CCRL2 Antibody

What is CCRL2 and why is it important in immunological research?

CCRL2 is an atypical chemokine receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. Unlike typical chemokine receptors, CCRL2 does not appear to signal through G protein-dependent pathways. Its importance stems from its role in modulating immune responses, particularly in inflammatory conditions and cancer .

CCRL2 has been implicated in various pathophysiological processes, as evidenced by studies using CCRL2-deficient mice. These mice showed:

• Protection in IgE-induced anaphylaxis models

• Protection in ovalbumin-induced lung hypersensitivity

• Protection in experimental models of inflammatory arthritis

• Exacerbated inflammatory responses in experimental autoimmune encephalomyelitis

At the molecular level, CCRL2 functions primarily by binding chemerin (RARRES2) and potentially presenting it to CMKLR1, a functional signaling receptor .

What cells express CCRL2 and how is its expression regulated?

CCRL2 shows diverse expression patterns across immune cell populations:

CCRL2 expression is rapidly upregulated following stimulation with proinflammatory stimuli, such as lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) . This dynamic regulation suggests a role in acute inflammatory responses and potential utility as a biomarker for inflammatory activation.

How does CCRL2 differ from conventional chemokine receptors?

Unlike conventional chemokine receptors, CCRL2 exhibits several distinctive characteristics:

• Signaling capacity: CCRL2 lacks conventional G protein-dependent signaling capabilities observed in typical chemokine receptors .

• Internalization dynamics: CCRL2 shows weak, constitutive, ligand-independent internalization and recycling, with kinetics slower than those observed with ACKR3 (a prototypic atypical chemokine receptor) or other chemotactic signaling receptors .

• Cellular trafficking: Intracellularly, CCRL2 colocalizes with early endosome antigen 1-positive and Rab5-positive vesicles, and with recycling compartments characterized by Rab11-positive vesicles .

• Scavenging ability: Unlike other atypical chemokine receptors, CCRL2 does not appear to scavenge its ligands from the extracellular environment, despite its ability to bind chemerin .

These distinctions position CCRL2 as a unique member of the chemokine receptor family with specialized functions in immune regulation.

What are the current models explaining CCRL2's role in neutrophil function?

Current research presents two contrasting models for CCRL2's role in neutrophil function:

Model 1: CCRL2-CXCR2 Heterodimer Hypothesis
Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8. The proposed molecular mechanism suggests the formation of CCRL2 heterodimers with the chemokine receptor CXCR2 .

Model 2: Ligand-Independent Function Hypothesis
Neutralization of CCRL2 with specific antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. These findings led researchers to hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils .

The discrepancy between these models suggests that CCRL2 may have both ligand-dependent and ligand-independent functions, possibly depending on cellular context or activation state. This represents a critical area for future research using CCRL2 antibodies as experimental tools.

How does CCRL2 interact with JAK2/STAT signaling in myeloid malignancies?

Research has revealed a significant role for CCRL2 in JAK2/STAT signaling, particularly in the context of myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML):

• Expression pattern: CCRL2 is upregulated in primitive cells from patients with MDS and sAML compared to healthy controls .

• Functional impact: CCRL2 knockdown suppresses MDS92 and MDS-L cell growth and clonogenicity both in vitro and in vivo .

• Signaling mechanism: CCRL2 knockdown decreases JAK2/STAT3/STAT5 phosphorylation in response to IL-3 stimulation .

• Protein interactions: Coimmunoprecipitation experiments have shown that CCRL2 physically associates with JAK2, and CCRL2 knockdown suppresses the JAK2/STAT interaction following IL-3 treatment .

• Clinical correlations: CCRL2 expression positively correlates with STAT3 phosphorylation in CD34+ cells from patients with MDS and CD34+ AML blasts, independent of blast percentage and mutation allele frequency .

These findings suggest that targeting CCRL2 might represent a novel therapeutic approach in myeloid malignancies, particularly in combination with JAK2 inhibitors such as fedratinib .

What is the current evidence regarding CCRL2's ligand binding specificity?

The ligand binding specificity of CCRL2 has been a subject of debate. Current evidence supports:

Confirmed ligand:

• Chemerin (RARRES2): Flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments have confirmed that chemerin binds to CCRL2 .

Disputed ligands:

• CCL19: Some studies suggest CCL19 binds to CCRL2, while others question this interaction .

• CCL2, CCL5, CCL7, CCL8, CCL21: These were proposed as CCRL2 ligands in earlier studies, but these interactions have been questioned by subsequent research .

The current consensus favors a model where CCRL2 primarily functions by binding chemerin at its N-terminus, leaving the C-terminus accessible for interaction with CMKLR1, essentially acting as a chemerin-presenting molecule at the surface of barrier cells .

How should researchers select the appropriate anti-CCRL2 antibody for their experiments?

Selection of the appropriate anti-CCRL2 antibody requires consideration of multiple factors:

Application compatibility:
Different antibodies show varying performance across applications. Based on the search results:

Species reactivity:
Ensure the antibody recognizes CCRL2 from your species of interest. Commercial antibodies are available for:

• Human CCRL2 (ab235002, 66611-1-Ig)

• Mouse CCRL2 (MAB5519, clone S21013E)

Clone selection:
For studies requiring neutralizing activity, specifically choose antibodies validated for blocking chemerin binding to CCRL2. Research has identified both human and mouse CCRL2 antibodies that effectively neutralize this interaction .

Validation methods:
Request validation data specific to your application. For example, flow cytometry validation should show clear separation between positive and negative populations with appropriate controls .

What are the optimal methods for assessing CCRL2-ligand interactions?

Based on the research literature, several complementary approaches have been validated for studying CCRL2-ligand interactions:

Flow cytometry binding assays:

• Seed HEK-CCRL2 overexpressing cells in 384-well plates

• Incubate with serially diluted unlabeled anti-CCRL2 antibodies (45 min, 4°C)

• Add biotin-labeled chemerin to 10 nM final concentration (45 min, 4°C)

• Wash with PBS + 0.1% BSA

• Incubate with fluorophore-conjugated streptavidin (45 min, 4°C)

• Wash again and analyze by flow cytometry

• Calculate EC50 values using GraphPad Prism

Surface Plasmon Resonance microscopy (SPRm):

• Prepare HEK-CCRL2 or control HEK cells on SPRm 200 sensor chips

• Prime with running buffer (PBS + 0.1% BSA)

• Inject increasing concentrations of chemerin (10-400 nM) or CCRL2 antibodies (6-100 nM)

• Set flow rate at 150 μl/min, with 3 min contact time and 5 min dissociation time

• Wash between runs for multiple analyte testing

• Analyze binding data using appropriate software and fit to a 1:1 binding model

Mesoscale Discovery (MSD) competition assays:
For antibody-epitope mapping and competitive binding studies, MSD-based assays provide quantitative data on binding affinities and competition dynamics .

These methodologies provide complementary data on binding kinetics, specificity, and the ability of antibodies to block ligand-receptor interactions.

What controls should be included when using anti-CCRL2 antibodies in research?

Proper experimental controls are essential for reliable interpretation of results with anti-CCRL2 antibodies:

For Western blot/immunoblotting:

• Positive control: Lysates from cells with confirmed CCRL2 expression (e.g., HeLa cells, K-562 cells)

• Negative control: CCRL2 knockdown/knockout cell lysates

• Loading control: Probing for housekeeping proteins (e.g., β-actin, GAPDH)

• Specificity control: Pre-absorption with immunizing peptide

For flow cytometry:

• Isotype controls: Matching isotype antibodies (e.g., IgG2a for K097F7, IgG2b for 152254)

• Fluorescence minus one (FMO) controls: All antibodies in panel except anti-CCRL2

• Positive control cells: RAW 264.7 mouse monocyte/macrophage cell line shows detectable CCRL2 expression

• Negative control cells: Cell lines without CCRL2 expression

For immunohistochemistry:

• Positive tissue controls: Human spleen tissue shows detectable CCRL2 expression

• Negative controls: Secondary antibody only

• Antigen retrieval optimization: Test both TE buffer pH 9.0 and citrate buffer pH 6.0

For neutralization assays:

• Isotype control antibodies: At equivalent concentrations

• Dose-response curves: Multiple antibody concentrations to determine EC50 values

• Cross-validation: Confirm results using both cell-based and cell-free assay systems

How can researchers validate CCRL2 antibody specificity?

Validation of CCRL2 antibody specificity requires a multi-faceted approach:

Genetic validation:

• CCRL2 knockout/knockdown models: Test antibody reactivity in CCRL2-deficient systems. Specific antibodies should show no signal in properly validated knockout models .

• Overexpression systems: Confirm enhanced signal in CCRL2-overexpressing cell lines compared to parental lines.

Peptide competition:

• Pre-incubate antibody with excess immunizing peptide

• Apply to samples in parallel with unblocked antibody

• Specific antibodies will show reduced or eliminated signal when blocked with cognate peptide

Multiple antibody validation:

• Compare staining patterns across multiple antibodies targeting different CCRL2 epitopes

• Concordant results increase confidence in specificity

Cross-species reactivity:

• Test antibody against CCRL2 orthologs from multiple species

• Expect reactivity only with species included in the specificity claims

Application-specific validation:
For flow cytometry: Confirm that cell populations known to express CCRL2 (monocytes, dendritic cells, neutrophils) show positive staining, while negative populations show minimal background

How should researchers interpret discrepancies between CCRL2 knockout studies and antibody neutralization experiments?

The literature reveals notable discrepancies between CCRL2 knockout studies and antibody neutralization experiments, particularly regarding neutrophil responses to CXCL8:

Methodological interpretation:

• Developmental compensation: Genetic knockout models may trigger compensatory developmental changes absent in acute antibody neutralization.

• Structural vs. functional roles: CCRL2 may have structural roles in receptor complexes that remain intact despite blocked ligand binding.

• Binding epitope specificity: Neutralizing antibodies may block specific epitopes without affecting protein-protein interactions critical for CXCL8 signaling.

• Technical limitations: Antibody concentration, tissue penetration, or half-life may limit complete neutralization compared to genetic deletion.

Recommended approach:
Researchers should employ both genetic (CRISPR/Cas9, siRNA) and pharmacological (neutralizing antibodies) approaches in parallel studies. When discrepancies arise:

• Assess temporal aspects (acute vs. chronic effects)

• Examine compensatory changes in related pathways

• Consider potential protein-protein interactions beyond ligand binding

• Test multiple neutralizing antibodies targeting different epitopes

The hypothesis that "the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils" offers a framework for designing experiments to clarify this apparent contradiction .

What technical factors influence the detection of CCRL2 in flow cytometry experiments?

Accurate detection of CCRL2 by flow cytometry requires attention to several technical factors:

Cell preparation considerations:

• Fixation impact: Overfixation may mask epitopes. Validated protocols typically use mild fixation (0.5-2% paraformaldehyde) or live-cell staining .

• Permeabilization requirements: As CCRL2 localizes to both membrane and cytoplasmic compartments, different permeabilization protocols may reveal distinct pools of the receptor .

Antibody selection factors:

• Clone specificity: Different antibody clones (e.g., S21013E, 498321) may recognize distinct epitopes with varying accessibility depending on receptor conformation or interactions .

• Fluorophore brightness: PE conjugation often provides optimal signal-to-noise ratio for detecting moderate-to-low abundance receptors like CCRL2 .

Protocol optimization:

• Temperature: Binding studies show optimal results at 4°C to prevent receptor internalization during staining .

• Blocking: 5-10% serum from the same species as the secondary antibody reduces non-specific binding.

• Incubation time: 45-minute incubations at 4°C show optimal results for both primary and secondary antibodies .

Data analysis considerations:

• Gating strategy: First gate on viable cells, then on specific cell populations known to express CCRL2.

• Control selection: Use isotype controls matched to the primary antibody's host species and isotype (IgG2a for K097F7, IgG2b for 152254) .

• Signal interpretation: Due to heterogeneous expression, analyze both percentage of positive cells and mean fluorescence intensity.

How do different experimental conditions affect CCRL2 expression and detection?

CCRL2 expression and detection are significantly influenced by experimental conditions:

Inflammatory stimuli:

• LPS and TNF-α rapidly upregulate CCRL2 expression on multiple cell types .

• Time-course experiments show peak expression typically occurring 6-24 hours post-stimulation.

Cell culture variables:

• Serum factors: Chemerin present in serum may saturate CCRL2 binding sites.

• Cell density: Confluency can affect receptor expression and internalization dynamics.

• Passage number: Higher passage cells may show altered receptor expression profiles.

Sample processing effects:

• Isolation methods: Density gradient separation vs. magnetic isolation may affect receptor expression or accessibility.

• Time delays: CCRL2 expression and membrane localization may change during prolonged sample processing.

• Temperature fluctuations: Receptor internalization rates vary with temperature.

Detection method considerations:

• Antibody concentrations: Titration is essential as both insufficient and excessive antibody can yield suboptimal results.

• Blocking reagents: BSA (0.1%) in staining buffers improves signal-to-noise ratio .

• Washing steps: Insufficient washing may leave background signal, while excessive washing can disrupt low-affinity interactions.

Researchers should systematically optimize and standardize these conditions, particularly when comparing CCRL2 expression across different experimental groups or time points.

How can CCRL2 antibodies be used to investigate receptor dimerization and protein-protein interactions?

CCRL2 antibodies offer several approaches to study receptor dimerization and protein-protein interactions:

Proximity ligation assay (PLA):

• Fix and permeabilize cells expressing CCRL2 and potential interaction partners

• Incubate with primary antibodies from different host species against CCRL2 and partner protein

• Apply species-specific PLA probes with attached oligonucleotides

• When proteins are in close proximity (<40 nm), the oligonucleotides can interact

• Enzymatic ligation and rolling circle amplification create a fluorescent spot

• Quantify spots by fluorescence microscopy

This technique has potential for investigating the proposed CCRL2-CXCR2 heterodimer formation .

Co-immunoprecipitation (co-IP) with CCRL2 antibodies:
Research demonstrates that CCRL2 associates with JAK2, and this interaction can be detected through co-IP experiments. Immunoprecipitation with anti-CCRL2 antibodies followed by JAK2 detection (or vice versa) provides evidence of their physical association .

Förster resonance energy transfer (FRET):

• Label CCRL2 antibodies with donor fluorophore (e.g., Cy3)

• Label antibodies against potential interaction partners with acceptor fluorophore (e.g., Cy5)

• Apply to fixed cells or tissue sections

• Measure energy transfer between fluorophores when proteins are in close proximity

• Quantify FRET efficiency to assess interaction strength

Bioluminescence resonance energy transfer (BRET):
For live-cell studies of receptor interactions, BRET offers advantages over FRET for monitoring dynamic interactions.

These methodologies have potential applications in understanding how CCRL2 interacts with JAK2 to potentiate JAK2-STAT interactions, as well as exploring the hypothesized CCRL2-CXCR2 heterodimer formation implicated in neutrophil responses to CXCL8 .

What approaches can researchers use to study CCRL2 trafficking and internalization dynamics?

Despite having weaker internalization dynamics than other chemokine receptors, CCRL2 still exhibits constitutive, ligand-independent internalization and recycling that can be studied using various approaches:

Live-cell imaging with fluorescently-tagged antibodies:

• Label anti-CCRL2 antibodies with pH-sensitive fluorophores (e.g., pHrodo)

• Apply to live cells expressing CCRL2

• Monitor fluorescence changes as receptors move from neutral (cell surface) to acidic (endosomal) compartments

• Quantify internalization rates, recycling kinetics, and compartment transitions

Confocal microscopy for colocalization studies:
CCRL2 colocalizes with specific intracellular compartments:

• Early endosome antigen 1 (EEA1)-positive vesicles

• Rab5-positive vesicles

• Rab11-positive recycling compartments

Researchers can perform double immunostaining with anti-CCRL2 antibodies and markers for these compartments to track receptor trafficking pathways.

Flow cytometry-based internalization assays:

• Label cells with anti-CCRL2 antibodies at 4°C (prevents internalization)

• Shift to 37°C to permit internalization for various time periods

• Strip remaining surface antibodies with acid wash

• Quantify internalized fluorescence by flow cytometry

• Calculate internalization rates from time-course data

Biochemical surface biotinylation:

• Biotinylate surface proteins on CCRL2-expressing cells

• Allow internalization for various time periods

• Strip remaining surface biotin with membrane-impermeable reducing agent

• Immunoprecipitate CCRL2 and detect biotinylated fraction

• Quantify internalization and recycling rates

These approaches would help clarify the unusual trafficking properties of CCRL2, which shows weaker and slower internalization compared to other atypical chemokine receptors like ACKR3 or conventional chemokine receptors .

How can researchers investigate the role of CCRL2 in disease models using antibody-based approaches?

Anti-CCRL2 antibodies enable multiple strategies for investigating CCRL2's role in disease:

In vivo neutralization studies:
Neutralizing antibodies that block chemerin binding to CCRL2 can be administered in disease models to assess functional outcomes. Research has identified antibodies effective for this purpose in both human and mouse systems . Key considerations include:

• Antibody dosing: Based on pharmacokinetic properties and EC50 values from in vitro studies

• Administration route: Intraperitoneal injection shows efficacy in murine models

• Treatment schedule: Both prophylactic and therapeutic regimens should be tested

• Plasma level monitoring: MSD-based assays can quantify antibody concentrations (detection range 0.080 to 5.12 μg/ml in mouse plasma)

Correlative studies in patient samples:
Research demonstrates positive correlation between CCRL2 expression and STAT3 phosphorylation in cells from MDS and AML patients . Similar approaches can be applied to other diseases using:

• Flow cytometry for simultaneous detection of CCRL2 and phosphorylated signaling molecules

• Immunohistochemistry on patient tissue sections

• Correlation of CCRL2 expression with clinical parameters and outcomes

Ex vivo functional studies:

• Isolate primary cells from disease models or patients

• Treat with anti-CCRL2 neutralizing antibodies

• Assess functional outcomes (migration, degranulation, cytokine production)

• Compare with isotype control treatment

Imaging approaches:

• Use fluorescently labeled anti-CCRL2 antibodies for in vivo or ex vivo imaging

• Track CCRL2-expressing cells in disease models

• Correlate CCRL2 expression with disease progression

In myeloid malignancies, combining CCRL2 antibody approaches with JAK2 inhibitors (e.g., fedratinib) may reveal synergistic therapeutic effects, as suggested by studies showing that CCRL2 knockdown enhances fedratinib sensitivity in certain cell types .