IL 4 Human, HEK

What are the primary biological functions of human IL-4 in immunological research?

Human IL-4 functions as a key regulator of immune responses with several critical roles:

• Regulates differentiation of antigen-stimulated naive T helper cells toward a type 2 phenotype (Th2)

• Induces proliferation of T lymphocytes and inhibits apoptosis in B cells

• Mediates immunoglobulin class switching in B cells (particularly to IgE)

• Induces alternative activation of macrophages

• Stimulates production of collagen by fibroblasts

IL-4 is produced by activated Th2 cells, mast cells, basophils, and eosinophils. It has been implicated in various disease processes, including allergic disorders, asthma, parasitic infections, tuberculosis, pulmonary fibrosis, and systemic connective tissue diseases . These diverse functions make IL-4 an attractive therapeutic target for numerous immune-mediated conditions.

How does the IL-4 signaling pathway function at the molecular level?

IL-4 signaling occurs through binding to the IL-4 receptor alpha (IL-4Rα) subunit, which it shares with IL-13. The signaling pathway involves:

• IL-4 binding to IL-4Rα, which then combines with either:

  • The common gamma chain (γc) to form the Type I receptor

  • The IL-13Rα1 chain to form the Type II receptor

• Receptor dimerization activates JAK (Janus Kinase) proteins

• JAKs phosphorylate and activate STAT6 (Signal Transducer and Activator of Transcription 6)

• Activated STAT6 dimerizes and translocates to the nucleus to regulate gene transcription

The importance of this pathway is demonstrated by studies showing that "germ-line deficiency of mIL-4Rα or murine signal transducer and activator of transcription 6 (STAT6)" attenuated the effects of human IL-4 isoforms in mice, confirming that "these signalling molecules mediate the in vivo effects of hIL-4 isoforms in mice" .

What is IL-4δ2, and how does it differ functionally from conventional IL-4?

IL-4δ2 is a naturally occurring alternatively spliced variant of IL-4 that exhibits distinct functional properties:

• Conventional IL-4 is encoded by four exons, whereas IL-4δ2 is encoded by exons 1, 3, and 4 (missing exon 2)

• Both human IL-4 and IL-4δ2 cause pulmonary infiltration of T and B lymphocytes when expressed in mice, but unlike mouse IL-4, they do not recruit eosinophils

• IL-4δ2 induces higher levels of pro-inflammatory cytokines (TNF-α, IL-1, MCP-1) and Th1 cytokines (IL-12, IFN-γ) compared to conventional IL-4

• In an ovalbumin model of asthma, IL-4δ2 stimulates greater accumulation of lymphocytes than IL-4

These functional differences suggest that IL-4δ2 possesses more pronounced pro-inflammatory properties compared to conventional IL-4, offering potential for differential targeting in therapeutic approaches.

How do different expression systems affect recombinant human IL-4 characteristics?

Expression systems significantly impact the structure, post-translational modifications, and biological activity of recombinant human IL-4:

For applications requiring physiologically relevant protein characteristics, HEK293-expressed IL-4 is often preferred due to its native-like glycosylation and higher likelihood of proper folding and tertiary structure .

What are the optimal storage and handling conditions for maintaining recombinant human IL-4 activity?

Proper storage and handling of recombinant human IL-4 are critical for maintaining its biological activity:

Storage Conditions:

• Lyophilized protein: -20°C to -80°C until expiry date; stable at room temperature for up to 2 weeks

• Reconstituted protein: -20°C to -80°C for up to 6 months; 4°C for up to 1 week

• Avoid repeated freeze-thaw cycles as these significantly reduce activity

Reconstitution Protocol:

• Briefly centrifuge the vial before opening

• Reconstitute to 0.2 mg/mL in sterile 1x PBS pH 7.4

• Include 0.1% endotoxin-free recombinant human serum albumin (HSA) as a carrier protein

• Gently swirl or tap vial to mix; avoid vigorous shaking

Quality Control Parameters:

• Endotoxin levels: Ensure ≤0.1 EU/μg or <1 EU/μg

• Confirm sterility through 0.2 μm filtration

• Verify purity (>95% by SDS-PAGE)

Following these guidelines ensures optimal protein stability and experimental reproducibility across research applications.

How should researchers address potential endotoxin contamination in IL-4 preparations?

Endotoxin contamination is a critical concern in immunological experiments as it can activate innate immune responses and confound results:

Source Selection:

• Choose products specifically labeled as endotoxin-free (≤0.1 EU/μg) or low-endotoxin (<1 EU/μg)

• Consider expression system: HEK293-expressed products are generally endotoxin-free and animal-component free

• E. coli-expressed IL-4 requires thorough purification to remove endotoxins

Quality Control Testing:

• Verify endotoxin levels using the Limulus Amebocyte Lysate (LAL) assay

• Document batch-specific endotoxin levels in experimental protocols

• Commercial preparations should be 0.2 μm sterile-filtered

Experimental Controls:

• Include endotoxin inhibitors like polymyxin B in control experiments

• Use heat-inactivated IL-4 controls (endotoxin remains active after heat treatment)

• For reconstitution, use certified endotoxin-free water and buffers

These precautions are especially important for experiments involving dendritic cells, macrophages, and other cells sensitive to endotoxin stimulation.

How can recombinant human IL-4 be effectively applied in immunological research?

Recombinant human IL-4 has multiple applications in immunology research:

• Standard for detection and quantification assays:

  • Used as a calibration standard in ELISA or other IL-4 quantification assays

• Screening platform for anti-IL-4 therapies:

  • Used with reporter cell lines like HEK-Blue IL-4/IL-13 cells to screen inhibitory molecules such as Dupilumab

  • Essential for potency assays for therapeutic antibodies

• T cell differentiation studies:

  • Drives naive CD4+ T cells toward the Th2 phenotype in vitro

  • Required component for studying T cell polarization mechanisms

• B cell research:

  • Induces B cell proliferation and antibody isotype switching to IgE

  • Enables studies of B cell activation and differentiation

• Dendritic cell generation:

  • Combined with GM-CSF for efficient dendritic cell differentiation from monocytes

• Cross-species activity studies:

  • Enables pre-clinical testing of human IL-4-targeting therapies in animal models

  • Facilitates mechanistic studies of IL-4 and its receptors

These applications make recombinant human IL-4 an essential tool in both basic immunology research and therapeutic development pipelines.

What is the HEK-Blue IL-4/IL-13 reporter cell system and how is it optimally utilized?

The HEK-Blue IL-4/IL-13 cell line is an engineered reporter system specifically designed for IL-4 and IL-13 activity assessment:

System Design:

• HEK293 cells engineered to produce secreted embryonic alkaline phosphatase (SEAP) in response to IL-4 or IL-13 stimulation

• Cells express the receptors for IL-4 and IL-13 and the necessary signaling components

• When active cytokines bind to receptors, they trigger the JAK/STAT6 pathway, leading to SEAP expression

• SEAP activity can be quantified using colorimetric or chemiluminescent detection methods

Applications:

• Validation of recombinant IL-4 bioactivity:

  • Each lot of IL-4 can be tested for consistent activity

  • Activity range for typical assays: 0.07-0.4 ng/mL

• Screening of IL-4/IL-13 pathway inhibitors:

  • Testing antibodies targeting IL-4Rα (e.g., Dupilumab)

  • Evaluating small molecule inhibitors of signaling components

  • Assessing novel biologics that interrupt cytokine-receptor interactions

• Comparative analysis:

  • Testing wild-type vs. engineered IL-4 variants

  • Comparing potency of different antagonists

  • Assessing cross-species activity

This standardized reporting system provides a quantitative and reproducible method for studying IL-4 and IL-13 biology and evaluating potential therapeutic agents.

What experimental controls should be included when studying IL-4 signaling?

Robust experimental design for IL-4 signaling studies requires comprehensive controls:

Cytokine Specificity Controls:

• Structurally related but functionally distinct cytokines (e.g., IL-13, IL-2)

• Heat-inactivated IL-4 (denatured protein control)

• IL-4 neutralizing antibodies to confirm specificity

Receptor Engagement Controls:

• Anti-IL-4Rα blocking antibodies (like Dupilumab or analogues)

• Soluble IL-4Rα to compete for IL-4 binding

• IL-4 antagonist variants that bind but don't activate signaling

Signaling Pathway Controls:

• JAK inhibitors to block downstream signaling

• STAT6 inhibitors or dominant-negative STAT6 constructs

• Cells from IL-4Rα or STAT6 knockout models when possible

Splice Variant Controls:

• When studying conventional IL-4, include IL-4δ2 for comparison

• Important given the significant functional differences between these variants

Species-Specificity Controls:

• Include both human and mouse IL-4 when working across species

• Germ-line deficiency of mouse IL-4 had no effect on human IL-4 function in some models

Dose-Response Controls:

• Include a range of IL-4 concentrations (typically 0.07-0.4 ng/mL)

• Establish dose-dependence relationships for observed effects

These controls ensure experimental validity and facilitate accurate interpretation of results in IL-4 signaling studies.

Does human IL-4 show functional activity in mouse models, and what are the implications?

Human IL-4 does demonstrate functional activity in mice, despite previous concerns about species specificity:

Evidence Supporting Cross-Species Activity:

• Adenovirus-mediated gene delivery of human IL-4 to mouse lungs causes pulmonary infiltration of T and B lymphocytes

• Human IL-4's effects in mice are partially dependent on murine IL-4Rα and STAT6 signaling

• Structural analysis indicates that key residues of human IL-4 that define its binding to human IL-4Rα (Glu-9, Arg-88, Arg-53, Arg-85) are present in mouse IL-4

• Direct BIAcore experiments show that glycosylated mouse IL-4 binds to human IL-4Rα

• Commercial human IL-4 products specifically note cross-reactivity with mouse systems

Research Implications:

• Pre-clinical Testing: Cross-species activity "would not only offer possibilities for pre-clinical testing of novel hIL-4-targeting therapies in animals, but also suggests new opportunities for mechanistic studies of IL-4 and its receptors"

• Partial Conservation of Signaling: Human IL-4 effects were "attenuated, but not completely abrogated, by germ-line deficiency of mIL-4Rα or murine STAT6," suggesting partial rather than complete conservation of signaling mechanisms

• Isoform Differences: Human IL-4δ2 has more pronounced pro-inflammatory effects compared to conventional human IL-4 in mouse models

These findings enable at least limited testing of anti-human IL-4 therapies in mice, creating valuable opportunities for translational research.

What key differences exist between mouse and human IL-4 signaling?

Despite cross-species activity, researchers should be aware of several important differences between mouse and human IL-4 signaling:

Biological Effect Differences:

• Mouse IL-4 causes significant influx of eosinophils in mouse lungs, whereas human IL-4 and IL-4δ2 recruit lymphocytes but not eosinophils

• This suggests differences in chemokine induction or eosinophil recruitment mechanisms

Signaling Pathway Integration:

• Both depend on STAT6 and IL-4Rα, but human IL-4 effects in mice were "attenuated, but not completely abrogated" by deficiency of these molecules

• This indicates human IL-4 might activate additional pathways in mice that partially compensate for canonical signaling

Splice Variant Differences:

• Both species have IL-4δ2 splice variants, but their expression patterns and functional importance may differ

• Human IL-4δ2 demonstrates more pronounced pro-inflammatory effects compared to conventional IL-4

Independence from Endogenous Mouse IL-4:

• "Germ-line deficiency of mIL-4 had no effect on the BAL cell count, suggesting that endogenous mIL-4 was not involved in mediating the effects of human IL-4 isoforms on mouse lung"

• Human IL-4 functions independently of endogenous mouse IL-4 production

These differences are critical considerations when designing experiments and interpreting results from mouse models using human IL-4.

How can researchers optimally design experiments using human IL-4 in mouse models?

Based on the available evidence, optimal experimental design for human IL-4 in mouse models should consider:

Delivery Method Selection:

• "Replication-deficient adenovirus-mediated gene delivery of hIL-4 isoforms to mouse lungs" has been successfully demonstrated

• Alternative methods include recombinant protein administration, transgenic expression, or other viral vectors

• Each method has different kinetics and expression patterns that should match experimental goals

Appropriate Readouts:

• For lung models: Analyze bronchoalveolar lavage (BAL) cell composition using flow cytometry to identify T cells (CD4+, CD8+) and B cells (CD19+)

• Measure both Th1 (IL-12, IFN-γ) and Th2 cytokines along with pro-inflammatory mediators (TNF-α, IL-1, MCP-1)

• Perform histological examination for tissue changes and cellular infiltration

Essential Control Groups:

• Include species-matched controls (mouse IL-4 and IL-4δ2)

• Empty vector or vehicle controls

• Consider using knockout models (IL-4Rα-/-, STAT6-/-, IL-4-/-)

Combinatorial Approaches:

• Combining "with an ovalbumin model of asthma" enhanced effects in published studies

• Consider combining with other challenge models relevant to specific research questions

Dosage Considerations:

• For HEK293-expressed human IL-4, effective concentrations range from 0.07-0.4 ng/mL

• The specific activity is reported as "minimally 5 x 10^6 IU/mg"

These design considerations will maximize the translational value of experiments using human IL-4 in mouse models.

How do antagonistic antibodies against IL-4Rα work, and what are their design considerations?

Anti-IL-4Rα antagonistic antibodies block IL-4/IL-13 signaling by targeting the receptor rather than the cytokines themselves:

Mechanism of Action:

• Target the IL-4Rα subunit shared by both Type I (IL-4 specific) and Type II (IL-4 and IL-13) receptors

• Prevent binding of both IL-4 and IL-13 to their receptors

• Block downstream signaling cascade through JAK/STAT6 pathway

Design Considerations:

• Targeting Strategy:

  • Focus on IL-4Rα allows blocking of both IL-4 and IL-13 signaling simultaneously

  • Antibodies can be isolated "from a large yeast surface-displayed human Ab library"

  • Further engineering of "complementarity-determining regions to improve the affinity using yeast display technology" often required

• Epitope Selection:

  • "Both affinity and epitope are critical factors for the efficacy of anti-IL-4Rα antagonistic Abs"

  • Some antibodies (like 4R34.1.19) bind "mainly to IL-4 binding sites on IL-4Rα with different epitopes from those of dupilumab analogue"

  • Different epitopes may provide varying degrees of antagonism

• Screening Methods:

  • Reporter cells like "HEK-BlueTM IL-4/IL-13 cell line" detect inhibition of signaling

  • Test inhibition of "IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells"

  • Evaluate suppression of "differentiation of naïve CD4+ T cells from healthy donors and asthmatic patients into TH2 cells"

• Affinity Optimization:

  • Engineering to achieve high affinity (e.g., KD ≈ 178 pM)

  • Higher affinity often correlates with greater antagonistic activity

These considerations are essential for developing effective therapeutic antibodies targeting IL-4Rα for allergic diseases such as asthma and atopic dermatitis.

What methodological approaches are optimal for studying differential effects of IL-4 splice variants?

To effectively study the differential effects of IL-4 splice variants like IL-4 and IL-4δ2, researchers should employ these methodological approaches:

Expression Systems:

• Use "replication-deficient adenovirus-mediated gene delivery" to express specific isoforms in vivo

• Ensure comparable expression levels between splice variants for valid comparison

• Consider both recombinant protein application and gene delivery approaches

Cellular Analysis:

• Perform bronchoalveolar lavage (BAL) cell counting and differential analysis

• Use flow cytometry to identify specific cell populations (T cells, B cells, macrophages, eosinophils)

• Compare recruitment patterns: "only mIL-4 caused significant influx of eosinophils in the lungs of mice, whereas hIL-4, hIL-4δ2 and mIL-4δ2 recruited lymphocytes"

Cytokine Profiling:

• Measure "pro-inflammatory (tumour necrosis factor-α, IL-1, and monocyte chemotactic protein-1) and T helper type 1 (IL-12 and interferon-γ) cytokines"

• Note that "hIL-4δ2 induced higher levels" of these cytokines compared to conventional hIL-4

Genetic Approaches:

• Utilize mice with "germ-line deficiency of mIL-4Rα or murine signal transducer and activator of transcription 6" to assess dependency on canonical signaling

• Effects that are "attenuated, but not completely abrogated" suggest partial pathway dependency

Disease Model Integration:

• Combine with established disease models (e.g., "ovalbumin model of asthma")

• Compare variant effects in disease contexts: "hIL-4δ2 stimulated a greater accumulation of lymphocytes than did hIL-4"

These methodological approaches provide a comprehensive framework for understanding the distinct biological roles of IL-4 splice variants.

How can researchers effectively validate the bioactivity of recombinant human IL-4?

Validating recombinant human IL-4 bioactivity is essential for experimental reproducibility and meaningful results:

Reporter Cell Assays:

• Use "HEK-BlueTM IL-4/IL-13 cell line" which produces "secreted embryonic alkaline phosphatase (SEAP)" in response to active IL-4

• Commercial suppliers validate each lot using these reporter cells

• This system provides a quantitative readout of IL-4 bioactivity

Functional Cellular Assays:

• Test "IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells"

• Evaluate capacity to drive "differentiation of naïve CD4+ T cells from healthy donors and asthmatic patients into TH2 cells"

• These assays directly measure key biological activities of IL-4

Molecular Characterization:

• Assess glycosylation status by techniques like mass spectrometry

• Perform SDS-PAGE analysis under reducing and non-reducing conditions

• HEK293-expressed IL-4 shows "15 and 21 kDa reduced, 13 and 19 kDa non-reduced, monomer, glycosylated" forms

Activity Quantification:

• Express activity in International Units (IU/mg) for standardization

• HEK293-expressed IL-4 typically has "minimally 5 x 10^6 IU/mg" specific activity

• Effective concentration range: 0.07-0.4 ng/mL

Cross-Species Testing:

• Test activity in both human and mouse systems where appropriate

• Human IL-4 demonstrates activity in mouse models

These validation approaches ensure that the recombinant IL-4 being used possesses appropriate biological activity for the intended experimental applications.

What are the most promising future directions in IL-4 research?

The understanding of human IL-4 biology and its applications continues to evolve, with several promising research directions emerging from current knowledge:

• Therapeutic Development: The cross-species activity of human IL-4 "offers possibilities for pre-clinical testing of novel hIL-4-targeting therapies in animals" , facilitating development of new treatments for allergic and inflammatory conditions.

• Splice Variant Targeting: The distinct properties of IL-4δ2, which exhibits "more pronounced pro-inflammatory effect" compared to conventional IL-4 , may enable more selective therapeutic approaches that target specific variants.

• Receptor Antagonism Refinement: Developing antibodies with optimized "affinity and epitope" characteristics may yield more effective IL-4Rα antagonists than current options.

• Mechanistic Studies: Human IL-4's activity in mice "suggests new opportunities for mechanistic studies of IL-4 and its receptors" , potentially revealing novel insights into cytokine biology.

• Biomarker Development: Understanding IL-4 splice variant expression patterns could lead to new biomarkers for disease diagnosis, prognosis, or treatment selection.