Recombinant Mouse Granulocyte-macrophage colony-stimulating factor protein (Csf2) (Active)

What is recombinant mouse GM-CSF protein and what are its main biological functions?

Recombinant mouse GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), also known as Csf2, is a cytokine initially characterized for its ability to support the in vitro colony formation of granulocyte-macrophage progenitors. The E. coli-derived recombinant protein typically consists of amino acids Ala18-Lys141, with an N-terminal Met .

Biologically, mouse GM-CSF functions as:

• A growth factor for erythroid, megakaryocyte, and eosinophil progenitors

• A survival factor for mature hematopoietic cells

• An activator of effector functions in granulocytes, monocytes/macrophages, and eosinophils

• A promoter of Th1-biased immune responses, angiogenesis, and allergic inflammation

• A contributor to autoimmunity development

GM-CSF exerts its biological effects through a heterodimeric receptor complex composed of GM-CSF Rα/CD116 (the specific binding subunit) and the common β chain (CD131), which is also a component of the IL-3 and IL-5 receptors .

What are the structural characteristics of recombinant mouse GM-CSF?

Recombinant mouse GM-CSF has a molecular weight of approximately 14.3 kDa as analyzed by SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering), suggesting that the protein exists as a monomer . Under reducing SDS-PAGE conditions, it appears as a single band at approximately 14 kDa when visualized by silver staining . In its native form, mouse GM-CSF typically runs as a 15.5-19 kDa band in both reducing and non-reducing SDS-PAGE analysis .

Structurally, mouse GM-CSF contains 124 amino acid residues and, similar to IL-3 and IL-5, has a core of four bundled alpha-helices . Mature mouse GM-CSF shares 49%-54% amino acid sequence identity with canine, feline, human, and porcine GM-CSF, and 69% with rat GM-CSF .

What are the recommended storage and reconstitution protocols for recombinant mouse GM-CSF?

For optimal activity preservation, follow these guidelines:

Storage:

• Store at -80°C in a manual defrost freezer

• Avoid repeated freeze-thaw cycles

• Upon initial thawing, aliquot into polypropylene microtubes before refreezing

Reconstitution by formulation type:

• For lyophilized product with carrier protein (e.g., 415-ML):

  • Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For carrier-free lyophilized product (e.g., 415-ML/CF):

  • Reconstitute at 100 μg/mL in sterile PBS

• For dilution and long-term storage:

  • For in vitro biological assays: Use carrier protein concentrations of 0.5-1 mg/mL

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

  • Never dilute below 5 μg/mL for long-term storage

When should carrier-free versions of recombinant mouse GM-CSF be used?

Carrier-free (CF) formulations of recombinant mouse GM-CSF do not contain Bovine Serum Albumin (BSA) or other carrier proteins. They are typically lyophilized from 0.2 μm filtered solutions in PBS .

These formulations are specifically recommended for applications where the presence of BSA could interfere, such as:

• Studies requiring pure protein interactions without carrier protein interference

• Experimental systems sensitive to bovine proteins

• Conjugation or labeling applications where carrier proteins might compete for reactive sites

• Mass spectrometry or other analytical techniques where carrier proteins could confound results

Researchers should be aware that carrier proteins may have undesired influence on experimental results due to potential toxicity, high endotoxin levels, or possible blocking activity .

How is the biological activity of recombinant mouse GM-CSF measured?

The biological activity of recombinant mouse GM-CSF can be assessed through:

• Cell proliferation assays:

  • The standard assay uses the DA3 mouse myeloma cell line

  • The effective dose (ED50) typically ranges from 5-30 pg/mL

  • This remarkably low ED50 demonstrates the high potency of the protein

• Colony formation assays:

  • Using bone marrow progenitor cells to measure colony-forming units of granulocyte-macrophage lineage

• Functional activation assays:

  • Measuring respiratory burst in neutrophils or macrophages

  • Cytokine production by stimulated cells

  • Phagocytic activity enhancement

• Receptor binding assays:

  • Using cells expressing mouse GM-CSF receptor complex

For quality control purposes, recombinant mouse GM-CSF preparations should have endotoxin levels ≤ 0.1 ng per μg of protein, as measured by chromogenic LAL assay, and ≥ 95% purity as determined by SDS-PAGE and absorbance measurements .

How does GM-CSF contribute to autoimmune and inflammatory diseases in mouse models?

GM-CSF has been implicated in several autoimmune and inflammatory conditions through multiple mechanisms:

Experimental Autoimmune Prostatitis (EAP):

• Elevated GM-CSF mRNA levels have been detected in prostate tissue from EAP mice

• GM-CSF may contribute to both inflammatory responses in prostate tissue and pain development through neuronal mechanisms

• GM-CSF knockout mice show decreased symptom severity in EAP models

Atherosclerosis:

• In LDL-receptor-deficient (Ldlr-/-) mice, GM-CSF promotes advanced plaque progression

• The mechanism involves increasing macrophage apoptosis susceptibility through:

  • GM-CSF-mediated production of IL-23

  • IL-23-induced proteasomal degradation of the cell-survival protein Bcl-2

  • Increased oxidative stress

• GM-CSF-deficient mice crossed with Ldlr-/- mice show substantial decreases in lesional macrophage apoptosis and plaque necrosis

These findings highlight GM-CSF as a potential therapeutic target in various inflammatory and autoimmune conditions, with its specific role varying depending on the disease context and affected tissues.

What experimental approaches can be used to study GM-CSF function in disease models?

Researchers can employ several experimental approaches to investigate GM-CSF function:

Genetic Models:

• GM-CSF knockout mice (Csf2-/-) - Complete absence of GM-CSF allows assessment of its necessity in disease development

• Cell-specific conditional knockouts - Targeting GM-CSF or its receptor in specific cell populations

• Transgenic overexpression - To study effects of increased GM-CSF levels

Pharmacological Approaches:

• Neutralizing antibodies - For temporal and dose-dependent inhibition

• Recombinant GM-CSF administration - To supplement or rescue phenotypes

• Receptor antagonists - To block signaling without affecting protein levels

Analytical Methods:

• mRNA quantification - Using qRT-PCR to measure GM-CSF expression in tissue samples

• Protein detection - ELISA, Western blotting, or immunohistochemistry

• Functional assays - As described in question 1.5

• Signaling pathway analysis - Phospho-flow cytometry, Western blotting for active signaling molecules

Experimental Design Considerations:

• Include appropriate controls (isotype controls, vehicle treatments)

• Validate findings using multiple approaches

• Consider potential compensatory mechanisms in knockout models

• Account for strain-specific differences in GM-CSF responses

• Remember the species-specific nature of GM-CSF activity - mouse GM-CSF shows limited cross-reactivity with rat cells

How does GM-CSF influence macrophage function and apoptosis in disease models?

GM-CSF exerts significant effects on macrophage biology that impact disease pathogenesis:

Macrophage Differentiation and Polarization:

• Promotes differentiation of monocytes into inflammatory macrophages

• Influences the balance between M1 (pro-inflammatory) and M2 (tissue repair) phenotypes

• Affects expression of scavenger receptors and pattern recognition receptors

Apoptosis Regulation in Atherosclerosis:

• Increases macrophage susceptibility to apoptosis in advanced atherosclerotic plaques

• The mechanism involves:

  • GM-CSF-induced IL-23 production

  • IL-23-mediated downregulation of the anti-apoptotic protein Bcl-2

  • Enhanced sensitivity to atherosclerosis-relevant pro-apoptotic factors (7-ketocholesterol, oxidized-LDL)

• GM-CSF-deficient (Csf2-/-Ldlr-/-) mice show reduced lesional macrophage apoptosis and plaque necrosis

Inflammatory Activation:

• Enhances production of pro-inflammatory cytokines and chemokines

• Increases reactive oxygen species (ROS) generation

• Augments phagocytic activity and antigen presentation

Tissue-Specific Effects:

• In prostate tissue (EAP model), affects inflammatory cell recruitment and activation

• In arterial plaques, contributes to advanced lesion progression and vulnerability

• Effects may vary depending on the tissue microenvironment and disease stage

These diverse effects make GM-CSF a potent regulator of macrophage function in multiple disease contexts, with potential for therapeutic targeting.

What role does GM-CSF play in the experimental autoimmune prostatitis (EAP) mouse model?

In the experimental autoimmune prostatitis (EAP) mouse model, GM-CSF has been identified as a significant contributor to disease pathogenesis:

Expression Pattern:

• Elevated GM-CSF mRNA levels are detected in prostate tissue from EAP mice

• This mirrors findings in expressed prostatic secretions from human chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) patients

Receptor Distribution:

• GM-CSF receptors are expressed in both mouse prostate tissue and dorsal root ganglia

• This dual expression suggests potential roles in both local inflammation and pain signaling

Functional Impact:

• GM-CSF knockout mice (Csf2-/-) show reduced symptom severity when EAP is induced, including:

  • Decreased behavioral indicators of pain

  • Reduced prostatic inflammation scores

  • Lower expression of nociceptive and inflammatory markers

Mechanistic Insights:

• May contribute to immune cell recruitment and activation in prostate tissue

• Potentially modulates nociceptive pathways, given receptor expression in dorsal root ganglia

• Could represent a link between prostatic inflammation and pelvic pain in this model

These findings suggest that GM-CSF plays dual roles in EAP pathogenesis - contributing to both inflammatory responses in prostate tissue and potentially to pain development through neuronal mechanisms, making it a promising therapeutic target for CP/CPPS.

What are the signaling pathways downstream of GM-CSF receptor activation in mice?

GM-CSF receptor signaling in mice involves a complex network of pathways following binding to its heterodimeric receptor:

Receptor Complex:

• GM-CSF Rα/CD116: The specific binding subunit

• Common β chain (CD131): The signaling subunit shared with IL-3 and IL-5 receptors

Major Signaling Cascades:

• JAK-STAT Pathway:

  • JAK2 associates with the β chain and becomes activated

  • Leads to phosphorylation and activation of STAT5, and to a lesser extent STAT3

  • Activated STATs translocate to the nucleus and regulate gene expression

  • Critical for proliferation and survival signals

• PI3K/Akt Pathway:

  • Important for cell survival signals

  • Regulates anti-apoptotic proteins including Bcl-2 family members

  • Disruption in this pathway can enhance macrophage apoptosis susceptibility as observed in atherosclerosis models

• MAPK Pathways:

  • Activation of ERK1/2, p38 MAPK, and JNK

  • Contributes to proliferation, differentiation, and inflammatory responses

  • Plays a role in cytokine production by activated cells

• NF-κB Pathway:

  • Important for inflammatory gene expression

  • Regulates production of cytokines, including IL-23

  • IL-23 has been implicated in GM-CSF-mediated effects in atherosclerosis

Pathway Crosstalk:

• These pathways interact extensively and show context-dependent activation

• The balance between these signals determines cellular outcomes including survival, proliferation, differentiation, and activation

Understanding these signaling mechanisms provides potential points for therapeutic intervention in GM-CSF-mediated pathologies.

How does recombinant mouse GM-CSF interact with other cytokines in inflammatory networks?

Recombinant mouse GM-CSF functions within a complex cytokine network during inflammatory responses:

GM-CSF and the IL-23/IL-17 Axis:

• GM-CSF stimulates production of IL-23, which mediates some of GM-CSF's pro-inflammatory and pro-apoptotic effects in atherosclerosis models

• In turn, IL-23 can stimulate IL-17 production by T cells

• IL-17 may further enhance GM-CSF production, creating a positive feedback loop

Interactions with Pro-inflammatory Cytokines:

• Synergizes with TNF-α to enhance inflammatory activation of macrophages

• Amplifies IL-1β production and signaling in myeloid cells

• Interferon-γ can enhance GM-CSF receptor expression and signaling

Regulation by Anti-inflammatory Cytokines:

• IL-10 can suppress GM-CSF production and some downstream effects

• TGF-β may modulate GM-CSF-induced myeloid cell differentiation

• IL-4 can alter macrophage responses to GM-CSF

Methodological Approaches for Studying Cytokine Interactions:

• Multi-parameter flow cytometry to analyze cell-specific responses

• Cytokine co-stimulation experiments in vitro

• Multiplex cytokine profiling in disease models

• Combined cytokine blockade or knockout models

• Transcriptomic analysis to identify cytokine networks

What are the experimental challenges when working with recombinant mouse GM-CSF in research?

Researchers face several technical and interpretative challenges when studying GM-CSF activity:

Technical Challenges:

• Protein Stability Issues:

  • Limited stability in solution

  • Requires carrier proteins (e.g., BSA) for stabilization

  • Needs storage at -80°C and avoidance of freeze-thaw cycles

  • Concentration should not be diluted below 5 μg/mL for long-term storage

• Detection Sensitivity Requirements:

  • Biological activity is potent at very low concentrations (ED50 of 5-30 pg/mL)

  • Requires highly sensitive detection methods

  • Standard curves must cover wide concentration ranges

• Formulation Considerations:

  • Carrier proteins may cause experimental interference

  • Need to select between standard and carrier-free formulations based on application

  • Endotoxin contamination must be minimal (≤0.1 ng per μg)

Experimental Design Challenges:

• Biological Complexity:

  • Overlapping functions with IL-3 and IL-5 (shared β chain receptor)

  • Context-dependent activity across different cell types and tissues

  • Species specificity limiting translational applications

• Interpretation Difficulties:

  • Compensatory mechanisms in knockout models

  • Distinguishing direct vs. indirect effects through cytokine networks

  • Variations in receptor expression across cell populations

Methodological Solutions:

These considerations highlight the importance of rigorous experimental design and appropriate controls when working with recombinant mouse GM-CSF in research applications.

How can GM-CSF knockout mice be used to elucidate cytokine function in disease models?

GM-CSF knockout mice (Csf2-/-) serve as valuable tools for investigating this cytokine's role in various disease processes:

Experimental Design Strategies:

• Disease Model Crosses:

  • GM-CSF knockout mice can be crossed with disease-specific models (e.g., Ldlr-/- for atherosclerosis)

  • This approach allows assessment of GM-CSF's role in specific pathological contexts

• Induced Disease Models:

  • Direct application of disease induction in GM-CSF knockout background (e.g., EAP induction)

  • Comparing disease progression between knockout and wildtype mice

• Bone Marrow Chimeras:

  • Transplanting GM-CSF-deficient bone marrow into wildtype recipients (or vice versa)

  • Helps distinguish between hematopoietic and non-hematopoietic sources of GM-CSF

Parameter Assessment:

• Clinical Evaluation:

  • Disease-specific clinical scores

  • Physiological parameters (weight, temperature, behavior)

  • Functional assessments (e.g., pain sensitivity in EAP models)

• Cellular Analysis:

  • Flow cytometry to assess immune cell populations

  • Histopathological evaluation of affected tissues

  • Cell subset function and activation status

• Molecular Assessment:

  • Cytokine and inflammatory mediator profiling

  • Gene expression analysis of disease-relevant pathways

  • Signaling molecule activation status

Mechanistic Investigations:

• Differences in disease outcomes can be analyzed to elucidate downstream mechanisms

• Example: Decreased macrophage apoptosis in atherosclerotic plaques of Csf2-/-Ldlr-/- mice revealed GM-CSF's role in promoting cell death through IL-23 and Bcl-2 regulation

Validation Approaches:

• Rescue experiments with recombinant GM-CSF administration

• Antibody-mediated GM-CSF neutralization in wildtype mice

• Cell-specific conditional knockout models for refined mechanistic insights

When interpreting results from knockout models, researchers should consider potential compensatory mechanisms and developmental effects that might influence outcomes independently of GM-CSF's direct role in pathogenesis.