Amelioration of experimental arthritis by a calpain-inhibitory compound: regulation of cytokine production by E-64-d in vivo and in vitro
Hajime Yoshifuji1,
Hisanori Umehara2,
Hidenori Maruyama3,
Mari Itoh3,
Masao Tanaka1,
Daisuke Kawabata1,
Takao Fujii1 and
Tsuneyo Mimori1
1 Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
2 Division of Hematology and Immunology, Department of Internal Medicine, Kanazawa Medical University, Ishikawa, Japan
3 Discovery Pharmacology I Group, Pharmacology and Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd, Osaka, Japan
Correspondence to: H. Yoshifuji; E-mail: yoshifuj{at}kuhp.kyoto-u.ac.jp
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Abstract
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Calpain, a calcium-dependent cysteine proteinase, has been reported to participate in the pathophysiology of rheumatoid arthritis (RA). The aim of this study is to investigate the therapeutic efficacy of calpain-inhibitory compounds in an animal model of RA and to clarify the underlying mechanisms in vivo and in vitro. Arthritis was induced in BALB/c mice with anti-type II collagen mAbs and LPS, and the mice were treated intra-peritoneally with a high dose (9 mg kg1 per day) or low dose (3 mg kg1 per day) of E-64-d (a membrane-permeable cysteine proteinase inhibitor) or control diluent. As a result, a high dose of E-64-d significantly alleviated the clinical arthritis and the histopathological findings, compared with the control diluent, although a low dose of E-64-d did not have a significant effect. Next, we evaluated the effects of E-64-d on cytokine mRNA expression at the inflamed joints by quantitative reverse transcriptionPCR. High dose of E-64-d significantly decreased IL-6 and IL-1ß mRNA levels at the inflamed joints. The regulatory effects of E-64-d on cytokine production were also confirmed in vitro, using a synovial cell line (E11) and crude synoviocytes derived from RA patients. These results suggest the key roles of calpain in the pathophysiology of arthritis and that calpain-inhibitory compounds might be applicable to the treatment of arthritic diseases such as RA.
Keywords: anti-type II collagen antibody-induced arthritis, calpastatin, IL-6, rheumatoid arthritis
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Introduction
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Rheumatoid arthritis (RA) is a chronic refractory disease, whose main symptom is polyarthritis characterized by autonomous synovial proliferation and osteocartilaginous destruction. Although the pathogenesis of RA remains to be elucidated, studies have indicated the contribution of proteinases, cytokines and auto-antibodies to the progression of the disease. First, over-expression of proteinases such as calpains (13), cathepsins (4) and matrix metalloproteinases (MMPs) (5) at the disease-affected joints has been reported and assumed to contribute to the osteocartilaginous destruction. Second, cytokines are now widely recognized to play major roles in RA, since an increasing number of favorable outcomes has been reported in trials to treat RA with biological agents against tumor necrosis factor-
(TNF-
) (6), IL-1 and IL-6. Third, the involvement of various auto-antibodies such as antibodies to citrullinated proteins (7), glucose-6-phosphate isomerase (8) and follistatin-related protein (FRP) (911) has been reported in the pathophysiology of RA. For example, we have reported that anti-FRP antibodies were detected in synovial fluids of RA patients (9), and demonstrated that recombinant FRP inhibited the production of MMP-1 and MMP-3 in cultured synoviocytes (10) and ameliorated an animal model of RA (11).
We and another group have identified a new auto-antibody against calpastatin (12, 13), which was detected significantly more frequently in RA patients than in patients with other rheumatic diseases or in healthy controls (12, 14, 15). Recently, it was reported that sensitivity and specificity of anti-calpastatin antibodies were 83 and 96% for RA, respectively (14). The positivity of anti-calpastatin antibodies was correlated with serological markers of the disease activity or a recent-onset subset in RA patients (1517).
Calpastatin, an endogenous inhibitor of calpain, is a ubiquitous 70-kDa protein, comprising five domains, L, I, II, III and IV (18). Calpain, a Ca2+-dependent cysteine proteinase, is a ubiquitous enzyme that has two subtypes, µ- and m-calpain, both of which have two subunits of 80 and 28 kDa (18). Calpain exhibits proteinase activity toward a wide range of substrates, such as cytoskeletal proteins, kinases, phosphatases, membrane-associated proteins and transcription factors (18). Since it cleaves the substrates at a limited number of specific sites for their activation or inactivation, calpain is regarded as a biomodulator rather than a digestive enzyme (19). For example, calpain activates IL-1
or protein kinase C by cleaving it (20, 21). Through the modulation of these substrates, calpain is supposed to contribute to the pathophysiology of RA. It is worth mentioning that calpain is more highly expressed in the synovial membranes of RA patients than in those of healthy controls (2), and similarly, calpain is detected in the synovial membranes of mice with collagen (CL)-induced arthritis, while not detected in those of non-manipulated mice (22).
E-64-d (synonyms: loxistatin, EST and Ep453), a membrane-permeable cysteine proteinase inhibitor, has been used in order to inhibit calpain in vivo and in vitro (23, 24). In the present study, we examined the therapeutic efficacy of two calpain-inhibitory compounds, E-64-d and recombinant calpastatin (rCS), in mice with anti-type II CL antibody-induced arthritis. We also analyzed the underlying mechanisms in vivo and in vitro, using the mice with mAb-induced arthritis and a fibroblast-like synovial cell line (E11) or crude synoviocytes of a RA patient.
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Methods
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Reagents
E-64 [molecular weight (MW) 375.4, non-membrane permeable] and E-64-d (MW 342.4, membrane permeable) were purchased from Amresco (Solon, OH, USA) and Peptide Institute (Osaka, Japan), respectively. Escherichia coli-expressed recombinant human calpastatin (14 kDa, identical to domain I) was from Takara Shuzo (Shiga, Japan) and filtrated through an endotoxin cut filter (Zetapore-dispo 020SP, Cuno, Meriden, CT, USA) before use. Phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM) were from Wako Chemicals (Richmond, VA, USA) and EMD Biosciences (La Jolla, CA, USA), respectively.
Induction and treatment of murine arthritis
Institutional bioethical approval (no. 02004, Kyoto University Graduate School of Medicine) was acquired prior to the animal experiments. Arthritis was induced in 7-week-old female BALB/c mice (Japan SLC, Hamamatsu, Japan) with a mixture of anti-type II CL mAbs (Arthrogen-CIA mAb, Chondrex, Redmond, WA, USA) and LPS, according to the method of Terato et al. (25). Briefly, 2 mg per body of mAb mixture was injected intra-peritoneally (i.p.) into each mouse (day 2), and 48 h later, 50 µg per body of LPS was injected i.p. in order to enhance the arthritis (day 0). To prevent the development of the disease, a high dose (9 mg kg1 per day) or low dose (3 mg kg1 per day) of E-64-d, rCS (9 mg kg1 per day) or control diluent (sterilized saline containing 10% dimethylsulfoxide) was administered i.p. to the disease-affected mice (N = 8, 6, 9 and 9, respectively) for 10 days from the day before the injection of mAbs (days 3 to 6).
Clinical and histopathological assessment of murine arthritis
The severity of the arthritis was evaluated macroscopically for every paw with a scoring system as follows: 0 = intact, 1 = mild swelling, 2 = severe swelling and 3 = deformed or ankylosed (11), although no deformity or ankylosis was observed in the present study. The sum of the four paws gave a clinical arthritis score, ranging 012. The assessments were performed in a blind-test fashion. For histopathological evaluation, the mice were sacrificed on the 19th day after the LPS injection. The right hind paw was resected from each body, fixed with 10% formaldehyde, decalcified in 10% EDTA, embedded in paraffin and stained with hematoxylin and eosin. The histopathological severity was evaluated for the findings of cell infiltration, pannus formation and bone erosion with a scoring system as follows: 0 = no signs, 1 = slight, 2 = moderate and 3 = remarkable. The sum of the three items gave a histopathological arthritis score, ranging 09.
Quantification of cytokine mRNA levels at inflamed joints
To analyze the time course of cytokine mRNA expression, the arthritis was induced in 40 BALB/c mice, and 5 mice each were sacrificed 0, 2, 4, 8, 24, 48, 72 or 96 h after the LPS injection. To examine the effects of E-64-d on the cytokine mRNA expression, 32 BALB/c mice affected by the arthritis were treated i.p. with a high dose (9 mg kg1 per day) of E-64-d or control diluent from the day before the injection of mAbs (day 3), until they were sacrificed at the 4th or 48th h after the LPS injection (N = 8; given the same treatment and sacrificed at a time). Then the four paws of each mouse were resected from the body and homogenized altogether in 6 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was purified with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and the cDNA was synthesized by random hexamer priming with the TaqMan reverse transcription (RT) reagents (Applied Biosystems, Foster City, CA, USA). The amount of cytokine mRNA was determined with a quantitative real-time RTPCR system (ABI PRISM 7700 Sequence Detection System, Applied Biosystems). Briefly, the cDNA was amplified with the primers and probes for the targeted cytokine (IL-1
, IL-1ß, IL-6 or TNF-
) mRNA and control 18S rRNA, which were available commercially (Applied Biosystems). The data were analyzed by the Sequence Detector software (Applied Biosystems). Ct values (cycle numbers to the threshold) for each of the targeted mRNA were normalized by subtracting Ct values for 18S rRNA (
Ct), i.e. the quantities of the targeted mRNA were normalized by the quantities of control 18S rRNA. The normalized mRNA quantities (proportional to 2
Ct) were compared among the experimental groups (
·
Ct method). We fixed the mRNA quantities at the fourth hour after the LPS injection in the control group as 100%, and relative mRNA quantities were calculated.
Cell line and synoviocytes
E11, a fibroblast-like synovial cell line established from knee joint tissues of a RA patient (26), was kindly provided by Y. Tanaka (University of Occupational and Environmental Health, Kitakyushu, Japan). Crude synoviocytes were obtained from joint tissues of a RA patient who fulfilled the American College of Rheumatology criteria, as described elsewhere (9). Briefly, the synovial membrane was cut into small pieces and digested with 0.25% collagenase (type S-1, Nitta Gelatin, Osaka, Japan). E11 cells and the crude synoviocytes were cultured in RPMI 1640 medium containing 10% FCS, 100 IU ml1 penicillin G and 100 µg ml1 streptomycin at 37°C in a 5% CO2 incubator. The crude synoviocytes were subjected to the assays on the third passage. The following in vitro assays were triplicated.
Cytokine production assay
E11 cells (1 x 104 per 200 µl per well) were seeded in a 96-well microplate and incubated for 12 h. After the supernatants were exchanged for new medium, the cells were treated with various concentrations of E-64-d, E-64, rCS or control (2% dimethylsulfoxide) for 24 h in the presence or absence of 10 ng ml1 PMA plus 750 ng ml1 IM. Then, the supernatant cytokines were quantified by ELISA (Cytoscreen ELISA Kit, Biosource, Camarillo, CA, USA). To quantify the intracellular cytokines, E11 cells (1 x 105 ml1 per well) were seeded in a 24-well microplate, incubated for 12 h and treated with each of the calpain inhibitors for 24 h. Then, the supernatants were removed, and the cells were lysed with 500 µl of a solution containing 0.1% Triton X-100, 20 mM HEPESNaOH (pH 7.9), 300 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM phenylmethylsulfonylfluoride and 0.5 mM dithiothreitol. After centrifugation at 6000 x g for 5 min, the supernatant cytokines were quantified by ELISA.
Cell proliferation assay
A colorimetric cell viability assay was performed with a water-soluble tetrazolium salt-1 (WST-1) solution (Cell Counting Kits, Dojindo Laboratories, Kumamoto, Japan). Briefly, E11 cells (1 x 104 per 200 µl per well) were seeded in a 96-well microplate, incubated for 12 h and treated with each of the calpain inhibitors or 2.5% sodium azide for 20 h. Then, the WST-1 solution (10 µl per well) was added, and the culture was continued for an additional 4 h. The number of viable cells was estimated by measuring the optical density at 450 nm (corrected by subtracting the optical density at 600 nm) with a microplate reader (model 550, Bio-Rad, Hercules, CA, USA).
Statistical analysis
Cell counts, mRNA levels and cytokine concentrations were compared among the experimental groups by the unpaired Student's t-test, whereas clinical and histopathological arthritis scores were compared by the MannWhitney U-test. P-values <0.05 were considered significant.
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Results
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Efficacy of calpain inhibitors in murine arthritis
To examine the therapeutic effects of calpain inhibitors on arthritis in vivo, experimental arthritis was induced with anti-type II CL mAbs and LPS (25) in the BALB/c strain, which is highly susceptible to this system (11, 27). Similar to a previous report (11), signs of arthritis emerged around 48 h after the LPS injection, peaked at around the eightth day (Fig. 1D) and remitted gradually (Fig. 1A). To prevent the development of the disease, a high dose or low dose of E-64-d or rCS was administered. In the macroscopic examination, the high-dose E-64-d group exhibited significantly lower clinical arthritis scores than the untreated group, while the low-dose E-64-d group and the rCS-treated group showed no improvements (Fig. 1A). The paws of the mice treated with a high dose of E-64-d were less affected than those of the untreated mice (Fig. 1D and E). In the microscopic evaluation, the joints of the mice treated with a high dose of E-64-d showed lighter findings than those of the untreated mice, which exhibited typical arthritic findings such as inflammatory cell infiltration, pannus formation and bone erosion (Fig. 2C and D). The histopathological score for cell infiltration and the total histopathological arthritis score in the high-dose E-64-d group were significantly lower than those in the untreated group (Fig. 2A). E-64-d was considered to be safe since there were no significant differences in macroscopic findings or body weight between the high-dose E-64-d group and the untreated group, although both groups of mice lost weight transiently after the LPS injection (Fig. 1B).

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Fig. 1. Effects of calpain inhibitors on mice with anti-type II CL antibody-induced arthritis. Arthritis was induced in 7-week-old BALB/c mice with anti-type II CL mAbs (day 2) and LPS (day 0). To alleviate the arthritis, a high dose (9 mg kg1 per day) or low dose (3 mg kg1 per day) of E-64-d, rCS (9 mg kg 1 per day) or control diluent was administered for 10 days (days 3 to 6). (A and B) Time courses of the clinical arthritis scores and body weight of the mice, which were treated or not treated with calpain inhibitors. SEMs are indicated for the untreated group and the high-dose E-64-d group. *P < 0.05 between the untreated group and the high-dose E-64-d group. (CE) Representative macroscopic images of the hind paws photographed on day 8. (C) A non-manipulated mouse. (D) A disease-affected mouse, whose hind paws are swollen severely. (E) A disease-affected mouse that was treated with a high dose of E-64-d, whose hind paws are less affected than in (D).
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Fig. 2. Effects of calpain inhibitors on histopathological findings of the inflamed joints. The hind paws were sampled on the 19th day after the LPS injection, and hematoxylin- and eosin-stained specimens were prepared. (A) Histopathological arthritis scores are compared between the untreated group and the high-dose (9 mg kg1 per day) E-64-d group. (BD) Representative microscopic images of the joints. (B) A non-manipulated mouse. (C) A disease-affected mouse, exhibiting typical arthritic findings such as bone erosion (filled arrow), pannus formation (opened arrow), inflammatory cell infiltration (thick arrows) and synovial proliferation (thin arrows). (D) A disease-affected mouse that was treated with a high dose of E-64-d, exhibiting lighter findings than in (C).
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Effects of E-64-d on cytokine expression in vivo
We examined the time courses of IL-1
, IL-1ß, IL-6 and TNF-
mRNA expression at the inflamed joints of the disease-affected mice by quantitative RTPCR. The IL-6 and IL-1ß mRNA levels showed prominent surges around 4 h after the LPS injection, then dropped and gradually increased again from around the 48th to the 72nd h (Fig. 3A and B, solid lines). When we examined the effects of LPS alone, the second peaks were not observed (Fig. 3A and B, broken lines). Compared with the IL-6 and IL-1ß mRNA, the TNF-
and IL-1
mRNA levels showed different patterns, increasing around the fourth hour and remaining elevated for
24 h, and then gradually decreasing (Fig. 3C and D). Next, we administered E-64-d to the mice and examined its effects on the cytokine mRNA expression. At the fourth hour, none of the cytokine mRNA levels was affected by E-64-d (Fig. 4). At the 48th h, the IL-6 and IL-1ß mRNA levels were significantly decreased by E-64-d, compared with those of the untreated mice (Fig. 4A and B).

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Fig. 3. Time courses of cytokine mRNA expression at the inflamed joints of mice with anti-type II CL antibody-induced arthritis. The arthritis was induced in 40 BALB/c mice, and 5 mice each were sacrificed 0, 2, 4, 8, 24, 48, 72 or 96 h after the LPS injection (solid lines). Other 40 mice were treated with LPS alone, and sacrificed similarly (broken lines). The mRNA levels of IL-6 (A), IL-1ß (B), TNF- (C) and IL-1 (D) at the joints were determined by quantitative RTPCR. The quantities of the targeted mRNA were normalized by the quantities of control 18S rRNA. The normalized mRNA quantities were compared among the experimental groups. We fixed the mRNA quantities at the fourth hour in the mAbs + LPS group as 100%, and relative mRNA quantities were indicated. Values are means ± SEM.
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Effects of calpain inhibitors on cytokine production in vitro
To examine the effects of the calpain inhibitors in vitro, we used a fibroblast-like synovial cell line (E11) established from knee joint tissues of a RA patient (26). After E11 cells were treated with E-64-d, E-64 or rCS, the supernatant IL-6 was quantified by ELISA. We found that E11 cells spontaneously secreted considerable levels of IL-6 and that E-64-d significantly inhibited the production of IL-6 in a dose-dependent manner (Fig. 5A and B). The significantly effective concentration of E-64-d was 1030 µg ml1, consistent with those of other reports (24, 28). The production of IL-6 in E11 cells was markedly enhanced by PMA plus IM, and E-64-d also decreased the production of IL-6 on stimulation with PMA plus IM (Fig. 5C and D). In contrast to E-64-d, E-64 and rCS showed only marginal effects (Fig. 5AC). Although the MW of rCS (14 kDa) is much larger than that of E-64 (375.4 kDa) or E-64-d (342.4 kDa), even a high dose (4000 µg ml1) of rCS, equivalent to 100 µg ml1 of E-64-d in molar concentration, did not affect the production of IL-6 in E11 cells (supernatant IL-6 levels were 432 ± 78 and 403 ± 37 pg ml1 in the high-dose rCS-treated and the untreated E11 cells, respectively). Next, since IL-1
, IL-1ß and TNF-
could not be detected in the supernatants of E11 cells, we examined intracellular levels of the cytokines by ELISA. Among the three cytokines, IL-1
(mature form) could be detected in E11 cells. E-64-d decreased the production of IL-1
in E11 cells significantly in a dose-dependent manner (Fig. 6B), whereas E-64 and rCS did not show significant effects (Fig. 6A). Suppression of the production of IL-1
by E-64-d was also observed on stimulation with PMA plus IM (Fig. 6C and D).

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Fig. 5. Effects of calpain inhibitors on IL-6 production in E11 cells. After E11 cells were incubated with the indicated calpain inhibitors for 24 h in the presence (C and D) or absence (A and B) of 10 ng ml1 PMA plus 750 ng ml1 IM, the supernatant IL-6 was quantified by ELISA. (E and F) Effects of calpain inhibitors on viability of E11 cells. After E11 cells were cultured with the indicated calpain inhibitors for 24 h, a colorimetric cell viability assay was performed with WST-1 solution. Relative cell counts among the groups are indicated. In (A, C and E), concentrations of E-64-d, E-64 and rCS were 100 µg ml1. (B, D and F) Analysis of dose dependency. Values are means ± SEM. *P < 0.05, **P < 0.01 versus untreated, ***P < 0.01 versus PMA + IM treated.
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Since the inhibitory effects of E-64-d on cytokine production might have been due to its direct cytotoxicity, we examined the effects of E-64-d on the viability of E11 cells by a colorimetric WST-1 assay (29). As shown in Fig. 5(E and F), the three calpain inhibitors had neither cytotoxic nor proliferative effects on E11 cells. Finally, we examined the effects of the calpain inhibitors using crude synoviocytes, which were obtained from joint tissues of a disease-active RA patient. Consistent with the results for E11 cells, the crude RA synoviocytes spontaneously secreted IL-6, which was significantly decreased by E-64-d in a dose-dependent manner, while E-64 and rCS did not show significant effects (Fig. 7).

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Fig. 7. Effects of calpain inhibitors on IL-6 production in crude RA synoviocytes. Synoviocytes obtained from a disease-active RA patient were incubated with the indicated calpain inhibitors for 24 h, and then the supernatant IL-6 was quantified by ELISA. In (A), concentrations of E-64-d, E-64 and rCS were 100 µg ml1. (B) Analysis of dose dependency. Values are means ± SEM. **P < 0.01 versus untreated.
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Discussion
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We and another group have found a new auto-antibody against calpastatin in RA patients (12, 13). Calpain, the target molecule of calpastatin, is a Ca2+-dependent cysteine proteinase that cleaves a broad range of substrates (18), and is considered to participate in the pathogenesis of RA through the modulation of its substrates. First, calpain converts pro-IL-1
into its mature form (20). Second, it degrades inhibitor
B (I
B), leading to the activation of nuclear factor-
B (NF-
B) (30, 31). Third, it activates neutrophils and promotes granular exocytosis (21, 32).
To clarify the role of calpain in the pathophysiology of RA, we treated an animal model of RA with calpain-inhibitory compounds and examined their biological effects. We found that the administration of a high dose of E-64-d significantly ameliorated the clinical arthritis and the histopathological findings in murine mAb-induced arthritis (Figs 1 and 2). Cuzzocrea et al. have previously demonstrated that CL-induced arthritis was successfully treated with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal) in rats and that the administration of calpain inhibitor I reduced the levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the inflamed joints (33). Since calpain can activate NF-
B through the degradation of I
B (30, 31), the therapeutic efficacy of calpain inhibitor I was assumed to be brought about by the inhibition of iNOS and COX-2, whose gene expression is induced by NF-
B (34). The contribution of calpain to the activation of NF-
B has been clearly demonstrated by the work of Chen et al., in which the transduction of the calpastatin gene into a macrophage-like cell line inhibited the degradation of I
B and the activation of NF-
B in the cells (31).
IL-6 is recognized as an important factor in the pathophysiology of RA since it enhances the proliferation and differentiation of T and B cells, and IL-6 levels in synovial fluids correlate with the disease activity of RA patients (35). However, the relationship between calpain and cytokines such as IL-6 has not been well described. In the present study, we demonstrated that the expression of IL-6 mRNA at the inflamed joints of mice was decreased by the administration of E-64-d (Fig. 4A) and that the production of IL-6 in cultured synoviocytes was inhibited by E-64-d (Figs 5 and 7). Since the gene expression of IL-6 can be enhanced by NF-
B (36, 37), one of the reasons why E-64-d inhibited IL-6 might be its negative effect on NF-
B. However, Kagari et al. have reported that IL-6 was not as responsible for the progression of this experimental arthritis as TNF-
and IL-1ß were (27). There may be other mechanisms by which E-64-d ameliorated the arthritis.
In the present study, we employed anti-type II CL antibody-induced arthritis, a kind of antibody-transfer model of autoimmune diseases, since the reproducibility of the disease is high (25) and cytokine levels change sharply in this model (27). In our analysis of the time course of cytokine mRNA expression at the affected joints (Fig. 3), the IL-6 and IL-1ß mRNA levels showed elevations twice at around 4 and 4872 h after the LPS injection (Fig. 3A and B, solid lines), whereas the TNF-
and IL-1
mRNA levels showed early peaks and declined gradually (Fig. 3C and D). When we examined the effects of LPS alone, the second peaks of the IL-6 and IL-1ß mRNA levels were not observed (Fig. 3A and B, broken lines). These results suggest that the peaks at the fourth hour in all the cytokines, which were not affected by E-64-d (Fig. 4), are related to the direct effects of LPS, and the second elevations of IL-6 and IL-1ß mRNA levels around the 48th72nd h, which were significantly decreased by E-64-d (Fig. 4A and B), are associated with the local immunologic reactions induced by anti-type II CL mAbs. The results also imply that calpain may be responsible for the antibody-induced chronic local inflammatory reactions, but not for the LPS-induced acute systemic inflammation.
IL-1, which stimulates the degradation of cartilage matrices (28), is also an important mediator of RA. Interestingly, calpain is crucial for the activation of IL-1
since it processes pro-IL-1
into its mature form (20). This is confirmed by Fig. 6, which shows that E-64-d inhibited the production of mature IL-1
in E11 cells. Therefore, one of the mechanisms by which E-64-d ameliorated murine mAb-induced arthritis might be the suppression of conversion of IL-1
. Although we could not observe the negative effects of E-64-d on IL-1
mRNA expression in vivo (Fig. 4D), it is not inconsistent because the conversion of IL-1
occurs downstream of the mRNA transcription.
In the histopathological evaluation, the score for cell infiltration was significantly decreased by the administration of E-64-d (Fig. 2A). Calpain contributes to the migration of fibroblasts through the degradation of cytoskeletal proteins such as talin (38). Calpain is also associated with the spreading and polarization of neutrophils by regulating Rho-family GTPases (39). The chemotactic migration of cultured fibroblasts was inhibited by E-64-d (40), whereas the chemotactic movement of neutrophils was enhanced by calpain inhibitor I (39). Although the two results seem to be opposite, they at least make sure that calpain plays important roles in cellular migration. Taken together, these mechanisms can be applied to the ameliorative effects of E-64-d on murine mAb-induced arthritis.
Since an excessive amount of calpain is produced in synovial fluids of RA patients (1, 2) and calpain can degrade cartilage matrices including proteoglycans (41), the contribution of calpain to the pathogenesis of RA has been assumed to occur in the extracellular space. However, we found that non-membrane-permeable E-64 and rCS did not have significant effects on cytokine production in vivo and in vitro, compared with lipophilic E-64-d (Figs 1A, 57
). These results suggest that the intracellular calpain is more strongly related to the pathophysiology of RA than the extracellular calpain. We have previously described that anti-calpastatin antibodies of RA patients were able to block the physiological function of calpastatin in vitro (12). Theoretically, the blockade of calpastatin by the auto-antibodies results in the activation of calpain, leading to the above-mentioned induction of various inflammatory mediators. Although the mechanism by which the auto-antibodies can interfere with the intracellular calpaincalpastatin system remains unknown, Menard and el-Amine have pointed out the existence of extracellular calpastatin in RA patients, and speculated that the auto-antibodies can bind to the extracellular calpastatin and indirectly affect the inner balance of calpain and calpastatin (19).
E-64-d has been used in order to inhibit calpain in vivo and in vitro. Ray et al. applied E-64-d for the treatment of spinal cord injury in rats, demonstrating that 1 mg kg1 of intravenous E-64-d inhibited calpain and alleviated neuronal apoptosis at the injured spinal cord (23). Tram et al. found that parathormone-induced contraction of cultured osteoblasts was inhibited by E-64-d but not by non-membrane-permeable E-64-c (24). Since E-64-d and calpain inhibitor I can inhibit not only calpain but also cathepsin (42, 43), it cannot be excluded that the effects of the two compounds on the animal models of arthritis might also be due to the inhibition of cathepsin. To target calpain more selectively, a trial is ongoing to treat cultured synoviocytes or an animal model of RA by transfection of the calpastatin gene.
In conclusion, we demonstrated the ameliorative effects of E-64-d, a calpain-inhibitory compound, on an animal model of RA, and presented new evidence for the negative potentiality of E-64-d on cytokine production. These results suggest that calpain contributes to the pathophysiology of arthritis and that the regulation of the calpaincalpastatin system may be a new strategy to treat arthritic diseases such as RA.
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Acknowledgements
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The study was supported by a grant-in-aid (16390287 for TM) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. E11 cells and crude RA synoviocytes were generously donated by Yoshiya Tanaka (University of Occupational and Environmental Health) and Takashi Nakamura (Department of Orthopedic Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan), respectively. We also thank to Shio Kobayashi for assistance in the blind evaluation of mice.
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Abbreviations
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CL | collagen |
COX-2 | cyclooxygenase-2 |
Ct values | cycle numbers to the threshold |
FRP | follistatin-related protein |
I B | inhibitor B |
IM | ionomycin |
iNOS | inducible nitric oxide synthase |
i.p. | intra-peritoneally |
MMP | matrix metalloproteinase |
MW | molecular weight |
NF- B | nuclear factor- B |
PMA | phorbol 12-myristate 13-acetate |
RA | rheumatoid arthritis |
rCS | recombinant calpastatin |
RT | reverse transcription |
TNF- | tumor necrosis factor- |
WST-1 | water-soluble tetrazolium salt-1 |
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Notes
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Transmitting editor: M. Miyasaka
Received 5 February 2005,
accepted 20 July 2005.
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References
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- Fukui, I., Tanaka, K. and Murachi, T. 1989. Extracellular appearance of calpain and calpastatin in the synovial fluid of the knee joint. Biochem. Biophys. Res. Commun. 162:559.[CrossRef][ISI][Medline]
- Yamamoto, S., Shimizu, K., Suzuki, K., Nakagawa, Y. and Yamamuro, T. 1992. Calcium-dependent cysteine proteinase (calpain) in human arthritic synovial joints. Arthritis Rheum. 35:1309.[ISI][Medline]
- Yamamoto, S., Shimizu, K., Niibayashi, H., Yasuda, T. and Yamamuro, T. 1994. Immunocytochemical demonstration of calpain in synovial cells in human arthritic synovial joints. Biomed. Res. 15:77.[ISI]
- Hashimoto, Y., Kakegawa, H., Narita, Y. et al. 2001. Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis. Biochem. Biophys. Res. Commun. 283:334.[CrossRef][ISI][Medline]
- Yoshihara, Y., Nakamura, H., Obata, K. et al. 2000. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Ann. Rheum. Dis. 59:455.[Abstract/Free Full Text]
- Maini, R. N., Breedveld, F. C., Kalden, J. R. et al. 2004. Sustained improvement over two years in physical function, structural damage, and signs and symptoms among patients with rheumatoid arthritis treated with infliximab and methotrexate. Arthritis Rheum. 50:1051.[CrossRef][ISI][Medline]
- Suzuki, A., Yamada, R., Chang, X. et al. 2003. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat. Genet. 34:395.[CrossRef][ISI][Medline]
- Wipke, B. T., Wang, Z., Kim, J., McCarthy, T. J. and Allen, P. M. 2002. Dynamic visualization of a joint-specific autoimmune response through positron emission tomography. Nat. Immunol. 3:366.[CrossRef][ISI][Medline]
- Tanaka, M., Ozaki, S., Osakada, F., Mori, K., Okubo, M. and Nakao, K. 1998. Cloning of follistatin-related protein as a novel autoantigen in systemic rheumatic diseases. Int. Immunol. 10:1305.[Abstract]
- Tanaka, M., Ozaki, S., Kawabata, D. et al. 2003. Potential preventive effects of follistatin-related protein/TSC-36 on joint destruction and antagonistic modulation of its autoantibodies in rheumatoid arthritis. Int. Immunol. 15:71.[Abstract/Free Full Text]
- Kawabata, D., Tanaka, M., Fujii, T. et al. 2004. Ameliorative effects of follistatin-related protein/TSC-36/FSTL1 on joint inflammation in a mouse model of arthritis. Arthritis Rheum. 50:660.[CrossRef][ISI][Medline]
- Mimori, T., Suganuma, K., Tanami, Y. et al. 1995. Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral protease, calpain) in systemic rheumatic diseases. Proc. Natl Acad. Sci. USA 92:7267.[Abstract/Free Full Text]
- Despres, N., Talbot, G., Plouffe, B., Boire, G. and Menard, H. A. 1995. Detection and expression of a cDNA clone that encodes a polypeptide containing two inhibitory domains of human calpastatin and its recognition by rheumatoid arthritis sera. J. Clin. Invest. 95:1891.[ISI][Medline]
- Iwaki-Egawa, S., Matsuno, H., Yudoh, K. et al. 2004. High diagnostic value of anticalpastatin autoantibodies in rheumatoid arthritis detected by ELISA using human erythrocyte calpastatin as antigen. J. Rheumatol. 31:17.[ISI][Medline]
- Vittecoq, O., Salle, V., Jouen-Beades, F. et al. 2001. Autoantibodies to the 27 C-terminal amino acids of calpastatin are detected in a restricted set of connective tissue diseases and may be useful for diagnosis of rheumatoid arthritis in community cases of very early arthritis. Rheumatology (Oxford) 40:1126.[CrossRef][Medline]
- Lackner, K. J., Schlosser, U., Lang, B. and Schmitz, G. 1998. Autoantibodies against human calpastatin in rheumatoid arthritis: epitope mapping and analysis of patient sera. Br. J. Rheumatol. 37:1164.[CrossRef][ISI][Medline]
- Kanazawa, Y., Kaneshiro, Y., Sawa, M. et al. 2000. Domain reactivity of autoantibodies to calpastatin in patients with systemic rheumatic diseases. Mod. Rheumatol. 10:38.[CrossRef]
- Goll, D. E., Thompson, V. F., Li, H., Wei, W. and Cong, J. 2003. The calpain system. Physiol. Rev. 83:731.[Abstract/Free Full Text]
- Menard, H. A. and el-Amine, M. 1996. The calpain-calpastatin system in rheumatoid arthritis. Immunol. Today 17:545.[CrossRef][ISI][Medline]
- Watanabe, N. and Kobayashi, Y. 1994. Selective release of a processed form of interleukin 1 alpha. Cytokine 6:597.[CrossRef][ISI][Medline]
- Melloni, E., Pontremoli, S., Michetti, M. et al. 1985. Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase. Proc. Natl Acad. Sci. USA 82:6435.[Abstract/Free Full Text]
- Szomor, Z., Shimizu, K., Fujimori, Y., Yamamoto, S. and Yamamuro, T. 1995. Appearance of calpain correlates with arthritis and cartilage destruction in collagen induced arthritic knee joints of mice. Ann. Rheum. Dis. 54:477.[Abstract]
- Ray, S. K., Matzelle, D. C., Wilford, G. G., Hogan, E. L. and Banik, N. L. 2000. E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats. Brain Res. 867:80.[CrossRef][ISI][Medline]
- Tram, K. K., Murray, S. S., Lee, D. B. and Murray, E. J. 1993. PTH-induced osteoblast contraction is mediated by cysteine proteases. Kidney Int. 43:693.[ISI][Medline]
- Terato, K., Harper, D. S., Griffiths, M. M. et al. 1995. Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen. Autoimmunity 22:137.[ISI][Medline]
- Abe, M., Tanaka, Y., Saito, K. et al. 1997. Regulation of interleukin (IL)-1beta gene transcription induced by IL-1beta in rheumatoid synovial fibroblast-like cells, E11, transformed with simian virus 40 large T antigen. J. Rheumatol. 24:420.[ISI][Medline]
- Kagari, T., Doi, H. and Shimozato, T. 2002. The importance of IL-1 beta and TNF-alpha, and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis. J. Immunol. 169:1459.[Abstract/Free Full Text]
- Buttle, D. J., Saklatvala, J., Tamai, M. and Barrett, A. J. 1992. Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases. Biochem. J. 281(Pt 1):175.[ISI][Medline]
- Umehara, H., Huang, J. Y., Kono, T. et al. 1998. Co-stimulation of T cells with CD2 augments TCR-CD3-mediated activation of protein tyrosine kinase p72syk, resulting in increased tyrosine phosphorylation of adapter proteins, Shc and Cbl. Int. Immunol. 10:833.[Abstract]
- Shumway, S. D., Maki, M. and Miyamoto, S. 1999. The PEST domain of IkappaBalpha is necessary and sufficient for in vitro degradation by mu-calpain. J. Biol. Chem. 274:30874.[Abstract/Free Full Text]
- Chen, F., Lu, Y., Kuhn, D. C. et al. 1997. Calpain contributes to silica-induced I kappa B-alpha degradation and nuclear factor-kappa B activation. Arch. Biochem. Biophys. 342:383.[CrossRef][ISI][Medline]
- Pontremoli, S., Melloni, E., Damiani, G. et al. 1988. Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C. J. Biol. Chem. 263:1915.[Abstract/Free Full Text]
- Cuzzocrea, S., McDonald, M. C., Mazzon, E. et al. 2000. Calpain inhibitor I reduces the development of acute and chronic inflammation. Am. J. Pathol. 157:2065.[Abstract/Free Full Text]
- Barnes, P. J. 1997. Nuclear factor-kappa B. Int. J. Biochem. Cell Biol. 29:867.[CrossRef][ISI][Medline]
- Brozik, M., Rosztoczy, I., Meretey, K. et al. 1992. Interleukin 6 levels in synovial fluids of patients with different arthritides: correlation with local IgM rheumatoid factor and systemic acute phase protein production. J. Rheumatol. 19:63.[Medline]
- Libermann, T. A. and Baltimore, D. 1990. Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol. Cell. Biol. 10:2327.[ISI][Medline]
- Miyazawa, K., Mori, A., Yamamoto, K. and Okudaira, H. 1998. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-kappaB and CBF1. Am. J. Pathol. 152:793.[Abstract]
- Dourdin, N., Bhatt, A. K., Dutt, P. et al. 2001. Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J. Biol. Chem. 276:48382.[Abstract/Free Full Text]
- Lokuta, M. A., Nuzzi, P. A. and Huttenlocher, A. 2003. Calpain regulates neutrophil chemotaxis. Proc. Natl Acad. Sci. USA 100:4006.[Abstract/Free Full Text]
- Fukai, F., Ohtani, T., Ueki, M. and Katayama, T. 1993. Involvement of calcium-dependent cysteine protease in fibronectin-induced chemotactic migration of NIH-L13 fibroblasts. Biochem. Mol. Biol. Int. 30:225.[ISI][Medline]
- Suzuki, K., Shimizu, K., Hamamoto, T., Nakagawa, Y., Murachi, T. and Yamamuro, T. 1992. Characterization of proteoglycan degradation by calpain. Biochem. J. 285(Pt 3):857.[ISI][Medline]
- Tamai, M., Matsumoto, K., Omura, S., Koyama, I., Ozawa, Y. and Hanada, K. 1986. In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64. J. Pharmacobio-dyn. 9:672.[Medline]
- Sasaki, T., Kishi, M., Saito, M. et al. 1990. Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins. J. Enzyme Inhib. 3:195.[Medline]