Chinese Medical Journal 2003;116(9):1408-1412
Expression of C5aR (CD88) of synoviocytes isolated from patients with rheumatoid arthritis and osteoarthritis
YUAN Guohua 袁国华, WEI Jin 魏 锦, ZHOU Jingguo 周京国, HU Hong 胡 宏, TANG Zhong 唐 中, ZHANG Guoyuan 张国元
YUAN Guohua 袁国华 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
WEI Jin 魏 锦 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
ZHOU Jingguo 周京国 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
HU Hong 胡 宏 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
TANG Zhong 唐 中 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
ZHANG Guoyuan 张国元 (Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)Correspondence to:Wei Jin,Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China (Tel: 86-817-2180269. Fax:86-817-222856. E-mail:firstname.lastname@example.org)
Methods The expression of C5aR was assessed in synoviocytes isolated from 27 RA and 12 OA patients using reverse transcription-polymerase chain reactions (RT-PCR), flow cytometry, and immunofluorescence analysis. The effects of C5a on the release of tumor necrosis factor α (TNFα) from synoviocytes were assayed using enzyme-linked immunosorbent assays (ELISA).
Results C5aR mRNA was detected in 24 of 27 samples from RA patients, and 10 of 12 samples from OA patients. Flow cytometric analysis and immunofluorescence study demonstrated the cell surface expression of C5aR in a significant proportion of synoviocytes from both RA and OA patients, and the level of C5aR expression in synoviocytes was significantly correlated with joint swelling, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in RA patients. Finally, interaction of C5aR with its ligand C5a was shown to enhance lipopolysaccharide (LPS)-induced TNFα release from synoviocytes.
Conclusions The expression of C5aR in synoviocytes from inflammatory joint diseases and also the induction of TNFα release in activated synovicytes by the interaction of C5a and C5aR suggest that the C5a/C5aR system may play an important role in joint inflammation process.
C5aR was initially reported to be present in circulating peripheral leukocytes and in mast cells.［3,4］ However, some recent studies have suggested that this receptor is widely expressed on various kinds of cells, such as vascular smooth muscle cells, epithelial cells, hepatocytes and mesangial cells.［5］ Although it has been well-documented that complement anaphylatoxin C5a plays an important role in the inflammatory process in joint lesions of arthritic diseases such as rheumatoid arthritis(RA),［6,7］ there have been only a few reports on the expression of C5aR in joint tissues.It is also not clear whether synoviocytes express receptors for the complement components.
In this study, we investigated the functional expression of C5aR in synoviocytes isolated from patients with RA and osteoarthritis (OA). Our results clarified the expression of C5aR in synoviocytes and the engagement of C5a to its receptor C5aR enhanced lipopolysaccharide (LPS)-stimulated proinflammatory cytokine production in synoviocytes.
Twenty-seven patients with RA were envolved in this study, six men and 21 women, aged 22-69 years, with a mean of 38.7 years. The clinical and laboratory data of all patients were collected, including morning stiffness, grip strength, number of tender joints, number of swollen joints, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and titer of rheumatoid factor (RF). Another 12 patients with OA were selected as the control group, four men and eight women, aged 48-71 years, with a mean of 56.4 years.
Isolation and culture of synoviocytes
Synoviocytes were isolated from synovial tissues (9 RA and 4 OA) or synovial fluid (SF, 18 RA and 8 OA). Synovial tissue specimens, obtained from knee joints of RA or OA patients undergoing synovectomy or arthroscopic biopsy in the Affiliated Hospital of North Sichuan Medical College, were minced and digested overnight at 37℃ with rotation in RPMI1640 medium (Gibco, Grand Island, NY,USA) containing l mg/ml of bacterial collagenase (Sigma, USA). The released cells were filtered, washed and seeded onto 60 cm2 dishes (IWAKI, Tokyo, Japan) at a density of 2-4×106 cells/dish and cultivated in vitro at 37℃ at 5% CO2. The culture medium was RPMI 1640 containing 10% heat inactivated fetal calf serum (FCS), 100 μg/ml penicillin and 100 μg/ml streptomycin. The medium was replaced weekly, and cells were used between the second and fifth passages. Synoviocytes in synovial fluid obtained from RA and OA patients were cultured as previously described.［8］
Reverse transcription-polymerase chain reactions
Total RNA was extracted from the cultured synoviocytes using the single-step guanidinium-thiocyanate-phenol-chloroform method. RNA was precipitated in ethanol, recovered by centrifugation, resuspended in 10 μl of sterile water, and evaluated with a spectrophotometer for quantity and purity. First-strand cDNA was synthesized in a 20 μl reaction mixture containing 5 μg of total RNA, 2.5 mmol/L of each dNTP, 1 μmol/L of random hexamers, 40 units of RNase inhibitor(RNasin, Toyobo CO.Ltd., Tokyo, Japan) and 200 units of Superscript Ⅱ RT (Gibco), incubated at 42℃ for 2 hours. One microliter of the resulting cDNA was subjected to PCR using Taq DNA polymerase and the specific primers for the C5aR (sense 5’-ATG AAC TCC TTC AAT TAT ACC-3’, antisense 5’-TGG TGG AAA GTA CTC CTC CCG-3’) under the following condition: 30 cycles at 94℃ for 1 minute, 56℃ for 1 minute, and 72℃ for 1 min. In each experiment, amplification of cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sense 5’-CCA CCC ATG GCA AAT TCC ATG GCA-3’; antisense 5’-TCT AGA CGG CAG GTCAGG TCC ACC-3’) was used as an internal standard. PCR products were electrophoresed with a molecular weight marker on 1.5% agarose gel, visualized by ethidium bromide stain, and photographed under UV illumination.
Flow cytometric analysis
The cultured synoviocytes were collected by EDTA and washed with phosphate-baffered saline PBS. For detection of cell surface expression of C5aR, 105 cells were processed in 0.5 ml of PBS/2% FCS and incubated with mouse anti-human CD88 (C5aR) (Serotec Ltd, Oxford, UK) or with isotype control mouse lgG2a for 30 minutes. After three washing steps with PBS/2%FCS, cells were blocked with 10% normal goat serum and incubated with a FITC-conjugated goat anti-mouse IgG(H+L) for 30 minutes in the dark. After washing, the cells were immediately analyzed by FACSCalibur (Nippon Becton Dickinson Co, Ltd, Tokyo, Japan), and calculations were performed with CellQuest analysis software (Nippon Becton Dickinson).Immunofluorescence study
Synoviocytes were seeded in 8-well chamber slides (Nunc, Naperville, IL) at a concentration of 2×104 per well and cultured for 2-3 days. The slides were fixed using acetone for 15 minutes. After washing with PBS, slides were pre-incubated with heat-inactivated 10% normal goat serum for 30 minutes, and then incubated for 45 min with a mouse anti-human CD88 (C5aR) or with isotype control mouse lgG2a. The bound antibodies were detected using FITC-conjugated goat anti-mouse IgG (H+L).
Enzyme-linked immunosorbent assays (ELLSA)
Synoviocytes were seeded in 24-well plates and incubated with 5 μg/ml of lipopolysaccharide (LPS, Sigma) and/or recombinant human C5a (Sigma) at the indicated concentrations for 48 hours. Cultured supernatants were then collected and stored at -20℃ until used. The TNFα concentration in the cultured supernatants was evaluated by commercial ELISA kits following the manufacturer’s instructions (R and D Systems).Statistical analysis
The Student’s t test was used to analyze the differences
Expression of mRNA of C5aR by synoviocytes
As a first step, we investigated whether the mRNA of C5aR was expressed in human synoviocytes. The representative reverse transcription-polymerase chain reation (RT-PCR) is shown in Figure 1 . A distinct band with nucleotide length corresponding to C5aR primer setting was found in the synoviocyte samples. This band was detected in 24 out of 27 RA samples (7/9 in synoviocytes isolated from synovial tissues, and 17/18 in synoviocytes obtained from SF), and in 10 out of 12 OA samples tested (4/4 in synovial synoviocytes, and 6/8 in SF synoviocytes).
Cell surface expression of C5aR in synoviocytes
We investigated the cell surface expression of C5aR on synoviocytes using flow cytometry. Figure 2 shows the representative results of the flow cytometric analysis. Of the 27 RA and 12 OA patients, the percentage with C5aR-positive cells varied from 5.4%-36.3% (mean 22.7%±12.1%) and from 2.1%-29.3% (mean 12.2%±7.1%, P<0.05), respectively. No difference was found in the C5aR expression of synoviocytes from synovial tissue or from SF in RA, which was 7.6%-29.7% (mean 19.1%±7.4%) in synovial synoviocytes, and 5.4%-36.3% (mean 23.6%±12.8%, P>0.05) in SF synoviocytes. The cell surface expression of C5aR in synoviocytes from RA and OA patients was also confirmed by immunofluoresecnce study as shown in Figure 3A, B and C .
Analysis of the relationship between C5aR expression in synoviocytes and clinical data in RA showed that the level of C5aR expression was significantly correlated with the number of swollen joints (r=0.716, P<0.05), CRP (r=0.787, P<0.05) and ESR (r=7.23, P<0.05).
Induction of TNFα release by C5a in synoviocytes
The expression of C5aR in synoviocytes in our studies showed that C5a might play a role in snyoviocyte activation. To this end, we analyzed whether C5a modulates production of proinflammtory cytokine TNFα in snyoviocytes. Figure 4 shows that C5a alone can not induce release of TNFα from synoviocytes. However, it enhances LPS-induced release of this cytokine from synoviocytes in a dose-dependent manner.
The involvement of the complement system, especially of the anaphylatoxin C5a, in inflammatory joint diseases such as RA has been studied extensively, mostly concerning its inflammatory property on cells of the myeloid lineage, such as granulocytes and monocytes.
C5a is known to be generated upon activation of the complement system by either the classical or alternative pathway. It has been reported that there is activation of the complement system in joint lesions of RA, mainly caused by IgG aggregates and immune complexes.［9，10］ Therefore, C5a is expected to be produced in joint lesions in RA. In fact, Jose et al［6］ previously reported an increased C5a concentration in RA synovial fluid. C5a has been found to mediate chemotactic migration of neutrophilic granulocytes and monocytes by directing these cell types into diseased joints, thus causing the release of histamine and lysosomal contents from inflammatory cells, and increasing vascular permeability. These effects of C5a are mediated by its binding to C5aR expressed on target cells.
It has also been shown that the expression of the C5aR, which at first had been believed to be restricted to myeloid cells such as monocytes, and neurtophilic, basophilic and eosinophilic granulocytes, is also detectable on nonmyeloid cells, such as vascular smooth muscle cells, epithellial cells, hepatocytes and astrocytes.［4，5］
Although C5aR has been detected in inflammatory cells in synovial tissues from RA patients,［11］ the distribution of the complement receptor in synoviocytes has not been clarified. In this study we first demonstrated distinct expression of C5aR (CD88) in a significant proportion of synoviocytes from RA patients. Further analysis showed that the level of C5aR expression was correlated with the number of swollen joints, ESR and serum CRP levels in RA, indicating the involvement of the C5a/C5aR system in active disease.
The data concerning the functions of C5aR in tissue cells are limited, because it is only recently that this receptor has been recognized in cells other than those of myeloid lineage. In our study, it was shown that C5a alone had no effect on the release of proinflammtory cytokine TNFα from synoviocytes. However, a significantly increased amount of TNFα release was observed in the cells co-stimulated with C5a and LPS, as compared with that in cells stimulated with LPS alone, thus indicating that C5a elicits the effects on activated synoviocytes, which is evident in RA synovium in vivo. The release of proinflammatory cytokines induced by C5a has also been reported in myeloid cells such as mononuclear leukocytes.［12］ Notably, TNFα was found to enhance the synthesis of complement components in joint tissue and to increase the expression of complement receptors in tissue cells，［13，14］ suggesting the existence of amplification effects of C5a and C5aR interaction by inducing the release of TNFα in target cells.
It is interesting to find that C5aR was also expressed in OA synoviocytes. OA is generally a degenerative joint disease caused mainly by mechanical stress. But recent observations suggest the involvement of immune factors in the process of cartilage degradation. Expression of complement components has been detected in OA synovial tissue as well as cartilage, and active complement components has been found in damaged cartilage.［14］ Moreover, complement components have been reported to be able to degrade cartilage matrix.［15］ These are all evidence of the participation of the complement system in the pathogenesis of OA.
In conclusion, our results showed a subset of human synoviocyte-expressed C5aR and the interaction of C5aR with its ligand C5a enhanced the LPS-induced release of TNFα from synoviocytes. The C5a/C5aR system may play an important role in the inflammatory process in joint diseases.
1.Kohl J. Anaphylatoxine and infectious and non-infectious inflammatory diseases. Mol lmmunol 2001;38:175-187.
2.Gerard C, Gerard NP. C5a anaphylatoxin and its seven transmembrane-segment receptor. Ann Rev lmmunol 1994;12:775-808.
3. Nataf S, Davoust N, Ames RS, et al. Human T-cells express the C5a receptor and are chemoattractant to C5a. J Immunol 1999;162:4018-4023.
4. Rothermel E, Gotze O, Zahn S, et al. Analysis of the tissue distribution of the rat C5a receptor and inhibition of C5a-mediated effects through the use of two MoAbs. Scand J Immunol 2000;52:401-410.
5.Wilmer WA, Kaumaya PT, Ember JA, et al. Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells. J lmmunol 1998;160:5646-5652.
6.Jose PJ, Moss IK, Maini RN, et al. Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: role of C5a in the acute inflammatory phase. Ann Rheum Dis 1990;49:747-752.
7.Wang Y, Scott AR, Madri JA, et al. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc Natl Acad Sci U S A 1995;92:8955-8959.
8.Hayes ME, Bayley D, Still P, et al. Differential metabolism of 25-hydroxyvitamin D3 by cultured synovial fluid macrophages and fibroblast-like cells from patients with arthritis. Ann Rheum Dis 1992;51:220-226.
9. Watson WC, Crener MA, Wooley PH, et al. Assessment of the potential pathogenicity of type Ⅱ collagen autoantibodies in patients with rheumatoid arthritis. Evidence of restricted IgG3 subclass expression and activation of complement C5 to C5a. Arthritis Rheum 1986;29:131613-21.
10. Molenaar ET, Voskuyl AE, Familian A, et al. Complement activation in patients with rheumatoid arthritis mediated in part by C-reactive protein. Arthritis Rheum 2001;44:997-1002.
11. Kiener HP, Baghestanian M, Domindus M, et al. Expression of the C5a receptor(CD88)on synovial mast cells in patients with rheumatoid arthritis. Arthritis Rheum 1998;41:233-245.
12. Cavaillon JM, Fitting C, Haeffner-Cavaillon N. Recombinant C5a enhances interleukin-1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur J lmmunol 1990;20:253-260.
13. Nakagawa K, Sakiyama H, Tsuchida T, et al. Complement Cls activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody. Ann Rheum Dis 1999;58:175-181.
14.Stahel PF, Kariya K, Shohami E, et al. Intracerebral complement C5a receptor (CD88) expression is regulated by TNF and lymphotoxin-alpha following closed head injury in mice. J Neuroimmunol 2000;109:164-172.
15.Yamaguchi K, Sakiyama H, Matsumoto M, et al. Degradation of type Ⅰ and type Ⅱ collagen by human Cls. FEBS Lett 1990;268:206-208.