and LPS (50 µg/100 µl/body) in control (WT) mice (•), IL-1R-/- mice (), IL-6-/- mice (), and TNFR-/- mice (). A, Time course of arthritis development with photos of representative hind paws. Swelling and redness of hind paws was observed in IL-6-/- and control mice but not in IL-1R-/- and TNFR-/- mice. The severity of arthritis was judged and scored as described in Materials and Methods. The data were expressed as means ± SEM of five or six mice in each group. *, Values are significantly different from the control (WT) (p < 0.05). B, Representative histopathologies of tarsal joints stained with H&E. Mice were sacrificed for histopathological analyses 7 days after arthritis induction. Infiltration of neutrophils and macrophages, proliferation of fibroblast, and presence of debris in the joint cavity can be seen in control and IL-6-/- mice.

View this table: [in this window] [in a new window]   Table III. Reduced expression of IL-1 in IL-1R-/- and TNFR-/- mice, but not in IL-6-/- mice1

To identify the role of T and B cells in this arthritis model, SCID mice, which lack functionally mature T and B cells, were used. Arthritis was induced in SCID and control mice by injecting 2 mg of anti-CII Ab and 50 µg of LPS (Fig. 7). Both control mice and SCID mice developed arthritis in a similar way, and their arthritis scores reached almost the same value on day 7. There was no significant difference in the arthritis score between control and SCID mice throughout the evaluation period. This result suggests that T and B cells do not play a significant role in the development of arthritis in this model.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. No amelioration of arthritis development in SCID mice. Arthritis was induced by injecting anti-CII Ab (2 mg/500 µl/body) and LPS (50 µg/100 µl/body) in control (•) and SCID () mice. Arthritis scores are shown. The data were expressed as means ± SEM of eight mice in each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Correlation between the arthritis score and levels of all the cytokines measured was high, but, interestingly, IL-1 and TNF-, but not IL-6, turned out to play crucial roles in the development of arthritis in this model. It is well known that the expression of cytokines and chemokines is elevated in arthritis models like CIA (8, 9, 21). This was also observed in the anti-CII Ab-induced arthritis model. Among these cytokines, a blockade of IL-1 and TNF- was shown to inhibit arthritis in those animal models (33), which is consistent with our results. As for IL-6, anti-IL-6R Ab was shown to inhibit the development of arthritis in CIA mice (34), and partial or complete inhibition of arthritis development was reported in IL-6^-/- mice (35, 36), which differs from our results. Arthritis severity in zymosan-induced arthritis in IL-6^-/- mice was reported to be more severe than in WT mice (37). These differences are probably due to the different nature of arthritis models used. IL-6 is reported to play various roles, such as Ab production and proliferation of T and B cells. The level of anti-CII IgG Ab was decreased and the peak anti-CII IgG Ab level was reached later in CIA-induced IL-6^-/- mice. Anti-IL-6R Ab treatment was reported to be effective in inhibiting CIA development if injected early (by 3 days postimmunization of CIA) but ineffective if injected later than 7 days postimmunization (34). These results suggest that the most important role of IL-6 in the development of arthritis may be restricted to the immunization phase in CIA mice. Therefore, a depletion of IL-6 could not inhibit the arthritis induced by passively administering anti-CII Ab. The IL-1 level in hind paws was high in arthritis-induced IL-6^-/- mice. IL-1 may have induced the production of proteinases and osteoclast activation, and, as a result, led to joint destruction. IL-6 is regarded as one of the differentiation factors for osteoclast (19, 38), but osteoclast-like multinucleated cells were increased even in IL-6^-/- mice. Thus, IL-6 must not be an indispensable factor in the differentiation of osteoclast. The present study indicates that anti-CII Ab as well as IL-1 and TNF- are important in arthritis development. Therefore, inhibition of autoantibody production through IL-6 inhibition could be an effective treatment for human RA. Moreover, a humanized anti-IL-6R mAb has been reported to be effective in clinical phase studies. The arthritis model used in the present study reflects only one of the phases (probably the effector phase) or one aspect of human RA; thus, IL-6 could play a role outside of this phase.

As for chemokines, anti-MIP-1 Ab was effective in inhibiting arthritis significantly and anti-MIP-2 Ab showed slight arthritis inhibition (NS). However, anti-MCP-1 Ab was ineffective in our study. Multiple injections of neutralizing Abs were conducted for MIP-2 and MCP-1, but no significant inhibition of arthritis was observed (data not shown). Although the inhibition of chemokines by neutralizing Ab was reported to suppress arthritis development (26, 27), the possibility that anti-chemokine Abs are not effective in preventing arthritis due to insufficient amounts of neutralizing Abs in our study cannot be fully excluded. Injection of chemokines with anti-CII Ab did not induce arthritis. Thus, chemokines are not direct inducers of arthritis. The degree of contribution of chemokines in arthritis development is still controversial. Further investigation such as by the experiments using chemokine-deficient mice is required.

Histopathological analyses revealed that infiltration of neutrophils and macrophages and proliferation of fibroblasts in joints occurred first, followed by bone and cartilage damage. The maximum damage in bone and cartilage was seen on day 21 and the rate of deterioration decreased thereafter, but the damage was seen for >35 days (data not shown). An increase in the number of osteoclasts was determined, which was consistent with the increased expression of receptor activator of NF-B and receptor activator of NF-B ligand as observed in the DNA chip analyses (data not shown).

The participation of T and B cells in this Ab-induced arthritis model was excluded by the experiment using SCID mice, in which no amelioration of arthritic severity was observed. Histopathological analyses support this view, because no invasion of lymphocytes was observed in arthritic hind paws as shown in Table I. In arthritis development in the CIA model, T and B cells play important roles such as Ab production and cytokine production (39, 40, 41, 42). IL-6 acts on B cells and promotes Ab production. A deficiency in IL-6, which did not diminish the severity of arthritis, may reflect this. The main role that IL-6 plays in RA may be in the immunization phase. A depletion of IL-6 was not effective in inhibiting anti-CII Ab-induced arthritis, because there is no involvement by T and B cells in this model and because of the bypassing of the immunization process.

Histopathological analyses also revealed that the destruction of joints in this model is not as severe when compared with that in the CIA mice. In CIA mice, B cells are essential in producing anti-CII Ab. Furthermore, CII-specific T cells are known to play significant roles in the development of arthritis. Therefore, the comparatively low severity of joint destruction in anti-CII Ab-induced arthritic mice could be due to the lack of T and B cell participation.

	Acknowledgments

 
We thank Drs. Yoichiro Iwakura, Kensuke Miyake, and Kuniaki Terato for their significant comments and critical review of this manuscript. We also thank Drs. Futoshi Nara, Toshiyuki Takagi, Kazuyo Yamada, Mitsutoshi Uemori, and Azusa Seki for their technical support and helpful advice on the PCR, statistical, and histopathological analyses. We acknowledge the invaluable help in animal management by Tsutomu Yajima. We greatly thank Emelyne Uchiyama for looking over this manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Takashi Kagari, Biological Research Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome Shinagawa-ku, Tokyo 140-8710, Japan. E-mail address: tkagar{at}shina.sankyo.co.jp

² Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II collagen; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; RA, rheumatoid arthritis; WT, wild type.

Received for publication November 26, 2001. Accepted for publication May 15, 2002.

	References

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