Rheumatology

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Rheumatology 2001; 40: 267-273
© 2001 British Society for Rheumatology

Glucocorticoid-mediated repression of inflammatory cytokine production in fibroblast-like rheumatoid synoviocytes is independent of nuclear factor-B activation induced by tumour necrosis factor

C. W. Han, J. H. Choi¹, J. M. Kim, W. Y. Kim, K. Y. Lee¹ and G. T. Oh¹

Department of Orthopaedic Surgery, College of Medicine, The Catholic University of Korea, Seoul and
¹ Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon, Korea

	Abstract

Top Abstract Introduction Patients and methods Results Discussion References

 
Objective. To determine whether steroids inhibit the production of inflammatory cytokines by the inhibition of nuclear factor B (NF-B) activation in fibroblast-like rheumatoid synoviocytes (FLSs) under inflammatory conditions, and to determine whether steroids stimulate the induction of synthesis of the inhibitory protein IB- in the anti-inflammatory immune response of these cells.

Methods. Expression of the interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) genes was measured by semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR), and the secreted IL-6 was measured with the enzyme-linked immunosorbent assay. Inhibition of the NF-B activation was examined with the electrophoretic mobility shift assay (EMSA). In order to study dexamethasone (DEX)-dependent regulation of IB- expression, we performed Western blotting before and after stimulation with tumour necrosis factor (TNF-).

Results. The inflammatory cytokine study showed that DEX suppressed gene expression and the production of protein in FLSs. EMSA demonstrated that identical amounts of NF-B were present in the nucleus of the FLSs stimulated by TNF-, with or without pretreatment with DEX. Treatment of FLSs with DEX did not induce an increase in IB- sufficient to prevent nuclear translocation of NF-B on stimulation with TNF-.

Conclusion. DEX may suppress the production of inflammatory cytokines, such as IL-6 and IL-1ß, but it neither prevents the translocation of NF-B to the nucleus nor induces the synthesis of IB- protein in FLSs stimulated by TNF-.

KEY WORDS: Dexamethasone, Rheumatoid arthritis, Synoviocytes, NF-B, IB-, TNF-, IL-6, IL-1ß.

	Introduction

Top Abstract Introduction Patients and methods Results Discussion References

 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferative and invasive synovitis. Human fibroblast-like synoviocytes respond to several cytokines and growth factors, including interleukin 1 (IL-1), tumour necrosis factor (TNF-) and platelet-derived growth factor. Fibroblast-like rheumatoid synoviocytes (FLSs) exhibit characteristics of inflammatory cells that are critically involved in several aspects of rheumatoid pathophysiology [1]. The level of TNF- is known to be elevated in the synovial fluid of RA patients [2, 3], and TNF- plays an important role in synovial cell activation, and thus serves as a major molecular regulator in RA.

The NF-B/Rel family of proteins includes c-Rel, p65 (RelA), Rel B, p50/p105, p49/p100, p55/p98 and the Drosophila morphogen dorsal, and they exist as homo- or heterodimers, with p50/65 heterodimers the most commonly found [4]. NF-B has been suggested to play an important role in gene regulation during inflammatory and immune reactions. NF-B exists as a dimeric complex that is present in the cytosol in an inactive state bound to inhibitory proteins, collectively termed IB [5]. Several IB proteins have been identified, including IB- [6] and the more recently cloned IB-ß [7] and IB- [8]. When the cells are stimulated, specific kinases lead to the phosphorylation of IB, which is subsequently degraded in a ubiquitin-dependent step by the proteasome, a multicatalytic high molecular weight protease system [9]. The degradation of IB and the release of NF-B lead to the transactivation of genes, including the IB- gene. Therefore, this establishes an autoregulatory loop in which newly synthesized IB- is thought to restore the cytoplasmic pool of latent NF-B [10]. The activation of NF-B has been implicated in the regulation of transcription in a variety of genes, such as those encoding TNF- [11], IL-6 [12] and IL-1ß [13], which are considered to be involved in the pathophysiology of rheumatoid arthritis.

Glucocorticoids can down-regulate the expression of several inflammatory genes, including those encoding cytokines such as IL-1, TNF- and IL-6 [14]. However, it remains unclear whether the induction and activation of NF-B is an essential event in the repression of inflammatory cytokine production. It now appears to be a mechanism that works through a protein–protein interaction of NF-B subunits and the glucocorticoid receptor, as in endothelial [15] and mesangial [16] cells. Another theory holds that the mechanism involves glucocorticoid receptor-induced synthesis of IB-, as in lymphocytes and monocytes [17, 18]. Under the latter hypothesis, newly synthesized IB- would inactivate NF-B by sequestering it into a cytoplasmic form.

We investigated whether glucocorticoids can inhibit the translocation of NF-B to the nucleus and whether glucocorticoids can induce the production of IB- after stimulation by TNF- in primary cultured FLSs from RA patients.

	Patients and methods

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Patients
Synovial tissues were obtained, with consent, from nine RA patients who were undergoing total knee replacement or arthroscopic synovectomy. From these samples, three cell lines were studied. To avoid the effect of steroids on the synovial cells, the patients were instructed to cease steroid medication for 2 months before the operation. All patients satisfied the 1987 revised diagnostic criteria of the American College of Rheumatology [19].

Synoviocyte culture
Synovial tissues were minced and treated for 4 h with 4 mg/ml collagenase (type 1; Worthington Biochemical, Freehold, NJ, USA) in Dulbecco's modified Eagle medium (DMEM) at 37°C in 5% carbon dioxide. Dissociated cells were plated in DMEM supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were used between the third and fifth passages. Where indicated, cells were treated with human recombinant TNF- (Genzyme, Cambridge, MA, USA).

Determination of IL-6 and IL-1ß gene expression by semi-quantitative RT-PCR
The expression of IL-6 and IL-1ß was determined by semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR) using ß-actin as the internal control. We performed densitometric analysis with image analysis software (Bio 1D Version 99; Vilber Lourmat, France) to determine the relative band density. The internal control for the ß-actin was performed in the 25th cycle to avoid the saturation of the PCR product. FLSs (5x10⁶ cells per 100 mm culture dish) were either left untreated or treated with 1 µM dexamethasone (DEX) and stimulated with TNF- (10 ng/ml) for 12 h. Total RNA was extracted from the primary-cultured FLSs using Trizol (Gibco) reagent. Complementary DNA was synthesized from 250 ng total RNA in a 50-µl reaction. All reagents were obtained from Perkin-Elmer (Norwalk, CT, USA). The reaction proceeded at 45°C for 15 min then 99°C for 5 min. The PCR assay used 10 pmol primers. The forward primer for human IL-6, 5'-ATCCTCGACGGCATCTCAGCC-3', and the reverse primer, 5'-CTACATTTGCCGAAGAGCCCT-3', define an amplicon of 462 base pairs (bp). The forward primer for IL-1ß, 5'-GACACATGGGATAACGAGGCT-3', and the reverse primer, 5'-TTAGGAAGACACAAATTGCAT-3', define an amplicon of 565 bp. The forward primer for ß-actin, 5'-GCCATGTACGTTGCTATCCAGGCTG-3', and the reverse primer, 5'-AGCCGTGGCCATCTCTTGCTCGAAG-3', define an amplicon of 300 bp. The PCR was carried out in a total volume of 20 µl containing 5 µl cDNA template, 20 pmol primer, 250 µM dNTP, 1.5 mM MgCl₂ and 1 U Taq DNA polymerase. The PCR profile started with a denaturing phase at 94°C for 10 min. Subsequent cycles consisted of a 1-min elongation phase at 72°C, a 45-s denaturing phase at 94°C and a 1-min annealing phase at 57°C. The PCR reaction was concluded by a 10-min elongation phase at 72°C.

Detection of IL-6 production by FLSs
Equal numbers of cells (5x10⁶ cells per 100 mm culture dish) were either left untreated or incubated with DEX (1 µM) and stimulated with TNF- (10 ng/ml) for 12 h. After incubation, IL-6 was measured in the culture supernatants with a high-sensitivity enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Nuclear protein extraction
Equal numbers of FLSs (5x10⁶ cells per 100 mm culture dish) were either left untreated or pretreated with DEX (1 µM) for 2 h and then stimulated with TNF- (10 ng/ml) for 10 min, 1 h or 2 h. Cells were harvested with a hypotonic lysis buffer containing 1 M HEPES, 1 M MgCl₂ and then centrifuged at 3000 r.p.m. for 5 min. After removing the supernatant, containing cytosolic protein, the nuclear protein was lysed in hypertonic buffer containing 30 mM HEPES, 0.3 mM EDTA, 1.5 mM MgCl₂, 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mg/ml each of aprotinin and leupeptin, and 450 mM KCl. After 30 min incubation on ice, the lysate was centrifuged at 15 000 r.p.m. for 15 min. The supernatant was subjected to electrophoretic mobility shift assay (EMSA). All steps were performed on ice. Aliquots of the supernatant were stored at -70°C until analysis.

Electrophoretic mobility shift assay
Double-stranded oligonucleotides (5 pM) corresponding to the NF-B/Rel binding sequence were incubated with 3 µl of [-³²P]dATP, 2 µl of T4 kinase buffer (10x; NEB, Beverly, MA, USA), 1 µl T4 kinase (NEB) and 9 µl distilled water for 30 min at 37°C and applied to a Sephadex G-50 column. The sequence used for the NF-B was as follows: 5'-GATCCGAGGGGACTTTCCGCTGGGGACTTTCCAGG-3' [20]. Radiolabelled nucleotides were collected and used in EMSA. Five micrograms of nuclear protein was incubated with 2 µg poly(dI-dC), 2 µl radiolabelled probe (20000 c.p.m.), 13 µl binding buffer containing 100 mM NaCl, 30 mM HEPES, 1.5 mM MgCl₂, 0.3 mM EDTA, 10% glycerol, 1 mM PMSF, 1 µg/µl of aprotinin and 1 µg/µl of leupeptin for 20 min at room temperature. After incubation, the protein–DNA complexes were separated at 120 V for 2 h using a 4.8% polyacrylamide gel in 0.5x Tris-borate EDTA buffer. After electrophoresis, the gel was dried and the binding activity was visualized by autoradiography. Specificity was verified by the addition of a 100-fold excess of unlabelled oligonucleotide for NF-B or unrelated sequence as competitor. For the gel supershift assay, nuclear protein was preincubated for 30 min with rabbit polyclonal antibodies against human NF-B p50, p65, c-Rel and Rel-B subunits (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Western blotting for IB-
Equal numbers of FLSs (5x10⁶ cells per 100 mm culture dish) were either left untreated or incubated with DEX (1 µM) for 2 h and then stimulated with TNF- (10 ng/ml) for 10 min, 1 h or 2 h. After incubation, the cells were washed with PBS, collected and suspended in 50 µl distilled water. Whole cellular protein was obtained by repeated fast freezing and thawing. Thirty micrograms of total cellular protein was separated by the use of 6% polyacrylamide gel containing 10% sodium dodecyl sulphate (SDS), 3% sucrose, 10 mM urea, 7.5 mM Tris–HCl, 0.035% N, N, N', N'-tetramethylenediamine and 7 mg ammonium persulphate. Separated proteins were transferred to a nitrocellulose membrane (Millipore, Bradford, MA, USA) at 36 mA in a transfer buffer containing 39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol. The membranes were incubated with polyclonal rabbit anti-mouse IB- antibody at 1:200 dilution (Santa Cruz Biotechnology). The horseradish peroxidase-conjugated anti-rabbit immunoglobulin was used as a secondary antibody at 1:1500 dilution. The detection of IB- was performed using electrochemiluminescence detection reagent (Amersham, UK).

Statistics
The data are expressed as mean±S.E.M. Statistical significance was determined by Student's t-test, and P<0.01 was considered significant.

	Results

Top Abstract Introduction Patients and methods Results Discussion References

 
Dexamethasone represses the cytokine production and gene expression stimulated by TNF-
To study the effects of DEX on cytokine production and gene expression in the FLSs, we set up four groups of FLSs. Group I was the control group, group II was treated with DEX (1 µM) only, group III was treated with TNF- (10 ng/ml) only, and group IV was treated with both DEX and TNF-. After 12 h of incubation, the gene expression levels of IL-6 and IL-1ß were measured by semi-quantitative RT-PCR (Fig. 1). Additionally, we measured the IL-6 level in the culture supernatants by ELISA (Fig. 2). The amount of IL-1ß produced in FLSs is too low to detect in culture supernatants [21], so we did not measure the amount of IL-1ß by ELISA but determined the gene expression level for IL-1ß by semi-quantitative RT-PCR (Fig. 1A and C). FLSs from RA patients expressed inflammatory cytokine genes and produced the proteins of those genes, IL-6 and IL-1ß (group I). DEX significantly decreased inflammatory cytokine gene expression and protein production compared with untreated FLSs (group II). Additionally, DEX significantly decreased inflammatory cytokine gene expression and protein production induced by treatment with TNF- (groups III and IV). The concentrations of IL-6 in groups I, II, III and IV were 602.96±20.02, 67.62±3.46, 976.89±13.29 and 478.51±14.97 pg/ml respectively (Fig. 2). Semi-quantitative RT-PCR and ELISA were conducted with three primary cultures from three patients. The results from the different cultures showed same pattern (Figs 1 and 2).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Levels of IL-6 and IL-1ß gene expression as determined by semi-quantitative RT-PCR. (A) Results of one of the three experiments. (B) Relative level of IL-6 gene expression. (C) Relative level of IL-1ß gene expression. M, DNA size standards; I, untreated control; II, treated with DEX (1 µM) only; III, treated with TNF- (10 ng/ml) only; IV, treated with both DEX and TNF-. Semi-quantitative RT-PCR was performed as described in Patients and methods. Each bar represents the mean±S.E.M. of three experiments. *P<0.05 vs group I; **P<0.05 vs group III.

 

View larger version (10K): [in this window] [in a new window]   FIG. 2. Amount of IL-6 secreted into the culture supernatant as determined by ELISA. I, untreated control; II, treated with DEX (1 µM) only; III, treated with TNF- (10 ng/ml) only; IV, treated with both DEX and TNF-. ELISA was performed as described in Patients and methods. Results are shown as means of duplicate determinations in the three experiments. Bars represent S.D. *P<0.001 vs group I; **P<0.001 vs group III.

 

Dexamethasone treatment does not interfere with the formation of active NF-B in FLSs in primary culture
In order to investigate whether glucocorticoids can inhibit the translocation of NF-B to the nucleus after stimulation by TNF- in primary culture of FLSs, we examined the translocation of NF-B to the nucleus using EMSA. The potential influence of DEX on TNF--activated NF-B in FLSs was tested by EMSA (Fig. 3A). There were no significant changes in the intensity of the bands seen, provided the TNF- was kept in the medium for 10 min, 1 h or 2 h. Supershift assays using antibodies to NF-B subunits demonstrated that p65 and p50 were the predominant subunits involved (Fig. 3B). As is evident from Fig. 3A, DEX had no effect on the DNA-binding activity of NF-B at any time. Our results demonstrated that DEX did not prevent nuclear translocation of NF-B activated by TNF-.

View larger version (66K): [in this window] [in a new window]   FIG. 3. Effect of DEX on the DNA-binding activity of NF-B in nuclear extracts of stimulated FLSs. (A) FLSs were either left untreated or pretreated with DEX (1 µM) for 1 h, and then stimulated with TNF- (10 ng/ml) for the times indicated. Nuclear proteins were extracted and incubated with radiolabelled oligonucleotides containing a binding site for NF-B. The resulting complexes were analysed by EMSA. (B) Nuclear extracts from FLSs were incubated with the indicated antibodies before EMSA. Binding specificity was demonstrated by competition with unlabelled oligonucleotides of identical (Cold B) or unrelated sequences (Cold mB).

 

DEX treatment has no effect on TNF- activated IB- protein levels
In order to determine whether steroids stimulate the activation of IB- in FLSs, TNF--stimulated FLSs were left untreated or were treated with DEX (1 µM). After lysis of the cell, equal amounts of protein were loaded onto a SDS–polyacrylamide gel for Western blot analysis (Fig. 4). The degradation of IB- was first observed after 10 min of stimulation with TNF-, and had returned to 80% of the control level at 1 h. In the DEX pretreatment group, the basal amount of IB- was slightly increased but DEX did not prevent the significant loss of IB- protein at 10 min, 1 h and 2 h of incubation with TNF-. After 1 h, the IB- level started to increase again in the TNF--treated groups, due to the usually observed autoregulatory induction of IB- synthesis by activated NF-B (Fig. 4). Our results showed that the amount of IB- protein was not significantly up-regulated by DEX treatment compared with the TNF--treated groups.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Degradation of IB- protein in the presence of TNF- and/or DEX. FLSs were left untreated or were pretreated with DEX (1 µM) for 1 h and/or TNF- (10 ng/ml) for different lengths of time. After cell lysis, equal amounts of protein were loaded onto an SDS–polyacrylamide gel for Western blot analysis.

 

	Discussion

Top Abstract Introduction Patients and methods Results Discussion References

 
To guarantee that previous steroid treatment would not interfere with the FLS cultures, the RA patients in our study ended their steroid treatment 2 months before the operation. The FLSs used in our experiments expressed the inflammatory cytokines IL-6 and IL-1ß and were down-regulated by treatment with DEX (Figs 1 and 2). FLSs are involved in the infiltration and activation of other immune cells in the synovial tissue through the secretion of TNF-, IL-1 and IL-6, as well as by expressing both intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 [22, 23]. In addition, FLSs secrete matrix metalloproteinases such as stromelysin 1 (matrix metalloproteinase 3) and interstitial collagenase (matrix metalloproteinase 1), which are essential enzymes in the degradation of cartilage [24, 25]. Human FLSs, therefore, seem to be the crucial regulator of joint inflammation and destruction in RA.

The main transcription factor in the response to inflammatory cytokines is NF-B [12]. This factor is a heterodimer that typically consists of a p65 (RelA) and a p50 subunit. In latent form, NF-B is sequestered in the cytoplasm through the ankyrin repeats of inhibitory IB proteins [26]. Activation of NF-B may occur through a variety of extracellular signals that induce phosphorylation and ubiquitinylation events acting on IB-, resulting in proteolytic degradation [27, 28]. Subsequently, NF-B is released from the inhibitor molecule and enters the nucleus, where it activates gene transcription. One such gene is that coding for IB- itself, which is resynthesized and will resequester the transcriptionally active NF-B complexes, thus functioning in an autoregulatory fashion [26, 29].

Glucocorticoids are among the most potent and clinically important immunosuppressant drugs that inhibit proinflammatory events, such as TNF- production, by ill-defined mechanisms. It is generally accepted that their mechanism of action is based on the repression of particular inflammatory (mostly cytokine) genes by the activated complex formed by binding of the ligands to their corresponding intracellular receptors [13]. Also, previous studies have demonstrated that the glucocorticoid DEX inhibits TNF- translation [30]. Swantek et al. [31] suggested a potential anti-inflammatory mechanism for glucocorticoids and demonstrated that DEX inhibits the induction of JNK/SAPK (Jun N-terminal kinase/stress-activated protein kinase) activity by lipopolysaccharide (LPS), but not the activity of extracellular signal-related kinase 1 and ERK2, p38, or mitogen activated protein kinase 3, -4 and -6. However, as with other anti-inflammatory actions of glucocorticoids, the mechanism of this inhibition is not fully understood. Our study showed that incubation of FLSs with TNF- resulted in the appearance of shifted NF-B bands. However, DEX had no effect on the DNA-binding activity of NF-B at any time point. We found that, after treatment with DEX, the level of IB- was unchanged compared with untreated or TNF--stimulated cells, although there was clear evidence of repression of inflammatory cytokine production under these conditions. These results are consistent with previous studies using endothelial cells [15], murine fibrosarcoma cells [32] and rat mesangial cells [16]; steroids neither inhibited NF-B translocation from the cytosol to the nucleus nor affected the degradation of IB- after stimulation of these cells. However, contradictory results on the anti-inflammatory mechanism of steroids have been reported. Recently, Scheinman et al. [17], using HeLa cells, a monocytic cell line and murine T-cell hybridoma, showed that the synthetic glucocorticoid DEX induces transcription of the IB- gene, resulting in an increased rate of IB- protein synthesis. In the presence of DEX, the newly released NF-B quickly reassociates with newly synthesized IB-, thus markedly reducing the amount of NF-B that translocates to the nucleus. This decrease in nuclear NF-B has been predicted to decrease cytokine secretion markedly, and thus to effectively block the activation of the immune system. This anti-inflammatory mechanism of glucocorticoid may differ depending on the cell type. In the case of FLSs, Fujisawa et al. [20] used N-acetyl-L-cysteine (NAC), an oxidant scavenger, and demonstrated that NAC inhibited not only the production of inflammatory cytokines such as TNF-, IL-6 and intercellular adhesion molecule-1, but also the activation of NF-B after stimulation by TNF-. Unlike NAC, DEX either acts independently of NF-B or, more probably, interacts with NF-B after DNA binding has occurred [16]. Our results demonstrate that the removal of activated NF-B by enhanced expression of IB- cannot be the underlying mechanism of gene repression in the case of FLSs. This information may stimulate future research to discover how steroids act independently of NF-B to reduce inflammatory cytokine production in FLSs. Revealing the anti-inflammatory mechanism of steroids in FLSs will contribute to the future treatment of RA patients.

	Acknowledgments

 
The authors wish to thank Hae-Kyung Cho and Sun-Hee Kim for assistance with cell culture and Philip McElroy and Michelle Van Balkom for revision of the English. This work was supported by the Molecular Medicine Research Group Program (98-J03-01–01-A-05) of the Ministry of Science and Technology.

	Notes

 
Correspondence to: G. T. Oh, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305–600, Korea

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Submitted 14 April 2000; Accepted 18 September 2000

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