Recombinant Mouse Interleukin-4 protein (Il4) (Active)

What is the molecular structure of recombinant mouse IL-4?

Recombinant mouse IL-4 is a monomeric Th2 cytokine with a molecular weight of approximately 13.2 kDa as analyzed by SEC-MALS. The protein adopts a bundled four alpha-helix structure and contains three intrachain disulfide bridges that are critical for its biological activity. The mature mouse IL-4 protein spans from His23 to Ser140, and when expressed recombinantly, it often includes an N-terminal methionine. The protein is synthesized with a 24 amino acid signal sequence in vivo that is cleaved to produce the mature form .

How does mouse IL-4 compare to IL-4 from other species?

Mouse IL-4 shares limited sequence homology with IL-4 from other species: approximately 39% amino acid sequence identity with both bovine and human IL-4, and 59% with rat IL-4. This relatively low sequence conservation explains why IL-4 activity is highly species-specific. Human, mouse, and rat IL-4 demonstrate species-specific activities in their biological systems, meaning that mouse IL-4 should be used for experiments with mouse cells or in mouse models, as cross-species reactivity is poor . This species specificity must be considered when designing experiments and interpreting results from comparative studies.

What are the primary biological functions of mouse IL-4?

Mouse IL-4 is a pleiotropic cytokine secreted primarily by mast cells, T-cells, eosinophils, and basophils that plays multiple roles in immune regulation. It induces the expression of class II MHC molecules on resting B-cells and enhances both secretion and cell surface expression of IgE and IgG1. IL-4 regulates the expression of the low-affinity Fc receptor for IgE (CD23) on lymphocytes and monocytes, positively regulates IL31RA expression in macrophages, and stimulates autophagy in dendritic cells by interfering with mTORC1 signaling .

Beyond immune regulation, IL-4 plays a critical role in higher brain functions, including memory and learning. Upon binding to IL-4 receptor, it initiates dimerization with either the common IL2R gamma chain (forming the type 1 signaling complex primarily on hematopoietic cells) or with IL13RA1 (forming the type 2 complex expressed on nonhematopoietic cells). This leads to JAK3 and JAK1 phosphorylation, activating the STAT6 signaling pathway .

What expression systems are used for producing recombinant mouse IL-4?

Recombinant mouse IL-4 can be produced using various expression systems, each with distinct advantages:

• Mammalian expression (HEK 293 cells): Produces correctly folded, glycosylated protein with high purity (≥95%) and low endotoxin levels (≤0.005 EU/μg). This system is preferred when post-translational modifications are critical for the intended application .

• Bacterial expression (E. coli): Produces higher yields at lower cost, though typically as insoluble inclusion bodies requiring refolding. The E. coli-derived mouse IL-4 spans His23-Ser140 with an N-terminal Met. BL21(DE3)-CodonPlus E. coli strain is commonly used, with expression induced by IPTG. The recombinant protein is often expressed as an insoluble 17.5 kDa precursor requiring refolding with agents like guanidine hydrochloride and dithiothreitol .

The choice of expression system should depend on the specific research application, with mammalian systems preferred for applications requiring fully authentic structure and post-translational modifications.

What is the standard methodology for producing recombinant mouse IL-4 in a research setting?

The production of recombinant mouse IL-4 in a laboratory setting typically follows this methodology:

• RNA extraction: Total RNA is extracted from mouse spleen tissue, which naturally expresses IL-4.

• cDNA synthesis: Reverse transcription PCR (RT-PCR) is used to generate cDNA.

• Target amplification: PCR amplification of the IL-4 coding sequence using designed primers containing appropriate restriction sites.

• Cloning: The amplified sequence (approximately 360 bp) is ligated into an expression vector, such as pET21-b(+).

• Transformation: E. coli Top10 is transformed with the recombinant plasmid, with successful transformants verified by PCR and sequencing.

• Protein expression: BL21(DE3)-CodonPlus E. coli is transformed with the verified plasmid, and protein expression is induced with IPTG.

• Protein analysis: Expressed protein is analyzed by SDS-PAGE to confirm the correct size (17.5 kDa for the precursor form).

• Refolding and purification: The insoluble protein is solubilized, refolded, and purified using chromatography.

• Verification: Western blot analysis with anti-IL-4 antibodies confirms the identity of the purified protein .

This methodology can be adapted based on available resources and specific research needs.

How can researchers validate the purity and activity of recombinant mouse IL-4?

Comprehensive validation of recombinant mouse IL-4 requires multiple analytical approaches:

Purity Assessment:

• SDS-PAGE analysis under reducing conditions, visualized by silver staining, should show a single band at approximately 12-13 kDa .

• High-performance liquid chromatography (HPLC) can provide quantitative purity assessment .

• Mass spectrometry can confirm the exact molecular mass and purity.

Structural Integrity:

• SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering) analysis to confirm the monomeric nature and molecular weight (~13.2 kDa) .

Biological Activity Assessment:

• Cell proliferation assay using the HT-2 mouse T cell line, where active IL-4 should stimulate proliferation with an ED50 of 0.3-1.5 ng/mL .

• Induction of alternative activation markers in macrophages, such as upregulation of CD206 and CCL18 and downregulation of CD14 .

• Functional assays measuring the induction of class II MHC molecules on B cells or enhancement of IgE and IgG1 expression.

Endotoxin Testing:

• Limulus Amebocyte Lysate (LAL) assay to ensure endotoxin levels are below 0.005 EU/μg for cell culture applications .

What are the optimal storage conditions for maintaining recombinant mouse IL-4 activity?

To maintain the biological activity of recombinant mouse IL-4, the following storage conditions should be observed:

• Lyophilized Form:

  • Store at -20°C to -80°C.

  • Protected from light and moisture.

  • Remains stable for at least 12 months when properly stored.

• Reconstituted Protein:

  • Reconstitute in sterile PBS at a concentration of 100 μg/mL.

  • For proteins with carrier (BSA), include at least 0.1% human or bovine serum albumin in the reconstitution buffer.

  • For carrier-free preparations, use sterile PBS alone.

  • After reconstitution, aliquot to avoid repeated freeze-thaw cycles.

  • Store aliquots at -20°C to -80°C.

  • Use a manual defrost freezer to prevent temperature fluctuations .

• Working Solutions:

  • For short-term use (≤1 month), store at 2-8°C.

  • For long-term storage, prepare single-use aliquots and store at -20°C to -80°C.

  • Avoid more than 2 freeze-thaw cycles, as this can significantly reduce biological activity.

How is recombinant mouse IL-4 used in macrophage polarization studies?

Recombinant mouse IL-4 is extensively used to generate alternatively activated (M2) macrophages, often denoted as M(IL4), for various experimental applications:

Methodology for M(IL4) Generation:

• Isolate macrophages or monocytes from mouse bone marrow or peritoneal cavity.

• Culture cells in appropriate media (e.g., RPMI-1640 with 10% FBS).

• Add recombinant mouse IL-4 (typically 10-20 ng/mL) to the culture.

• Incubate for 24-48 hours to allow complete polarization.

• Verify polarization by assessing expression of M2 markers such as CD206, Arg1, and CCL18 .

Transcriptional Profile Analysis:
RNA-seq analysis has revealed that IL-4 treatment significantly alters the expression of 996 genes in macrophages, with 510 genes upregulated and 486 downregulated. M(IL4) macrophages display increased expression of markers associated with alternative activation and tissue repair pathways. Pathway analysis shows upregulation of networks related to IL-4 and IL-10 signaling, fatty acid metabolism, and degranulation .

Functional Characteristics of M(IL4) Macrophages:

• Promote epithelial wound healing, partly through TGF-β secretion.

• Reduce cytokine-driven loss of epithelial barrier function.

• Show hyporesponsiveness to LPS stimulation, with reduced production of pro-inflammatory cytokines like TNF-α, IL-6, GM-CSF, and MCP-1 .

These alternatively activated macrophages have therapeutic potential, particularly in inflammatory conditions like inflammatory bowel disease (IBD), where systemic delivery of human M(IL4) macrophages has shown efficacy in reducing disease severity in experimental models .

What are the considerations for using recombinant mouse IL-4 in in vivo experimental models?

When using recombinant mouse IL-4 in vivo, researchers should consider several important factors:

Dosage and Administration:

• The effective dose varies by application but typically ranges from 0.1-10 μg per mouse.

• Administration routes include intraperitoneal (IP), intravenous (IV), or subcutaneous (SC) injection.

• For localized effects, consider site-specific administration.

• Sustained delivery may require multiple injections or use of slow-release formulations.

Potential Immunomodulatory Effects:

• IL-4 can suppress cytolytic responses of natural killer (NK) cells and cytotoxic T lymphocytes (CTL).

• It can inhibit the expression of gamma interferon by CTL cells.

• Even in genetically resistant or previously immunized mice, virus-expressed IL-4 can result in significant mortality due to suppression of immune responses .

Experimental Design Considerations:

• Include appropriate controls to account for non-specific effects of protein administration.

• Consider the half-life of IL-4 in circulation (relatively short, often requiring repeated administration).

• Monitor for potential side effects related to Th2 polarization of immune responses.

• In disease models, timing of administration relative to disease onset is critical.

• Consider genetic background of mice, as some strains may have different sensitivities to IL-4.

Validation of In Vivo Activity:

• Measure serum cytokine levels to confirm systemic effects.

• Assess immune cell polarization in relevant tissues.

• Evaluate target gene expression changes in tissues of interest.

• Consider pharmacokinetic and pharmacodynamic studies for novel applications .

How can recombinant mouse IL-4 be used to study the IL-4/IL-13 signaling axis?

The IL-4/IL-13 signaling axis is central to type 2 immune responses, and recombinant mouse IL-4 provides a valuable tool for dissecting these pathways:

Receptor Complex Formation:
Mouse IL-4 can engage two distinct receptor complexes:

• Type 1 complex: IL-4 binds to IL-4Rα, which then dimerizes with the common IL-2Rγ chain (γc). This complex is primarily expressed on hematopoietic cells.

• Type 2 complex: IL-4 binds to IL-4Rα, which then dimerizes with IL-13Rα1. This complex is expressed on both hematopoietic and non-hematopoietic cells and can also be engaged by IL-13 .

Experimental Approaches:

• Differential Signaling Analysis: Compare IL-4 and IL-13 effects on different cell types expressing various receptor components.

• Receptor Blocking Studies: Use antibodies against specific receptor components (IL-4Rα, IL-2Rγ, IL-13Rα1) to dissect pathway contributions.

• JAK/STAT Activation: Monitor phosphorylation of JAK3, JAK1, and STAT6 using phospho-specific antibodies and flow cytometry or western blotting.

• Gene Expression Profiling: Analyze changes in target gene expression using qPCR or RNA-seq approaches .

Methodological Approach for JAK/STAT Signaling Analysis:

• Treat cells with recombinant mouse IL-4 (10-20 ng/mL) for various time points (5-60 minutes).

• Fix and permeabilize cells for flow cytometry or prepare cell lysates for western blotting.

• Probe with phospho-specific antibodies against pJAK1, pJAK3, and pSTAT6.

• For downstream effects, extend treatment time (2-24 hours) and analyze expression of STAT6 target genes.

What methodologies are recommended for studying IL-4's role in neural functions?

IL-4's roles in higher brain functions such as memory and learning represent an emerging research area. The following methodological approaches are recommended:

In Vitro Neural Culture Systems:

• Primary Neuron Cultures: Treat with recombinant mouse IL-4 (1-50 ng/mL) and assess:

  • Neuronal viability and morphology

  • Neurite outgrowth and synapse formation

  • Electrophysiological properties using patch-clamp techniques

  • Expression of IL-4 receptors and downstream signaling components

• Glial Cell Responses: Examine how IL-4 affects:

  • Microglial polarization (shift toward anti-inflammatory phenotype)

  • Astrocyte reactivity and neuroprotective functions

  • Oligodendrocyte development and myelination

In Vivo Approaches:

• Intracerebroventricular (ICV) Administration: Deliver recombinant IL-4 directly to the brain and assess:

  • Cognitive performance in learning and memory tasks

  • Neuroinflammatory markers

  • Neurogenesis and synaptic plasticity

• Conditional Knockout Models: Use Cre-lox systems to delete IL-4 or IL-4R specifically in:

  • Neurons (using Syn1-Cre or CaMKII-Cre)

  • Microglia (using Cx3cr1-Cre)

  • Astrocytes (using GFAP-Cre)

• Behavioral Testing: Following IL-4 administration or genetic manipulation:

  • Morris water maze for spatial learning and memory

  • Fear conditioning for associative learning

  • Novel object recognition for working memory

  • Long-term potentiation (LTP) measurements for synaptic plasticity

What are common issues encountered when using recombinant mouse IL-4 and how can they be addressed?

Researchers often encounter several challenges when working with recombinant mouse IL-4. Here are common issues and their solutions:

Loss of Biological Activity:

• Problem: Reduced or absent biological response in functional assays.

• Solutions:

  • Minimize freeze-thaw cycles by preparing single-use aliquots.

  • Reconstitute with carrier protein (0.1% BSA) to enhance stability.

  • Store at appropriate temperatures (-20°C to -80°C for long-term).

  • Check for protein aggregation by visual inspection or SEC analysis .

Batch-to-Batch Variation:

• Problem: Different potency between production lots.

• Solutions:

  • Always validate new lots with a biological activity assay (e.g., HT-2 cell proliferation).

  • Calculate exact ED50 values for each batch (expected range: 0.3-1.5 ng/mL for HT-2 cells).

  • Adjust dosing based on actual potency rather than protein concentration alone .

Species-Specific Activity Issues:

• Problem: Lack of cross-reactivity between species.

• Solutions:

  • Ensure mouse IL-4 is used only with mouse cells or in mouse models.

  • For cross-species studies, obtain species-specific IL-4 for each system .

Endotoxin Contamination:

• Problem: Cellular responses due to endotoxin rather than IL-4.

• Solutions:

  • Use preparations with certified low endotoxin levels (≤0.005 EU/μg).

  • Include polymyxin B in cell cultures to neutralize potential endotoxin.

  • Include proper controls (heat-inactivated IL-4) to distinguish specific from non-specific effects .

Protein Adsorption to Surfaces:

• Problem: Loss of protein due to binding to tubes or culture plates.

• Solutions:

  • Use low-binding tubes and plates.

  • Add carrier protein (0.1-0.5% BSA) to diluted working solutions.

  • Pre-coat surfaces with BSA before adding diluted IL-4 .

How can researchers optimize experiments to study IL-4-induced alternative macrophage activation?

Optimizing studies of IL-4-induced alternative macrophage activation requires attention to several key parameters:

Cell Source and Preparation:

• Bone marrow-derived macrophages (BMDMs):

  • Harvest bone marrow from femurs and tibias of mice.

  • Culture in media containing M-CSF (typically 20-40 ng/mL) for 6-7 days.

  • Use cells between days 7-10 for IL-4 stimulation experiments.

• Peritoneal macrophages:

  • Obtain by peritoneal lavage, with or without thioglycollate elicitation.

  • Purify by adhesion or magnetic bead separation.

IL-4 Treatment Optimization:

• Concentration: Titrate IL-4 concentrations (typically 5-50 ng/mL) to determine optimal dose for each experimental system.

• Timing: Test various treatment durations (6, 24, 48, 72 hours) as different markers may have different induction kinetics.

• Media conditions: Use serum-free or reduced serum conditions during IL-4 treatment to minimize background stimulation.

• Refreshing cytokine: For longer treatments, consider adding fresh IL-4 every 24-48 hours .

Validation of M(IL4) Phenotype:
Create a comprehensive panel of markers for thorough characterization:

Functional Assays:

• Wound healing: Use conditioned media from M(IL4) macrophages in epithelial scratch assays.

• Anti-inflammatory activity: Measure suppression of LPS-induced cytokine production.

• Phagocytosis: Assess phagocytic capacity using fluorescent beads or apoptotic cells.

• Transcriptional profiling: Perform RNA-seq to capture the full spectrum of gene expression changes .

What analytical methods are recommended for studying IL-4-induced signaling dynamics?

Advanced analytical techniques can provide deeper insights into IL-4 signaling dynamics:

Phosphoproteomic Analysis:

• Treat cells with recombinant mouse IL-4 for various timepoints (5 min to 24 hours).

• Lyse cells and enrich for phosphopeptides using TiO2 or IMAC techniques.

• Analyze by LC-MS/MS to identify and quantify phosphorylation sites.

• Use pathway analysis tools to map phosphorylation cascades activated by IL-4.

Live-Cell Imaging and Biosensors:

• Generate cells expressing FRET-based biosensors for key signaling nodes (JAK/STAT, MAPK, PI3K).

• Perform time-lapse imaging following IL-4 stimulation.

• Quantify signaling dynamics at single-cell resolution.

• Correlate signaling patterns with phenotypic outcomes.

Single-Cell RNA Sequencing:

• Treat heterogeneous cell populations with IL-4.

• Perform scRNA-seq to identify cell type-specific responses.

• Map trajectories of cellular differentiation or activation.

• Identify previously unrecognized IL-4-responsive cell subsets.

ChIP-Seq and ATAC-Seq:

• Use ChIP-seq for STAT6 and other transcription factors to map genome-wide binding sites.

• Combine with ATAC-seq to assess chromatin accessibility changes.

• Integrate with transcriptomic data to build comprehensive gene regulatory networks.

These advanced techniques can reveal the complex signaling networks and transcriptional programs initiated by IL-4, providing deeper insights than traditional western blotting or PCR approaches.

How can researchers quantitatively assess the impact of IL-4 on macrophage metabolism?

IL-4 significantly affects macrophage metabolism, shifting cells toward oxidative phosphorylation and fatty acid oxidation. The following analytical approaches can quantitatively assess these metabolic changes:

Seahorse XF Analysis:

• Seed macrophages in Seahorse XF plates and treat with recombinant mouse IL-4 (20 ng/mL, 24-48 hours).

• Perform Mitochondrial Stress Test to measure:

  • Basal respiration

  • ATP production

  • Maximal respiratory capacity

  • Spare respiratory capacity

• Perform Glycolysis Stress Test to assess:

  • Basal glycolysis

  • Glycolytic capacity

  • Glycolytic reserve

Metabolomic Analysis:

• Treat macrophages with IL-4 for various timepoints.

• Extract metabolites using appropriate protocols.

• Analyze using LC-MS or GC-MS platforms.

• Focus on TCA cycle intermediates, fatty acid metabolites, and amino acids.

Stable Isotope Tracing:

• Culture IL-4-treated macrophages with 13C-labeled glucose, glutamine, or fatty acids.

• Extract metabolites and analyze isotopomer distribution.

• Determine relative flux through different metabolic pathways.

• Compare flux patterns between M0 and M(IL4) macrophages.

Gene Expression Analysis of Metabolic Enzymes:
Create a panel of metabolic genes known to be affected by IL-4: