Recombinant Mouse Interleukin-33 protein (Il33) (Active)

What is the molecular structure of recombinant mouse IL-33 protein?

Recombinant mouse IL-33 typically refers to the mature form spanning amino acids 109-266 of the full-length protein. The protein has a molecular weight of approximately 18-23 kDa depending on processing state and post-translational modifications. The amino acid sequence includes: SIQGTSLLTQSPASLS TYNDQSVSFVLENGCYVINVDDSGKDQEQDQVLLRYYESPCPASQSGDGVDGKKLMVNMSPIKDTDIWLHANDKDYSVELQRGDVSPPEQAFFVLHKKSSDFVSFECKNLPGTYIGVKDNQLALVEEKDESCNNIMFKLSKI . This mature form shares approximately 55% amino acid sequence identity with human IL-33 and 90% with rat IL-33, while sharing less than 25% identity with other IL-1 family proteins .

How does proteolytic processing affect mouse IL-33 activity?

Proteolytic processing significantly enhances mouse IL-33 activity. Full-length IL-33 can be processed by various proteases including:

• Exogenous allergen proteases

• Endogenous calpains from damaged airway epithelial cells

• Serine proteases from immune cells

This processing can increase IL-33's alarmin activity up to ~60-fold compared to the full-length protein. Processed forms of IL-33 with apparent molecular weights of approximately 18, 20, 22, and 23 kDa have been detected in human lungs, consistent with specific processing sites . Importantly, while proteolytic processing activates IL-33, subsequent oxidation of cysteine residues followed by further proteolytic degradation can inactivate the protein, suggesting a self-limiting regulatory mechanism .

What expression systems are used for producing active recombinant mouse IL-33?

Recombinant mouse IL-33 can be produced using different expression systems, each with distinct advantages:

For most functional studies, the choice between these systems depends on the specific experimental requirements. HEK293-expressed protein may better replicate naturally occurring IL-33 for studies focusing on subtle regulatory mechanisms, while E. coli-derived protein is often sufficient for broader functional studies and in vivo applications.

What are the primary signaling mechanisms of mouse IL-33?

Mouse IL-33 signals primarily through binding to the IL1RL1/ST2 receptor, which then forms a complex with IL-1 receptor accessory protein (IL-1RAcP). This receptor complex activation triggers several downstream signaling cascades:

• NF-κB pathway activation - critical for inflammatory gene expression

• MAPK signaling pathways - regulating cellular stress responses and differentiation

• In specific contexts, IL-33 can induce a signaling cascade via TNFα → IL-1β → IFNγ → ET-1 → PGE2, particularly in hypernociception models

The activation of these pathways ultimately leads to the synthesis and release of various inflammatory mediators, particularly Th2-associated cytokines like IL-5 and IL-13 .

How does IL-33 function as an alarmin in tissue injury?

IL-33 acts as an "alarmin" during tissue injury through several mechanisms:

• Constitutive expression in barrier tissues and rapid release during necrosis or tissue damage

• Sensing of the proteolytic and oxidative microenvironment during injury

• Proteolytic processing by endogenous calpains and other proteases that enhance its activity up to 60-fold

• Chemoattraction of Th2 cells to sites of injury

• Amplification of immune responses in damaged tissues

The alarmin function of IL-33 represents a critical link between tissue damage and the initiation of innate and adaptive immune responses. In barrier tissues, IL-33 is constitutively expressed and stored in the nucleus, ready for immediate release upon cellular damage, thus serving as an early warning system that activates immune cells without requiring de novo synthesis .

What is the role of IL-33 in mitochondrial function and metabolic reprogramming?

IL-33 induces rapid UCP2-dependent mitochondrial rewiring that:

• Attenuates the generation of reactive oxygen species

• Preserves the integrity of the Krebs cycle

• Supports persistent production of itaconate

• Promotes GATA3-dependent differentiation of inflammation-resolving alternatively activated macrophages

This mitochondrial reprogramming represents a novel mechanism by which IL-33 influences cellular metabolism to shape immune responses, particularly in alternatively activated macrophages. This function highlights IL-33's role not only in initiating inflammatory responses but also in orchestrating resolution and tissue repair processes through metabolic regulation.

What are the optimal storage and handling conditions for recombinant mouse IL-33?

To maintain optimal activity of recombinant mouse IL-33:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, store at -80°C in small aliquots to avoid repeated freeze-thaw cycles

• Reconstitute in sterile buffer (PBS or manufacturer's recommended buffer) containing carrier protein (0.1-1% BSA) to prevent adsorption to tubes

• Avoid oxidizing conditions that can inactivate processed IL-33 through cysteine modification

• Work quickly when preparing dilutions and keep on ice

• Monitor activity periodically using functional assays appropriate for your experimental system

Proper storage and handling are essential as processed forms of IL-33 can be particularly susceptible to degradation by proteases after cysteine residue oxidation .

How can researchers assess IL-33 activity in functional assays?

Several approaches can be used to measure recombinant mouse IL-33 activity:

• Cell-based assays:

  • Stimulation of mast cells, ILC2s, or basophils and measurement of IL-5/IL-13 production

  • Activation of ST2-expressing reporter cell lines

  • Assessment of alternatively activated macrophage polarization

• Molecular readouts:

  • NF-κB activation using reporter constructs

  • MAPK phosphorylation by Western blotting

  • Gene expression analysis of IL-33 responsive genes

  • Measurement of preproET-1 mRNA expression in target tissues

• In vivo functional tests:

  • Measurement of cutaneous or articular hypernociception

  • Assessment of Th2 cytokine induction (IL-5, IL-13)

  • Quantification of IgE and IgA production

  • Evaluation of tissue inflammation markers

For rigorous assessment, combine multiple readouts and include appropriate controls (IL-33 neutralizing antibodies, soluble ST2, or experiments in ST2-null mice).

What are effective dosing strategies for in vivo studies with recombinant mouse IL-33?

Based on published research, effective dosing strategies for in vivo studies include:

The ED50 for some effects can be as low as 0.0125-0.05 ng/mL in highly sensitive in vitro systems, indicating the potency of this cytokine . Dosing should be carefully optimized for each specific experimental model.

How can recombinant mouse IL-33 be utilized in studying cardioprotection mechanisms?

Recombinant mouse IL-33 has emerged as a potential cardioprotective agent. Researchers can use IL-33 to study cardioprotection through the following approaches:

• In vitro cardiomyocyte protection studies:

  • Pretreatment of cultured cardiomyocytes with IL-33 before hypoxia challenge

  • Assessment of apoptosis markers (caspase activation, TUNEL staining)

  • Evaluation of cellular survival pathways (Akt, ERK signaling)

• In vivo ischemia/reperfusion models:

  • Administration of IL-33 subcutaneously after experimental myocardial infarction

  • Quantification of infarct size, cardiomyocyte apoptosis, and echocardiographic parameters of left ventricular function

  • Comparative studies in wild-type versus ST2-null mice to confirm receptor specificity

• Mechanistic investigations:

  • Analysis of how IL-33 administration affects inflammatory infiltrates in cardiac tissue

  • Assessment of cardiomyocyte-specific versus immune cell-mediated effects

  • Investigation of temporal requirements for IL-33 administration relative to ischemic insult

This research direction is particularly promising as no currently available pharmacotherapies effectively interrupt cardiomyocyte death pathways during acute ischemic insult or subsequent reperfusion injury .

What approaches can be used to study the role of IL-33 in allergic inflammation?

To investigate IL-33's role in allergic inflammation, researchers can employ several strategies:

• Processing and activation studies:

  • Examine how allergen proteases process full-length IL-33 to enhance its activity

  • Investigate the kinetics of IL-33 release and processing during allergen exposure

  • Study how the oxidative environment affects IL-33 stability and activity

• Cellular mechanism investigations:

  • Analysis of IL-33's effects on mast cells, basophils, and ILC2s in allergic contexts

  • Assessment of IL-33-dependent Th2 cell recruitment and activation

  • Investigation of how IL-33 coordinates with other allergic mediators

• Intervention approaches:

  • Use of soluble ST2 as a decoy receptor to block IL-33 signaling

  • Application of proteolytic inhibitors to prevent IL-33 activation

  • Testing of antioxidants to promote IL-33 inactivation through cysteine oxidation

These approaches can be integrated into established models of allergic asthma, atopic dermatitis, or food allergy to better understand IL-33's contribution to disease pathogenesis .

How does IL-33 contribute to inflammatory hypernociception and what are the experimental approaches?

IL-33 plays a significant role in inflammatory hypernociception through a complex cytokine cascade. Researchers can study this phenomenon through:

• Signaling cascade analysis:

  • Demonstration of the IL-33 → TNFα → IL-1β → IFNγ → ET-1 → PGE2 signaling pathway

  • Use of specific inhibitors or genetic knockout models at each step of the cascade

  • Measurement of each mediator's production following IL-33 administration

• Behavioral testing:

  • Assessment of mechanical hypernociception using von Frey filaments after IL-33 administration

  • Comparison of cutaneous versus articular hypernociceptive responses

  • Time-course studies to determine the onset and resolution of IL-33-induced hypernociception

• Molecular readouts:

  • Quantification of preproET-1 mRNA expression in target tissues

  • Measurement of PGE2 production in response to IL-33

  • Analysis of TNFα, IL-1β, and IFNγ production kinetics

This research area is particularly interesting as it demonstrates IL-33's capacity to induce Th1-associated inflammatory responses, highlighting its pleiotropic nature beyond the conventional view of IL-33 as a Th2-inducing cytokine .

What are common problems in detecting mouse IL-33 expression and how can they be addressed?

Detection of mouse IL-33 expression presents several challenges:

• Nuclear versus extracellular localization:

  • In quiescent cells, IL-33 is predominantly nuclear and functions as a chromatin-associated factor

  • During cell damage, IL-33 is released extracellularly as an alarmin

  • Solution: Use both nuclear and cytoplasmic/extracellular extraction methods for comprehensive analysis

• Processing artifacts:

  • Full-length IL-33 can be rapidly processed during sample preparation

  • Solution: Include protease inhibitors in all buffers and process samples quickly at cold temperatures

• Cross-reactivity concerns:

  • Some antibodies may cross-react with other IL-1 family members

  • Solution: Validate antibody specificity using IL-33 knockout tissues or cells

• Low expression levels:

  • Constitutive expression may be below detection thresholds in some tissues

  • Solution: Consider enrichment steps or more sensitive detection methods like digital PCR or highly sensitive ELISA

How can researchers distinguish between different processed forms of IL-33?

To distinguish between different processed forms of IL-33:

• High-resolution gel electrophoresis:

  • Use gradient gels (10-20%) for better separation of processed forms

  • Employ Western blotting with antibodies targeting different epitopes

  • Include molecular weight standards in the 15-25 kDa range for accurate sizing

• Mass spectrometry approaches:

  • Perform LC-MS/MS analysis of purified protein samples

  • Use targeted approaches to identify specific N-terminal cleavage sites

  • Compare experimental data with known processed forms (18, 20, 22, and 23 kDa variants)

• Functional discrimination:

  • Compare activity of different molecular weight fractions in bioassays

  • Use recombinant proteins with defined cleavage sites as references

  • Assess differential susceptibility to oxidation-induced inactivation

These approaches can help researchers characterize the specific IL-33 forms present in their experimental systems, which is critical since different processed forms can exhibit up to 60-fold differences in biological activity .

What controls should be included in IL-33 functional studies?

Rigorous IL-33 functional studies should include the following controls:

• Receptor specificity controls:

  • Soluble ST2 (sST2) as a decoy receptor to confirm IL-33-specific effects

  • Comparison between wild-type and ST2-null mice or cells

  • IL-33 neutralizing antibodies

• Pathway validation controls:

  • Specific inhibitors for downstream mediators (IL-1ra, TNF antagonists, ET receptor antagonists)

  • Experiments in knockout mice for key pathway components (TNFR1-/-, IFNγ-/-)

• Processing controls:

  • Comparison between full-length and processed recombinant IL-33

  • Protease inhibitors to prevent uncontrolled processing

  • Oxidized versus reduced IL-33 to assess activity modulation by redox state

• Technical controls:

  • Heat-inactivated IL-33 for non-specific protein effects

  • Fc control when using Fc-fusion proteins

  • Endotoxin testing to exclude LPS contamination effects

Including these controls ensures that observed effects are specifically attributable to IL-33 signaling through its canonical receptor and helps to delineate the exact mechanisms involved in the biological response being studied.