Recombinant Rhesus Macaque Interleukin-4 protein (IL4) (Active)

What is the molecular structure and key properties of Rhesus Macaque IL4?

Rhesus Macaque Interleukin-4 (IL4) is a 14.9 kDa cytokine expressed as a mature protein spanning amino acids 25-153 of the full-length sequence. The protein sequence is: HNCHIALREI IETLNSLTEQ KTLCTKLTIT DILAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE DFLERLKTIM REKYSKCSS . The recombinant protein is typically produced in E. coli expression systems as a tag-free protein with purity greater than 96% as determined by SDS-PAGE and HPLC analysis . The protein is characterized by its ability to maintain full biological activity in appropriate assay systems and contains less than 1.0 EU/μg of endotoxin as determined by the LAL method .

How does Rhesus Macaque IL4 compare structurally to human IL4?

While the search results don't provide direct comparison data between Rhesus Macaque and human IL4, it's worth noting that Rhesus IL4 shares significant sequence homology with human IL4. This conservation is typical of interleukins across closely related species. For context, other interleukins like IL-18 show 96% amino acid sequence identity between rhesus and human variants . The high degree of conservation typically allows for cross-reactivity in certain experimental systems, though species-specific differences in receptor binding affinity and downstream signaling may exist. Researchers should validate cross-reactivity empirically when considering rhesus IL4 as a surrogate for human studies or vice versa.

What are the recommended reconstitution and storage conditions for lyophilized Rhesus Macaque IL4?

The lyophilized Rhesus Macaque IL4 protein should be briefly centrifuged prior to opening to bring contents to the bottom of the vial. For reconstitution, it is recommended to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and aliquoting the solution is advised. These aliquots should be stored at -20°C to -80°C to maintain stability . Repeated freeze-thaw cycles significantly degrade protein activity and should be strictly avoided. Working aliquots can be stored at 4°C for up to one week . The protein is originally lyophilized from a 0.2 μm filtered 2× PBS buffer at pH 7.4 containing 5% trehalose as a cryoprotectant .

How can researchers optimize protein stability during experimental workflows?

To maintain optimal protein stability during experimental workflows, researchers should implement several critical practices. First, always work with small aliquots of the reconstituted protein to avoid repeated freeze-thaw cycles. When designing multi-day experiments, create working dilutions that will be entirely consumed within a week while stored at 4°C . For concentration-dependent experiments, prepare serial dilutions immediately before use rather than storing diluted solutions. Additionally, researchers should consider the specific buffer components when diluting into assay media—avoid exposing the protein to extreme pH conditions or detergents that could disrupt protein folding. When handling the protein, use low-binding microcentrifuge tubes and pipette tips to minimize protein loss due to adsorption to plastic surfaces. Finally, include appropriate carrier proteins (such as BSA at 0.1-1%) in very dilute working solutions to prevent non-specific binding losses.

How is the biological activity of Rhesus Macaque IL4 quantitatively determined?

The biological activity of Recombinant Rhesus Macaque IL4 is primarily determined through cell proliferation assays using TF-1 cells (a human erythroleukemic cell line responsive to various cytokines). The protein is considered fully biologically active when compared to standard preparations . The ED50 (effective dose that induces a 50% maximal response) in TF-1 cell proliferation assays is less than 1.0 ng/mL, which corresponds to a specific activity of approximately 1.0 × 10^6 IU/mg . This high specific activity indicates the potent biological effect of properly folded and active IL4. Researchers can verify activity of their specific lot by performing their own TF-1 proliferation assays and comparing the dose-response curve to a known standard.

What cellular responses can be used to validate IL4 activity in experimental settings?

Multiple cellular responses can be used to validate IL4 activity beyond standard proliferation assays:

• B-cell activation markers: IL4 induces class II MHC molecule expression on resting B-cells, which can be measured by flow cytometry .

• Antibody class switching: IL4 enhances both secretion and cell surface expression of IgE and IgG1, which can be quantified through ELISA or flow cytometry .

• CD23 expression: IL4 regulates the expression of the low-affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes, providing another flow cytometry-based readout .

• STAT6 phosphorylation: IL4 receptor engagement triggers JAK-STAT signaling, specifically STAT6 phosphorylation, which can be detected via Western blotting or phospho-flow cytometry within minutes of stimulation.

• Gene expression changes: IL4 induces specific gene expression profiles in responsive cells, which can be measured through qPCR of known IL4-responsive genes.

These diverse readouts allow researchers to validate IL4 activity in various experimental contexts and cell types relevant to their specific research questions.

How can Rhesus Macaque IL4 be effectively used in immunological research models?

Rhesus Macaque IL4 serves as a valuable tool in immunological research due to its central role in type 2 immune responses. For effective implementation in research models, the protein can be used to:

• Induce Th2 differentiation: When added to naive CD4+ T cell cultures at 10-20 ng/mL along with TCR stimulation, IL4 drives Th2 cell differentiation, which can be confirmed by intracellular cytokine staining for IL4, IL5, and IL13 .

• Modulate macrophage polarization: Treatment of macrophages with 20-50 ng/mL IL4 for 24-48 hours induces an M2 (alternatively activated) phenotype, characterized by increased expression of mannose receptor (CD206) and reduced inflammatory cytokine production .

• Enhance B cell responses: IL4 at 5-10 ng/mL promotes B cell proliferation and antibody class switching to IgE and IgG1, making it useful for studying humoral immunity in rhesus models of allergic and parasitic diseases .

• Counterbalance Th1 responses: In co-stimulation experiments, IL4 can be used to suppress Th1 cell activity and IFN-γ production, allowing researchers to study immune balance and regulation .

When designing experiments, researchers should consider that IL4 functions in concert with other cytokines and stimuli. For instance, IL4's effects on B cells are enhanced when combined with CD40 ligation, while its impact on T cell differentiation is influenced by the strength of TCR signaling and the presence of other polarizing cytokines.

What considerations should be made when designing cross-species experiments with Rhesus Macaque IL4?

When designing cross-species experiments with Rhesus Macaque IL4, researchers should consider several critical factors:

• Receptor compatibility: While IL4 receptors show conservation across primates, subtle differences in binding affinity may exist. Preliminary dose-response experiments should be conducted to determine optimal concentrations when using rhesus IL4 on human cells or vice versa .

• Downstream signaling variations: Even with receptor binding, differences in adapter protein recruitment or signaling pathway activation may occur. Validation of key signaling events (such as STAT6 phosphorylation) is essential when working across species .

• Context-dependent effects: IL4's effects can vary depending on the cellular microenvironment and concurrent stimuli. When transitioning experimental designs between species, the entire cytokine milieu should be considered, not just the IL4 component .

• Readout selection: Choose functional readouts that are conserved between species. For example, while gene names may differ slightly, focus on conserved biological processes like M2 macrophage polarization or Th2 differentiation that show similar markers across primate species.

• Control experiments: Include appropriate controls with species-matched IL4 whenever possible to establish baseline responses and properly interpret cross-species results.

By carefully addressing these considerations, researchers can design robust cross-species experiments that leverage the high conservation of IL4 biology among primates while accounting for potential species-specific variations.

How can Rhesus Macaque IL4 be utilized in neuroimmunology research beyond classical immune functions?

Recent evidence indicates that IL4 plays critical roles beyond classical immune functions, particularly in neuroimmunology. Researchers can leverage Rhesus Macaque IL4 in several advanced applications:

• Memory and learning studies: IL4 has been implicated in higher brain functions, including memory and learning processes . Researchers can utilize rhesus IL4 in ex vivo brain slice cultures or in vivo models to investigate how this cytokine modulates synaptic plasticity, long-term potentiation, and memory formation in primate models.

• Microglial polarization: Similar to peripheral macrophages, microglia respond to IL4 by adopting an alternative activation state. Treatment of microglial cultures with 10-50 ng/mL IL4 can be used to study neuroprotective versus neurotoxic microglial phenotypes in the context of neuroinflammation .

• Neuron-glia interactions: Co-culture systems incorporating neurons, astrocytes, and microglia can be treated with IL4 to examine how cytokine-mediated glial responses influence neuronal health, synaptic pruning, and circuit function.

• Blood-brain barrier studies: IL4 affects blood-brain barrier permeability and endothelial cell function. Researchers can examine how IL4 modulates the expression of tight junction proteins and transport systems in primate brain microvascular endothelial cell models.

• Neurological disease models: In models of neurodegenerative or neuroinflammatory conditions, IL4 administration or neutralization can help elucidate the role of type 2 immune responses in disease progression or resolution .

When designing these experiments, researchers should carefully consider delivery methods that ensure adequate penetration of IL4 into relevant brain regions, as well as appropriate timepoints for examining acute versus chronic effects on neural function.

What are the methodological approaches for studying IL4 receptor signaling dynamics in primary rhesus macaque cells?

Studying IL4 receptor signaling dynamics in primary rhesus macaque cells requires sophisticated methodological approaches:

• Single-cell phospho-flow cytometry: This technique allows temporal resolution of IL4 receptor signaling by fixing cells at various timepoints after stimulation (30 seconds to 60 minutes) and staining for phosphorylated STAT6, key JAK proteins, and other downstream mediators. This approach preserves information about cell subpopulations within heterogeneous primary samples.

• Live-cell imaging with fluorescent biosensors: Custom FRET-based biosensors for JAK-STAT pathway activation can be introduced into primary rhesus cells via nucleofection or viral transduction to monitor signaling in real-time with subcellular resolution.

• Quantitative proteomics: Phosphoproteomics using techniques like SILAC or TMT labeling coupled with mass spectrometry enables comprehensive mapping of phosphorylation events downstream of IL4 receptor activation, revealing both canonical and non-canonical signaling nodes.

• Proximity ligation assays: These assays can visualize protein-protein interactions within the IL4 receptor signalosome in fixed primary cells, providing spatial information about receptor complex formation.

• CRISPR-mediated tagging of endogenous proteins: Introducing small epitope tags or fluorescent proteins at endogenous loci allows tracking of native receptor components without overexpression artifacts.

For optimal results with primary rhesus macaque cells, researchers should carefully consider cell isolation methods that preserve receptor expression, appropriate media formulations that support primary cell viability, and stimulation protocols that mimic physiological IL4 concentrations and kinetics.

How do mouse and rhesus macaque IL4 systems differ, and what are the implications for translational research?

Understanding the differences between mouse and rhesus macaque IL4 systems is crucial for translational research:

• Sequence homology: While mouse IL4 shares only 60-76% amino acid sequence identity with rhesus IL4 (compared to rhesus IL4's 96% identity with human IL4) , this translates to significant functional differences in receptor-ligand interactions and downstream signaling.

• Receptor distribution: The distribution of IL4 receptors across immune cell subsets differs between mice and primates, particularly on innate lymphoid cells and memory T cell populations. This affects how experimental results translate between models.

• Signaling pathway variations: Despite conservation of core JAK-STAT signaling, species-specific differences exist in adapter protein recruitment, signaling kinetics, and negative regulation mechanisms.

• Functional outcomes: Key IL4-mediated processes like antibody class switching show species differences; mouse IL4 promotes IgG1 and IgE, while primate IL4 drives switching to IgG4 and IgE.

• Neuronal IL4 effects: IL4's roles in brain function show species-specific variations, with primate models demonstrating more complex integration with cognitive functions than murine models .

These differences highlight why rhesus macaque models often provide more translatable insights for human applications than murine models, particularly for complex immune-mediated diseases and neuroinflammatory conditions.

What experimental systems best model IL4 biology in rhesus macaques compared to human systems?

For optimal modeling of IL4 biology across rhesus and human systems, researchers should consider these experimental approaches:

• Primary peripheral blood mononuclear cells (PBMCs): Freshly isolated PBMCs from both species treated with species-matched IL4 provide the most physiologically relevant comparison of immediate signaling events and short-term responses like STAT6 phosphorylation and early gene induction.

• Monocyte-derived macrophages: Culturing monocytes with M-CSF for 5-7 days before IL4 stimulation allows comparison of macrophage polarization responses between species, including transcriptional, metabolic, and functional changes.

• Memory T cell recall responses: IL4's modulation of memory T cell activation can be compared by stimulating antigen-experienced T cells from both species with cognate antigens in the presence of IL4.

• Organoid models: Increasingly, 3D organoid cultures derived from either species provide complex tissue architecture for studying IL4's effects on cellular interactions within structured microenvironments.

• Ex vivo tissue explants: Short-term culture of lymphoid tissue explants (tonsil, lymph node) or lung tissue from both species offers a system to study IL4's impact on tissue-resident immune cells within native stroma.

For each system, validation experiments should establish comparable baseline responsiveness to IL4 across species before testing experimental hypotheses. Key readouts should include both universal markers (like phospho-STAT6) and species-specific optimal readouts. This dual approach allows researchers to bridge findings between rhesus macaque models and human translational applications while accounting for species-specific biology.

What are common pitfalls in IL4 functional assays and how can they be addressed?

When working with IL4 functional assays, researchers frequently encounter several challenges that can impact experimental outcomes:

• Loss of activity during storage: IL4 activity can diminish during improper storage. Solution: Strictly follow reconstitution guidelines, prepare single-use aliquots in LoBind tubes, and add carrier protein (0.1% BSA) to dilute solutions .

• Endotoxin contamination: Even low levels of endotoxin can confound IL4 assays by independently activating immune cells. Solution: Use certified low-endotoxin IL4 preparations (<1.0 EU/μg) and include polymyxin B controls in sensitive assays to neutralize potential LPS effects .

• Cell receptor desensitization: Continuous exposure to high IL4 concentrations can downregulate receptors and reduce cellular responsiveness. Solution: For long-term assays, consider intermittent IL4 replenishment rather than single high-dose addition.

• Matrix effects in complex samples: Serum or tissue components can interfere with IL4 detection or activity. Solution: Validate assays with spike-recovery experiments in the specific matrix being used .

• Non-specific binding in ELISAs: When measuring IL4 production, non-specific binding can produce false positives. Solution: Optimize blocking conditions and include isotype control antibodies to establish specific signal thresholds .

The table below summarizes linearity assessment data from an IL4 ELISA, demonstrating how sample dilution can affect measurement accuracy:

This data highlights how proper sample dilution can improve assay performance, with 1:2 dilutions showing optimal recovery percentages .

How can researchers troubleshoot unexpected results when comparing IL4 responses across different cell types?

When comparing IL4 responses across different cell types, unexpected results may stem from several factors:

• Differential receptor expression: IL4 receptor levels vary dramatically between cell types and activation states. Solution: Quantify IL4Rα and common gamma chain expression by flow cytometry on each cell population before comparative studies.

• Context-dependent signaling: The same IL4 concentration can trigger different signaling outcomes depending on the cell's microenvironment. Solution: Standardize culture conditions, including serum lot, cell density, and plate coating materials, when comparing across cell types.

• Temporal response variations: Different cell types exhibit unique temporal patterns of IL4 response. Solution: Perform time-course experiments (30 minutes to 72 hours) for each cell type to identify optimal assessment timepoints for comparable pathway activation.

• Antagonistic pathway activation: Some cell types may simultaneously activate pathways that counteract IL4 signaling. Solution: Profile key negative regulators (SOCS proteins, phosphatases) across cell types to identify differential regulatory mechanisms.

• Technical variation in assessment methods: Flow cytometry, Western blotting, and qPCR each introduce different technical biases. Solution: Validate key findings using multiple methodological approaches.

When unexpected results persist despite these troubleshooting steps, consider that genuine biological differences in IL4 response mechanisms may exist between cell types. These differences can be further investigated through comprehensive phosphoproteomic or transcriptomic profiling to identify cell type-specific signaling networks activated by IL4.

What are emerging areas of research involving Rhesus Macaque IL4 in neurological disorders?

Emerging research suggests several promising directions for investigating Rhesus Macaque IL4 in neurological disorders:

• Neurodegenerative disease modeling: IL4 has demonstrated neuroprotective properties that warrant further investigation in rhesus models of Alzheimer's and Parkinson's diseases. The protein's ability to modulate microglial phenotypes could potentially mitigate neuroinflammation and promote neural repair .

• Cognitive enhancement pathways: Beyond its role in pathology, IL4's involvement in learning and memory processes opens avenues for exploring cognitive enhancement strategies. Investigating how IL4 signaling integrates with established memory consolidation pathways could reveal novel therapeutic targets .

• Blood-brain barrier modulation: IL4's effects on endothelial function suggest potential applications in disorders characterized by blood-brain barrier dysfunction, such as multiple sclerosis or stroke. Controlled delivery of IL4 could potentially restore barrier integrity in these conditions.

• Neuropsychiatric disorder mechanisms: Emerging evidence links immune dysregulation to disorders like depression and schizophrenia. Rhesus models allow investigation of how IL4 signaling affects neurotransmitter systems implicated in these conditions.

• Neuroimmune development: IL4's role in normal brain development, particularly in microglia-mediated synaptic pruning and circuit refinement, represents an understudied area with implications for neurodevelopmental disorders.

These research directions benefit significantly from the close homology between rhesus and human IL4 systems, increasing translational relevance . Future studies would benefit from combining advanced neuroimaging techniques with targeted IL4 pathway manipulation to establish causal relationships between IL4 signaling and neurological outcomes.

How might systems biology approaches enhance our understanding of IL4 signaling networks in primates?

Systems biology approaches offer powerful frameworks for understanding the complex signaling networks initiated by IL4 in primates:

• Multi-omics integration: Combining transcriptomics, proteomics, metabolomics, and epigenomics data from IL4-stimulated cells can reveal coordinated molecular programs beyond traditional pathway analyses. This integration identifies regulatory hubs that may not be apparent from single-platform studies.

• Network modeling: Computational modeling of IL4 signaling networks, incorporating temporal dynamics and feedback loops, can predict emergent system behaviors and identify critical nodes for experimental validation. These models become particularly valuable when comparing network architectures between species.

• Single-cell multi-parametric analysis: Technologies like CITE-seq, which combines surface protein measurement with transcriptomics at single-cell resolution, can map IL4 response heterogeneity across immune cell populations and reveal how cellular context shapes signaling outcomes.

• Spatial systems immunology: Emerging spatial transcriptomics and proteomics techniques allow mapping of IL4 responses within tissue microenvironments, providing critical context for understanding how cellular neighborhoods influence signaling.

• Perturbation biology: Systematic CRISPR screens targeting IL4 pathway components, coupled with multi-parameter readouts, can identify previously unrecognized regulators and establish their functional hierarchy within the signaling network.

These approaches are particularly powerful in rhesus macaque systems, where genetic and physiological similarity to humans enhances translational relevance. By applying systems approaches across species, researchers can distinguish conserved "core" IL4 signaling modules from species-specific regulatory features, thereby improving preclinical-to-clinical translation of IL4-targeted therapeutics.