Recombinant Human Interleukin-3 protein (IL3) (Active)

What is the molecular structure of recombinant human IL-3?

Recombinant human IL-3 is a monomeric protein comprising 134 amino acids with a molecular weight of 15.2 kDa. The protein has a specific amino acid sequence: MAPMTQTTPL KTSWVNCSNM IDEIITHLKQ PPLPLLDFNN LNGEDQDILM ENNLRRPNLE AFNRAVKSLQ NASAIESILK NLLPCLPLAT AAPTRHPIHI KDGDWNEFRR KLTFYLKTLE NAQAQQTTLS LAIF . The recombinant protein is typically produced in E. coli expression systems and undergoes purification to achieve ≥95% purity as determined by SDS-PAGE under both reducing and non-reducing conditions .

How does IL-3 signal through its receptor complex?

IL-3 signals through a heterodimeric receptor complex consisting of the IL-3 receptor α-chain (CD123) and the common receptor β-chain (CD131). CD123 provides specificity for IL-3 binding, while CD131 is essential for signaling and receptor complex assembly. Due to the low affinity of IL-3 for CD123 alone, heterodimerization with CD131 is crucial as it creates a high-affinity receptor complex . Interestingly, there are species-specific differences in receptor composition, as mice express an additional IL-3-specific β chain that differs from CD131 in its ability to bind murine IL-3 directly, although CD123 is still required for signaling .

What cell types express the IL-3 receptor?

The IL-3 receptor is predominantly expressed on hematopoietic cells, including:

• Hematopoietic stem and progenitor cells (HSPCs)

• Basophils, eosinophils, and mast cells

• Non-classical monocytes and macrophages

• Human plasmacytoid dendritic cells (pDCs)

• Activated T and B cells

Additionally, CD123 expression extends to non-hematopoietic cells such as endothelial cells, epithelial cells, osteoblasts, and osteoclasts . This diverse expression pattern explains the pleiotropic effects of IL-3 in both immune and non-immune settings.

What are the primary biological functions of IL-3 in hematopoiesis?

IL-3 functions as a multi-lineage growth factor that promotes the proliferation, differentiation, and survival of various hematopoietic stem cells toward myeloid progenitors . Specifically, IL-3:

• Induces differentiation of hematopoietic stem cells into myeloid precursor cells, including erythrocytes, megakaryocytes, granulocytes, monocytes, and dendritic cells

• Supports the development and survival of basophils, mast cells, and eosinophils

• Acts as a key regulator in emergency hematopoiesis during inflammatory conditions

For experimental applications, IL-3 is commonly used in cell culture to stimulate the differentiation and maturation of human induced pluripotent stem cells towards mast cells, basophils, neutrophils, eosinophils, monocytes, and megakaryocytes .

How can I verify the bioactivity of recombinant IL-3 in my experiments?

The bioactivity of recombinant human IL-3 is typically assessed through a TF-1 cell proliferation assay. In this assay:

• Culture TF-1 cells (a human erythroleukemic cell line dependent on IL-3 or GM-CSF)

• Starve cells of growth factors for 24 hours

• Treat cells with serial dilutions of recombinant IL-3

• Measure cell proliferation after 48-72 hours using MTT/XTT assay or [3H]-thymidine incorporation

• Calculate the ED50 (effective dose inducing 50% maximal response)

Fully bioactive human IL-3 typically demonstrates an ED50 of less than 2 ng/ml in this assay, corresponding to an expected specific activity of approximately 5.0 × 10^5 units/mg . When establishing this assay in your laboratory, include appropriate positive controls and standard curves for accurate quantification.

What is the role of IL-3 in inflammation regulation?

Recent evidence has expanded our understanding of IL-3 beyond hematopoiesis, identifying it as a critical orchestrator of inflammation in various diseases . IL-3 demonstrates remarkable context-dependent effects:

• In systemic lupus erythematosus, IL-3 levels correlate with disease progression, and administration of IL-3 aggravates lupus nephritis in mouse models, while antibodies against IL-3 reduce disease severity

• In viral infections such as SARS-CoV-2, IL-3 appears protective by promoting plasmacytoid dendritic cell recruitment to the lungs and enhancing interferon responses

• In bacterial infections, IL-3 shows contradictory effects: it can enhance pro-inflammatory responses to LPS and potentiate inflammation during sepsis, but also protects against certain bacterial infections like S. Typhimurium

• In inflammatory bowel disease, increased IL-3 levels are observed in inflamed mucosa, and IL-3 receptor signaling regulates T cell trafficking and mechanical properties in intestinal inflammation

This dual nature (pro- or anti-inflammatory depending on context) makes IL-3 an intriguing but complex target for immunomodulatory research.

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

For optimal stability and activity of recombinant human IL-3:

• Storage: Store lyophilized protein at -20°C to -80°C.

• Reconstitution:

  • Centrifuge the vial before opening to prevent loss of material

  • Reconstitute in sterile water or buffer (typically PBS with 0.1% BSA or HSA as a carrier)

  • For a stock concentration of 100 μg/ml, add 10 μl of reconstitution buffer to a 1 μg vial

  • Allow the lyophilized protein to dissolve completely by gentle agitation

• Working solutions: Further dilute in cell culture medium containing serum or serum-free medium with appropriate carrier protein

• Storage after reconstitution: Store reconstituted protein in working aliquots at -20°C to -80°C to avoid repeated freeze-thaw cycles, which can compromise bioactivity

The formulation of commercial recombinant IL-3 typically contains 10 mM sodium phosphate and 50 mM sodium chloride at pH 7.5 , but consult the specific product information for your particular source.

How can I develop robust protocols for IL-3-dependent cell culture systems?

When establishing IL-3-dependent cell culture systems:

• Dose optimization:

  • Perform dose-response experiments (typically 1-20 ng/ml) to determine optimal IL-3 concentration

  • Monitor proliferation, viability, and differentiation markers

  • Be aware that excessive IL-3 may alter differentiation patterns

• Timing considerations:

  • For short-term cultures (<7 days), add IL-3 at the beginning

  • For long-term cultures, supplement fresh IL-3 every 2-3 days due to protein degradation

• Synergy with other factors:

  • Test combinations with complementary cytokines (e.g., SCF, IL-6, GM-CSF) for optimal results

  • Use factorial design experiments to identify optimal cytokine combinations

• Quality control measures:

  • Regularly test batch-to-batch consistency using reference bioassays

  • Include appropriate positive and negative controls in experiments

  • Monitor for endotoxin contamination, which can confound inflammatory readouts

These practices will help ensure reproducible results when working with IL-3 in cell culture applications.

What advanced methods can be used to study IL-3 receptor signaling?

To investigate IL-3 receptor signaling mechanisms:

• Phosphoproteomic analysis:

  • Use phospho-specific antibodies to detect activation of JAK/STAT, MAPK, and PI3K/AKT pathways

  • Employ mass spectrometry-based phosphoproteomics for unbiased discovery of novel signaling nodes

• CRISPR/Cas9 gene editing:

  • Generate knockout or knockin cell lines for specific signaling components

  • Create reporter cell lines to monitor pathway activation in real-time

• Proximity labeling techniques:

  • BioID or TurboID fusions to IL-3 receptor components to identify proximal interacting proteins

  • APEX2-based approaches for temporal resolution of signaling complex assembly

• Live-cell imaging:

  • FRET-based biosensors to monitor kinase activation or second messenger production

  • Single-molecule tracking to analyze receptor clustering and diffusion dynamics

• Mechanobiology approaches:

  • Real-time deformability cytometry and atomic force microscopy to assess IL-3-induced changes in cell mechanical properties

  • Fluorescence recovery after photobleaching (FRAP) to study cytoskeletal dynamics

These advanced techniques can provide insights beyond traditional biochemical assays into the spatiotemporal dynamics of IL-3 signaling.

How does IL-3 contribute to inflammatory bowel disease pathophysiology?

Recent studies have revealed that IL-3 plays a complex role in inflammatory bowel disease (IBD):

• IL-3 levels are increased in the inflamed mucosa of IBD patients

• Experimental chronic colitis is exacerbated in the absence of IL-3 or IL-3 receptor signaling, suggesting a protective role

• IL-3 receptor signaling induces changes in kinase phosphorylation and actin cytoskeleton structure, resulting in:

  • Increased mechanical deformability of T cells

  • Enhanced egress of regulatory T cells (Tregs) from the inflamed colon mucosa

• IL-3 controls mechanobiology in human Tregs and is associated with increased mucosal Treg abundance in IBD patients

These findings suggest that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation. For researchers studying IBD models, monitoring IL-3 levels and receptor expression on different T cell subsets can provide valuable insights into disease mechanisms and potential therapeutic targets.

What are the contradictory roles of IL-3 in asthma and how can researchers address this complexity?

Studies investigating IL-3 in asthma pathogenesis show inconsistent results, presenting a research challenge:

Contradictory findings include:

• IL-3 levels reported as increased in sputum and BALF of asthmatic patients in some studies

• Reduced IL-3 in nasopharyngeal fluid of asthmatic children in other studies

• Similar IL-3 levels in bronchial biopsies between asthmatic and non-asthmatic patients in yet others

• IL-3-deficient mice showing either increased or similar pulmonary inflammation during asthma

To address this complexity, researchers should:

• Standardize parameters:

  • Clearly define patient populations (age, asthma phenotype, disease stage)

  • Standardize sampling methods and processing

  • Use consistent IL-3 detection methods

• Design time-course studies:

  • Investigate IL-3 levels at different stages of disease

  • Monitor temporal relationship between IL-3 and other inflammatory markers

• Analyze cell-specific effects:

  • Identify specific cellular sources and targets of IL-3 in asthmatic airways

  • Investigate differential effects on distinct immune cell populations

• Use multiple models:

  • Compare findings across different animal models

  • Validate in human primary cells and tissue samples

These approaches can help reconcile contradictory findings and elucidate the true role of IL-3 in asthma pathophysiology.

How might IL-3 be involved in viral infection responses?

IL-3 appears to play a significant role in antiviral immunity:

• IL-3 serves as a predictive marker for clinical outcome and disease severity during SARS-CoV-2 infections

• During viral infections, IL-3 is primarily produced by CD4+ T cells and can be induced by viral proteins (e.g., ORF7a in SARS-CoV-2)

• In mouse models of pulmonary HSV-1 infection, IL-3 protects against viral pneumonia by promoting:

  • Recruitment of plasmacytoid dendritic cells (pDCs) into lung parenchyma

  • CXCL12-dependent immune cell trafficking

• In humans, IL-3 enhances T cell priming by pDCs during viral exposure

• Plasma IL-3 and IFNλ levels correlate in patients with SARS-CoV-2 infections and in septic patients with pulmonary viral infections

For researchers studying viral infections, these findings suggest several experimental approaches:

• Monitor IL-3 production during different phases of viral infection

• Investigate IL-3-dependent recruitment of antiviral immune cells

• Explore the relationship between IL-3 and interferon responses

• Evaluate IL-3 as a biomarker for disease severity and outcome prediction

How can I investigate the mechanobiological effects of IL-3 on immune cells?

Based on recent findings about IL-3's impact on cell mechanics and migration , researchers can employ several sophisticated approaches:

• Real-time deformability cytometry (RT-DC):

  • Measures mechanical properties of thousands of cells per minute

  • Can detect IL-3-induced changes in cell deformability

  • Compare IL-3-treated vs. untreated cells or wild-type vs. IL-3R-deficient cells

• Atomic force microscopy (AFM):

  • Provides detailed mechanical measurements of individual cells

  • Quantifies Young's modulus, membrane tension, and cytoskeletal stiffness

  • Can map mechanical properties across the cell surface

• Scanning electron microscopy (SEM):

  • Visualizes surface morphology changes induced by IL-3

  • Detects alterations in membrane protrusions and cell shape

• Fluorescence recovery after photobleaching (FRAP):

  • Evaluates cytoskeletal dynamics and protein mobility

  • Can detect IL-3-induced changes in actin polymerization and turnover

• In vitro and in vivo cell trafficking assays:

  • Transwell migration assays to assess chemotactic responses

  • Adoptive transfer experiments with labeled cells to track migration in vivo

  • Intravital microscopy to visualize cell movement in real-time

These approaches have revealed that IL-3 receptor signaling affects T cell mechanical properties and migration patterns, particularly for regulatory T cells in inflammatory conditions .

What are the key considerations when investigating IL-3 in different species models?

Important species-specific differences must be considered when studying IL-3:

• Receptor structure differences:

  • Mice express an additional IL-3-specific β chain that differs from CD131

  • This murine-specific β chain can bind IL-3 directly, unlike human CD131

  • Human IL-3 does not cross-react with the mouse IL-3 receptor

• Experimental design implications:

  • Use species-matched IL-3 for in vitro and in vivo studies

  • Consider humanized mouse models for studies relevant to human disease

  • Validate findings across species when possible

• Expression pattern variations:

  • Cell type-specific expression of IL-3 and its receptor may differ between species

  • Quantify receptor expression on target cells of interest

  • Be cautious when extrapolating across species

• Functional conservation assessment:

  • Compare signaling pathways activated by IL-3 across species

  • Determine if cellular responses are conserved or divergent

  • Consider evolutionary aspects when interpreting results

These species differences may partly explain discrepancies in experimental outcomes between mouse models and human studies.

How can I address contradictory data in IL-3 research?

The IL-3 field contains numerous examples of seemingly contradictory findings . To navigate these contradictions:

• Context-dependent analysis:

  • Evaluate the specific disease model, cell types, and tissue microenvironment

  • Consider the timing of IL-3 action relative to disease progression

  • Analyze concentration-dependent effects that may explain opposing outcomes

• Methodological standardization:

  • Standardize protein sources, concentrations, and administration routes

  • Use multiple complementary techniques to verify findings

  • Develop robust positive and negative controls

• Biological heterogeneity consideration:

  • Account for genetic background effects in animal models

  • Consider patient heterogeneity in clinical samples

  • Analyze IL-3 effects on multiple cell types simultaneously

• Systems biology approaches:

  • Integrate multi-omics data to understand network effects

  • Develop computational models to predict context-dependent outcomes

  • Use single-cell analysis to resolve population heterogeneity

By addressing these aspects, researchers can develop more nuanced and accurate models of IL-3 function in different biological contexts.