Recombinant Human Interleukin-4 (IL4) (Active)

What is recombinant human IL-4 and how is it produced?

Recombinant human IL-4 is a 20 kilodalton, 129 amino acid lymphokine that can be produced through various expression systems. One established method involves cloning a synthetic gene coding for human IL-4 and expressing it in Saccharomyces cerevisiae as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence. This approach results in secretion of mature IL-4 into the culture medium at concentrations of approximately 0.6-0.8 μg/ml . Other production systems exist, but the yeast expression system offers advantages for producing a protein that closely resembles native human IL-4.

When purifying recombinant IL-4, researchers typically employ a multi-step process involving:

• Initial capture from cell-free conditioned medium using concanavalin A-Sepharose affinity chromatography

• Further purification via S-Sepharose Fast Flow cation exchange

• Final polishing using C18 reverse-phase HPLC

This protocol typically yields highly purified IL-4 (0.3-0.4 mg per liter of culture) with approximately 51% recovery .

What are the structural characteristics of recombinant human IL-4?

Recombinant human IL-4 contains no free cysteine residues, indicating the presence of three disulfide bonds that contribute to its tertiary structure . The C-terminal N-glycosylation site is largely unmodified in yeast-derived recombinant IL-4, and the N-terminus matches that of authentic human IL-4 . These structural characteristics are critical for biological activity, as proper folding and disulfide bond formation are essential for receptor recognition and subsequent signaling.

How is the biological activity of recombinant IL-4 assessed?

Multiple assays can be used to evaluate the biological activity of recombinant IL-4:

• TF-1 cell proliferation assay: Recombinant human IL-4 induces the proliferation of TF-1 human erythroleukemic cells in a dose-dependent manner. The ED50 typically ranges from 0.04-0.2 ng/mL . This assay serves as a standard for determining specific activity.

• B-cell co-stimulator assays: IL-4 enhances B-cell activation in the presence of appropriate co-stimulatory signals .

• T-cell proliferation assays: IL-4 stimulates T-cell proliferation under specific conditions .

• CD23 induction: IL-4 induces cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells, with half-maximal biological activity achieved at approximately 120 pM .

• Comparison to reference standards: The specific activity of recombinant human IL-4 can be compared against the WHO International Standard for Human Interleukin-4 (NIBSC code: 88/656), with typical values around 1.02 × 104 IU/μg .

How can recombinant IL-4 be used to generate alternative activated macrophages (M2) in vitro?

Human monocytes can be differentiated into IL-4-treated macrophages (hM(IL4)) exhibiting an alternatively activated phenotype. A standard protocol involves:

• Isolate human monocytes from peripheral blood

• Culture in appropriate medium with recombinant human IL-4 (typically 20 ng/mL)

• Incubate for 5-7 days, refreshing medium and cytokines every 2-3 days

The resulting hM(IL4)s display a characteristic CCL18+CD14low/− phenotype . RNA sequencing reveals that IL-4 treatment significantly alters the expression of approximately 996 genes, with 510 genes up-regulated and 486 down-regulated . These cells show up-regulation of signaling networks related to IL-4 and IL-10 signaling, fatty acid metabolism, and degranulation pathways .

To confirm successful polarization, researchers should assess:

• Increased expression of CD206 and CCL18 by qPCR, ELISA, or flow cytometry

• Down-regulation of CD14 by flow cytometry

• Hyporesponsiveness to LPS stimulation (reduced TNFα, IL-6, GM-CSF, and MCP-1 production)

What considerations are important when designing experiments using IL-4 in cell culture systems?

Several factors influence experimental outcomes when using recombinant IL-4:

• Dosage optimization: Biological responses to IL-4 are dose-dependent. For instance, TF-1 cell proliferation shows an ED50 of 0.04-0.2 ng/mL , while induction of CD23 expression on B-cells shows half-maximal response at approximately 120 pM .

• Receptor expression: Target cells must express adequate levels of IL-4 receptors. Human B-cell lines like Raji cells express approximately 1100 high-affinity IL-4 receptors per cell (Kd = 100 pM) .

• Stability considerations: Recombinant IL-4 should be stored according to manufacturer recommendations, typically at -20°C to -80°C for long-term storage, with aliquoting to avoid freeze-thaw cycles.

• Specificity controls: Consider using anti-IL-4 neutralizing antibodies to confirm observed effects are IL-4-specific. Monoclonal antibodies against human IL-4 that block both biological activity and receptor binding provide excellent controls .

• Cross-species reactivity: Human IL-4 exhibits limited cross-reactivity with murine systems and vice versa, necessitating species-matched reagents.

How can recombinant IL-4 be radiolabeled for receptor binding studies?

Radioiodination of recombinant IL-4 can be performed without loss of biological activity, making it valuable for receptor binding studies . A typical protocol involves:

• Using the chloramine-T or lactoperoxidase method for iodination with 125I

• Purifying the labeled protein on a gel filtration column

• Confirming retained biological activity before use in binding assays

For equilibrium binding studies:

• Incubate target cells (e.g., Raji B-cells) with increasing concentrations of 125I-labeled IL-4

• Include parallel samples with excess unlabeled IL-4 to determine non-specific binding

• After reaching equilibrium, separate bound from free ligand by centrifugation through oil or filtration

• Analyze data using Scatchard plots or non-linear regression to determine binding parameters

This approach has revealed that IL-4 binds to a single class of high-affinity receptors on Raji cells (Kd = 100 pM) with approximately 1100 receptors per cell .

What are the potential therapeutic applications of recombinant IL-4 and IL-4-treated cells?

Research suggests several potential therapeutic applications:

• Inflammatory bowel disease (IBD) treatment: IL-4-treated human macrophages promote epithelial wound healing and can potentially serve as a cell transfer treatment for IBD. Conditioned media from freshly generated or cryopreserved hM(IL4)s promotes epithelial wound healing partly through TGF-mediated mechanisms and reduces cytokine-driven loss of epithelial barrier function in vitro .

• Animal models of colitis: Systemic delivery of hM(IL4) to dinitrobenzene sulfonic acid (DNBS)-treated Rag1−/− mice significantly reduced disease severity, providing proof-of-concept support for developing autologous M(IL4) transfer as a cellular immunotherapy .

• Hematological malignancies: Clinical trials have investigated recombinant IL-4 (5μg/kg thrice weekly for 3 weeks) in patients with B-CLL or low-grade B-cell lymphoma. While toxicity was generally mild, efficacy was limited, with only 3 partial responses observed in lymphoma patients and none in CLL patients .

What are the molecular mechanisms underlying IL-4-mediated signaling and gene regulation?

IL-4 activates complex signaling networks that result in significant transcriptional changes:

• Receptor binding: IL-4 binds to a cell surface receptor with an apparent molecular mass of 124 kDa . The receptor complex typically involves IL-4Rα chain paired with either common gamma chain (γc) or IL-13Rα1.

• Transcriptional regulation: RNA sequencing of IL-4-treated human macrophages reveals regulation of 996 genes - 510 up-regulated and 486 down-regulated . This includes markers indicative of alternatively activated macrophages and genes associated with immune signaling and tissue repair.

• Pathway activation: Gene pathway analytics shows IL-4 treatment up-regulates signaling networks related to:

  • IL-4 and IL-10 signaling

  • Fatty acid metabolism

  • Degranulation processes

• Cross-regulation: IL-4 down-regulates IL-6 (a growth factor for B-cells) and inhibits IL-6 secretion by activated monocytes . It also blocks B-cell progression in or into the G1 stage of the cell cycle and inhibits DNA synthesis in B-cells .

What considerations should be addressed when translating IL-4-based therapies to clinical applications?

Several considerations must be addressed before IL-4-based therapies can be effectively translated to clinical practice:

• Safety concerns:

  • Potential fibrosis development (although Rag1−/− mice treated with hM(IL4) displayed no overt fibrosis)

  • Susceptibility to bacterial infection (a concern with any immunosuppressive therapy)

  • Potential cancer promotion (immunosuppressive macrophages can promote cancer development)

• Longevity of effect: The duration of therapeutic effect following hM(IL4) administration requires characterization. Determining whether cells persist long-term or initiate a sustained program in the recipient is crucial for defining treatment protocols .

• Optimization strategies:

  • Cell tracking studies to determine the localization of transferred cells

  • Enhancement of mucosal or gut-specific addressins to improve targeting in IBD applications

  • Assessment of combined therapy approaches

• Patient selection factors: As with many cell therapies, factors such as sex, disease location, and concomitant therapy may influence treatment efficacy and should be addressed in rigorous clinical studies .

How can researchers optimize the production and purification of recombinant IL-4 for research purposes?

Optimizing production and purification involves several considerations:

• Expression system selection: While S. cerevisiae has been successfully used to produce recombinant human IL-4 with yields of 0.6-0.8 μg/ml in culture medium , other systems like E. coli, mammalian cells, or insect cells may offer advantages depending on research needs.

• Purification strategy: A multi-step purification process typically achieves the best results:

  • Initial capture using concanavalin A-Sepharose affinity chromatography

  • Intermediate purification via S-Sepharose Fast Flow cation exchange

  • Final polishing with C18 reverse-phase HPLC

  This approach yields highly purified IL-4 with approximately 51% recovery .

• Quality control assessments:

  • Confirmation of N-terminal sequence authenticity

  • Verification of glycosylation status

  • Thiol titration to confirm proper disulfide bond formation

  • Functional assays to ensure biological activity (e.g., TF-1 cell proliferation)

What are the key experimental controls needed when studying IL-4-mediated effects?

Proper experimental controls are essential when studying IL-4-mediated effects:

• Cytokine specificity controls:

  • Include alternative cytokines (e.g., IFN-γ) to confirm the specificity of IL-4-induced responses

  • Use neutralizing anti-IL-4 antibodies to block IL-4 effects

  • Include isotype control antibodies when using neutralizing antibodies

• Concentration-response relationships:

  • Test multiple IL-4 concentrations to establish dose-dependency of observed effects

  • Determine EC50/ED50 values for specific responses (e.g., 0.04-0.2 ng/mL for TF-1 proliferation)

• Receptor expression verification:

  • Confirm target cells express IL-4 receptors before conducting experiments

  • Consider quantifying receptor expression using binding assays with radiolabeled IL-4

• Time course studies:

  • Establish appropriate time points for measuring IL-4-mediated effects, as responses may be time-dependent

  • Include both early and late time points to capture the full spectrum of IL-4-induced changes

How can researchers address variability in IL-4 responsiveness across primary human cell samples?

Primary human cells often show significant donor-to-donor variability in IL-4 responsiveness. Strategies to address this include:

• Donor screening and characterization:

  • Assess IL-4 receptor expression levels on donor cells

  • Consider genotyping for IL-4 receptor polymorphisms that may affect signaling

  • Evaluate baseline activation state of cells, as this may influence IL-4 responsiveness

• Normalization approaches:

  • Use paired experimental designs where possible (comparing treated vs. untreated cells from the same donor)

  • Calculate fold changes rather than absolute values for measured parameters

  • Consider normalizing to a standardized response to a reference stimulus

• Statistical considerations:

  • Increase sample size to account for inter-donor variability

  • Use appropriate statistical methods for paired data

  • Consider mixed-effects models that account for both fixed (treatment) and random (donor) effects

• Technical standardization:

  • Standardize isolation procedures to minimize variability in starting populations

  • Use consistent lot numbers of recombinant IL-4 when possible

  • Implement rigorous quality control for all reagents and culture conditions

How can researchers resolve issues with diminished IL-4 bioactivity?

If recombinant IL-4 shows reduced bioactivity, consider the following approaches:

• Storage and handling:

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Store according to manufacturer recommendations (typically -20°C to -80°C)

  • Use low-binding tubes to prevent protein adsorption

  • Add carrier protein (e.g., BSA) for dilute solutions

• Verification assays:

  • Confirm bioactivity using standardized assays like TF-1 cell proliferation

  • Compare activity to reference standards (e.g., WHO International Standard for Human IL-4)

  • Assess activity in multiple bioassays, as some may be more sensitive than others

• Receptor functionality:

  • Verify target cells express functional IL-4 receptors

  • Consider testing a known responsive cell line as a positive control

  • Assess expression of signaling components downstream of the IL-4 receptor

What are common pitfalls in designing experiments with IL-4-treated macrophages?

When working with IL-4-treated macrophages, researchers should be aware of these common pitfalls:

• Insufficient characterization:

  • Always confirm M(IL4) polarization by assessing multiple markers (e.g., CD206, CCL18) and functional properties (e.g., hyporesponsiveness to LPS)

  • Do not rely on a single marker to define polarization state

• Cross-contamination issues:

  • Maintain strict separation between differently polarized macrophage cultures

  • Use dedicated pipettes and reagents to prevent cross-contamination

  • Include appropriate polarization controls in each experiment

• Timing considerations:

  • Allow sufficient time for complete polarization (typically 5-7 days)

  • Be aware that some IL-4-induced changes occur rapidly while others develop over several days

  • Consider the stability of the polarized phenotype if long-term experiments are planned

• Species differences:

  • Human and mouse macrophages respond differently to IL-4

  • Do not assume findings from murine systems will translate directly to human cells

  • Use appropriate species-matched reagents and protocols