Recombinant Mouse Interleukin-3 protein (Il3), partial (Active)

What is the structural composition of recombinant mouse IL-3 protein?

Recombinant mouse IL-3 is a disulfide-linked monomeric protein consisting of 135-140 amino acid residues with a molecular weight of approximately 15-17.2 kDa as determined by SDS-PAGE analysis under both reducing and non-reducing conditions . Commercial preparations typically include the protein fragment spanning amino acids 33-166, which represents the mature, bioactive form of the cytokine . The protein contains critical disulfide bonds that maintain its tertiary structure and are essential for biological activity. The primary sequence is not highly conserved across species, with only 29% homology between human and mouse IL-3, which explains the observed species specificity of its biological actions .

How is recombinant mouse IL-3 produced for research applications?

Recombinant mouse IL-3 for research applications is typically produced using an Escherichia coli expression system with an optimized DNA sequence encoding the mature IL-3 chain . Following expression, the protein undergoes purification processes including chromatography techniques to achieve >95-98% purity as verified by SDS-PAGE and HPLC analysis . Commercial preparations are generally supplied as either lyophilized protein (formulated from a 0.2 μm filtered PBS solution) or as frozen liquid comprised of sterile-filtered aqueous buffered solution containing glycerol and bovine serum albumin . Endotoxin levels are controlled to <0.1 ng/μg (1 EU/μg) as determined by the LAL gel clot method to prevent experimental interference .

What are the primary biological functions of mouse IL-3?

Mouse IL-3 functions as a pleiotropic cytokine with multiple roles in hematopoiesis and immune regulation:

• Controls the production and differentiation of hematopoietic progenitor cells into lineage-restricted cells, acting on progenitors of nearly every lineage except those committed to the lymphoid lineage

• Stimulates mature basophils, eosinophils, and monocytes to become functionally activated, enhancing their immune effector functions

• Plays important roles in neural cell proliferation and survival through alternative signaling mechanisms

• Participates in bone homeostasis by inhibiting osteoclast differentiation through prevention of NF-kappa-B nuclear translocation and activation

• Contributes to cell survival under oxidative stress conditions in non-hematopoietic systems by activating pathways mediated by PI3K/AKT and ERK

Which cell types produce IL-3 naturally, and what stimulates its production?

IL-3 is produced predominantly by:

• Activated T-lymphocytes (particularly CD4+ T cells) following antigenic or mitogenic stimulation, with Th1 cells expressing approximately 4-fold higher levels compared to Th0 and Th2 cells as demonstrated in reporter mouse studies

• Mast cells, which can both produce and respond to IL-3 in a potential autocrine feedback loop

• Keratinocytes, NK cells, endothelial cells, and monocytes, serving as secondary sources

• Osteoblastic cells, contributing to the role of IL-3 in bone homeostasis

Production is primarily stimulated by T cell receptor engagement and co-stimulatory signals during immune responses, particularly in the context of certain infections such as helminth parasites like Nippostrongylus brasiliensis .

What are the signal transduction mechanisms of mouse IL-3?

Mouse IL-3 exerts its biological effects through complex signaling cascades:

• The IL-3 receptor complex consists of:

  • IL-3 receptor alpha subunit (IL-3Rα) - provides ligand specificity

  • Signal transducing beta subunit (IL-3Rβ) - shared with GM-CSF and IL-5 receptors

• Receptor engagement activates multiple signaling pathways:

  • JAK2 kinase activation leads to STAT5 phosphorylation and nuclear translocation, initiating a transcriptional program that promotes cell survival, proliferation, and differentiation

  • Secondary pathways involve PI3K/AKT and ERK activation, which are particularly important in non-hematopoietic systems under oxidative stress conditions

• Receptor-ligand complexes have dissociation constants (Kd) of 10^-9 to 10^-10 M, indicating high-affinity binding

• Binding of IL-3 to its receptor causes specific phosphorylation of a 150 kDa membrane glycoprotein, initiating the intracellular signaling cascade

How is IL-3 receptor expression regulated, and what is the role of MARCH proteins?

IL-3 receptor expression and signaling are regulated through multiple mechanisms, with MARCH family proteins playing a critical role:

• MARCH3 functions as a key negative regulator of IL-3 signaling through:

  • Constitutive association with IL-3Rα in cells expressing both proteins

  • Mediating K48-linked polyubiquitination of IL-3Rα, which targets the receptor for proteasomal degradation

  • Being induced by IL-3 itself, creating a negative feedback regulatory mechanism to control signaling duration and intensity

• MARCH2 also downregulates IL-3Rα levels, but with weaker effects compared to MARCH3

• MARCH8 can downregulate IL-3Rα when overexpressed but may not be physiologically relevant in certain cell types

• The E3 ubiquitin ligase activity of MARCH3 is essential for its regulatory function, as demonstrated by experiments with ligase-inactive mutants (C71S, C74S, and C87S) that fail to enhance polyubiquitination of IL-3Rα

This regulatory system provides precise control over IL-3 signaling duration and intensity, preventing excessive inflammatory responses.

What phenotypes are observed in IL-3 knockout mice, and what do they reveal about IL-3 function?

Studies with IL-3 gene-deficient (knockout) mice have revealed complex and sometimes contradictory roles for IL-3 in various disease models:

• Normal steady-state hematopoiesis:

  • IL-3 knockout mice display intact steady-state hematopoiesis

  • Normal numbers of tissue mast cells and basophils are maintained

  • These observations suggest that IL-3 may be dispensable for normal hematopoietic development or is part of a redundant cytokine network

• Disease-specific alterations:

  • Impaired T cell-dependent contact hypersensitivity responses to haptens

  • Increased accumulation of eosinophils during ragweed-induced allergic peritonitis, suggesting a potential regulatory role in allergic inflammation

  • Attenuated mast cell and basophil responses to gastrointestinal nematode infection, resulting in compromised worm expulsion

• Low detectability:

  • IL-3 levels in blood or tissues are typically low or undetectable in normal mice, complicating analysis of its physiological roles

These findings indicate that while IL-3 may be dispensable for steady-state hematopoiesis, it plays critical context-dependent roles in certain immune and inflammatory responses.

What are the optimal storage and handling conditions for recombinant mouse IL-3?

To maintain optimal biological activity of recombinant mouse IL-3, researchers should follow these evidence-based protocols:

• Long-term storage:

  • Store lyophilized protein at -80°C for maximum stability

  • Upon initial thawing, aliquot the protein into polypropylene microtubes to avoid freeze-thaw cycles and store at -80°C

• Working solution preparation:

  • For biological assays: Dilute in sterile neutral buffer containing 1-2 mg/mL carrier protein (e.g., human or bovine serum albumin)

  • For ELISA standards: Use carrier protein concentrations of 5-10 mg/mL

  • Do not dilute to less than 2 μg/mL for long-term storage to prevent activity loss

• Precautions:

  • Pre-screen carrier proteins for potential effects in your experimental system

  • Be aware that carrier proteins may influence results due to toxicity, endotoxin content, or blocking activity

  • Add carrier protein immediately after thawing to prevent activity loss

How can the biological activity of recombinant mouse IL-3 be measured?

Several validated methodologies can be employed to assess the biological activity of recombinant mouse IL-3:

• Cell proliferation assays:

  • The gold standard involves measuring dose-dependent stimulation of mouse M-NFS-60 cells

  • Typical ED50 values for high-quality preparations are <0.05 ng/ml

  • Calculate specific activity as units/mg based on the dose-response curve

• Signaling pathway activation:

  • Western blot analysis of phosphorylated STAT5, JAK2, PI3K/AKT, or ERK in responsive cells following IL-3 treatment

  • Flow cytometry for phospho-proteins can provide single-cell resolution of signaling events

• Gene expression analysis:

  • qRT-PCR to measure IL-3-induced genes such as Pim1, Cd69, and Id1

  • RNA-seq for genome-wide transcriptional responses to IL-3 stimulation

• Functional assays:

  • Colony formation assays using bone marrow progenitors

  • Basophil and mast cell activation markers

  • Osteoclast differentiation inhibition assays

What experimental approaches can be used to study IL-3 production in vivo?

Studying IL-3 production in vivo presents significant challenges due to its low expression levels. Several advanced approaches have been developed:

• Reporter mouse models:

  • CRISPR/Cas-engineered mice containing bicistronic mRNA linking enhanced green fluorescent protein (ZsGreen1) to IL-3 expression

  • These models allow direct visualization and quantification of IL-3-producing cells via flow cytometry or microscopy

  • Validated through in vitro T cell subset analysis and in vivo infection models with Nippostrongylus brasiliensis

• Quantification methods:

  • ELISA-based detection of IL-3 in serum or tissue homogenates, though often near detection limits

  • Intracellular cytokine staining following ex vivo stimulation with PMA/ionomycin or specific antigens

  • Single-cell RNA-seq to identify IL-3-expressing cells within heterogeneous populations

• Experimental triggers for IL-3 production:

  • Helminth infections (e.g., Nippostrongylus brasiliensis)

  • T cell-dependent contact hypersensitivity models

  • Allergic inflammation models (e.g., ragweed-induced peritonitis)

How can researchers manipulate IL-3 signaling in experimental models?

Several approaches are available to modulate IL-3 signaling in experimental systems:

• Genetic manipulation:

  • IL-3 knockout mice for loss-of-function studies

  • Receptor component (IL-3Rα, IL-3Rβ) knockout models

  • MARCH3 knockout or overexpression to modulate receptor levels

  • CRISPR/Cas9-mediated editing of specific signaling components

• Pharmacological interventions:

  • JAK2 inhibitors (e.g., AG490, ruxolitinib) to block downstream signaling

  • PI3K inhibitors (e.g., wortmannin, LY294002) to inhibit alternative signaling pathways

  • Proteasome inhibitors (e.g., MG132) to prevent MARCH3-mediated receptor degradation

• Protein-based approaches:

  • Neutralizing antibodies against IL-3 or its receptor

  • Recombinant soluble IL-3 receptor components as decoys

  • Structure-based design of IL-3 variants with altered signaling properties

• Cellular manipulation:

  • Adoptive transfer of specific IL-3-producing or IL-3-responsive cell populations

  • Ex vivo expansion and modification of hematopoietic progenitors with IL-3

What are the current knowledge gaps and future research directions for mouse IL-3?

Despite significant advances in understanding mouse IL-3 biology, several important questions remain unanswered:

• Contextual regulation of IL-3 production:

  • How do the nature, strength, site, and stage of infection determine the cellular sources of IL-3?

  • What are the molecular mechanisms underlying the expression of IL-3, and are these mechanisms cell-type and/or tissue-specific?

• Functional heterogeneity:

  • Does the type of IL-3-producing cells determine the nature, quality, duration, and outcome of immune responses?

  • What accounts for the apparently contradictory roles of IL-3 in different disease models?

• Receptor regulation:

  • How is the balance between different MARCH family members regulated to control IL-3Rα levels?

  • Are there additional post-translational modifications beyond K48-linked ubiquitination that regulate receptor function?

• Therapeutic implications:

  • Can targeted manipulation of IL-3 signaling be exploited for treating inflammatory disorders or hematological conditions?

  • What are the compensatory mechanisms that maintain normal hematopoiesis in the absence of IL-3?