Objective To investigate the interplay between IL-32 and tumour necrosis factor alpha (TNFα) during the chronic inflammation of rheumatoid arthritis (RA) and to assess whether anti-TNFα treatment of RA patients modulates synovial IL-32 expression.
Methods Induction of IL-32γ by Pam3Cys, lipopolysaccharide, IL-1β or TNFα was investigated in human fibroblast-like synoviocytes (FLS). Stimulation of TNFα production by IL-32γ was studied by adenoviral overexpression of IL-32γ (AdIL-32γ) and lipopolysaccharide stimulation of THP1 cells. Silencing of endogenous IL-32 was employed to study cytokine regulation in FLS. AdIL-32γ followed by TNFα stimulation was performed in FLS to investigate cytokine induction. Immunohistochemistry was applied to study IL-32 expression in synovial biopsies from RA patients.
Results TNFα potently induced IL-32γ expression in FLS. Increased TNFα, IL-1β, IL-6 and CXCL8 production was observed after IL-32γ overexpression and lipopolysaccharide stimulation of THP1 cells. TNFα stimulation of FLS after silencing IL-32γ resulted in diminished IL-6 and CXCL8 production, whereas IL-32γ overexpression resulted in enhanced IL-6 and CXCL8 levels. Remarkably, the mechanism through which IL-32γ overexpression induced TNFα, IL-1β and CXCL8 was by counteracting messenger RNA decay. Importantly, treatment of RA patients with anti-TNFα resulted in significant reduction of IL-32 protein in synovial tissue.
Conclusions TNFα is a potent inducer of endogenous IL-32 expression and IL-32 itself contributes to prolonged TNFα production, thus inducing an important auto-inflammatory loop. Treatment of RA patients with anti-TNFα antibodies diminished IL-32 expression in synovial tissue. The potent anti-inflammatory effect of TNFα blockade in RA patients may be partly due to the reduction of synovial IL-32 expression.
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Tumour necrosis factor alpha (TNFα) plays an essential role in the pathogenesis of rheumatoid arthritis (RA), in view of the fact that anti-TNFα treatment is successful in controlling chronic inflammation in RA.1,–,3 It has previously been shown that the cytokine IL-32 is a potent inducer of TNFα.4 Moreover, expression levels of this novel proinflammatory cytokine in synovial biopsies isolated from RA patients correlates with the severity of inflammation and TNFα expression.5 Stimulation with recombinant human IL-32 resulted in enhanced expression of TNFα in human THP1 cells, human peripheral blood mononuclear cells (PBMC), murine macrophages and murine peritoneal macrophages.4,–,8 In addition, overexpression of intracellular IL-32γ in human synovial fibroblasts followed by TLR-2/NOD2 activation showed potent induction of TNFα messenger RNA, demonstrating the proinflammatory properties of IL-32.9 Suppression of endogenous IL-32 resulted in diminished production of TNFα after lipopolysaccharide stimulation in human monocytes10 and prevented apoptosis in human HeLa cells, although involvement of TNFα was not demonstrated.11 Finally, silencing endogenous IL-32 showed impaired TNFα production in human monocytes after Mycobacterium tuberculosis infection12 and it was demonstrated that M tuberculosis induced intracellular IL-32 production in human PBMC,13 indicating a fundamental role for endogenous IL-32 in regulating proinflammatory cytokine production.
In line with the results demonstrating that IL-32 is a potent inducer of TNFα in vitro, it was shown that the induction of joint inflammation by IL-32 in the mouse was TNFα dependent, as TNFα-deficient mice did not develop joint swelling and expressed less influx of inflammatory cells.5 These data lead to the hypothesis that a potent inflammatory loop between TNFα and IL-32 may play an important role in the chronic inflammation of RA. The aim of the present study was to assess the potential auto-inflammatory loop between IL-32γ and TNFα by using a set of complementary approaches: (1) we assessed the induction of IL-32γ by inflammatory stimuli, with a focus on TNFα; (2) we silenced endogenous IL-32γ expression in fibroblast-like synoviocytes (FLS) to investigate the production of IL-6 and CXCL8; (3) we enhanced endogenous IL-32γ protein expression in FLS and THP1 cells by using an adenoviral approach and examined the mechanisms through which IL-32 increased TNFα stimulation; and (4) in order to elucidate the interaction between IL-32 and TNFα in humans, we investigated IL-32 protein expression in synovial tissue specimens from RA patients receiving anti-TNFα therapy.
Materials and methods
FLS were cultured in Dulbecco's modified Eagle's medium–Glutamax medium (Gibco-Invitrogen, Paisley, UK) and THP1 cells in RPMI-1640 (Gibco-Invitrogen), both containing 10% fetal calf serum, pyruvate and penicillin/streptomycin. FLS were isolated from arthritis patients as described by Heinhuis et al.9
Stimulation of FLS with TLR agonists and cytokines
FLS were seeded in 24-well plates and cultured at 37°C with 5% carbon dioxide until 70–90% confluency was reached and subsequently stimulated with 1 µg/ml Pam3Cys-SKKKK (Pam3Cys) from EMC Microcollections (Tuebingen, Germany), 1 µg/ml Escherichia coli serotype 055:B5 lipopolysaccharide (Sigma-Aldrich, St Louis, Missouri, USA), 1 ng/ml IL-1β (R&D Systems, Minneapolis, Minnesota, USA), or 10 ng/ml TNFα (R&D Systems) in serum-free Dulbecco's modified Eagle's medium–Glutamax medium for 6 h and 24 h. After the stimulation, RNA was isolated9 and used for determining IL-32γ, IL-6 and CXCL8 mRNA. Quantitative real-time PCR was performed using the ABI/PRISM sequence detection system with SYBR Green Mastermix (Applied Biosystems, Foster City, California, USA) as a fluorescent dye. Primers were designed with Primer Express 2.0 (Applied Biosystems) and manufactured by Biolegio (Nijmegen, The Netherlands).
IL-32γ overexpression in human THP1 cells followed by lipopolysaccharide stimulation
Monocytes are normally difficult to transduce with adenoviral vectors, however Mayne et al14 described a protocol to improve transduction efficiency in primary monocytes. Based on that protocol, we successfully transduced THP1 cells (human acute monocytic leukaemia cell line) with an adenoviral vector expressing enhanced green fluorescent protein (AdControl) and obtained high transduction efficiencies of approximately 70–80% (data not shown). Briefly, 0.5 million THP1 cells were seeded in 24-well plates per well and incubated with 10 ng/ml macrophage colony-stimulating factor purchased from R&D Systems. After 24 h of incubation, cells were transduced with 50 multiplicity of infection (MOI) AdIL-32γ or AdControl as described by Heinhuis et al9 and subsequently centrifuged at 2000×g for 2 h (37°C) and placed in an incubator (37°C with 5% carbon dioxide). Twenty-four hours after transduction, cells were stimulated with lipopolysaccharide (100 ng/ml) to induce proinflammatory mediators such as TNFα. Culture media were isolated 48 h post-stimulation and used to measure TNFα, IL-1β, IL-6 and CXCL8 production with Luminex multianalyte technology (BioPlex system; Bio-Rad Laboratories, California, USA).
Silencing of endogenous IL-32γ by siRNA
Synovial fibroblasts were electroporated by using Amaxa Nucleofector technology (Lonza, Basel, Switzerland) with 30 pmol small interfering RNA (Dharmacon, Lafayette, Colorado, USA) per transfection, specific for silencing the γ isoform (5′-CCTGGGTCTCAGCGTGTGA-3′) of IL-32 (siIL-32γ) or control siRNA (siControl). Twenty-four hours post-transfection, FLS were stimulated with 10 ng/ml TNFα in serum-free medium. Culture media were isolated 24 h after TNFα stimulation and IL-6 and CXCL8 were determined by Luminex technology. The efficacy of IL-32γ silencing was determined by quantitative real-time PCR.
IL-32γ overexpression in human FLS followed by TNFα stimulation
FLS were transduced with 10 MOI AdIL-32γ or AdControl as described by Heinhuis et al9 and incubated for 24 h followed by TNFα (10 ng/ml) stimulation. Twenty-four hours post-stimulation, culture media were isolated and used to determine IL-6 and CXCL8 protein levels by Luminex technology. Cells were used for RNA isolation and transformed into complementary DNA as described by Heinhuis et al.9 Transduction efficacy was investigated by determining endogenous IL-32γ mRNA expression by quantitative real-time PCR.
TNFα, IL-1β, IL-6 and CXCL8 mRNA transcript stability
THP1 cells were transduced with 50 MOI AdIL-32γ or AdControl as previously described and incubated for 24 h followed by Pam3Cys (1 µg/ml) stimulation for 4 h. Subsequently, actinomycin D (2 µg/ml) purchased from Sigma-Aldrich was added to stop mRNA transcription. RNA samples were isolated at different time points. Transcript stability was investigated by determining TNFα, IL-1β, IL-6 and CXCL8 mRNA expression using quantitative real-time PCR.
IL-32 expression in RA synovial biopsies before and after anti-TNFα therapy
Synovial knee joint biopsies isolated from 16 RA patients previously employed in several studies15,–,17 were used for determining IL-32 protein expression. Informed consent and ethical approval of the medical ethics committee of the Radboud University Nijmegen Medical Centre were acquired. Synovial biopsies were isolated before and 2 weeks after anti-TNFα therapy (Humira) and stained with goat-anti-IL-32 (AF3040) antibody as described by Joosten et al5 that recognises all isoforms of IL-32 (R&D Systems).
TNFα is a potent inducer of IL-32γ expression in synovial fibroblasts
To study the induction of IL-32γ mRNA expression, human synovial fibroblasts were stimulated with TLR ligands or proinflammatory cytokines for 6 h or 24 h. Figure 1A shows that Pam3cys (TLR-2 ligand), lipopolysaccharide (TLR-4 ligand), or IL-1β mildly induced IL-32γ mRNA expression. Between 6 h and 24 h of stimulation, we observed equal or lower IL-32γ expression during Pam3Cys, lipopolysaccharide, or IL-1β stimulation. Induction of other IL-32 isoforms (α, β and δ) followed that of IL-32γ (data not shown). Of great interest, TNFα potently induced IL-32γ mRNA expression at 6 h, but predominantly at 24 h after TNFα exposure. In contrast to TLR-2/4 ligands or IL-1β, TNFα strongly increased the IL-32γ mRNA expression. Induction of IL-6 (figure 1B) and CXCL8 (figure 1C) was significantly enhanced by IL-1β stimulation, whereas TNFα or TLR ligands were less potent. Figure 1D shows that TNFα strongly enhanced the relative expression of IL-32γ, in contrast to IL-6 or CXCL8 expression.
Overexpression of intracellular IL-32γ followed by lipopolysaccharide stimulation results in strong TNFα, IL-1β, IL-6 and CXCL8 protein production in human monocytes
Besides synovial fibroblasts, monocytes and macrophages are present in synovial tissue. Isolation of these synovial monocytes/macrophages is possible, although culturing is difficult because these cells do not divide. For that reason, we used a monocytic cell line (THP1) and transduced them with AdIL-32γ or AdControl. In our previous study, TLR-2/NOD2 activation was necessary to show the full potential of IL-32γ,9 therefore we included lipopolysaccharide stimulation together with medium control. Figure 2 shows significant amplification of lipopolysaccharide-stimulated TNFα production induced by IL-32γ compared with the other groups. Furthermore, IL-1β, IL-6 or CXCL8 production was greatly enhanced by the overexpression of intracellular IL-32γ followed by lipopolysaccharide stimulation (figure 2). Without lipopolysaccharide stimulation, no differences between AdIL-32γ or AdControl were observed, as shown in figure 2.
Silencing of endogenous IL-32γ leads to reduction of IL-6 and CXCL8 after TNFα stimulation in human synovial fibroblasts
To investigate whether endogenous IL-32 is involved in TNFα-induced production of proinflammatory cytokines, we silenced intracellular IL-32γ expression significantly by siRNA technology as shown in figure 3A, to interfere with the possible inflammatory loop between IL-32 and TNFα. Silencing of endogenous IL-32γ in FLS cells resulted in significant suppression of IL-6 or CXCL8 production triggered by TNFα stimulation (figure 3A).
IL-32γ primes human synovial fibroblasts towards TNFα responsiveness leading to enhanced production of IL-6 and CXCL8
To examine whether elevated endogenous IL-32 results in an enhanced TNFα-driven cytokine production, human FLS were efficiently transduced with AdIL-32γ as shown in figure 3B or AdControl followed by TNFα stimulation. Figure 3B shows enhanced production of IL-6 after TNFα exposure by AdIL-32γ transduced FLS compared with control vector. Furthermore, increased CXCL8 production by IL-32γ overexpression was observed after TNFα stimulation (figure 3B). In addition, enhanced expression of IL-1β, CCL20, MMP1 and MMP3 mRNA was observed (data not shown).
IL-32γ prevents TNFα, IL-1β and CXCL8 mRNA transcript decay
To investigate whether enhanced intracellular IL-32γ expression could lead to stabilising of mRNA, coding for proinflammatory mediators, we transduced human THP1 cells with AdIL-32γ or AdControl. Next, cells were stimulated with Pam3Cys to induce mRNA transcription. After the stimulation, actinomycin D was added to block transcription of mRNA and the decay of transcripts was determined. Overexpression of IL-32γ prevented TNFα, IL-1β and CXCL8 mRNA transcript decay compared with the controls as shown in figure 4. In contrast, IL-6 mRNA stability was not regulated by enhanced intracellular IL-32γ expression (figure 4). These data reveal that IL-32γ is capable of delaying TNFα, IL-1β and CXCL8 mRNA decay, resulting in higher concentrations of mRNA, which leads finally to enhanced protein production of these inflammatory cytokines.
Suppression of endogenous IL-32 protein expression in RA synovial tissue after anti-TNFα therapy
As we noted that TNFα is a potent inducer of endogenous IL-32γ in synovial fibroblasts, we explored whether anti-TNFα treatment of RA patients influenced synovial IL-32 expression. Therefore, synovial knee biopsies were taken before and after anti-TNFα treatment from 16 RA patients and IL-32 protein expression was determined. Immunohistochemistry showed significant suppression of IL-32 protein in synovial biopsies from RA patients after anti-TNFα treatment (figure 5A). Before anti-TNFα treatment, synovial tissue from RA patients showed cytoplasmatic IL-32 expression in synovial fibroblasts, infiltrating cells and synovial endothelium (Figure 5B,C). After anti-TNFα treatment, suppression of synovial (figure 5B,C) IL-32 protein expression was observed (figure 5B,C).
In this study, human synovial fibroblasts were stimulated with Pam3cys, lipopolysaccharide, IL-1β or TNFα. We observed that predominantly TNFα was capable of inducing IL-32γ at 24 h, whereas IL-1β showed decreased expression of IL-32γ. These findings are in line with previous studies, in which it was shown that TNFα is capable of inducing IL-32.18 ,19 Here we showed for the first time that TNFα can induce IL-32γ or other IL-32 isoforms (data not shown) in a time-dependent manner, induction that is different from either TLR-2, TLR-4, or IL-1β-induced IL-32γ expression. The induction of IL-32γ by TNFα is specific because the exposure of FLS to IL-1β does not lead to prolonged IL-32γ expression, whereas IL-6 and CXCL8 were significantly upregulated by IL-1β.
IL-32-induced TNFα production was first described by Kim et al.4 They demonstrated that recombinant IL-32 could induce TNFα in murine Raw macrophages and in human THP1 cells. We previously observed that recombinant IL-32 induces TNFα in murine peritoneal macrophages and that IL-32 expression correlated with the severity of inflammation in synovial biopsies of RA patients.5 Moreover, the injection of recombinant IL-32γ into mouse knee joints recruited inflammatory cells, as observed by joint swelling and histology.5 In TNFα-deficient mice the IL-32γ-induced joint swelling was completely absent. However, cartilage proteoglycan depletion was still present in these mice after the administration of IL-32γ, indicating that the induction of other cytokines, for example IL-1, is more important in cartilage destruction.5 Previously, we demonstrated that intracellular overexpression of IL-32γ and TLR-2/NOD2 activation resulted in potent induction of TNFα mRNA and other proinflammatory mediators in human synovial fibroblasts, indicating an essential role for IL-32 in promoting the production of proinflammatory cytokines.9 In line with these findings, it was shown that recombinant IL-32 strongly synergise with NOD1/NOD2 ligands for the production of IL-1β in human PBMC.7 However, no synergy was found for TNFα production, indicating that IL-32-induced TNFα induction is not enhanced by the NOD2–Rick pathway. This study demonstrated for the first time that in AdIL-32γ transduced THP1 cells a potent induction of TNFα production in response to lipopolysaccharide exposure was observed. However, in the absence of lipopolysaccharide, TNFα production was comparable with the viral control group, demonstrating that elevated intracellular IL-32γ expression is not sufficient for TNFα production and that a second signal is required. By enhancing intracellular IL-32γ expression, we observed increased production of IL-1β, IL-6 and CXCL8 by THP1 cells after lipopolysaccharide stimulation, showing that IL-32 promotes the inflammatory status of cells through aggravated lipopolysaccharide responses. This confirms that intracellular IL-32γ plays an important role in the amplification of proinflammatory cytokines and chemokines in inflamed tissues.
The role of endogenous IL-32 as an intracellular amplifier of cytokine responses was investigated by silencing endogenous IL-32. This study showed for the first time that silencing of IL-32γ in human synovial fibroblasts resulted in significant downregulation of IL-6 and CXCL8 production after TNFα stimulation. Apparently, endogenous IL-32 induced by TNFα stimulation or other stimuli plays an essential role in controlling the expression of proinflammatory mediators. This hypothesis is also confirmed in human umbilical vein endothelial cells,20 PBMC21 and in human THP1 cells.10 These data confirm that endogenous IL-32 plays a pivotal role in synovial fibroblasts and other human cells in controlling proinflammatory cytokines and chemokines.
It was recently shown that recombinant IL-32γ exposure resulted in enhanced production of IL-6 and CXCL8 in FLS.22 We showed that overexpression of intracellular IL-32γ in human synovial fibroblasts followed by TLR-2/NOD2 stimulation resulted in the potent induction of IL-6, CXCL8 and other proinflammatory mediators by the upregulation of TLR-2 and NOD2 receptors.9 Similar to the regulation of TLR-2 and NOD2 receptors, we investigated whether the modulation of TNF receptor I or II by the enhanced intracellular expression of IL-32γ was responsible for the augmented TNFα sensitivity. Upregulation of these TNF receptors might explain the observed increase in IL-6 and CXCL8 production after TNFα stimulation. However, the expression of TNF receptors was not regulated by IL-32γ overexpression (data not shown).
An alternative explanation for the enhanced and extended TNFα production is that IL-32 may modulate TNFα mRNA stability. Intracellular overexpression of IL-32γ in human THP1 cells showed a prolonged TNFα mRNA stability compared with the control group, which showed significant downregulation. In addition, stability of IL-1β and CXCL8 mRNA was also enhanced by the overexpression of intracellular IL-32γ, whereas IL-6 mRNA stability was not influenced by IL-32. The fact that IL-32γ stabilises mRNA of several cytokines and chemokines might be the explanation that elevated intracellular IL-32γ levels change the inflammatory status of several cell types. However, the increase in IL-6 protein by intracellular overexpression of IL-32γ followed by TNFα or lipopolysaccharide stimulation is not induced by enhanced stability of IL-6 mRNA, because overexpression of IL-32γ did not change the IL-6 mRNA stability. A possible explanation for the increased IL-6 production could be the enhanced IL-1β production, as it is known that IL-1β is a potent inducer of IL-6 production.23 Another explanation could be that IL-6 potentially acts like an autocrine factor upregulating its mRNA levels24 or that the half-life of IL-6 mRNA is 30 min25 or longer.
It has been demonstrated earlier that IL-32β could induce IL-10 production in monocytic cell lines or monocyte-derived macrophages.26 However, this effect could not explain the aggravated TNFα production after increasing the intracellular IL-32γ expression, because it is known that IL-10 downmodulates TNFα production mainly by inhibition of the activating p38/MAPK-activated protein kinase-2 pathway.27
A hypothesis of the possible mechanisms of the auto-inflammatory loop between IL-32 and TNFα is shown in figure 6. TNFα is a potent IL-32 inducer and IL-32 upregulates TNFα, especially when a second signal is provided. This results in an auto-inflammatory cascade leading to the production of IL-1β, IL-6 and CXCL8 that can be blocked by silencing IL-32. Another way to interfere with the auto-inflammatory loop is to neutralise TNFα activity. In fact, in synovial tissue isolated from RA patients who received anti-TNFα therapy, a remarkable decrease in IL-32 protein expression was observed. We demonstrated that TNFα drives intracellular IL-32 expression and IL-32 itself modulates TNFα responsiveness and production in RA synovial tissue. Targeting of IL-32 might be a novel therapy to counteract the auto-inflammatory cascade of TNFα–IL-32–TNFα present in the chronic inflamed synovial tissue of RA patients.
The authors would like to thank Monique M. Helsen, Birgitte Walgreen, Liduine van den Bersselaar, Elly L. Vitters and Miranda B. Bennink for their technical support.
Funding BH was supported by a research grant from the Dutch Arthritis Association (06-1-301) and CAD was supported by a grant from the National Institutes of Health AI-15614. MGN was supported by a Vici grant from The Netherlands Organization for Scientific Research.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was obtained from the medical ethics committee of the Radboud University Nijmegen Medical Centre.
Provenance and peer review Not commissioned; externally peer reviewed.
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