IL 13 Rat 109 a.a.

What is the molecular structure of IL-13 Rat 109 a.a.?

Recombinant rat IL-13 is a single, non-glycosylated polypeptide chain containing 109 amino acids with a molecular mass of 11.9 kDa. The protein is produced in E. coli expression systems and purified using chromatographic techniques . The amino acid sequence is: VRRSTSPPVA LRELIEELSN ITQDQKTSLC NSSMVWSVDL TAGGFCAALE SLTNISSCNA IHRTQRILNG LCNQKASDVA SSPPDTKIEV AQFISKLLNY SKQLFRYGH . Structurally, IL-13 closely resembles other cytokines including IL-3, IL-4, IL-5, and GM-CSF, with the gene encoding IL-13 located in a cytokine gene cluster on chromosome 5q, particularly close to IL-4 gene . This structural homology reflects shared evolutionary origins and suggests potential functional overlaps between these cytokines.

What are the primary biological functions of rat IL-13?

IL-13 serves as a multifunctional immunoregulatory cytokine with several key roles in immune responses. It exerts anti-inflammatory effects on monocytes and macrophages by inhibiting the production of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-8 . IL-13 is also involved in allergen-induced asthma through mechanisms independent of IgE and eosinophils . Additionally, IL-13 regulates several stages of B cell maturation and differentiation, up-regulates CD23 and MHC class II expression, and promotes IgE isotype switching of B cells . In experimental models, targeted deletion of IL-13 in mice resulted in impaired Th2 cell development and indicated an important role for IL-13 in the expulsion of gastrointestinal parasites . The varied functions of IL-13 make it a critical mediator in allergic inflammation, parasitic infections, and tissue remodeling processes.

What are the main cellular sources of IL-13 in rat models?

IL-13 is produced primarily by activated T helper type 2 (Th2) cells, which are central orchestrators of type 2 immune responses . Additional significant cellular sources include mast cells and natural killer (NK) cells . In rat models of allergic inflammation such as ovalbumin-induced asthma, these immune cells become activated and produce IL-13 as part of the orchestrated immune response . The production of IL-13 by these cell types contributes to various physiological and pathological processes, including allergic inflammation, parasitic immunity, and tissue remodeling. Understanding the cellular sources of IL-13 in specific disease contexts is crucial for designing targeted experimental approaches and interpreting results accurately.

How is the biological activity of recombinant rat IL-13 measured?

The biological activity of recombinant rat IL-13 is typically assessed through its ability to stimulate the proliferation of human TF-1 cells, a cytokine-dependent erythroleukemic cell line . The effective dose at 50% maximal response (ED50) is typically less than 5 ng/ml, corresponding to a specific activity greater than 2 × 10^5 units/mg . This standardized bioassay serves as a reliable method for confirming the functionality of recombinant IL-13 preparations prior to experimental use. Alternative functional assays include measuring STAT6 phosphorylation in responsive cell types, quantifying upregulation of IL-13-induced genes such as CD23 in B cells, or evaluating the polarization of macrophages toward an M2 phenotype. When conducting bioactivity measurements, researchers should include appropriate positive and negative controls to ensure assay specificity and reliability.

How can IL-13 Rat 109 a.a. be effectively used in asthma research models?

IL-13 plays a critical role in asthma pathophysiology, making recombinant rat IL-13 valuable for studying this condition in experimental models. In ovalbumin-induced bronchial asthma models, IL-13 receptors and IL-13R genes show increased expression in bronchial tissues . When designing asthma research experiments, researchers should consider several key methodological aspects:

• Administration routes: Intranasal, intratracheal, or systemic delivery depending on the specific research question

• Dosing regimens: Acute versus chronic exposure protocols to model different disease phases

• Timing relative to allergen challenge: Pre-treatment, concurrent administration, or post-challenge intervention

• Measurement parameters: Bronchial hyperresponsiveness, mucus production, inflammatory cell infiltration, and cytokine profiles

• Receptor analysis: Expression of IL-13Rα1 and IL-13Rα2 in bronchial tissues varies by anatomical location, with higher expression in bronchi containing intramural ganglia

Blocking IL-13 activity using neutralizing antibodies or receptor antagonists has been shown to inhibit asthma pathophysiology, providing a therapeutic research avenue . Additionally, treatments such as sodium cromoglycate (a mast cell stabilizer) can decrease IL-13R mRNA expression in bronchial tissues, offering insights into potential regulatory mechanisms .

What are the optimal conditions for reconstitution and storage of IL-13?

Proper handling of recombinant rat IL-13 is essential for maintaining its biological activity. The lyophilized protein should be reconstituted according to manufacturer recommendations, typically using sterile buffer solutions . The following protocol optimizes stability and activity:

• Reconstitution: Dissolve lyophilized protein in sterile water, PBS, or other appropriate buffer to a concentration of 0.1-1.0 mg/ml

• Sterile filtration: Use a 0.22 μm filter for cell culture applications

• Aliquoting: Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

• Storage temperature: Store reconstituted protein at -20°C or -80°C for long-term stability

• Carrier protein addition: Consider adding 0.1-0.5% BSA as a carrier to prevent adsorption to tubes

• Working solution preparation: Dilute to working concentration immediately before use

Stability studies indicate that reconstituted IL-13 maintains most of its activity for at least 6 months when stored properly at -80°C. Each freeze-thaw cycle can significantly reduce activity, so single-use aliquots are strongly recommended. For critical experiments, freshly reconstituted protein may provide optimal activity.

What methods can be used to detect IL-13 expression in rat tissue samples?

Several complementary techniques can be employed to analyze IL-13 expression in rat tissues:

• Real-time PCR (qPCR): Quantifies IL-13 mRNA expression with high sensitivity. This approach was used to measure IL-13R gene expression in bronchial tissues of rats with ovalbumin-induced asthma . Reference genes must be validated for the specific tissue type being analyzed.

• Immunohistochemistry (IHC): Visualizes protein expression and localization within tissue context. This technique can reveal cell-specific expression patterns and has been used to detect IL-13 receptor expression in bronchial tissues .

• ELISA: Measures secreted IL-13 in tissue homogenates, serum, or other biological fluids. Commercial kits specific for rat IL-13 are available with detection limits typically in the pg/ml range.

• Western blotting: Confirms protein expression and molecular weight (11.9 kDa for rat IL-13).

• Flow cytometry: Identifies cellular sources of IL-13 through intracellular cytokine staining, often combined with surface markers to characterize producing cells.

When analyzing tissues such as bronchi with intramural ganglia, expression levels may vary significantly compared to tissues without ganglia, as observed in studies of ovalbumin-induced asthma in rats . Therefore, precise anatomical localization and standardized tissue sampling are crucial for reproducible results.

What controls should be included when using IL-13 in experimental setups?

Robust experimental design for IL-13 studies should include several types of controls:

• Negative controls:

  • Vehicle/buffer only (solvent control)

  • Heat-inactivated IL-13 (protein control)

  • Isotype control or irrelevant cytokine (specificity control)

• Positive controls:

  • IL-4 (related cytokine with overlapping functions)

  • Known IL-13-responsive cell line or tissue

  • Previously validated target genes or pathways

• Mechanistic controls:

  • IL-13 receptor blocking antibodies

  • JAK/STAT pathway inhibitors

  • IL-13Rα2 (decoy receptor) to distinguish receptor-specific effects

• Technical controls:

  • Endotoxin testing of recombinant IL-13 preparations

  • Freshly reconstituted versus stored IL-13 aliquots

  • Multiple concentrations to establish dose-response relationships

How does IL-13 signaling integrate with other cytokine pathways in rat disease models?

IL-13 signaling operates within a complex cytokine network with significant pathway integration:

• Receptor sharing: IL-13 and IL-4 share signaling components, specifically the type II receptor heterodimer comprised of IL-4Rα and IL-13Rα1 . This creates both functional redundancy and cooperative interactions between these cytokines.

• Signaling cascade overlap: Both IL-13 and IL-4 activate JAK1/STAT6 via IL-4Rα, while IL-13 can additionally activate TYK2/STAT3/STAT6 via IL-13Rα1 . This overlap in downstream signaling contributes to shared biological functions.

• Counter-regulatory mechanisms: IL-13 exerts anti-inflammatory effects by inhibiting pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-8 . This creates a balance between pro- and anti-inflammatory mediators in disease settings.

• Receptor regulation: IL-13Rα2, initially considered only a decoy receptor, can inhibit IL-4 signaling through physical interaction between cytoplasmic domains of IL-13Rα2 and IL-4Rα . This adds another layer of complexity to the IL-13/IL-4 signaling network.

• Cross-pathway activation: IL-13Rα2 can activate the AP-1 pathway upstream of TGF-β transactivation, independent of the canonical JAK/STAT pathway .

In cardiac injury models, IL-4/IL-13 signaling influences repair processes, with IL-13 serum levels post-myocardial infarction correlating with improved left ventricular ejection fraction in patients . Understanding these complex interactions is essential for accurately interpreting experimental results and designing interventions that target specific nodes within the integrated cytokine network.

What are the implications of IL-13Rα2 acting beyond a simple decoy receptor?

The evolving understanding of IL-13Rα2 function has significant research implications:

• Non-canonical signaling: While IL-13Rα2 was classically considered a decoy receptor due to its short intracellular domain and lack of signaling kinase tail, recent evidence suggests it can elicit transmembrane signaling in response to IL-13 binding . IL-13Rα2 does not activate JAK1 or STAT6 pathways but can stimulate the activator protein 1 (AP-1) pathway upstream of transforming growth factor-β (TGF-β) transactivation .

• Pathway regulation: IL-13Rα2 can inhibit IL-4 downstream signaling via physical interaction between cytoplasmic domains of IL-13Rα2 and IL-4Rα . This unexpected regulatory mechanism adds complexity to interpreting IL-13 signaling experiments.

• Experimental design implications: Studies targeting IL-13 must consider both IL-13Rα1 and IL-13Rα2 receptors. Simple IL-13 neutralization may not block IL-13Rα2-specific effects, potentially leading to incomplete pathway inhibition.

• Therapeutic relevance: The dual nature of IL-13Rα2 suggests that selective targeting of specific receptor subtypes may produce different therapeutic outcomes than general IL-13 neutralization.

Researchers should assess both IL-13Rα1 and IL-13Rα2 expression in their experimental systems and consider the potential for non-canonical signaling when interpreting IL-13-mediated effects. This dual functionality challenges simplistic models of IL-13 biology and necessitates more nuanced experimental approaches.

How can contradictory findings regarding IL-13 levels in disease models be reconciled?

Contradictory results regarding IL-13 levels and functions in disease models are not uncommon in the literature. For example, in arthritis research, some studies reported increased IL-13 concentrations in sera of early rheumatoid arthritis patients with positive correlation to disease activity, while others found no difference or decreased levels . These apparent contradictions can be reconciled by considering several key factors:

• Disease stage specificity: IL-13 may play different roles during initiation, acute, and chronic phases of disease. In cardiac models, IL-13Rα1/IL-13Rα1 expression is significantly downregulated in patients with heart failure, while IL-13 itself may be elevated as a compensatory mechanism .

• Compartment-specific regulation: IL-13 levels may differ between systemic circulation and local tissue environments. In arthritis studies, elevated levels of IL-13 were found in both serum and synovial fluid of rheumatoid arthritis patients in some studies .

• Methodological differences: Detection techniques, antibody specificity, sample processing, and storage conditions can significantly impact measured IL-13 levels.

• Biological variability: Animal strain differences, age, sex, and environmental factors contribute to variable cytokine responses.

• Receptor regulation: Changes in receptor expression and ratio between IL-13Rα1 and IL-13Rα2 may occur independently of ligand levels.

To address such contradictions, researchers should clearly define disease stage and model parameters, measure IL-13 in multiple compartments, assess both protein and mRNA levels, evaluate receptor expression alongside ligand levels, and focus on functional outcomes rather than absolute concentrations. This comprehensive approach provides a more nuanced understanding of IL-13's role in complex disease processes.

What are the key differences between IL-13 and IL-4 signaling pathways in rat models?

Though IL-13 and IL-4 share signaling components and have overlapping functions, several important distinctions exist:

• Receptor utilization: IL-4 can signal through both type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1) receptor complexes, while IL-13 signals primarily through the type II receptor . The type II receptor is the major canonical receptor for IL-13, with IL-13 binding to IL-13Rα1 followed by recruitment of IL-4Rα .

• Binding kinetics: IL-13 binds with moderate affinity to IL-13Rα1, whereas IL-4 binding to IL-4Rα has equal affinity for recruitment of either γc or IL-13Rα1 . This means that the availability of each chain on the cell surface largely determines the resultant signaling pathway.

• Additional receptor component: IL-13 uniquely binds to IL-13Rα2, which has more than 35% homology to IL-13Rα1 but binds IL-13 with higher affinity . This receptor was traditionally considered a decoy receptor but may have signaling capabilities.

• Downstream signaling: While both cytokines activate JAK1/STAT6 via their shared type II receptor components, IL-13 additionally activates TYK2/STAT3/STAT6 via IL-13Rα1 . IL-13Rα2 activates neither JAK1 nor STAT6 pathways but can stimulate the AP-1 pathway upstream of TGF-β transactivation .

• Biological outcomes: Despite signaling overlaps, IL-13 and IL-4 can produce distinct biological effects depending on the cellular context, receptor expression patterns, and timing of exposure.

Understanding these differential signaling mechanisms is crucial for designing experiments that distinguish IL-13-specific effects from those that might be mediated by either cytokine through shared pathways. When studying IL-13, researchers should consider parallel assessment of IL-4 responses as an important comparison.

What are the typical dosing ranges for IL-13 in rat in vivo and in vitro studies?

Appropriate IL-13 dosing varies based on experimental model and desired outcome:

In vitro applications:

• Cell proliferation assays: 1-20 ng/ml

• Macrophage polarization: 10-50 ng/ml

• B cell activation studies: 5-25 ng/ml

• TF-1 cell bioassay: 0.1-10 ng/ml (ED50 < 5 ng/ml)

In vivo applications:

• Intranasal/intratracheal administration for asthma models: 1-10 μg per animal

• Systemic administration: 0.5-5 μg/kg body weight

• Local tissue injection: 0.1-2 μg per site

Dose-response studies should be conducted for each specific application, as effective concentrations may vary depending on the rat strain, age, disease model, and specific readout. When using recombinant rat IL-13 to stimulate human TF-1 cells, the ED50 is typically less than 5 ng/ml, corresponding to a specific activity greater than 2 × 105 units/mg . This standardized bioassay provides a reference point for determining appropriate concentrations in other experimental systems.

How do you determine the optimal concentration of IL-13 for stimulating specific cell types?

Determining the optimal IL-13 concentration requires a systematic approach:

• Literature review: Begin with concentrations reported for similar cell types and assays

• Dose-response analysis: Test a logarithmic range of concentrations (typically 0.1-100 ng/ml)

• Time-course experiments: Evaluate responses at multiple time points (4, 24, 48, 72 hours)

• Multiple parameter assessment:

  • Receptor phosphorylation (short-term response)

  • Gene expression changes (intermediate response)

  • Functional alterations (long-term response)

• Receptor expression analysis: Quantify IL-13Rα1 and IL-13Rα2 levels on target cells

• Positive control inclusion: Compare with IL-4 or other known stimulators

How might residual endotoxin in recombinant IL-13 preparations affect experimental results?

Endotoxin contamination is a critical concern in cytokine research that can significantly confound experimental results:

• Potential confounding effects:

  • Activation of TLR4 signaling pathway

  • Pro-inflammatory cytokine induction (IL-1β, TNF-α, IL-6)

  • Macrophage activation opposing IL-13's anti-inflammatory effects

  • Altered cell proliferation or differentiation

  • Masking or enhancing IL-13-specific effects

• Prevention and detection strategies:

  • Use endotoxin-tested recombinant proteins (<0.1 EU/μg protein)

  • Perform LAL (Limulus Amebocyte Lysate) testing on preparations

  • Include polymyxin B (10-50 μg/ml) in experiments to neutralize potential endotoxin

  • Test heat-inactivated IL-13 controls (proteins denature while endotoxin remains active)

  • Monitor endotoxin-responsive genes alongside IL-13 targets

For studies examining IL-13's anti-inflammatory effects, endotoxin contamination is particularly problematic as it directly counteracts the cytokine's natural activity. Commercial recombinant rat IL-13 products should be selected from manufacturers that provide endotoxin testing certification . When planning critical experiments, consider additional purification steps or endotoxin neutralization strategies to ensure results reflect true IL-13 activity.

How should IL-13 be used in models of ovalbumin-induced bronchial asthma?

Ovalbumin-induced bronchial asthma is a well-established model for studying allergic airway inflammation where IL-13 plays a crucial role. Research has shown that under conditions of asthma development in the tissues of rat bronchi, there is pronounced expression of IL-13R genes and significant expression of the molecular receptor for interleukin-13 . When designing experiments with this model:

• Anatomical considerations: Studies have found that in bronchi with an intramural ganglion (bifurcation zone), the level of IL-13R gene expression and the level of expression of the IL-13R molecular receptor were significantly higher than in tracheal samples without ganglia . This suggests that sampling location is critical for accurate assessment.

• Intervention timing: Treatment interventions should be carefully timed. For example, sodium cromoglycate administered to rats 5 hours after the last inhalation of ovalbumin led to a decrease in IL-13R mRNA content compared to untreated animals .

• Methodological approach:

  • Use both qPCR for gene expression and immunohistochemistry for protein detection

  • Compare different anatomical regions of the bronchial tree

  • Include time-course analyses to capture dynamic changes

  • Consider both IL-13 and its receptors (both IL-13Rα1 and IL-13Rα2)

• Experimental readouts:

  • Airway hyperresponsiveness measurements

  • Inflammatory cell infiltration assessment

  • Mucus production quantification

  • Cytokine profile analysis

  • IL-13 receptor expression mapping

This model provides valuable insights into IL-13's role in allergic inflammation and allows for testing of interventions targeting various components of the IL-13 signaling pathway.

What are emerging applications for IL-13 in cardiac repair and regeneration research?

Recent research has revealed intriguing roles for IL-13 in cardiac regeneration and repair, opening new avenues for investigation:

• Clinical correlations: In humans, IL-13 serum levels post-myocardial infarction correlate with improved left ventricular ejection fraction, suggesting a protective role . Conversely, IL-4Rα/IL-13Rα1 expression is significantly downregulated in patients with heart failure, indicating a potential link between reduced IL-13 signaling and cardiac dysfunction .

• Experimental models: Several animal models have been developed to study IL-13's role in cardiac repair, including:

  • IL-13Rα1 knockout mice subjected to myocardial infarction

  • IL-13 knockout mice with cardiac injury

  • Neonatal mouse cardiac apical resection models

  • Conditional knockout models targeting specific cell types

• Signaling mechanisms: Both IL-4 and IL-13 signal through the type II receptor heterodimer (IL-4Rα/IL-13Rα1), activating JAK1/STAT6 via IL-4Rα and TYK2/STAT3/STAT6 via IL-13Rα1 . Additionally, IL-13Rα2 may activate the AP-1 pathway upstream of TGF-β transactivation, potentially influencing fibrotic remodeling .

• Research opportunities: Future studies could explore:

  • Cell-specific effects of IL-13 on cardiomyocytes, fibroblasts, and immune cells

  • Temporal dynamics of IL-13 signaling during different phases of cardiac injury and repair

  • Therapeutic potential of IL-13 administration or pathway modulation

  • Interaction between IL-13 and other cardioprotective or pathological factors

This emerging field connects IL-13's immunomodulatory functions with tissue repair processes, potentially revealing new therapeutic targets for cardiac diseases.

What are the current challenges in targeting IL-13 signaling in rat disease models?

Despite significant advances in understanding IL-13 biology, several challenges remain in effectively targeting this pathway in rat disease models:

• Signaling complexity:

  • Redundancy with IL-4 pathway

  • Dual receptor system with distinct functions (IL-13Rα1 vs. IL-13Rα2)

  • Context-dependent signaling outcomes

  • Integration with multiple inflammatory and repair pathways

• Receptor heterogeneity: Expression of IL-13 receptors varies significantly across tissues and even within specific anatomical regions. For example, in bronchi with intramural ganglia, IL-13R expression levels are significantly higher than in tracheal samples without ganglia . This heterogeneity complicates targeted interventions.

• Temporal dynamics: IL-13 may play different roles during acute versus chronic disease phases, requiring careful timing of experimental interventions. The dynamic regulation of receptor expression further complicates this timing.

• Technical limitations:

  • Limited availability of rat-specific reagents compared to mouse models

  • Challenges in developing receptor-selective compounds

  • Difficulties in achieving tissue-specific targeting

  • Limited genetic manipulation tools for rats

• Translational considerations:

  • Species differences in IL-13 signaling between rats and humans

  • Varied responses across different rat strains

  • Complex pathophysiology of human diseases not fully recapitulated in models

Addressing these challenges requires integrated approaches combining molecular, cellular, and in vivo techniques, along with the development of more selective tools for targeting specific components of the IL-13 signaling pathway.

How might genetic engineering approaches enhance IL-13 research in rat models?

Genetic engineering technologies offer promising opportunities to advance IL-13 research in rat models:

• CRISPR/Cas9 gene editing:

  • Generation of IL-13 knockout rat strains

  • Creation of receptor-specific knockouts (IL-13Rα1 or IL-13Rα2)

  • Introduction of reporter tags for tracking IL-13 expression

  • Engineering human receptor variants to study species differences

• Conditional expression systems:

  • Cell-specific IL-13 or receptor deletion using Cre-loxP technology

  • Inducible expression systems for temporal control

  • Tissue-specific promoters to target expression to relevant tissues

• Reporter systems:

  • IL-13 promoter-driven fluorescent proteins to visualize expression

  • Receptor-activity sensors to monitor signaling in real-time

  • STAT6 response element reporters to track downstream activation

• Humanized models:

  • Replacement of rat IL-13 or receptors with human counterparts

  • Creation of chimeric signaling components

  • Models for testing human-specific therapeutics

• Precision disease modeling:

  • Introduction of disease-associated polymorphisms

  • Modeling specific human pathologies with greater fidelity

  • Combined genetic and environmental models

These genetic engineering approaches would facilitate more precise investigation of IL-13 biology, overcoming some limitations of traditional pharmacological or antibody-based interventions. They would enable clearer distinction between direct and indirect effects of IL-13 and provide platforms for testing targeted therapeutics with greater translational relevance.