IL1R2 Antibody

What is IL1R2 and what are its primary biological functions?

IL1R2 (Interleukin 1 Receptor Type 2) is a cytokine receptor belonging to the interleukin 1 receptor family. The canonical IL1R2 protein in humans is 398 amino acids in length with a molecular weight of approximately 45.4 kDa. It exists in both membrane-bound and soluble forms, and functions primarily as a decoy receptor that binds interleukin-1 alpha (IL1A), interleukin-1 beta (IL1B), and interleukin 1 receptor type I (IL1R1/IL1RA) without initiating signaling cascades. Unlike IL1R1, IL1R2 lacks the intracellular TIR domain required for signal transduction, making it the first identified example of a decoy receptor. Its primary function is to serve as a negative regulator of IL-1 signaling, thereby suppressing inflammatory responses by rapidly scavenging free IL-1 from circulation .

Recent research has shown that IL1R2 resolves inflammation by inhibiting IL-1 activity through multiple mechanisms: competing with IL1R1 for IL-1 at the cell surface, binding IL-1 in its soluble form, competing for IL1RAcP away from IL1R1, and an intracellular form of IL1R2 that prevents IL-1α activation .

What types of IL1R2 antibodies are available for research applications?

Researchers have access to over 450 anti-IL1R2 antibodies from more than 30 different suppliers. These antibodies are available in both monoclonal and polyclonal formats, designed for various applications including Western blot, ELISA, Flow Cytometry, Immunohistochemistry (IHC), and Immunofluorescence (IF/ICC) .

For instance, Proteintech offers the 60262-1-Ig IL1R2 monoclonal antibody with validated reactivity against human and mouse samples, suitable for Western blot (1:500-1:2000 dilution), Immunofluorescence (1:20-1:200 dilution), and ELISA applications. This specific antibody is a mouse IgG2a monoclonal that recognizes the full IL1R2 protein (45 kDa observed molecular weight) .

When selecting an IL1R2 antibody, researchers should consider:

• Host species and isotype (mouse, rabbit, etc.)

• Clonality (monoclonal vs. polyclonal)

• Validated applications (WB, IF, ELISA, etc.)

• Species reactivity (human, mouse, rat, etc.)

• Recognition epitope within the protein

How does IL1R2's structure relate to its function as a decoy receptor?

IL1R2's ability to act as a decoy receptor stems directly from its structural characteristics. While IL1R2 shares similar extracellular domains with IL1R1 and binds IL-1 with comparable affinity, it critically lacks the intracellular Toll/IL-1R (TIR) domain necessary for signal transduction. This structural difference allows IL1R2 to effectively bind IL-1 without initiating downstream signaling cascades .

The protein exists in both membrane-bound and soluble forms. The soluble form is generated either through alternative splicing or through metalloprotease-dependent shedding from the cell surface, which can be induced by various stimuli including LPS, TNF-α, and PMA. This soluble form circulates in the serum where it can rapidly scavenge free IL-1, as demonstrated in knockout mouse models where IL-1α clearance from circulation was significantly reduced in IL1R2-deficient mice .

Post-translational modifications, particularly proteolytic cleavage and glycosylation, further influence the protein's function by affecting its binding capabilities and circulation half-life .

How does IL1R2 expression in T follicular regulatory cells influence germinal center reactions?

Recent research has uncovered a critical role for IL1R2 in T follicular regulatory (Tfr) cells during germinal center (GC) reactions. Tfr cells produce IL1R2 that antagonizes IL-1 activation of T follicular helper (Tfh) cells. Experimental models using conditional knockout mice with Tfr cell-specific deletion of IL1R2 have revealed significant alterations in GC dynamics .

Following sheep red blood cell (sRBC) immunization, mice lacking IL1R2 specifically in Tfr cells showed:

These findings demonstrate that Tfr cell-derived IL1R2 functions to restrain germinal center reactions and antibody production. Interestingly, this regulatory mechanism appears most crucial during primary immune responses, as the effects were less pronounced during booster immunizations .

The GC-restraining effect of IL1R2 was confirmed to work through IL-1 antagonism, as treatment with anakinra (an IL-1 receptor antagonist) reversed the enhanced GC responses seen in Tfr-specific IL1R2 knockout mice .

What are the key considerations when validating IL1R2 antibody specificity in knockout/knockdown models?

Validating IL1R2 antibody specificity using genetic models is crucial for ensuring experimental rigor. Based on published approaches, researchers should consider the following methodological steps:

• Genetic model verification: Confirm gene deletion at the DNA level through genotyping PCR targeting the deleted exon (typically exon 3 for IL1R2 knockout models) .

• Transcript level validation: Perform qRT-PCR to verify absence of IL1R2 mRNA in knockout tissues or cells, or reduced expression in knockdown models.

• Protein level confirmation: Use multiple techniques to verify absence or reduction of IL1R2 protein:

  • Western blot analysis of tissues from wild-type versus knockout animals

  • Flow cytometry of cells known to express IL1R2 (e.g., neutrophils show measurable IL1R2 expression that is absent in knockout models)

  • ELISA for soluble IL1R2 in serum samples

• Functional validation: Implement assays that measure IL1R2-dependent processes:

  • IL-1α clearance assays (injected IL-1α persists longer in circulation of IL1R2 knockout mice)

  • IL-1-induced inflammatory responses (e.g., IL-6 production remains elevated longer in IL1R2 knockout mice)

  • Neutrophil recruitment in sterile peritonitis models (increased in IL1R2 knockout mice)

• Cross-reactivity testing: Test antibody with closely related family members (particularly IL1R1) to ensure specificity.

Of note, research has shown that germline IL1R2 knockout mice may develop compensatory mechanisms that mask phenotypes, whereas conditional knockout models in adult mice reveal more pronounced effects, suggesting developmental adaptation can occur .

How does the soluble versus membrane-bound form of IL1R2 affect experimental design when studying inflammatory regulation?

The dual existence of IL1R2 in both membrane-bound and soluble forms creates important experimental design considerations when studying inflammatory regulation. Based on published findings:

Soluble IL1R2 is generated through either alternative splicing or metalloprotease-dependent shedding from the cell surface. This soluble form circulates in serum and plays a dominant role in scavenging free IL-1, thereby rapidly resolving inflammatory responses. In experimental models, IL1R2 knockout mice show significantly reduced clearance of injected IL-1α from circulation, leading to prolonged inflammatory responses measured by sustained IL-6 production .

In contrast, membrane-associated IL1R2 appears to have less effect on a given cell's sensitivity to IL-1. This suggests distinct regulatory mechanisms depending on the form being studied .

Key experimental design considerations include:

• Compartment-specific analysis: Distinguish between IL1R2 effects in circulation versus tissue-specific effects by:

  • Measuring serum IL1R2 levels via ELISA

  • Analyzing membrane IL1R2 expression by flow cytometry on specific cell types (neutrophils express higher levels than monocytes/macrophages)

  • Using cell-specific conditional knockout models to isolate effects

• Stimulus-dependent regulation: Account for factors that regulate IL1R2 expression and shedding:

  • Glucocorticoids, IL-4, and IL-13 induce IL1R2 transcripts

  • LPS and IFN-γ decrease IL1R2 expression

  • LPS, TNF-α, and PMA induce IL1R2 shedding from the cell surface

• Timing considerations: IL1R2's primary role appears to be in resolution of inflammation rather than initial response:

  • Short-term experiments (≤2 hours) may show minimal differences between wild-type and IL1R2-deficient models

  • Longer timepoints (≥4 hours) reveal IL1R2's role in resolving inflammatory responses

What are the optimal protocols for using IL1R2 antibodies in Western blot applications?

Based on validated protocols for IL1R2 antibodies in Western blot applications, researchers should consider the following methodological approach:

Sample Preparation:

• Extract total protein from tissues or cells using RIPA buffer containing protease inhibitors

• Quantify protein concentration (BCA or Bradford assay)

• Prepare samples with loading buffer containing reducing agent (β-mercaptoethanol)

• Heat samples at 95°C for 5 minutes to denature proteins

Gel Electrophoresis and Transfer:

• Load 20-50 μg protein per lane on 10-12% SDS-PAGE gels (appropriate for IL1R2's 45.4 kDa size)

• Run gels at 80-120V until sufficient separation

• Transfer to PVDF or nitrocellulose membranes (0.45 μm pore size) at 100V for 60-90 minutes in cold transfer buffer or overnight at 30V

Antibody Incubation:

• Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary anti-IL1R2 antibody at 1:500-1:2000 dilution (e.g., 60262-1-Ig) overnight at 4°C

• Wash membranes 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (anti-mouse IgG for mouse monoclonals) at 1:5000-1:10000 dilution for 1 hour at room temperature

• Wash membranes 3-5 times with TBST

Detection and Verification:

• Develop using ECL substrate and capture images using appropriate imaging system

• Verify expected molecular weight (~45 kDa for full-length IL1R2)

• Include appropriate controls:

  • Positive control: HeLa cells are known to express IL1R2

  • Negative control: Samples from IL1R2 knockout mice/cells when available

  • Loading control: Probing for housekeeping proteins like β-actin or GAPDH

Troubleshooting Common Issues:

• Multiple bands: May indicate post-translational modifications (particularly proteolytic cleavage and glycosylation) or non-specific binding

• No signal: Verify antibody reactivity with your species of interest (human and mouse are confirmed for many IL1R2 antibodies)

• High background: Increase blocking time/concentration or adjust antibody dilutions

How can researchers effectively study the interaction between IL1R2 and its ligands in different experimental systems?

Studying IL1R2-ligand interactions requires techniques that can detect binding events and functional outcomes. Based on published approaches, researchers can implement the following methodological strategies:

1. Binding Assays:

• Surface Plasmon Resonance (SPR): Determine binding kinetics and affinity between purified IL1R2 (soluble form) and IL-1α, IL-1β, or IL1R1

• Co-immunoprecipitation: Use anti-IL1R2 antibodies to pull down protein complexes from cell lysates or biological fluids, followed by detection of associated IL-1 family members

• ELISA-based binding assays: Coat plates with recombinant IL1R2 and detect binding of labeled IL-1 ligands, or use competitive binding assays to compare affinity with IL1R1

2. Functional Interaction Studies:

• IL-1 Scavenging Assays: Measure clearance of exogenously added IL-1 from culture media or circulation in the presence or absence of IL1R2

• Reporter Cell Assays: Use cells expressing an IL-1-responsive reporter gene to measure IL-1 activity in the presence of varying concentrations of soluble IL1R2

• Neutrophil Recruitment Models: Implement sterile peritonitis models to assess IL-1-dependent neutrophil recruitment in WT versus IL1R2-deficient mice

3. Cell-Specific Analysis:

• Flow Cytometry: Measure surface IL1R2 expression on specific cell populations like neutrophils and monocytes/macrophages

• Immunofluorescence Microscopy: Visualize co-localization of IL1R2 with IL-1 ligands or IL1R1 in cells or tissues

• Single-Cell RNA-Seq: Determine cell types expressing IL1R2 and IL-1 family members simultaneously

4. In Vivo Models:

• Conditional Knockout Systems: Use cell-specific Cre recombinase systems (e.g., Foxp3-Cre for Tfr cells) to delete IL1R2 in specific cell populations and study outcomes

• Germinal Center Models: Immunize mice with antigens like sheep red blood cells to study how IL1R2 loss affects GC formation, Tfh/Tfr ratios, and antibody production

• Inflammation Resolution Models: Compare wild-type and IL1R2-deficient mice for their ability to resolve IL-1-induced inflammatory responses over time

5. Compensation and Redundancy Analysis:

• Compare germline knockout versus conditional knockout models to identify developmental compensation mechanisms

• Use combined inhibition of multiple IL-1 family decoy receptors to address functional redundancy

What methodological approaches can resolve contradictory findings about IL1R2 function in different disease models?

Resolving contradictory findings about IL1R2 function across disease models requires systematic approaches that account for biological complexity and experimental variables. Based on published research, the following methodological strategies can help reconcile discrepancies:

1. Development-Dependent Compensation Analysis:
Research has revealed that germline IL1R2 knockout mice may not show the same phenotypes as conditional IL1R2 knockout models where deletion occurs in adulthood. This suggests that compensatory mechanisms develop during embryonic and postnatal development that can mask the effects of IL1R2 loss . To address this:

• Compare germline versus inducible/conditional knockout models

• Analyze expression of other IL-1 family receptors/antagonists in response to IL1R2 deletion

• Implement time-course studies following inducible deletion to identify adaptive changes

2. Cell Type-Specific Function Analysis:
IL1R2 expression and function varies across cell types. For example, neutrophils express higher levels of surface IL1R2 than monocytes/macrophages . To address cell-specific contributions:

• Use conditional knockout models targeting specific cell lineages

• Perform adoptive transfer experiments with wild-type or IL1R2-deficient cells

• Conduct parallel in vitro studies with purified cell populations

3. Context-Dependent Signaling Assessment:
The regulatory role of IL1R2 may be more critical in certain inflammatory contexts than others. For example, IL1R1 knockout mice show normal germinal center reactions despite IL1R2's demonstrated role in regulating these processes . To address context-dependency:

• Compare acute versus chronic disease models

• Test different inflammatory triggers (sterile, infectious, autoimmune)

• Analyze IL1R2 function at different stages of disease progression

4. Human-Animal Model Comparison:
Findings from animal models may not always translate to human biology. Research showed that patients with ulcerative colitis or Crohn's disease had lower serum levels of IL1R2 , suggesting disease-specific regulation. To bridge this gap:

• Analyze IL1R2 levels in relevant human patient samples

• Compare expression patterns between human tissues and animal models

• Conduct parallel experiments in human and animal cells

5. Systematic Review and Meta-Analysis:
To resolve contradictions across multiple studies:

• Perform systematic comparison of experimental methods

• Analyze differences in genetic backgrounds, housing conditions, and microbiota

• Implement standardized reporting of key experimental variables

• Consider sex and age differences in IL1R2 function

6. Systems Biology Approaches:

• Use computational modeling to predict context-dependent outcomes

• Implement multi-omics approaches to identify network-level compensation

• Analyze IL1R2 in the broader context of inflammatory regulation pathways