IL-33 Reporter HEK 293 Cells

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IL-33 responsive NF-κB/AP1-SEAP reporter assay

Signaling pathway in HEK-Blue™ IL-33 cells

HEK-Blue™ IL-33 cells are designed to monitor human IL-33-induced NF-κB/AP1 stimulation or inhibition. This colorimetric cytokine bioassay can be used to screen activatory or inhibitory molecules, such as engineered cytokines and neutralizing antibodies, respectively.

IL-33 reporter cells respond strongly to recombinant human IL-33 (see figures). At higher doses, they also detect mouse (m)IL-33. Of note, they do not respond to human and mIL-1β, nor to mTNF-α cells. Their reliable and consistent performance makes them suitable for release assays of activatory and inhibitory molecules such as Astegolimab, a monoclonal antibody that targets the IL-33 receptor (IL-33R or ST2) and prevents IL-33 signaling (see figures).

Key features

• Readily assessable NF-κB/AP-1-SEAP reporter activity
• Convenient readout using QUANTI-Blue™ Solution
• High sensitivity to human IL-33 activity
• No response to human (h) and murine (m) IL-1β
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

IL-33 is a member of the IL-1 family, a group of cytokines that play important roles in host defense, immune regulation and inflammation [1, 2].

More details

References:

1. Arend W. et al., 2008. IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev. 223:20-38.
2. Mantovani, A., et al., 2019. Interleukin-1 and Related Cytokines in the Regulation of Inflammation and Immunity. Immunity. 50(4): p. 778-795.

Figures

Dose-response of HEK-Blue™ IL-33 cells to recombinant human IL-33. HEK-Blue™ IL-33 cells (cat code: hkb-hil33) were stimulated with increasing concentrations of recombinant human IL-33. After overnight incubation, the NF-kB/ AP1-induced SEAP activity was determined using QUANTI-Blue™, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-dependent inhibition of HEK-Blue™ IL-33 cell response using Astegolimab biosimilar. HEK-Blue™ IL-33 cells were incubated with increasing concentrations of Anti-hIL-33R-hIgG2 (0.3 ng - 10 µg) for 1 h prior to the addition of recombinant human IL-33 (30 pg/ml). After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown in percentage of activity (mean ± SEM).

Response of HEK-Blue™ IL-33 cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines: 1 ng/ml of hIL-33, 100 ng/ml mIL-33, 10 ng/ml hIL-1β, mIL-1β, hIL-2, hIL-4, hIL-17, hIL-18, hTNF-γ, hTNF-α, mTNF-α, or 100 U/ml hIFN-α2a or IFN-β. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. The OD at 650 nm is shown as mean ± SEM.

Specifications

Antibiotic resistance: blasticidin, hygromycin B, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Guaranteed mycoplasma-free

Specificity: Detects human IL-33

Detection range: 0.5 - 100 ng/ml

Contents

HEK-Blue™ IL-33 Cells (hkb-hil33)

• 1 vial containing 3-7 x 10⁶ cells
• 2 x 1 ml of HEK-Blue™ Selection (250x concentrate)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

HEK-Blue™ IL-33 vial (hkb-hil33-av)

• 1 vial containing 3-7 x 10⁶ cells

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Notification:  Reference #hkb-hil33-av can only be ordered together with reference #hkb-hil33.

Details

Cell line description

HEK-Blue™ IL-33 cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with the gene encoding the IL-1R4 chain (also known as IL-33 receptor IL-33R, or ST2) of the IL-33 receptor to obtain a fully active IL-33 signaling pathway. The other receptor subunit, IL-1R3 (aka IL-1RAcP), is naturally expressed in these cells. They were further transfected with an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter.

The binding of IL-33 to its receptor triggers a signaling cascade leading to NF-κB/AP-1 activation and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. HEK-Blue™ IL-33 cells detect human (h) and, at high doses, mouse (m) IL-33. Of note, these cells do not respond to other AP-1/ NF-κB-signaling cytokines, TNF-α and IL-1β (see figures).

IL-33 background

Interleukin-33 (IL-33; also known as IL-1F11, DVS22, NF-HEV)) is a member of the IL-1/Toll-like receptor cytokine superfamily, a group of cytokines that play important roles in host defense, immune regulation, and inflammation [1, 2].

IL-33 is constitutively expressed in lymphoid tissues, epithelial cells, fibroblasts, mucosal tissues, tumor cells, and vascular tissues [3]. It plays a central role in type 2 innate and adaptive immunity and inflammation, modulating Th2, ILC2 and M2 macrophage responses [2]. It is involved in responses to type 2 infections (e.g. parasites), tissue repair, as well as harmful allergic responses (e.g. asthma) [2].

IL-33 mediates its biological effects through the IL-1R4 receptor (also known as ST2, IL1RL1, IL-33R) and the IL-1R3 accessory protein (also known as IL-1RAcP or IL-1RAP) [2, 4, 5].

Upon ligand binding, IL-1Rs dimerize through their Toll/interleukin-1 resistance (TIR) domains to recruit the MyD88 adaptor protein, which then couples to IL-1R-associated kinases (IRAKs) and tumor necrosis factor receptor-associated factor 6 (TRAF6). This leads to the activation of key transcription factors, including NF-KB, AP-1, and IRFs [2, 4].

IL-33 can function both as a traditional cytokine and as a nuclear factor regulating gene transcription. Following pro-inflammatory stimulation, IL-33 can induce Th2-biased immune responses, such as the production of IL-4, IL-5, and IL-13 [6]. In addition, as IL-33 is constitutively expressed in endothelial and epithelial cells, it can act as an endogenous danger signal, or damage-associated molecular pattern (DAMP; also called alarmins), in response to tissue damage [7, 8].

IL-33 has emerged as a key regulatory cytokine in barrier tissues, making it an important target for inhibition therapy in autoimmune diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), asthma, and allergies.

1. Arend W. et al., 2008. IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev. 223:20-38.
2. Mantovani, A., et al., 2019. Interleukin-1 and Related Cytokines in the Regulation of Inflammation and Immunity. Immunity. 50(4): p. 778-795.
3. Catalan-Dibene, J. et al., 2018. Interleukin 30 to Interleukin 40. J Interferon Cytokine Res. 38(10):423-439.
4. Teufel, L.U., et al., 2022. IL-1 family cytokines as drivers and inhibitors of trained immunity. Cytokine. 150: p. 155773.
5. Gaballa, J.M., et al., 2024. International nomenclature guidelines for the IL-1 family of cytokines and receptors. Nature Immunology. 25(4): p. 581-582.
6. Schiering C. et al., 2014. The alarmin IL-33 promotes regulatory T-cell function in the intestine. Nature. 513(7519):564-8.
7. Cayrol C. & Girard JP., 2014. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr Opin Immunol. 31-7.
8. Oboki K. et al., 2011. IL-33 and airway inflammation. Allergy Asthma Immunol Res. 3(2): 81–88. 

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