31 Antibody

What is IL-31 and what is its role in immune responses?

IL-31 is a 24 kDa, short-chain member of the alpha-helical family of cytokines. The human IL-31 cDNA encodes a 164 amino acid precursor containing a 23 amino acid signal peptide and a 141 amino acid mature protein. The mature region forms four alpha-helices with a typical up-up-down-down topology. IL-31 is predominantly produced by T helper 2 (Th2) cells and activated mast cells in response to antimicrobial peptides .

IL-31 functions primarily in skin immunity and has been strongly associated with pruritus (itching) in atopic dermatitis. It activates STAT3 and possibly STAT1 and STAT5 through its heterodimeric receptor, and enhances myeloid progenitor cell survival in vitro. Additionally, IL-31 induces RETNLA and serum amyloid A protein expression in macrophages .

How does the IL-31 receptor complex function in signal transduction?

IL-31 signals through a heterodimeric receptor complex composed of:

• IL-31 receptor A (IL-31RA, also known as GPL or GLM-R) - a 120 kDa gp130-related molecule

• Oncostatin M receptor beta (OSMR β) - a 180 kDa protein

Within this complex, IL-31 directly binds to GPL (IL-31RA), not OSMR. The signaling cascade involves:

• Jak/STAT pathway activation

• PI3 kinase/AKT cascade

• MAP kinase pathway

It's important to note that while multiple isoforms of IL-31RA exist, only forms containing the entire length of the cytoplasmic domain are capable of signal transduction .

What techniques are most reliable for detecting IL-31 in experimental samples?

Several validated techniques for IL-31 detection include:

ELISA: Anti-IL-31 antibodies such as MAB28241 can detect human IL-31 in direct ELISAs with high specificity. When selecting antibodies for ELISA, verify that they show no cross-reactivity with other species variants (e.g., mouse IL-31) .

Western Blot: Antibodies like MAB28241 demonstrate effective detection in Western blot applications. For optimal results, ensure proper sample preparation and protein denaturation protocols .

Immunofluorescence/Immunocytochemistry: Some IL-31 antibodies have been validated for immunofluorescence applications, allowing visualization of IL-31 expression in cell lines such as A375 human melanoma cells .

How can I validate the specificity of an IL-31 antibody for my research?

A methodical approach to antibody validation should include:

• Cross-reactivity testing: Compare reactivity against related proteins. For example, human IL-31 antibodies should be tested against recombinant mouse IL-31 to ensure specificity .

• Positive and negative controls: Use cell lines with known IL-31 expression levels as positive controls, and IL-31 knockout or low-expressing lines as negative controls.

• Neutralization assays: Employ functional assays where IL-31 activity is measured (e.g., IL-31-dependent BaF3 cell proliferation). The antibody should inhibit this activity in a dose-dependent manner .

• Multiple detection methods: Validate antibody performance across different techniques (ELISA, Western blot, immunofluorescence) to ensure consistent specificity.

What evidence demonstrates IL-31's role in atopic dermatitis pathogenesis?

Multiple lines of evidence support IL-31's critical role in atopic dermatitis:

• Elevated expression: IL-31 is expressed at higher levels in lesional AD skin compared to non-lesional skin .

• Correlation with disease severity: Serum IL-31 levels increase in AD patients and show significant correlation with disease severity .

• Animal models: IL-31 transgenic mice develop spontaneous pruritus and skin lesions resembling AD. In NC/Nga mice (which spontaneously develop AD), IL-31 levels correlate with scratching behavior .

• Cellular source: IL-31 is predominantly expressed by skin-homing cutaneous lymphocyte antigen-positive T-cells in AD lesions .

• Receptor expression: The IL-31 receptor is constitutively expressed by keratinocytes and upregulated by IFN-gamma on monocytes .

How do anti-IL-31 antibody and anti-IL-31RA antibody mechanisms differ in experimental models?

Research shows that anti-IL-31 receptor α-neutralizing antibodies can reduce ear swelling and dermatitis scores in chronic pruritus-inducing AD-like models, even when administered after disease development, suggesting therapeutic potential beyond prevention .

What animal models are most appropriate for studying IL-31 antibody efficacy?

Several validated animal models have demonstrated utility in IL-31 antibody research:

• BALB/c mice with IL-31 injection: Intravenous injection of IL-31 induces scratching behavior that can be inhibited by pretreatment with anti-IL-31RA neutralizing antibody but not by antihistamines, immunosuppressants, or μ-opioid receptor antagonists .

• Chronic AD-like model: This model induces steady scratching behavior and dermatitis, including hemorrhage, edema, and crust formation. Anti-IL-31RA antibody reduces ear swelling and dermatitis scores in this model .

• Contact sensitivity (CS) reaction model: Used to assess antigen-specific and T-cell-dependent immune responses, though continuous pruritus is not induced in this model .

• Cynomolgus monkey model: Administration of cynomolgus IL-31 induces transient scratching behavior in cynomolgus monkeys. A single subcutaneous injection of nemolizumab (1 mg/kg), a humanized anti-human IL-31RA monoclonal antibody, suppressed IL-31-induced scratching for approximately 2 months .

How do bispecific antibodies targeting IL-31RA compare with monoclonal antibodies in efficacy and mechanism?

Recent research has explored bispecific antibodies targeting both IL-4Rα and IL-31Rα for atopic dermatitis treatment. This novel approach offers several advantages over monoclonal antibodies:

What experimental evidence demonstrates the pruritogenic mechanism of IL-31?

The precise pruritogenic mechanism of IL-31 has been investigated through several experimental approaches:

• Direct vs. indirect effects: Studies investigating the effects of existing drugs on IL-31-induced scratching behavior in mice showed that while anti-IL-31RA antibodies inhibited this behavior, antihistamines (terfenadine), immunosuppressants (dexamethasone and tacrolimus), and μ-opioid receptor antagonists (naloxone) did not significantly inhibit IL-31-induced scratching. This suggests IL-31 induces pruritus through direct mechanisms rather than via histamine release or indirect pathways .

• Neuronal expression: IL-31RA is expressed on dorsal root ganglia neurons, suggesting direct neuronal activation by IL-31 as a mechanism for itch induction .

• Temporal relationship: In experimental models, IV administration of IL-31 induces scratching behavior that can be blocked by pretreatment with anti-IL-31RA antibodies, demonstrating a direct causal relationship .

• Therapeutic effects: Anti-IL-31RA antibody treatment shows dual effects, reducing both scratching behavior and dermatitis scores in AD models, suggesting IL-31 is not only pruritogenic but also contributes to skin inflammation .

What is the current status of clinical development for anti-IL-31 pathway therapeutics?

Several anti-IL-31 pathway therapeutics have progressed to clinical development:

• Nemolizumab (CIM331): A humanized anti-IL-31RA monoclonal antibody that has been evaluated in clinical trials for atopic dermatitis. Data from the global Phase II study (XCIMA study) published in The New England Journal of Medicine demonstrated safety and efficacy at 12 weeks . Nemolizumab has been approved in Japan for the treatment of itch associated with AD in adults and children over 13 years of age .

• BMS-981164: An IL-31 monoclonal antibody that has been investigated in Phase 1 clinical trials for atopic dermatitis .

• Bispecific antibodies: Novel bispecific antibodies targeting both IL-4Rα and IL-31Rα are in preclinical development, showing promising results for potential enhanced efficacy compared to monoclonal antibodies .

The clinical evidence suggests that anti-IL-31 receptor antibodies represent a promising therapeutic approach for breaking the itch-scratch cycle in atopic dermatitis, with the potential for broader applications in other pruritic conditions .

What are the optimal protocols for using IL-31 antibodies in neutralization assays?

A standardized protocol for IL-31 neutralization assays includes:

• Cell preparation: Culture IL-31-responsive cells (e.g., human aortic endothelial cells pre-stimulated with recombinant human IFN-gamma at 10 ng/ml or hIL31Rα/hOSMR BaF3 cells) .

• Antibody dilution: Prepare serial dilutions of anti-IL-31 or anti-IL-31RA antibody at a 1:3 ratio in assay medium (typically RPMI 1640 with 10% FBS) starting at 100 nM concentration .

• IL-31 preparation: Dilute human recombinant IL-31 to working concentration (typically 4 ng/ml for cell proliferation assays or 50 ng/ml for IL-6 production assays) .

• Assay setup:

  • For proliferation assays: Seed 10,000 hIL31Rα/hOSMR BaF3 cells per well in 96-well plates

  • For IL-6 production: Use human aortic endothelial cells pre-stimulated with IFN-gamma

  • Add 25 μL of antibody dilutions and 25 μL of IL-31 solution to each well

• Incubation: Incubate at 37°C with 5% CO₂ for 48 hours (proliferation assay) or overnight (IL-6 production) .

• Readout: Measure cell proliferation using appropriate assays (e.g., MTT) or IL-6 production by ELISA .

• Analysis: Calculate the neutralizing capacity and ND50 (neutralization dose providing 50% inhibition) from the dose-response curve .

How should researchers account for IL-31 isoforms and receptor variants in experimental design?

When designing experiments involving IL-31 or its receptors, researchers should consider:

• IL-31RA isoform variability: Twelve alternatively spliced human IL-31RA isoforms are known, ranging from 356-745 amino acids. The predominant forms are a long (745 aa) and short (560 aa) transmembrane form .

• Functional differences: The long form signals by recruiting STAT3, 5, or 1, while the short form does not recruit STATs and actually inhibits IL-31 signaling. Therefore, consider:

  • Verifying which isoforms are expressed in your experimental system

  • Using isoform-specific primers for PCR analysis

  • Performing Western blots that can distinguish between isoforms by size

• Co-receptor requirements: IL-31 requires both IL-31RA and OSMR for signaling. Verify expression of both receptor components in your cell system .

• Cell type variability: The ratio of long and short IL-31RA forms and their co-expression with OSMR determines a cell's response to IL-31. Document receptor expression profiles in your experimental system .

• Species differences: Human and mouse IL-31 share only 24% amino acid sequence identity in the mature region, and human IL-31 receptors do not respond to mouse IL-31. Use species-matched ligands and receptors in your experiments .