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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

FR167653 is a potent inhibitor of p38 mitogen-activated protein kinase (MAPK) and inhibits tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) production in inflammatory cells. In this study we investigated the effect of FR167653 on collagen-induced arthritis (CIA).

Methods

Rats with CIA were subcutaneously injected with FR167653 (32 mg/kg/day) starting on the day of the booster injection (day 7) in the prophylactic treatment group and after the onset of arthritis (day 21) in the therapeutic treatment group. Hind-paw swelling, body weight, radiographic and histologic scores, and osteoclast number were evaluated. Cytokine levels in the serum and tissue were assessed by enzyme-linked immunosorbent assays. Flow cytometric analysis of T lymphocytes from bone marrow was performed. The effect of FR167653 on in vitro osteoclast formation induced by soluble receptor activator of nuclear factor κB ligand (sRANKL) and TNFα was examined.

Results

Swelling of hind paws and loss of weight occurred in the CIA rats, but this was not evident in the prophylactic treatment group. Therapeutic treatment also significantly reduced paw swelling. The mean radiographic and histologic scores as well as the osteoclast numbers were significantly lower in the treatment group than in the CIA rats without treatment. FR167653 treatment reduced the serum levels of TNFα and IL-1β, lowered the IL-1β concentration in the ankle joints, and decreased the CD4−,CD8a+ T cell population in bone marrow. Furthermore, FR167653 inhibited the osteoclast-like cell differentiation induced by both sRANKL and TNFα in vitro.

Conclusion

FR167653 prevents the onset of arthritis in a prophylactic treatment model and suppresses the progression of joint destruction in a therapeutic treatment model, suggesting that p38 MAPK is a potential therapeutic target for rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized as a chronic and progressive inflammatory process that leads to systemic immunologic abnormalities of the joints (1). It has been suggested that proinflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-6, and IL-8, which are linked in a cascade, are important in the etiology of RA (2–5). Histopathologic characterization of bone erosions in patients with RA and in animal models of inflammatory arthritis has provided strong evidence that osteoclasts play an important role in focal, marginal, and subchondral bone loss in inflammatory arthritis (6). Recently, the roles of receptor activator of nuclear factor κB ligand (RANKL), a central regulator of osteoclast recruitment and activation, and TNFα have been shown to be crucial in the pathogenesis of rheumatoid joint destruction (7–11).

Although no conventional medications effectively suppress such joint destruction, TNFα blockers (soluble TNFα receptor fusion protein and TNFα blocking antibody), IL-1 blockers (soluble IL-1 receptor and IL-1 receptor antagonist), and monoclonal antibodies that neutralize IL-6 have all successfully decreased the intensity of synovitis and prevented or retarded the progression of cartilage destruction, both in experimental models and in human trials (12–26). However, these protein products are limited, due to host immune response, rebound of symptoms, a short half-life, and cost (27–29).

FR167653 was first discovered as a potent inhibitor of TNFα and IL-1β production in lipopolysaccharide-stimulated human monocytes and phytohemagglutinin-M–stimulated human lymphocytes (30, 31). FR167653 inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by suppressing the phosphorylation of p38 MAPK, preferentially in the α isoform, but not in the γ isoform (32–34). In addition, p38 MAPK is involved in production of IL-6 and IL-8 induced by TNFα and IL-1β (1, 35).

Collagen-induced arthritis (CIA) is a widely used experimental model of polyarthritis that has many histopathologic features in common with RA. TNFα, IL-1β, IL-6, and IL-8 also play an important role in the pathogenesis of CIA (5, 36–39). In this study we subcutaneously injected FR167653 into rats with CIA. This study is the first to demonstrate that this p38 MAPK inhibitor effectively prevents the onset and progression of CIA in rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Materials.

FR167653 (Figure 1) was kindly provided by Fujisawa Pharmaceutical (Osaka, Japan). Lewis rats were purchased from Clea Japan (Tokyo, Japan). We used 77 rats in this study, all of which were included in 7 in vivo experiments. Bovine type II collagen was purchased from Cosmo Bio (Tokyo, Japan), and Freund's incomplete adjuvant was purchased from Difco (Detroit, MI).

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Figure 1. Chemical structure of 1-(7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo(5,1-c)(1,2,4)triazin-2-yl)-2-phenylethanedione sulfate monohydrate, or FR167653, a p38 mitogen-activated protein kinase inhibitor.

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Induction of CIA in rats.

CIA was induced using the modified method described by Trentham et al (40). Briefly, 6-week-old female Lewis rats were immunized intradermally with 0.5 mg of bovine type II collagen, which was dissolved in 0.5 ml of 0.1M acetic acid at 4°C and emulsified in 0.5 ml of cold Freund's incomplete adjuvant. On day 7, the rats received an intradermal booster injection of half the volume of the first immunization (41). Onset of arthritis in the ankle joints could usually be visually recognized between days 18 and 21.

Experimental protocol.

To investigate whether FR167653 prevents arthritis and suppresses joint destruction in vivo, we developed 2 experimental protocols. In the prophylactic treatment model, 8 rats with CIA were subcutaneously injected with 32 mg/kg of FR167653 in sterilized water (FR167653-treated rats in the prophylactic treatment model; FR167653-P rats) every day starting on the day of the collagen booster injection (day 7). Four normal rats (normal rats in the prophylactic treatment model; normal-P rats) and 6 rats with CIA (untreated CIA rats in the prophylactic treatment model; CIA-P rats) were used as controls. All rats in the prophylactic treatment model were killed on day 35.

In the therapeutic treatment model, 7 rats with CIA were subcutaneously injected with 32 mg/kg of FR167653 (FR167653-treated rats in the therapeutic model; FR167653-T rats) every day after the onset of arthritis (day 21). Seven rats with CIA (untreated CIA rats in the therapeutic model; CIA-T rats) and 7 normal rats (normal rats in the therapeutic model; normal-T rats) were used as controls. All rats in the therapeutic treatment group were killed on day 49.

Swelling in the hind paws was measured every 7 days using a plethysmometer (model TK-101 CMP; UNICOM, Chiba, Japan). The body weight of rats was also measured every 7 days. All procedures complied with the standards described in the Osaka University Medical School Guidelines for the Care and Use of Laboratory Animals.

Radiologic and histologic studies.

At the end of the experiments (day 35 and day 49), the hind paws of rats were imaged on high-speed radiographic film (Fuji Photo Film, Tokyo, Japan), using the MX-20 Specimen Radiography system (Faxitron X-ray, Wheeling, IL). Radiographic scoring criteria were assessed according to the method reported previously (42), on the following scale: 0 = no bone damage, 1 = tissue swelling and edema, 2 = joint erosion, and 3 = bone erosion and osteophyte formation. The ankle joints were fixed in 4% paraformaldehyde, decalcified with EDTA, and embedded in paraffin; 4-μm sections were prepared. The extent of arthritis was assessed according to the method reported previously (43), on sections stained with hematoxylin and eosin, using the following scale: 0 = normal synovium, 1 = synovial membrane hypertrophy and cell infiltrates, 2 = pannus and cartilage erosion, 3 = major erosion of cartilage and subchondral bone, and 4 = loss of joint integrity and ankylosis. The assessment was performed by 2 of the authors (AM and TT), who were blinded to the identity of the specimens, and the average of the 2 scores was used.

To investigate the in vivo activity of osteoclastic bone resorption, sections were stained with a tartrate-resistant acid phosphate (TRAP) staining kit (Hokudo, Sapporo, Japan). The number of osteoclasts, or TRAP-positive multinucleated cells containing 3 or more nuclei, was counted in 10 areas of each ankle (at 200× magnification).

Cytokine level in peripheral blood serum and ankle joints.

Peripheral blood samples were collected from rats in the prophylactic model (normal-P, FR167653-P, and CIA-P rats) on day 35. Ankle joints and surrounding tissue (∼500 mg, excluding skin) were isolated on day 21 and homogenized with 700 μl of phosphate buffered saline (PBS). After centrifugation, the supernatant was collected from the ankles. The levels of TNFα and IL-1β in the serum and ankle samples were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Camarillo, CA). In addition, to investigate whether a humoral response to type II collagen was altered by FR167653 treatment, we also measured serum levels of interferon-γ (IFNγ) and IL-4 using the ELISA kit (Biosource International) in the prophylactic treatment model on day 21. The cytokine concentration in serum and ankle joints was expressed in pg/ml of serum and pg/mg of ankle tissue, respectively. The detection limits of the assay were 15.6 pg/ml for TNFα, 31.2 pg/ml for IL-1β, 21.8 pg/ml for IFNγ, and 7.8 pg/ml for IL-4.

Flow cytometric analysis of bone marrow cells.

The effect of FR167653 on the T lymphocyte population was analyzed with bone marrow cells prepared from femurs of rats in the prophylactic treatment model (normal-P, FR167653-P, and CIA-P rats). Cells were collected on day 21 by flushing the bone marrow cavity with minimum essential medium, alpha modification (α-MEM; Invitrogen, Grand Island, NY). After lysing erythrocytes with a lysing buffer (10 mM Tris HCl, pH 7.4, 0.83% ammonium chloride), cells were suspended to a concentration of 106 cells/ml in PBS containing 0.1% bovine serum albumin. Then, 100 μl of this cell suspension was exposed to Fc Block (BD PharMingen, San Diego, CA) at 4°C for 5 minutes and stained with fluorescein-conjugated anti-CD4 antibody and phycoerythrin-conjugated anti-CD8a antibody (BD PharMingen) at 4°C for 30 minutes. Two-color flow cytometry was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with an argon laser at 488 nm. All specimens were analyzed on the day of collection. To exclude debris or dead cells, the cells were gated on the basis of forward and right angle scatter. Each test used 20,000 cells, and the number of positive cells was expressed as a percentage of the total cell count.

Osteoclast differentiation assay.

In vitro osteoclast differentiation induced by macrophage colony-stimulating factor (M-CSF) and soluble RANKL (sRANKL) or TNFα was analyzed using the modified method described by Takeshita et al (44) to investigate the effect of FR167653 on osteoclast formation. After elimination of erythrocytes, bone marrow cells prepared from femurs of 5-week-old female Lewis rats were seeded at 5 × 106 cells per 10-cm petri dish and cultured in α-MEM containing 10% fetal bovine serum (TRACE Scientific, Melbourne, Australia), 1% penicillin/streptomycin (Invitrogen), and 100 ng/ml recombinant human M-CSF (PeproTech EC, London, UK). After 6 days, cells were trypsinized and reseeded at 1 × 104 cells/well in 48-well culture plates in the presence or absence of 100 ng/ml M-CSF, 100 ng/ml recombinant human sRANKL (PeproTech EC), and FR167653, and cultivated for an additional 6 days. Then, cells were washed and stained with the TRAP staining kit.

An in vitro osteoclast formation assay induced by M-CSF and 100 ng/ml murine TNFα (R&D Systems, Minneapolis, MN) was also performed by replacing sRANKL with TNFα in the presence of 200 ng/ml recombinant human osteoprotegerin (Wako Pure Chemical, Osaka, Japan), an endogenous inhibitor of RANKL. Osteoclast-like TRAP-positive multinucleated cells containing 3 or more nuclei were counted. In addition, calcified matrix resorption activity of the osteoclast-like cells was tested using BD BioCoat osteologic calcium hydroxyapatite–coated slides (BD Biosciences, Bedford, MA). At the end of osteoclast formation culture, cells were removed by vigorous washing, and microphotographs (at 100× magnification) of 4 randomly selected fields were taken. The total resorption area on the photographs was calculated by using image analysis software, Mac scope version 2.51 (Mitani, Fukui, Japan).

Statistical analysis.

Statistical analysis was performed using the unpaired t test, Mann-Whitney U test, and analysis of variance with Fisher's protected least significant difference test. Analyses were carried out with STATVIEW, version 4.5 software (SAS Institute, Cary, NC). The statistical significance level was set at a P value of 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Effect of prophylactic treatment with FR167653 in CIA rats.

In the FR167653 prophylactic treatment model, CIA-P rats had maximum paw swelling (mean hind paw volume 2.22 cm3) by day 21, which gradually reduced after day 21. Most FR167653-P rats showed no paw swelling (mean 1.42 cm3) on day 21. From day 21 to day 35, the paw volume of the FR167653-P rats was significantly reduced compared with that of the CIA-P rats (P < 0.0001) (Figure 2A). The average weight of CIA-P rats on day 21 (142.8 gm) was reduced compared with that of FR167653-P rats (161.1 gm) (P = 0.0087). This difference in weight continued through day 35 (CIA-P rats 149.0 gm versus FR167653-P rats 174.1 gm; P < 0.0039) (Figure 2B).

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Figure 2. Clinical findings in rats with collagen-induced arthritis (CIA), showing the prophylactic effect (A and B) and therapeutic effect (C and D) of FR167653. A, Maximum paw swelling was observed on day 21 in untreated CIA rats (CIA-P; n = 6), but no increase in paw volume over time was found in CIA rats treated prophylactically with FR167653 (FR167653-P; n = 8). B, An apparent weight loss was seen on day 21 in CIA-P rats compared with FR167653-P rats and normal rats in the prophylactic treatment model (normal-P; n = 4). The weight loss in CIA-P rats continued through day 35. C, Paw swelling in rats treated therapeutically with FR167653 (FR167653-T; n = 7) was significantly less than in untreated CIA rats (CIA-T; n = 7). D, There was no significant weight difference between FR167653-T and CIA-T rats during the entire study period, except for a single time point that showed a marginal difference (day 35). Normal-T = normal rats in the therapeutic treatment model (n = 7). Bars show the mean ± SD. = P < 0.05; ∗∗ = P < 0.01; # = P < 0.0001, versus CIA rats without treatment.

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Radiographic examination of the hind paws of CIA rats revealed severe bone matrix resorption and erosion that suggested active arthritis and joint destruction. Histologically, CIA rats had severe infiltration of neutrophils, lymphocytes, and TRAP-positive osteoclasts, with disruption and loss of articular cartilage. In the rats prophylactically treated with FR167653 before the onset of arthritis, those radiographic and histologic findings were markedly improved (Figures 3 and 4). The mean radiographic score of 3.0 in CIA-P rats was significantly higher than the mean score of 0.3 in FR167653-P rats and 0.0 in normal-P rats (both P < 0.0001 versus CIA-P rats). The mean histologic score of 1.0 in FR167653-P rats was significantly reduced (P < 0.0001) compared with the mean score of 3.2 in CIA-P rats. The number of osteoclasts around the ankle joints was significantly fewer in FR167653-P rats (mean 20.9) than in CIA-P rats (mean 107.5) (P = 0.0002) (Table 1).

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Figure 3. Gross appearance (A,C, and E) and macroradiographs (B,D, and F) of rat hind paws in the prophylactic treatment model. Severe paw swelling, bone matrix resorption, and erosion were seen in collagen-induced arthritis (CIA) rats (A and B), suggesting the presence of active arthritis and joint destruction. When FR167653 was administered prophylactically to CIA rats, those arthritic changes were absent (C and D). Neither paw swelling nor joint damage were seen in normal rats (E and F).

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Figure 4. Histologic analysis in the prophylactic treatment model. Histologic features of ankle joints (stained with hematoxylin and eosin in A–C; stained with tartrate-resistant acid phosphate [TRAP] in D–F) on day 35 in rats in the prophylactic treatment model. A, Rats with collagen-induced arthritis (CIA) showed marked infiltration of neutrophils and lymphocytes, with disruption and loss of articular cartilage. B, FR167653 prophylactically treated CIA rats showed almost intact articular cartilage and subchondral bone. C, Normal rats showed normal articular cartilage and absence of infiltrate in the synovium. D, CIA rats showed active periarticular bone resorption with marked infiltration of TRAP-positive osteoclasts. E, TRAP-positive multinuclear cell formation was almost completely abolished in FR167653 prophylactically treated CIA rats. F, Normal rats showed few TRAP-positive multinuclear cells. (Original magnification × 40.)

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Table 1. Radiographic score, histologic score, and osteoclast number in the collagen-induced arthritis (CIA) prophylactic treatment model*
GroupNo. of jointsRadiographic scoreHistologic scoreOsteoclast number
  • *

    Except where otherwise indicated, values are the mean ± SD. FR167653-P = CIA rats treated prophylactically with FR167653; CIA-P = untreated CIA rats in the prophylactic model; normal-P = normal rats in the prophylactic model.

  • P < 0.0001 versus CIA-P rats.

  • P < 0.01 versus CIA-P rats.

  • §

    P = 0.0001 versus CIA-P rats.

FR167653-P rats160.3 ± 0.71.0 ± 0.820.9 ± 38.7
CIA-P rats123.0 ± 0.03.2 ± 0.5107.5 ± 55.0
Normal-P rats80.0 ± 0.00.0 ± 0.0§1.8 ± 2.2

Effect of therapeutic treatment with FR167653 in CIA rats.

Although the treatment in the therapeutic model was started after the onset of arthritis, FR167653-T rats had significantly reduced paw swelling (1.73 cm3 at day 28 and 1.59 cm3 at day 35) compared with CIA-T rats (2.05 cm3 at day 28 [P = 0.0015] and 1.96 cm3 at day 35 [P < 0.0001]) (Figure 2C). However, there was no statistically significant difference in weight between the FR167653-T rats and CIA-T rats, except at the day 35 time point, which showed a marginal difference (P = 0.042) (Figure 2D).

The radiographic severity of joint destruction and histologic findings of abnormalities in the ankle joints of FR167653-T rats were markedly reduced when compared with those in the CIA-T rats (Figures 5 and 6). The mean radiographic and histologic scores in CIA-T rats (2.9 and 3.3, respectively) were significantly higher than those in FR167653-T rats (1.3 and 1.2, respectively) and normal-T rats (0.0 for both) (P < 0.0001). The number of osteoclasts around the ankle joints was significantly smaller in FR167653-T rats (9.7) than in CIA-T rats (90.0) (P < 0.0001) (Table 2).

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Figure 5. Gross appearance (A,C, and E) and macroradiographs (B,D, and F) of rat hind paws in the therapeutic treatment model. Severe paw swelling, bone matrix resorption, and erosion were seen in collagen-induced arthritis (CIA) rats (A and B), suggesting the presence of active arthritis and joint destruction. When FR167653 was administered therapeutically to rats after the onset of arthritis (C and D), those arthritic changes were markedly reduced. Neither paw swelling nor joint damage were seen in normal rats (E and F).

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Figure 6. Histologic analysis in the therapeutic treatment model. Histologic features of ankle joints (stained with hematoxylin and eosin in A–C; stained with tartrate-resistant acid phosphate [TRAP] in D–F) on day 49 in rats in the therapeutic treatment model. A, Rats with collagen-induced arthritis (CIA) showed marked infiltration of neutrophils and lymphocytes, with disruption and loss of articular cartilage. B, FR167653 therapeutically treated CIA rats showed slight thinning of articular cartilage and intact subchondral bone with mild inflammatory cell infiltration into synovium. C, Normal rats showed normal articular cartilage and absence of infiltrate in the synovium. D, CIA rats showed active periarticular bone resorption with marked infiltration of TRAP-positive osteoclasts. E, TRAP-positive multinuclear cell formation was almost completely abolished in FR167653 therapeutically treated CIA rats. F, Normal rats showed few TRAP-positive multinuclear cells. (Original magnification × 40.)

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Table 2. Radiographic score, histologic score, and osteoclast number in the collagen-induced (CIA) therapeutic treatment model*
GroupNo. of jointsRadiographic scoreHistologic scoreOsteoclast number
  • *

    Except where otherwise indicated, values are the mean ± SD. FR167653-T = CIA rats treated therapeutically with FR167653; CIA-T = untreated CIA rats in the therapeutic model; normal-T = normal rats in the therapeutic model.

  • P < 0.0001 versus CIA-T rats.

  • P = 0.0001 versus CIA-T rats.

FR167653-T rats101.3 ± 0.81.2 ± 0.99.7 ± 8.9
CIA-T rats132.9 ± 0.33.3 ± 0.690.0 ± 44.5
Normal-T rats140.0 ± 0.00.0 ± 0.01.0 ± 1.8

Cytokine level in peripheral blood serum and ankle joints.

The serum concentrations of TNFα (3.16 pg/ml) (P = 0.0051) and IL-1β (not detectable) (P = 0.0041) in FR167653-P rats were significantly lower than those in CIA-P rats (TNFα 66.7 pg/ml and IL-1β 116.6 pg/ml) (Table 3). The tissue concentration of IL-1β (0.450 pg/mg) (P < 0.0001) in the ankle joints of FR167653-P rats was significantly lower than that in CIA-P rats (2.448 pg/mg). However, tissue TNFα levels were similar among the 3 groups (Table 4). IFNγ and IL-4 levels were lower than the detection limit in all groups.

Table 3. Cytokine levels in the peripheral blood serum in the CIA prophylactic model*
GroupNo. of ratsTNFα, pg/mlIL-1β, pg/ml
  • *

    Except where otherwise indicated, values are the mean ± SD. TNFα = tumor necrosis factor α; IL-1β = interleukin-1β; ND = not detectable (see Table 1 for other definitions).

  • P < 0.01 versus CIA-P rats.

  • The values under the detectable limit were treated as 0.0 for statistical analysis.

FR167653-P rats53.16 ± 7.1ND
CIA-P rats666.7 ± 25.2116.6 ± 35.8
Normal-P rats4NDND
Table 4. Cytokine levels in the ankle joints in the CIA prophylactic model*
GroupNo. of jointsTNFα, pg/mgIL-1β, pg/mg
  • *

    Except where otherwise indicated, values are the mean ± SD. See Tables 1 and 3 for definitions.

  • P < 0.0001 versus CIA-P rats.

FR167653-P rats60.056 ± 0.0120.450 ± 0.067
CIA-P rats60.060 ± 0.0132.448 ± 0.610
Normal-P rats60.052 ± 0.0450.187 ± 0.090

Effect of FR167653 on the T lymphocyte population.

The percentages of CD4+,CD8a− and CD4+,CD8a+ T cells in the bone marrow were similar among normal-P, FR167653-P, and CIA-P rats. However, the mean percentage of CD4−,CD8a+ T cells in CIA-P rats (16.7%) (Figure 7A) was significantly greater than that in FR167653-P rats (10.6%) (P = 0.0434) (Figure 7B) and normal-P rats (8.4%) (P < 0.006) (Figure 7C).

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Figure 7. T cell population of bone marrow cells on day 21. Two-color flow cytometric analysis with fluorescein-conjugated anti-CD4 antibody and phycoerythrin-conjugated anti-CD8a antibody was performed using a FACScan equipped with an argon laser at 488 nm on bone marrow cells collected from rats in the prophylactic treatment model. A, Rats with collagen-induced arthritis (CIA). B, FR167653 prophylactically treated CIA rats. C, Normal rats. The proportion of CD4+,CD8a− and CD4+,CD8a+ T cells was similar among the 3 groups of rats. However, the percentage of CD4−,CD8a+ T cells in the untreated CIA rats was significantly greater than that in the FR167653-treated rats and normal rats. Data are the mean ± SD (n = 5).

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Inhibition of osteoclast differentiation and maturation of bone marrow cells induced by M-CSF and sRANKL or TNFα, following treatment with FR167653.

In vitro TRAP-positive multinucleated cell formation induced by sRANKL was almost completely abolished by FR167653 (P < 0.0001 for all concentrations of FR167653 compared with no FR167653) (Figures 8A and B). FR167653 also inhibited the osteoclast-like cell differentiation induced by TNFα independent of the RANKL/RANK pathway, in a dose-dependent manner (P < 0.0001 for all concentrations of FR167653 compared with no FR167653) (Figures 8C and D). Calcified matrix resorption by sRANKL-induced osteoclast-like cells was also inhibited by FR167653 (P = 0.0433) (Figure 9).

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Figure 8. Effect of FR167653 on osteoclast differentiation induced in vitro by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor κB ligand (sRANKL) or tumor necrosis factor α (TNFα). Rat bone marrow cells were incubated for 6 days in the presence or absence of M-CSF (100 ng/ml), sRANKL (100 ng/ml), and FR167653 (10−8M, 10−7M, or 10−6M) (A and B) or M-CSF (100 ng/ml), TNFα (100 ng/ml), osteoprotegerin (OPG) (200 ng/ml), and FR167653 (10−8M, 10−7M, or 10−6M) (C and D). After incubation, cells were stained with the tartrate-resistant acid phosphatase (TRAP) staining kit. Representative microphotographs of TRAP staining induced by M-CSF and sRANKL (A) and by M-CSF, OPG, and TNFα (C) are shown (original magnification × 40). FR[−] = without FR167653; FR[10−6] = with 10−6M FR167653. The mean and SD number of TRAP-positive multinuclear cells (MNC) containing 3 or more nuclei is also shown (B and D) (n = 6). # = P < 0.0001 versus the M-CSF and sRANKL group in B and versus the M-CSF, OPG, and TNFα group in D.

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Figure 9. Calcified matrix resorption by osteoclast-like cells, induced by soluble receptor activator of nuclear factor κB ligand (sRANKL). Calcified matrix resorption activity of the osteoclast-like cells was tested using BD BioCoat osteologic calcium hydroxyapatite–coated slides. Rat bone marrow cells were incubated with macrophage colony-stimulating factor (M-CSF; 100 ng/ml) and sRANKL (100 ng/ml) in the presence or absence of FR167653 (10−6M). Bars show the mean and SD (n = 4) total resorption area on 4 randomly selected microscopic fields (100× magnification), calculated using an image analysis system. = P < 0.05 versus the M-CSF and sRANKL group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Because p38 MAPK regulates inflammatory cytokines such as TNFα and IL-1β, several researchers have used inhibitors of p38 MAPK in inflammatory disease models in vivo, including adjuvant-induced arthritis, and their results have confirmed the effectiveness of these inhibitors (30–34, 45–49). However, the role of p38 MAPK in the CIA model has not been previously investigated. The current study is the first to elucidate the effects of FR167653, a potent p38 MAPK inhibitor, which completely prevented the onset of CIA and also markedly improved the symptoms of inflammatory changes even after the onset of arthritis, with significant reductions in radiographic and histologic degrees of joint injury.

By what mechanism does FR167653 protect the joint against inflammatory injury? There is evidence that the proinflammatory cytokines TNFα and IL-1β help to propagate the extension of a local or systemic inflammatory process. We confirmed that the inflammatory process in the untreated CIA rats led to substantial increases in the serum levels of TNFα and IL-1β and in the ankle-joint concentration of IL-1β. The serum and ankle-joint concentrations of these proinflammatory cytokines were significantly lower in the rats treated prophylactically with FR167653, suggesting that FR167653 inhibits the polyarticular inflammation process and joint destruction by inhibiting the production of TNFα and IL-1β.

The involvement of CD8+ T cells in autoimmune disease is multifaceted. CD8+ T cells and major histocompatibility complex class I participate in disease onset in murine and rat autoimmune disease models (50–55). CD8+ T cells may play an important role in initiating CIA (56), but their role is not fully understood (57, 58). In the present study, we found a significant increase of CD4−,CD8a+ T cells in the local bone marrow of untreated CIA rats when compared with FR167653-treated rats and normal rats in the prophylactic treatment model. These findings suggest that CD4−,CD8+ T cells play an important role in initiating CIA, and that FR167653 may possibly inhibit the accumulation of inflammation initiators, such as CD4−,CD8a+ T cells, in local bone marrow.

Since macrophage inflammatory protein 1α (MIP-1α) and monocyte chemoattractant protein 1 (MCP-1), which are chemokines that may be responsible for CD8a+ T cell and macrophage infiltration (59, 60), are regulated by cytokines such as IL-1α, IL-1β, and TNFα (61), these chemokines may be involved in the mechanism of CD4−,CD8a+ cell accumulation in bone marrow. Therefore, FR167653 may possibly inhibit cytokines via the inhibition of IL-1β and/or TNFα, result-ing in inhibition of inflammatory cell infiltration. How-ever, in our immunohistochemical study, the difference in local expression of MIP-1α and MCP-1 in the periarticular region was marginal between the groups (data not shown).

TNFα and IL-1β are potent inducers of osteoclastic bone resorption. Accordingly, FR167653 inhibition of TNFα and IL-1β production may suppress the periarticular osteolysis in RA. It was recently reported that activation of the p38 MAPK pathway plays an important role in RANKL-induced and TNF-mediated osteoclast differentiation of mouse bone marrow cells (62, 63). In the present study, we found that the number of osteoclastic TRAP-positive multinuclear cells in both the prophylactic and therapeutic FR167653-treated rats was significantly smaller than that in their respective untreated CIA groups. Furthermore, in the rat bone marrow culture assay, an FR167653 concentration of 10−6M almost completely inhibited the differentiation and maturation of osteoclast-like cells induced by both sRANKL and TNFα. This concentration is equivalent to the serum concentration in rats 8 hours after receiving 32 mg/kg daily by subcutaneous injection (Fujisawa Pharmaceutical: unpublished data). Thus, FR167653 multilaterally inhibits joint destruction by suppressing joint inflammation, reducing serum osteoclastic cytokine levels, and directly inhibiting osteoclast formation and maturation.

In addition to FR167653, other inhibitors of p38 MAPK, SB203580 and SB242235, have been used in several inflammatory disease models, and obvious adverse events were not seen (30–34, 45–49). In our in vivo study, we found no adverse events caused by 32 mg/kg/day of FR167653, a dosage that was used in another inflammation model and found to be safe and effective (64). Although most of the in vivo investigations found no adverse effects, one study demonstrated increased plasma creatine levels and lactate dehydrogenase levels in rats (65). The toxicity of FR167653 should be studied extensively.

In conclusion, FR167653, a potent p38 MAPK inhibitor, not only prevents the onset of arthritis by prophylactic treatment, but also suppresses the progression of joint destruction by therapeutic treatment of rats with CIA. FR167653 appears to protect the joints from inflammation injury through the inhibition of TNFα and IL-1β production, recruitment of CD4−,CD8a+ T cells, and osteoclastic bone resorption. These findings suggest that p38 MAPK is a potential therapeutic target for RA.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
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