Recombinant Human Interleukin-1 receptor antagonist protein (IL1RN) (Active)

What is Recombinant Human Interleukin-1 Receptor Antagonist Protein (IL1RN) and what are its primary physiological functions?

Recombinant Human Interleukin-1 Receptor Antagonist (rhIL-1ra) is a protein that competitively inhibits the binding of interleukin-1 (IL-1) to its cell surface receptors. Physiologically, IL-1Ra serves as a natural regulator of IL-1-mediated inflammatory responses by preventing IL-1 (both IL-1α and IL-1β) from interacting with IL-1 receptors. This inhibition blocks downstream signaling cascades that would otherwise lead to inflammation, making IL-1Ra an important endogenous anti-inflammatory mediator. The protein is encoded by the IL1RN gene and can be produced recombinantly for research and therapeutic purposes . In clinical settings, rhIL-1ra has been evaluated for treating conditions characterized by excessive IL-1 activity, including sepsis syndrome where it showed differential efficacy depending on organ dysfunction status and mortality risk .

How can researchers assess IL1RN activity in experimental settings?

Researchers can employ several methodological approaches to assess IL1RN activity:

• Functional inhibition assays: IL-1Ra activity can be measured by its ability to inhibit IL-1β-induced phosphorylated JNK (phospho-JNK) nuclear translocation in cell lines such as KB epithelial cells. In this method, varying concentrations of IL-1Ra are added to cells prior to stimulation with IL-1β. After fixation, permeabilization, and immunostaining, the inhibitory effect can be quantified by measuring nuclear versus cytoplasmic fluorescence intensity using imaging systems like Cellomics ArrayScan VTI .

• Western blotting: Protein expression can be analyzed using specific antibodies directed against the IL-1Ra N-terminus. This approach enables quantification of IL-1Ra protein levels in cellular or tissue samples, with recombinant IL-1Ra serving as a positive control .

• Quantitative real-time RT-PCR: mRNA expression levels of IL1RN can be assessed using TaqMan probes specific for IL1RN, normalized to endogenous controls such as 18S rRNA. This technique allows researchers to evaluate gene expression regulation under different experimental conditions .

• Competitive binding assays: Researchers can measure the competitive binding of labeled IL-1 and IL-1Ra to IL-1 receptors on target cells to determine binding kinetics and inhibitory potency.

What genetic variations in IL1RN have been associated with disease pathophysiology?

Several genetic variations in IL1RN have been identified and associated with disease susceptibility:

• Variable Number Tandem Repeat (VNTR): This polymorphism in the IL1RN gene consists of 86-base pair repeats that vary in number (2-6 repeats). The VNTR*2 allele (with only two repeats) has been significantly associated with idiopathic pulmonary fibrosis (IPF) susceptibility .

• Single Nucleotide Polymorphisms (SNPs): Several SNPs including rs408392, rs419598, and rs2637988 have been investigated in relation to disease. These SNPs are in strong linkage disequilibrium with the VNTR polymorphism (D′ > 0.90) .

• Haploblock associations: A haploblock comprising VNTR*2 and the minor alleles of rs408392 and rs419598 has been associated with IPF susceptibility, with an odds ratio of 1.42 (p = 0.002) in allelic models and 1.60 (p = 0.002) in carriership models .

A meta-analysis of five case-control studies revealed the following data on IL1RN polymorphisms in IPF patients:

The data demonstrates a consistent association between IL1RN polymorphisms and IPF across different populations .

What factors affect the stability and aggregation of rhIL-1ra in high-concentration formulations?

The stability and aggregation of rhIL-1ra in high-concentration formulations are influenced by several physicochemical factors that researchers should consider when designing experiments:

• Ionic strength: At high protein concentrations (50-100 mg/mL) and 37°C, low solution ionic strength significantly accelerates aggregation of rhIL-1ra. Specifically, nucleation rates increase at low ionic strength, while growth rates remain relatively unaffected .

• Protein concentration: Aggregation behavior differs dramatically between low (<1 mg/mL) and high (≥50 mg/mL) protein concentrations. At high concentrations, the monomer-dimer equilibrium shifts significantly to favor dimerization, especially at low ionic strength. This shift is accompanied by subtle conformational changes detectable by circular dichroism and second-derivative FTIR spectra .

• Temperature: At 37°C, rhIL-1ra undergoes conformational changes that promote the formation of both reversible and irreversible dimers through second-order kinetics. The irreversible dimer, while stable, does not significantly participate in further aggregation processes .

• Aggregation mechanism: The loss of native protein due to aggregation follows third-order kinetics with respect to protein thermodynamic activity. This results from the rate-limiting formation of an aggregation-prone trimer, which forms through interactions between monomers and reversible dimers .

• Activity coefficients: The activity coefficient of rhIL-1ra estimated from aggregation rates is approximately 50% higher at 100 mg/mL protein concentration than at 50 mg/mL, aligning with predictions from hard-sphere models for activity coefficients .

These findings have important implications for formulation design in both research and therapeutic applications of rhIL-1ra.

How should researchers design clinical trials involving rhIL-1ra based on past studies?

Design of clinical trials with rhIL-1ra should incorporate lessons from previous investigations to optimize study parameters and patient selection:

• Patient stratification: Previous clinical trials have demonstrated that rhIL-1ra efficacy may vary across patient subgroups. For example, in sepsis syndrome treatment, retrospective analyses showed increased survival time with rhIL-1ra treatment specifically among patients with dysfunction of one or more organs (p = 0.009) and those with a predicted risk of mortality of 24% or greater (p = 0.005) . Stratification based on disease severity and specific organ dysfunction patterns is therefore critical.

• Dosing considerations: Previous studies employed an intravenous loading dose (e.g., 100 mg) followed by continuous infusion (1.0-2.0 mg/kg per hour) . Researchers should design dose-finding studies to establish optimal therapeutic windows for specific conditions.

• Study design elements:

  • Use randomized, double-blind, placebo-controlled designs

  • Conduct multicenter trials to ensure adequate power and generalizability

  • Define primary endpoints precisely (e.g., 28-day all-cause mortality for sepsis trials)

  • Pre-specify subgroup analyses to identify responsive patient populations

  • Consider genetic testing for IL1RN polymorphisms that might influence treatment response

• Statistical considerations: Include appropriate interim analyses and adaptive design elements to allow for early identification of treatment effects or futility. Primary analyses should be specified a priori, with clear distinction between primary and secondary analyses .

What techniques can be used to study IL1RN mRNA expression in relation to polymorphisms?

Researchers investigating the relationship between IL1RN polymorphisms and mRNA expression should consider these methodological approaches:

• Quantitative real-time RT-PCR: This technique allows precise quantification of IL1RN mRNA levels. RNA should be prepared from appropriate tissue sources (e.g., peripheral blood collected in PAXgene tubes) and converted to complementary DNA. TaqMan probes specific for IL1RN (e.g., Hs00277299_m1) can be used with appropriate endogenous controls for normalization .

• Genotyping methodologies:

  • PCR amplification followed by gel electrophoresis for VNTR polymorphisms, using appropriate size ladders to discern the number of repeats

  • Sanger sequencing for SNPs such as rs408392, rs419598, and rs2637988

  • High-throughput genotyping platforms for larger population studies

• Linkage disequilibrium analysis: Software like Haploview can be used to determine linkage disequilibrium between polymorphisms within the IL1RN gene. This is particularly important given the tight linkage observed between various polymorphisms (D′ > 0.99 for VNTR, rs408392, and rs419598) .

• Haplotype construction and analysis: For complex genetic analyses, constructing haploblocks of linked polymorphisms (e.g., VNTR*2 with minor alleles of rs408392 and rs419598) allows for more comprehensive genotype-phenotype analyses .

• Functional validation: After establishing genotype-expression correlations, functional studies can elucidate the impact of differential expression on protein function and cellular responses. This may include protein quantification, cellular activation assays, and cytokine production measurements .

Research has demonstrated that specific IL1RN polymorphisms influence mRNA expression levels, with rs2637988 significantly affecting expression (p < 0.001). Carriers of the minor GG genotype exhibit lower IL1RN mRNA levels, potentially predisposing to inflammatory conditions like IPF .

What methodological considerations are important when analyzing IL1RN in protein aggregation studies?

Researchers investigating IL1RN protein aggregation should consider these methodological aspects:

• Population balance modeling: Apply continuous mixed suspension, mixed product removal (MSMPR) reactor systems at steady-state to determine aggregate nucleation and growth rates. This approach enables quantitative assessment of aggregation kinetics under varying solution conditions .

• Spectroscopic techniques: Employ multiple complementary methods including:

  • Circular dichroism to detect subtle conformational changes

  • Second-derivative FTIR spectroscopy to monitor protein secondary structure

  • Size-exclusion chromatography to separate and quantify protein species (monomers, dimers, oligomers)

• Thermodynamic analysis: Determine free energies of unfolding (ΔG_unf) across different solution conditions to assess protein stability. These measurements should be conducted at physiologically relevant temperatures (e.g., 37°C) .

• Oligomerization analysis: Monitor the formation of different protein species (monomers, dimers, trimers) over time to establish reaction orders and rate constants. The monomer-dimer equilibrium should be carefully characterized, as it significantly influences aggregation propensity at higher protein concentrations .

• Second osmotic virial coefficient measurements: Quantify protein-protein interactions using techniques such as light scattering to determine second osmotic cross virial coefficients, which provide insight into attractive and repulsive forces between protein molecules .

• Activity coefficient determination: Compare experimental activity coefficients with theoretical predictions from hard-sphere models to validate aggregation mechanisms and thermodynamic models .

These methodological considerations are essential for developing stable formulations of rhIL-1ra and understanding the fundamental principles that govern protein aggregation in high-concentration therapeutic protein formulations.