Recombinant Human Granulocyte-macrophage colony-stimulating factor receptor subunit alpha (CSF2RA), partial (Active)

What is the molecular structure of CSF2RA and how does it contribute to GM-CSF receptor function?

CSF2RA (CD116) forms one portion of the heterodimeric GM-CSF receptor. The complete receptor consists of an α chain (CSF2RA) and a β chain, with the latter also participating in IL-3 and IL-5 receptors. The α subunit contains the binding site for GM-CSF but associates with the ligand only with low affinity. High-affinity binding and receptor activation require association of both α and β subunits .

Structurally, CSF2RA is an 80kDa type I transmembrane protein composed of three domains: extracellular, transmembrane, and cytoplasmic. The mature polypeptide contains 378 amino acids with 298 amino acids in the extracellular domain, 26 in the transmembrane domain, and 54 in the cytoplasmic tail, plus a 22 amino acid signal peptide that is cleaved during translation . The extracellular domain contains the cytokine receptor domain responsible for ligand binding.

How is CSF2RA expression regulated at the genetic and cellular levels?

The CSF2RA gene is located in the pseudoautosomal region (PAR) of X and Y chromosomes, specifically near the telomere regions. The gene contains several transcription regulatory binding sites with common binding motifs for transcription factors such as GATA, C/EBP, and NF-κB .

At the cellular level, CSF2RA is primarily expressed on neutrophils, eosinophils, and monocytes/macrophages. It is also present on CD34+ progenitor cells (myeloblasts) and precursors for erythroid and megakaryocytic lineages, but only in the early stages of their development . Expression levels can be modulated in response to inflammatory stimuli, as GM-CSF itself is secreted by various cell types including T cells, macrophages, endothelial cells, epithelial cells, and fibroblasts in response to pro-inflammatory signals such as LPS, IL-1, and TNF-α .

What are the primary research applications for recombinant CSF2RA protein?

Recombinant CSF2RA has several key research applications:

• Receptor-ligand interaction studies: Investigating binding kinetics and structural requirements for GM-CSF interaction with its receptor complex.

• Signal transduction research: Examining downstream signaling pathways activated following GM-CSF receptor engagement.

• Neutralization experiments: Using soluble recombinant CSF2RA to neutralize GM-CSF in experimental systems.

• Structure-function relationship studies: Understanding how specific domains of CSF2RA contribute to receptor assembly and function.

• Therapeutic development: Designing receptor antagonists or agonists that can modulate GM-CSF signaling in inflammatory conditions.

• Immunological research: Studying how CSF2RA contributes to myeloid cell functions, including phagocytosis, cytokine production, and antigen presentation .

How can CSF2RA knockout models be utilized to understand inflammatory diseases?

CSF2RA knockout (KO) models provide valuable insights into inflammatory pathologies. Studies have demonstrated that Csf2ra KO mice show attenuated inflammatory responses in experimental models of acute lung injury (ALI) .

When implementing CSF2RA knockout models:

• Survival analysis: Csf2ra KO mice challenged with ricin toxin (RT) showed significantly improved survival (70% vs. 10% in wild-type) after 21 days, demonstrating the role of GM-CSF signaling in exacerbating inflammatory damage .

• Inflammatory pathway assessment: Transcriptomic analysis of Csf2ra KO mice revealed decreased activity in pro-inflammatory pathways, including TNF signaling and NF-κB signaling after RT exposure .

• Immune cell infiltration studies: Flow cytometry analyses showed reduced neutrophil chemotaxis and recruitment in Csf2ra KO mice following inflammatory challenge, suggesting mechanisms by which GM-CSF signaling contributes to tissue damage .

• Histopathological evaluation: H&E staining of lung tissues from Csf2ra KO mice showed reduced alveolar damage, vascular leakage, and cellular infiltration, providing visual confirmation of the protective effect of CSF2RA deletion .

These models are particularly useful for studying inflammatory conditions where GM-CSF signaling plays a pathogenic role and for evaluating potential therapeutic approaches targeting this pathway.

What are the recommended protocols for evaluating CSF2RA-mediated cell activation?

When evaluating CSF2RA-mediated cell activation, researchers should consider the following methodology:

• Cellular morphology assessment: Monitor morphological changes in neutrophils and eosinophils after GM-CSF stimulation. Documentation should include temporal changes, as these typically occur within 6-9 hours of stimulation .

• Surface marker expression analysis: Use flow cytometry to measure upregulation of functional antigens. Key markers include granulocyte functional antigens 1 and 2, and the Mo1 antigen, which are selectively enhanced by GM-CSF signaling .

• Functional assays:

  • Cytotoxicity assays against antibody-coated targets

  • Phagocytosis assays using serum-opsonized yeast particles

  • Iodination assays in the presence of zymosan

  • Degranulation measurements following FMLP stimulation of Cytochalasin B-pretreated neutrophils

  • Superoxide production assays following FMLP stimulation

• Cell survival quantification: Implement trypan blue exclusion or annexin V/PI staining to monitor enhanced cell survival, which is a key biological outcome of GM-CSF receptor activation .

• Western blot analysis: Assess phosphorylation status of downstream signaling molecules including p65, IκB, and other NF-κB pathway components to confirm receptor activation .

What are the best practices for isolating and analyzing CSF2RA-expressing primary cells?

For optimal isolation and analysis of CSF2RA-expressing primary cells:

• Tissue processing:

  • For lung tissue: Immerse in tissue digestion solution (1.5 mg/ml collagenase A + 0.4 mg/ml DNase I + 1.5 U/ml dispase II in HBSS containing 5% FBS and 10 mM HEPES) for 30 minutes

  • Mechanically disrupt tissues, filter through cell strainers, and centrifuge at 3500 rpm for 10 minutes at 4°C

  • Lyse red blood cells using appropriate buffer and filter through a 40 μm nylon cell filter

• Cell counting and viability assessment:

  • Use automated cell counters for consistent quantification

  • Ensure viability >90% before proceeding with experiments

• Flow cytometry staining protocol:

  • Block FcγR using CD16/CD32 antibody for 20 minutes at 4°C

  • Stain cell suspensions with fluorochrome-conjugated antibodies for 30 minutes

  • Key markers: CD45 (leukocyte common antigen), Ly6G (neutrophils), CD116/CSF2RA

  • Include viability dye (e.g., Fixable Viability Stain 510)

• RNA isolation for gene expression analysis:

  • Extract total RNA using RNAprep Pure Tissue Kit or similar

  • Verify RNA quality using NanoPhotometer and Agilent 2100 Bioanalyzer

  • Proceed with RT-qPCR or RNA-Seq as needed

• Protein extraction and analysis:

  • Lyse cells in RIPA buffer containing protease and phosphatase inhibitors

  • Perform SDS-PAGE and Western blotting with antibodies against CSF2RA and associated signaling molecules

  • Include appropriate loading controls (actin, GAPDH)

How should transcriptomic data from CSF2RA studies be analyzed and interpreted?

Transcriptomic analysis of CSF2RA studies requires systematic approaches to ensure valid interpretation:

• Time-series analysis: Implement Bioconductor packages like maSigPro for temporal gene expression pattern analysis, especially when comparing CSF2RA-deficient and wild-type samples. This two-step regression strategy first identifies significant differentially expressed genes (DEGs) among treatment groups and then groups genes with similar expression patterns for visualization .

• Pathway enrichment analysis: Focus on inflammation-related genes in GM-CSF signaling contexts, particularly examining:

  • TNF signaling pathway components

  • NF-κB signaling pathway genes

  • Neutrophil chemotaxis and recruitment genes

  • Cytokine/chemokine expression patterns

• Immune cell profiling: Utilize computational methods like ImmuCellAI to estimate the abundance of immune cell populations from RNA-Seq data. This gene set signature-based approach helps identify shifts in immune cell distribution resulting from CSF2RA manipulation .

• Validation approaches: Confirm key transcriptomic findings with:

  • RT-qPCR for selected genes (using primers as shown in Table 1)

  • Flow cytometry for cell population changes

  • Protein expression analysis for critical pathway components

• Integration with phenotypic data: Correlate transcriptomic changes with functional outcomes, survival data, and histopathological findings to establish meaningful biological connections .

What statistical considerations are important when analyzing CSF2RA functional studies?

When analyzing data from CSF2RA functional studies, researchers should address these statistical considerations:

• Sample size determination: Calculate appropriate sample sizes for in vivo studies based on expected effect sizes. For example, survival studies comparing Csf2ra KO and WT mice typically require 10 animals per group to detect significant differences with adequate power .

• Time-point selection: Carefully select experimental time points to capture the dynamics of GM-CSF-mediated responses. Studies examining acute lung injury models have identified 0h, 4h, 12h, and 72h as critical time points for observing the evolution of inflammatory responses and gene expression changes .

• Multiple testing correction: When analyzing high-dimensional data like transcriptomics, apply appropriate corrections for multiple hypothesis testing (e.g., Benjamini-Hochberg procedure) to control false discovery rates.

• Paired analyses for pre/post-stimulation: Use paired statistical tests when examining the same cells or tissues before and after GM-CSF stimulation to increase statistical power.

• Semi-quantitative scoring systems: For histopathological assessments, implement validated scoring systems with defined parameters. For example, lung injury can be scored on parameters including:

  • Alveolar septal thickening

  • Perivasculitis

  • Peribronchiolitis

  • Immune cell infiltration (neutrophils, lymphocytes, monocytes)

  • Vascular leakage

  • Alveolar edema

  • Hyaline membrane formation

  • Bleeding

  • Bronchial epithelial damage

  • Endothelial injury

  Each parameter can be graded on a scale from 0 to 4, where 0 = normal; 1 = mild; 2 = moderate; 3 = severe; 4 = very severe injury .

How does CSF2RA function differ between intestinal inflammation and acute lung injury models?

CSF2RA function exhibits tissue-specific differences that are important for understanding its role in different inflammatory conditions:

Intestinal Inflammation:

• In the intestine, GM-CSF signaling through CSF2RA has been primarily studied in intestinal epithelial cells (IECs), where it promotes epithelial barrier function and homeostasis .

• GM-CSF is secreted by various cell types within the intestinal microenvironment in response to inflammatory stimuli and contributes to host defense against gastrointestinal infections .

• Unlike in lung models, GM-CSF in the intestine appears to have protective effects in many contexts, supporting epithelial regeneration and antimicrobial immunity .

• GM-CSF deficiency in intestinal models may lead to increased susceptibility to infection rather than protection from inflammatory damage .

Acute Lung Injury Models:

• In acute lung injury models, GM-CSF signaling through CSF2RA appears to exacerbate inflammatory damage, as evidenced by the improved survival of Csf2ra KO mice after ricin toxin exposure .

• Transcriptomic analysis shows reduced activity of pro-inflammatory pathways in the lungs of Csf2ra KO mice compared to wild-type after inflammatory challenge .

• In the lung, CSF2RA signaling promotes neutrophil recruitment and activation, contributing to tissue damage through release of reactive oxygen species and proteolytic enzymes .

• CSF2RA is also associated with surfactant metabolism dysfunction type 4, highlighting its importance in lung-specific functions beyond inflammation .

These tissue-specific differences suggest that therapeutic approaches targeting GM-CSF signaling may need to be tailored to specific anatomical contexts.

What are the current technical challenges in developing CSF2RA-targeted therapeutics?

Developing therapeutics targeting CSF2RA presents several technical challenges:

• Receptor complex heterogeneity: The GM-CSF receptor functions as a heterodimer with shared components (β chain) also present in IL-3 and IL-5 receptors. This creates challenges for developing highly specific inhibitors that don't affect signaling by these related cytokines .

• Tissue-specific effects: The divergent roles of GM-CSF signaling in different tissues (protective in intestine vs. pathogenic in lung inflammation) complicate therapeutic development, requiring approaches that can selectively target specific tissues or inflammatory contexts .

• Functional redundancy: Immune cells often respond to multiple cytokines with overlapping functions, potentially limiting the effectiveness of single-pathway inhibition strategies.

• Timing considerations: The temporal dynamics of GM-CSF signaling differ across disease models, with early inhibition potentially beneficial in acute inflammatory conditions but detrimental in chronic settings where tissue repair mechanisms predominate.

• Delivery methods: For lung-specific targeting, inhalation-based delivery systems may be most effective but require careful optimization of formulation, particle size, and stability parameters.

• Preclinical model translation: While Csf2ra KO mice show protection in acute lung injury models, human genetic variations in CSF2RA may have more subtle effects, requiring careful biomarker development to identify patients most likely to benefit from CSF2RA-targeted therapies .

• Safety concerns: Given the role of GM-CSF in myeloid cell development and host defense, complete blockade of CSF2RA signaling may compromise antimicrobial immunity, necessitating careful dosing strategies and patient monitoring.

What are the optimal conditions for studying GM-CSF/CSF2RA interactions in vitro?

When designing experiments to study GM-CSF/CSF2RA interactions:

• Cell types: Select appropriate cell models based on research questions:

  • Primary neutrophils and eosinophils for functional studies

  • CD34+ progenitor cells for differentiation studies

  • Monocytes/macrophages for cytokine production analysis

  • Cell lines expressing recombinant CSF2RA for mechanistic studies

• Recombinant protein quality: Use highly purified recombinant human GM-CSF (rH GM-CSF) to ensure reproducible results. Verify protein activity through established bioassays before experimental use .

• Dose-response relationships: Implement dose-response experiments to determine optimal GM-CSF concentrations. Studies have shown that GM-CSF enhances phagocytosis and other functions in a dose-dependent manner .

• Temporal considerations: Monitor time-dependent effects, as GM-CSF induces morphological changes and enhances survival of neutrophils and eosinophils by 6 and 9 hours, respectively .

• Combinatorial stimulation: For certain functional assays, combine GM-CSF with secondary stimuli such as N-formylmethionylleucylphenylalanine (FMLP) to assess potentiation effects on degranulation and superoxide production .

• Controls: Include appropriate controls such as:

  • Unstimulated cells

  • Isotype-matched control antibodies for blocking experiments

  • Positive controls using established stimuli (e.g., PMA for respiratory burst)

  • Heat-inactivated GM-CSF to confirm specificity

• Pretreatment protocols: For certain assays like degranulation studies, pretreat neutrophils with Cytochalasin B before FMLP stimulation to enhance measurable responses .

How can researchers effectively model CSF2RA-related diseases in laboratory settings?

To effectively model CSF2RA-related diseases:

• Genetic modification approaches:

  • Generate Csf2ra knockout models using CRISPR/Cas9 or traditional gene targeting

  • Create conditional knockout models using Cre-loxP systems to study tissue-specific effects

  • Develop knock-in models with specific human mutations to study pathogenic variants

• Acute lung injury models:

  • Intratracheal inoculation of ricin toxin (RT) at appropriate doses (e.g., 2× LD50) provides a reproducible model for studying acute inflammatory lung damage

  • Monitor survival, histopathological changes, and molecular responses at key timepoints (4h, 12h, 72h)

• Tissue processing and analysis:

  • For lung tissue analysis, follow established protocols for tissue fixation (≥48h), paraffin embedding, and sectioning (4μm thickness)

  • Implement semi-quantitative scoring systems with defined parameters to ensure consistent evaluation of tissue damage

• Multi-omics approach:

  • Combine transcriptomics, proteomics, and functional assays to comprehensively characterize disease models

  • Use RNA-Seq with paired-end strategy on platforms like NovaSeq 6000 for high-quality transcriptomic data

  • Validate key findings using RT-qPCR and protein-level assays

• Immune cell profiling:

  • Analyze tissue-infiltrating immune cells using flow cytometry with markers such as CD45, Ly6G, and cell type-specific markers

  • Use computational methods to estimate immune cell populations from transcriptomic data

• Signaling pathway analysis:

  • Assess activation of downstream pathways like NF-κB using phospho-specific antibodies against p65, IκB, and other components

  • Monitor inflammatory mediators such as TNF-α using appropriate antibodies and detection methods

These approaches allow researchers to create robust models of CSF2RA-related pathologies and test potential therapeutic interventions in preclinical settings.