Recombinant Rhesus Macaque Interleukin-3 protein (IL3) (Active)

Basic Research Questions

• What is Recombinant Rhesus Macaque IL-3 protein and what are its functions?

  Recombinant Rhesus Macaque Interleukin-3 (IL-3) is a cytokine produced through E. coli expression systems that functions as a biological signal primarily produced by activated T-cells, thymic epithelial cells, and other immune cells . It is a full-length mature protein consisting of 124 amino acids that acts in hematopoiesis by controlling the production, differentiation, and function of two related white cell populations: granulocytes and monocytes/macrophages in the blood . The protein has been characterized as a multilineage hematopoietic growth factor and a weak inflammatory mediator .

  IL-3 is encoded by the IL3 gene and classified as a hematopoietic growth factor that stimulates mast cell growth and functions as a multipotential colony-stimulating factor . In rhesus macaques, IL-3 has demonstrated important roles in immune regulation, particularly in radiation response and allergic conditions .

• What are the key physical and biochemical properties of Rhesus Macaque IL-3?

  Property	Specification	Reference
  Molecular Weight	14.0 kDa
  Amino Acid Length	124 amino acids (full-length mature protein)
  Expression Range	20-143aa
  Purity	>98% by SDS-PAGE and HPLC
  Endotoxin Level	<1.0 EU/μg by LAL method
  Source	E. coli expression system
  Structure	Single non-glycosylated polypeptide chain (monomer)
  Complete Sequence	APMTQTTSLK TSWAKCSNMI DEIITHLNQP PLPSPDFNNL NEEDQTILVE KNLRRSNLEA FSKAVKSLQN ASAIESILKN LPPCLPMATA APTRPPIRIT NGDRNDFRRK LKFYLKTLEN EQAQ

  The protein is typically provided as tag-free (no fusion tags) to ensure native functionality . The complete amino acid sequence corresponds to the mature, active protein found naturally in rhesus macaques .

• How should Recombinant Rhesus Macaque IL-3 be stored and reconstituted for optimal stability?

  Proper handling of Recombinant Rhesus Macaque IL-3 is critical for maintaining its biological activity. The recommended protocol includes:

  Storage:

  • Store lyophilized protein at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles

  • Use a manual defrost freezer for long-term storage

  Reconstitution:

  1. Briefly centrifuge the vial prior to opening to bring contents to the bottom

  2. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  3. For increased stability, reconstitute in PBS (pH 7.4) containing at least 0.1% human or bovine serum albumin

  4. For long-term storage of reconstituted protein, add 5-50% glycerol (final concentration) and aliquot before storing at -20°C to -80°C

  The protein is typically lyophilized from a 0.2 μm filtered PBS solution (pH 7.4) containing 5% trehalose as a cryoprotectant . Working aliquots can be stored at 4°C for up to one week, but longer storage requires freezing with appropriate cryoprotectants .

• What methods are used to determine the biological activity of Recombinant Rhesus Macaque IL-3?

  Biological activity assessment of Recombinant Rhesus Macaque IL-3 typically employs multiple methodologies:

  Cell Proliferation Assays:

  1. Murine NFS-60 Cell Assay: The ED50 (effective dose for 50% maximum response) is determined by dose-dependent stimulation of NFS-60 cell proliferation. Activity is considered optimal when ED50 is less than 5.0 ng/mL, corresponding to a specific activity of >2×10^5 IU/mg .

  2. Human TF-1 Cell Assay: The ED50 is determined using TF-1 erythroleukemic cells. Activity is considered optimal when ED50 is less than 0.05 ng/mL, corresponding to a specific activity of >2.0×10^7 IU/mg .

  Additional Activity Assessment Methods:

  • Measurement of induced cytokine production (IL-6, IL-10) following IL-3 stimulation

  • Evaluation of signal transduction pathway activation (JAK/STAT, MAPK) in target cells

  • Assessment of dose-dependent effects on eosinophil, basophil, and neutrophil counts in vivo

  These complementary approaches provide a comprehensive evaluation of both the potency and mechanisms of IL-3 biological activity, ensuring that the recombinant protein functions comparably to the native cytokine.

• What are the primary cellular targets of IL-3 in rhesus macaques?

  IL-3 targets multiple cell lineages in the rhesus macaque hematopoietic system:

  Hematopoietic Progenitors:

  • CD34-positive bone marrow cells (hematopoietic stem and progenitor cells)

  • Multipotential colony-forming cells

  Mature Immune Cells:

  • Eosinophils - IL-3 regulates CCR3 expression and IL-5Rα, affecting their survival and migration

  • Basophils - IL-3 can stimulate basophil production and function

  • Macrophages/Monocytes - IL-3 influences differentiation and functional activity

  • Mast cells - IL-3 can promote mast cell growth and development

  • T cells - IL-3 affects regulatory T cell (Treg) induction and function

  • Innate lymphoid cells (ILC2s) - IL-3 treatment can alter their cytokine production profile

  Studies on rhesus macaque bone marrow have identified both high-affinity IL-3 receptors (25-80 sites/cell with Kd of 3-8 pM/L) and low-affinity receptors (1070-1290 sites/cell with Kd of 1200-2600 pM/L) . This distribution of receptors across various cell types explains the pleiotropic effects of IL-3 in the hematopoietic and immune systems.

Advanced Research Questions

• How does the species specificity of IL-3 impact cross-species experimental design?

  Species specificity is a critical consideration when designing experiments involving IL-3:

  Binding Affinity Differences:

  • The affinity of human IL-3 for rhesus monkey bone marrow cells is 25-50 fold lower than that of homologous rhesus IL-3

  • Interestingly, only small differences exist between human and rhesus IL-3 in binding affinity for human acute myelogenous leukemia (AML) cells, indicating largely unidirectional species specificity

  Experimental Design Considerations:

  1. Appropriate Protein Selection: When working with rhesus macaque cells or animal models, researchers must use recombinant rhesus macaque IL-3 rather than human IL-3 to achieve optimal biological effects

  2. Dose Adjustment: Due to different binding affinities, dose adjustments may be necessary when comparing results across species

  3. Receptor Analysis: Confirmation of receptor expression and binding characteristics should be performed when establishing new experimental systems

  4. Control Selection: In comparative studies, species-matched controls are essential to account for these specificity differences

  The biological significance of these binding differences has been demonstrated through direct in vivo comparison of high-dose recombinant rhesus monkey and human IL-3 effects . This species specificity pattern is consistent with the evolutionary divergence of cytokines and their receptors, and must be factored into experimental design, particularly in preclinical studies using rhesus macaques as models for human diseases.

• What are the implications of IL-3 signaling in radiation response studies using rhesus macaque models?

  Transcriptome analysis of rhesus macaques exposed to total body irradiation has revealed significant insights about IL-3 signaling in radiation response:

  Differential Expression Patterns:

  • IL-3 signaling is upregulated in both survivors and non-survivors at 24 hours post-irradiation

  • In survivors, IL-3 signaling remains upregulated through day 3 post-exposure

  • Non-survivors show progressive decrease in expression of IL-3 signaling pathway genes after initial upregulation

  Key Signaling Components:
  In survivors, genes including MAPK3, PAK1, PRKCE, PRKCH, PRKD3, RALB, RRAS, and STAT5B of the IL-3 signaling pathway remain significantly upregulated . Non-survivors initially show upregulation of genes including JAK1, MAPK3, PAK1, PRKCE, PRKCH, PRKD3, PTPN6, RALB, RRAS, and STAT5B at day 1, followed by decreased expression in subsequent days .

  Therapeutic Implications:

  • Sustained IL-3 signaling may contribute to recovery from radiation injury

  • IL-3 has been shown to elevate IL-1 levels, which is protective against ionizing radiation

  • IL-3 has been used as a radiation countermeasure for treatment of radiation exposed accident victims

  These findings suggest that IL-3 signaling pathway modulation could represent a potential therapeutic approach for radiation injury. The persistence of IL-3 signaling appears to be a critical factor distinguishing survivors from non-survivors following radiation exposure, highlighting its importance in hematopoietic recovery mechanisms .

• How can Recombinant Rhesus Macaque IL-3 be used in studies of allergic asthma?

  Recombinant IL-3 has demonstrated significant immunoregulatory effects in allergic asthma models, with timing of administration being particularly critical:

  Experimental Approaches:

  1. Administration during different phases:

     • Sensitization phase administration showed limited benefit

     • Challenge phase administration significantly reduced airway inflammation and pathology

  2. Outcome Measurements:

     • Lung function assessment through methacholine challenge (measuring respiratory resistance)

     • Histological analysis of lung inflammation and mucus production using H&E and PAS staining

     • Flow cytometry analysis of inflammatory cells, particularly eosinophil subpopulations

     • Quantification of immunoregulatory cytokines like IL-10 and TGF-beta

  Observed Effects in Challenge Phase Administration:

  • Inhibition of airway eosinophilic inflammation

  • Reduction in mucus production

  • Increased TGF-beta production by lung cells

  • Enhanced IL-10 release from ILC2 cells

  • Induction of Foxp3+CD25+CD4+ regulatory T cells

  Mechanistic Insights:
  IL-3 administration during the challenge phase downregulates CCR3 chemokine receptor expression on eosinophils and negatively regulates IL-5Rα expression, resulting in reduced eosinophil survival and migration . This regulatory effect, combined with increased immunosuppressive cytokine production, makes IL-3 a potential therapeutic approach for seasonal allergen exposure, consistent with the use of inhaled steroids during disease exacerbations .

• What methodological approaches can be used to study IL-3 receptor expression and binding in rhesus macaque tissues?

  Several complementary techniques can be employed to characterize IL-3 receptors in rhesus macaque tissues:

  Binding Assays:

  • Radiolabeled IL-3 binding studies using 125I-labeled rhesus IL-3

  • Scatchard analysis to determine receptor numbers per cell and binding affinities (Kd values)

  • Competition binding assays to assess receptor specificity and cross-reactivity with other cytokines

  Receptor Characterization Results:

  Receptor Type	Sites/Cell	Equilibrium Dissociation Constant (Kd)
  High-affinity	25-80	3-8 pM/L
  Low-affinity	1070-1290	1200-2600 pM/L

  Cell-Specific Analysis:

  • Flow cytometry to identify and quantify receptor expression on specific cell populations

  • Cell sorting to isolate receptor-positive populations (e.g., CD34+ cells)

  • Immunohistochemistry to visualize receptor distribution in tissues

  Signaling Studies:

  • Western blotting to detect activation of downstream signaling molecules (JAK/STAT, MAPK)

  • Phospho-flow cytometry to measure signaling at single-cell resolution

  • Functional assays to correlate receptor expression with biological responses

  An interesting finding from competition studies is that human GM-CSF does not compete with IL-3 for binding to high- or low-affinity receptors on rhesus monkey peripheral blood and bone marrow cells . This suggests that the growth factor-specific alpha-subunits of the GM-CSF and IL-3 receptors are expressed predominantly on different cell types in rhesus macaques, an important consideration when designing experiments involving these cytokines .

• How does the concentration and timing of IL-3 administration affect biological outcomes in experimental models?

  The concentration and timing of IL-3 administration significantly impact experimental outcomes, with distinct effects observed across different model systems:

  Dose-Dependent Effects:

  • In clinical studies with human IL-3, increasing doses (60-500 μg/m²) produced proportionally greater increases in white blood cell counts

  • At higher doses, neutrophil counts increased up to 3-fold, while eosinophil and basophil counts increased 10-50 fold

  • Platelet counts showed more modest increases of up to 2-fold

  • Higher IL-3 doses have been associated with increased regulatory T cell induction

  Timing-Dependent Effects in Asthma Models:

  Administration Phase	Airway Eosinophilia	Mucus Production	Regulatory T Cell Induction	IL-10 Production
  Sensitization Phase	No significant effect	No significant effect	Minimal	Low
  Challenge Phase	Significant reduction	Significant reduction	Substantial increase	High

  Administration Route Considerations:

  • Intravenous (IV) bolus administration of IL-3 results in a short serum half-life of approximately 20±3 minutes

  • Subcutaneous (SC) administration provides more sustained levels with a half-life of 210±15 minutes

  • Intranasal administration has shown efficacy in asthma models when timed appropriately relative to allergen challenge

  Experimental Design Implications:

  1. Dose-response relationships should be established for each experimental model

  2. Timing relative to challenge or injury is critical, particularly in models of allergic inflammation or radiation response

  3. Administration route should be selected based on the desired pharmacokinetic profile and target tissue accessibility

  4. Multiple dosing regimens may be necessary to maintain therapeutic levels for extended periods

  These considerations highlight the importance of careful experimental design when using IL-3 in research models, as both concentration and timing can dramatically alter biological outcomes and experimental interpretations .

• What are the key differences between rhesus macaque IL-3 and human IL-3 in terms of structure and function?

  Rhesus macaque IL-3 and human IL-3 exhibit important structural and functional differences that impact their experimental and potential therapeutic applications:

  Structural Differences:

  • While both are monomeric proteins, human IL-3 spans amino acids Ala20-Phe152, while rhesus macaque IL-3 consists of amino acids 20-143

  • The molecular weight of rhesus macaque IL-3 is approximately 14.0 kDa, compared to human IL-3 at 14.6 kDa

  • Both are produced as non-glycosylated proteins when expressed in E. coli systems

  Functional Differences:

  • The relative affinity of human IL-3 for rhesus monkey bone marrow cells is 25-50 fold lower than that of homologous rhesus IL-3

  • In contrast, only small differences exist in binding affinity for human acute myelogenous leukemia (AML) cells

  • These binding differences translate to functional differences in potency when tested across species

  Receptor Interaction:

  • Both interact with the IL-3 receptor complex consisting of an IL-3-specific alpha chain and a common beta chain shared with IL-5 and GM-CSF receptors

  • The species specificity is primarily due to differences in interaction with the alpha chain of the receptor

  • Despite these differences, both activate similar downstream signaling pathways including JAK/STAT, MAPK, and PI3K/Akt

  These differences are significant for researchers using rhesus macaques as models for human diseases, as they necessitate the use of species-appropriate IL-3 to achieve physiologically relevant results. The unidirectional nature of the species specificity (human IL-3 works poorly in macaques, but macaque IL-3 works relatively well on human cells) is an important consideration for translational research .