Expression and regulation of interleukin-33 in human monocytes

Christopher J Nile; Emma Barksby; Paiboon Jitprasertwong; Philip M Preshaw; John J Taylor

doi:10.1111/j.1365-2567.2009.03221.x

. 2010 Jun;130(2):172–180. doi: 10.1111/j.1365-2567.2009.03221.x

Expression and regulation of interleukin-33 in human monocytes

Christopher J Nile ¹, Emma Barksby ¹, Paiboon Jitprasertwong ¹, Philip M Preshaw ¹, John J Taylor ¹

PMCID: PMC2878462 PMID: 20070408

Abstract

Interleukin-33 (IL-33) is an IL-1 family cytokine that has a role in regulating T helper type 2 cytokines and mast cell development. Expression of IL-33 is also associated with chronic inflammatory conditions such as rheumatoid arthritis. However, there is little information regarding IL-33 in myeloid cell immune responses, which are important in immunity and inflammation. We therefore investigated the expression, intracellular location and regulation of myeloid cell IL-33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingivalis. We detected IL-33 messenger RNA in the human promonocytic cell line THP-1, in monocytes derived from these cells and in primary human monocytes. However, IL-33 was not expressed in primary monocyte-derived dendritic cells. Stimulation of monocytes with E. coli LPS (Toll-like receptor 4 agonist) and LPS from P. gingivalis (Toll-like receptor 2 agonist) up-regulated IL-33 at both the messenger RNA and protein levels but IL-1β and tumour necrosis factor-α had no effect. The IL-33 protein was mainly found in the cytoplasm of monocytes with no evidence of nuclear translocation in stimulated cells. Furthermore, no IL-33 secretion was detected after stimulation with LPS and/or ATP. These data indicate that the function, if any, of IL-33 in activated monocytes is primarily intracellular. Interestingly, immunofluorescence analysis indicated that IL-33 was sequestered in the nucleus of monocytes undergoing apoptosis but released into the extracellular milieu by LPS-stimulated cells in which necrosis had been induced by freeze–thawing. Therefore, this endorses the view that IL-33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.

Keywords: cytokines, interleukin-1 family, monocytes, myeloid immune cells

Introduction

The interleukin-1 (IL-1) family of cytokines are important mediators of destructive inflammatory disorders such as rheumatoid arthritis and periodontitis and are important therapeutic targets, for example through the use of neutralizing monoclonal antibodies.¹ The most recently discovered member of the IL-1 family is IL-33 (IL-F11).² Interleukin-33 exhibits structural similarity to IL-18 and is synthesized as a 30 000 molecular weight precursor protein that lacks a signal peptide. The IL-33 precursor has been found to be cleaved to a form with a molecular weight of between 20 000 and 22 000 by both caspase-1²^,³ and caspase-3.³ The receptor for IL-33 has recently been identified and has a structure analogous to other IL-1 cytokine receptors: hence IL-33 binds to a heterodimer comprising the IL-1 accessory protein IL-1RAcP and the Toll-interleukin-1 receptor superfamily member ST2.²^,⁴^,⁵

Studies of complementary DNA (cDNA) libraries indicated constitutive expression of IL-33 in bronchial and arterial smooth muscle cells and epithelial cells from the bronchus and small airways.² Activation of primary lung or dermal fibroblasts and keratinocytes by IL-1β and tumour necrosis factor-α (TNF-α) resulted in expression of IL-33.² The IL-33 is also expressed in the central nervous system and in particular the astrocytes of central nervous system glia.²^,⁶ Significantly, IL-33 is expressed in high endothelial venules associated with human tonsil, Crohn’s disease intestine and rheumatoid arthritis synovium.⁷ and may be involved in endothelial cell activation, for example during angiogenesis.⁸ Other recent reports indicate that IL-33 is expressed at the site of immune-mediated pathologies; hence IL-33 was found to be expressed in synovial fibroblasts isolated from patients with rheumatoid arthritis where it is also up-regulated by IL-1β and TNF-α⁹ and IL-33 was increased in the brain tissues of mice infected with Theiler’s murine encephalomyelitis virus.⁶ Significantly, Moussion et al.¹⁰ reported that IL-33 is constitutively expressed in endothelial cells from both small and large blood vessels, in the fibroblastic reticular cells of lymphoid tissues as well as in a number of epithelial cells including epidermal keratinocytes; this further endorses the potential role of IL-33 in immune–inflammatory reactions. In their screen of a cDNA library, Schmitz et al.² did report modest levels of IL-33 expression in lipopolysaccharide (LPS) -activated human monocytes and dendritic cells (of unknown phenotype); there is no other detailed information on the expression and regulation of IL-33 in myeloid immune cells. There is strong evidence for a role of IL-33 in regulating T helper type 2 (Th2) cytokines and in stimulating mast cell development and associated pathologies.²^,¹¹^–¹⁶ Hence, ST2 is strongly expressed on Th2 cells and IL-33 stimulates the production of IL-5 and IL-13 in these cells in vitro.² When IL-33 is administered to mice, increased levels of immunoglobulin E (IgE) and the Th2 cytokines (IL-4, IL-5 and IL-13) ensue; this is associated with eosinophilia, splenomegaly and pathological changes in arteries, lungs and intestine of the mice; consistent with a Th2-driven pathology.² Furthermore, IL-33 has also been found to be a chemoattractant for human Th2 cells.¹⁴

Mast cells are activated by IgE and IgG and play a key role in mediating Th2 pathologies. Significantly, mast cells express ST2 and IL-1RAcpP⁵ and there is evidence now from several studies that IL-33 can also drive mast cell maturation, survival, adhesion and cytokine production.⁵^,¹¹^–¹³^,¹⁵ Furthermore, a model of IL-33 involvement in the pathogenesis of collagen-induced arthritis recently proposed by Xu et al.⁹ suggests that pro-inflammatory cytokines derived from IL-33-activated mast cells have a pivotal role in this chronic inflammatory pathology. Although studies of soluble (s)ST2 (reviewed by Arend et al.¹⁷), in experiments with ST2 knockout mice (e.g. Xu et al.⁹) as well as studies with recombinant IL-33²^,⁹ support a role for IL-33 in inflammatory pathologies, there is very limited information concerning the identification of the cellular source of IL-33 and measurement of IL-33 secretion. As a result, although IL-33 was reported to be secreted from phorbol 12-myristate 13-acetate-stimulated rat cardiac fibroblasts¹⁸ and also from adenosine triphosphate (ATP) -stimulated mixed glial cell cultures and astrocyte-enriched cultures⁶ there are no reports of measurement of IL-33 secretion from other cell types and in particular immune cells relevant to chronic inflammation.

Perhaps significantly, IL-33 has been reported to be identical to nuclear factor of high endothelial venules (NF-HEV), which is a peptide that is associated with chromatin and has transcriptional repressor activity in vitro.⁷^,¹⁹ These authors propose that IL-33 may be a ‘dual function’ cytokine and, like IL-1α and HMGB1, may therefore have an intracellular function (for example in high endothelial venules, where it is highly expressed) as well as mediating pro-inflammatory responses as an extracellular cytokine.⁷ Indeed, recent published evidence demonstrates that biologically active IL-33 is released from damaged endothelial cells suggesting that this cytokine may function as an ‘alarmin’, providing an endogenous signal activating innate immunity during tissue damage and infection.³

In this study we report that intracellular IL-33 expression is up-regulated by THP-1 monocytes and primary monocytes but not by dendritic cells in response to stimulation with LPS from Escherichia coli and LPS from the periodontal pathogen, Porphyromonas gingivalis. However, IL-1β or TNF-α had no effect on IL-33 messenger RNA (mRNA) expression in monocytes. Our data suggest that in monocytes, IL-33 is primarily an intracellular protein: we have not observed secretion of IL-33 by viable cells stimulated with LPS, either alone or in combination with ATP. We present preliminary evidence to suggest that IL-33 is sequestered in the nucleus during apoptosis and demonstrate IL-33 release by cells undergoing necrosis. Therefore, IL-33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.

Materials and methods

Cell culture

The THP-1 promonocytic cell line was obtained from the European Collection of Cell Cultures (Salisbury, UK). The cells were cultured in RPMI-1640 medium (Sigma, Poole, UK), supplemented with 10% (volume/volume) heat-inactivated fetal bovine serum (Sigma), 0·1 mm l-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37°. THP-1 promonocytes were converted to monocytes by incubation with 100 nm 1α,25-dihydroxy-vitamin D₃ (Merck Chemicals, Nottingham, UK) for 48 hr. Cell differentiation was confirmed by adherent capabilities, visual morphology and CD14 expression.²⁰

Primary monocytes were obtained from buffy coat fractions of human blood (National Blood Service, Newcastle, UK). The peripheral blood mononuclear cell (PBMC) population was extracted from the buffy coat using Histopaque-1077 (Sigma). In some experiments, PBMCs were cultured in complete RPMI-1640 medium for 24 hr at 37° with 5% CO₂ and after 24 hr the non-adherent cell population was removed by repeated washes in RPMI-1640 to reveal adherent primary monocytes. Alternatively, primary (CD14⁺) monocytes were isolated by positive magnetic selection with anti-CD14 conjugated magnetic beads using a commercially available kit (EasySep, StemCell Technologies, Grenoble, France). Primary monocytes were converted to dendritic cells by incubating in supplemented RPMI-1640 medium containing granulocyte–macrophage colony-stimulating factor (10 ng/ml) and IL-4 (50 ng/ml) for 7 days, changing the medium every other day. Differentiation was confirmed by observing morphological changes and analysis of expression of CD1a, CD11C, CD14, HLA-DR and CD83 by fluorescence-acitvated cell sorting (data not shown).

Cell stimulation experiments

THP-1 monocytes, primary monocytes and primary monocyte-derived dendritic cells (MDDCs) were cultured at a density of 10⁶ cells/ml. Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr. An unstimulated control for each time-point was also included. After incubation the culture supernatant was removed for analysis by enzyme-linked immunosorbent assay (ELISA) and RNA was isolated from cells using the RNeasy mini kit (Qiagen, Crawley, UK).

Reverse transcription and quantitative real-time polymerase chain reaction

Reverse transcription (RT) was performed using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Warrington, UK). The RT reactions were carried out in an Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems), under the following reaction conditions; 25° for 10 min, 37° for 2 hr and 85° for 5 seconds. Levels of cDNA transcript were determined using real-time polymerase chain reaction (PCR) with pre-designed primers and Taqman probes (Assays on Demand; Applied Biosystems). The sequences for these probes were as follows: IL-33: Hs00369211_m1; ST2L: Hs00545033_m1; HMGB1: Hs01923466_g1; RNA polymerase II: Hs00172187_m1. Each PCR consisted of 2·5 μl cDNA, 12·5 μl 2× Sensimix (5 mm final MgCl₂) (Quantace, Watford, UK), 1·25 μl TaqMan Primer (900 nm)/Probe (250 nm) and 8·75 μl H₂O. Each sample was assayed in triplicate over 40 cycles and the reactions were conducted in a 96-well plate format using the ABI 7900 instrument (Applied Biosystems). RNA polymerase II gene expression was used as a control for cDNA input. The data were analysed using SDS 2.2 software (Applied Biosystems) and normalized against RNA Polymerase II expression; levels of specific mRNA in stimulated cells were presented as relative expression compared with control cultures using the ΔΔCt method.²¹

Cytokine ELISA

Cytokines in cell culture supernatants were quantified using specific ELISA kits for IL-33 (Axxorra, Nottingham, UK) and TNF-α (R&D Systems); the sensitivities for these ELISAs were 3·56 and 1·27 pg/ml pg/ml, respectively. Interleukin-18 was measured by sandwich ELISA using commercially available antibodies and recombinant IL-18 (R&D Systems); the sensitivity of the IL-18 ELISA was 5·19 pg/ml.

Apoptosis and necrosis

Necrosis was induced by subjecting cells to five cycles of freezing to − 70° and thawing at 38°.²² Cell viability in necrotic cell preparations was analysed by Trypan blue exclusion and was consistently < 5% (data not shown). Apoptosis was induced by exposing monocytes to a 62 mJ/cm² dose of UVB irradiation using a bank of four Philips TL 20W/12 RS lamps (Philips, Guilford, UK). The extent of apoptosis was analysed by fluorescence microscopy with 4′-6′,diamidino-2-phenylindole (DAPI) staining of nuclei: nuclei in these preparations exhibited condensation characteristic of nuclear fragmentation and apoptosis (Fig. 4b).

Immunocytochemistry

For immunocytochemical analysis, cells were cultured on glass cover-slips and then fixed in ice-cold methanol. The cover-slips were washed with phosphate-buffered saline (PBS) before permeabilizing cells using Triton X-100 (Sigma). The cells were then probed with 5 μg/ml of a mouse monoclonal antibody to IL-33 (Nessy-1, Axxora, Nottingham, UK) for 60 min at room temperature. After repeat washes with PBS, the cells were then probed with 5 μg/ml secondary polyclonal antibody to mouse IgG1 conjugated to fluorescein isothiocyanate (FITC; Axxora) or tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma) for 40 min. The cells were again washed with PBS and their nuclei were counterstained by exposure to DAPI (Sigma) at a concentration of 10 μg/ml for 15 min. The cover-slips were washed in PBS before finally being mounted on microscope slides with Dako fluorescent mounting medium (DAKO, Ely, UK). The cells were examined using a Leica TCS SP UV confocal laser scanning microscope (Leica, Wetzlar, Germany). A series of Z-stack images were taken and each image was analysed individually using Leica LCS Lite software (Leica) and findings were verified by an independent expert observer. Specificity of the IL-33 monoclonal antibody was confirmed by complete abrogation of fluorescent labelling after pre-incubation with a fivefold concentration of blocking peptide (IL-33_112–270) (Axxora) (data not shown).

Statistical analysis

Statistical analysis was performed using Mann–Whitney tests (cytokine expression analysis) and Student’s t-test (real-time RT-PCR).²¹ A P-value of < 0·05 was considered to be statistically significant.

Results

IL-33 expression by myeloid cells

Data from analysis of a cDNA library indicated that modest levels of IL-33 mRNA are expressed by human monocytes and dendritic cells.² However, to date, the expression of IL-33 by myeloid cells has not been fully characterized. We therefore investigated the expression of IL-33 by monocytes in response to bacterial LPS and the cytokines IL-1β and TNF-α using quantitative real-time PCR (Fig. 1). We found that IL-33 mRNA was significantly up-regulated in LPS-stimulated THP-1 monocytes (Fig. 1a) and primary monocytes isolated from three separate donors (Fig. 1b), in comparison to unstimulated controls, with maximal transcript levels observed after 6–9 hr of incubation with LPS. Conversely, real-time PCR performed on cDNA isolated from MDDCs derived from primary macrophages of three separate donors showed a lack of IL-33 expression (data not shown). In contrast to IL-33, the ST2 and HMGB1 genes exhibited no up-regulation in expression levels in myeloid cells upon stimulation with bacterial LPS (data not shown). The up-regulation of IL-33 in monocytes by LPS may be the result of transcriptional up-regulation via Toll-like receptor (TLR) signalling or may be an indirect, secondary effect of LPS-stimulated TNF-α or IL-1β secretion. To address this issue, THP-1 monocytes were stimulated with TNF-α and IL-1β and IL-33 mRNA was quantified by real-time RT-PCR; the results do not provide any evidence to suggest that these cytokines up-regulate IL-33 (Fig. 1c).

Interleukin-33 (IL-33) messenger RNA (mRNA) expression is up-regulated by monocytes in response to lipopolysaccharide (LPS) but not tumour necrosis factor-α (TNF-α) and IL-1β. Quantitative real-time polymerase chain reaction analysis was used to determine IL-33 expression by THP-1 monocytes (a) and primary human monocytes (b) in response to stimulation with 100 ng/ml *Escherichia coli* or *Porphyromonas gingivalis* LPS. IL-33 mRNA expression by THP-1 monocytes stimulated with 100 ng/ml TNF-α and 100 pg/ml IL-1β was also measured (c). Levels of mRNA were normalized against RNA polymerase II mRNA levels and fold changes (‘IL-33 expression’) were calculated relative to unstimulated cells at each individual time-point using the ΔΔCt method.²⁰ The data represent the mean values (± SD) from three independent experiments. Significant differences we determined using Student’s t-test on the ΔCt values.²⁰*P < 0·05 relative to unstimulated cells.

IL-33 is not secreted from viable cells

The LPS-stimulated IL-33 mRNA levels were maximal after 6–9 hr of monocyte culture but we consistently failed to detect any IL-33 secretion from viable cells either at 9 hr (Fig. 2) or during the full time–course of our experiments (0–48 hr) (data not shown). There is a suggestion that pro-IL-33 is processed by the action of caspase-1³^,⁴^,⁶ and ATP has been shown to enhance the activity of caspase-1, enhancing the processing and secretion of IL-1β and IL-18.²³ We therefore investigated whether ATP stimulated IL-33 secretion, but we were again unable to detect any extracellular IL-33 in monocytes exposed to ATP either alone or in combination with LPS (Table 1). Similar experiments confirmed ATP enhancement of IL-18 secretion in LPS-stimulated cells over the same time–course, confirming that ATP-stimulated caspase-1 was functional in this system (Table 1). Furthermore, IL-33 was not detected in the culture supernatants of LPS-stimulated cells incubated with ATP for 9–24 hr, when IL-33 mRNA expression was maximal (not shown). Previous reports have indicated that IL-33 is primarily an intracellular cytokine⁷ and HMGB1 has been shown to be released by cells undergoing necrosis²² so we therefore induced necrosis in LPS-stimulated cells and investigated IL-33 release (Fig. 2). There was no IL-33 released by necrotic monocytes that had not been stimulated by LPS, suggesting that this protein was not constitutively synthesized by these cells or at least was below the level of detection of the ELISA (Fig. 2). However, IL-33 was released into the culture medium by necrotic cells stimulated by LPS and the levels of IL-33 protein detected reflected the kinetics of IL-33 mRNA stimulation by LPS (Fig. 2a) with maximal levels of IL-33 protein being released by monocytes stimulated with LPS for 9 hr (Fig. 2). Stimulation of the cells in these experiments was confirmed by detection of TNF-α in the isolated supernatants using ELISA (data not shown). Immunocytochemistry confirmed that IL-33 protein was barely expressed in unstimulated THP-1 monocytes and was not expressed at all in unstimulated primary monocytes but there was a prominent expression of intracellular IL-33 protein by LPS-stimulated monocytes (Fig. 3a,b). Counterstaining of cells with the nuclear stain DAPI indicated that IL-33 was predominantly located in the cytoplasmic compartment in LPS-stimulated monocytes as confirmed by the absence of co-localization of green and blue fluorescence (Fig. 3a,b).

Interleukin-33 (IL-33) is not secreted by viable THP-1 monocytes but is released from necrotic cells. THP-1 monocytes were either stimulated with 100 ng/ml *Escherichia coli* or *Porphyromonas gingivalis* lipopolysaccharide (LPS) for 9 hr (live control), or stimulated with 100 ng/ml *E. coli* or *P. gingivalis* LPS for 3–48 hr followed by induction of necrosis by five rounds of freezing (− 70°) and thawing (38°). Stimulation of the cells in these experiments was confirmed by detection of tumour necrosis factor-α in the isolated supernatants using enzyme-linked immunosorbent assay (not shown). ND = not detected. The data represent the mean values (± SD) from three independent experiments.

Table 1.

Effect of adenosine triphosphate (ATP) on interleukin-33 (IL-33) and IL-18 secretion by THP-1 monocytes

Treatment	IL-33 (pg/ml)	IL-18 (pg/ml)
Unstimulated	0	27·0 ± 10·7
ATP (6 mm)	0	110·1 ± 52·0
E. coli LPS (1 ng/ml)	0	228·2 ± 96·5
E. coli LPS (1 ng/ml) + ATP (6 mm)	0	1635·5 ± 524·5^*
E. coli LPS (1 ng/ml) + freeze–thawing	66·37 ± 8·1	–

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IL-33 secretion from THP-1 monocytes is not activated by ATP. THP-1 monocytes were stimulated with 1 ng/ml Escherichia coli lipopolysaccharide (LPS) for 3 hr in the presence of 6 mm ATP for the final 30 min of this incubation. Cells without additives (unstimulated) and with ATP or LPS alone served as controls. The IL-33 and IL-18 were measured by specific enzyme-linked immunosorbent assay. The data represent the median values (± interquartile range) from three independent experiments. IL-18 secretion was significantly increased by stimulation of the monocytes with E. coli LPS in combination with ATP as compared with LPS alone; Mann–Whitney test

P < 0·05.

Interleukin-33 (IL-33) protein is expressed in the cytoplasm of lipopolysaccharide (LPS) -stimulated THP-1 monocytes (a) and primary monocytes (b). Immunocytochemistry was performed on monocytes (control) and cells stimulated with 100 ng/ml *Escherichia coli* (Ec) or *Porphyromonas gingivalis* (Pg) LPS for 9 hr. The IL-33 was located using an IL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green) or tetramethyl rhodamine iso-thiocyanate (TRITC; red) and the nuclei were stained with 4′-6′,diamidino-2-phenylindole (DAPI; blue). A series of Z-stack images were taken to confirm the cellular location of IL-33 and the image depicted is representative of findings throughout the Z stack. The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (red TRITC) and nuclei (blue DAPI) in combination. The results are representative of three independent stimulation experiments and have been verified by an independent observer. Scale as depicted on images.

IL-33 is sequestered in the nucleus of apoptotic monocytes

In cells induced to undergo programmed cell death HMGB1 was found to remain closely associated with nuclear chromatin.²⁴ We therefore investigated the effect of apoptosis on IL-33 in monocytes. The LPS-stimulated monocytes induced to undergo apoptosis by UVB irradiation did not release any IL-33 into the culture medium over a 0–48-hr time–course (data not shown). Immunocytochemistry was then used to determine the intracellular location of IL-33 in stimulated cells (Fig. 4). Nuclear morphology in control cell cultures confirms the presence of apoptotic cells (Fig. 4a) and staining for IL-33 using FITC-conjugated antibodies clearly shows that the intracellular IL-33 protein is tightly associated with the nucleus of cells undergoing apoptosis (Fig. 4b).

Discussion

The IL-1 family of cytokines have an established role in immune regulation and inflammatory processes.¹^,¹⁷ Inappropriate activity of IL-1 cytokines is a feature of a number of immune-mediated pathologies and therefore pathways activated by these cytokines are rational therapeutic targets.¹ Interleukin-33 is a recently described member of the IL-1 family and has attracted attention because, uniquely among IL-1 cytokines, it stimulates the production of Th2 cytokines such as IL-5 and IL-13, activates Th2 cells, and has a role in mast cell development and function.²^,¹¹^–¹⁶ As a result, IL-33 has been implicated in the pathogenesis of immune-mediated disorders which feature Th2 responses and mast cell dysregulation, and in particular those which involve pulmonary and mucosal inflammation.²^,¹⁶^,²⁵ Studies of the action of recombinant IL-33 and the expression of ST2 (a subunit of the IL-33 receptor) suggest that IL-33 has roles in immune responses and immunopathogenesis in addition to those mediated by mast cells and Th2 cells. Hence, eosinophils and basophils express ST2 and are stimulated by exogenous IL-33.²⁶^–²⁹ Interleukin-33 also enhanced cytokine production by invariant natural killer (NK) T cells and human NK cells.²⁹ Also, IL-33 and ST2 are widely expressed in vascular cells and tissues, in fibroblasts and in the central nervous system.⁶^,⁷^,⁹^,¹⁸

We have investigated the expression, intracellular location and regulation of IL-33 in monocytes and MDDCs. In experiments using both conventional RT-PCR (not shown) and quantitative RT-PCR, IL-33 was not expressed in MDDCs and stimulation with LPS had no effect on IL-33 expression in MDDCs. This is in contrast to Schmitz et al.² who reported no expression in ‘resting’ MDDC but IL-33 expression (albeit at very modest levels as compared with smooth muscle cells and fibroblasts) was detected when these cells were activated by LPS. Similarly, whereas Schmitz et al.² only detected IL-33 mRNA in LPS-activated monocytes, we detected IL-33 mRNA in the human promonocytic cell line THP-1, in monocytes derived from these cells and in primary human monocytes by both conventional RT-PCR (not shown) and quantitative RT-PCR. However, analysis of protein expression using immunocytochemistry demonstrated only very low levels of constitutive IL-33 expression in THP-1 monocytes and none in primary cells. The differences between our findings and those of Schmitz et al.² with respect to mRNA expression may reflect technical differences or differences in the sources of cDNA.

We investigated regulation of IL-33 expression in monocytes: LPS from E. coli as well as LPS from the periodontal pathogen P. gingivalis both enhanced IL-33 expression. The LPS from E. coli and P. gingivalis represent different pathogen-associated molecular patterns (PAMPs): LPS from these species stimulate host cells via TLR4 and TLR2, respectively, which results in overlapping but distinct cytokine responses.³⁰^,³¹ Our data suggest that engagement of both TLR2 and TLR4 pathways stimulates IL-33 indicating a potential role for this cytokine in immune responses to diverse pathogens and PAMPs.

We also investigated the regulation of IL-33 in monocytes by both TNF-α and IL-1β but were unable to demonstrate any effect of these cytokines on IL-33 expression. Previously, expression of IL-33 in human primary lung fibroblasts as well as in dermal fibroblasts and keratinocytes was shown to be enhanced by a combination of TNF-α and IL-1β.² Also, TNF-α either alone or in combination with IL-1β enhances IL-33 expression in human primary synovial fibroblasts.⁹^,³² However, TNF-α and IL-1β had no effect on IL-33 expression in rat cardiac fibroblasts or rat cardiomyocytes.¹⁸ The reasons for these inconsistencies are not clear, although the data may reflect variable responsiveness of different cell types.

The function, if any, of IL-33 in the myeloid lineage remains obscure but a key question is whether or not IL-33 is secreted or remains in the intracellular compartment. In terms of myeloid cell IL-33, we consistently failed to detect IL-33 in culture supernatants even after LPS stimulation. Although there is much indirect evidence for an immunological role of IL-33 in vitro and in vivo, only a limited number of reports have described the detection of soluble, endogenous IL-33.⁶^,¹⁸ Hence, although rat cardiac fibroblasts synthesized IL-33 protein, unstimulated cells did not secrete this protein but IL-33 secretion (as detected by Western blot of culture supernatants) was induced by stimulation with phorbol 12-myristate 13-acetate.¹⁸ Similarly, in a study of mixed glial cell cultures and astrocyte-enriched cultures, IL-33 was only found to be secreted in cells stimulated with PAMPs and ATP together.⁶ Recently, the release of IL-33 from primary human endothelial cells in conditions of mechanical stress or injury was demonstrated.³ The physiological relevance of these data remains unclear, but this does suggest that under certain circumstances cells may secrete IL-33 in response to the appropriate signals.

It is possible that IL-33 may be secreted in a form that is not detectable by ELISA. For example, the soluble form of the IL-33 receptor subunit ST2, sST2 interacts with IL-33 and blocks IL-33 signalling¹⁸^,²⁵ and sST2 is induced by LPS and cytokines in monocytes.³³ Although we have not analysed sST2 in our cultures, soluble IL-33 was readily detectable by ELISA after induction of necrosis in monocyte cultures previously stimulated by LPS.

Interleukin-33 may require other signals, in addition to LPS, to induce efficient extracellular release. For example, ATP is known to enhance both IL-1β and IL-18 release via activation of a multiprotein complex (the inflammasome) containing caspase-1, the enzyme responsible for intracellular processing of the pro- form of these cytokines.²³ It is not yet clear whether IL-33 is processed in vivo via a caspase-1 pathway: although there is evidence that in-vitro-translated IL-33 is cleaved by incubation with caspase-1,² this was not confirmed by experiments analysing intracellular IL-33.⁷ Furthermore, the predicted cleavage site for caspase-1-induced IL-33 is not conserved in orthologues from other species.⁷ Recently, it was demonstrated that IL-33 is probably cleaved by both caspase-1 and caspase-3 to yield a 20 000–22 000 molecular weight form of IL-33 and that this cleavage took place at an amino acid residue (Asp₁₇₈) distinct from that previously proposed (Ser₁₁₁).³ Significantly, unlike the full-length IL-33_1–270, the 20 000–22 000 form was not biologically active (in terms of binding and activation of the ST2 receptor), suggesting that IL-33 was inactivated by caspase cleavage.³ The antibodies used for the ELISA and immunocytochemistry in the present report do not distinguish between the different forms of IL-33 described by Cayrol et al.;³ it will therefore be interesting to investigate in detail the molecular structures of IL-33 expressed in monocytes, the effect of LPS on IL-33 maturation, and the specific forms of IL-33 released from necrotic monocytes.

Addition of ATP to mixed glial cell cultures and astrocyte-enriched cultures stimulated the release of IL-33 from these cells whereas these reagents had no effect in isolation.⁶ Significantly, we failed to demonstrate any effect of ATP on IL-33 secretion from monocytes, either alone or in combination with LPS, although enhancement of IL-18 secretion was measured in the same experiments (Table 1). Intriguingly, it has been speculated that IL-33 may be processed by an inflammasome distinct from that which processes IL-1β and IL-18.³⁴ There is evidence that IL-33 may function as a so-called dual function cytokine having a role as an (extracellular) pro-inflammatory cytokine and an (intracellular) nuclear factor with transcriptional regulatory action.⁷

These authors draw analogy with other ‘dual function’ cytokines such as IL-1α and HMGB1.⁷ In support of this hypothesis they found that in high endothelial venules, IL-33 was found to be primarily an intracellular protein that closely associates with eukaryotic chromatin⁷ The nuclear localization was found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix (HTH) motif which is located within the N-terminal portion of the IL-33 precursor protein.⁷ More recent data provide a molecular model for the chromatin interactions of IL-33 and suggest that IL-33 binds to the histone dimer H2A-H2B and hence regulates chromatin compaction by influencing nucleosome–nucleosome interactions.³⁵ Interleukin-33 is therefore shown to confer potent transcriptional repressor activity although the target genes remain to be characterized.⁷

Significantly, it was demonstrated that endothelial cells in inflamed tissues from patients with Crohn’s disease and rheumatoid arthritis express high levels of IL-33 and constitute the major source of IL-33 in these tissues.⁷ The high expression of IL-33 in the endothelial cells of normal and inflamed synovial tissue has recently been confirmed.³² Xu et al.⁹ also confirmed the high expression of IL-33 (and ST2) in the rheumatoid synovium and in fibrobasts isolated from this tissue. Although we have demonstrated that IL-33 is not secreted from stimulated monocytes, IL-33 is predominantly associated with the cytoplasmic compartment in viable monocytes and we did not observe prominent nuclear translocation of IL-33. Palmer et al.³² reported that IL-33 was expressed in both the nucleus and cytoplasm of synovial fibroblasts and ‘mononuclear inflammatory cells’. The sub-cellular localization of IL-33 may reflect intrinsic differences between cell types. Also, there are differences in IL-33 expression in smooth muscle cells cultured in vitro and IL-33 expression in these cells in vivo.²^,¹⁰ Significantly, the expression pattern of IL-33 changes in different cell culture conditions.⁸ Therefore, data from in vitro culture models should be interpreted with care and it will be important to extend studies of IL-33 expression in immune cells in vitro to investigate the expression of this cytokine in tissue sections from patients with inflammatory disease.

We also generated preliminary evidence to suggest that during apoptosis, IL-33 is associated with the nucleus in monocytes. In this regard it is interesting to note that, apoptosis in endothelial cells stimulated IL-33 cleavage (into a form which does not bind the extracellular ST2 receptor) suggesting that this reaction existed to inactivate IL-33 after apoptosis.³ In addition, we demonstrated release of IL-33 as the result of necrosis in the same cells. In similar experiments it was recently demonstrated that necrosis in primary endothelial cells was associated with the release of biologically active IL-33.³ The behaviour of IL-33 during apoptosis and necrosis is similar to the so-called ‘alarmins’, which are a group of biologically active mediators that include the dual function cytokines HMGB1 and IL-1α.³⁶ Alarmins are endogenous signalling molecules that, like exogenous PAMPs, trigger similar pathways and have a common purpose as damage-associated molecule patterns (DAMPS).³⁶ These findings endorse the view that IL-33 has functions beyond that of a conventional extracellular, immunoregulatory cytokine and may be classified as a DAMP.³⁴ The recent finding of widespread expression of IL-33 in the vasculature, in the skin and mucosal epithelial cells supports this hypothesis.¹⁰ Interestingly, in addition to IL-1α and IL-33, an isoform of IL-7 (IL-7b), also an IL-1 family cytokine, has recently been shown to translocate to the nucleus in activated monocytes and to down-regulate the synthesis of pro-inflammatory cytokines.³⁷

In summary, the function of IL-33 in activated monocytes is hypothesized to be primarily intracellular; and as such IL-33 may act as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.

Acknowledgments

This work was supported by a grant from the Newcastle Healthcare charity and a UK Department of Health Clinician Scientist Fellowship DHCS/03/G121/46 awarded to Philip Preshaw.

Disclosures

The authors declare no conflict of interest.

References

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22.Rovere-Querini P, Capobianco A, Scaffidi P, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep. 2004;5:825–30. doi: 10.1038/sj.embor.7400205. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Sutterwala FS, Ogura Y, Flavell RA. The inflammasome in pathogen recognition and inflammation. J Leukoc Biol. 2007;82:259–64. doi: 10.1189/jlb.1206755. [DOI] [PubMed] [Google Scholar]
24.Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 2002;418:191–5. doi: 10.1038/nature00858. [DOI] [PubMed] [Google Scholar]
25.Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J Biol Chem. 2007;282:26369–80. doi: 10.1074/jbc.M704916200. [DOI] [PubMed] [Google Scholar]
26.Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol. 2008;121:1484–90. doi: 10.1016/j.jaci.2008.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Suzukawa M, Iikura M, Koketsu R, et al. An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor. J Immunol. 2008;181:5981–9. doi: 10.4049/jimmunol.181.9.5981. [DOI] [PubMed] [Google Scholar]
28.Suzukawa M, Koketsu R, Iikura M, et al. Interleukin-33 enhances adhesion, CD11b expression and survival in human eosinophils. Lab Invest. 2008;88:1245–53. doi: 10.1038/labinvest.2008.82. [DOI] [PubMed] [Google Scholar]
29.Smithgall MD, Comeau MR, Yoon BR, Kaufman D, Armitage R, Smith DE. IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells. Int Immunol. 2008;20:1019–30. doi: 10.1093/intimm/dxn060. [DOI] [PubMed] [Google Scholar]
30.Chen C, Coats SR, Bumgarner RE, Darveau RP. Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli. Cell Microbiol. 2007;9:1028–38. doi: 10.1111/j.1462-5822.2006.00849.x. [DOI] [PubMed] [Google Scholar]
31.Barksby HE, Nile CJ, Jaedicke KM, Taylor JJ, Preshaw PM. Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide. Clin Exp Immunol. 2009;156:479–87. doi: 10.1111/j.1365-2249.2009.03920.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Palmer G, Talabot-Ayer D, Lamacchia C, et al. Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis. Arthritis Rheum. 2009;60:738–49. doi: 10.1002/art.24305. [DOI] [PubMed] [Google Scholar]
33.Trajkovic V, Sweet MJ, Xu D. T1/ST2 – an IL-1 receptor-like modulator of immune responses. Cytokine Growth Factor Rev. 2004;15:87–95. doi: 10.1016/j.cytogfr.2004.02.004. [DOI] [PubMed] [Google Scholar]
34.Gadina M, Jefferies CA. IL-33: a sheep in wolf’s clothing? Sci STKE. 2007;2007 doi: 10.1126/stke.3902007pe31. [DOI] [PubMed] [Google Scholar]
35.Roussel L, Erard M, Cayrol C, Girard JP. Molecular mimicry between IL-33 and KSHV for attachment to chromatin through the H2A-H2B acidic pocket. EMBO Rep. 2008;9:1006–12. doi: 10.1038/embor.2008.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol. 2007;81:1–5. doi: 10.1189/jlb.0306164. [DOI] [PubMed] [Google Scholar]
37.Sharma S, Kulk N, Nold MF, et al. The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines. J Immunol. 2008;180:5477–82. doi: 10.4049/jimmunol.180.8.5477. [DOI] [PubMed] [Google Scholar]

[b1] 1.Barksby HE, Lea SR, Preshaw PM, Taylor JJ. The expanding family of interleukin-1 cytokines and their role in destructive inflammatory disorders. Clin Exp Immunol. 2007;149:217–25. doi: 10.1111/j.1365-2249.2007.03441.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b2] 2.Schmitz J, Owyang A, Oldham E, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity. 2005;23:479–90. doi: 10.1016/j.immuni.2005.09.015. [DOI] [PubMed] [Google Scholar]

[b3] 3.Cayrol C, Girard JP. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci USA. 2009;106:9021–6. doi: 10.1073/pnas.0812690106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b4] 4.Chackerian AA, Oldham ER, Murphy EE, Schmitz J, Pflanz S, Kastelein RA. IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex. J Immunol. 2007;179:2551–5. doi: 10.4049/jimmunol.179.4.2551. [DOI] [PubMed] [Google Scholar]

[b5] 5.Ali S, Huber M, Kollewe C, Bischoff SC, Falk W, Martin MU. IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc Natl Acad Sci USA. 2007;104:18660–5. doi: 10.1073/pnas.0705939104. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b6] 6.Hudson CA, Christophi GP, Gruber RC, Wilmore JR, Lawrence DA, Massa PT. Induction of IL-33 expression and activity in central nervous system glia. J Leukoc Biol. 2008;84:631–43. doi: 10.1189/jlb.1207830. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b7] 7.Carriere V, Roussel L, Ortega N, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci USA. 2007;104:282–7. doi: 10.1073/pnas.0606854104. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b8] 8.Kuchler AM, Pollheimer J, Balogh J, et al. Nuclear interleukin-33 is generally expressed in resting endothelium but rapidly lost upon angiogenic or proinflammatory activation. Am J Pathol. 2008;173:1229–42. doi: 10.2353/ajpath.2008.080014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b9] 9.Xu D, Jiang HR, Kewin P, et al. IL-33 exacerbates antigen-induced arthritis by activating mast cells. Proc Natl Acad Sci USA. 2008;105:10913–8. doi: 10.1073/pnas.0801898105. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b10] 10.Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’? PLoS ONE. 2008;3:e3331. doi: 10.1371/journal.pone.0003331. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b11] 11.Allakhverdi Z, Smith DE, Comeau MR, Delespesse G. Cutting Edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol. 2007;179:2051–4. doi: 10.4049/jimmunol.179.4.2051. [DOI] [PubMed] [Google Scholar]

[b12] 12.Ho LH, Ohno T, Oboki K, et al. IL-33 induces IL-13 production by mouse mast cells independently of IgE-FcεRI signals. J Leukoc Biol. 2007;82:1481–90. doi: 10.1189/jlb.0407200. [DOI] [PubMed] [Google Scholar]

[b13] 13.Iikura M, Suto H, Kajiwara N, et al. IL-33 can promote survival, adhesion and cytokine production in human mast cells. Lab Invest. 2007;87:971–8. doi: 10.1038/labinvest.3700663. [DOI] [PubMed] [Google Scholar]

[b14] 14.Komai-Koma M, Xu D, Li Y, McKenzie AN, McInnes IB, Liew FY. IL-33 is a chemoattractant for human Th2 cells. Eur J Immunol. 2007;37:2779–86. doi: 10.1002/eji.200737547. [DOI] [PubMed] [Google Scholar]

[b15] 15.Moulin D, Donze O, Talabot-Ayer D, Mezin F, Palmer G, Gabay C. Interleukin (IL)-33 induces the release of pro-inflammatory mediators by mast cells. Cytokine. 2007;40:216–25. doi: 10.1016/j.cyto.2007.09.013. [DOI] [PubMed] [Google Scholar]

[b16] 16.Humphreys NE, Xu D, Hepworth MR, Liew FY, Grencis RK. IL-33, a potent inducer of adaptive immunity to intestinal nematodes. J Immunol. 2008;180:2443–9. doi: 10.4049/jimmunol.180.4.2443. [DOI] [PubMed] [Google Scholar]

[b17] 17.Arend WP, Palmer G, Gabay C. IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev. 2008;223:20–38. doi: 10.1111/j.1600-065X.2008.00624.x. [DOI] [PubMed] [Google Scholar]

[b18] 18.Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT. IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system. J Clin Invest. 2007;117:1538–49. doi: 10.1172/JCI30634. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b19] 19.Baekkevold ES, Roussigne M, Yamanaka T, et al. Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules. Am J Pathol. 2003;163:69–79. doi: 10.1016/S0002-9440(10)63631-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b20] 20.Foster N, Cheetham J, Taylor JJ, Preshaw PM. VIP inhibits Porphyromonas gingivalis LPS-induced immune responses in human monocytes. J Dent Res. 2005;84:999–1004. doi: 10.1177/154405910508401106. [DOI] [PubMed] [Google Scholar]

[b21] 21.Yuan JS, Reed A, Chen F, Stewart CN., Jr Statistical analysis of real-time PCR data. BMC Bioinformatics. 2006;7:85. doi: 10.1186/1471-2105-7-85. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b22] 22.Rovere-Querini P, Capobianco A, Scaffidi P, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep. 2004;5:825–30. doi: 10.1038/sj.embor.7400205. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b23] 23.Sutterwala FS, Ogura Y, Flavell RA. The inflammasome in pathogen recognition and inflammation. J Leukoc Biol. 2007;82:259–64. doi: 10.1189/jlb.1206755. [DOI] [PubMed] [Google Scholar]

[b24] 24.Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 2002;418:191–5. doi: 10.1038/nature00858. [DOI] [PubMed] [Google Scholar]

[b25] 25.Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J Biol Chem. 2007;282:26369–80. doi: 10.1074/jbc.M704916200. [DOI] [PubMed] [Google Scholar]

[b26] 26.Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol. 2008;121:1484–90. doi: 10.1016/j.jaci.2008.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b27] 27.Suzukawa M, Iikura M, Koketsu R, et al. An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor. J Immunol. 2008;181:5981–9. doi: 10.4049/jimmunol.181.9.5981. [DOI] [PubMed] [Google Scholar]

[b28] 28.Suzukawa M, Koketsu R, Iikura M, et al. Interleukin-33 enhances adhesion, CD11b expression and survival in human eosinophils. Lab Invest. 2008;88:1245–53. doi: 10.1038/labinvest.2008.82. [DOI] [PubMed] [Google Scholar]

[b29] 29.Smithgall MD, Comeau MR, Yoon BR, Kaufman D, Armitage R, Smith DE. IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells. Int Immunol. 2008;20:1019–30. doi: 10.1093/intimm/dxn060. [DOI] [PubMed] [Google Scholar]

[b30] 30.Chen C, Coats SR, Bumgarner RE, Darveau RP. Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli. Cell Microbiol. 2007;9:1028–38. doi: 10.1111/j.1462-5822.2006.00849.x. [DOI] [PubMed] [Google Scholar]

[b31] 31.Barksby HE, Nile CJ, Jaedicke KM, Taylor JJ, Preshaw PM. Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide. Clin Exp Immunol. 2009;156:479–87. doi: 10.1111/j.1365-2249.2009.03920.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b32] 32.Palmer G, Talabot-Ayer D, Lamacchia C, et al. Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis. Arthritis Rheum. 2009;60:738–49. doi: 10.1002/art.24305. [DOI] [PubMed] [Google Scholar]

[b33] 33.Trajkovic V, Sweet MJ, Xu D. T1/ST2 – an IL-1 receptor-like modulator of immune responses. Cytokine Growth Factor Rev. 2004;15:87–95. doi: 10.1016/j.cytogfr.2004.02.004. [DOI] [PubMed] [Google Scholar]

[b34] 34.Gadina M, Jefferies CA. IL-33: a sheep in wolf’s clothing? Sci STKE. 2007;2007 doi: 10.1126/stke.3902007pe31. [DOI] [PubMed] [Google Scholar]

[b35] 35.Roussel L, Erard M, Cayrol C, Girard JP. Molecular mimicry between IL-33 and KSHV for attachment to chromatin through the H2A-H2B acidic pocket. EMBO Rep. 2008;9:1006–12. doi: 10.1038/embor.2008.145. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b36] 36.Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol. 2007;81:1–5. doi: 10.1189/jlb.0306164. [DOI] [PubMed] [Google Scholar]

[b37] 37.Sharma S, Kulk N, Nold MF, et al. The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines. J Immunol. 2008;180:5477–82. doi: 10.4049/jimmunol.180.8.5477. [DOI] [PubMed] [Google Scholar]

PERMALINK

Expression and regulation of interleukin-33 in human monocytes

Christopher J Nile

Emma Barksby

Paiboon Jitprasertwong

Philip M Preshaw

John J Taylor

Abstract

Introduction