Recombinant Mouse Interleukin-1 beta protein (Il1b)

What are the structural and functional characteristics of recombinant mouse IL-1β protein?

Recombinant mouse IL-1β (also known as IL-1F2) is a 17.5 kDa proinflammatory cytokine primarily produced by monocytes, tissue macrophages, keratinocytes, and other epithelial cells . The commercially available recombinant protein typically consists of amino acids Val118-Ser269 with an N-terminal methionine, expressed in E. coli or mammalian expression systems like HEK293 cells .

Functionally, IL-1β promotes T cell proliferation and cytokine production while attenuating regulatory T cell function, enabling CD4+CD25- autoreactive effector T cells . The protein demonstrates potent biological activity with an ED50 (effective dose for 50% maximum response) of 2-10 pg/mL in cell proliferation assays using D10.G4.1 mouse helper T cell lines . IL-1β is directly involved in neuronal injury in neurodegenerative disorders and stimulates mitogenic FGF-like activity, bone resorption, and promotes the release of collagenase and prostaglandin from synovial cells .

Recent research has identified its critical role in age-associated decline of beta cell function, with evidence that myeloid cell-specific IL-1β knockout can preserve glucose-stimulated insulin secretion during aging .

Reconstitution protocols

The reconstitution procedure differs based on the formulation of the recombinant protein:

For carrier-containing formulations (e.g., 401-ML):

• The protein is typically lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein

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

• Gently mix after reconstitution as the protein may appear as a film at the bottom of the vial

For carrier-free formulations (e.g., 401-ML/CF):

• Supplied as a 0.2 μm filtered solution in PBS

• Does not contain bovine serum albumin (BSA)

Storage recommendations

Researchers should select the appropriate formulation based on their experimental needs. The carrier-containing version (with BSA) is generally recommended for cell/tissue culture applications and as ELISA standards, while carrier-free versions are preferable for applications where BSA could interfere with experimental outcomes .

How does IL-1β influence pancreatic beta cell function and glucose homeostasis in aging models?

Research has demonstrated that IL-1β plays a significant role in age-associated decline of beta cell function . Key findings include:

• Age-related expression patterns: IL-1β expression is selectively induced in islet immune cells during aging, while expression of cell-cycle genes (Mki67, E2f1, and Ccnd1) is reduced in beta cells from aged mice (52-week-old) compared to young mice (12-week-old) .

• Impact on glucose homeostasis:

  • 52-week-old mice show impaired glucose disposal compared to 16-week-old mice

  • Aged mice exhibit increased fasting insulin but diminished glucose-stimulated insulin secretion

  • The insulin secretion ratio (15 min/0 min) is lower in aged mice, reflecting impaired function

• Effects of IL-1β knockout:

  • IL-1β whole-body knockout mice show elevated insulin levels at 52 weeks of age

  • Improved glycemia in old age despite insulin resistance

  • Increased mean beta cell mass in 52-week-old IL-1β knockout mice compared to age-matched controls

  • Larger islet size distribution and higher percentage of islets with Ki67-positive beta cells

• Myeloid-specific knockout effects:

  • Myeloid cell-specific IL-1β knockout (Lyz2 Cre+/− Il1b fl/fl) shows 96.3% knockout efficiency in peritoneal macrophages, 94.8% in liver tissue, and 68% in islets

  • Preserved glucose-stimulated insulin secretion during aging

These findings suggest that IL-1β may act as a brake for the expansion of islet size and number during aging, and targeting IL-1β specifically in myeloid cells could represent a therapeutic approach for age-related metabolic dysfunction .

Dose considerations

When designing experiments with recombinant mouse IL-1β, researchers should consider that:

• The ED50 for cell proliferation effects is typically 2-10 pg/mL

• Dose-response curves should be generated for each specific cell type and readout

• Biological responses may vary significantly between different cell types and experimental conditions

Carrier protein implications

The presence of carrier proteins (typically BSA) can impact experimental outcomes:

• For most cell culture applications, carrier-containing formulations enhance protein stability

• For applications where BSA might interfere (e.g., certain binding assays, mass spectrometry), carrier-free versions should be used

• Control experiments should include carrier protein alone to rule out carrier-dependent effects

Validation considerations

Researchers should validate protein activity and specificity:

• Confirm protein identity by SDS-PAGE (appears as a single band at approximately 17-19 kDa)

• Validate biological activity using established assays (e.g., T cell proliferation)

• Consider including IL-1 receptor antagonist (IL-1Ra) controls to confirm specificity of observed effects

Experimental controls

For knockout or inhibition studies:

• Include appropriate littermate wild-type controls

• Validate knockout efficiency in relevant tissues (e.g., peritoneal macrophages showed 96.3% efficiency, islets showed 68% efficiency in myeloid-specific knockout models)

• Consider age as a critical variable, as IL-1β effects may differ significantly between young and aged animals

What are the key differences between carrier-free and carrier-containing recombinant mouse IL-1β formulations?

Researchers should select the appropriate formulation based on their specific experimental requirements . The carrier protein (BSA) enhances stability and increases shelf-life but may interfere with certain downstream applications. When publishing results, researchers should clearly specify which formulation was used to ensure reproducibility.

How can researchers differentiate between the effects of exogenous recombinant IL-1β and endogenous IL-1β in experimental systems?

Differentiating between exogenous and endogenous IL-1β effects requires careful experimental design:

Blocking strategies

• Use specific IL-1β neutralizing antibodies to block both endogenous and exogenous IL-1β

• Employ IL-1 receptor antagonist (IL-1Ra) to block signaling from both sources

• Utilize receptor knockout models to eliminate all IL-1β signaling

Genetic approaches

• Compare IL-1β knockout models with wild-type controls treated with recombinant IL-1β

• Use tissue-specific or inducible knockout systems (e.g., myeloid-specific IL-1β knockout showed 68% efficiency in islets)

• Consider compensatory effects (e.g., no compensatory increase in IL-1α was observed in IL-1β knockout mice)

Technical considerations

• Tag recombinant IL-1β to distinguish it from endogenous protein

• Monitor endogenous IL-1β expression levels via qPCR before adding recombinant protein

• Utilize mouse models with humanized IL-1β receptors and human IL-1β to distinguish signaling

These approaches are particularly important when studying tissues with high endogenous IL-1β expression or in inflammatory conditions where IL-1β is upregulated.

What are the critical factors to consider when comparing findings across studies using different recombinant mouse IL-1β preparations?

When comparing studies using different recombinant IL-1β preparations, researchers should consider:

Source and expression system

• E. coli-derived vs. mammalian cell-expressed proteins

• Potential differences in post-translational modifications

• HEK293-expressed proteins (≥95% purity) may have different activity profiles than bacterial systems

Protein sequence and structure

• Confirm identical amino acid sequences (typically Val118-Ser269 with N-terminal Met)

• Assess potential differences in tertiary structure or aggregation state

• Verify molecular weight (approximately 17.5 kDa)

Activity standardization

• Compare ED50 values (typically 2-10 pg/mL for cell proliferation assays)

• Normalize doses based on biological activity rather than protein concentration

• Consider lot-to-lot variations in activity, even from the same manufacturer

Experimental context

• Cell types used may respond differently to various preparations

• Presence of carrier proteins can affect results

• Buffer composition and additives may influence protein activity

Researchers should explicitly report these details in methods sections to enable proper comparison and reproducibility of findings across different studies.

How does IL-1β signaling intersect with other inflammatory pathways in metabolic disease models?

IL-1β signaling intersects with multiple inflammatory pathways in metabolic disease contexts:

Beta cell function and insulin secretion

• IL-1β expression is selectively induced in islet immune cells during aging

• IL-1β knockout improves glycemia in old age despite insulin resistance through enhanced insulin secretion

• IL-1β activity is counterbalanced by endogenous IL-1Ra, with deletion of IL-1Ra in beta cells decreasing insulin secretion via targeting of E2f1 and Kir6.2

Macrophage polarization

• Islet macrophages are constitutively M1-polarized, suggesting chronic IL-1 activity

• Myeloid-specific IL-1β knockout preserved glucose-stimulated insulin secretion during aging

Cell proliferation and mass regulation

• IL-1β appears to act as a brake for islet size and number expansion

• IL-1β knockout mice show increased mean beta cell mass with larger islet size distribution

• Higher percentage of islets with Ki67-positive beta cells observed in IL-1β knockout mice

Compensatory mechanisms

• No compensatory increase in IL-1α gene expression was observed in IL-1β knockout mice

• Expression of the protective IL-1Ra was higher in aged IL-1β knockout islets than in wild-type islets

These interactions highlight the complex role of IL-1β in metabolic regulation and suggest potential therapeutic approaches targeting specific inflammatory pathways in age-related metabolic dysfunction.

What advanced analytical techniques are recommended for detecting and quantifying recombinant mouse IL-1β in complex biological samples?

For researchers working with complex biological samples, several advanced analytical techniques can be employed:

Protein detection methods

Functional assays

• D10.G4.1 mouse helper T cell proliferation assays (standard for activity)

• Gene expression analysis of IL-1β-responsive genes

• Phosphorylation of downstream signaling molecules (e.g., NF-κB pathway components)

• Calcium flux assays for rapid signaling responses

In vivo tracking

• Use of tagged recombinant proteins

• Tissue-specific reporter systems for IL-1β signaling

• Monitoring of physiological responses (e.g., insulin secretion in pancreatic studies)

When analyzing samples from knockout models, researchers should verify knockout efficiency across different tissues, as this can vary significantly (e.g., 96.3% in peritoneal macrophages vs. 68% in islets for myeloid-specific knockout) .

How should researchers design longitudinal studies to investigate IL-1β's role in age-related metabolic dysfunction?

Based on current research findings, effective longitudinal study designs should incorporate:

Age cohorts and timeline

• Include multiple age points (e.g., 12, 24, 52, and 67 weeks) to capture progressive changes

• Consider both young (16-24 weeks) and aged (52+ weeks) cohorts for comparative analyses

• Design sampling protocols that minimize interference with aging processes

Comprehensive metabolic phenotyping

• Monitor glucose tolerance via intraperitoneal glucose tolerance tests (ipGTT)

• Assess insulin secretion capacity and insulin resistance throughout aging

• Track body weight changes (mean body weight increases from 29.7g at 16 weeks to 41.5g at 52 weeks)

Tissue-specific analyses

• Isolate islets for ex vivo functional studies

• Perform FACS purification of specific cell populations (e.g., beta cells, immune cells)

• Analyze gene expression changes in different cell fractions over time

Genetic models

• Compare whole-body IL-1β knockout with wild-type littermates

• Utilize tissue-specific knockout models (e.g., myeloid-specific using Lyz2 Cre)

• Consider inducible systems to distinguish developmental from adult-onset effects

Molecular and histological endpoints

• Quantify beta cell mass and proliferation (Ki67 staining)

• Assess islet size distribution and number

• Measure expression of key genes (Il1b, Il1a, Il1rn, Mki67, E2f1, Kir6.2)

This comprehensive approach would allow researchers to delineate the specific contributions of IL-1β to age-related metabolic dysfunction and identify potential therapeutic intervention points.