Recombinant Mouse Interleukin-13 (Il13), partial (Active)

What is the molecular structure and basic function of recombinant mouse IL-13?

Recombinant mouse IL-13 is a cytokine consisting of 113 amino acids (typically spanning positions 19-131 of the full sequence), with a molecular weight of approximately 11.0 kDa when analyzed by SEC-MALS, indicating its monomeric structure . The protein plays crucial roles in allergic inflammation and immune response to parasite infection . IL-13 functions as a Th2 cytokine that synergizes with IL-2 in regulating interferon-gamma synthesis and stimulates B-cell proliferation and activation of eosinophils, basophils, and mast cells . It also demonstrates the capacity to antagonize Th1-driven proinflammatory immune responses by downregulating the synthesis of proinflammatory cytokines including IL-1, IL-6, IL-10, IL-12, and TNF-alpha, partially through suppression of NF-kappa-B signaling mechanisms .

How does mouse IL-13 signal through its receptors?

Mouse IL-13 exerts its biological effects through a receptor complex comprising the IL-4R chain and the IL-13RA1 chain . This binding activates Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), leading to the activation of signal transducer and activator of transcription 6 (STAT6) . In addition to IL-13RA1, another receptor called IL-13RA2 acts as a high-affinity decoy for IL-13, mediating internalization and depletion of extracellular IL-13 . The complete signaling cascade occurs through the JAK/STAT pathway, which is essential for the downstream effects of IL-13 . This dual receptor system provides regulatory complexity for IL-13 function and offers multiple potential targets for experimental manipulation.

What are the common sources and preparations of recombinant mouse IL-13?

Recombinant mouse IL-13 is typically produced using either mammalian expression systems like HEK293 cells or bacterial expression systems such as E. coli . The HEK293-expressed protein (such as ab270080) offers advantages in terms of post-translational modifications and proper folding, with purity levels typically ≥95% and endotoxin levels <0.005 EU/μg . The E. coli-derived protein (like R&D Systems' 413-ML) typically spans amino acids Ser26-Phe131 of the native sequence . Commercial preparations are available in both carrier-containing and carrier-free formats. Carrier-containing preparations include bovine serum albumin (BSA) to enhance protein stability, increase shelf-life, and allow storage at more dilute concentrations . Carrier-free versions are recommended for applications where BSA might interfere with experimental results .

What is the optimal reconstitution procedure for lyophilized recombinant mouse IL-13?

The optimal reconstitution procedure depends on the specific preparation and intended application. For carrier-containing formulations (lyophilized from a 0.2 μm filtered solution in PBS with BSA), reconstitution at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin is recommended . For carrier-free formulations, 5 μg vials should be reconstituted at 50 μg/mL in sterile PBS, while 25 μg or larger vials should be reconstituted at 100 μg/mL in sterile PBS . It is critical to avoid introducing excessive physical agitation during reconstitution to prevent protein denaturation. After reconstitution, the solution should be allowed to sit for at least 15 minutes at room temperature to ensure complete solubilization before aliquoting for storage or experimental use.

How should reconstituted mouse IL-13 be stored to maintain optimal activity?

To maintain optimal activity, reconstituted mouse IL-13 should be stored according to the following guidelines: for immediate use, store at 2-8°C for up to one month; for longer-term storage, prepare aliquots and store at ≤-20°C for up to one year or at ≤-70°C for optimal stability . It is crucial to use a manual defrost freezer and avoid repeated freeze-thaw cycles as these can significantly reduce protein activity . Each aliquot should be of sufficient volume for a single experiment to eliminate the need for multiple freeze-thaw cycles. If slight turbidity or particulates are observed upon thawing, these can often be removed by microcentrifugation before use. Activity testing after extended storage periods is recommended to verify retention of functional properties.

What cell-based assays can be used to validate the activity of recombinant mouse IL-13?

The biological activity of recombinant mouse IL-13 can be validated using several cell-based assays. A commonly used method is the TF-1 cell proliferation assay, where IL-13 stimulates the proliferation of TF-1 human erythroleukemic cells with an ED50 (effective dose for 50% maximal response) typically in the range of 0.75-3 ng/mL . Additionally, mouse IL-13 can be evaluated through its ability to induce STAT6 phosphorylation in responsive cell lines such as THP-1 or A549 cells transfected with IL-13 receptors. Other functional assays include measuring IL-13-induced expression of adhesion molecules like VCAM1 on endothelial cells, assessment of B-cell activation markers, or quantification of IgE production in appropriate B-cell models . Validation should include both positive controls (known active IL-13) and negative controls (buffer only) to ensure assay specificity.

How can recombinant mouse IL-13 be used to study allergic airway disease models?

Recombinant mouse IL-13 is instrumental in studying allergic airway disease models, particularly asthma. In experimental designs, IL-13 can be administered intranasally or via nebulization to induce asthma-like phenotypes characterized by airway hyperresponsiveness, goblet cell metaplasia, and eosinophilic inflammation . Studies have shown that IL-13 is crucial for the development of allergic airway disease, as it promotes mucus hypersecretion and airway remodeling . For instance, research by Sehra et al. demonstrated that IL-13 regulates goblet cell metaplasia through periostin in a model of allergic airway inflammation . Furthermore, Starkey et al. showed that constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease . In these experimental models, typical dosing ranges from 1-5 μg of IL-13 per mouse, administered over varying schedules depending on the specific disease features being investigated.

What role does IL-13 play in parasitic infection models, and how can recombinant IL-13 enhance these studies?

IL-13 plays a critical role in the immune response to parasitic infections, particularly helminth infections. Recombinant mouse IL-13 can be used to study the mechanisms underlying this response in various models . In Nippostrongylus brasiliensis infection models, IL-13 is essential for the expulsion of parasites through its effects on goblet cell hyperplasia, mucus production, and intestinal contractility . Morimoto et al. demonstrated that IL-13 receptor alpha2 regulates both the immune and functional responses to N. brasiliensis infection . Researchers can use recombinant IL-13 to:

• Examine dose-dependent effects on parasite clearance

• Study the interplay between IL-13 and other cytokines in orchestrating anti-parasitic immunity

• Investigate the role of IL-13 in tissue repair following parasitic infection

• Analyze IL-13-dependent mechanisms for alternative activation of macrophages

Typical experimental designs include administration of recombinant IL-13 (0.5-2 μg daily) before or during infection to assess its ability to accelerate parasite clearance or modulate the immune response.

How can recombinant IL-13 be used to investigate fibrotic conditions?

Recombinant mouse IL-13 is valuable for investigating fibrotic conditions due to its profibrotic properties. In skin fibrosis models relevant to atopic dermatitis, IL-13 induces fibrosis through thymic stromal lymphopoietin as demonstrated by Oh et al. . For experimental approaches, researchers can:

• Administer recombinant IL-13 intradermally (typically 0.5-1 μg per site) to induce localized fibrosis

• Use IL-13 in combination with TGF-β to potentiate fibrotic responses

• Apply IL-13 to cultured fibroblasts to study direct profibrotic effects, including collagen synthesis and myofibroblast differentiation

• Employ IL-13 in lung explant cultures to examine tissue-specific fibrotic responses

In cardiac repair models, Cho et al. showed that macrophages play a crucial role in the transition to myofibroblasts after myocardial infarction, a process influenced by IL-13 . These experimental approaches allow for mechanistic studies of IL-13-mediated fibrosis across multiple organ systems and disease contexts.

How do different expression systems affect the activity and post-translational modifications of recombinant mouse IL-13?

The choice of expression system significantly impacts the structural and functional characteristics of recombinant mouse IL-13. HEK293-expressed IL-13 (such as ab270080) generally exhibits mammalian-type post-translational modifications, including proper glycosylation patterns that may influence protein stability and receptor binding kinetics . In contrast, E. coli-expressed IL-13 (like R&D Systems' 413-ML) lacks these modifications, which can affect certain aspects of protein function . Comparative studies have shown that while both forms maintain core biological activities, such as the ability to stimulate TF-1 cell proliferation, HEK293-expressed IL-13 may demonstrate enhanced stability in certain experimental conditions and potentially more closely resemble the native cytokine's tertiary structure.

A systematic comparison of different preparations reveals the following differences:

These differences make selection of the appropriate preparation critical depending on the specific research questions being addressed.

What are the experimental challenges in distinguishing IL-13 effects from those of IL-4 given their overlapping functions?

Distinguishing IL-13 effects from IL-4 effects presents significant experimental challenges due to their overlapping functions and shared receptor components. Both cytokines signal through the IL-4Rα chain, leading to activation of similar downstream pathways, particularly STAT6 . To address this challenge, researchers should consider:

• Using receptor-specific blocking antibodies that selectively inhibit either IL-4Rα/γc (IL-4 specific) or IL-4Rα/IL-13Rα1 (shared IL-4/IL-13) interactions

• Employing genetic models with targeted deletions of IL-13Rα1 (to eliminate IL-13 but not IL-4 signaling) or IL-4Rα (to eliminate both)

• Utilizing chimeric receptors or mutant ligands with altered binding specificities

• Implementing temporal expression studies to identify differences in kinetics between IL-4 and IL-13 responses

• Conducting comparative transcriptomic or proteomic analyses to identify genes/proteins uniquely regulated by each cytokine

Additionally, researchers should consider cell-type specific responses; for example, IL-13 functions more prominently on non-hematopoietic cells, including endothelial cells where it induces VCAM1 expression, which is important for eosinophil recruitment .

How can researchers effectively study the interaction between IL-13 and IL-13Rα2 as a regulatory mechanism?

The interaction between IL-13 and IL-13Rα2 represents a complex regulatory mechanism, as IL-13Rα2 acts as a high-affinity decoy receptor that mediates internalization and depletion of extracellular IL-13 . To effectively study this interaction, researchers can employ several strategies:

• Quantitative binding assays using surface plasmon resonance or biolayer interferometry to determine binding kinetics between recombinant IL-13 and soluble IL-13Rα2

• Fluorescently labeled IL-13 to track receptor-mediated internalization in live-cell imaging studies

• Pulse-chase experiments with biotinylated IL-13 to measure the rate of clearance in systems with varying levels of IL-13Rα2 expression

• CRISPR/Cas9 gene editing to manipulate IL-13Rα2 expression levels in relevant cell types

• Development of IL-13 mutants with altered binding affinity for IL-13Rα2 but preserved signaling through IL-13Rα1/IL-4Rα

Morimoto et al. demonstrated the importance of IL-13Rα2 in regulating immune and functional responses to Nippostrongylus brasiliensis infection, highlighting the physiological relevance of this regulatory mechanism . Understanding this interaction is particularly important when designing experimental systems to study IL-13 functions, as variations in IL-13Rα2 expression across different cell types and under different conditions can significantly impact the effective concentration of IL-13 available for signaling.

What are common causes of decreased activity in recombinant IL-13 preparations, and how can they be mitigated?

Several factors can contribute to decreased activity in recombinant IL-13 preparations, with corresponding mitigation strategies:

Regular quality control testing using bioassays such as the TF-1 cell proliferation assay with an expected ED50 of 0.75-3 ng/mL can help monitor activity levels over time . SDS-PAGE analysis under reducing conditions can also be used to assess protein integrity, with active IL-13 appearing as a single band at approximately 9-11 kDa .

How can researchers determine the optimal dose of IL-13 for specific experimental models?

Determining the optimal dose of IL-13 for specific experimental models requires a systematic approach that balances physiological relevance with experimental objectives. Researchers should:

• Perform preliminary dose-response experiments covering a broad range (typically 0.1-100 ng/mL for in vitro studies or 0.1-10 μg for in vivo applications)

• Measure multiple parameters, including:

  • Activation of signaling pathways (STAT6 phosphorylation)

  • Target gene expression (e.g., VCAM1, mucin genes)

  • Functional responses (cell proliferation, differentiation, or migration)

• Compare results to documented ED50 values from similar studies (e.g., 0.75-3 ng/mL for TF-1 cell proliferation)

• Consider the temporal dimension by testing different exposure durations

• Validate findings against physiological IL-13 concentrations reported in relevant disease states

For in vivo models, successful studies have used doses ranging from 0.5-5 μg per mouse, with timing and route of administration (intranasal, intraperitoneal, subcutaneous) optimized based on the specific model and research question . It's important to note that optimal doses may vary significantly between different experimental systems and readouts, necessitating model-specific optimization.

What are the best approaches for detecting and quantifying IL-13-induced STAT6 activation?

STAT6 activation is a critical indicator of functional IL-13 signaling. Several techniques can be employed for detection and quantification:

• Western Blotting: Using phospho-specific antibodies against tyrosine 641 (Y641) of STAT6, researchers can detect the activated form. This approach provides a semi-quantitative assessment of activation.

• Flow Cytometry: Phospho-flow techniques allow for single-cell resolution of STAT6 activation, enabling analysis of heterogeneous cell populations and identification of responsive subtypes.

• ELISA-based Methods: Commercial phospho-STAT6 ELISA kits provide quantitative measurements of activated STAT6 levels in cell lysates.

• Reporter Assays: Cells transfected with STAT6-responsive elements driving luciferase or fluorescent protein expression can provide dynamic, real-time readouts of activation.

• Immunofluorescence Microscopy: This allows visualization of STAT6 nuclear translocation, a hallmark of its activation.

For optimal results, time-course experiments should be conducted, as STAT6 activation typically occurs rapidly (15-30 minutes) after IL-13 stimulation, with signal decay over several hours. Positive controls (IL-4 stimulation) and negative controls (JAK inhibitors) should be included to validate assay specificity. When comparing different experimental conditions, quantification of the ratio between phosphorylated and total STAT6 provides a normalized measure of activation that accounts for variations in total protein levels.

How is recombinant IL-13 being used to investigate the role of IL-33 in inflammatory pathways?

Recombinant IL-13 is increasingly being employed to explore the complex interplay between IL-13 and IL-33 in inflammatory pathways. Research indicates that IL-13 plays an important role in controlling IL-33 activity by modulating the production of transmembrane and soluble forms of interleukin-1 receptor-like 1 (IL1RL1) . Hudson et al. demonstrated that IL-13 can induce IL-33 expression and activity in central nervous system glia, suggesting a feedback mechanism between these cytokines in neuroinflammatory conditions . To investigate these interactions, researchers typically:

• Treat target cells with recombinant IL-13 (5-20 ng/mL) and measure changes in IL-33 and IL1RL1 expression using qPCR and ELISA

• Utilize IL-13 in combination with other stimuli to assess synergistic effects on IL-33 pathway activation

• Employ IL-13 receptor blocking antibodies to determine the dependency of IL-33 responses on IL-13 signaling

• Compare wild-type and IL-13-deficient models in their IL-33 responses to inflammatory challenges

This research direction is particularly relevant for understanding allergic inflammation, tissue repair, and host defense against parasites, where both IL-13 and IL-33 play significant roles.

What insights can recombinant IL-13 provide in cancer immunotherapy research?

Recombinant IL-13 is providing valuable insights into cancer immunotherapy research, particularly in understanding tumor microenvironment immunomodulation. Recent work by Shen et al. demonstrated that IL-13 signaling interacts with Notch pathway components to influence the efficacy of immune checkpoint blockade in triple-negative breast cancer models . Experimental approaches using recombinant IL-13 in cancer research include:

• Ex vivo treatment of tumor-associated macrophages to induce M2-like polarization, followed by functional characterization

• Study of IL-13-induced changes in tumor cell expression of immune checkpoint molecules

• Development of IL-13-based chimeric antigen receptor (CAR) T cells targeting IL-13Rα2-expressing tumors

• Investigation of IL-13 neutralization as an adjunct to existing immunotherapies

The dual role of IL-13—potentially promoting tumor growth through immunosuppressive effects while also offering targeting opportunities through receptor expression on certain tumors—makes it a complex but promising focus for cancer immunotherapy research. When designing such experiments, researchers should consider both direct effects on tumor cells and indirect effects mediated through immune cell modulation.

How can recombinant IL-13 contribute to understanding cardiac repair mechanisms after myocardial infarction?

Recombinant IL-13 is emerging as a valuable tool for investigating cardiac repair mechanisms following myocardial infarction. Cho et al. demonstrated that IKKβ-deficient macrophages impede cardiac repair after myocardial infarction by enhancing the macrophage-myofibroblast transition, a process influenced by IL-13 . To leverage recombinant IL-13 in cardiac repair research, investigators can:

A typical experimental design might involve administering 1-2 μg of recombinant IL-13 via intramyocardial injection at the border zone of the infarct, followed by comprehensive assessment of inflammatory markers, fibrosis, and cardiac function over a 1-4 week period.