IL 1RA Human His

What is IL-1RA and what is its fundamental role in human immunology?

Interleukin-1 receptor antagonist (IL-1Ra) is an acute phase protein belonging to the Interleukin-1 family of cytokines . It functions as a natural IL-1 inhibitor with anti-inflammatory properties by competitively binding to the Interleukin-1 receptor type 1 without triggering intracellular signaling . IL-1Ra specifically blocks Interleukin-1 alpha and beta (IL-1α and IL-1β) from binding to their receptors, thereby reducing inflammation and fever responses . This mechanism is critical in modulating both adaptive and innate immunity, as the IL-1 family plays a central role in promoting inflammatory responses . In research contexts, understanding IL-1Ra's inhibitory function is essential for designing experiments targeting inflammatory pathways. When using His-tagged IL-1RA in experimental systems, researchers should consider that the tag, while useful for purification, might affect binding kinetics in some experimental conditions, though structural studies generally show minimal interference with receptor binding.

What are the different isoforms of IL-1RA and how are they expressed in human tissues?

IL-1Ra exists in multiple isoforms derived from the same gene: one secreted form (sIL-1Ra) and three intracellular forms (icIL-1Ra1, icIL-1Ra2, and icIL-1Ra3) . Human articular chondrocytes predominantly produce the secreted form, especially when stimulated with IL-1β . The expression of different isoforms is tissue-specific and stimulus-dependent. IL-1Ra is secreted by various cell types including immune cells, epithelial cells, and adipocytes . In experimental contexts, researchers should be aware that human synovial fibroblasts show enhanced synthesis of IL-1Ra when exposed to IL-1, TNF-alpha, or PDGF . Additionally, IL-4 and IL-13 amplify the stimulatory effect of IL-1β on the production of both soluble and intracellular forms of IL-1Ra . When designing experiments with His-tagged IL-1RA, it's important to determine which isoform the construct represents, as this affects cellular localization and functional studies.

How can IL-1RA be quantitatively measured in experimental samples?

Quantitative measurement of IL-1RA in research samples can be accomplished through several validated methodologies:

• ELISA-based detection systems: The Q-Plex technology offers a sensitive sandwich assay format with an assay range of 1,100–1.51 pg/mL and a lower limit of detection (LLD) of 0.05 pg/mL . This method requires minimal sample volumes (25μL) and can measure IL-1Ra in approximately 2.25 hours .

• Chemiluminescent assays: These provide high sensitivity for IL-1Ra detection in human serum and EDTA plasma samples .

• Multiplex platforms: For researchers investigating multiple cytokines simultaneously, customizable assays can measure up to 18 biomarkers including IL-1Ra in a single experiment .

For His-tagged IL-1RA specifically, researchers can employ:

• Western blotting with anti-His antibodies

• Affinity-based detection using nickel or cobalt resins

• Combined approaches that leverage both the His-tag for purification and IL-1RA-specific antibodies for detection

Sample preparation is critical, with proper consideration of potential matrix effects in complex biological samples. When measuring IL-1Ra in conditioned media from cell cultures, it's important to note that baseline levels may be undetectable in unstimulated cells but significantly increased following cytokine stimulation such as IL-1β .

How do recombinant IL-1RA and genetically engineered cell-produced IL-1RA compare in inhibitory efficacy?

A direct comparative analysis between recombinant IL-1Ra (rIL-1Ra) and transgenic IL-1Ra (tIL-1Ra) produced by genetically modified cells revealed important insights for researchers designing IL-1 inhibition experiments:

Under static culture conditions, both sources demonstrated similar inhibitory potency when evaluated by their ability to suppress IL-1β-induced prostaglandin E₂ (PGE₂) production in human synovial fibroblasts . Specifically, 50% IL-1β inhibition required approximately 230 ng/mL IL-1Ra from either source, while complete inhibition was achieved at approximately 800 ng/mL .

• Pre-incubation effect: When human synovial fibroblasts were pre-treated with IL-1Ra 24 hours before IL-1β stimulation, the inhibitory potency increased markedly. Under these conditions, 50% inhibition required only 50 ng/mL for rIL-1Ra and between 10-50 ng/mL for tIL-1Ra, while complete inhibition required approximately 950 ng/mL for rIL-1Ra versus 400-700 ng/mL for tIL-1Ra .

• Long-term dynamics: Under extended static culture conditions (96 hours), tIL-1Ra from genetically modified cells maintained protective effects better than rIL-1Ra, which showed diminished inhibitory capacity by day 4 .

These findings highlight critical methodological considerations for researchers using His-tagged IL-1RA in experimental systems. The mode of delivery (recombinant protein vs. gene therapy approach) should be selected based on the temporal requirements of the experimental system. For acute inhibition studies, recombinant protein may be sufficient, while for sustained inhibition models, expression-based approaches may offer advantages.

What cytokine interactions regulate IL-1RA expression and how can this be leveraged in experimental systems?

IL-1RA expression is subject to complex cytokine-mediated regulation that researchers can manipulate experimentally:

• IL-1β stimulation: Human articular chondrocytes produce undetectable levels of IL-1Ra at baseline, but significantly upregulate IL-1Ra production following IL-1β stimulation . This primarily involves the secreted isoform (sIL-1Ra) rather than intracellular forms .

• Synergistic effects: IL-6 alone does not induce IL-1Ra production in chondrocytes but significantly enhances the stimulatory effect of IL-1β when applied in combination . This synergism represents an important experimental consideration when designing studies of inflammatory cascades.

• Anti-inflammatory mediators: Dexamethasone inhibits IL-1β-induced IL-1Ra production, providing a useful experimental tool for studying glucocorticoid effects on this pathway .

• Th2 cytokine amplification: IL-4 and IL-13 enhance IL-1β-stimulated production of both soluble and intracellular forms of IL-1Ra .

• Growth factor influence: In synovial fibroblasts, PDGF markedly enhances IL-1Ra synthesis, offering another experimental variable for manipulating expression levels .

When working with His-tagged IL-1RA constructs, researchers should consider how these regulatory interactions might affect experimental readouts, particularly in co-culture systems or when studying complex inflammatory networks. Strategic application of these cytokine combinations can allow fine-tuning of IL-1RA expression in experimental systems.

What methodological considerations are critical when using IL-1RA in gene therapy approaches?

Gene therapy approaches utilizing IL-1RA present unique methodological considerations for researchers:

How does IL-1RA function in disease models and what experimental parameters affect therapeutic efficacy?

IL-1RA has demonstrated significant therapeutic potential in various disease models, with several experimental parameters affecting its efficacy:

• Rheumatoid arthritis applications: A modified version of IL-1Ra is used clinically to treat rheumatoid arthritis . In experimental models, IL-1Ra gene therapy has shown promising results across numerous animal models of rheumatoid arthritis and osteoarthritis . A phase I human study of IL-1Ra gene therapy in rheumatoid arthritis has been successfully completed .

• Neuropsychiatric connections: IL-1Ra dysregulation has been associated with schizophrenia, with elevated levels commonly found in schizophrenia patients . This provides an experimental rationale for investigating IL-1Ra in neuropsychiatric disease models.

• Dose-response relationships: Complete inhibition of IL-1β effects requires approximately 800 ng/mL IL-1Ra under simultaneous administration conditions, while pretreatment with IL-1Ra reduces this threshold . This dose-response relationship is critical for designing preclinical efficacy studies.

• Dynamic delivery advantage: Under conditions that progressively dilute culture media (more closely resembling in vivo conditions), continuous production of IL-1Ra by genetically modified cells shows striking advantages over bolus administration of recombinant protein .

• Temporal considerations: The timing of IL-1Ra administration relative to inflammatory stimulus significantly impacts efficacy, with pre-treatment showing enhanced inhibitory effects compared to simultaneous or post-treatment administration .

When using His-tagged IL-1RA in disease models, researchers should consider how the tag might affect biodistribution, half-life, and immunogenicity. Control experiments comparing tagged and untagged versions are advisable when moving from in vitro to in vivo systems to ensure comparable therapeutic efficacy.

What are the molecular mechanisms of IL-1RA competitive inhibition and how can these be quantitatively assessed?

IL-1RA functions through competitive inhibition of IL-1 receptor binding, with several quantitative approaches available to assess this activity:

• Competitive binding mechanism: IL-1Ra binds to IL-1 receptors with high affinity but does not elicit intracellular responses upon binding . This competitive antagonism prevents IL-1α and IL-1β from activating their signaling cascades.

• PGE₂ production assay: A well-established quantitative method for assessing IL-1Ra inhibitory activity involves measuring PGE₂ production by human synovial fibroblasts stimulated with IL-1β . This provides a functional readout of IL-1 signaling and allows construction of inhibition curves.

• Dose-response assessment: Quantitative comparison of inhibitory potency can be performed by plotting IL-1Ra concentration versus percent inhibition of IL-1β-induced responses . The table below summarizes key inhibitory concentrations determined in human synovial fibroblast models:

• Time-course analysis: Extended time-course experiments (up to 96 hours) reveal important differences in the sustained inhibitory activity of different IL-1Ra delivery methods .

• Receptor occupancy studies: Direct assessment of receptor binding competition can be performed using radiolabeled ligands or fluorescently tagged receptor constructs.

For His-tagged IL-1RA specifically, binding kinetics studies should be conducted to ensure the tag does not alter receptor interactions. Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) techniques can provide quantitative binding parameters (kon, koff, KD) for comparison with untagged protein.

What are the optimal experimental conditions for studying IL-1RA in primary human cell cultures?

Optimizing experimental conditions for IL-1RA studies in primary human cells requires attention to several methodological details:

• Cell culture systems: Human synovial fibroblasts and articular chondrocytes serve as relevant primary cell models for studying IL-1Ra responses, particularly in the context of inflammatory joint diseases .

• Media composition: For human synovial fibroblast cultures, DMEM supplemented with 10% FBS and antibiotics provides appropriate conditions . For human articular chondrocytes, specific culture conditions may need optimization based on the research question.

• Cell density considerations: Experimental protocols typically use 5 × 10^5 human synovial fibroblasts per well or 40,000 chondrocytes per well in 96-well plates .

• Cytokine stimulation protocols:

  • IL-1β: Typically applied at 5 ng concentration to stimulate IL-1-dependent responses

  • IL-6: While ineffective alone, can enhance IL-1β effects when used in combination

  • Dexamethasone: Can be employed as an inhibitory control for IL-1Ra induction studies

• Temporal considerations: IL-1Ra detection is typically performed 48 hours after cytokine stimulation, but the exact timing should be optimized for specific experimental questions .

• Detection methodologies: ELISA remains the gold standard for quantifying secreted IL-1Ra in culture supernatants, with cell lysates analyzed for intracellular isoforms .

When incorporating His-tagged IL-1RA into these experimental systems, researchers should consider running parallel experiments with untagged protein to confirm comparable biological activity. Additionally, the His-tag provides opportunities for specific pulldown experiments to identify novel binding partners in primary cell lysates.

How can IL-1RA be effectively incorporated into complex in vitro disease models?

Incorporating IL-1RA into complex in vitro disease models requires strategic experimental design:

• Co-culture systems: Studies comparing recombinant IL-1Ra with IL-1Ra produced by genetically modified cells have employed co-culture approaches, placing IL-1Ra-producing cells (e.g., HIG-82-IL-1Ra+) together with target cells (e.g., human synovial fibroblasts) . This mimics paracrine delivery in tissue microenvironments.

• Dynamic culture conditions: To better represent in vivo situations where cytokines are chronically produced and diluted over time, experiments can be designed with progressive dilution of culture media . This approach reveals important differences between bolus administration and continuous production of IL-1Ra.

• Sequential cytokine challenges: Experimental designs that include pretreatment, simultaneous treatment, or post-treatment with IL-1Ra relative to inflammatory stimuli provide insights into therapeutic timing considerations .

• Three-dimensional culture systems: Advanced disease models may incorporate IL-1Ra into 3D culture systems like spheroids, organoids, or bioprinted tissues to better recapitulate tissue architecture and cellular interactions.

• Inflammatory network models: Given that IL-1Ra expression is regulated by multiple cytokines (IL-1β, IL-6, IL-4, IL-13, TNF-α, PDGF), complex models incorporating these cytokine networks can provide insights into IL-1Ra regulation under physiologically relevant conditions .

His-tagged IL-1RA offers particular advantages in complex models, as the tag enables tracking of protein distribution within multi-cellular systems using immunohistochemistry or live imaging with fluorescently labeled anti-His antibodies. This can help researchers understand spatial aspects of IL-1RA activity in disease microenvironments.

What are the critical quality control parameters for validating IL-1RA functionality in research applications?

Researchers working with IL-1RA should implement several quality control measures to ensure functional integrity:

• Protein concentration determination: Accurate quantification of IL-1Ra concentration is essential for dose-response studies. ELISA techniques with a validated lower limit of detection (0.05 pg/mL for some commercial assays) provide reliable measurements .

• Bioactivity assessment: Functional validation through PGE₂ inhibition assays using IL-1β-stimulated human synovial fibroblasts confirms that the IL-1Ra preparation maintains its inhibitory capacity .

• Stability testing: IL-1Ra stability under experimental conditions should be verified, particularly for time-course studies extending beyond 48 hours .

• Isoform characterization: RT-PCR analysis can confirm which IL-1Ra isoforms (secreted vs. intracellular) are being produced in response to experimental stimuli .

• Batch-to-batch consistency: For recombinant protein preparations, consistency between batches should be verified through both concentration and functional assays.

• Receptor binding validation: Competitive binding assays can confirm that IL-1Ra preparations maintain their ability to bind IL-1 receptors with appropriate affinity.

For His-tagged IL-1RA specifically, additional quality control parameters include:

• Tag integrity verification using anti-His Western blots

• Purification homogeneity assessment through SDS-PAGE

• Potential tag interference evaluation by comparing activity with untagged protein

• Proper folding confirmation through circular dichroism or thermal stability assays

These quality control measures ensure experimental reproducibility and valid interpretation of results in IL-1Ra research applications.