Recombinant Mouse Interleukin-33 (Il33), partial (Active)

How does the processing of IL-33 affect its biological activity?

Processing of IL-33 significantly enhances its biological activity. Research has demonstrated that proteolytic processing can increase IL-33's alarmin activity up to ~60-fold . Various proteases can cleave full-length IL-33, including:

• Allergen-derived proteases

• Endogenous calpains from damaged airway epithelial cells

• Serine proteases from immune cells (neutrophils, mast cells, cytotoxic lymphocytes)

In human lungs, processed forms of IL-33 have been detected with apparent molecular weights of ~18, 20, 22, and 23 kDa, corresponding to different processing sites . Importantly, oxidation of cysteine residues in IL-33 can lead to its degradation by allergen proteases, suggesting that IL-33 may sense both the proteolytic and oxidative microenvironment during tissue injury to regulate the duration of its alarmin function .

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

Recombinant mouse IL-33 binds to and signals through the IL1RL1/ST2 receptor, which subsequently activates two main signaling pathways:

• Nuclear factor-kappa B (NF-κB) pathway

• Mitogen-activated protein kinase (MAPK) pathway

Upon binding to the ST2 receptor, IL-33 induces the formation of a ternary signaling complex by association with IL-1 receptor accessory protein (IL-1RAcP) . This complex formation initiates downstream signaling cascades that regulate various cellular responses, including:

• Cytokine production

• Cell activation and proliferation

• Inflammatory gene expression

• Cell survival and apoptosis regulation

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

For optimal results with recombinant mouse IL-33:

Reconstitution protocol:

• Reconstitute lyophilized protein with 100 μl sterile distilled water to achieve a concentration of approximately 0.1 mg/ml

• For further dilutions, use medium containing 5% fetal calf serum to stabilize the protein

• Filter-sterilize solutions if required for cell culture applications

Storage recommendations:

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

• Store reconstituted protein in small aliquots at -20°C to -80°C to avoid repeated freeze-thaw cycles

• Use reconstituted protein within 3 months for optimal activity

What dosing regimens are effective for in vitro and in vivo experiments?

In vitro applications:

• Cell stimulation: 0.0125-0.05 ng/ml for inducing biological effects in sensitive cell types

• Mast cell activation: 1-100 ng/ml for inducing IL-6 production in P815 mastocytoma cells and bone marrow-derived mast cells

• T cell and ILC2 stimulation: 10-50 ng/ml for inducing IL-5 and IL-13 production

In vivo applications:

• Intracerebral ventricular (i.c.v.) injection: 500 ng for microglial studies

• Systemic administration: Varies by model, typically 0.5-1 μg per mouse per day

• Wound healing models: Administration directly to wound site, with dosing dependent on wound size and model specifics

When designing experiments, consider that IL-33 may have different effects in different disease contexts and may interact with other inflammatory stimuli, such as viral infections .

How is recombinant mouse IL-33 utilized in asthma and allergy research?

Recombinant mouse IL-33 is a valuable tool in asthma and allergy research due to its role in type 2 immune responses. Key experimental approaches include:

• Co-exposure models with allergens or viruses:
  Research has shown that IL-33 selectively augments rhinovirus (RV)-induced type 2 immune responses in cells from people with allergic asthma. In a study using peripheral blood mononuclear cells (PBMCs), IL-33 enhanced RV-induced IL-5 and IL-13 release in cells from asthmatic individuals but had no effect on cells from healthy donors .

• Receptor expression analysis:
  IL-33 has been shown to enhance mRNA and surface protein expression of ST2 (the IL-33 receptor) in asthmatic individuals while having no effect on ST2 expression in healthy individuals .

• Cellular source identification:
  Flow cytometry analysis has revealed that ST2+ innate lymphoid cells (ST2+ILC) are the predominant source of IL-33-augmented IL-13 release in asthmatic individuals, while natural killer cells are the predominant source of IFN-γ in healthy individuals .

This differential regulation provides insight into how IL-33 might contribute to asthma pathogenesis and suggests targeting IL-33 could be therapeutically beneficial.

What is the role of recombinant IL-33 in wound healing research?

Recombinant IL-33 has shown promising results in wound healing research, particularly in diabetic wound models:

• Cellular mechanisms:

  • IL-33 accelerates scratch-healing of keratinocytes and fibroblasts at the cellular level

  • In diabetic mice, exogenous administration of recombinant human mature IL-33 (rhmatIL-33) increases wound healing

• Endogenous regulation:

  • Wild-type mice show up-regulation of endogenous IL-33 mRNA after injury

  • Diabetic mice show decreased IL-33 mRNA after injury

  • Exogenous IL-33 administration increased endogenous IL-33 mRNA in diabetic mice but decreased IL-33 mRNA expression in wild-type mice, suggesting a balancing role in wound healing

• Immune cell recruitment:

  • IL-33 administration elevates ILC2 cells in wounds of both diabetic and non-diabetic mice

  • IL-33 improves the transcript levels of YM1, a marker of M2 macrophages, suggesting it accelerates the transformation of macrophages from M1 to M2 phenotype

These findings suggest that recombinant IL-33 may have therapeutic potential for enhancing wound healing, particularly in diabetic conditions where endogenous IL-33 production is impaired.

How does IL-33 affect microglial function in neuroinflammation research?

Recombinant IL-33 has been shown to coordinate a microglial phagocytic response in the central nervous system:

• Epigenetic regulation:

  • IL-33 induces significant epigenetic changes in microglia

  • Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq reveal that IL-33 increases accessibility at promoter regions and induces novel enhancers

• Transcription factor activation:

  • IL-33 induces robust de novo enhancer peaks

  • Motif enrichment analysis shows significant enrichment for binding sites of adaptive-response type transcription factors, including AP-1 and NF-κB-p65

  • IL-33 markedly increases accessibility to stimulus-responsive transcription factors, particularly the AP-1 transcription factor complex

• Synaptic effects:

  • CNS-derived IL-33 acting on myeloid cells increases excitatory/inhibitory ratio by both restricting excitatory synapse numbers and promoting inhibitory synapse numbers

  • Knockout of IL-33 (IL-33cKO) in mice results in higher frequency of miniature excitatory post-synaptic currents (mEPSC) and reduced frequency of miniature inhibitory postsynaptic currents (mIPSCs)

These findings suggest IL-33 plays a crucial role in microglial regulation of neuroinflammation and synaptic function, making it an important target for neurological disease research.

How can researchers ensure optimal activity of recombinant mouse IL-33?

To maintain optimal activity of recombinant mouse IL-33:

• Quality control parameters:

  • Verify purity (≥95% by SDS-PAGE)

  • Check endotoxin levels (<0.1 EU/μg purified protein)

  • Confirm protein identity by Western blot or mass spectrometry

• Stability considerations:

  • Avoid repeated freeze-thaw cycles

  • Consider the oxidation status of cysteine residues, as oxidation can lead to degradation by proteases

  • Use carrier proteins (e.g., BSA) for dilute solutions to prevent loss due to adsorption

• Bioactivity verification:

  • Test ability to induce IL-6 production in mast cells

  • Confirm ST2 receptor binding

  • Verify activation of downstream signaling pathways (NF-κB and MAPK)

What are common pitfalls in experimental design when using recombinant IL-33?

Researchers should be aware of several potential pitfalls when designing experiments with recombinant mouse IL-33:

• Differential responses in disease states:

  • IL-33 has different effects in healthy versus diseased states (e.g., asthmatic versus healthy individuals)

  • Consider the disease context of your experimental model

• Proteolytic processing:

  • Full-length versus mature IL-33 have different activities

  • Environmental factors (proteases, oxidative stress) can modify IL-33 activity and stability

  • Consider the potential for endogenous proteolytic processing in your experimental system

• Cell-type specificity:

  • Different cell populations respond differently to IL-33

  • In asthmatic individuals, ST2+ innate lymphoid cells are the predominant source of IL-33 augmented IL-13

  • In healthy individuals, NK cells are the predominant source of IL-33 augmented IFNγ

  • Use appropriate cell-specific markers when analyzing responses

• Receptor regulation:

  • IL-33 itself can alter expression of its receptor (ST2)

  • This effect varies between disease states (increased in asthmatic cells, unchanged in healthy cells)

  • Consider measuring receptor expression as part of your experimental design

How is IL-33 being studied in the context of sepsis and immunosuppression?

Recent research has investigated the immunoprotective activity of IL-33 in sepsis models:

• Survival benefits:

  • IL-33 has been shown to improve survival in mouse models of sepsis

  • Recombinant IL-33 protein infused at 1h and 6h after cecal ligation and puncture surgery enhanced bacterial clearance and attenuated the severity of organ damage

• Cytokine modulation:

  • IL-33 decreased the levels of pro-inflammatory cytokines IL-6, IL-10, IFN-γ, and TNF-α

  • Simultaneously, IL-33 increased levels of IL-17

• Apoptosis inhibition:

  • IL-33 inhibited the apoptosis of CD4+ and CD8+ T lymphocytes and CD19+ B cells in the spleen

  • The number of CD3+ T cells was higher and the expression of active caspase-3, caspase-8, and caspase-9 was lower in IL-33-treated septic mice

  • Expression of anti-apoptotic Bcl-2 was higher in the IL-33 group than in the sepsis-only group

These findings suggest that IL-33 may have therapeutic potential in preventing immunosuppression during sepsis by inhibiting lymphocyte apoptosis.