Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433, 1 Centre for Transfusion Medicine, Health Sciences Authority, Singapore and 2 Present address: Department of Physiology, National University of Singapore, Singapore.
Correspondence to: H. S. Howe, Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433. E-mail: Hwee_Siew_Howe{at}ttsh.com.sg
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Abstract |
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Methods. Serum levels of TGFß1 were measured by enzyme-linked immunosorbent assay (ELISA). The ability of PBMCs to synthesize TGFß1 and other cytokines was assessed by in vitro cultures stimulated with mitogen. Genomic DNA was extracted from PBMCs of AS patients (n = 72) or unrelated healthy controls (n = 96). The codon 10 and 25 polymorphisms in the TGFB1 gene were analysed using standard polymerase chain reaction-based methods.
Results. AS patients had significantly higher serum TGFß1 levels than controls (P<0.001). There was no difference in the distribution of codon 10 and 25 TGFB1 genotypes between AS patients and controls. Incubation of AS and control PBMC with phytohaemagglutinin (PHA) led to upregulation of TGFß1, interleukin-10, tumour necrosis factor-alpha (TNF) and interferon-
(IFN
) assessed by ELISA. Importantly, PHA-induced TGFß1 production was significantly enhanced in AS patients compared with normal controls whereas the production of the pro-inflammatory cytokines TNF
and IFN
was reduced.
Conclusions. Our results show that AS patients express significantly higher levels of serum TGFß1 independent of the codon 10 and 25 genotype. Activation of AS PBMCs led to enhanced TGFß1 production accompanied by reduction of TNF and IFN
while the converse was observed in normal controls.
KEY WORDS: Ankylosing spondylitis, Transforming growth factor beta-1, Cytokines, Polymorphisms
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Introduction |
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TGFß1 is a pleiotropic cytokine belonging to a family of dimeric polypeptide growth factors that includes bone morphogenic proteins and activins. It regulates embryonic development, the proliferation and differentiation of cells, apoptosis, angiogenesis and wound healing [4, 5]. The expression of TGFß1 and its receptors is widespread, having been detected in both innate and specific immune response cells as well as on non-immune cells. Locally, TGFß1 has been shown to have pro-inflammatory properties, whereas systemically it has an immunosuppressive effect [4]. Altered levels of TGFß1 have been linked to numerous disease states including atherosclerosis and fibrotic disease of the kidneys, liver and lungs. TGFß1 is important for bone development and fracture healing, and has been shown to cause excessive proliferation of fibroblasts in mice with progressive ankylosis [5, 6].
There are comparatively few studies of cytokines in AS and information on serum levels and expression of TGF in peripheral blood mononuclear cells (PBMCs) in this disease is rather limited. Thus the purpose of our study was to determine protein expression of TGFß1 both in serum and in in vitro assays and their association with codon 10 and 25 TGFB1 gene polymorphisms in our population of southern Chinese AS patients.
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Patients and methods |
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Preparation of PBMCs and in vitro stimulation assays
PBMCs from AS patients and healthy donors were isolated by gradient centrifugation over Histopaque-1077 (Sigma, St Louis, MO) according to the manufacturer's recommendations and resuspended in complete RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU penicillin and 100 mg/ml streptomycin (all obtained from Invitrogen Gibco, Carlsbad, CA). PBMCs (2 x 105/well) were placed in triplicate in 96-well flat-bottom plates (Nunc, Roskilde, Denmark) in complete RPMI 1640 medium supplemented with 5% fetal calf serum (FCS) (Invitrogen Gibco) at 37°C 5% CO2 and stimulated with phytohaemagglutinin (PHA, Sigma) at 10 µg/ml for 3 days. Culture supernatants were harvested and stored at 20°C until estimated by ELISA.
ELISA
TGFß1, tumour necrosis factor-alpha (TNF), interferon-
(IFN
) and interleukin-10 (IL-10) levels in culture supernatants were detected using paired antibodies from OptEIA systems (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. The detection limit was: for TNF
, IFN
and IL-10 <10 pg/ml; for TGFß1 <20 pg/ml. Latent TGFß1 in supernatants was activated by 1 N HCl for 10 min followed by neutralization (1.2 N NaOH/0.5 M HEPES). Given the significant levels of latent TGFß1 found in bovine serum, background levels in cell-free RPMI 1640 medium containing 5% FCS were measured and subtracted from samples of in vitro assays in order to determine genuine TGFß1 production. Total TGFß1 in serum samples was measured by acid activation (2.5 N acetic acid/10 M urea) for 10 min followed by neutralization (2.7 N NaOH/1 M HEPES). Samples were then diluted 10-fold in PBS/0.05% Tween 20 and measured as described above. The TGFß1 assay used in this study has no cross-reactivity with TGFß2, TGFß3 or other cytokines (BD Biosciences).
DNA extraction and TGFß1 genotyping
Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood using the Puregene DNA purification kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's instructions. TGFß1 genotyping was by amplification refractory mutation site polymerase chain reaction (ARMS-PCR) analysis for codon 10 and 25 as previously described [8]. Products were separated and visualized by ethidium bromide-stained agarose gel electrophoresis. To avoid incorrect assessment of genotype, all tests were performed in duplicate independently. Negative and positive controls were included in each run to ensure specificity.
Statistical analysis
Data are shown as mean ± S.D. Serum TGFß1 levels were analysed with the non-parametric MannWhitney U-test. Cytokine levels were compared between stimulated and control cultures using the Student's t-test. P values less than 0.05 were considered significant. The distribution of codon 10 and 25 TGFB1 polymorphisms was analysed using Fisher's exact test to identify significant departures from the HardyWeinberg equilibrium. Finally, based on the power calculation for the size of our study population, the odds ratio of the difference in genotype frequency had to be less than 0.20.25 in order to detect a significant difference (P<0.05).
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Results |
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In vitro stimulation assays
Incubation of AS and normal PBMCs with PHA led to upregulation of TGFß1, IL-10, TNF and IFN
production as assayed by ELISA. When cultured in medium alone, PBMCs from AS patients produced more TGFß1 than those from normal donors, although this was not significant (P>0.05). Furthermore, PHA-induced TGFß1 production was significantly increased in AS patients compared with normal controls (1066 ± 250 pg/ml vs 238 ± 134 pg/ml, respectively; mean ± S.D.; P<0.05, Fig. 1A). AS PBMCs also secreted more IL-10 upon activation by PHA. In contrast, PHA induced only low and variable levels of TNF
and IFN
synthesis in AS PBMCs compared with normal controls (Figs 1BD). These results suggest that AS PBMCs exhibit an up-regulated response to TGFß1 production but a down-regulated response to TNF
and IFN
production.
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Discussion |
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TGFß1 plays a critical role in the regulation of inflammatory events, extracellular matrix synthesis and bone modelling and may be important in the pathogenesis of AS and other inflammatory diseases [1, 3, 4]. TGFß1 stimulates several processes that are critical for tissue repair, including the reduction of pro-inflammatory cytokine production from macrophages, promotion of cell growth and stimulation of extracellular matrix deposition [4, 5]. However, Toussirot et al. [9] failed to observe any association of serum TGFß1 levels and bone mass in their study, suggesting that TGFß1 may serve rather as an immunomodulator to counteract the autoimmune nature of the disease. As this cytokine directs IgA switching in B cells the raised IgA levels in AS may be a result of elevated serum TGFß1 levels [3]. In addition, most TGFß in serum appears to be derived from platelets, which contain two pools of latent TGFß1. One pool, containing the latent TGFß binding protein and the mature TGFß1 dimer, is released into the serum during clotting [10]. It is not clear whether the differences in the circulating concentration of TGFß1 among our study populations with various genotypes are correlated with the platelet TGFß1 levels. Interestingly, our study showed that PBMCs from AS patients produced significantly higher levels of TGFß1 following PHA stimulation than healthy controls, suggesting that these cells exhibit an up-regulated response in TGFß1 production. We also observed an increase in PHA-induced IL-10 production in AS patients. However, only low and variable levels of TNF and IFN
synthesis in AS PBMCs were detected compared with normal controls (Fig. 1). Similar findings were observed by Rudwaleit et al. [11], whose study demonstrated lower T-cell production of TNF
and IFN
in this disease, suggesting that there may be a systemic dysregulation of the cell-mediated immune response in AS.
In contrast to AS, lymphocyte production of total and active forms of TGFß1 has been shown to be decreased in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [12]. As the mechanisms involved in this cytokine imbalance in AS are not clear, we compared the frequency of codon 10 and 25 TGFB1 polymorphisms between AS patients and healthy controls but found no difference in genotype distribution between the two groups. We chose to study the codon 25 TGFB1 polymorphisms because of their association with diseases such as systemic sclerosis, lung and hepatic fibrosis [13] and codon 10 TGFB1 polymorphisms because of their association with osteoporosis, spinal osteoarthritis and RA [14]. The TGFB1 genotype distribution for AS patients in our study was similar to that for the controls (Table 1). Thus, although our AS patients had significantly higher serum levels of TGFß1 than healthy controls, no correlation was found with codon 10 and 25 polymorphisms of the gene, possibly as a result of ethnic differences. However, given the limited sample size and statistical power in our current study, a much larger cohort is required to confirm our findings. It is of interest that a recent study in the Finnish population with 437 individuals reported only a weak association between the rare TGFB1 + 1632 T allele and AS (P = 0.04), thus the authors conclusion that TGFB1 polymorphism plays at most a minor role in the pathogenesis of AS and that other genes encoded on chromosome 19 are involved in disease susceptibility [15].
In summary, our data show that AS patients have significantly elevated serum levels of TGFß1, independent of codon 10 and 25 TGFB1 gene polymorphisms, and a reduced ability to secrete TNF and IFN
in vitro. The mechanisms behind such cytokine imbalance are currently unclear and merit further investigation.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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