Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS.
FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase–polymerase chain reaction, and small interfering RNA (siRNA) transfection.
IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor α (TNFα). TNFα-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cδ (PKCδ), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCδ mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCδ.
The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCδ-mediated signaling events.
Interleukin-32 (IL-32) is a recently described cytokine that is induced by IL-18 and is formally known as natural killer cell transcript 4 (1). Production of IL-32 by T lymphocytes, natural killer cells, epithelial cells, and blood monocytes has also been reported (2, 3). Besides inducing tumor necrosis factor α (TNFα) secretion in human monocyte and mouse macrophage cell lines, IL-32 exhibits many properties of proinflammatory cytokines and has been demonstrated to be associated with disease severity in arthritis and Crohn's disease (4).
Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease that predominantly affects multiple peripheral joints. The exact mechanisms that contribute to disease pathogenesis are largely unknown. However, accumulating evidence suggests the participation of inflammation-associated cells such as T cells, B cells, fibroblast-like synoviocytes (FLS), and antigen-presenting cells (5), as well as the production of proinflammatory mediators such as TNFα and IL-1, from these cells. FLS have been noted to be a critical contributor to the pathologic process of RA (6, 7). Among the proinflammatory cytokines implicated in the pathogenesis of RA (8, 9), IL-32 is a recently described candidate. It is highly expressed in FLS and synovial tissue (ST) from patients with RA but not those with osteoarthritis (OA) (10, 11). The intensity of immunohistochemical staining for IL-32 in the synovium has been found to correlate closely with RA severity. For example, it has been shown to correlate with the erythrocyte sedimentation rate as well as with the intensity of staining for TNFα, IL-1, and IL-18 in RA patients (11). Furthermore, injection of human IL-32 into the knee joints of mice resulted in joint swelling, infiltration of immune cells (predominantly neutrophils), and cartilage damage (11). In addition, transgenic mice that overexpressed human IL-32 exhibited increased levels of TNFα, IL-1, and IL-6 produced by splenocytes in response to lipopolysaccharide stimulation, and increased levels of TNFα in serum (3).
Collectively, these findings suggest that IL-32 might be associated with the pathogenesis of RA. However, little is known about the presence of free IL-32 in human synovial fluid (SF) and the regulation of IL-32 production in RA FLS. In this study, we demonstrated for the first time that free IL-32 is present at high levels in SF from the joints of RA patients and that its secretion and expression are positively regulated via a Syk/protein kinase Cδ (PKCδ)/JNK/c-Jun signaling pathway in TNFα-stimulated human RA FLS.
MATERIALS AND METHODS
Recombinant human TNFα was purchased from BioSource International (Camarillo, CA). The following specific inhibitors were obtained from Calbiochem (La Jolla, CA): PD98059 (ERK inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), Gö6976 (PKCα/β inhibitor), and rottlerin (PKCδ inhibitor). In the absence of reliable specific antibodies to all 4 IL-32 isoforms (α, β, γ, δ), the anti–IL-32 antibody, which is highly specific to the IL-32 α isoform, was used in this study. Rabbit anti-human IL-32 antibody and goat anti-human IL-32 antibody were provided by one of the authors (SHK). Antibodies directed against the phosphorylated forms of PKCα/βII (Thr-638/641), PKCδ (Ser-643), JNK (Thr-183/Tyr-185), and c-Jun (Ser-63) were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Collection of human SF and isolation of RA FLS.
SF and ST was obtained from 10 patients with RA and 8 patients with OA, at the time of therapeutic arthrocentesis (SF) or total knee replacement (ST). RA patients fulfilled the criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (12). Informed consent was obtained from all patients, and the experimental protocol was approved by the Asan Medical Center Institutional Review Board. Clinical characteristics of the patients are shown in Table 1.
Table 1. Clinical characteristics of the patients with RA and OA*
OA or RA FLS were isolated from the ST according to a previously described protocol (13), with minor modifications. Briefly, ST specimens were washed thoroughly with RPMI 1640 (Gibco BRL, Gaithersburg, MD), minced, and digested for 90 minutes at 37°C in RPMI 1640 containing 1 mg/ml collagenase (Gibco BRL). The digested tissue was filtered with a 70-μm cell strainer (Becton Dickinson, Franklin Lakes, NJ), and the resulting cell suspension was centrifuged at 250g for 10 minutes. The cell pellet was resuspended in RPMI 1640, washed 3 times by centrifugation at 250g for 10 minutes, and suspended in α-minimum essential medium (α-MEM; Irvine Scientific, Santa Ana, CA) containing 10% heat-inactivated fetal bovine serum (Gibco BRL). The cells were then subcultured for 3–6 passages before use.
Cell stimulation and immunoblotting.
RA FLS were harvested by trypsinization, transferred to 6-well (2 × 105 cells in 3 ml/well) cluster plates, and incubated for 2 days. Cells were washed, followed by replacement of medium with 1% L-glutamine and 1% antibiotics in α-MEM. RA FLS were incubated with inhibitors for 30 minutes, after which some samples were stimulated with 20 ng/ml recombinant human TNFα. RA FLS were washed twice with ice-cold phosphate buffered saline (PBS) and lysed in 50 μl of ice-cold lysis buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 1% Nonidet P40, 10% glycerol, 60 mM octyl β-glucoside, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 μg/ml pepstatin, and a protease inhibitor cocktail tablet). The lysates were kept on ice for 30 minutes, followed by centrifugation at 15,000g for 10 minutes at 4°C. The cell lysates were denatured by boiling in 2× Laemmli buffer for 5 minutes at 95°C (14). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. After blocking in Tris buffered saline–Tween buffer (10 mM Tris HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) containing 5% skim milk powder or bovine serum albumin (BSA), the membrane was incubated with individual specific antibodies. Immunoreactive proteins were detected with the use of HRP-coupled secondary antibodies and enhanced chemiluminescence, according to the protocol of the manufacturer (Amersham Biosciences, Piscataway, NJ).
Extraction of RNA and reverse transcriptase–polymerase chain reaction (RT-PCR).
RA FLS were harvested, washed, and treated with 1% L-glutamine and 1% antibiotics in α-MEM as described above. Some samples were then stimulated for 4 hours with 20 ng/ml recombinant human TNFα. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and reverse transcribed with the Superscript First-Strand Synthesis System according to the protocol recommended by the manufacturer (Invitrogen). PCR was performed under the following conditions: 94°C for 20 seconds, 56°C for 10 seconds, and 70°C for 40 seconds, for 32 cycles (IL-32 α and γ isoforms) or 30 cycles (IL-32 β and δ isoforms). The following primers were used: IL-32α forward 5′-CTGAAGGCCCGAATGCACCA-3′, reverse 5′-CCGTAGGACTTGTCACAAAA-3′; IL-32β forward 5′-CTGAAGGCCCGAATGCACCAG-3′, reverse 5′-GCAAAGGTGGTGTCAGTATC-3′; IL-32γ forward 5′-GTAATGCTCCTCCCTACTTC-3′, reverse 5′-GCAAAGGTGGTGTCAGTATC-3′; IL-32δ forward 5′-TCTCTGGTGACATGAAGAAGCT-3′, reverse 5′-GCAAAGGTGGTGTCAGTATC-3′.
Transfection with small interfering RNA (siRNA).
Using the Human Dermal Fibroblast Nucleofector kit (NHDF-adult) with program no. U-23 (Amaxa, Cologne, Germany), 5 × 105 cells were transfected with 100 nM of each siRNA smart pool against Syk, PKCδ, JNK-1, JNK-2, or c-Jun, or control siRNA, according to the instructions of the manufacturer (Dharmacon, Chicago, IL). Immediately after electroporation, the cells were suspended in 500 μl of culture medium and transferred to a 6-well culture plate. Cells were incubated for 96 hours before use in experiments.
Enzyme-linked immunosorbent assay (ELISA) for IL-32.
For analysis of IL-32 concentrations in culture medium or SF, rabbit polyclonal antibody against human IL-32α (0.5 μg in 100 μl/well) was incubated overnight at 4°C in a 96-well plate, and wells were then washed with 300 μl blocking buffer (3% BSA in PBS) for 2 hours at room temperature. Culture medium or SF (100 μl) was added to each well and incubated for 2 hours at room temperature. Subsequently, 100 μl of biotinylated goat anti-human IL-32 polyclonal antibody (50 ng/ml) was added to each well, followed by 1-hour incubation at room temperature. Then, 100 μl streptavidin–HRP (150 ng/ml; BioSource International) was added, with incubation for 40 minutes, followed by addition of 100 μl tetramethylbenzidine solution (BioSource International), with incubation for 30 minutes. After addition of stop solution, absorbance at 450 nm was measured. Recombinant IL-32α was used to quantify the concentration of free IL-32 in the solution.
Data are presented as the mean ± SEM from ≥3 independent experiments. Statistical significance was tested using SigmaPlot 2000 (Jandel Scientific, Corte Madera, CA). P values less than 0.05 were considered significant.
Measurement of IL-32 in RA and OA SF and production of IL-32 in human RA FLS.
Concentrations of IL-32 in SF from OA and RA patients were determined by measuring the level of free IL-32 by ELISA. Levels of free IL-32 were higher in SF from RA patients (mean ± SEM 107.5 ± 50.9 pg/ml) than in SF from OA patients (14.4 ± 5.9 pg/ml) (Figure 1A). We next investigated whether IL-32 was secreted by RA FLS upon treatment with TNFα, a cytokine that stimulates an inflammatory reaction in RA FLS. As shown in Figure 1B, TNFα treatment induced secretion of significant amounts of IL-32 in culture media, in a dose-dependent manner. Next, the expression of IL-32 in RA FLS was verified by immunoblotting and RT-PCR analysis. In FLS from 5 RA patients, IL-32 expression was significantly enhanced by stimulation with TNFα (Figure 1C). It has previously been reported that at least 4 isoforms of IL-32 are generated by alternate splicing (2, 15). In this study, we investigated whether the 4 known spliced isoforms (α, β, γ, and δ) were expressed in RA FLS. Messenger RNA (mRNA) for all 4 of these IL-32 isoforms was expressed in response to TNFα stimulation (Figure 1D). The expression of IL-32 was evident within 4 hours and reached its maximum in 8–16 hours (Figure 1E). Optimal expression was achieved with 5–20 ng/ml of TNFα (Figure 1F).
These data confirm the previous finding that IL-32 is expressed in RA FLS. Further, they demonstrate for the first time that IL-32 expression in RA FLS is stimulated to a significant degree by TNFα and that IL-32 is also present in RA SF.
Suppression of IL-32 expression by treatment with specific inhibitors of PKCδ and JNK.
Since little is known regarding the regulation of IL-32 production in RA FLS, we next examined which signaling molecules are involved in production of IL-32 by TNFα-stimulated RA FLS. We observed that TNFα induced activating phosphorylation of PKCδ, p38 MAPK, ERK, JNK, c-Jun, and activating transcription factor 2 (ATF-2) in RA FLS, in a time-dependent manner (Figure 2A). The activating phosphorylation of PKCδ was increased at 7 minutes, with a maximum response at 15 minutes, and this persisted through 30 minutes. Of relevance to subsequent studies was the finding that phosphorylation of JNK was also apparent at 7 minutes and reached a maximum at 15 minutes. ATF-2 and c-Jun are downstream molecules in JNK signaling pathways and are activated by proinflammatory cytokines, such as TNFα, in FLS (16), as was confirmed in the present studies. In contrast, the presence or absence of TNFα stimulation did not alter the phosphorylation of PKCα/β (Figure 2A).
Various kinase inhibitors were tested to identify the relevant signaling molecules in expression of IL-32. In these experiments, the PKCδ inhibitor rottlerin and the JNK inhibitor SP600125 suppressed TNFα-induced IL-32 expression in the cells (Figure 2B). The inhibition of IL-32 was dose-dependent and was apparent with as little as 1.0 μM rottlerin (Figure 2C) or 5 μM SP600125 (Figure 2D). Gö6976, SB203580, and PD98059 (which specifically inhibit PKCα/β, p38 MAPK, and ERK, respectively) were inactive (Figure 2B), indicating that PKCδ and JNK are critical for the expression of IL-32 in TNFα-stimulated RA FLS. Furthermore, activating phosphorylation of JNK and c-Jun, but not of ATF-2, was specifically inhibited by the PKCδ inhibitor rottlerin (Figures 2B and C), suggesting that PKCδ is an upstream kinase for the activation of those molecules.
Inhibition of IL-32 expression by down-regulation of PKCδ, JNK, and c-Jun with specific siRNA.
To further verify the physiologic relevance of PKCδ, JNK, and c-Jun in IL-32 production by RA FLS, cells were transfected with siRNA against these specific signaling molecules. Transfection with siRNA targeted against PKCδ, JNK, or c-Jun resulted in the suppression of expression of the targeted molecules and IL-32 (Figure 3). Transfection with siRNA against PKCδ produced inhibitory effects on the activating phosphorylation of JNK (but not p38 MAPK and ERK) and c-Jun (but not ATF) (Figure 3A). Overexpression of siRNA that down-regulated JNK inhibited the expression of IL-32 and the activating phosphorylation of c-Jun (Figure 3B). Finally, the transfection of siRNA against c-Jun suppressed the expression of IL-32, but not the activating phosphorylation of ATF (Figure 3C). In summary, 3 different siRNA were similarly effective in suppressing IL-32 expression. Consistent with findings of the inhibitor studies (Figure 2), the effects observed in the siRNA experiments strongly suggested that PKCδ is an upstream signaling molecule involved in the activation of the JNK/c-Jun pathway and, subsequently, in the production of IL-32 in RA FLS.
Importance of Syk in IL-32 expression via its activation of PKCδ and JNK.
The kinase Syk has recently been demonstrated to have an important role in cytokine and matrix metalloproteinase (MMP) production by RA FLS (17). We therefore investigated whether Syk and Src family kinase (a well-recognized upstream kinase in Syk activation) were expressed in RA FLS. The presence of both kinases was confirmed by RT-PCR and immunoblotting (results not shown). Next, we examined the effects of additional inhibitors, including the Syk inhibitor piceatannol and the Src family kinase inhibitor PP2, in order to investigate their participation in IL-32 expression. Piceatannol was found to inhibit the expression of IL-32 and activating phosphorylation of PKCδ, JNK, and c-Jun in TNFα-stimulated RA FLS (Figure 4A), whereas PP2 had no effect (results not shown). Bay61-3606, another Syk-specific inhibitor, also suppressed the expression of IL-32 in TNFα-stimulated RA FLS (Figure 4A). Further confirmation of the role of Syk in production of IL-32 was obtained from experiments showing that transfection of cells with a specific siRNA against Syk also suppressed IL-32 expression (Figure 4B).
Suppression of IL-32 secretion in RA FLS by specific inhibitors.
Finally, we investigated the effects of specific inhibitors on the secretion of IL-32 by TNFα-stimulated RA FLS. IL-32 secretion was consistently inhibited by the Syk inhibitor piceatannol, the PKCδ inhibitor rottlerin, and the JNK inhibitor SP600125, in a dose-dependent manner (Figure 5). These results confirmed that Syk, PKCδ, and JNK are essential for the expression and secretion of IL-32 in TNFα-stimulated RA FLS.
IL-32, which has been suggested to be an RA-associated cytokine, is highly expressed in RA ST and FLS (3, 10), and is known to be associated with severity of the disease (11). In addition, it has been reported that the expression of IL-32 mRNA in CD4+ T cells, dendritic cells, and RA FLS is stimulated by TNFα (3). However, little is known about the presence of IL-32 in RA SF and the regulation of its expression and secretion in RA FLS. In the present study, we have demonstrated for the first time that IL-32 is present in SF from the joints of RA patients, and that expression and secretion of IL-32 are positively regulated by the Syk/PKCδ/JNK pathway in TNFα-stimulated RA FLS.
Various cytokines are believed to be involved in cartilage degeneration (8, 9), and it has been proposed that IL-32 participates specifically in the amplification of inflammatory reactions that lead to cartilage and tissue damage in RA and Crohn's disease (4). The notion that IL-32 plays such a role has been supported by the observation that overexpression or injection of human IL-32 exacerbated collagen antibody–induced arthritis in mice (3, 11). IL-32 is highly expressed in RA ST and FLS (3,11). Of the various types of cells in synovium, FLS are thought to have an underlying role in induction of RA (6, 7). To investigate the source and mechanism of IL-32 secretion in ST, we isolated FLS from RA patients in order to determine whether these cells secreted IL-32 upon TNFα stimulation and, if so, to investigate how IL-32 expression and secretion are regulated in these cells. We indeed found that TNFα stimulated the expression and secretion of IL-32 in RA FLS, thus implicating RA FLS as a source of free IL-32 in SF from RA patients.
In the present study we also demonstrated for the first time, by ELISA, that levels of IL-32 in SF from RA patients were much higher than those in SF from OA patients. This result is of interest and potential significance in relation to the search for possible markers of RA. Although the difference in IL-32 levels between OA and RA SF was not statistically significant, it neared significance (P = 0.072). We did not, however, identify any meaningful correlations between IL-32 levels and clinical characteristics of the RA patients. Further studies involving a larger number of patients will be needed in order to establish any correlations with different stages of RA.
TNFα is one of the master inflammatory cytokines that initiate synovial inflammation and joint destruction in RA. In addition to inducing the synthesis of other cytokines, TNFα activates a broad array of intracellular signaling pathways in FLS. As described herein, TNFα activates PKCδ, p38 MAPK, ERK, and JNK in RA FLS. These kinases are reported to regulate production of various proinflammatory mediators in RA FLS, with the synthesis and secretion of these mediators and specific cytokines being individually regulated by different signaling pathways (18–22). For example, JNK and MKK-7 regulate secretion of MMPs in RA FLS (19), whereas p38 MAPK is important for the production of IL-6 and IL-8 in TNFα-stimulated RA FLS (22).
With specific regard to the expression and secretion of IL-32 in RA FLS, we have demonstrated a critical role of the PKCδ/JNK/c-Jun pathway. JNK was regulated by PKCδ whereas ERK and p38 MAPK were not, thus implicating the signaling connection of PKCδ and JNK in IL-32 production. Of further note, c-Jun, as opposed to ATF, appeared to be the downstream target of JNK that regulated expression of IL-32 in RA FLS. It was recently reported that phosphatidylinositol 3-kinase/Akt signals are important for IL-32 mRNA expression in human pancreatic periacinar myofibroblasts (23). These results suggest that RA FLS and pancreatic cells each have a specific signaling pathway for expression of IL-32.
Another novel finding of our study was that the PKCδ/JNK/c-Jun pathway was regulated by the tyrosine kinase Syk. Syk is a known signaling molecule in IgE-mediated activation of mast cells (24–26) and, as reported recently (17), its activation is necessary for TNFα-induced IL-6 and MMP-3 production in RA FLS. However, the downstream signaling events in that study were not defined. In the present investigation we showed that activation of Syk by TNFα results in activation of the PKCδ/JNK/c-Jun signaling pathway and that this pathway is essential for the expression and secretion of IL-32 in RA FLS. This is the first indication of a link between Syk and PKCδ in RA FLS. Given the association of FLS and IL-32 with RA, our findings have relevance with regard to the pathogenesis of this disease.
We thank Dr. Michael A. Beaven for critical reading and comments.
Dr. Wahn Soo Choi had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Young Mi Kim, Wahn Soo Choi.
Acquisition of data. Mun, Jie Wan Kim, Nah, Do Kyun Kim, Ji Da Choi, Hyung Sik Kim.
Analysis and interpretation of data. Soo Hyun Kim, Chang Keun Lee, Park, Bo Kyung Kim.
Manuscript preparation. Ko, Jun Ho Lee, Wahn Soo Choi.
Statistical analysis. Ju Dong Kim, Hyuk Soon Kim.
Collection of RA synovial fluid and FLS. Nah, Chang Keun Lee.