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Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 406453, 9 pages
http://dx.doi.org/10.1155/2014/406453
Research Article
Attenuation of
Sayuri Yoshimura, Krisana Asano, and Akio Nakane
Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, 5
Correspondence should be addressed to Akio Nakane;
Received 20 February 2014; Accepted 4 May 2014; Published 22 May 2014
Academic Editor: Shigeru Kotake
Copyright © 2014 Sayuri Yoshimura et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Rheumatoid arthritis (RA) is a serious autoimmune disease caused by chronic inflammation of connective tissues. The basic principle of RA treatment is aimed to reduce joint inflammation. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in different mouse inflammatory diseases. In this study, we investigated the prophylactic effect of PG on the progression of RA using an experimental mouse model,
1. Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic inflammation of synovial joints, subsequently with progressive, erosive destruction of articu- lar tissues [1]. It affects 1% of population and is associated with significant morbidity and mortality [2]. In the synovial tissues of RA, numerous cytokines are expressed and are functionally active. T hey are directly implicated in the immune processes that are thought to play crucial roles in the pathology of RA. In many rodent models, the cytokine modulation alters the outcome of arthritis [3].
Proteoglycans (PGs) are widely distributed in connecting tissues such as skin, bone, and cartilage by forming a complex with collagen, fibronectin, laminin, hyaluronic acid, and other glycoproteins
attached with one or more glycosaminoglycan(s). Our previ- ous studies have shown that PG extracted from salmon carti- lage has the immunomodulatory effect. It suppresses inflam- matory response of macrophages induced by stimulation with
In this study, the immunomodulatory effect of PG on the progression of arthritis was investigated. Mice with collagen- induced arthritis (CIA) were administered with PG per os
2
daily. Our results demonstrated that immune response of splenocytes to collagen stimulation and proinflammatory cytokine and chemokine expression in the joints were modu- lated by oral administration of PG. These data suggested that PG has the prophylactic effect which is able to attenuate the severity of several inflammatory diseases not only colitis and EAE but also arthritis which is an important autoimmune disease.
2. Materials and Methods
2.1.Mice. DBA/1J mice were purchased from CLEA Japan, Inc., Tokyo, Japan. They were housed under specific-
2.2.Preparation and Administration of PG. Salmon cartilage PG was purchased from Kakuhiro Co., Ltd., Aomori, Japan. Lyophilized PG powder was dissolved in
2.3.Induction of Arthritis. Arthritis was induced as described previously [11]. Briefly, 8- to
2.4.Scoring for Evaluating Arthritis Severity in CIA. Mice were monitored for arthritis daily from day 18 after immu- nization. Each paw was scored for clinical signs of arthritis as follows: 0: no evidence of erythema and swelling, 1: erythema and swelling confined to digits, 2: erythema and mild swelling extending from ankle to tarsals, 3: erythema and moderate swelling extending from ankle to metatarsal joints, and 4: erythema and severe swelling encompass ankle, foot, and digits [11].
2.5.Histological Analysis. After completing the experiment, mice were sacrificed and paws were fixed in 10% neutral- buffered formalin and decalcified with 10% EDTA, pH 7.4. The paws were then embedded in paraffin and cut into
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hematoxylin and eosin and observed under light microscope. For immunostaining, the sections were incubated for 1 h at room temperature (RT) with monoclonal antibody (mAb) to mouse
2.6.Determination of
with 8 N H2SO4. The absorbance was measured at 490 nm with reference of 655 nm wavelength using microplate reader model 680
2.7.Quantitative
(0.05) for all data analysis. Threshold cycle (C��) of each target product was determined and set in relation to the ampli- fication plot of
(GAPDH). Difference in C�� values ( C��) of two genes was used to calculate the relative expression (relative expression
= 2−(���� of target genes−���� of GAPDH) =2−Δ����).
2.8.Ex Vivo Cytokine Production of CII Restimulated Spleno- cytes.
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cultured in Dulbecco’s modified Eagle medium (DMEM, Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS) and 0.03%
[12].
2.9.Statistical Analysis. Statistical significance was deter- mined by a
3. Results
3.1.Attenuation of CIA Severity by Daily Oral Administration of PG. In order to investigate the effect of PG on CIA, CII- immunized mice were administered with 2 mg of PG per os daily by starting on the day of the first CII immunization. Clinical scores of CIA were recorded between day 18 and day 56 after the first immunization. Percent incidence and clinical scores of CIA in the
3.2.Decrease in Macrophages, Neutrophils, and Osteoclasts in the Joint of
3.3.Reduction of Chemokine Expression in the Joints of PG- Administered Mice. Osteoclast activation and infiltration of inflammatory cells are regulated by chemokines. Thus, we then examined the chemokine production in the ankle joints of
3
mice was determined by quantitative
3.4.PG Administration Reduced Cytokine Expression in the Joints of
3.5.Administration of PG Suppressed the Cytokine Production from Splenocytes in CII Restimulation. The immune response to CII in CIA is characterized by the stimulation of collagen- specific T cells [11]. We examined the response of splenocytes to restimulation with CII ex vivo.
3.6.PG Administration Did Not Affect Antibody Responses.
4. Discussion
To assess the efficacy of immunomodulatory agents for RA therapy in humans, CIA is a promising experimental model
[15].Chronic inflammation of joints is a main characteristic of RA and CIA [1, 16]. It has been reported that autoantibodies to CII and
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100 |
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90 |
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∗ |
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80 |
∗ ∗ |
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(%) |
70 |
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60 |
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Score |
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Incidence |
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50 |
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40 |
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30 |
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20
10
0 18 21 24 27 30 33 36 39 42 45 48 51 54 57
Days after immunization
Control (n = 8)
(a)
Control |
250 ��m
(c)
12 |
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10 |
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8 |
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6 |
∗∗ ∗∗ |
∗∗ |
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4 |
∗ |
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2 |
∗∗ |
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0 18 21 24 27 30 33 36 39 42 45 48 51 54 57
Days after immunization
Control (n = 8)
(b)
250 ��m |
250 ��m |
Figure 1: Oral administration of PG attenuated severity of arthritis in
Table 1: Primers and PCR conditions used in this study.
Product |
Primer sequence |
|
|
Forward |
Reverse |
||
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|||
GAPDH |
TGAAGGTCGGTGTGAACGGATTTGG |
ACGACATACTCAGCACCGGCCTCAC |
|
CCL2 |
ACTGAAGCCAGCTCTCTCTTCCTC |
TTCCTTCTTGGGGTCAGCACAGAC |
|
CXCL1 |
GGATTCACCTCAAGAACATCCAGAG |
CACCCTTCTACTAGCACAGTGGTTG |
|
CXCL2 |
GAACAAAGGCAAGGCTAACTGA |
AACATAACAACATCTGGGCAAT |
|
CCTCAAAGCTCAGCGTGTCC |
GAGCTCACTTTTGCGCCAAG |
||
GGCAGGTCTACTTTGGAGTCATTGC |
ACATTCGAGGCTCCAGTGAATTCGG |
||
TGGAGTCACAGAAGGAGTGGCTAAG |
TCTGACCACAGTGAGGAATGTCCAC |
||
AAGGAGAACCAAGCAACGACAAAA |
TGGGGAACTCTGCAGACTCAAACT |
||
AGCGGCTGACTGAACTCAGATTGTAG |
GTCACAGTTTTCAGCTGTATAGGG |
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5 |
Control |
Macrophages
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100��m |
100��m |
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100��m |
||||
(a) |
(b) |
(c) |
Neutrophils
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|
100��m |
100��m |
100��m |
|||
(d) |
(e) |
(f) |
Osteoclasts
Number of macrophages (cells/mm2)
100��m
(g)
60∗∗
50
40
30
20
10
0
Control
(j)
Number of neutrophils (cells/mm2)
100��m
(h)
600∗∗
500 |
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osteoclastsof |
/20mm |
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) |
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2 |
400 |
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300 |
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Number |
(cells |
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100 |
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200 |
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0 |
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Control |
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(k) |
|
100��m
(i)
25∗∗
20
15
10
5
0
Control
(l)
Figure 2: Continued.
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Relative CCL2 mRNA expression
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
∗∗
Relative CXCL1 mRNA expression
Control
(m)
0.012
0.01
0.008
0.006
0.004
0.002
0
∗
Relative CXCL2 mRNA expression
Control
(n)
∗
0.012
0.01
0.008
0.006
0.004
0.002
0
Control
(o)
Figure 2: Administration of
Relative
Relative
|
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∗∗ |
0.007 |
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expression |
0.006 |
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|
0.005 |
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6mRNA |
0.003 |
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0.004 |
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IL- |
0.002 |
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Relative |
0.001 |
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0 |
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Control |
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(a) |
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∗∗ |
0.25 |
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expression |
0.2 |
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0.15 |
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0.1 |
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IFN |
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0.05 |
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Relative |
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0 |
||
Control |
(c)
∗
0.25
0.2
0.15
0.1
0.05
0
Control
(b)
∗
0.001
0.0008
0.0006
0.0004
0.0002
0
Control
(d)
Figure 3: Administration of PG reduced cytokine expression in joints of
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Relative
2500 |
∗∗ |
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2000 |
��(pg/mL)- |
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1500 |
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1000 |
IFN |
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500 |
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0 |
CII 50 ��g/mL |
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No stimulation |
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Control |
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(a) |
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NS |
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1 |
production |
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0.7 |
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0.9 |
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0.8 |
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0.6 |
2a |
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IgG |
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0.5 |
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0.4 |
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0.3 |
anti |
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0.1 |
Relative |
|
0.2 |
|
≤0.001
Control
(c)
∗∗
3000
2500
2000
1500
1000
500
0
No stimulation |
CII 50 ��g/mL |
Control
(b)
NS
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
≤0.001
ControlCII-PBS
(d)
Figure 4: Administration of PG suppressed
associates with CCL2 [17] and CXCL1 and CXCL2 derived from macrophages act as neutrophil chemoattractants [18, 19]. Inflammatory cells migrated into the joints produce several proinflammatory cytokines including tumor necrosis
proinflammatory cytokines are thought to be responsible for bone erosion [3, 20].
We presumed that salmon PG displays a potential to reduce the pathogenesis of RA because of its immunomod- ulating activities
8
models. Oral administration of PG showed no influence on
In addition to macroscopic evaluation, the effect of PG on severity of CIA was investigated by histopathological analysis. In the joints of
[28].Thus,
Our previous studies demonstrated that PG reduces TNF- �� production and induces Foxp3+ Treg cells in colitis and EAE mouse models [8, 9]. However, in this CIA model, TNF- �� and Foxp3 expression in the joints was not significantly altered by PG administration (data not shown). Although the reason of these results remained unclear, overall of our data indicated that daily administration of salmon PG attenuates inflammation and severity in CIA. This finding implied that salmon PG is a promising prophylactic agent. Continuous consumption of PG from salmon might be able to reduce the progression of inflammatory and autoimmune diseases.
Conflict of Interests
The authors have no conflict of interests to report.
Acknowledgments
This study was supported by the City Area Program for Promotion of Science and Technology in Regional Areas from Ministry of Education, Culture, Sports, Science and Technology.
References
[1]M. Feldmann, F. M. Brennan, and R. N. Maini, “Role of cytokines in rheumatoid arthritis,” Annual Review of Immunol- ogy, vol. 14, pp.
BioMed Research International
[2]G. S. Firestein, “Evolving concepts of rheumatoid arthritis,” Nature, vol. 423, no. 6937, pp.
[3]I. B. McInnes and G. Schett, “Cytokines in the pathogenesis of rheumatoid arthritis,” Nature Reviews Immunology, vol. 7, no. 6, pp.
[4]R.
[5]J. A. Buckwalter and L. C. Rosenberg, “Electron microscopic studies of cartilage proteoglycans,” Journal of Biological Chem- istry, vol. 257, no. 16, pp.
[6]M. Hook, L. Kjellen, S. Johansson, and J. Robinson, “Cell- surface glycosaminoglycans,” Annual Review of Biochemistry, vol. 53, pp.
[7]H. Sashinami, K. Takagaki, and A. Nakane, “Salmon cartilage proteoglycan modulates cytokine responses to Escherichia coli in mouse macrophages,” Biochemical and Biophysical Research Communications, vol. 351, no. 4, pp.
[8]T. Mitsui, H. Sashinami, F. Sato et al., “Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3+ regulatory T cells,” Biochemical and Bio- physical Research Communications, vol. 402, no. 2, pp.
[9]H. Sashinami, K. Asano, S. Yoshimura, F. Mori, K. Wakabayashi, and A. Nakane, “Salmon proteoglycan suppresses progression of mouse experimental autoimmune encephalomyelitis via regulation of Th17 and Foxp3+ regulatory T cells,” Life Sciences, vol. 91, no.
[10]K. Asano, S. Yoshimura, and A. Nakane, “Alteration of intestinal microbiota in mice orally administered with salmon cartilage proteoglycan, a prophylactic agent,” PLoS ONE, vol. 8, no. 9, pp.
[11]E. F. Rosloniec, M. Cremer, A. H. Kang, L. K. Myers, and D. D. Brand,
[12]H. Sashinami, A. Nakane, Y. Iwakura, and M. Sasaki, “Effective induction of acquired resistance to Listeria monocytogenes by immunizing mice with in
[13]K. Okamoto and H. Takayanagi, “Osteoclasts in arthritis and Th17 cell development,” International Immunopharmacology, vol. 11, no. 5, pp.
[14]Y. Iwakura, S. Nakae, S. Saijo, and H. Ishigame, “The roles of
[15]D. D. Brand, K. A. Latham, and E. F. Roslonic,
[16]M. Seeling, U. Hillenhoff, J. P. David et al., “Inflammatory mon- ocytes and Fc��receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice,” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 26, pp.
[17]S. Shahrara, S. R. Pickens, A. M. Mandelin et al., “IL-
[18]K. D. Filippo, A. Dudeck, M. Hasenberg et al., “Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage
BioMed Research International |
9 |
of neutrophil recruitment during tissue inflammation,” Blood, vol. 121, no. 24, pp.
[19]J. Ha, H. S. Choi, Y. Lee, H. J. Kwon, Y. W. Song, and H. H. Kim, “CXC chemokine ligand 2 induced by receptor activator of
[20]G. Schett and E. Gtravallese, “Born erosion in rheuma- toid arthritis: mechanisms, diagnosis and treatment,” Nature Reviews Rheumatology, vol. 8, pp.
[21]H. Kellner, “Targeting
[22]M. Hashizume, N. Hayakawa, and M. Mihara,
[23]T. T. Glant, A. Finnegan, and K. Mikecz, “Proteoglycan- induced arthritis: immune regulation, cellular mechanisms, and genetics,” Critical Reviews in Immunology, vol. 23, no. 3, pp. 199– 250, 2003.
[24]T. T. Glant and K. Mikecz, “Proteoglycan
[25]S. Nakae, A. Nambu, K. Sudo, and Y. Iwakura, “uppression of immune induction of
[26]M. J. Park, H. S. Park, H. J. Oh et al.,
[27]S. Sarkar, L. A. Cooney, P. White et al., “Regulation of pathogenic
[28]M. C. Boissier, G. Chiocchia, N. Bessis et al., “Biphasic effect of