Recombinant Mouse Interleukin-13 (Il13) (Active)

How does Recombinant Mouse IL-13 compare structurally and functionally with IL-13 from other species?

Recombinant Mouse IL-13 exhibits specific sequence homology patterns with IL-13 from other species that impact cross-reactivity studies. Mature mouse IL-13 shares 57% amino acid sequence identity with human IL-13, 75% with rat IL-13, and 58% with rhesus IL-13 . Despite this relatively low sequence homology, especially between mouse and human variants, mouse IL-13 demonstrates significant cross-species activity with human and rat systems .

When designing experiments requiring cross-species applications, researchers should conduct preliminary validation studies to confirm the anticipated activity of mouse IL-13 in their specific experimental system, particularly when working with human cell lines or tissues.

What cellular sources produce IL-13 naturally and what are its primary biological functions?

IL-13 is produced by multiple cell types in vivo, which informs experimental design when modeling physiological conditions. The primary cellular sources include:

• Th2 CD4+ T cells

• NK cells

• Visceral smooth muscle cells

• Eosinophils

• Mast cells

• Basophils

The biological activities of IL-13 are diverse and cell-type dependent:

Functionally, IL-13 plays critical roles in:

• Allergic inflammation and asthma pathogenesis

• Expulsion of gastrointestinal parasites

• Atopy and other inflammatory responses

• Tissue fibrosis, particularly in models of atopic dermatitis

Targeted deletion studies in mice have demonstrated impaired Th2 cell development, confirming IL-13's essential role in the immune response pathway .

What receptors does Recombinant Mouse IL-13 interact with and how does receptor binding influence experimental design?

Understanding IL-13 receptor interactions is crucial for experimental design. Recombinant Mouse IL-13 engages with a complex receptor system:

• IL-13Rα1 (Low-affinity interaction): Initial binding triggers IL-13Rα1 association with IL-4Rα to form a high-affinity receptor complex. This heterodimeric complex also functions as the type 2 IL-4 receptor complex, explaining the overlap in biological activities between IL-4 and IL-13 .

• IL-13Rα2 (High-affinity interaction): This receptor exists in multiple forms - intracellularly, on the cell surface, and as a soluble molecule. IL-13Rα2 primarily functions as a negative regulator by sequestering IL-13, thereby controlling its bioavailability. This receptor also indirectly regulates IL-4 activity .

The expression pattern of these receptors varies significantly between cell types and pathological states. For example, IL-13Rα2 is overexpressed in glioma and several bronchial pathologies, making it a potential therapeutic target .

When designing blocking experiments, researchers should consider that neutralizing antibodies against either IL-13 or its receptors may have distinct effects on downstream signaling. Additionally, the R110Q variant of IL-13 (associated with atopy) elicits increased responsiveness from eosinophils that express low levels of IL-13Rα2, demonstrating how genetic variants can influence receptor interactions .

How should Recombinant Mouse IL-13 be reconstituted, stored, and handled to maintain optimal activity?

Proper handling of Recombinant Mouse IL-13 is essential for maintaining its biological activity. Following these methodological guidelines will ensure consistent experimental results:

Reconstitution Protocol:

• For standard preparations containing BSA as a carrier protein: Reconstitute at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin .

• For carrier-free preparations: Reconstitute 5 μg vials at 50 μg/mL in sterile PBS, or 25 μg or larger vials at 100 μg/mL in sterile PBS .

Storage Recommendations:

• Upon receipt, store the lyophilized product immediately at -20°C to -70°C .

• After reconstitution, the protein can be stored:

  • For 1 month at 2-8°C under sterile conditions

  • For 3 months at -20°C to -70°C under sterile conditions

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these significantly reduce protein activity .

Critical Handling Considerations:

• For long-term storage, do not dilute to less than 5 μg/ml .

• If preparing stock solutions, use sterile neutral buffer containing carrier protein (human or bovine serum albumin) at concentrations of:

  • 0.5-1 mg/ml for biological assays

  • 5-10 mg/ml for ELISA standards

• When aliquoting the reconstituted protein, use polypropylene microtubes rather than glass containers, as some proteins may adhere to glass surfaces .

Failure to follow these guidelines may result in loss of biological activity and compromise experimental results.

How can the bioactivity of Recombinant Mouse IL-13 be accurately measured and validated?

Validating the bioactivity of Recombinant Mouse IL-13 preparations is essential for reliable experimental outcomes. The standard bioassay method involves:

TF-1 Cell Proliferation Assay:

• The TF-1 human erythroleukemic cell line responds to mouse IL-13 by proliferating in a dose-dependent manner .

• The expected ED50 (effective dose for 50% maximal response) for this effect typically ranges from 0.75-3 ng/mL .

• Protocol overview:

  • Culture TF-1 cells in appropriate medium

  • Serum-starve cells for 24 hours

  • Add serial dilutions of recombinant mouse IL-13

  • Incubate for 48-72 hours

  • Measure proliferation using standard methods (MTT, BrdU, or similar proliferation assays)

  • Calculate ED50 by plotting dose-response curve

Additional Validation Methods:

• SDS-PAGE Analysis: Under reducing conditions, 1 μg/lane of Recombinant Mouse IL-13 should show a single band at approximately 9 kDa when visualized by silver staining .

• SEC-MALS Analysis: This technique confirms the monomeric state of the protein with a molecular weight of approximately 11.0 kDa .

• Functional Assays: Depending on research focus, functional assays might include:

  • Measuring upregulation of IL-6 in fibroblasts

  • Assessing suppression of proinflammatory cytokines in macrophages

  • Monitoring MHC class II upregulation on appropriate cell types

When benchmarking a new lot of recombinant protein, always include a previously validated preparation as a positive control to ensure consistency between experiments.

What are the optimal conditions for using Recombinant Mouse IL-13 in cell culture experiments?

Optimizing conditions for Recombinant Mouse IL-13 in cell culture requires careful consideration of multiple factors:

Concentration Range Guidance:
The effective concentration of IL-13 varies by cell type and readout:

• For TF-1 cell proliferation assays: 0.75-3 ng/mL (ED50 range)

• For macrophage activation studies: 5-20 ng/mL

• For B cell immunoglobulin class switching: 10-50 ng/mL

• For fibroblast and endothelial cell experiments: 1-20 ng/mL

Medium Composition Considerations:

• Carrier protein: Include 0.1-0.5% BSA or human serum albumin to minimize non-specific protein adsorption to plastic surfaces and maintain stability .

• Serum levels: Lower serum concentrations (0.5-2%) often enhance cytokine effects by reducing interference from serum components.

• For long-term cultures (>72 hours), consider refreshing IL-13 every 48-72 hours as the protein may degrade over time.

Experimental Timing:

• Pre-treatment periods: Allow 24-48 hours for maximal receptor expression before IL-13 treatment in some cell types.

• Response kinetics: Different IL-13-induced responses have different optimal timepoints:

  • Morphological changes in macrophages: 24-48 hours

  • Gene expression changes: 2-24 hours (early vs. late response genes)

  • Protein secretion changes: 24-72 hours

Control Recommendations:

• Include carrier-matched vehicle controls to account for any effects from the buffer components.

• Consider including anti-IL-13 neutralizing antibodies or receptor blockers as negative controls to confirm specificity.

• Include IL-4 treatments as comparative controls due to overlapping signaling pathways.

When investigating cell types that express both IL-13 receptors (IL-13Rα1 and IL-13Rα2), consider pre-treatment with blocking antibodies against IL-13Rα2 to prevent sequestration of IL-13, especially when studying signaling through the IL-13Rα1/IL-4Rα complex.

What are the differences between carrier-free and BSA-containing preparations of Recombinant Mouse IL-13?

The choice between carrier-free and BSA-containing preparations of Recombinant Mouse IL-13 has significant implications for experimental design:

Selection Guidance Based on Application:

• Cell culture applications: BSA-containing preparations are generally preferred due to enhanced stability and reduced non-specific binding to culture vessels .

• Mechanistic studies of receptor binding: Carrier-free preparations eliminate potential interference from BSA in binding kinetics experiments .

• Mass spectrometry or proteomic applications: Carrier-free preparations avoid overwhelming the analysis with BSA peptides .

• Antibody production: Carrier-free preparations prevent generation of anti-BSA antibodies that could confound results .

Important methodological consideration: When switching between carrier-free and BSA-containing preparations, validation experiments should be performed to ensure comparable bioactivity, as the absence of carrier protein may affect protein conformation and activity in some experimental systems.

How can Recombinant Mouse IL-13 be effectively used in mouse models of allergic airway disease?

Recombinant Mouse IL-13 has been instrumental in developing mouse models of allergic airway disease, particularly asthma. The following methodological approach outlines best practices:

Administration Methods:

• Intranasal delivery: 0.5-5 μg of IL-13 in 25-50 μL PBS, administered for 3-7 consecutive days.

• Intratracheal instillation: 1-10 μg of IL-13 in 50-100 μL PBS, typically requiring anesthesia.

• Nebulization: IL-13 solution (5-50 μg/mL) aerosolized for inhalation over 20-30 minutes.

Experimental Design Considerations:

• Timing: Acute models typically involve 3-7 days of exposure, while chronic models may extend to 4-8 weeks with intermittent dosing.

• Strain selection: BALB/c mice generally develop stronger Th2 responses than C57BL/6 mice.

• Combination approaches: IL-13 can be used in conjunction with allergens (e.g., ovalbumin, house dust mite extract) to enhance allergic responses.

Key Readouts and Assessments:

• Airway hyperresponsiveness: Measured by whole-body plethysmography or forced oscillation techniques.

• Inflammatory cell infiltration: Assessed in bronchoalveolar lavage fluid (BALF) and lung tissue.

• Goblet cell hyperplasia and mucus production: Evaluated by PAS staining and MUC5AC expression.

• Airway remodeling: Measured by collagen deposition, smooth muscle thickening, and fibrotic changes.

Research Findings from Animal Models:
Studies have demonstrated that early-life exposure to IL-13 promotes Chlamydia respiratory infection and subsequent allergic airway disease, indicating a mechanistic link between early immune responses and later susceptibility to allergic conditions . IL-13 has also been shown to be crucial for murine asthma development, in contrast to Th17 cell-derived IL-17 or IL-17F, highlighting the specific role of IL-13 in allergic inflammation pathways .

A recent study has also demonstrated that IL-13 induces skin fibrosis in atopic dermatitis through thymic stromal lymphopoietin, providing insights into the mechanisms of IL-13-mediated tissue remodeling beyond the respiratory system .

What experimental approaches can be used to study IL-13 signaling pathways?

Understanding IL-13 signaling pathways requires sophisticated experimental approaches spanning multiple techniques:

Receptor-Ligand Interaction Analysis:

• Surface Plasmon Resonance (SPR): For measuring binding kinetics between IL-13 and its receptors (IL-13Rα1 and IL-13Rα2).

• Flow Cytometry: To quantify receptor expression levels on cell surfaces before and after IL-13 stimulation.

• Immunoprecipitation: To detect receptor dimerization (IL-13Rα1/IL-4Rα) following IL-13 binding.

Downstream Signaling Analysis:

• Phosphorylation Assays:

  • Western blotting to detect phosphorylation of STAT6, the primary transcription factor activated by IL-13

  • Phospho-flow cytometry for single-cell analysis of signaling events

  • Kinase activity assays for JAK1/JAK2/TYK2 activation

• Transcriptional Regulation:

  • Chromatin immunoprecipitation (ChIP) to identify STAT6 binding sites

  • Reporter gene assays using STAT6-responsive elements

  • RNA-seq or microarray analysis to identify IL-13-regulated genes

Functional Outcome Assessment:

• Protein Expression Analysis:

  • ELISA or multiplex cytokine assays to measure secreted factors

  • Flow cytometry for cell surface marker changes

  • Immunohistochemistry for tissue-level protein expression changes

• Cellular Response Quantification:

  • Proliferation assays (e.g., TF-1 cell assay)

  • Migration assays for studying chemotactic responses

  • Immunoglobulin class switching in B cells

Genetic and Pharmacological Manipulation Approaches:

• siRNA or CRISPR-Cas9: To knockdown or knockout components of the IL-13 signaling pathway.

• Dominant-negative constructs: To disrupt specific signaling interactions.

• Small molecule inhibitors: JAK inhibitors (e.g., ruxolitinib) or STAT6 inhibitors to block downstream signaling.

• Blocking antibodies: Against IL-13 or its receptors to prevent initiation of signaling.

Integration of Multiple Approaches:
For comprehensive understanding of IL-13 signaling, researchers should integrate multiple approaches. For example, combining receptor binding analysis with phosphorylation assays and functional readouts provides a more complete picture of how IL-13 variants (like the R110Q variant) might differentially activate signaling pathways that contribute to disease phenotypes .

How can the atopy-associated R110Q variant of IL-13 be studied in comparison to wild-type IL-13?

The R110Q variant of IL-13 (where arginine at position 110 is replaced by glutamine) is associated with atopic conditions and demonstrates distinct functional properties compared to wild-type IL-13. Methodological approaches to study this variant include:

Comparative Functional Analysis:

• Receptor binding assays: The R110Q variant shows altered binding affinity, particularly to IL-13Rα2, which can be quantified using surface plasmon resonance or radioligand binding assays .

• Cell responsiveness assays: Eosinophils expressing low levels of IL-13Rα2 show increased responsiveness to the R110Q variant compared to wild-type IL-13 .

Experimental Design for Variant Comparison:

• Parallel testing: Always test wild-type and R110Q variant IL-13 in parallel under identical conditions to accurately compare their effects.

• Dose-response curves: Generate complete dose-response curves rather than single concentrations to identify shifts in potency or efficacy.

• Time-course studies: The variant may exhibit different kinetics of receptor binding, signaling activation, or signal termination.

Key Considerations for R110Q Studies:

• Cell selection: Choose cell types relevant to atopic conditions (eosinophils, mast cells, bronchial epithelial cells) that express varying levels of IL-13Rα2.

• Receptor expression analysis: Quantify IL-13Rα1 and IL-13Rα2 expression levels in your experimental system, as the ratio significantly affects the differential response to the variant.

• Downstream signaling focus: Pay particular attention to STAT6 phosphorylation kinetics and magnitude, as this is a key differentiator between variant and wild-type responses.

Translational Research Applications:

• Genotype-phenotype correlation studies: Compare responses to the R110Q variant in cells from individuals with different atopic phenotypes.

• Therapeutic targeting considerations: The R110Q variant may respond differently to IL-13 neutralizing antibodies or receptor antagonists, informing personalized medicine approaches.

• Biomarker development: Differential responses to the variant could serve as biomarkers for atopic risk or treatment response.

Studies have shown that the R110Q variant elicits increased responsiveness specifically in cells with low IL-13Rα2 expression, suggesting that the variant may partially escape the negative regulatory function of this receptor . This finding has significant implications for understanding individual susceptibility to atopic conditions and may inform therapeutic strategies targeting the IL-13 pathway.