IMMUNOLOGY                   ORIGINAL ARTICLEExpression and regulation of interleukin-33 in human monocytesChristopher J. ...
IL-33 in monocytesof IL-33.2 The IL-33 is also expressed in the central ner-    secreted from phorbol 12-myristate 13-acet...
C. J. Nile et al.   Primary monocytes were obtained from buffy coat frac-     cDNA, 12Á5 ll 2· Sensimix (5 mM final MgCl2) ...
IL-33 in monocytesmounted on microscope slides with Dako fluorescent              (a) 200                                  ...
C. J. Nile et al.                70                   Unstimulated   E. coli LPS     P. gingivalis LPS                nata...
IL-33 in monocytes                                                        (a)         Control                     Ec LPS  ...
C. J. Nile et al.targets.1 Interleukin-33 is a recently described member of    onstrate any effect of these cytokines on I...
IL-33 in monocytesand caspase-3 to yield a 20 000–22 000 molecular weight           cytoplasmic compartment in viable mono...
C. J. Nile et al.Disclosures                                                                                    17 Arend W...
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  1. 1. IMMUNOLOGY ORIGINAL ARTICLEExpression and regulation of interleukin-33 in human monocytesChristopher J. Nile, Emma Barksby, SummaryPaiboon Jitprasertwong, Philip M. Interleukin-33 (IL-33) is an IL-1 family cytokine that has a role in regu-Preshaw and John J. Taylor lating T helper type 2 cytokines and mast cell development. Expression ofPeriodontal Immunobiology Research Group, IL-33 is also associated with chronic inflammatory conditions such asInstitute of Cellular Medicine, NewcastleUniversity, Newcastle upon Tyne, UK 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 loca- tion and regulation of myeloid cell IL-33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingi- valis. 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-1b and tumour necrosis factor-a 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 thatdoi:10.1111/j.1365-2567.2009.03221.x IL-33 was sequestered in the nucleus of monocytes undergoing apoptosisReceived 6 March 2009; revised 30 October but released into the extracellular milieu by LPS-stimulated cells in which2009; accepted 18 November 2009. necrosis had been induced by freeze–thawing. Therefore, this endorses theCorrespondence: Dr J. J. Taylor, PeriodontalImmunobiology Research Group, Institute of view that IL-33 may function as an ‘alarmin’ and have a role in signallingCellular Medicine, School of Dental Sciences, cellular damage and inflammatory disease pathogenesis through releaseNewcastle University, Framlington Place, from damaged or necrotic cells.Newcastle upon Tyne, NE2 4BW, UK.Email: Keywords: cytokines; interleukin-1 family; monocytes; myeloid immuneSenior author: John J. Taylor cells weight of between 20 000 and 22 000 by both caspase-12,3Introduction and caspase-3.3 The receptor for IL-33 has recently beenThe interleukin-1 (IL-1) family of cytokines are important identified and has a structure analogous to other IL-1mediators of destructive inflammatory disorders such as cytokine receptors: hence IL-33 binds to a heterodimerrheumatoid arthritis and periodontitis and are important comprising the IL-1 accessory protein IL-1RAcP and thetherapeutic targets, for example through the use of neu- Toll-interleukin-1 receptor superfamily member ST2.2,4,5tralizing monoclonal antibodies.1 The most recently dis- Studies of complementary DNA (cDNA) libraries indi-covered member of the IL-1 family is IL-33 (IL-F11).2 cated constitutive expression of IL-33 in bronchial andInterleukin-33 exhibits structural similarity to IL-18 and arterial smooth muscle cells and epithelial cells from theis synthesized as a 30 000 molecular weight precursor bronchus and small airways.2 Activation of primary lungprotein that lacks a signal peptide. The IL-33 precursor or dermal fibroblasts and keratinocytes by IL-1b andhas been found to be cleaved to a form with a molecular tumour necrosis factor-a (TNF-a) resulted in expression172 Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180
  2. 2. IL-33 in monocytesof IL-33.2 The IL-33 is also expressed in the central ner- secreted from phorbol 12-myristate 13-acetate-stimulatedvous system and in particular the astrocytes of central rat cardiac fibroblasts18 and also from adenosine triphos-nervous system glia.2,6 Significantly, IL-33 is expressed in phate (ATP) -stimulated mixed glial cell cultures andhigh endothelial venules associated with human tonsil, astrocyte-enriched cultures6 there are no reports of mea-Crohn’s disease intestine and rheumatoid arthritis surement of IL-33 secretion from other cell types and insynovium.7 and may be involved in endothelial cell acti- particular immune cells relevant to chronic inflammation.vation, for example during angiogenesis.8 Other recent Perhaps significantly, IL-33 has been reported to bereports indicate that IL-33 is expressed at the site of identical to nuclear factor of high endothelial venulesimmune-mediated pathologies; hence IL-33 was found to (NF-HEV), which is a peptide that is associated withbe expressed in synovial fibroblasts isolated from chromatin and has transcriptional repressor activitypatients with rheumatoid arthritis where it is also in vitro.7,19 These authors propose that IL-33 may be aup-regulated by IL-1b and TNF-a9 and IL-33 was ‘dual function’ cytokine and, like IL-1a and HMGB1,increased in the brain tissues of mice infected with may therefore have an intracellular function (for exampleTheiler’s murine encephalomyelitis virus.6 Significantly, in high endothelial venules, where it is highly expressed)Moussion et al.10 reported that IL-33 is constitutively as well as mediating pro-inflammatory responses as anexpressed in endothelial cells from both small and large extracellular cytokine.7 Indeed, recent published evidenceblood vessels, in the fibroblastic reticular cells of lym- demonstrates that biologically active IL-33 is releasedphoid tissues as well as in a number of epithelial cells from damaged endothelial cells suggesting that this cyto-including epidermal keratinocytes; this further endorses kine may function as an ‘alarmin’, providing an endo-the potential role of IL-33 in immune–inflammatory genous signal activating innate immunity during tissuereactions. In their screen of a cDNA library, Schmitz damage and infection.3et al.2 did report modest levels of IL-33 expression in In this study we report that intracellular IL-33 expres-lipopolysaccharide (LPS) -activated human monocytes sion is up-regulated by THP-1 monocytes and primaryand dendritic cells (of unknown phenotype); there is no monocytes but not by dendritic cells in response to stimu-other detailed information on the expression and regula- lation with LPS from Escherichia coli and LPS from thetion of IL-33 in myeloid immune cells. There is strong periodontal pathogen, Porphyromonas gingivalis. However,evidence for a role of IL-33 in regulating T helper type IL-1b or TNF-a had no effect on IL-33 messenger RNA2 (Th2) cytokines and in stimulating mast cell develop- (mRNA) expression in monocytes. Our data suggest thatment and associated pathologies.2,11–16 Hence, ST2 is in monocytes, IL-33 is primarily an intracellular protein:strongly expressed on Th2 cells and IL-33 stimulates the we have not observed secretion of IL-33 by viable cellsproduction of IL-5 and IL-13 in these cells in vitro.2 stimulated with LPS, either alone or in combination withWhen IL-33 is administered to mice, increased levels of ATP. We present preliminary evidence to suggest thatimmunoglobulin E (IgE) and the Th2 cytokines (IL-4, IL-33 is sequestered in the nucleus during apoptosis andIL-5 and IL-13) ensue; this is associated with eosino- demonstrate IL-33 release by cells undergoing necrosis.philia, splenomegaly and pathological changes in arteries, Therefore, IL-33 may function as an ‘alarmin’ and have alungs and intestine of the mice; consistent with a role in signalling cellular damage and inflammatory diseaseTh2-driven pathology.2 Furthermore, IL-33 has also been pathogenesis through release from damaged or necrotic cells.found to be a chemoattractant for human Th2 cells.14 Mast cells are activated by IgE and IgG and play a key Materials and methodsrole in mediating Th2 pathologies. Significantly, mast cellsexpress ST2 and IL-1RAcpP5 and there is evidence now Cell culturefrom several studies that IL-33 can also drive mast cellmaturation, survival, adhesion and cytokine produc- The THP-1 promonocytic cell line was obtained from thetion.5,11–13,15 Furthermore, a model of IL-33 involvement European Collection of Cell Cultures (Salisbury, UK).in the pathogenesis of collagen-induced arthritis recently The cells were cultured in RPMI-1640 medium (Sigma,proposed by Xu et al.9 suggests that pro-inflammatory Poole, UK), supplemented with 10% (volume/volume)cytokines derived from IL-33-activated mast cells have a heat-inactivated fetal bovine serum (Sigma), 0Á1 mMpivotal role in this chronic inflammatory pathology. L-glutamine, 100 U/ml penicillin and 100 U/ml strepto-Although studies of soluble (s)ST2 (reviewed by Arend mycin. Cultures were maintained in a humidified atmo-et al.17), in experiments with ST2 knockout mice (e.g. Xu sphere with 5% CO2 at 37°. THP-1 promonocytes wereet al.9) as well as studies with recombinant IL-332,9 sup- converted to monocytes by incubation with 100 nMport a role for IL-33 in inflammatory pathologies, there is 1a,25-dihydroxy-vitamin D3 (Merck Chemicals, Notting-very limited information concerning the identification of ham, UK) for 48 hr. Cell differentiation was confirmedthe cellular source of IL-33 and measurement of IL-33 by adherent capabilities, visual morphology and CD14secretion. As a result, although IL-33 was reported to be expression.20Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180 173
  3. 3. C. J. Nile et al. Primary monocytes were obtained from buffy coat frac- cDNA, 12Á5 ll 2· Sensimix (5 mM final MgCl2) (Quan-tions of human blood (National Blood Service, Newcastle, tace, Watford, UK), 1Á25 ll TaqMan Primer (900 nM)/UK). The peripheral blood mononuclear cell (PBMC) Probe (250 nM) and 8Á75 ll H2O. Each sample waspopulation was extracted from the buffy coat using Hist- assayed in triplicate over 40 cycles and the reactions wereopaque-1077 (Sigma). In some experiments, PBMCs were conducted in a 96-well plate format using the ABI 7900cultured in complete RPMI-1640 medium for 24 hr at instrument (Applied Biosystems). RNA polymerase II37° with 5% CO2 and after 24 hr the non-adherent cell gene expression was used as a control for cDNA input.population was removed by repeated washes in RPMI- The data were analysed using SDS 2.2 software (Applied1640 to reveal adherent primary monocytes. Alternatively, Biosystems) and normalized against RNA Polymerase IIprimary (CD14+) monocytes were isolated by positive expression; levels of specific mRNA in stimulated cellsmagnetic selection with anti-CD14 conjugated magnetic were presented as relative expression compared with con-beads using a commercially available kit (EasySep, Stem- trol cultures using the DDCt method.21Cell Technologies, Grenoble, France). Primary monocyteswere converted to dendritic cells by incubating in supple- Cytokine ELISAmented RPMI-1640 medium containing granulocyte–macrophage colony-stimulating factor (10 ng/ml) and Cytokines in cell culture supernatants were quantifiedIL-4 (50 ng/ml) for 7 days, changing the medium every using specific ELISA kits for IL-33 (Axxorra, Nottingham,other day. Differentiation was confirmed by observing UK) and TNF-a (R&D Systems); the sensitivities for thesemorphological changes and analysis of expression of ELISAs were 3Á56 and 1Á27 pg/ml pg/ml, respectively.CD1a, CD11C, CD14, HLA-DR and CD83 by fluorescence- Interleukin-18 was measured by sandwich ELISA usingacitvated cell sorting (data not shown). commercially available antibodies and recombinant IL-18 (R&D Systems); the sensitivity of the IL-18 ELISA was 5Á19 pg/ml.Cell stimulation experimentsTHP-1 monocytes, primary monocytes and primary Apoptosis and necrosismonocyte-derived dendritic cells (MDDCs) were culturedat a density of 106 cells/ml. Cells were stimulated with Necrosis was induced by subjecting cells to five cycles of100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, freezing to ) 70° and thawing at 38°.22 Cell viability inCalne, UK), 100 ng/ml P. gingivalis (from strain W50, a necrotic cell preparations was analysed by Trypan bluegift from M. Rangarajan, Queen Mary’s School of Medi- exclusion and was consistently < 5% (data not shown).cine and Dentistry, London, UK), 100 ng/ml TNF-a Apoptosis was induced by exposing monocytes to a 62 mJ/(R&D Systems, Abingdon, UK), 100 pg/ml IL-1b (R&D cm2 dose of UVB irradiation using a bank of four PhilipsSystems) or ATP (Sigma) for between 0Á5 and 48 hr. An TL 20W/12 RS lamps (Philips, Guilford, UK). The extentunstimulated control for each time-point was also of apoptosis was analysed by fluorescence microscopy withincluded. After incubation the culture supernatant was 40 -60 ,diamidino-2-phenylindole (DAPI) staining of nuclei:removed for analysis by enzyme-linked immunosorbent nuclei in these preparations exhibited condensation char-assay (ELISA) and RNA was isolated from cells using the acteristic of nuclear fragmentation and apoptosis (Fig. 4b).RNeasy mini kit (Qiagen, Crawley, UK). ImmunocytochemistryReverse transcription and quantitative real-time For immunocytochemical analysis, cells were cultured onpolymerase chain reaction glass cover-slips and then fixed in ice-cold methanol. TheReverse transcription (RT) was performed using the ABI cover-slips were washed with phosphate-buffered salineHigh Capacity cDNA Reverse Transcription Kit (Applied (PBS) before permeabilizing cells using Triton X-100Biosystems, Warrington, UK). The RT reactions were car- (Sigma). The cells were then probed with 5 lg/ml of aried out in an Applied Biosystems GeneAmp PCR System mouse monoclonal antibody to IL-33 (Nessy-1, Axxora,9700 (Applied Biosystems), under the following reaction Nottingham, UK) for 60 min at room temperature. Afterconditions; 25° for 10 min, 37° for 2 hr and 85° for repeat washes with PBS, the cells were then probed with5 seconds. Levels of cDNA transcript were determined 5 lg/ml secondary polyclonal antibody to mouse IgG1using real-time polymerase chain reaction (PCR) with conjugated to fluorescein isothiocyanate (FITC; Axxora)pre-designed primers and Taqman probes (Assays on or tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma)Demand; Applied Biosystems). The sequences for these for 40 min. The cells were again washed with PBS andprobes were as follows: IL-33: Hs00369211_m1; ST2L: their nuclei were counterstained by exposure to DAPIHs00545033_m1; HMGB1: Hs01923466_g1; RNA poly- (Sigma) at a concentration of 10 lg/ml for 15 min. Themerase II: Hs00172187_m1. Each PCR consisted of 2Á5 ll cover-slips were washed in PBS before finally being174 Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180
  4. 4. IL-33 in monocytesmounted on microscope slides with Dako fluorescent (a) 200 Unstimulated E. coli LPS P. gingivalis LPSmounting medium (DAKO, Ely, UK). The cells were exam- 175 * IL-33 mRNA expressionined using a Leica TCS SP UV confocal laser scanning 150 *microscope (Leica, Wetzlar, Germany). A series of Z-stack 125images were taken and each image was analysed individu- 100 * *ally using Leica LCS LITE software (Leica) and findings were 75verified by an independent expert observer. Specificity of 50the IL-33 monoclonal antibody was confirmed by complete 25abrogation of fluorescent labelling after pre-incubation 0with a fivefold concentration of blocking peptide 0·5 3 6 9 24 48 Time (hr)(IL-33112–270) (Axxora) (data not shown). (b) 50 Unstimulated E. coli LPS P. gingivalis LPS 45 IL-33 mRNA expressionStatistical analysis 40Statistical analysis was performed using Mann–Whitney 35 * 30tests (cytokine expression analysis) and Student’s t-test 25 *(real-time RT-PCR).21 A P-value of < 0Á05 was consid- 20ered to be statistically significant. 15 10 5Results 0 3 9 24 Time (hr)IL-33 expression by myeloid cells (c) 50Data from analysis of a cDNA library indicated that mod- 45 Unstimulated TNF-a IL-1b IL-33 mRNA expressionest levels of IL-33 mRNA are expressed by human mono- 40 35cytes and dendritic cells.2 However, to date, the expression 30of IL-33 by myeloid cells has not been fully characterized. 25We therefore investigated the expression of IL-33 by 20monocytes in response to bacterial LPS and the cytokines 15IL-1b and TNF-a using quantitative real-time PCR 10 5(Fig. 1). We found that IL-33 mRNA was significantly up- 0regulated in LPS-stimulated THP-1 monocytes (Fig. 1a) 0·5 1 3 6 24and primary monocytes isolated from three separate Time (hr)donors (Fig. 1b), in comparison to unstimulated controls, Figure 1. Interleukin-33 (IL-33) messenger RNA (mRNA) expressionwith maximal transcript levels observed after 6–9 hr of is up-regulated by monocytes in response to lipopolysaccharideincubation with LPS. Conversely, real-time PCR per- (LPS) but not tumour necrosis factor-a (TNF-a) and IL-1b. Quanti-formed on cDNA isolated from MDDCs derived from pri- tative real-time polymerase chain reaction analysis was used to deter-mary macrophages of three separate donors showed a lack mine IL-33 expression by THP-1 monocytes (a) and primary humanof IL-33 expression (data not shown). In contrast to IL-33, monocytes (b) in response to stimulation with 100 ng/ml Escherichiathe ST2 and HMGB1 genes exhibited no up-regulation in coli or Porphyromonas gingivalis LPS. IL-33 mRNA expression byexpression levels in myeloid cells upon stimulation with THP-1 monocytes stimulated with 100 ng/ml TNF-a and 100 pg/mlbacterial LPS (data not shown). The up-regulation of IL-1b was also measured (c). Levels of mRNA were normalizedIL-33 in monocytes by LPS may be the result of transcrip- against RNA polymerase II mRNA levels and fold changes (‘IL-33tional up-regulation via Toll-like receptor (TLR) signalling expression’) were calculated relative to unstimulated cells at each individual time-point using the DDCt method.20 The data representor may be an indirect, secondary effect of LPS-stimulated the mean values (± SD) from three independent experiments. Signif-TNF-a or IL-1b secretion. To address this issue, THP-1 icant differences we determined using Student’s t-test on the DCtmonocytes were stimulated with TNF-a and IL-1b and values.20 *P < 0Á05 relative to unstimulated cells.IL-33 mRNA was quantified by real-time RT-PCR; theresults do not provide any evidence to suggest that thesecytokines up-regulate IL-33 (Fig. 1c). 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 aIL-33 is not secreted from viable cells suggestion that pro-IL-33 is processed by the action ofThe LPS-stimulated IL-33 mRNA levels were maximal caspase-13,4,6 and ATP has been shown to enhanceafter 6–9 hr of monocyte culture but we consistently the activity of caspase-1, enhancing the processing andÓ 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180 175
  5. 5. C. J. Nile et al. 70 Unstimulated E. coli LPS P. gingivalis LPS natants of LPS-stimulated cells incubated with ATP for 60 9–24 hr, when IL-33 mRNA expression was maximal (not 50 shown). Previous reports have indicated that IL-33 is pri- marily an intracellular cytokine7 and HMGB1 has beenIL-33 (pg/ml) 40 shown to be released by cells undergoing necrosis22 so we 30 therefore induced necrosis in LPS-stimulated cells and 20 investigated IL-33 release (Fig. 2). There was no IL-33 10 released by necrotic monocytes that had not been stimu- 0 ND ND ND ND ND ND ND ND ND ND lated by LPS, suggesting that this protein was not consti- Live control 3 6 9 24 48 tutively synthesized by these cells or at least was below Time (hr) the level of detection of the ELISA (Fig. 2). However,Figure 2. Interleukin-33 (IL-33) is not secreted by viable THP-1 IL-33 was released into the culture medium by necroticmonocytes but is released from necrotic cells. THP-1 monocytes cells stimulated by LPS and the levels of IL-33 proteinwere either stimulated with 100 ng/ml Escherichia coli or Porphyro- detected reflected the kinetics of IL-33 mRNA stimulationmonas gingivalis lipopolysaccharide (LPS) for 9 hr (live control), or by LPS (Fig. 2a) with maximal levels of IL-33 proteinstimulated with 100 ng/ml E. coli or P. gingivalis LPS for 3–48 hr fol- being released by monocytes stimulated with LPS for 9 hrlowed by induction of necrosis by five rounds of freezing () 70°) (Fig. 2). Stimulation of the cells in these experiments wasand thawing (38°). Stimulation of the cells in these experiments was confirmed by detection of TNF-a in the isolated super-confirmed by detection of tumour necrosis factor-a in the isolated natants using ELISA (data not shown). Immunocyto-supernatants using enzyme-linked immunosorbent assay (not chemistry confirmed that IL-33 protein was barelyshown). ND = not detected. The data represent the mean values expressed in unstimulated THP-1 monocytes and was not(± SD) from three independent experiments. expressed at all in unstimulated primary monocytes but there was a prominent expression of intracellular IL-33Table 1. Effect of adenosine triphosphate (ATP) on interleukin-33 protein by LPS-stimulated monocytes (Fig. 3a,b). Count-(IL-33) and IL-18 secretion by THP-1 monocytes erstaining of cells with the nuclear stain DAPI indicated that IL-33 was predominantly located in the cytoplasmic IL-33 IL-18 compartment in LPS-stimulated monocytes as confirmedTreatment (pg/ml) (pg/ml) by the absence of co-localization of green and blue fluorescence (Fig. 3a,b).Unstimulated 0 27Á0 ± 10Á7ATP (6 mm) 0 110Á1 ± 52Á0E. coli LPS (1 ng/ml) 0 228Á2 ± 96Á5 IL-33 is sequestered in the nucleus of apoptoticE. coli LPS (1 ng/ml) + ATP (6 mm) 0 1635Á5 ± 524Á5* monocytesE. coli LPS (1 ng/ml) + freeze–thawing 66Á37 ± 8Á1 – In cells induced to undergo programmed cell deathIL-33 secretion from THP-1 monocytes is not activated by ATP. HMGB1 was found to remain closely associated withTHP-1 monocytes were stimulated with 1 ng/ml Escherichia coli lipo- nuclear chromatin.24 We therefore investigated the effectpolysaccharide (LPS) for 3 hr in the presence of 6 mm ATP for the of apoptosis on IL-33 in monocytes. The LPS-stimulatedfinal 30 min of this incubation. Cells without additives (unstimu- monocytes induced to undergo apoptosis by UVB irradia-lated) and with ATP or LPS alone served as controls. The IL-33 and tion did not release any IL-33 into the culture mediumIL-18 were measured by specific enzyme-linked immunosorbentassay. The data represent the median values (± interquartile range) over a 0–48-hr time–course (data not shown). Immuno-from three independent experiments. IL-18 secretion was signifi- cytochemistry was then used to determine the intracellu-cantly increased by stimulation of the monocytes with E. coli LPS in lar location of IL-33 in stimulated cells (Fig. 4). Nuclearcombination with ATP as compared with LPS alone; Mann–Whitney morphology in control cell cultures confirms the presencetest, *P < 0Á05. of apoptotic cells (Fig. 4a) and staining for IL-33 using FITC-conjugated antibodies clearly shows that the intra- cellular IL-33 protein is tightly associated with thesecretion of IL-1b and IL-18.23 We therefore investigated nucleus of cells undergoing apoptosis (Fig. 4b).whether ATP stimulated IL-33 secretion, but we wereagain unable to detect any extracellular IL-33 in mono- Discussioncytes exposed to ATP either alone or in combination withLPS (Table 1). Similar experiments confirmed ATP The IL-1 family of cytokines have an established roleenhancement of IL-18 secretion in LPS-stimulated cells in immune regulation and inflammatory processes.1,17over the same time–course, confirming that ATP-stimu- Inappropriate activity of IL-1 cytokines is a feature of alated caspase-1 was functional in this system (Table 1). number of immune-mediated pathologies and thereforeFurthermore, IL-33 was not detected in the culture super- pathways activated by these cytokines are rational therapeutic176 Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180
  6. 6. IL-33 in monocytes (a) Control Ec LPS Pg LPS IL-33 47·62 µm 47·62 µm 47·62 µmFigure 3. Interleukin-33 (IL-33) protein isexpressed in the cytoplasm of lipopolysaccha-ride (LPS) -stimulated THP-1 monocytes (a) IL-33 +and primary monocytes (b). Immunocyto- DAPIchemistry was performed on monocytes (con-trol) and cells stimulated with 100 ng/ml 47·62 µm 47·62 µm 47·62 µmEscherichia coli (Ec) or Porphyromonas gingiva-lis (Pg) LPS for 9 hr. The IL-33 was located (b) Control Ec LPS Pg LPSusing an IL-33 antibody and secondary anti-body conjugated to fluorescein isothiocyanate(FITC; green) or tetramethyl rhodamine iso-thiocyanate (TRITC; red) and the nuclei werestained with 40 -60 ,diamidino-2-phenylindole IL-33(DAPI; blue). A series of Z-stack images weretaken to confirm the cellular location of IL-33and the image depicted is representative of 47·62 µm 47·62 µm 47·62 µmfindings throughout the Z stack. The upperpanels (a) show IL-33 expression alone (greenFITC) and the lower panels (b) show IL-33(red TRITC) and nuclei (blue DAPI) in com- IL-33 +bination. The results are representative of three DAPIindependent stimulation experiments and havebeen verified by an independent observer. Scale 47·62 µm 47·62 µm 47·62 µmas depicted on images. (a) Control Ec LPS Pg LPS IL-33 12·30 µm 8·7 µm 11·50 µm (b) IL-33 + DAPI 12·30 µm 8·7 µm 11·50 µmFigure 4. Interleukin-33 (IL-33) protein is closely associated with the nucleus in THP-1 monocytes undergoing apoptosis. Immunocytochemistrywas performed on unstimulated THP-1 monocytes (control) and on cells stimulated with 100 ng/ml Escherichia coli (Ec) or Porphyromonasgingivalis (Pg) lipopolysaccharide (LPS) for 9 hr followed by exposure to UVB-irradiation to induce apoptosis. The IL-33 was located using anIL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green). The nuclei were stained with 40 -60 ,diamidino-2-phenylindole (DAPI; blue). The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (green FITC)and nuclei (blue DAPI) in combination. Arrows indicate examples of apoptotic cells. The results are representative of three independent stimu-lation experiments and have been verified by an independent observer. Scale as depicted on the images.Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180 177
  7. 7. C. J. Nile et al.targets.1 Interleukin-33 is a recently described member of onstrate any effect of these cytokines on IL-33 expression.the IL-1 family and has attracted attention because, Previously, expression of IL-33 in human primary lunguniquely among IL-1 cytokines, it stimulates the produc- fibroblasts as well as in dermal fibroblasts and keratino-tion of Th2 cytokines such as IL-5 and IL-13, activates cytes was shown to be enhanced by a combination ofTh2 cells, and has a role in mast cell development and TNF-a and IL-1b.2 Also, TNF-a either alone or in combi-function.2,11–16 As a result, IL-33 has been implicated in nation with IL-1b enhances IL-33 expression in humanthe pathogenesis of immune-mediated disorders which primary synovial fibroblasts.9,32 However, TNF-a andfeature Th2 responses and mast cell dysregulation, and in IL-1b had no effect on IL-33 expression in rat cardiacparticular those which involve pulmonary and mucosal fibroblasts or rat cardiomyocytes.18 The reasons for theseinflammation.2,16,25 Studies of the action of recombinant inconsistencies are not clear, although the data mayIL-33 and the expression of ST2 (a subunit of the IL-33 reflect variable responsiveness of different cell types.receptor) suggest that IL-33 has roles in immune The function, if any, of IL-33 in the myeloid lineageresponses and immunopathogenesis in addition to those remains obscure but a key question is whether or notmediated by mast cells and Th2 cells. Hence, eosinophils IL-33 is secreted or remains in the intracellular compart-and basophils express ST2 and are stimulated by exoge- ment. In terms of myeloid cell IL-33, we consistentlynous IL-33.26–29 Interleukin-33 also enhanced cytokine failed to detect IL-33 in culture supernatants even afterproduction by invariant natural killer (NK) T cells and LPS stimulation. Although there is much indirecthuman NK cells.29 Also, IL-33 and ST2 are widely evidence for an immunological role of IL-33 in vitro andexpressed in vascular cells and tissues, in fibroblasts and in vivo, only a limited number of reports have describedin the central nervous system.6,7,9,18 the detection of soluble, endogenous IL-33.6,18 Hence, We have investigated the expression, intracellular loca- although rat cardiac fibroblasts synthesized IL-33 protein,tion and regulation of IL-33 in monocytes and MDDCs. unstimulated cells did not secrete this protein but IL-33In experiments using both conventional RT-PCR (not secretion (as detected by Western blot of culture super-shown) and quantitative RT-PCR, IL-33 was not natants) was induced by stimulation with phorbolexpressed in MDDCs and stimulation with LPS had no 12-myristate 13-acetate.18 Similarly, in a study of mixedeffect on IL-33 expression in MDDCs. This is in contrast glial cell cultures and astrocyte-enriched cultures, IL-33to Schmitz et al.2 who reported no expression in ‘resting’ was only found to be secreted in cells stimulated withMDDC but IL-33 expression (albeit at very modest levels PAMPs and ATP together.6 Recently, the release of IL-33as compared with smooth muscle cells and fibroblasts) from primary human endothelial cells in conditions ofwas detected when these cells were activated by LPS. Sim- mechanical stress or injury was demonstrated.3 The physio-ilarly, whereas Schmitz et al.2 only detected IL-33 mRNA logical relevance of these data remains unclear, but thisin LPS-activated monocytes, we detected IL-33 mRNA in does suggest that under certain circumstances cells maythe human promonocytic cell line THP-1, in monocytes secrete IL-33 in response to the appropriate signals.derived from these cells and in primary human mono- It is possible that IL-33 may be secreted in a form thatcytes by both conventional RT-PCR (not shown) and is not detectable by ELISA. For example, the solublequantitative RT-PCR. However, analysis of protein form of the IL-33 receptor subunit ST2, sST2 interactsexpression using immunocytochemistry demonstrated with IL-33 and blocks IL-33 signalling18,25 and sST2 isonly very low levels of constitutive IL-33 expression in induced by LPS and cytokines in monocytes.33 AlthoughTHP-1 monocytes and none in primary cells. The differ- we have not analysed sST2 in our cultures, soluble IL-33ences between our findings and those of Schmitz et al.2 was readily detectable by ELISA after induction of necro-with respect to mRNA expression may reflect technical sis in monocyte cultures previously stimulated by LPS.differences or differences in the sources of cDNA. Interleukin-33 may require other signals, in addition to We investigated regulation of IL-33 expression in LPS, to induce efficient extracellular release. For example,monocytes: LPS from E. coli as well as LPS from the peri- ATP is known to enhance both IL-1b and IL-18 releaseodontal pathogen P. gingivalis both enhanced IL-33 via activation of a multiprotein complex (the inflamma-expression. The LPS from E. coli and P. gingivalis repre- some) containing caspase-1, the enzyme responsible forsent different pathogen-associated molecular patterns intracellular processing of the pro- form of these cyto-(PAMPs): LPS from these species stimulate host cells via kines.23 It is not yet clear whether IL-33 is processedTLR4 and TLR2, respectively, which results in overlapping in vivo via a caspase-1 pathway: although there is evidencebut distinct cytokine responses.30,31 Our data suggest that that in-vitro-translated IL-33 is cleaved by incubation withengagement of both TLR2 and TLR4 pathways stimulates caspase-1,2 this was not confirmed by experiments analy-IL-33 indicating a potential role for this cytokine in sing intracellular IL-33.7 Furthermore, the predicted cleav-immune responses to diverse pathogens and PAMPs. age site for caspase-1-induced IL-33 is not conserved in We also investigated the regulation of IL-33 in mono- orthologues from other species.7 Recently, it was demon-cytes by both TNF-a and IL-1b but were unable to dem- strated that IL-33 is probably cleaved by both caspase-1178 Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180
  8. 8. IL-33 in monocytesand caspase-3 to yield a 20 000–22 000 molecular weight cytoplasmic compartment in viable monocytes and weform of IL-33 and that this cleavage took place at an did not observe prominent nuclear translocation of IL-33.amino acid residue (Asp178) distinct from that previously Palmer et al.32 reported that IL-33 was expressed in bothproposed (Ser111).3 Significantly, unlike the full-length the nucleus and cytoplasm of synovial fibroblasts andIL-331–270, the 20 000–22 000 form was not biologically ‘mononuclear inflammatory cells’. The sub-cellular locali-active (in terms of binding and activation of the ST2 zation of IL-33 may reflect intrinsic differences betweenreceptor), suggesting that IL-33 was inactivated by caspase cell types. Also, there are differences in IL-33 expressioncleavage.3 The antibodies used for the ELISA and in smooth muscle cells cultured in vitro and IL-33 expres-immunocytochemistry in the present report do not distin- sion in these cells in vivo.2,10 Significantly, the expressionguish between the different forms of IL-33 described by pattern of IL-33 changes in different cell culture condi-Cayrol et al.;3 it will therefore be interesting to investigate tions.8 Therefore, data from in vitro culture modelsin detail the molecular structures of IL-33 expressed in should be interpreted with care and it will be importantmonocytes, the effect of LPS on IL-33 maturation, and the to extend studies of IL-33 expression in immune cellsspecific forms of IL-33 released from necrotic monocytes. in vitro to investigate the expression of this cytokine in Addition of ATP to mixed glial cell cultures and astro- tissue sections from patients with inflammatory disease.cyte-enriched cultures stimulated the release of IL-33 We also generated preliminary evidence to suggest thatfrom these cells whereas these reagents had no effect in during apoptosis, IL-33 is associated with the nucleus inisolation.6 Significantly, we failed to demonstrate any monocytes. In this regard it is interesting to note that,effect of ATP on IL-33 secretion from monocytes, either apoptosis in endothelial cells stimulated IL-33 cleavagealone or in combination with LPS, although enhancement (into a form which does not bind the extracellular ST2of IL-18 secretion was measured in the same experiments receptor) suggesting that this reaction existed to inactivate(Table 1). Intriguingly, it has been speculated that IL-33 IL-33 after apoptosis.3 In addition, we demonstratedmay be processed by an inflammasome distinct from that release of IL-33 as the result of necrosis in the same cells.which processes IL-1b and IL-18.34 There is evidence that In similar experiments it was recently demonstrated thatIL-33 may function as a so-called dual function cytokine necrosis in primary endothelial cells was associated withhaving a role as an (extracellular) pro-inflammatory cyto- the release of biologically active IL-33.3 The behaviour ofkine and an (intracellular) nuclear factor with transcrip- IL-33 during apoptosis and necrosis is similar to the so-tional regulatory action.7 called ‘alarmins’, which are a group of biologically active These authors draw analogy with other ‘dual function’ mediators that include the dual function cytokinescytokines such as IL-1a and HMGB1.7 In support of this HMGB1 and IL-1a.36 Alarmins are endogenous signallinghypothesis they found that in high endothelial venules, molecules that, like exogenous PAMPs, trigger similarIL-33 was found to be primarily an intracellular protein pathways and have a common purpose as damage-associ-that closely associates with eukaryotic chromatin7 The ated molecule patterns (DAMPS).36 These findingsnuclear localization was found to be mediated by an evo- endorse the view that IL-33 has functions beyond that oflutionarily conserved homeodomain-like helix-turn-helix a conventional extracellular, immunoregulatory cytokine(HTH) motif which is located within the N-terminal por- and may be classified as a DAMP.34 The recent finding oftion of the IL-33 precursor protein.7 More recent data widespread expression of IL-33 in the vasculature, in theprovide a molecular model for the chromatin interactions skin and mucosal epithelial cells supports this hypo-of IL-33 and suggest that IL-33 binds to the histone thesis.10 Interestingly, in addition to IL-1a and IL-33, andimer H2A-H2B and hence regulates chromatin compac- isoform of IL-7 (IL-7b), also an IL-1 family cytokine, hastion by influencing nucleosome–nucleosome interac- recently been shown to translocate to the nucleus in acti-tions.35 Interleukin-33 is therefore shown to confer potent vated monocytes and to down-regulate the synthesis oftranscriptional repressor activity although the target genes pro-inflammatory cytokines.37remain to be characterized.7 In summary, the function of IL-33 in activated mono- Significantly, it was demonstrated that endothelial cells cytes is hypothesized to be primarily intracellular; and asin inflamed tissues from patients with Crohn’s disease such IL-33 may act as an ‘alarmin’ and have a role in sig-and rheumatoid arthritis express high levels of IL-33 and nalling cellular damage and inflammatory disease patho-constitute the major source of IL-33 in these tissues.7 The genesis through release from damaged or necrotic cells.high expression of IL-33 in the endothelial cells of normaland inflamed synovial tissue has recently been con- Acknowledgementsfirmed.32 Xu et al.9 also confirmed the high expression ofIL-33 (and ST2) in the rheumatoid synovium and in This work was supported by a grant from the Newcastlefibrobasts isolated from this tissue. Although we have Healthcare charity and a UK Department of Health Clini-demonstrated that IL-33 is not secreted from stimulated cian Scientist Fellowship DHCS/03/G121/46 awarded tomonocytes, IL-33 is predominantly associated with the Philip Preshaw.Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180 179
  9. 9. C. J. Nile et al.Disclosures 17 Arend WP, Palmer G, Gabay C. IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev 2008; 223:20–38. 18 Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT. IL-33 and ST2The authors declare no conflict of interest. comprise a critical biomechanically induced and cardioprotective signaling system. J Clin Invest 2007; 117:1538–49. 19 Baekkevold ES, Roussigne M, Yamanaka T et al. Molecular characterization of NF-HEV,References a nuclear factor preferentially expressed in human high endothelial venules. Am J Pathol 1 Barksby HE, Lea SR, Preshaw PM, Taylor JJ. The expanding family of interleukin-1 2003; 163:69–79. cytokines and their role in destructive inflammatory disorders. Clin Exp Immunol 2007; 20 Foster N, Cheetham J, Taylor JJ, Preshaw PM. VIP inhibits Porphyromonas gingivalis 149:217–25. LPS-induced immune responses in human monocytes. J Dent Res 2005; 84:999–1004. 2 Schmitz J, Owyang A, Oldham E et al. IL-33, an interleukin-1-like cytokine that signals 21 Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cyto- BMC Bioinformatics 2006; 7:85. kines. Immunity 2005; 23:479–90. 22 Rovere-Querini P, Capobianco A, Scaffidi P et al. HMGB1 is an endogenous immune 3 Cayrol C, Girard JP. The IL-1-like cytokine IL-33 is inactivated after maturation by adjuvant released by necrotic cells. EMBO Rep 2004; 5:825–30. caspase-1. Proc Natl Acad Sci USA 2009; 106:9021–6. 23 Sutterwala FS, Ogura Y, Flavell RA. The inflammasome in pathogen recognition and 4 Chackerian AA, Oldham ER, Murphy EE, Schmitz J, Pflanz S, Kastelein RA. IL-1 recep- inflammation. J Leukoc Biol 2007; 82:259–64. tor accessory protein and ST2 comprise the IL-33 receptor complex. J Immunol 2007; 24 Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic 179:2551–5. cells triggers inflammation. Nature 2002; 418:191–5. 5 Ali S, Huber M, Kollewe C, Bischoff SC, Falk W, Martin MU. IL-1 receptor accessory 25 Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin-33 protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc signaling in allergic airway inflammation. J Biol Chem 2007; 282:26369–80. Natl Acad Sci USA 2007; 104:18660–5. 26 Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL- 6 Hudson CA, Christophi GP, Gruber RC, Wilmore JR, Lawrence DA, Massa PT. Induc- 33, potently activates human eosinophils. J Allergy Clin Immunol 2008; 121:1484–90. tion of IL-33 expression and activity in central nervous system glia. J Leukoc Biol 2008; 27 Suzukawa M, Iikura M, Koketsu R et al. An IL-1 cytokine member, IL-33, induces 84:631–43. human basophil activation via its ST2 receptor. J Immunol 2008; 181:5981–9. 7 Carriere V, Roussel L, Ortega N et al. IL-33, the IL-1-like cytokine ligand for ST2 28 Suzukawa M, Koketsu R, Iikura M et al. Interleukin-33 enhances adhesion, CD11b receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci USA 2007; expression and survival in human eosinophils. Lab Invest 2008; 88:1245–53. 104:282–7. 29 Smithgall MD, Comeau MR, Yoon BR, Kaufman D, Armitage R, Smith DE. IL-33 8 Kuchler AM, Pollheimer J, Balogh J et al. Nuclear interleukin-33 is generally expressed amplifies both Th1- and Th2-type responses through its activity on human basophils, in resting endothelium but rapidly lost upon angiogenic or proinflammatory activation. allergen-reactive Th2 cells, iNKT and NK cells. Int Immunol 2008; 20:1019–30. Am J Pathol 2008; 173:1229–42. 30 Chen C, Coats SR, Bumgarner RE, Darveau RP. Hierarchical gene expression profiles of 9 Xu D, Jiang HR, Kewin P et al. IL-33 exacerbates antigen-induced arthritis by activat- HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingi- ing mast cells. Proc Natl Acad Sci USA 2008; 105:10913–8. valis and Escherichia coli. Cell Microbiol 2007; 9:1028–38.10 Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expres- 31 Barksby HE, Nile CJ, Jaedicke KM, Taylor JJ, Preshaw PM. Differential expression of sed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’? immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and PLoS ONE 2008; 3:e3331. Escherichia coli lipopolysaccharide. Clin Exp Immunol 2009; 156:479–87.11 Allakhverdi Z, Smith DE, Comeau MR, Delespesse G. Cutting Edge: the ST2 ligand 32 Palmer G, Talabot-Ayer D, Lamacchia C et al. Inhibition of interleukin-33 signaling IL-33 potently activates and drives maturation of human mast cells. J Immunol 2007; attenuates the severity of experimental arthritis. Arthritis Rheum 2009; 60:738–49. 179:2051–4. 33 Trajkovic V, Sweet MJ, Xu D. T1/ST2 – an IL-1 receptor-like modulator of immune12 Ho LH, Ohno T, Oboki K et al. IL-33 induces IL-13 production by mouse mast cells responses. Cytokine Growth Factor Rev 2004; 15:87–95. independently of IgE-FceRI signals. J Leukoc Biol 2007; 82:1481–90. 34 Gadina M, Jefferies CA IL-33: a sheep in wolf’s clothing? Sci STKE 2007; 2007:pe31.13 Iikura M, Suto H, Kajiwara N et al. IL-33 can promote survival, adhesion and cytokine 35 Roussel L, Erard M, Cayrol C, Girard JP. Molecular mimicry between IL-33 and KSHV production in human mast cells. Lab Invest 2007; 87:971–8. for attachment to chromatin through the H2A-H2B acidic pocket. EMBO Rep 2008;14 Komai-Koma M, Xu D, Li Y, McKenzie AN, McInnes IB, Liew FY. IL-33 is a chemo- 9:1006–12. attractant for human Th2 cells. Eur J Immunol 2007; 37:2779–86. 36 Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leu-15 Moulin D, Donze O, Talabot-Ayer D, Mezin F, Palmer G, Gabay C. Interleukin (IL)-33 koc Biol 2007; 81:1–5. induces the release of pro-inflammatory mediators by mast cells. Cytokine 2007; 37 Sharma S, Kulk N, Nold MF et al. The IL-1 family member 7b translocates to the 40:216–25. nucleus and down-regulates proinflammatory cytokines. J Immunol 2008; 180:5477–82.16 Humphreys NE, Xu D, Hepworth MR, Liew FY, Grencis RK. IL-33, a potent inducer of adaptive immunity to intestinal nematodes. J Immunol 2008; 180:2443–9.180 Ó 2010 Blackwell Publishing Ltd, Immunology, 130, 172–180