Recombinant Human Interleukin-4 (IL4), partial (Active) (GMP)

What is Recombinant Human IL-4 and how is it produced?

Recombinant Human IL-4 is an E. coli-derived cytokine protein spanning amino acids His25-Ser153 with an N-terminal methionine. GMP-grade recombinant IL-4 is produced using non-animal reagents in animal-free laboratory conditions and manufactured under current Good Manufacturing Practice (cGMP) guidelines . The production process ensures high purity and consistency required for research applications. The recombinant protein undergoes rigorous quality control testing, including biological activity assays, where it demonstrates specific activity of >1.0 x 10^7 IU/mg, calibrated against the human IL-4 WHO International Standard (NIBSC code: 88/656) .

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

Recombinant human IL-4 is a small protein with a molecular mass of approximately 15,094 Da as confirmed by MALDI-TOF analysis . The protein appears as a single band at 14 kDa when resolved with SDS-PAGE under reducing conditions . Structurally, IL-4 belongs to the four-helix bundle cytokine family, with its biological activity dependent on proper folding and disulfide bond formation. When properly folded, it maintains the three-dimensional conformation necessary for receptor binding and subsequent signal transduction.

What is the difference between carrier-free (CF) and regular recombinant IL-4?

Carrier-free (CF) recombinant IL-4 does not contain bovine serum albumin (BSA) or other carrier proteins. While regular recombinant proteins typically include BSA to enhance stability, increase shelf-life, and allow storage at more dilute concentrations, the CF version is free from these additions . CF formulations are recommended for applications where the presence of BSA might interfere with experimental outcomes. For most cell culture applications and ELISA standards, the BSA-containing version is generally preferred due to enhanced stability .

What are the primary cellular sources and targets of IL-4 in the immune system?

IL-4 is primarily secreted by mast cells, Th2 cells, eosinophils, and basophils . Natural killer T (NKT) cells also produce significant amounts of IL-4 rapidly upon stimulation in vivo . The primary targets of IL-4 include B cells, T cells, mast cells, basophils, and eosinophils . IL-4 promotes cell proliferation, survival, and immunoglobulin class switching to IgG4 and IgE in human B cells . It also induces the acquisition of the Th2 phenotype by naïve CD4+ T cells and mediates the priming and chemotaxis of mast cells, eosinophils, and basophils .

How does IL-4 signaling differ between type I and type II receptor complexes?

IL-4 can signal through two distinct receptor complexes: type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1). Recent research using engineered IL-4 mimetics (Neo-4) has revealed distinct functional outcomes between these signaling pathways . While natural IL-4 can activate both type I and type II receptor complexes, Neo-4 signals exclusively through the type I complex . This selectivity provides researchers with a powerful tool to dissect the specific contributions of type I receptor signaling in various biological contexts.

Type I signaling predominantly occurs in hematopoietic cells and strongly activates STAT6 and STAT5, while type II signaling occurs in non-hematopoietic cells and primarily activates STAT6. These differential signaling cascades lead to distinct gene expression profiles and cellular responses that may be exploited therapeutically.

What is the role of IL-4 in neurological functions and pathologies?

Beyond its well-established role in immunity, IL-4 plays critical functions in the normal brain, particularly in memory and learning processes . Research has demonstrated that IL-4 knockout mice exhibit cognitive defects, suggesting its essential role in cognitive function . Mechanistically, IL-4 affects astrocytes, which respond to IL-4 signaling by producing brain-derived neurotrophic factor (BDNF) . BDNF promotes neuronal growth, survival, and increased dendritic arborization, which positively impacts learning capabilities .

In neurological pathologies, IL-4 levels decrease in aging animals, corresponding with increased inflammatory cytokines like IL-1β and IL-6 in the hippocampus . Direct intracerebroventricular administration of IL-4 can rescue long-term potentiation (LTP) defects observed in aged mice, highlighting its potential neuroprotective properties . IL-4 can also counteract the effects of inflammatory cytokines like IL-1β on LTP, suggesting potential therapeutic applications in age-related cognitive decline .

How do basophils and NKT cells function as primary IL-4 producing cells?

Basophils and NKT cells represent critical sources of "primary IL-4" in immune responses. NKT cells (CD4+NK1.1+ T cells) produce substantial amounts of IL-4 within 30-90 minutes after stimulation . These cells comprise approximately 1% of splenic cells and express specific TCRs (Vα14 and Vβ8.2) that recognize MHC class I-like molecules CD1 .

Basophils serve dual functions as primary IL-4 producers and as antigen-presenting cells (APCs) that preferentially induce Th2 cells both in vivo and in vitro . This dual functionality places basophils at a critical junction in the initiation and development of Th2 responses. Understanding the regulation of IL-4 production by these cell types provides important insights into the mechanisms driving Th2-mediated immune responses and potential therapeutic targets for allergic and inflammatory conditions.

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

Proper reconstitution and storage are crucial for maintaining the biological activity of recombinant IL-4. GMP-grade recombinant human IL-4 is typically supplied as a lyophilized powder from a 0.2 μm filtered solution in PBS . For reconstitution, it should be dissolved at a concentration of 100-200 μg/mL in PBS . After reconstitution, the solution should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly reduce biological activity.

For long-term storage, reconstituted IL-4 should be kept in a manual defrost freezer. The following storage conditions are recommended:

Researchers should avoid repeated freeze-thaw cycles as these significantly impact protein stability and biological activity .

What are the optimal conditions for assessing IL-4 biological activity in vitro?

The biological activity of recombinant human IL-4 can be assessed through several established assays. The most common approach is evaluating its ability to stimulate proliferation of TF-1 human erythroleukemic cells . In this assay, the effective dose (ED50) typically ranges from 0.05-0.2 ng/mL . When designing experiments to assess IL-4 activity:

• Use low-passage TF-1 cells maintained in proper growth conditions

• Starve cells of growth factors for 12-24 hours before the assay

• Prepare serial dilutions of IL-4 spanning at least 2 logs around the expected ED50

• Include appropriate positive controls (e.g., GM-CSF) and negative controls

• Incubate cells with IL-4 for 48-72 hours at 37°C, 5% CO2

• Quantify proliferation using established methods (e.g., MTT assay, thymidine incorporation)

Researchers should note that the specific activity of recombinant human IL-4 (>1.0 x 10^7 IU/mg) is calibrated against the human IL-4 WHO International Standard, ensuring consistency across experimental settings .

How can IL-4 be effectively incorporated into biomaterials for tissue engineering applications?

The development of hyperstable IL-4 mimetics (Neo-4) has opened new possibilities for incorporating bioactive IL-4 into biomaterials . Unlike natural IL-4, Neo-4 can withstand the heat processing required for certain biomaterial fabrication techniques, including 3D printing . For successful incorporation:

• Select an appropriate biomaterial matrix compatible with the intended application

• Determine the optimal concentration of Neo-4 required for biological effect

• For direct incorporation methods:

  • Mix Neo-4 with the biomaterial precursor solution

  • Process the mixture according to standard protocols for the chosen biomaterial

  • Validate protein retention and activity post-processing

• For surface immobilization approaches:

  • Functionalize the biomaterial surface with appropriate chemical groups

  • Conjugate Neo-4 using biocompatible crosslinkers

  • Confirm surface density and orientation of the immobilized protein

The thermal stability of Neo-4 (maintaining activity after exposure to temperatures required for 3D printing) makes it particularly valuable for developing advanced tissue engineering scaffolds with immunomodulatory properties .

How should researchers interpret conflicting IL-4 signaling data in different cell types?

Interpreting conflicting IL-4 signaling data requires careful consideration of several factors:

• Receptor expression profile: Different cell types express varying levels of IL-4Rα, γc, and IL-13Rα1, affecting the balance between type I and type II signaling . Quantify receptor expression in your experimental system.

• Cell activation state: Pre-activated cells may respond differently to IL-4 than naïve cells. For example, IL-1β-pretreated primary mouse astrocytes show enhanced IL-6 production when subsequently treated with IL-4, whereas untreated astrocytes may not .

• Timing of IL-4 exposure: IL-4 can act as an anti-inflammatory agent when administered concurrently with inflammatory stimuli, but priming cells with IL-4 before pro-inflammatory stimulation can enhance inflammatory responses .

• Concentration-dependent effects: Dose-response experiments across a wide range of IL-4 concentrations should be performed to identify potential biphasic responses.

To resolve conflicting data, researchers should implement mechanistic models parameterized by IL-4 signaling data that account for the sequential nature of receptor binding and activation . Using tools like Neo-4, which signals exclusively through type I receptors, can help disambiguate the specific contributions of different signaling pathways .

What are the key considerations when analyzing IL-4's effects in complex neurological models?

Analyzing IL-4's effects in neurological models requires special considerations due to the complex interplay between immune and nervous systems:

• Blood-brain barrier (BBB) dynamics: The BBB strictly regulates IL-4 entry into the brain parenchyma. Researchers must clarify whether observed effects result from direct IL-4 action on neurons or indirect effects mediated by glial cells .

• Cell-specific responses: Different neural cell types respond uniquely to IL-4. For example, IL-4 affects astrocytic production of BDNF, which subsequently impacts neuronal function . Cell-type specific analyses (e.g., single-cell transcriptomics) provide valuable insights.

• Regional heterogeneity: Brain regions differ in their response to IL-4. The hippocampus, crucial for learning and memory, shows particular sensitivity to IL-4 signaling .

• Age-dependent effects: Microglia become less responsive to IL-4 in aged mice, contributing to increased inflammation and impaired LTP . Age-matched controls are essential for meaningful comparisons.

• Integration with other neuroimmune signals: IL-4 functions within a complex network of neuroimmune signals. Comprehensive analysis should include assessment of related cytokines and their receptors.

When designing experiments to study IL-4 in neurological contexts, researchers should consider these factors and implement appropriate controls to distinguish direct IL-4 effects from secondary consequences of altered immune function.