Recombinant Rhesus Macaque Interleukin-1 beta protein (IL1B) (Active)

What are the optimal storage and reconstitution conditions for maintaining IL-1β activity?

For optimal stability, lyophilized Rhesus Macaque IL-1β should be stored at -20°C or -80°C . After reconstitution in PBS (pH 7.4), it is recommended to aliquot and store at -20°C or -80°C for up to one month to prevent activity loss from repeated freeze-thaw cycles . Storage in frost-free freezers is not recommended due to temperature fluctuations that may affect protein stability .

How does the biological activity of Rhesus Macaque IL-1β compare to human IL-1β?

Mature rhesus macaque IL-1β shares 96% amino acid sequence identity with human IL-1β, making it highly similar in structure and function . The activity is typically determined by its ability to induce cell proliferation in specific cell lines, with the ED50 ranging from 3-12 pg/mL in standardized bioassays . Due to its high homology with human IL-1β, it often demonstrates cross-reactivity with human IL-1 receptors, making it valuable for comparative studies .

What are the most reliable methods for measuring IL-1β activity in experimental settings?

Rhesus Macaque IL-1β activity can be reliably measured through:

• Cell proliferation assays: Using D10.G4.1 or similar responsive cell lines, with typical ED50 values <8 pg/ml

• Inflammatory response assays: Measuring secondary mediator production (NO, PGE2) from target cells

• Receptor binding assays: Quantifying interaction with IL-1R using competitive binding techniques

• Signaling pathway activation: Assessing NF-κB translocation or phosphorylation of downstream kinases

• Ex vivo tissue response: Measuring cytokine production in tissue explants or primary cell cultures

For consistent results, researchers should include appropriate positive controls and establish dose-response curves within their experimental system .

How can IL-1β be effectively used in rhesus macaque disease models?

For effective use in disease models:

• Dosing considerations: Start with physiologically relevant concentrations (picogram to nanogram range) based on the disease context

• Administration route: For CNS studies, consider intrathecal or stereotactic injections to bypass blood-brain barrier limitations

• Timing protocols: For inflammatory studies, implement time-course experiments to capture both acute and chronic phases

• Combination approaches: Examine IL-1β in conjunction with other cytokines (TNF-α, IL-6) to model complex inflammatory cascades

• Measurement endpoints: Include both molecular/cellular readouts and behavioral/physiological outcomes

When studying neuroinflammatory conditions like EAE, the timing of IL-1β administration relative to disease induction is critical for capturing relevant pathophysiological processes .

What is the specific role of IL-1β in neuroinflammatory processes in rhesus macaque models?

In rhesus macaque models of neuroinflammation, IL-1β demonstrates prominent staining in MHC class II+ cells within perivascular infiltrates and at the edges of large demyelinating lesions . Interestingly, IL-1β expression is primarily detected in resident microglia or differentiated macrophages rather than infiltrating monocytes, suggesting that IL-1β expression is induced within the central nervous system (CNS) itself rather than being imported by peripheral cells .

This pattern differs somewhat from human multiple sclerosis (MS) lesions, where IL-1β staining is less pronounced and more focal, suggesting species-specific differences in neuroinflammatory responses that researchers should consider when translating findings .

How does IL-1β contribute to experimental autoimmune encephalomyelitis (EAE) pathogenesis?

IL-1β plays several critical roles in EAE pathogenesis:

• Disease initiation: Inhibition of IL-1-induced signaling ameliorates EAE development in both rats and mice

• Cellular activation: Promotes activation of microglia and astrocytes within the CNS

• Blood-brain barrier (BBB) disruption: Contributes to increased BBB permeability

• T-cell responses: Enhances Th17 differentiation, critical for EAE pathogenesis

• Inflammatory cascade: Triggers secondary inflammatory mediators like prostaglandins

Studies have confirmed that mice deficient in NLRP3, ASC, or caspase-1 (components of the inflammasome that processes IL-1β) show delayed disease onset and less severe clinical symptoms . Expression levels of IL-1β increase in brain and spinal cord during disease progression, and treatments that reduce IL-1β activation (like caspase-1 inhibitors) attenuate clinical signs of EAE .

How can researchers distinguish between the activities of pro-IL-1β and mature IL-1β in experimental systems?

Distinguishing between pro-IL-1β and mature IL-1β requires specialized techniques:

• Western blotting with specific antibodies: Pro-IL-1β appears at ~31-35 kDa while mature IL-1β appears at ~17-19 kDa

• Caspase-1 inhibition studies: Using specific inhibitors like Ac-YVAD-CMK to block processing

• Inflammasome activation assays: Monitoring ASC speck formation and caspase-1 activation

• Mass spectrometry: Identifying specific cleavage products

• Activity assays: Only mature IL-1β activates the IL-1 receptor complex

When designing experiments, researchers should consider that rhesus IL-1β precursor contains a 116 amino acid propeptide that is cleaved intracellularly by caspase-1/ICE to generate the active cytokine . This processing step is critical for biological activity and can be a target for experimental manipulation.

What methodological approaches are most effective for studying IL-1β in MHC class II+ microglia nodules in neuroinflammatory conditions?

For studying IL-1β in MHC class II+ microglia nodules:

• Immunohistochemical double-staining: Combine IL-1β staining with MHC class II markers, using DAB and alkaline phosphatase for different visualization

• Laser capture microdissection: Isolate specific nodules for molecular analysis

• Single-cell RNA sequencing: Profile gene expression in nodule vs. non-nodule microglia

• Spatial transcriptomics: Map IL-1β expression patterns relative to lesion boundaries

• In vivo imaging: Track nodule formation and IL-1β expression over time in animal models

Research has shown that in MS brain tissue, IL-1β is found in parenchyma of active lesions and in nodules of MHC class II+ microglia in otherwise normal appearing white matter, but expression is detected in only a minority of nodules . These IL-1β+ nodules cannot be distinguished by other pro- or anti-inflammatory markers, suggesting complex regulatory mechanisms worthy of further investigation .

What key differences should researchers consider between rhesus macaque IL-1β and mouse IL-1β in experimental design?

When comparing rhesus macaque IL-1β with mouse IL-1β:

The mature rhesus IL-1β shares 67-78% amino acid sequence identity with mouse IL-1β, which can result in different binding affinities and biological responses that should be considered when translating findings between species .

How do the expression patterns of IL-1β differ between rhesus macaque EAE models and human multiple sclerosis?

Significant differences exist in IL-1β expression between rhesus EAE and human MS:

• Expression intensity: IL-1β staining is prominent in rhesus EAE but much less pronounced in MS brain tissue

• Cellular distribution: In rhesus EAE, IL-1β is found mainly in MHC class II+ cells within perivascular infiltrates and at lesion edges, while in MS it appears in active lesion parenchyma and microglial nodules

• Temporal dynamics: Rhesus models show more acute and synchronized IL-1β expression compared to the heterogeneous patterns in human MS lesions

• Relationship to demyelination: More direct spatial correlation between IL-1β expression and demyelinating activity in rhesus models than in human MS

• Response to therapy: IFNβ, a registered therapeutic for MS, decreases IL-1β levels and caspase-1 activation, effects that can be more consistently monitored in rhesus models

These differences highlight the importance of careful interpretation when translating findings between species and suggest that while rhesus macaque models provide valuable insights, they do not fully recapitulate the complexity of human MS pathology .

What are the most common pitfalls when working with recombinant IL-1β in cell culture systems?

Common pitfalls and solutions when working with recombinant IL-1β include:

• Loss of activity due to improper handling: Minimize freeze-thaw cycles and maintain cold chain during experiments

• Endotoxin contamination: Verify endotoxin levels (<1.0 EU/μg) when interpreting inflammatory responses

• Receptor desensitization: Use pulsed rather than continuous exposure for certain experiments

• Species cross-reactivity issues: Validate activity in your specific cell system before full experiments

• Inadequate controls: Include both positive controls (known IL-1β responsive cells) and negative controls (IL-1Ra or neutralizing antibodies)

For optimal results, researchers should reconstitute lyophilized protein according to manufacturer recommendations and conduct preliminary dose-response studies to determine the optimal concentration range for their specific experimental system .

How can researchers effectively distinguish IL-1β-specific effects from other inflammatory mediators in complex biological systems?

To isolate IL-1β-specific effects:

• Use specific antagonists: IL-1 receptor antagonist (IL-1Ra) blocks IL-1α and IL-1β but not other cytokines

• Employ neutralizing antibodies: Anti-IL-1β antibodies provide specific blockade

• Implement genetic approaches: siRNA knockdown of IL-1R or CRISPR/Cas9 receptor deletion

• Design competition assays: Use excess inactive IL-1β mutants to compete for receptor binding

• Conduct parallel studies: Compare IL-1β with TNF-α, IL-6, and other inflammatory mediators using the same readouts

In rhesus macaque studies, it's important to note that therapeutic approaches used for MS treatment (IFNβ, Copaxone, or steroid treatment) lead to increased levels of IL-1 receptor antagonist (IL-1RA) in the blood, which can confound interpretation of IL-1β-specific effects .