Recombinant Horse Interleukin-1 beta protein (IL1B) (Active)

What is the molecular weight of recombinant equine IL-1β?

Recombinant equine IL-1β has a molecular weight of approximately 21-22.6 kDa as determined by SDS-PAGE analysis, which aligns with its predicted molecular mass. This is notably different from human IL-1β, which has a molecular mass of approximately 32-34 kDa, highlighting important species-specific structural differences .

How is recombinant equine IL-1β produced for research applications?

Recombinant equine IL-1β is typically produced by amplifying the entire coding region of equine IL-1β mRNA using reverse transcriptase polymerase chain reaction (RT-PCR). This cDNA is then cloned into an expression vector and expressed in bacterial systems (commonly E. coli). The protein is often engineered with a hexahistidine tag to facilitate purification using Ni²⁺ affinity chromatography methods, which yields highly pure protein (>95% as determined by SDS-PAGE) .

How stable is reIL-1β under laboratory conditions?

Recombinant equine IL-1β maintains optimal stability when stored as lyophilized powder at -20°C to -80°C, where it typically remains stable for up to 12 months. After reconstitution in sterile PBS (pH 7.4), aliquoting is recommended to avoid repeated freeze-thaw cycles that can compromise biological activity .

What are the optimal concentrations of reIL-1β for in vitro experiments?

For in vitro experiments with equine chondrocytes, reIL-1β demonstrates significant biological activity in the range of 1-10 ng/mL, with effects on gene expression appearing saturable at higher concentrations. A dose-dependent but saturable increase in gene expression is typically observed at reIL-1β doses in the 1 to 10 ng/mL range, depending on the specific gene being studied .

How does reIL-1β affect matrix metalloproteinase (MMP) expression in equine chondrocytes?

Recombinant equine IL-1β induces a marked up-regulation of MMP gene expression (MMP-1, MMP-3, MMP-13) in equine chondrocytes. Northern blot analyses reveal dose-dependent increases in MMP expression that appear saturable at reIL-1β concentrations between 1-10 ng/mL. This transcriptional activation translates to increased enzymatic activities in conditioned media from treated cultures .

What culture systems are appropriate for studying reIL-1β effects on equine tissues?

Multiple culture systems have been validated for studying reIL-1β effects:

• Monolayer chondrocyte cultures: Optimal for gene expression studies with treatment periods of 3-6 hours for RNA extraction

• Cartilage explant cultures: More physiologically relevant for studying tissue-level matrix degradation over 24-48 hours

• Three-dimensional collagen gel cultures: Particularly useful for tenocyte studies, allowing assessment of contraction responses over extended periods (up to 14 days)

Does reIL-1β affect cell viability or proliferation in equine cell cultures?

Research indicates that exposure of equine cells to reIL-1β at concentrations ranging from 0.1 to 10 ng/mL does not significantly affect cell proliferation or reduce viability. Cell survival and proliferation experiments show that equine cells from multiple lines grow at expected rates even with IL-1β challenge .

What primary signaling pathways are activated by reIL-1β in equine cells?

Recombinant equine IL-1β primarily activates the nuclear factor kappa B (NF-κB) signaling pathway in equine cells. This activation leads to nuclear translocation of NF-κB transcription factors, resulting in the transcriptional upregulation of target genes including MMPs, COX-2, and inflammatory cytokines. The pathway activation appears to be similar in both chondrocytes and tenocytes .

How does reIL-1β influence nitric oxide production in equine cartilage?

Recombinant equine IL-1β stimulates the activity of inducible nitric oxide synthase (iNOS) in equine chondrocytes, resulting in increased nitric oxide production that can be measured as nitrite accumulation in conditioned media. This increase in nitric oxide parallels the augmentation of MMP activities, suggesting coordinated regulation of multiple catabolic pathways in cartilage degradation .

Can reIL-1β effects be inhibited by anti-inflammatory compounds?

Yes, the effects of reIL-1β on gene expression can be inhibited by concurrent administration of dexamethasone (10⁻⁵ M). Research demonstrates that dexamethasone treatment effectively blocks the IL-1β-stimulated expression of MMPs, TIMP-1, and COX-2 genes in equine chondrocytes, providing a useful positive control for inhibition studies .

How do equine and human IL-1β compare in structure and function?

Equine and human IL-1β show limited sequence identity, which impacts cross-species applications. The molecular weight difference (21-22.6 kDa for equine vs. 32-34 kDa for human) reflects structural variations that affect receptor binding. Research suggests that species-specific cytokines are important when modeling inflammatory responses in equine tissues, as receptor-ligand specificity influences signaling efficiency .

How does reIL-1β impact gene expression in equine tenocytes compared to chondrocytes?

While both cell types show upregulation of catabolic enzymes (MMPs) and inflammatory mediators upon IL-1β stimulation, there appear to be cell type-specific responses. Research on equine tenocytes in 3D collagen cultures shows that IL-1β stimulation affects NF-κB signaling and impacts IL-6 secretion, with specific effects on gel contraction that may differ from the responses observed in chondrocytes .

What control measures should be included in experiments using reIL-1β?

Comprehensive controls for reIL-1β experiments should include:

• Vehicle controls containing the same buffer composition as the reIL-1β preparation

• Dexamethasone (10⁻⁵ M) as a positive control for inhibition of IL-1β effects

• Time-course controls to establish optimal response windows for specific outcome measures

• Biological replicates from multiple animals to account for individual variation in responsiveness

Why might researchers observe variable responses to reIL-1β between different equine cell populations?

Variable responses may result from:

• Donor-specific variations in IL-1 receptor expression

• Differences in cellular passage number and dedifferentiation status

• Variations in experimental conditions including serum content

• Age-related differences in cellular responsiveness

• Joint-specific or tissue-specific variations when studying cells from different anatomical locations

How can researchers verify the biological activity of reIL-1β preparations?

Verification of reIL-1β activity should employ multiple approaches:

• Dose-dependent induction of known target genes (MMPs, COX-2) via Northern blot or qPCR analysis

• Functional enzyme activity assays measuring collagenase/gelatinase or stromelysin activities

• Quantification of downstream mediators such as nitric oxide production (measured as nitrite)

• Visual assessment of 3D collagen gel contraction in tenocyte cultures

How might reIL-1β be utilized in developing in vitro models of equine osteoarthritis?

Recombinant equine IL-1β serves as a critical tool for developing in vitro models of equine osteoarthritis through:

• Dynamic loading systems combining mechanical stress with IL-1β stimulation

• Co-culture models incorporating multiple cell types to study cell-cell communication

• Three-dimensional tissue-engineered cartilage constructs for studying matrix degradation

• Comparative studies examining the interplay between mechanical forces and inflammatory cytokines

What emerging technologies might enhance our understanding of reIL-1β effects in equine joint tissues?

Advanced transcriptomic and proteomic approaches offer opportunities to comprehensively characterize the effects of reIL-1β on equine joint tissues. RNA-seq analysis could reveal genome-wide expression changes, identifying novel target genes beyond previously studied candidates. Proteomics could identify changes in the secretome of IL-1β-stimulated tissues, potentially revealing new biomarkers of joint inflammation .