Recombinant Human C-C motif chemokine 24 protein (CCL24), partial (Active)

What is the molecular structure and basic properties of recombinant human CCL24?

Recombinant human CCL24, also designated as eotaxin-2 and Macrophage inflammatory protein 3 (MIP-2), is a chemokine with a molecular weight of 8834.41 Daltons . The mature form (amino acids 1-78) is secreted from cells after cleavage of the signal peptide. Its amino acid sequence is:

VVIPSPCCMFFVSKRIPENRVVSYQLSSRSTCLKAGVIFTTKKGQQFCGDPKQEWVQRYMKNLDAKQKKASPRARAVA

The protein is typically produced in E. coli expression systems, purified to high homogeneity (>95%), and supplied in lyophilized form. Recombinant CCL24 exerts its biological functions by binding to and activating its primary receptor, CCR3, a G protein-coupled receptor that mediates chemotaxis of specific immune cell populations .

What are the optimal storage and handling conditions for maintaining CCL24 activity?

To maintain the biological activity of recombinant human CCL24, researchers should adhere to specific storage and handling protocols. The lyophilized protein exhibits the longest shelf life when stored at -20°C to -80°C (12 months), while reconstituted protein should be stored at 4°C for medium-term use (6 months) or room temperature for short-term use (1 month) .

When working with CCL24, it's critical to avoid repeated freeze-thaw cycles of reconstituted protein, as this can lead to denaturation and loss of biological activity . For reconstitution, sterile water is recommended as CCL24 is freely soluble in aqueous solutions. When designing experiments requiring extended incubation periods, researchers should account for potential activity loss over time and consider replenishing the protein at appropriate intervals.

How can I verify the biological activity of recombinant CCL24 in my experimental system?

Validating the biological activity of recombinant CCL24 is crucial before initiating comprehensive experiments. Multiple approaches can be employed:

• Chemotaxis assays: Measure the migration of CCR3-expressing cells (e.g., eosinophils or transfected cell lines) in response to concentration gradients of CCL24. Effective concentrations typically range from 1-100 ng/ml .

• CCR3 binding assays: Utilize fluorescently-labeled CCL24 to measure direct binding to CCR3-expressing cells, or perform competitive binding assays with labeled reference ligands.

• Signal transduction analysis: Monitor CCR3-mediated signaling events such as calcium flux, ERK phosphorylation, or β-arrestin recruitment following CCL24 stimulation.

• Functional cellular assays: For liver research, assess the proliferative response of hepatic stellate cells and cholangiocytes to CCL24 stimulation (10-100 ng/ml range shows significant effects) .

• Cell recruitment in vivo: In mouse models, intraperitoneal injection of CCL24 selectively recruits neutrophils and monocytes, which can be quantified by flow cytometry or immunohistochemistry .

When establishing dose-response relationships, testing a concentration range (typically 0.1-100 ng/ml) is recommended to determine optimal working concentrations for your specific experimental system .

How can I effectively design experiments to study CCL24-mediated immune cell recruitment?

Designing robust experiments to investigate CCL24-mediated immune cell recruitment requires careful consideration of multiple factors:

• Cell type selection: Focus on cells expressing CCR3, including eosinophils, basophils, Th2 cells, and certain subsets of macrophages. For comprehensive analysis, consider evaluating multiple immune cell types simultaneously using multiparameter flow cytometry.

• In vitro migration assays: Utilize transwell systems with CCL24 gradients (1-100 ng/ml) in the lower chamber and immune cells in the upper chamber. Include positive controls (known CCR3 ligands like CCL11) and negative controls (non-chemotactic proteins) .

• In vivo trafficking models: Administer CCL24 via intraperitoneal injection to assess physiological recruitment patterns. Based on published research, CCL24 injection selectively recruits neutrophils, monocytes, and natural killer (NK) cells, a pattern distinct from other chemokines like CCL11 .

• Specificity controls: Include CCR3 receptor blocking antibodies or small molecule antagonists to confirm receptor-specific effects. Control experiments with related chemokines (e.g., CCL11) help distinguish CCL24-specific responses .

• Temporal considerations: Design time-course experiments to capture both immediate (0-6 hours) and delayed (24-72 hours) recruitment phases, as different cell populations may exhibit distinct temporal response patterns.

Single-cell RNA sequencing analysis following CCL24 administration has identified distinct changes in the immune cell compartment, characterized by selective recruitment of neutrophils, monocytes, and NK cells—effects not observed with related chemokines like CCL11 .

What are the methodological approaches for studying CCL24 in liver fibrosis and inflammation models?

Investigating CCL24's role in liver fibrosis and inflammation requires specialized methodological approaches:

• Animal models selection: Multiple models are suitable for CCL24 research in liver pathology:

  • Mdr2-/- mice (model for primary sclerosing cholangitis)

  • α-naphthylisothiocyanate (ANIT)-induced cholestasis model

  • Carbon tetrachloride (CCl₄)-induced fibrosis model

  • Bile duct ligation (BDL) model

• Intervention strategies:

  • Administer recombinant CCL24 (typically 1-10 μg per injection) to exacerbate disease

  • Apply CCL24-neutralizing antibodies (e.g., CM-101) to inhibit endogenous CCL24 activity

  • Utilize genetic approaches (siRNA knockdown or CRISPR/Cas9) to modulate CCL24 or CCR3 expression

• Assessment parameters:

  • Histological evaluation: H&E, Masson's trichrome, Sirius Red staining for fibrosis quantification

  • Immunohistochemistry for inflammatory cell infiltration (neutrophils, macrophages)

  • Biochemical markers: ALT, AST, ALP, bilirubin levels

  • Fibrosis markers: hydroxyproline content, α-SMA, collagen expression

  • Inflammatory mediators: cytokine/chemokine profiles using multiplex assays

• Advanced analytical techniques:

  • Spatial transcriptomics to assess microenvironmental changes following CCL24 manipulation

  • Single-cell RNA sequencing to characterize cell-specific responses

  • Proximity extension assays to analyze protein signatures in response to CCL24

Studies have demonstrated that CM-101, a CCL24-neutralizing antibody, significantly reduced peribiliary neutrophil and macrophage accumulation while diminishing biliary hyperplasia and fibrosis in mouse models of cholestasis . These findings provide a methodological framework for investigating therapeutic approaches targeting the CCL24/CCR3 axis.

What concentration ranges of CCL24 should be used for different in vitro applications?

Selecting appropriate CCL24 concentrations is critical for generating physiologically relevant and reproducible results. Concentration requirements vary based on the specific application:

When designing dose-response experiments, include at least 4-5 concentration points within the relevant range to establish accurate dose-response relationships. For proliferation studies specifically, 10 ng/ml has been shown to significantly increase DSC proliferation, while simultaneously affecting apoptosis in a concentration-dependent manner .

How can I effectively neutralize CCL24 activity in experimental systems to study its biological functions?

Several approaches can be employed to neutralize CCL24 activity for investigating its biological significance:

• Neutralizing antibodies: Anti-CCL24 neutralizing antibodies have been effectively used at concentrations ranging from 0.08-2 μg/ml in cell culture systems . CM-101, a specific CCL24-neutralizing monoclonal antibody, has demonstrated efficacy in both in vitro and in vivo models, significantly reducing inflammation, fibrosis, and cholestasis-related markers in the biliary area of disease models .

• Receptor antagonism: Blocking the CCR3 receptor using neutralizing antibodies (effective at 0.32-8 μg/ml in cell culture) prevents CCL24 signaling . Small molecule CCR3 antagonists can also be employed for more sustained inhibition in both in vitro and in vivo systems.

• Genetic approaches:

  • siRNA or shRNA targeting CCL24 or CCR3 (effective knockdown typically achieves >70% reduction)

  • CRISPR/Cas9-mediated knockout of CCL24 or CCR3 in relevant cell types

  • Conditional knockout models for tissue-specific CCL24/CCR3 deletion

• Soluble receptor decoys: Recombinant CCR3 extracellular domains can sequester CCL24 and prevent receptor activation.

For validation of neutralization efficiency, researchers should include functional readouts such as chemotaxis inhibition, proliferation assays, or signaling pathway analyses. When studying cellular interactions, as demonstrated with decidual stromal cells and trophoblasts, blocking CCL24 or CCR3 by neutralizing antibodies significantly abolished the effects induced by trophoblast-derived CCL24 .

What are the methodological considerations for measuring CCL24 expression and secretion in different biological samples?

Accurate quantification of CCL24 expression and secretion requires careful selection of methodologies based on sample type and research objectives:

• Protein-level detection:

  • ELISA: Commercially available kits offer detection sensitivity of approximately 2.5 pg/ml, suitable for culture supernatants, serum, plasma, and tissue homogenates .

  • Western blotting: For semi-quantitative analysis of cellular CCL24 protein, using validated antibodies with appropriate positive controls.

  • Immunohistochemistry/Immunofluorescence: For spatial localization in tissue sections, enabling identification of CCL24-producing cells.

  • Proximity extension assay: For multiplex protein analysis in complex biological samples .

• mRNA expression analysis:

  • RT-qPCR: For quantitative assessment of CCL24 transcript levels in cells and tissues.

  • RNA-Seq/single-cell RNA-Seq: For comprehensive transcriptomic profiling and cell-type-specific expression patterns.

  • In situ hybridization: For spatial localization of CCL24 mRNA in tissue sections.

• Sample collection considerations:

  • Cell culture supernatants: Collect at multiple timepoints (24, 48, 72, and 96 hours) to establish secretion kinetics .

  • Serum/plasma: Standardize collection protocols to minimize pre-analytical variability.

  • Tissue samples: Rapid processing and appropriate preservation methods (flash freezing for protein/RNA, fixation for histology).

• Analytical validation:

  • Include recombinant CCL24 standards for calibration curves.

  • Assess recovery and matrix effects for complex biological samples.

  • Include biological controls (samples with known CCL24 expression patterns).

Studies examining CCL24 secretion in co-culture systems (e.g., DSCs and trophoblasts) have demonstrated that measuring supernatants at multiple timepoints (24-96 hours) provides valuable insights into the dynamics of CCL24 production and accumulation .

How do I interpret contradictory findings regarding CCL24 functions in different experimental systems?

Resolving contradictory findings in CCL24 research requires systematic analysis of experimental variables and biological context:

• Cell-type specificity: CCL24 exhibits diverse effects depending on cell type. For example, CCL24 promotes both proliferation and apoptosis in decidual stromal cells, with the net effect being increased cell numbers . In contrast, its effects on hepatic stellate cells may differ, emphasizing the importance of cell-type context in data interpretation.

• Concentration-dependent effects: CCL24 can exhibit biphasic or concentration-dependent responses. Lower concentrations may promote certain cellular processes, while higher concentrations may inhibit or alter the same processes. For instance, CCL24's effect on apoptosis decreases with increasing concentration .

• Temporal considerations: Short-term versus long-term exposure to CCL24 may yield different or even opposing effects. Early signaling events may trigger different pathways than sustained receptor activation, leading to apparently contradictory findings.

• Experimental system differences:

  • In vitro versus in vivo studies may yield different results due to the complex microenvironment in vivo.

  • Different animal models (e.g., Mdr2-/- mice versus ANIT-induced cholestasis) may exhibit model-specific responses.

  • Primary cells versus cell lines may respond differently to CCL24 stimulation.

• Receptor expression levels: Varying levels of CCR3 expression can significantly impact cellular responses to CCL24. Factors like cell-cell contact and hormonal environments can modulate CCR3 expression, as demonstrated in DSCs-trophoblasts co-culture systems and hormone treatment experiments .

When encountering contradictory findings, researchers should systematically evaluate these variables and design experiments with appropriate controls to address specific hypotheses about context-dependent effects.

How is CCL24 implicated in liver pathologies, and what methodologies are used to study its therapeutic targeting?

CCL24 plays significant roles in various liver pathologies, particularly in inflammatory and fibrotic diseases:

• Primary Sclerosing Cholangitis (PSC):

  • CCL24 is highly expressed in damaged bile ducts and liver biopsies of PSC patients .

  • Serum CCL24 levels correlate with Enhanced Liver Fibrosis score, particularly in patients with elevated alkaline phosphatase levels .

  • CCL24 is associated with upregulation of monocyte and neutrophil chemotaxis pathways in PSC patients .

• Non-alcoholic Steatohepatitis (NASH) and Hepatocellular Carcinoma (HCC):

  • Both CCL24 and its receptor CCR3 are upregulated in these conditions .

  • CCL24 contributes to inflammatory cell recruitment and fibrosis progression.

Methodological approaches for studying CCL24 as a therapeutic target include:

• Preclinical evaluation:

  • Neutralizing antibody studies: CM-101, a CCL24-neutralizing monoclonal antibody, has shown significant efficacy in reducing inflammation, fibrosis, and cholestasis-related markers in the Mdr2-/- mouse model and the ANIT-induced cholestasis model .

  • Mechanism studies: Spatial transcriptomics analysis has revealed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization .

  • Cell-specific investigations: In vitro studies with primary human cholangiocytes, macrophages, and hepatic stellate cells have demonstrated that CCL24 induces proliferation of these cells, which can be attenuated by CCL24 inhibition .

• Clinical biomarker development:

  • Correlation analyses between CCL24 serum levels and clinical parameters (e.g., Enhanced Liver Fibrosis score, alkaline phosphatase levels) .

  • Protein signatures in CCL24-treated hepatic stellate cells that differentiate patients by disease severity .

  • Proximity extension assays to analyze proteomic changes associated with disease presence, fibrosis severity, and CCL24 levels .

These methodologies have collectively provided strong evidence supporting CCL24 as a potential therapeutic target in liver diseases, particularly PSC, where blocking CCL24 may reduce liver inflammation, fibrosis, and cholestasis .

What techniques are most effective for studying the interaction between CCL24 and its receptor CCR3 in different cellular contexts?

Investigating CCL24-CCR3 interactions requires specialized techniques tailored to address specific research questions:

• Binding and affinity studies:

  • Surface Plasmon Resonance (SPR): Provides real-time binding kinetics and affinity constants for purified CCL24 and CCR3.

  • Radioligand binding assays: Using labeled CCL24 to determine binding parameters on cells expressing CCR3.

  • Flow cytometry: For cell-based binding assays and competitive binding studies with other CCR3 ligands.

  • BRET/FRET approaches: For studying molecular proximity and conformational changes during ligand-receptor interaction.

• Receptor expression analysis:

  • Flow cytometry: Quantifies CCR3 expression levels on different cell populations.

  • Immunohistochemistry/Immunofluorescence: Provides spatial information about CCR3 expression in tissues.

  • RT-qPCR and Western blotting: Assess transcript and protein expression levels.

• Functional response characterization:

  • Calcium mobilization assays: Measures immediate signaling responses upon CCL24 binding to CCR3.

  • Phosphorylation studies: Detects activation of downstream signaling proteins (ERK, Akt, etc.).

  • Gene expression analysis: Identifies transcriptional changes induced by CCL24-CCR3 signaling.

  • Migration/chemotaxis assays: Evaluates functional cellular responses to CCL24 gradients.

• Contextual modulation studies:

  • Co-culture systems: As demonstrated with DSCs and trophoblasts, co-culture can significantly increase CCR3 expression on target cells .

  • Hormone treatments: Estrogen, progesterone, and hCG have been shown to up-regulate CCR3 expression on DSCs at appropriate concentrations, highlighting the importance of hormonal context .

  • Inflammatory conditions: Proinflammatory cytokines can modulate CCR3 expression and responsiveness to CCL24.

• Receptor signaling manipulation:

  • Biased ligand screening: Identifies compounds that selectively activate specific CCR3 signaling pathways.

  • Receptor mutagenesis: Determines critical residues for CCL24 binding and signaling.

  • Signaling pathway inhibitors: Elucidates the relative contributions of different downstream pathways.

These approaches have revealed that the CCL24-CCR3 axis is significantly influenced by cellular context, with factors like cell-cell contact and hormonal environment substantially modulating receptor expression and downstream responses .

What are the emerging technologies for studying CCL24's role in intercellular communication and tissue microenvironments?

Several cutting-edge technologies are transforming our understanding of CCL24's functions in complex biological systems:

• Spatial multi-omics approaches:

  • Spatial transcriptomics: Maps gene expression patterns within tissue contexts, revealing microenvironmental changes following CCL24 manipulation. This approach has already demonstrated reduced proliferation and senescence of cholangiocytes following CCL24 neutralization in liver disease models .

  • Imaging mass cytometry: Simultaneously visualizes multiple proteins, including CCL24, CCR3, and downstream effectors, within tissue sections with subcellular resolution.

  • Digital spatial profiling: Quantifies protein and RNA levels with spatial context, enabling precise mapping of CCL24-responsive cellular neighborhoods.

• Single-cell analysis techniques:

  • Single-cell RNA sequencing: Characterizes cell-specific responses to CCL24 stimulation or inhibition. This approach has identified distinct immune cell recruitment patterns following CCL24 administration, specifically neutrophils, monocytes, and NK cells .

  • Single-cell proteomics: Profiles protein expression and post-translational modifications at single-cell resolution.

  • Single-cell secretomics: Measures secreted factors from individual cells, revealing heterogeneous responses to CCL24.

• Advanced imaging technologies:

  • Intravital microscopy: Visualizes CCL24-mediated cell recruitment and interactions in live animals.

  • Expansion microscopy: Provides super-resolution imaging of CCL24-CCR3 interactions and signaling complexes.

  • Multiplexed ion beam imaging (MIBI): Simultaneously detects multiple proteins in tissue sections with high resolution.

• Organ-on-chip and organoid systems:

  • Liver-on-chip models: Recapitulates complex liver architecture for studying CCL24's role in hepatobiliary diseases.

  • Co-culture organoids: Creates three-dimensional tissue models incorporating multiple cell types for studying intercellular communication mediated by CCL24.

  • Microfluidic gradient systems: Establishes stable chemokine gradients for studying cell migration dynamics.

• Systems biology approaches:

  • Multi-omics integration: Combines transcriptomics, proteomics, and metabolomics data to comprehensively map CCL24 signaling networks.

  • Network modeling: Predicts intercellular communication patterns and feedback mechanisms involving CCL24.

  • Machine learning algorithms: Identifies complex patterns in large datasets that may reveal novel functions of CCL24 in tissue homeostasis and disease.

These emerging technologies promise to provide unprecedented insights into how CCL24 orchestrates cellular behaviors within complex tissue microenvironments, potentially revealing new therapeutic opportunities.

How can genetic and epigenetic regulation of CCL24 expression be effectively studied in different disease contexts?

Investigating the genetic and epigenetic regulation of CCL24 expression requires multifaceted approaches tailored to specific disease contexts:

• Genetic regulatory mechanisms:

  • Promoter analysis: Characterize the CCL24 promoter region to identify transcription factor binding sites and regulatory elements.

  • ChIP-seq: Map transcription factor binding patterns at the CCL24 locus under different disease conditions.

  • Genome-wide association studies (GWAS): Identify genetic variants associated with altered CCL24 expression in relevant diseases.

  • CRISPR screening: Systematically identify genes that regulate CCL24 expression using genome-wide or targeted CRISPR libraries.

  • Reporter assays: Evaluate CCL24 promoter activity in response to disease-relevant stimuli or genetic perturbations.

• Epigenetic regulatory mechanisms:

  • DNA methylation analysis: Assess methylation patterns at the CCL24 locus using bisulfite sequencing or methylation arrays.

  • Histone modification profiling: Characterize chromatin states at the CCL24 locus using ChIP-seq for histone marks (H3K27ac, H3K4me3, H3K27me3, etc.).

  • ATAC-seq: Map chromatin accessibility at the CCL24 locus and identify potential regulatory regions.

  • 3D chromatin organization: Investigate topologically associating domains and enhancer-promoter interactions affecting CCL24 using Hi-C or related techniques.

  • Non-coding RNA regulation: Identify microRNAs or long non-coding RNAs that modulate CCL24 expression.

• Disease-specific approaches:

  • For liver diseases: Study epigenetic changes in hepatocytes, cholangiocytes, and hepatic stellate cells isolated from disease models or patient samples.

  • For reproductive biology: Investigate hormonal regulation of CCL24 expression in trophoblasts and decidual stromal cells, as these cell types have demonstrated CCL24 production and response capabilities .

  • For inflammatory conditions: Examine epigenetic reprogramming of immune cells that affects CCL24 expression or CCR3 responsiveness.

• Integrative analysis:

  • Multi-omics integration: Combine genetic, epigenetic, transcriptomic, and proteomic data to comprehensively map CCL24 regulatory networks.

  • Single-cell multi-omics: Simultaneously profile genetic, epigenetic, and transcriptional states in individual cells to capture regulatory heterogeneity.

  • Trajectory analysis: Map dynamic changes in CCL24 regulation during disease progression or cellular differentiation.

These approaches provide complementary insights into the complex regulatory mechanisms controlling CCL24 expression, potentially revealing disease-specific regulatory abnormalities that could be targeted therapeutically.