Innate immunity triggers IL-32 expression by fibroblast-like synoviocytes in rheumatoid arthritis

Human FLSs were isolated from synovial tissues from four different RA and OA (osteoarthritis) patients at the time of knee-joint arthroscopic synovectomy, as described previously [19]. The diagnosis conformed to the revised criteria of the American College of Rheumatology [20]. Normal FLSs were isolated from synovial tissues obtained with arthroscopic biopsy. Informed consent was provided according to the Declaration of Helsinki and obtained from all patients. Approval by the ethical committee of the Hopitaux Universitaires de Strasbourg was obtained. FLS cultures were made as previously described [21]. Experiments were performed between the third and the ninth passages. Cell number and cell viability were checked by the MTT test, as described elsewhere [22].

FLSs (10⁶ cells) were stimulated with 2 ml of medium alone or medium containing IFN-γ (0.1 ng/ml), TNF-α (10 ng/ml) (R&D Systems, Lille, France), LPS from Salmonella abortus equi (Sigma, St. Quentin Fallavier, France) (1 μg/ml), BLP (EMC Microcollections GMBH, Tübigen, Germany) (1 μg/ml), and poly I:C (Invivogen, Toulouse, France) (10 μg/ml). After a 4- or 24-h incubation period, total RNA was extracted by using TRIzol according to the manufacturer's instructions. FLS (2 × 10⁵ cells) were stimulated with 1 ml of complete medium containing the various activators for 24 h. An IL-32α- specific ELISA test was obtained from Biolegend (Ozyme, Saint Quentin en Yveline, France). IL-32 release was measured with ELISA with the monoclonal antibody KU32-56 as a capture antibody and the biotinylated monoclonal antibody KU32-52 as the detection antibody, according to the manufacturer's instructions (Biolegend, Ozyme, Saint Quentin en Yveline, France). The IL-6-specific ELISA test was from R&D Systems.

Total RNA isolated from FLSs was reverse transcribed by using the First Strand cDNA Synthesis Kit, according to the manufacturer's instructions (In Vitrogen). Real-time quantitative RT-PCR was performed in a total volume of 20 μl by using a SensiMix Plus SYBR (Quantace; Corbett Life Science, Sydney, Australia) and gene-specific primers:

and 5'-CCGTAGGACTGGAAAGAGGA-3'

and 5'-CCGTAGGACTTGTCACAAAA-3'

and 5'-GCAAAGGTGGTGTCAGTATC-3'

and 5'-CATGACCTTGTCACAAAAGCTC-3'

and 5'-GCAAAGGTGGTGTCAGTATC-3'

and 5'-GAGGGATCTCGCTCGCTCCTGGAAGA-3'

and 5'-CATCCGGTACACTCGCACAG-3'

and 5'-AGAGGTGTCTGGCTGGGAAA-3'

IL-32 isoforms were reverse transcribed and amplified. Amplification products were detected as an increased fluorescent signal of SYBRGreen during the amplification cycles. Results were obtained by using SDS Software (Perkin Elmer) and evaluated by using Excel (Microsoft). Melting-curve analysis was performed to assess the specificity of PCR products. Results were normalized to GAPDH and expressed as the fold change compared with samples from cells incubated in medium.

FLSs (5 × 10⁴ cells/well; IbiTreat slides) were stimulated with medium containing the various activators. After a 16-h incubation-period, cells were fixed with paraformaldehyde, 4%, at 4°C, washed with PBS, and permeabilized with 0.2% Triton X100 in PBS, pH 7.4, for 10 min. FLSs were incubated with goat anti-IL-32 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C and then with FITC rabbit anti-goat antibodies for 1 h at 25°C. Fluorescence was analyzed with confocal microscopy.

FLSs (2 × 10⁴ cells) were seeded into 96-well plates and then incubated for 16 h and 24 h in 200 μl of complete medium containing the different activators. Cells were then fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min. Free aldehyde groups were quenched with NH₄Cl, 50 mM, in PBS, pH 7.4, for 20 min. Nonspecific binding was blocked by incubation in PBS containing 0.2% bovine serum albumin and 0.05% saponin for 30 min at 37°C. The cells were then incubated with biotinylated anti-IL-32α antibodies (Biolegend; Ozyme, Saint Quentin en Yveline, France) for 2 h. Absorbance was measured at 450 nm.

The siRNA duplexes used in our study were designed to target sequences for human IRF-1 [GenBank: NM_002198] gene. Four selected siRNA oligonucleotides consisting of sequences of 21 nucleotides were supplied by Dharmacon (Perbio Science, France). Transient transfection of FLSs with siRNA (100 nM) was performed by using the Human Dermal Fibroblast Nucleofactor kit from Amaxa, as previously described [23]. FLSs were then plated in 24-well plates (2 × 10⁵ cells per well). All assays were performed 48 h after transfection. The control was carried out with the Dharmacon siControl nontargeting siRNA consisting of a four-oligonucleotide pool. Transfection efficiency was evaluated with the PmaxGFP control vector.

Stimulation of RA FLSs with IFN-γ induced a dose-dependent production of IL-32 transcripts, which were detectable within 4 h (Figure 1a). IL-32mRNA expression was also compared in normal, OA, and RA FLSs activated with 0.1 ng/ml of IFN-γ. As shown in Figure 1a, amounts of IL-32 transcripts were higher in stimulated RA FLSs compared with normal and OA FLSs.

We next investigated the expression of the different isotypes in RA FLSs. Treatment with IFN-γ at a concentration of 0.1 ng/ml resulted in an increasing amount of IL-32β, γ, and δ transcripts, which were detectable within 4 h (Figure 1b), with a mean increase of seven-, three-, and fivefold after stimulation for 24 h, respectively (Figure 1c). IL-32α mRNA was not expressed at 4 and 24 h (Figure 1b, c). To determine whether increased IL-32 mRNA synthesis led to IL-32 protein expression, immunostaining was performed with a polyclonal anti-human IL-32 antibody, which detected all isoforms (α, β, γ, δ). By using confocal microscopy, no basal expression of IL-32 was observed in unstimulated FLS (Figure 1d/a). IL-32 was strongly expressed after a 24-h incubation with IFN-γ (Figure 1d/c). By using an ELISA test that detected IL-32, we observed that IFN-γ induced IL-32 release by FLSs at 24 (55 pg/ml ± 18 pg/ml) and 48 h (125 pg/ml ± 30 pg/ml), compared with nonactivated cells (Figure 1e). The α isoform of IL-32 (IL-32α) protein was neither detected intracellularly in IFN-γ-activated FLSs (Figure 1f) nor released (data not shown).

Stimulation of RA FLSs with TNF-α induced a dose-dependent production of IL-32 mRNA, which was detectable within 4 h (Figure 2a). By using normal, OA, and RA FLSs activated with 5 ng/ml of TNF-a, we observed that amounts of IL-32 transcripts were higher in stimulated RA FLSs compared with normal and OA FLSs (Figure 2a).

We then assessed the expression of the different isotypes. After stimulation with TNF-α, IL-32α, β, γ, and δ mRNA were detectable within 4 h, with a mean increase of four-, nine-, five- and eightfold after stimulation for 24 h, respectively (Figure 2b, c). Protein expression and release by TNF-α-activated FLSs was demonstrated by immunostaining (Figure 2d/c) and by ELISA (Figure 2e). Mature IL-32α was detected intracellularly (Figure 2f) but not released (data not shown).

IL-32 mRNA expression was also compared in normal, OA, and RA FLSs activated with TLR ligands (LPS, BLP, and poly I:C). As shown in Figure 3a, amounts of IL-32 transcripts were higher in stimulated FLSs isolated from RA patients compared with OA and normal FLSs.

IL-32 β, γ, and δ mRNA expression was induced in RA FLSs in response to either BLP or poly I:C after 4 h (Figure 3b), with a lower induction after a 24-h stimulation (Figure 3c). LPS induced only IL-32β and δ isoforms (Figure 3b). An increase (fivefold) of IL-32α mRNA was detected only in poly I:C-activated FLS (Figure 3b, c). Immunostaining and ELISA demonstrated the presence and release of mature IL-32 protein in FLSs stimulated by LPS, BLP, and poly I:C (Figure 3d/c,/d,/e and 3e). IL-32α was also expressed intracellularly in response to poly I:C (Figure 3f) but not released (data not shown).

The inflammatory environment of the synovial cavity is complex in RA, because most of cytokines are present in the synovial cavity and can interact with each other. We therefore investigated whether a combination of TNF-α (10 ng/ml) and IFN-γ (0.1 ng/ml) could modulate levels of IL-32 mRNA expression by FLSs, because FLS are exposed to both cytokines in the synovium during RA. Concomitant stimulation of FLS with IFN-γ and TNF-α resulted in a strong induction of IL-32 mRNA expression as compared with stimulation with only one of these cytokines. This synergistic effect was observed in IL-32 mRNA 4 h and 24 h after activation with TNF-α and IFN-γ (Figure 4a) but was not observed at the protein level by using ELISA (Figure 4b). However, a concordant effect on mRNA and protein stimulation with both cytokines was demonstrated for the α isoform of IL-32 (Figure 4c, d).

We also studied the effect of a concomitant stimulation of IFN-γ (0.1 ng/ml) and either BLP, LPS, or poly I:C. Stimulation of FLS with IFN-γ (0.1 ng/ml) and either of these PAMPs strongly induced the transcript levels of IL-32 mRNA as compared with FLSs activated with the same amount of IFN-γ alone (Figure 5a). This effect was observed after 4 h of stimulation but not after 24 h. A synergistic effect on IL-32 release was observed after activation with IFN-γ and either LPS, BLP, or poly I:C (Figure 5b). Moreover, LPS, BLP, and poly I:C also exert a synergistic effect on IL-32α mRNA synthesis and IL-32α intracellular expression, when combined with IFN-γ (Figure 5c, d).

We subsequently analyzed the mechanisms responsible for the synergistic effect of PAMPs or TNF-α on IFN-γ related IL-32 induction. Previous studies showed that nuclear concentrations of IFN regulatory factor-1 (IRF-1) were found to increase after stimulation with IFN-γ and TNF-α compared with stimulation with individual cytokines [24, 25]. We therefore evaluated IRF-1 mRNA expression in RA FLSs, stimulated with either TNF-α or IFN-γ alone or in combination. Stimulation with IFN-γ induced IRF-1 mRNA expression in activated RA FLSs, and concomitant stimulation of FLSs with IFN-γ and TNF-α resulted in a stronger increase of IRF-1 mRNA expression (Figure 6a). These results indicate that a simultaneous stimulation with TNF-α has a synergistic effect on IFN-γ-induced IRF-1 transcription in FLSs.

To determine whether IRF-1 is necessary for IL-32 mRNA synthesis by stimulated FLSs, cells were transfected with siRNA targeting IRF-1 or control siRNA for 48 h and then stimulated with TNF-α and IFN-γ alone or in combination. Transfection with siRNAs did not affect cell viability, assessed by the MTT test. We first confirmed that transfection of siRNA targeting IRF-1 impaired endogenous IRF-1mRNA expression, as compared with IRF-1 expression in cells transfected with a nontargeting, control siRNA (Figure 6b). Inhibition of IRF-1 significantly reduced IL-32 mRNA expression after TNF-α, IFN-γ, and TNF-α + IFN-γ stimulation of FLSs (Figure 6c). This role of IRF-1 in the synergy between TNF-α and IFN-γ was specific to IL-32, because the release of IL-6, another proinflammatory cytokine, was not modified after inhibition of IRF-1 (Figure 6d). Stimulation with either LPS, BLP or poly I:C did not induce IRF-1 mRNA expression but IRF-3 mRNA (Figure 6e), and no synergic effect either on IRF-1 or IRF-3 mRNA expression was observed when FLSs were concomitantly stimulated with IFN-γ and LPS, BLP, or poly I:C (Figure 6e). Likewise, by using IRF-1 siRNA, the synergy between LPS and IFN-γ was not modified (Figure 6f), converse to that observed after stimulation with IFN-γ and TNF-α. Thus, these results indicate that IRF-1 is involved in the synergistic effect of TNF-α and IFN-γ on IL-32 mRNA expression by activated FLSs, but IRF-1 and IRF-3 are not implicated in the synergy for the induction of IL-32 observed between IFN-γ and the PAMPs studied.