E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 2 5 2 7– 2 53 7 available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: Functional responses and identification of signaling pathways Nadine Tare, Hongli Li, Andrew Morschauser, Javier Cote-Sierra, Grace Ju, Louis Renzetti, Tai-An Lin⁎ Pharma Research and Early Development, Hoffmann-La Roche Inc., Nutley, NJ 07110-1199, USA A R T I C L E I N F O R M A T I O N AB S TR AC T Article Chronology: Activation of interleukin-1 family receptor ST2L by its ligand interleukin-33 (IL-33) is an important Received 5 March 2010 component in inflammatory responses. Peripheral blood basophils, recognized as major effector Revised version received 1 April 2010 cells in allergic inflammation that play a role in both innate and adaptive immunity, are activated Accepted 10 April 2010 by IL-33 through ST2L. However, studies are challenging due to the paucity of this cell population, Available online 18 April 2010 representing less than 1% of peripheral blood leukocytes. We identified a basophil-like chronic myelogenous leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates Keywords: functional responses to IL-33 stimulation. IL-33 induced production of multiple inflammatory ST2 mediators in KU812 cells that were blocked by anti-ST2L and anti-IL-33 antibodies. The interaction IL-33 of IL-33 and ST2L activated NF-κB, JNK, and p38 MAPK, but not ERK1/2 signaling pathways. Studies IKK-2 using pharmacological inhibitors to IKK-2 and MAP kinases revealed that one of the functional NF-κB responses, IL-33-induced IL-13 production, was regulated through NF-κB, but not JNK or p38 Cytokine MAPK signaling. The requirement of NF-κB was confirmed by IKK-2 knockdown using shRNA. Basophil KU812 represents the first human cell line-based in vitro model of the IL-33/ST2L axis and provides a valuable tool to aid in understanding the mechanism and significance of IL-33 and ST2L interaction and function. © 2010 Elsevier Inc. All rights reserved. Introduction Interleukin-33 (IL-33), a cytokine expressed primarily in smooth muscle, endothelial, and epithelial cells, is the ligand for the IL-1 receptor-related protein ST2 [1,2]. The ST2 proteins consist of two isoforms: a transmembrane form (ST2L) which is highly expressed in mast cells [3,4], and a secreted soluble form (sST2) that can serve as a decoy receptor of IL-33 [5]. The interaction of IL-33 and ST2 plays an important role in human inflammatory diseases as evidenced by their high levels of expression in diseased tissues. Elevated IL-33 levels are observed in human rheumatoid arthritis (RA) synovium [6,7], lung biopsy specimens from severe asthma patients [8], sera of patients during anaphylactic shock, and in inflamed skin tissue [9]. Both IL-33 and ST2 are overproduced in fibrotic livers [10] and are abnormally expressed in dermal fibroblast and inflammatory cells of patients with systemic ⁎ Corresponding author. Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA. Fax: +1 973 235 2981. E-mail address: tai-an.lin@roche.com (T.-A. Lin). Abbreviations: CIA, collagen-induced arthritis; IL-33, interleukin-33; MEF, mouse embryonic fibroblasts; RA, rheumatoid arthritis; shRNA, small hairpin RNA; sST2, secreted soluble form of the IL-33 receptor ST2; ST2, interleukin-1 receptor-like 1 or IL-33 receptor; ST2L, transmembrane form of the IL-33 receptor ST2; Th1, T-helper type 1; Th2, T-helper type 2; TIR, Toll-like/IL-1 receptor 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.04.007 2528 E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 2 5 27 – 2 53 7 sclerosis [11]. Single nucleotide polymorphisms of the ST2 gene, which have higher ST2 transcriptional activity, are associated with atopic dermatitis [12]. In addition, elevated sST2 levels are observed in the serum and/or bronchoalveolar lavage fluid of human patients with acute eosinophilic pneumonia [13], acute exacerbation of asthma [14], idiopathic pulmonary fibrosis [15], sepsis and trauma [16], and autoimmune diseases [17]. The level of sST2 protein is also elevated in cerebrospinal fluid after subarachnoid hemorrhage [18]. In contrast to its inflammatory role, IL-33/ ST2L signaling also appears to have a cardioprotective effect [19]. Soluble ST2 is reported to be a biomarker of heart failure [20] and its levels in serum may predict mortality and clinical outcome in acute myocardial infarction [21]. Similar to its role in human biology, the IL-33/ST2L axis has been shown to be critical in murine inflammatory disease models. Levels of IL-33 are increased in lung tissue and synovium in experimental mouse inflammation models of allergic airway hyperreactivity [22] and arthritis [7], respectively. Treatment of mice with IL-33 leads to induction of Th2-related cytokines, increased serum IgE and IgA levels, and histological changes in the lungs and GI tract such as eosinophilic and mononuclear infiltrates, increased mucus production, and epithelial cell hyperplasia and hypertrophy [1]. In addition, recombinant IL-33 exacerbates collagen-induced arthritis (CIA) [23] and induces mechanical hypernociception in mice [24]. Disruption of the IL-33/ST2L pathway using an ST2-IgG fusion protein or a blocking antibody to ST2L results in reduced allergic inflammation and resolution of airway hyperreactivity [22,25]. Similarly, the severity of CIA and antigen-induced hypernociception can be attenuated by a blocking antibody to ST2L and recombinant sST2 or sST2-Fc fusion protein [7,24,26]. These observations indicate that interruption of IL-33/ST2L signaling may be beneficial for the resolution of inflammatory diseases. Mast cells are the primary target cells of IL-33 in both humans and mice. The IL-33/ST2L axis appears to play a role in driving effector functions of both T helper type 1 (Th1) and type 2 (Th2) cells [27,28], although it was originally shown to predominantly promote Th2 responses. Human basophils and eosinophils, but not resting Th2 cells, are reported to be direct target leukocytes of IL-33 even though surface expression of the ST2L protein is not detectable in either cell type [29–31]. Depending on the cell type, IL-33 appears to activate the NF-κB and MAP kinase signaling pathways to promote its cellular functions, which include producing cytokines, chemokines, and growth factors, altering cellular morphology, and promoting cell migration, adhesion, maturation and survival. Investigations of the IL-33/ST2L system in human cell lines are lacking although two IL-33-responsive mouse cell lines, p815 and MC/9, have been reported previously [32–34]. ST2L protein is expressed in the human mast cell-derived leukemia cell line LAD2 [12] and in the megakaryoblastic leukemia cell line UT-7 [35]; however, the function and signaling of IL-33/ST2L in these leukemia cells are not known. Studies of IL-33 and ST2L in primary basophils are challenging and time-consuming due to the extremely low percentage of this cell population in peripheral blood. In addition, donor-to-donor variability as well as the inconsistency inherent within a single human donor can lead to inconclusive or conflicting results. In the present study, we identify a human leukemia cell line, KU812, that constitutively expresses ST2L. The KU812 cell line was established from the peripheral blood of a patient in blastic crisis of chronic myelogenous leukemia and the cells have been characterized as basophil precursors [36]. We profile the production of various cytokines, chemokines, and growth factors from KU812 cells in response to IL-33 and reveal that NF-κB is the major signaling pathway involved in these functional responses. Materials and methods Cell line The human chronic myelogenous leukemia cell line KU812 was obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA) at 37 °C in 5% CO2. Imaging flow cytometry analysis KU812 cells (1 × 106 cells) were incubated with 10 μg/ml anti-ST2 antibody (MAB523, R&D Systems, Minneapolis, MN) or IgG1 isotype control (MAB002, R&D Systems) at room temperature for 15 min in 100 μl D-PBS. The cells were washed, and stained with rat PE-conjugated anti-mouse IgG1 (340270, BD Bioscience, at 1:10 dilution) at 4 °C for 20 min. The stained cells were washed and analyzed by the imaging flow cytometer ImageStream (Amnis Corporation, Seattle, WA) with the 488 nm laser set at 200 mW. Measurements of cytokines, chemokines, and growth factors KU812 cells (6× 105 cells/well) were incubated with IL-33 (3625-IL010/CF, R&D Systems) or culture media at 37 °C for appropriate times as indicated in the figures or figure legends in 0.24 ml final volume in 96-well round bottom plates (Falcon 353077, BD Bioscience, Franklin Lakes, NJ). For studies using antibodies or kinase inhibitors, cultures were pre-incubated for 20 min with antibodies at 20 µg/ml or with various concentrations of inhibitors. The antibodies used were anti-ST2L antibody (AF523, R&D Systems), anti-IL-33 antibody (AF3625, R&D Systems), and normal goat IgG (AB-108-C, R&D Systems). Protein kinase inhibitors were purchased from EMD Chemicals, Inc. (San Diego, CA). They include IKK-2 inhibitor TPCA-1 [37] (Calbiochem 401481), p38 MAP kinase inhibitor SD-169 [38] (Calbiochem 506158), JNK inhibitor SP600125 [39] (Calbiochem 420119), and ERK1/2 inhibitor FR180204 [40] (Calbiochem 38007). Levels of cytokines, chemokines, and growth factors in the cellfree culture supernatants were measured by multiplex Luminex xMAP™ technology (Luminex Corporation, Austin, TX) with STarStation software (Applied Cytometry Systems, Sacramento, CA). The human cytokine LINCOplex premixed kit (21-plex, LINCO Research, St. Charles, MO) was employed using the procedure provided by the manufacturer for multiplex detection. For some studies, IL-13 levels in the cell-free culture media were determined using Instant ELISA assay kits (Bender MedSystems, Burlingame, CA). Phosphoprotein detection The Bio-Plex™ multiplex detection system (Bio-Rad, Hercules, CA) was employed to detect phosphoprotein levels in KU812 cells. The following 14 kinases or substrate proteins were chosen: IκBα, JNK, c-JUN, p38 MAPK, HSP27, ERK1/2, p90RSK, MEK1, Akt, STAT3, E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 2 5 2 7– 2 53 7 STAT6, Tyk2, p70 S6 kinase, and Src. They were assembled by the manufacturer as a multiplex detection kit for assessing phosphorylated protein and the corresponding total cellular protein level for each kinase or substrate. The cells were cultured in RPMI 1640 medium containing 0.1% bovine serum albumin (BSA) at 37 °C for 24 h. For time course studies, cells were reconstituted in the same serum-free medium (1.5 × 106 cells/0.3 ml) and incubated with 50 ng/ml of IL-33 at 37 °C for various time points as indicated in the figures. For studies using antibodies, the cells (or the ligand IL-33) were pre-incubated with 20 μg/ml of anti-ST2L (or anti-IL-33) antibody before the stimulation. The cells were then centrifuged and lysed in 0.3 ml lysing solution. The composition of lysing solution, lysate preparation, and the procedure for multiplex detection were essentially the same as recommended by the manufacturer (Bio-Rad, Hercules, CA). shRNA knockdown of IKK-2 Lentivirus expressing small hairpin RNA (shRNA) was used to suppress the expression of IKK-2 in KU812 cells. The following MISSION® shRNA lentiviral particles, which are based on the pLKO.1-puromycin vector, were purchased from Sigma-Aldrich (St. Louis, MO) and used to infect KU812 cells: Control (nontarget) shRNA (product No. SHC002V) with the sequence CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT, IKK-2 shRNA-1 (clone ID TRCN0000018915) with the sequence CCGGGCACTGGGAAAGTATCTGAAACTCGAGTTTCAGATACTTTCCCAGTGCTTTTT, IKK-2 shRNA-2 (clone ID TRCN0000018916) with the sequence CCGGCCAGCCAAGAAGAGTGAAGAACTCGAGTTCTTCACTCTTCTTGGCTGGTTTTT, and IKK-2 shRNA-3 (clone ID TRCN0000018917) with the sequence CCGGGCTGGTTCATATCTTGAACATCTCGAGATGTTCAAGATATGAACCAGCTTTTT. The manufacturer's suggested procedure to generate cells that expressed shRNA was slightly modified as follows. KU812 cells (1 × 105 cells) were incubated with lentiviral particles at MOI of 5 in 0.5 ml culture medium containing 8 μg/ml polybrene (sc-134220, Santa Cruz Biotechnology, Santa Cruz, CA). After incubation at 37 °C in 5% CO2 for 24 h, polybrene was removed from the medium and the infected cells were cultured for one additional day before adding 2 μg/ml puromycin to the medium to select for cells that expressed the transduced vectors. IL-33-induced IL-13 production and cellular IKK-2 and β-actin protein levels were examined after culturing the cells for 3 weeks in the presence of 2 μg/ml puromycin. 2529 ml, #31437, Pierce, Rockford, IL). The blot was stripped and reprobed with HRP-conjugated anti-β-actin antibody (1:1000, 13E5, #5125, Cell Signaling Technology, Boston, MA). Results KU812 cells express ST2L protein and produce multiple cytokines, chemokines, and growth factors in response to IL-33 We examined the expression of the ST2L protein on the surface of KU812 cells using imaging flow cytometry. Unlike freshly isolated human peripheral blood basophils, which have no detectable expression of ST2L protein on their surface [30], we detected significant surface expression of ST2L on KU812 cells (Fig. 1A). The merged images derived from the bright field microscopy and antiST2 antibody-stained fluorescent signal reveal that the expression of ST2L protein tends to be concentrated in specific surface areas of the cells (Fig. 1B). These image data suggest that the ST2L protein may be expressed as localized clusters, rather than broadly distributed, on the surface of KU812 cells. To determine whether the ST2L protein on KU812 cells is functional, we applied multiplex technology to analyze 21 soluble proteins in the culture media after incubating the cells with various concentrations (0.1 to 1000 ng/ml) of IL-33 for 2 to 24 h at 37 °C. As shown in Fig. 2A, IL-33 induced a dose- and timedependent secretion of multiple cytokines, chemokines, and growth factors into the culture media. These included G-CSF, GM-CSF, IL-1α, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IP-10, MCP-1, and MIP-1α (Fig. 2A). The levels of IL-1β, TNFα, eotaxin, and IL-7 in the culture media were not increased by IL-33 stimulation (Fig. 2B). Of the 21 proteins examined, the levels in the culture media of 5 analytes, IL-2, IL-12p70, IL-15, IL-17, and IFNγ, were below the detection limit under all experimental conditions. The maximum fold increase and EC50 value of IL-33 for each analyte are summarized in Table 1. The soluble proteins with the greatest maximum fold increases induced by IL-33 in KU812 cells were the Th2 cytokines IL-5 and IL-13, the chemoattractants MIP-1α and IP-10, the hematopoietic growth factors GM-CSF and G-CSF, and the inflammatory cytokine IL-6. These all exhibited production at least 10-fold above that of the medium control. The potency (EC50) of IL-33 for these functional responses ranged from 5 to 30 ng/ml. Western blotting IL-33-induced functional responses are dependent on its interaction with ST2L KU812 cells were lysed in PBS containing 1 mM EDTA, 10% glycerol, 1% NP-40, and protease inhibitors (Complete Protease Inhibitor Tablets, Roche Diagnostics, Indianapolis, IN). After centrifugation at 10,000 ×g for 20 min, the supernatant protein samples (10 μg) were dissolved in LDS sample buffer and fractionated in NuPAGE® 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA). The proteins were transferred to a polyvinylidene difluoride membrane using the iBlot™ Dry Blotting System (Invitrogen, Carlsbad, CA) and probed with mouse monoclonal antibody to IKK-2 (0.5 μg/ml, IMG-159A, IMGENEX, San Diego, CA) or IκBα (1 μg/ml, 39-7700, ZYMED Laboratories, Invitrogen) as the primary antibody. The proteins were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (40 ng/ To verify that the production of multiple cytokines in response to IL-33 in KU812 cells is mediated through the interaction of IL-33 and ST2L, we utilized antibodies against ST2L or IL-33 to disrupt the interaction. Pre-incubation of the cells with an anti-ST2L antibody (20 μg/ml) effectively blocked IL-33-induced increases in IL-5 and IL-13 levels, whereas normal goat IgG had no inhibitory effect (Fig. 3). Similarly, the production of IL-5 and IL-13 were effectively reduced when the IL-33 ligand was first incubated with an anti-IL-33 antibody (20 μg/ml) before adding it to KU812 cells. The efficacy of anti-ST2L and anti-IL-33 antibodies in blocking IL-33-induced IL-5 and IL-13 production was not observed when a high concentration of IL-33 (1000 ng/ml) was used (Fig. 3). This could be due to relative low affinity of the commercially available 2530 E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 2 5 27 – 2 53 7 anti-ST2L antibody to ST2L, or an insufficient amount of anti-IL-33 antibody to bind and neutralize a high concentration of IL-33. Similar suppressive effects by antibodies to ST2L and IL-33 were observed for other cytokines, chemokines, and soluble proteins induced by IL-33 (data not shown). These observations suggest that the IL-33-induced production of multiple soluble proteins in KU812 cells is mediated through its interaction with ST2L. IL-33 activates NF-κB, JNK, and p38 MAPK signaling pathways in KU812 cells Depending on the cell type and species, the interaction of IL-33 and ST2L has been shown to activate the NF-κB signaling pathway, as well as the canonical MAP kinases including ERK1/2, p38 MAPK, and JNK [1,31,41,42]. We examined the signaling pathways triggered by IL-33 in KU812 cells by monitoring the phosphorylation status of 14 cellular proteins following IL-33 (50 ng/ml) stimulation for 5 to 120 min. As shown in Fig. 4A, IL-33 induced transient increases in the levels of phospho-IκBα (IKK-2 substrate) and phospho-c-JUN (JNK substrate) with peak times at 5 and 30 min, respectively. Both phosphoproteins returned to un-stimulated levels after 120 min. The increase in the level of the p38 MAPK substrate HSP27 was less pronounced and there was no change in the level of ERK1/2 substrate p90RSK upon IL-33 stimulation. The phosphorylation of MAPKs exhibited similar trends as those of their substrate proteins following IL-33 stimulation. Both phospho-JNK and phospho-p38 MAPK levels were increased, and there was no change in the level of phosphoERK1/2 (Fig. 4B and Table 2). We did not observe any increase in the phosphorylation of MEK1, Akt, p70 S6 kinase, STAT3, STAT6, Tyk2, and Src in KU812 cells after IL-33 stimulation for 5 to 120 min. Similar to cytokine production, IL-33-induced phosphorylation of IκB, c-JUN, and HSP27 could be blocked by antibodies against ST2L and IL-33 (Fig. 5), verifying that the signaling cascades are activated via the interaction of IL-33 and ST2L. These observations indicate that NF-κB, JNK, and p38 MAPK signaling cascades are activated by IL-33 in KU812 cells. Unlike human blood basophils [31], the ERK1/2 signaling pathway is not triggered by IL-33 in KU812 cells. NF-κB signaling is required for IL-33-induced IL-13 production Fig. 1 – ST2L is expressed in KU812 cells. KU812 cells were stained with 10 μg/ml mouse monoclonal antibody against ST2 or mouse IgG1 (isotype control). The cells were then incubated with a rat anti-mouse IgG1 conjugated with PE and analyzed using imaging flow cytometry (ImageStream). (A) Histogram of fluorescent intensity of PE. (B) Images of three representative cells from anti-ST2 antibody-stained cells (upper panel) and control mouse IgG1-stained cells (lower panel). The cells are taken from the populations indicated by arrows in (A). We made use of pharmacological kinase inhibitors to determine whether the activation of NF-κB, JNK and p38 MAPK signaling is all required for a functional response, i.e. IL-33-induced IL-13 production. As shown in Fig. 6, the IKK-2 inhibitor TPCA-1 dosedependently inhibited IL-33-induced IL-13 production with an IC50 value of 60 nM. The effect of the JNK inhibitor SP600125 was less pronounced with 39% inhibition observed at the highest concentration (3 μM) tested. Neither the p38 MAPK inhibitor SD-169 nor the ERK1/2 inhibitor FR180204 had significant impact on IL-33induced IL-13 production. The data obtained from the kinase inhibitor study suggest that the NF-κB signaling pathway plays a major role in IL-13 production induced by IL-33 in KU812 cells. Fig. 2 – IL-33 induces production of multiple cytokines and chemokines from KU812 cells. KU812 cells were stimulated with various concentrations (0.1 ng/ml–1 µg/ml) of IL-33 for 2 to 24 h as indicated. The levels of 21 cytokines/chemokines in the cell-free culture media were analyzed using Luminex technology as described in “Materials and methods.” Detectable cytokines/chemokines are shown. (A) Cytokines/chemokines that are induced by IL-33 stimulation. (B) Cytokines that are not induced by IL-33 stimulation. The means ± SD of triplicate samples from one experiment are shown. Similar results were observed in a second independent experiment. E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 2 5 2 7– 2 53 7 2531 2532 E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 2 5 27 – 2 53 7 Table 1 – IL-33 induces cytokine/chemokine production in KU812 cells. Cytokine/chemokine Maximum fold increase a EC50 (ng/ml) IL-5 IL-13 MIP-1α IP-10 GM-CSF IL-6 G-CSF MCP-1 IL-8 IL-4 IL-10 IL-1α IL-1β TNFα Eotaxin IL-7 IL-2 b IL-12p70 b IL-15 b IL-17 b IFNγ b 158.3 56.7 56.3 39.6 19.8 14.4 12.9 4.9 3.3 2.8 2.7 2.5 0.4 0.4 0.1 0.1 NA NA NA NA NA 5.6 ± 1.3 16 ± 6.5 4.8 ± 2.1 30.2 ± 2.3 8.4 ± 2.3 11.8 ± 0.8 12.3 ± 6.7 5.3 ± 0.5 27.2 ± 17.0 7.3 ± 5.2 4.7 ± 3.3 19.7 ± 14 NA NA NA NA NA NA NA NA NA KU812 cells were stimulated with various concentrations (0.1 ng/ml– 1 μg/ml) of IL-33 for 24 h and the levels of cytokines/chemokines in the cell-free culture media were determined using a multiplex assay. EC50 values were estimated using the GraphPAD Prism program, and values shown are means ± SEM from two independent experiments. NA, not applicable. a Maximum fold increase calculated in comparison to vehicle control. b Below the limit of detection. Despite their activation by IL-33 in KU812 cells, MAP kinases p38 and JNK do not appear to be essential in the production of IL-13. To confirm the critical role of NF-κB signaling in mediating IL-33-induced IL-13 production, we suppressed the expression of IKK-2 in KU812 cells via the introduction of IKK-2 shRNAs using lentivirus infection. Among the three IKK-2 shRNAs tested, shRNA3 exhibited the greatest knockdown efficacy in KU812 cells as indicated by significant reduction of IKK-2 protein and induction of IκBα protein levels detected by Western blot analysis (Fig. 7A). Similarly, the production of IL-13 induced by IL-33 was reduced significantly in KU812 cells expressing IKK-2 shRNA-3 (Fig. 7B). The suppressive effect was less pronounced in cells expressing IKK-2 shRNA-1 and shRNA-2 correlating with the IKK-2 protein level in these cells. There was no significant change in IL-33induced IL-13 production in cells expressing a control shRNA. These data provide further evidence that IL-33-induced IL-13 production is mediated through the NF-κB signaling pathway in KU812 cells. Discussion Elucidation of the cytokine production profile induced by activation of the IL-33/ST2L axis in human basophils and basophilic leukemia cells may help in understanding the potential pathophysiological roles of IL-33 and ST2L in human disease. However, in vitro means to study the interactions of ST2L and IL-33 are challenging and labor- and time-intensive, and studies performed on primary cells often yield inconsistent results. We Fig. 3 – Inhibition of IL-33-induced IL-5 and IL-13 production by anti-ST2L and anti-IL-33 antibodies. KU812 cells were incubated with 20 μg/ml anti-ST2L antibody or normal goat IgG (control IgG) for 20 min before being stimulated with various concentrations of IL-33 for 24 h. For testing with anti-IL-33 antibody, the antibody (20 μg/ml) was first incubated with IL-33 for 20 min before being added to the cells. The levels of IL-5 (A) and IL-13 (B) in the cell-free culture media were analyzed using Luminex technology. The data are means ± SD of quadruplicate determinations from one representative experiment. have identified a basophilic leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates functional responses following stimulation with IL-33. Studies in primary blood-derived human basophils have demonstrated that IL-33 induces modest production of multiple cytokines, including IL-4, IL-5, IL-6, IL-8, IL-13 and GM-CSF [27,31]. Additional mediators such as IL-10, TNF, and CCL1 were induced by IL-33 in CD34+ progenitor-derived human mast cells [4]. Our studies revealed that IL-33 was a strong inducer of chemokines including MIP-1α (56fold over background), IP-10 (40-fold), and MCP-1 (5-fold) in KU812 cells. Compared to blood-derived basophils, KU812 cells generated relatively greater amounts of inflammatory mediators when stimulated with IL-33. For example, high levels of Th2 cytokines IL-5 (158-fold) and IL-13 (57-fold), inflammatory cytokine IL-6 (14-fold), and hematopoietic growth factors GMCSF (20-fold) and G-CSF (13-fold) were observed in the cell-free culture media 24 h after KU812 stimulation with IL-33. We hypothesize that these potent effects are due to the relatively high surface expression levels of ST2L on KU812 cells. This is supported by our observation that ST2L protein was significantly E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 2 5 2 7– 2 53 7 2533 Fig. 4 – IL-33 activates IKK-2, JNK and p38 MAPK in KU812 cells. KU812 cells were cultured in serum-free medium for 24 h before being stimulated with 50 ng/ml IL-33 (or medium for controls) for 5 to 120 min as indicated. The levels of 14 phosphoproteins and their corresponding cellular proteins were determined using multiplex technology as described in “Materials and methods” and were expressed as mean fluorescence intensity (MFI). (A) The MFI ratio was calculated by dividing the MFI of phosphoprotein by that of the corresponding cellular protein (total) and is shown for IκBα, c-JUN, HSP27, and p90RSK as indicated. (B) Fold increases in the levels of phospho-p38 MAPK, phospho-JNK, and phospho-ERK1/2 were calculated by comparison of the MFI ratios of IL-33-treated samples to those of medium-treated samples. Data are means ± SD of duplicate determinations from one experiment. Similar results were observed in two other independent studies. expressed by KU812 cells, whereas surface expression of the protein was not readily detectable in human blood-derived basophils [27,30,31]. The mechanism of ST2L up-regulation in KU812 cells remains to be elucidated. The human ST2 gene (IL1RL1) is located at chromosome 2q11.2 and is tightly linked to the IL1R1 and IL18R1 locus [43,44]. Interestingly, IL-18 has been reported to prime KU812 cells for higher leukotriene synthesis [45]. Therefore, it is possible that the levels of IL1R1 and IL18R1 may also be up-regulated in KU812 cells, as compared to the levels of these proteins in primary basophils. IL-33 is a chemoattractant for human Th2 cells [46], enhances chemotaxis of purified basophils toward eotaxin [30], and is implicated as a potent regulator of migration and activation of human basophils [47]. We observed that IL-33 is also a chemoattractant for KU812 cells (data not shown). The inflammatory mediator production profile induced by IL-33 in both KU812 cells and primary blood basophils suggests that IL-33, produced during inflammatory insults or tissue injury, initially attracts and activates basophils, Th2 cells, and other ST2L-expressing cells such as eosinophils. These ST2L-expressing leukocytes initiate innate immune responses at the sites of injury or inflammation and produce mediators such as G-CSF and GM-CSF to sustain their own survival. In addition, they produce chemokines/cytokines such as MCP-1, IP-10, MIP-1α, and Th2 cytokines to attract and activate other non-ST2L-expressing leukocytes, including macrophages, monocytes, dendritic cells, and T and B cells. This second wave of leukocytes then amplifies the inflammatory reaction and further promotes both innate and adaptive immune responses. The signal transduction mechanism following IL-33 binding to ST2L is believed to be similar to that of other Toll-like/IL-1 2534 E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 2 5 27 – 2 53 7 Table 2 – IL-33 induces increases of phosphoprotein levels in KU812 cells. Phosphoprotein IκBα JNK c-JUN P38 MAPK HSP27 ERK1/2 p90RSK MEK1 Akt STAT3 STAT6 Tyk2 p70 S6 kinase Src Fold increase a Peak time (min) 11.2 ± 7.4 3.2 ± 0.7 4.8 ± 2.2 4.1 ± 1.1 0.7 ± 0.3 NI NI NI NI NI NI NI NI NI 5 15 30 5 5 NA NA NA NA NA NA NA NA NA KU812 cells were cultured in serum-free medium for 24 h before being treated with IL-33 (50 ng/ml) for 5 min to 2 h. Values are means ± SEM from three independent experiments. Peak times were used to calculate fold increases. NI, no increase. NA, not applicable. a Fold increase calculated in comparison to vehicle control. receptor (TIR) family members, which includes the recruitment of MyD88, IRAK1, IRAK4, and TRAF6 with the formation of a signaling complex [48]. This signaling complex then activates downstream pathways such as NF-κB and canonical MAP kinases including ERK1/2, p38 MAPK, and JNK. We observed robust activation of NF- Fig. 6 – Effects of pharmacological kinase inhibitors on IL-33-induced IL-13 production. KU812 cells were incubated with various concentrations of kinase inhibitors for 20 min before being stimulated by IL-33 (50 ng/ml) for 24 h. IL-13 levels in the cell-free culture media were determined by ELISA. Data are expressed as percent of vehicle control and are means± SD of duplicate determinations from one representative experiment. Fig. 5 – IL-33-induced phosphorylation of cellular proteins is blocked by anti-ST2L and anti-IL-33 antibodies. This experiment was done essentially the same way as the study for Fig. 4 except that KU812 cells (or the ligand IL-33) were pre-incubated with 20 μg/ml anti-ST2L antibody (or anti-IL-33 antibody) for 20 min prior to stimulation. Normal goat IgG was used as a control antibody. The MFI ratios at peak times are shown for IκBα (at 5 min), c-JUN (at 30 min), HSP27 (at 5 min), and p90RSK (at 5 min). Data are means ± SD of duplicate determinations from one representative experiment. *P < 0.05 and **P < 0.005 vs control (IL-33-treated) by t test. E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 2 5 2 7– 2 53 7 Fig. 7 – Knockdown of IKK-2 reduces IL-33-induced IL-13 production. KU812 cells were infected with lentiviral particles containing various shRNA sequences as indicated. Lentivirus/ shRNA infected cells were selected by culturing them in the presence of 2 μg/ml puromycin for 3 weeks. (A) Western blot analysis of IKK-2, IκBα, and β-actin in KU812 cells expressing various shRNAs as indicated. (B) KU812 cells expressing various shRNAs were stimulated with 100 ng/ml IL-33 for 24 h. IL-13 in culture supernatants was quantified by ELISA and normalized by the amounts of cellular protein in the cultures. Data are means ± SD of duplicate determinations from one experiment. Similar results were obtained from another independent experiment. *P < 0.05 and **P < 0.005 vs IL-33-treated non-infected cells by t test. κB, p38 MAPK and JNK by IL-33 in KU812 cells. Phosphorylation of the p38 MAPK substrate HSP27 was less pronounced, suggesting that HSP27 may not be the major downstream substrate of p38 MAPK activation induced by IL-33 in these cells. Unlike the conventional TIR signaling observed in primary blood basophils and CD34+ progenitor-derived mast cells, we were not able to detect activation of ERK1/2 following IL-33 stimulation in KU812 cells. Previous studies using TRAF-6-deficient mouse embryonic fibroblasts (MEFs) suggested that the activation of NF-κB, p38 MAPK, and JNK induced by IL-33 is dependent on the MyD88IRAK-TRAF6 pathway. On the other hand, IL-33-induced ERK1/2 activation is independent of this pathway in MEFs [41]. The inability of IL-33 to induce ERK1/2 activation in KU812 cells also indicates that the ERK1/2 pathway resides separately and is not downstream of the MyD88-IRAK-TRAF6 pathway. The upstream signaling pathway of IL-33-induced ERK1/2 activation in MEFs and in human blood basophils and CD34+ progenitor-derived mast cells remains to be clarified. Although activation of the ERK1/2, p38, JNK, and NF-κB pathways are observed in basophils and mast cells following 2535 IL-33 stimulation, their requirement for promoting IL-33-induced production of cytokines and chemokines in these cells is not wellcharacterized. Previous studies in mouse splenocytes concluded that the JNK pathway, but not the NF-κB pathway, is a key mediator for IL-4 production induced by ST2L activation [49]. In cultured mouse bone marrow-derived basophils, IL-33-mediated Th2 cytokine production appears to be dependent on p38α signaling [50]. The identification of human basophilic leukemia cell line KU812 as a responder to IL-33 through interaction with ST2L allows for more extensive investigations of IL-33/ST2L signaling pathways in a homogeneous cell population. Our studies using pharmacological inhibitors to MAPK pathways did not support a key role for JNK or p38 MAPK in mediating KU812 IL33-induced IL-13 production. Instead, we found that the NF-κB signaling pathway plays a critical role in regulating this functional response, as demonstrated when the inhibition and reduced expression of IKK-2 using inhibitor TPCA-1 and shRNA-3, respectively, resulted in diminished IL-33-induced IL-13 production in KU812 cells. Our current studies together with previous reports suggest that the signal transduction mechanisms of IL-33/STL2 may vary in different cell types, as well as by species of origin. In conclusion, we have revealed a functional role of the IL-33/ ST2L axis in the human chronic myelogenous leukemia cell line KU812, and showed that at least one such response is mediated through the NF-κB signaling pathway. KU812 represents the first human cell line-based in vitro model of IL-33/ST2L interaction and function. Studies utilizing a well-characterized cell line are a safe alternative that eliminates the variability typically seen in primary cells and allows for analyses that may be impossible on sparse cell populations. With its homogeneous nature and accessibility, the KU812 cell line is a valuable tool to aid in further understanding the significance of the IL-33/ST2L axis in leukemia cells of basophilic lineage, and provides a convenient in vitro model in which to evaluate pharmaceutical agents that may disrupt that system. Acknowledgment We thank Dr. Alexandra Hicks for critically reviewing the manuscript and making valuable suggestions. REFERENCES [1] J. Schmitz, A. 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