Transforming growth factor beta-1 and gene polymorphisms in oriental ankylosing spondylitis

H. S. Howe, P. L. Cheung, K. O. Kong, H. Badsha, B. Y. H. Thong, K. P. Leong, E. T. Koh, T. Y. Lian, Y. K. Cheng, S. Lam1, D. Teo1, T. C. Lau and B. P. Leung2

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


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To study serum levels of transforming growth factor beta-1 (TGFß1) and the expression of TGFß1 in in vitro peripheral blood mononuclear cell (PBMC) cultures in oriental ankylosing spondylitis (AS) patients, and to determine their association with codon 10 and 25 TGFB1 gene polymorphisms.

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{alpha}) and interferon-{gamma} (IFN{gamma}) 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{alpha} and IFN{gamma} 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{alpha} and IFN{gamma} while the converse was observed in normal controls.

KEY WORDS: Ankylosing spondylitis, Transforming growth factor beta-1, Cytokines, Polymorphisms


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Ankylosing spondylitis (AS) is characterized by axial skeletal involvement beginning with inflammation in the sacroiliac joints. The majority of patients are histocompatibility antigen HLA-B27 positive, supporting the hypothesis that disease might initially be triggered by bacterial infections that provoke a subsequent T-cell response. Importantly, both CD4+ and CD8+ T cells can be located in sacroiliac joints and entheseal structures in these patients [1, 2]. Transforming growth factor beta-1 (TGFß1) plays a critical role in the balance of inflammatory processes and has been linked to extracellular matrix synthesis, bone remodelling and fibrosis in AS [3].

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.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and controls
Seventy-two southern Chinese patients who attended the Tan Tock Seng Hospital Department of Rheumatology, Allergy and Immunology in Singapore were recruited in this study which was approved by our hospital Research Ethics committee. All cases had primary AS that met the modified New York criteria for AS [7]; patients with other clinical conditions such as psoriasis and inflammatory bowel disease were excluded. Ninety-six race-matched unrelated healthy subjects were randomly selected as controls. All blood samples from AS patients and controls were collected by venous puncture with the informed consent of the subjects. Serum samples were stored at –80°C until assayed by enzyme-linked immunosorbent assay (ELISA).

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{alpha}), interferon-{gamma} (IFN{gamma}) 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{alpha}, IFN{gamma} 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 Mann–Whitney 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 Hardy–Weinberg 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 0.2–0.25 in order to detect a significant difference (P<0.05).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Serum levels of TGFß1
We found that serum levels of TGFß1 in AS patients were significantly higher than in controls (12.08 ± 3.56 ng/ml vs 4.95 ± 1.20 ng/ml, respectively; mean ± S.D.; P<0.001). Median serum concentrations of TGFß1 in our AS patients did not differ significantly between codon 10 and codon 25 genotypes (data not shown).

In vitro stimulation assays
Incubation of AS and normal PBMCs with PHA led to upregulation of TGFß1, IL-10, TNF{alpha} and IFN{gamma} 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{alpha} and IFN{gamma} synthesis in AS PBMCs compared with normal controls (Figs 1B–D). These results suggest that AS PBMCs exhibit an up-regulated response to TGFß1 production but a down-regulated response to TNF{alpha} and IFN{gamma} production.



View larger version (30K):
[in this window]
[in a new window]
 
FIG. 1. Production of cytokines by PBMC following stimulation with PHA in both AS and normal controls. PBMCs were purified from healthy individuals (n = 5) or AS patients (n = 6) and cultured for 72 h at 37°C with PHA (10 µg/ml). Cytokine concentrations in the culture supernatant were determined by ELISA. Experiments from each donor were performed in triplicate and graphs show mean ± S.D. from all donors. *P<0.05, Student's t-test.

 
TGFB1 gene polymorphisms and susceptibility to AS
TGFB1 genotyping data are presented in Table 1. No significant difference in codon 25 genotypes was found between AS patients and controls, as both groups predominantly had the GG genotype for codon 25 (95.8 and 96.9% respectively, P = 0.516). No significant difference was found also for codon 10 polymorphisms between patients and controls. Finally, we failed to observe any significant association between TGFB1 codons 10, 25 and disease susceptibility in our local population (data not shown).


View this table:
[in this window]
[in a new window]
 
TABLE 1. TGFB1 gene polymorphism and susceptibility to AS

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This study demonstrates that our local southern Chinese AS patients have significantly elevated levels of TGFß1 in their serum, and also that AS PBMCs have an up-regulated response in TGFß1 production. Our results do not support the hypothesis of an association between a polymorphism in the signal sequence of the TGFB1 gene and susceptibility to AS. The significance of the elevated serum TGFß1 levels observed in our study is not known and the precise role of TGFß1 in AS pathogenesis and disease perpetuation is still not clear.

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{alpha} and IFN{gamma} 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{alpha} and IFN{gamma} 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{alpha} and IFN{gamma} in vitro. The mechanisms behind such cytokine imbalance are currently unclear and merit further investigation.


    Acknowledgments
 
This study was supported by Tan Tock Seng Hospital Research Grant RI 99/04, the Seah Cheng Siang Research Fellowship, Singapore and Biomedical Research Council grant 01/1/28/18/016. B. P. Leung was supported by grant NHG RCP/01005 from the National Healthcare Group, Singapore.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Sieper J, Braun J, Rudwaleit M, Boonen A, Zink A. Ankylosing spondylitis: an overview. Ann Rheum Dis 2002;61 (Suppl 3):iii8–iii18.[Medline]
  2. Braun J, Bollow M, Neure L et al. Use of immunohistologic and in situ hybridization techniques in the examination of sacroiliac joint biopsy specimens from patients with ankylosing spondylitis. Arthritis Rheum 1995;38:499–505.[ISI][Medline]
  3. Archer JR. Ankylosing spondylitis, IgA, and transforming growth factors. Ann Rheum Dis 1995;54:544–6.[ISI][Medline]
  4. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 2000;342:1350–8.[Free Full Text]
  5. Ramoshebi LN, Matsaba TN, Teare J, Renton L, Patton J, Ripamonti U. Tissue engineering: TGF-beta superfamily members and delivery systems in bone regeneration. Expert Rev Mol Med 2002;4:1–11.
  6. Krug HE. Fibroblasts from mice with progressive ankylosis proliferate excessively in response to transforming growth factor-beta 1. J Investig Med 1998;46:134–9.[ISI][Medline]
  7. Van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:361–8.[ISI][Medline]
  8. Perrey C, Turner SJ, Pravica V, Howell WM, Hutchinson IV. ARMS-PCR methodologies to determine IL-10, TNF-alpha, TNF-beta and TGF-beta 1 gene polymorphisms. Transpl Immunol 1999;7:127–8.[CrossRef][ISI][Medline]
  9. Toussirot E, Racadot E, Nguyen NU, Dumoulin G, Wendling D. Absence of relation between TGF beta 1 serum levels and bone mass in ankylosing spondylitis patients. Clin Exp Rheumatol 2000;18:111.[ISI][Medline]
  10. Grainger DJ, Wakefield L, Bethell HW, Farndale RW, Metcalfe JC. Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin. Nat Med 1995;1:932–7.[ISI][Medline]
  11. Rudwaleit M, Siegert S, Yin Z et al. Low T cell production of TNFalpha and IFNgamma in ankylosing spondylitis: its relation to HLA-B27 and influence of the TNF-308 gene polymorphism. Ann Rheum Dis 2001;60:36–42.[Abstract/Free Full Text]
  12. Ohtsuka K, Gray JD, Stimmler MM, Horwitz DA. The relationship between defects in lymphocyte production of transforming growth factor-beta1 in systemic lupus erythematosus and disease activity or severity. Lupus 1999;8:90–4.[CrossRef][ISI][Medline]
  13. Ohtsuka T, Yamakage A, Yamazaki S. The polymorphism of transforming growth factor-beta1 gene in Japanese patients with systemic sclerosis. Br J Dermatol 2002;147:458–63.[CrossRef][ISI][Medline]
  14. Sugiura Y, Niimi T, Sato S et al. Transforming growth factor beta1 gene polymorphism in rheumatoid arthritis. Ann Rheum Dis 2002;61:826–8.[Abstract/Free Full Text]
  15. Jaakkola E, Crane AM, Laiho K et al. The effect of transforming growth factor-beta 1 gene polymorphisms in ankylosing spondylitis. Rheumatology 2004;43:32–8.[Abstract/Free Full Text]
Submitted 13 April 2004; revised version accepted 7 September 2004.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
44/1/51    most recent
keh426v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Howe, H. S.
Articles by Leung, B. P.
PubMed
PubMed Citation
Articles by Howe, H. S.
Articles by Leung, B. P.
Related Collections
Other Rheumatology