The -308 polymorphism in the tumour necrosis factor (TNF) gene promoter region and ex vivo lipopolysaccharide-induced TNF expression and cytotoxic activity in Chilean patients with rheumatoid arthritis

J. Cuenca1, M. Cuchacovich2, C. Pérez1, L. Ferreira1,2, A. Aguirre1, I. Schiattino3, L. Soto2, A. Cruzat1, F. Salazar-Onfray1 and J. C. Aguillón1,

1 Disciplinary Program of Immunology, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile,
2 Rheumatology Section, Department of Medicine, University of Chile Clinical Hospital and
3 School of Public Health, Faculty of Medicine, University of Chile, Santiago, Chile


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate the association of the -308 polymorphism in the promoter region of the tumour necrosis factor (TNF) gene with susceptibility to the development of RA. We also explored the expression and cytotoxicity of TNF in relation to the -308 polymorphism.

Methods. We recruited 92 RA patients and 42 healthy control subjects. Genotyping for the TNF promoter was performed by polymerase chain reaction–restriction fragment length polymorphism analysis. To study the overexpression of TNF we used a whole-blood culture system. TNF cytotoxicity was assessed in the L929 cell line.

Results. The TNF2 allele was found in 23% of RA patients and 10% of controls. Although both groups showed high variability in serum TNF concentration, in the lipopolysaccharide-induced TNF level and in the cytotoxicity of the cytokine in the L929 cell line, these differences were not associated with the -308 TNF polymorphism.

Conclusion. No associations were found between the -308 TNF promoter polymorphism, serum and ex vivo TNF levels and the cytotoxic activity of TNF in RA patients.

KEY WORDS: TNF, Polymorphism, Gene, Rheumatoid arthritis.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints, hyperplasia and overgrowth of synoviocytes, with ensuing destruction of articular cartilage [1]. Cumulative studies suggest that RA occurs in patients whose genetic background includes multiple common genetic risk factors that have been inherited. The association with the major histocompatibility complex (MHC) DRß1 alleles that share the amino acid sequence at position 70–74 in the binding groove of the MHC has been well recognized in Caucasian patients with RA. Approximately 30% of the genetic susceptibility to RA has been attributed to these alleles [2].

The importance of the unbalanced production of cytokines such as tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6 in affected tissues and the successful introduction of an anti-TNF monoclonal antibody into therapeutic use have suggested that TNF is involved in the pathogenesis of the disease [3, 4]. It has been suggested that variability in the promoter and coding regions [single-nucleotide polymorphisms (SNPs)] of the TNF gene may modulate the magnitude of the secretory response of this cytokine [57]. Numerous studies on the possible functional relevance of these polymorphisms as part of transcriptionally functional motifs have been carried out using transient transfection of reporter genes controlled by allelic variants of the TNF promoter [6, 7] or investigating the expression of TNF by cells derived from individuals with different TNF promoter genotypes [5, 8]. Polymorphisms in the TNF promoter gene may affect transcriptional regulation by modifying the binding site of specific transcription factors [9, 10].

Several SNPs have been identified in the human TNF gene promoter to date [11]. One of those is a guanine (G) to adenine (A) transition at position -308, which generates the TNF1 and TNF2 alleles respectively [12]. Studies of TNF expression have produced conflicting results. Some studies using reporter constructs have concluded that the TNF2 allele is associated with a high level of in vitro TNF expression [6, 7], while other studies have concluded that it is not [13, 14]. The TNF2 allele has also been linked to increased susceptibility to and severity of a variety of illnesses, including cerebral malaria [15], inflammatory bowel disease [5, 16], systemic lupus erythematosus [17] and ankylosing spondylosis [18]. However, association between the -308 TNF polymorphism and RA is still controversial [1924].

In this study, the frequency of the -308 TNF promoter polymorphism, the serum TNF concentration, the lipopolysaccharide (LPS)-induced TNF level and the cytotoxic activity of TNF were compared between patients with RA and healthy control subjects. We also analysed the possible association of this polymorphism with the serum and ex vivo levels of TNF and with the cytotoxicity of TNF in the two groups of subjects.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and controls
Ninety-two patients diagnosed with RA (in accordance with the 1987 revised criteria of the American College of Rheumatology) (85 women and seven men, average age 51.1±11.7 yr) and 42 healthy individuals (28 women and 14 men, average age 36.3±16.5 yr) were studied. All RA patients and controls and two generations of ancestors for each group were born in Chile. None of the RA patients received TNF-neutralizing therapy during the study or had received it previously. Medical and dental status were determined in both groups.

Genotyping of -308 TNF promoter polymorphism
The genotype was analysed by a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique [12]. A 107 base pair region of the TNF gene was amplified using the following primers: 5'-AGG CAA TAG GTT TTG AGG GCC AT-3' and 5'-TCC TCC CTG CTC CCG GAT TTC CG-3'. DNA was amplified for 30 cycles at 94°, 60° and 72°C for 30 s, 35 s and 1 min respectively, ending with a 10-min extension cycle at 72°C.

Whole-blood culture system (WBCS) for TNF expression
Briefly, blood samples from 92 RA patients and 42 controls were diluted five times in RPMI-1640 medium, supplemented with L-glutamine, penicillin and streptomycin. Each diluted blood sample was analysed in triplicate. After 4 h of incubation, LPS (Escherichia coli, serotype 026:B6; Sigma, St Louis, MO, USA) was added to each culture well to a final concentration of 10 µg/ml. After an additional 12 h of incubation, the supernatants of each culture well were centrifuged, and TNF was measured with an immunoradiometric assay (IRMA) [25].

Cytotoxic assay for TNF
As described [26], 100 µl of a suspension of 4x105 L929 fibroblasts (ATCC CCL 1; American Type Culture Collection, Rockville, MD, USA) was seeded in 10% FBS (fetal bovine serum) in DMEM (Dulbecco's Modified Eagle Medium) in 96-well flat-bottomed microtitration plates. The culture medium was removed and 100 µl of each supernatant in triplicate, obtained from the ex vivo LPS-stimulated WBCS, was added (42 supernatants from each group of individuals). All supernatants contained 1 ng/ml of TNF, and 0–5 ng/ml dilutions of recombinant TNF were used as standards. Next, 100 µl of medium containing actinomycin D was added to each well, yielding a final concentration of 5 µg/ml. After incubation, the culture medium was removed and 0.05% crystal violet in 20% ethanol was added. To wash the stained cells, 100% methanol was added and the absorbance at 600 nm was determined.

Immunoradiometric assay for TNF
IRMA was performed as described by Aguillón et al. [27], using an anti-human TNF monoclonal antibody generated in our laboratory.

Detection of TNF in serum
An ultra-sensitive enzyme-linked immunosorbent assay (ELISA) kit (BioSource International, Camarillo, CA, USA; quantification limit 0.5 pg/ml) was used according to the manufacturer's instructions.

Statistical analysis
Comparison of proportions between groups was performed with the {chi}2 test or Fisher's exact test. When comparing two groups, we used Wilcoxon's non-parametric two-sample test. Student's t-test was used for statistical comparison of data from patients and controls. A P value of <=0.05 was considered statistically significant. Odds ratios (OR), as estimates of the relative risk, were calculated with 95% confidence intervals (CI). Stata 5.0 [28] software was used for the analysis of relationships or associations between variables.

Volunteer human blood donors provided informed written consent. Trained medical personnel carried out venipunctures in humans. The ethics review board of the University of Chile approved this study.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
-308 TNF promoter polymorphism in RA patients and controls
Table 1Go shows the frequencies of -308 TNF promoter polymorphism in RA patients and control individuals. The homozygous TNF1 allele was present in 77% of the RA patients and 90% of controls, while the TNF2 allele (heterozygous and homozygous) was found in 23% of the RA patients and 10% of controls (OR=2.8, P=0.051). The homozygous TNF2 allele was detected in only one RA patient.


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TABLE 1. Genotype distribution of the -308 TNF promoter polymorphism in RA patients and healthy controls

 

Spontaneous and LPS-stimulated TNF expression and circulating TNF levels in RA patients and controls
As shown in Fig. 1Go, the spontaneous and LPS-induced TNF levels did not differ significantly between RA patients and healthy controls (P=0.710 and P=0.158 for spontaneous and LPS-induced TNF levels respectively). A wide range of inter-individual variability in spontaneous and induced-TNF levels in RA patients and controls was found (Table 2Go).



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FIG. 1. Spontaneous and LPS-induced TNF expression in patients with RA and healthy controls. TNF levels, measured by IRMA, were detected in supernatants obtained from LPS-stimulated and non-stimulated whole blood cultures. Bars show the mean TNF concentration of each group and vertical lines the S.D.

 

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TABLE 2. Differences in spontaneous and LPS-induced TNF concentrations (pg/ml) in RA patients and healthy controls

 
As depicted in Fig. 2Go, we did not observe differences between spontaneous and LPS-stimulated or over-expressed TNF levels either in RA patients or in controls when they were compared according to -308 TNF gene promoter genotype. Although the LPS-induced TNF levels were higher in control individuals heterozygous for the TNF2 allele than in RA patients, no significant differences were found (P>0.05). When both groups were divided into subgroups according to -308 TNF promoter genotype (G/G and G/A), broad inter-individual variability in the spontaneous and induced TNF expression capabilities in RA patients and controls was observed (Table 3Go).



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FIG. 2. Spontaneous and LPS-induced TNF expression by genotypes of the -308 TNF gene promoter polymorphism in patients with RA and healthy controls. TNF levels, measured by IRMA, were detected in supernatants obtained from LPS-stimulated and non-stimulated whole-blood cultures. Bars show the mean concentration of each group and vertical lines the S.D.

 

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TABLE 3. Differences in spontaneous and LPS-induced TNF concentrations (pg/ml) according to -308 TNF promoter polymorphism in RA patients and healthy controls

 
In addition, a high level of inter-individual variation in serum TNF concentration was observed in RA patients and controls. The mean serum TNF concentration in RA patients (18.9 pg/ml) was 6-fold higher than that in controls (3.2 pg/ml) (P=0.000). In both groups, no differences were found among serum TNF concentration when the comparison was done according to -308 TNF promoter genotype.

Cytotoxic TNF activity in RA patients and controls
The biological activity of the TNF present in the supernatants from the ex vivo assays was determined in L929 murine fibroblasts. The biological activity of TNF, at equivalent concentrations, varied among individuals in RA patients and in controls (results not shown). No differences were observed between RA patients and controls when compared for mean cell survival. Similarly, cell survival did not differ according to -308 TNF genotype in either RA patients or controls.



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FIG. 3. Cytotoxic activity of TNF in supernatants of ex vivo, LPS-induced, whole-blood cultures from patients with RA and healthy controls. L929 murine cells were treated with supernatants from individuals or human recombinant TNF as standard. Mean cell survival for controls and patients pooled (open bars), and for genotypes G/G (grey bars) and G/A (black bars). Vertical lines show S.D.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Several studies indicate that individuals display different capabilities for TNF production, which is independent of age and does not show a cyclic rhythm [29]. Most evidence suggests a strong association with specific genetic components, such as HLA and polymorphisms affecting the TNF promoter gene [5, 8]. The -308 TNF promoter polymorphism has been related to increased transcriptional activity of the TNF gene [6, 7]. However, the association of the TNF2 allele with susceptibility to RA and/or severity of the disease has been controversial [1924].

Our evaluation of the -308 TNF polymorphism in Chilean RA patients and controls demonstrated that the TNF2 allele occurred in the heterozygous state in 22 and 10% of individuals respectively. Although the odds ratio (2.8) suggests an association between the presence of the polymorphism and the disease, this was not statistically significant (P=0.051). This agrees with results obtained in several studies that have indicated a lack of association between -308 TNF promoter polymorphism and susceptibility to RA [2124]. In contrast, Danis et al. [19] reported a higher frequency of the TNF2 allele in Caucasian RA patients than in healthy individuals.

One aspect to be considered in the interpretation of these studies is the way the -308 TNF polymorphism frequencies are distributed in the different ethnic groups. As reported by us [30], significant differences in the -308 TNF genotype frequencies of the Chilean population were detected when compared with Caucasian -308 TNF genotypes.

On the other hand, numerous studies have investigated the relationship of TNF promoter polymorphisms and the clinical and radiological state of the disease. Unlike the -308 TNF promoter polymorphism [2123, 31], the -238 polymorphism has been demonstrated to play a central role in the development of a more severe form of RA [22, 23, 32, 33]. However, Cvetkovic et al. [20] have also recently reported an association of the -308 TNF polymorphism with the severity of the disease.

The search for a function for the -308 polymorphism in the expression of TNF has produced controversial results [58, 11, 13, 14]. Our approach for measuring the TNF production was based on detecting its ex vivo overexpression by LPS-stimulated peripheral mononuclear cells present in whole-blood culture [25]. The WBCS is a useful ex vivo technique for the study of cytokine production because it maintains the microenvironment of the blood and avoids the extraction procedure associated with modifying cell ratios and activation. It is the technique of choice for exploring inter-individual variation in TNF production and its relationship to the genetic background [8, 25].

In agreement with previous reports [25, 34], we found wide inter-individual variability in TNF expression capability both in controls and in RA patients (Table 2Go). This behaviour was also observed for serum TNF concentration. No significant differences were detected in either RA patients or controls when the means of spontaneous and LPS-induced TNF levels were compared (Fig. 1Go). A similar result was obtained when inter- and intra-group comparisons were performed with respect to genotype (Fig. 2Go and Table 3Go). Our results are consistent with recent studies using transcriptional activity assays in cells from RA patients, in which no functional differences for the allelic forms of the -308 TNF polymorphism were reported [13, 14]. However, as chromosome 6 is highly polymorphic and characterized by extensive linkage disequilibrium, the possibility exists that SNPs are haplotype markers rather than being themselves responsible for the disease association [13].

However, the presence of pharmacological treatment for all of the RA patients could have been responsible for the absence of differences in the LPS-induced TNF levels between patients and controls. It is well established that anti-inflammatory and immunosuppressive drugs have an inhibitory effect on proinflammatory cytokine expression, generating a hyporeactive state of the activated mononuclear cells [35]. In order to discard this possibility, a group of untreated patients should have been included. However, due to the progression of the disease and the consequent detriment of the patient's quality of life, this would have been ethically unacceptable.

Multiple studies have shown that the TNF levels in serum and synovial fluid and tissue from RA patients are higher than those measured in healthy individuals [36]. As expected, the RA patient group had a higher serum TNF concentration (approximately six times greater than that of the controls).

The biological activities of TNF are mediated through two kinds of receptors, TNFRI and TNFRII [37]. An association between TNFRII polymorphism with systemic lupus erythematosus and with the cytotoxic activity of TNF induced by the receptor was reported recently [38]. In the present study, we observed that biological TNF activity in L929 fibroblasts varied over a wide range, with cell survival of 10–88 and 3–92% for RA patients and healthy individuals respectively. Thus, given the same amount of TNF, the cytokine from distinct individuals displays differential capacity to induce cytotoxicity. Unlike the correlation between TNF concentration and TNF cytotoxicity reported for type 2 diabetics [39], we did not find any difference in the cytotoxic activity of TNF between RA patients and controls.

Because the TNF concentration in RA patients is elevated, our results suggest that native TNF cytotoxic activity is independent of its concentration, and biologically inactive TNF molecules may exist in supernatants from individuals whose cytotoxic TNF activity is diminished. However, although nucleotide differences in the coding region of the TNF gene have been described, they do not correlate with changes in the TNF amino acid sequence that could have explained the differential cytotoxic activity of the cytokine [40].

In summary, the -308 TNF promoter polymorphism may not be associated with the presence of RA, an increase in the circulating TNF concentration, the capacity to produce TNF in the WBCS, or the cytotoxic activity of TNF. Thus, other factors may be important in determining the circulating levels of TNF in RA.


    Acknowledgments
 
This research was supported by FONDECYT-CHILE grant 1990936, ID-University of Chile grant ENL-02/13 and Aventis Laboratories. We thank Ms Juana Orellana and Ruth Mora for excellent technical help.


    Notes
 
Correspondence to: J. C. Aguillón, Disciplinary Program of Immunology, ICBM, Faculty of Medicine, University of Chile, Independencia 1027, Santiago, Chile. E-mail: jaguillo{at}machi.med.uchile.cl Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 25 March 2002; Accepted 8 August 2002





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