Recombinant Human Interleukin-19 protein (IL19), partial (Active)

What is Interleukin-19 and what protein family does it belong to?

Interleukin-19 (IL-19) is a secreted protein that belongs to the IL-10 family of cytokines, alongside IL-10, IL-20, IL-22, IL-24, IL-26, and several virus-encoded cytokines. While these cytokines share partial homology in their amino acid sequences, they exhibit dissimilar biological functions. Human and murine IL-19 share approximately 71% amino acid sequence identity, indicating evolutionary conservation with species-specific variations .

What are the molecular characteristics of recombinant human IL-19?

Recombinant human IL-19 protein typically has a predicted molecular weight of 18 kDa. When produced in E. coli expression systems, the purified protein demonstrates >97% purity by SDS-PAGE with silver stain analysis. The protein should contain minimal endotoxin contamination (<0.1 EU/μg as determined by LAL method) to ensure experimental validity. The amino acid sequence begins with MLRRCLI followed by the full sequence, which is critical for proper folding and biological activity .

How is the biological activity of recombinant human IL-19 determined?

The biological activity of recombinant human IL-19 is primarily determined by its ability to induce proliferation of mouse BaF/3 cells co-transfected with human IL-20 Rα and IL-20 Rβ receptor subunits. The expected ED₅₀ for this proliferative effect typically ranges from 0.5-1.5 ng/mL. This standardized bioassay provides a functional measurement of protein activity that correlates with its potential efficacy in experimental applications .

How should I design experiments to investigate IL-19 expression in response to cellular stress?

When designing experiments to study IL-19 expression in response to cellular stress, consider the following methodology:

• Cell Line Selection: Use multiple cell lines (at least three distinct human cell lines) that express the IL-20R subunits necessary for IL-19 signaling

• Stress Induction Protocol: Apply various stress inducers such as ionizing radiation (IR), etoposide (TOPO II inhibitor), taxol (microtubule stabilizer), or carboplatin (DNA cross-linking agent)

• Time Course Analysis: Measure IL-19 mRNA abundance at multiple timepoints, starting at 24 hours post-treatment and continuing for at least 5 days, as IL-19 expression peaks 2-3 days after treatment

• Quantification Method: Use qRT-PCR to measure IL-19 mRNA levels relative to housekeeping genes

• Controls: Include appropriate vehicle controls and positive controls (known inducers of cellular stress responses)

This design enables comprehensive characterization of the IL-19 expression kinetics and magnitude in response to various cellular stressors.

What controls should be included when studying the effects of recombinant IL-19 on experimental models?

To ensure experimental rigor when studying recombinant IL-19 effects, implement the following control strategy:

The experimental design should include an untreated control group alongside the control conditions listed above to accurately assess IL-19-mediated effects .

How does IL-19 modulate macrophage function in inflammatory conditions?

IL-19 significantly modulates macrophage function through multiple mechanisms:

• Recruitment Inhibition: IL-19 treatment inhibits macrophage recruitment following inflammatory stimuli, as demonstrated in murine models using thioglycolate injection

• Phenotype Modulation: In classically activated (M1) or alternatively activated (M2) macrophages, IL-19 downregulates mRNA expression of:

  • Inducible nitric oxide synthase (iNOS)

  • Chemokine C-C motif ligand 2 (CCL2)

  • Matrix metalloproteinases 2 and 9 (MMP-2, MMP-9)

• Inflammatory Mediator Regulation: IL-19 treatment alters the production of key inflammatory mediators without apparent effect on cytokine-expressing helper or cytotoxic T-cell differentiation

• Tissue-Protective Effects: In pathological contexts like abdominal aortic aneurysms (AAAs), IL-19 attenuates medial elastin degradation, smooth-muscle depletion, leukocyte infiltration, and neoangiogenesis

These effects collectively position IL-19 as an immunomodulatory cytokine with potential therapeutic applications in inflammatory disorders.

What techniques can be used to investigate the IL-19 signaling pathway in immune cells?

To comprehensively investigate IL-19 signaling pathways, researchers should employ the following methodological approaches:

• Receptor Expression Analysis:

  • Flow cytometry to quantify IL-20Rα and IL-20Rβ surface expression

  • qRT-PCR to measure receptor mRNA levels

  • Immunofluorescence to visualize receptor distribution

• Signaling Pathway Activation Assessment:

  • Western blotting for phosphorylated STAT3 and STAT1 (primary downstream effectors)

  • Immunoprecipitation to detect receptor-adaptor protein interactions

  • Kinase activity assays for JAK family members

• Functional Outcome Measurements:

  • Gene expression profiling using RNA-seq or microarrays

  • Cytokine/chemokine production using multiplex ELISA

  • Cell migration, proliferation, and differentiation assays

• Pathway Perturbation Approaches:

  • Small molecule inhibitors targeting specific pathway components

  • siRNA/shRNA knockdown of signaling intermediates

  • CRISPR-Cas9 gene editing of pathway components

Integration of these techniques provides a comprehensive understanding of IL-19 signaling mechanisms in specific immune cell populations.

How does IL-19 expression differ from other cytokines in response to DNA damage?

IL-19 expression exhibits distinct characteristics compared to other cytokines following DNA damage:

• Temporal Dynamics: While IL-19 expression begins to increase within 24 hours of ionizing radiation (IR) treatment similar to other SASP (Senescence-Associated Secretory Phenotype) factors, it reaches peak expression 2-3 days later, achieving up to 200-fold increase over basal levels

• Pathway Independence: Unlike other DNA damage-induced cytokines, IL-19 expression operates independently of:

  • p38 MAPK signaling (inhibiting p38 MAPK has no effect on IL-19 expression)

  • IL-1 receptor engagement (IL-1α neutralizing antibodies don't affect IL-19 expression)

  • Inflammasome activation (caspase-1 inhibition doesn't suppress IL-19 induction)

• Regulatory Relationship: IL-19 appears to function upstream of other cytokines, as IL-19 knockdown substantially limits IR-mediated increases in IL-1, IL-6, and IL-8 expression

• ATM/DNA-PK Independence: While many DNA damage-induced cytokines depend on ATM (Ataxia Telangiectasia Mutated) kinase, IL-19 expression is not significantly affected by inhibition of either ATM or DNA-PK

These distinctive characteristics suggest IL-19 operates through a unique regulatory pathway in DNA damage responses, potentially representing a novel target for intervention in radiation-induced inflammation.

What methodological approaches can assess IL-19's role in the DNA damage response?

To investigate IL-19's role in DNA damage responses, implement the following methodological framework:

• IL-19 Expression Manipulation:

  • RNA interference: Use at least three distinct shRNAs targeting IL-19 to establish knockdown models

  • CRISPR-Cas9: Create frameshift-inducing deletions in the IL-19 gene

  • Recombinant protein: Supplement experimental systems with exogenous IL-19

• DNA Damage Induction Methods:

  • Ionizing radiation: Apply controlled dosages (typically 2-10 Gy)

  • Topoisomerase inhibitors: Etoposide treatment

  • Other DNA-damaging agents: Taxol, carboplatin

• Pathway Analysis:

  • Selective inhibitors: Use small molecule inhibitors targeting ATM, DNA-PK, p38 MAPK

  • Downstream mediator assessment: Measure phosphorylation of H2AX, MAPKAPK2 (MK2)

  • Secretome analysis: Quantify IL-1α, IL-6, IL-8 protein levels

• Temporal Resolution:

  • Early response: 0-24 hours post-treatment

  • Peak response: 2-5 days post-treatment

  • Resolution phase: 5+ days post-treatment

This systematic approach enables comprehensive characterization of IL-19's functional role within the complex network of DNA damage-induced cytokine responses.

How can recombinant IL-19 be applied in vascular disease research models?

Recombinant IL-19 has demonstrated significant potential in vascular disease research, particularly in abdominal aortic aneurysm (AAA) models, through the following methodological applications:

• Preventive Treatment Protocol:

  • Administer recombinant IL-19 before AAA initiation via elastase infusion

  • Monitor aneurysm development using serial ultrasonography

  • Assess endpoints including medial elastin degradation, smooth-muscle depletion, and matrix metalloproteinase expression

• Therapeutic Intervention Protocol:

  • Initiate IL-19 treatment after AAA creation

  • Evaluate limitation of further aneurysmal degeneration

  • Compare with established aneurysm therapies

• Mechanism Investigation:

  • Analyze leukocyte infiltration via flow cytometry

  • Assess neoangiogenesis through histopathological techniques

  • Quantify MMP-2 and MMP-9 expression through qRT-PCR and gelatin zymography

• Alternative Model Validation:

  • Test IL-19 effects in angiotensin II-induced AAA in hyperlipidemic mice

  • Compare findings with human AAA specimens obtained during repair surgery

These approaches provide comprehensive insights into IL-19's potential as both a biomarker and therapeutic target in vascular disease research.

What are advanced approaches for studying IL-19's effects on gene expression profiles?

To conduct sophisticated analysis of IL-19's impact on gene expression profiles, researchers should implement these advanced methodological approaches:

• Comprehensive Transcriptomic Analysis:

  • RNA-seq of IL-19-treated versus control cells at multiple timepoints

  • Single-cell RNA-seq to resolve cell population heterogeneity

  • Alternative splicing analysis to identify IL-19-dependent transcript variants

• Integrative Multi-Omics:

  • Combine transcriptomics with proteomics and metabolomics

  • Integrate with IL-19-dependent epigenetic modifications (ChIP-seq)

  • Correlate with chromatin accessibility patterns (ATAC-seq)

• Network Analysis:

  • Construct gene regulatory networks using statistical approaches

  • Identify key transcriptional hubs through machine learning algorithms

  • Validate predicted interactions using perturbation studies

• Translation to Human Disease:

  • Compare IL-19-regulated gene networks with human disease transcriptomes

  • Identify conserved IL-19-responsive gene signatures across model systems

  • Correlate with clinical parameters in disease cohorts

• Computational Validation:

  • In silico prediction of IL-19 binding sites in promoter regions

  • Molecular dynamics simulations of IL-19-receptor interactions

  • Pharmacological modeling of IL-19 pathway inhibition

These sophisticated approaches enable systems-level understanding of IL-19's biological effects, facilitating translation to therapeutic applications.