Recombinant Human Interleukin-4 protein (IL4) (Active)

What is the structural composition of recombinant human IL-4?

Recombinant human IL-4 is a monomeric glycosylated polypeptide with a molecular weight of approximately 13-18 kDa. The protein contains 129 amino acid residues (specifically His25-Ser153, with an N-terminal Met in E. coli-derived versions) and features three intrachain disulfide bridges that contribute to its bundled four alpha-helix tertiary structure . Human IL-4 is naturally synthesized with a 24 amino acid signal sequence, and alternative splicing can generate an isoform with a 16 amino acid internal deletion . Commercial preparations of recombinant human IL-4 typically have a purity of ≥95% as determined by SDS-PAGE analysis .

How does recombinant human IL-4 exert its biological effects?

IL-4 functions through two distinct receptor complexes. The type I receptor, predominantly expressed on hematopoietic cells, is a heterodimer consisting of IL-4 receptor alpha (IL-4Rα) and the common gamma chain (γc), which is shared with receptors for IL-2, IL-7, IL-9, IL-15, and IL-21 . The type II receptor, found on non-hematopoietic cells, comprises IL-4Rα and IL-13Rα1, and can also transduce IL-13 mediated signals .

Upon receptor binding, IL-4 activates multiple signaling pathways that regulate gene transcription. RNA sequencing studies have revealed that IL-4 treatment significantly alters the expression of nearly 1,000 genes (510 up-regulated and 486 down-regulated), particularly those involved in immune signaling, tissue repair, fatty acid metabolism, and degranulation pathways .

What is the specific activity of recombinant human IL-4?

The biological activity of recombinant human IL-4 is typically assessed through its ability to stimulate the proliferation of TF-1 human erythroleukemic cells. The effective dose (ED50) for this stimulation ranges between 0.04-0.2 ng/mL . When compared against the 1st WHO International Standard for Human Interleukin-4 (NIBSC code: 88/656), recombinant human IL-4 has a specific activity of approximately 1.02 × 104 IU/μg . This standardized measurement ensures consistency in experimental applications across different research settings.

How should recombinant human IL-4 be prepared and stored for optimal activity?

Recombinant human IL-4 is typically supplied as a lyophilized powder from a 0.2 μm filtered solution in PBS, either with or without bovine serum albumin (BSA) as a carrier protein . For reconstitution:

• Carrier-containing formulations: Reconstitute at 100-200 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin .

• Carrier-free formulations: Reconstitute at 100-200 μg/mL in sterile PBS .

For long-term storage:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Upon initial thawing, aliquot into polypropylene microtubes and store at -80°C

• For diluted solutions, maintain a concentration of at least 50 μg/mL

• Add carrier protein (0.5-10 mg/mL) for stability during storage

For specific applications:

• In vitro biological assays: Carrier-protein concentrations of 1-2 mg/mL are recommended

• ELISA standards: Carrier-protein concentrations of 5-10 mg/mL are recommended

What are the optimal conditions for using IL-4 in macrophage polarization studies?

For studies examining alternative macrophage activation (M(IL4) polarization), researchers should consider the following protocol-based guidance:

• Isolation of monocytes/macrophages: Standard protocols using density gradient centrifugation or magnetic separation can be employed to isolate primary human monocytes or established macrophage cell lines.

• IL-4 concentration: Effective polarization typically occurs at 10-20 ng/mL of recombinant human IL-4, although dose-response studies should be performed for each experimental system .

• Treatment duration: 24-48 hours of IL-4 exposure is generally sufficient to induce robust M(IL4) phenotype development.

• Confirmation markers: Successful M(IL4) polarization can be confirmed by measuring increased expression of CD206 and CCL18 and decreased expression of CD14 at both mRNA and protein levels using qPCR, ELISA, or flow cytometry .

• Specificity controls: Include IFN-γ treated controls, as IFN-γ does not evoke the same response from macrophages, confirming the specificity of IL-4 effects .

• Functional assays: Assess the hyporesponsiveness of M(IL4) to LPS by measuring reduced production of TNFα, IL-6, GM-CSF, and MCP-1 .

How can recombinant human IL-4 be used in wound healing research models?

IL-4-treated human macrophages (hM(IL4)) have demonstrated significant potential in promoting epithelial wound repair. For experimental design in wound healing studies:

• Macrophage preparation: Human monocytes can be isolated from peripheral blood and differentiated into macrophages before IL-4 treatment (10-20 ng/mL for 24-48 hours) .

• Wound healing assay setup:

  • Epithelial cell scratch assays or transwell co-culture systems can be employed

  • Conditioned media from IL-4-treated macrophages (M(IL4)-CM) can be collected and applied to wounded epithelial monolayers

  • Time-lapse microscopy can be used to quantify wound closure rates

• Assessment parameters:

  • Percentage of epithelial wound healing

  • Correlation analysis between wound healing and expression of repair-associated factors (e.g., TGFβ, CD206)

• Mechanistic studies: RNA sequencing can be employed to identify downstream mediators of repair. Previously identified pathways include up-regulation of signaling networks related to IL-4 and IL-10 signaling, fatty acid metabolism, and degranulation .

How can RNA sequencing be employed to investigate IL-4-induced transcriptional changes?

RNA sequencing is a powerful approach for comprehensive analysis of IL-4-induced transcriptional regulation. Based on published protocols:

• Experimental design:

  • Compare IL-4-treated cells (e.g., M(IL4)) with non-stimulated controls (M(0))

  • Include biological replicates from multiple donors

  • Design appropriate time points (e.g., 24-48 hours post-treatment)

• Library preparation and sequencing:

  • Use standard RNA extraction protocols with RIN >8

  • Prepare libraries using established kits (e.g., Illumina TruSeq Stranded mRNA LT)

  • Sequence on appropriate platforms (e.g., 75-cycle high-output NextSeq 500)

• Bioinformatic analysis:

  • Quantify transcripts using tools like Kallisto 0.43.1 with appropriate genome references (e.g., Homo sapiens GRCh38)

  • Perform differential expression analysis using packages like Sleuth 0.30.0

  • Include treatment as the main effect and donor as a covariate

  • Use Wald test for comparisons, selecting differentially expressed genes based on q-value cutoff of 0.05

  • Conduct pathway analysis using tools like clusterProfiler and ReactomePA for KEGG and Reactome pathways, respectively

• Validation:

  • Confirm key findings using qPCR with appropriate primers

  • Validate protein-level changes with techniques such as ELISA or flow cytometry

What are the key considerations when using recombinant human IL-4 in inflammation models?

When designing experiments to study IL-4's anti-inflammatory effects:

• Cell type selection:

  • Endothelial cells (HUVECs, HLMVECs, MLMVECs) are suitable models for studying vascular inflammation

  • Macrophages are ideal for studying alternative activation and resolution of inflammation

  • Neuronal models can be used for studying neuroinflammation

• Inflammation induction:

  • Lipopolysaccharide (LPS) treatment (typically 250 ng/mL for 30 minutes) is commonly used to induce inflammatory responses

  • Other stimuli such as TNFα or IL-1β can be used depending on the research question

• IL-4 treatment strategy:

  • Concentration dependence: Test multiple concentrations (25-150 μg/mL) to establish dose-response relationships

  • Timing: IL-4 can be administered before (preventive) or after (therapeutic) inflammatory stimuli

  • Duration: 23.5-24 hours is typically sufficient to observe anti-inflammatory effects

• Readout parameters:

  • Pro-inflammatory cytokine production (e.g., IL-6) measured by ELISA

  • Gene expression analysis using RT-qPCR

  • Functional assays specific to the cell type under investigation

• Controls:

  • Include IL-4-only treatment groups to assess baseline effects

  • Consider receptor knockout models (e.g., CD44−/−) to investigate receptor specificity

What differences should researchers consider between carrier-free and carrier-containing IL-4 formulations?

The choice between carrier-free and carrier-containing (typically BSA) IL-4 formulations depends on the specific research application:

Researchers should pre-screen carrier proteins for possible effects in their experimental systems, as these may influence results due to toxicity, high endotoxin levels, or possible blocking activity .

How does human IL-4 compare to IL-4 from other species in research applications?

Researchers should be aware of important species-specific differences when designing cross-species studies:

• Sequence homology:

  • Human IL-4 shares 55% amino acid sequence identity with bovine IL-4

  • Human IL-4 shares only 39% and 43% amino acid sequence identity with mouse and rat IL-4, respectively

• Species specificity:

  • Human, mouse, and rat IL-4 demonstrate strong species-specific activities

  • Cross-reactivity between species is limited, making human IL-4 unsuitable for mouse studies and vice versa

• Experimental implications:

  • Use species-matched IL-4 for in vitro and in vivo studies

  • Human IL-4 should be used with human cells or humanized models

  • When comparing studies across species, consider potential differences in signaling and cellular responses

• Common gene regulation:

  • Despite sequence differences, some core IL-4 regulated genes are conserved

  • Comparison of murine M(IL4) with human RNA sequence data revealed significant changes in 18 common genes, with 12 showing similar directional regulation

How can researchers address variability in IL-4 responses across different experimental systems?

Variability in IL-4 responses is a common challenge in research. To address this issue:

• Standardize recombinant IL-4 quality:

  • Use preparations with defined specific activity

  • Compare against WHO International Standards (e.g., NIBSC code: 88/656)

  • Maintain consistent lot numbers when possible

• Account for donor variability in primary cells:

  • Include donor as a covariate in statistical analyses

  • Increase sample size to account for heterogeneity

  • Consider pooling samples when appropriate

• Establish dose-response relationships:

  • Perform concentration titrations (typically 0.04-0.2 ng/mL for proliferation assays)

  • Determine the optimal concentration for each specific cell type and readout

• Control experimental conditions:

  • Standardize cell density, passage number, and culture conditions

  • Define appropriate positive and negative controls

  • Ensure consistent timing of IL-4 administration and sample collection

• Validation with multiple readouts:

  • Combine mRNA and protein measurements

  • Use both functional and phenotypic assays

  • Correlate different parameters (e.g., TGFβ mRNA, CD206 mRNA, and % epithelial wound healing)

What emerging applications of recombinant IL-4 show promise in neurodegenerative and inflammatory disease research?

Recent research highlights several promising directions for IL-4 in disease models:

• Neuroinflammation modulation:

  • IL-4 has demonstrated potential in regulating neuroinflammation and improving neurological outcomes in both in vitro and in vivo models

  • Studies suggest IL-4 can induce phenotypic changes in neural cells that may be neuroprotective

• Sepsis management:

  • Recombinant human proteoglycan-4 (PRG4) working in concert with IL-4 pathways has emerged as a potential adjunct therapy for sepsis patients

  • IL-4 signaling manipulation could help modulate the excessive inflammatory response characteristic of sepsis

• Wound healing applications:

  • IL-4–treated human macrophages (hM(IL4)) promote epithelial wound repair

  • Cell transfer treatments using IL-4-conditioned macrophages show promise for enhancing tissue regeneration

• Cancer immunotherapy:

  • Understanding IL-4's role in tumor microenvironments may lead to novel immunotherapeutic approaches

  • IL-4's interaction with dendritic cells could be leveraged to enhance cancer vaccination strategies

• Autoimmune disease modulation:

  • IL-4's ability to shift immune responses away from pro-inflammatory phenotypes has potential applications in autoimmune conditions

  • Targeted delivery of IL-4 to specific tissues might help address localized inflammation while minimizing systemic effects

What methodological advances might improve the research applications of recombinant human IL-4?

Several technological and methodological innovations could enhance IL-4 research:

• Controlled release systems:

  • Development of sustained-release formulations could better mimic physiological IL-4 signaling

  • Biomaterial-based delivery systems might improve the stability and targeted activity of IL-4 in vivo

• Gene expression technologies:

  • Single-cell RNA sequencing could provide higher resolution insights into heterogeneous responses to IL-4

  • CRISPR-based screens might identify novel regulators of IL-4 signaling pathways

• Advanced imaging techniques:

  • Live cell imaging of IL-4 receptor dynamics could clarify signaling mechanisms

  • Intravital microscopy might illuminate IL-4's effects in complex tissue environments

• Receptor-specific variants:

  • Engineered IL-4 variants with selective activity on type I or type II receptor complexes

  • Super-agonists or partial antagonists could provide more precise control over downstream signaling events

• Multiomics integration:

  • Combining transcriptomics, proteomics, and metabolomics approaches could provide comprehensive understanding of IL-4 effects

  • Integration of epigenetic analyses might reveal long-term consequences of IL-4 exposure