Recombinant Rat Chemokine (C-C motif) ligand 24 protein (Ccl24), partial (Active)

What is the primary function of CCL24 in inflammatory processes?

CCL24 functions as a chemotactic protein that mediates immune cell recruitment and trafficking in inflammatory settings. Research demonstrates that CCL24 selectively recruits neutrophils and monocytes to sites of inflammation, as evidenced by murine models where intraperitoneal injection of CCL24 induced significant recruitment of these cell types. Unlike other chemokines such as CCL11, CCL24 shows a distinctive pattern of immune cell recruitment, particularly affecting neutrophils, monocytes, and natural killer (NK) cells . In inflammatory conditions, CCL24 contributes to the accumulation of peribiliary neutrophils and macrophages, with studies showing up to 44% and 20% reduction in these cell populations, respectively, when CCL24 is neutralized with monoclonal antibodies .

The inflammatory role of CCL24 extends beyond simple recruitment, as it is also associated with multiple inflammatory pathways, including granulocyte and agranulocyte adhesion and diapedesis, Th1 and Th2 activation, and pathogen-induced cytokine storm mechanisms . Proteomic analysis of patient samples reveals that CCL24 is strongly associated with an inflammatory-fibrotic signature involving TNF, IFNγ, IL1β, TGFβ, and IL4 .

How can researchers effectively use recombinant rat CCL24 in experimental models?

When designing experiments with recombinant rat CCL24, researchers should consider the following methodological approaches:

• Dose determination: Studies have shown that 5 μg of CCL24 administered intraperitoneally in mice is sufficient to induce substantial immune cell recruitment . For rat models, appropriate dose scaling should be performed based on body weight differences.

• Vehicle selection: Phosphate-buffered saline (PBS) has been successfully used as a vehicle control in CCL24 injection experiments .

• Administration route: Intraperitoneal administration has been validated for studying immune cell trafficking, while subcutaneous administration may be preferred for other applications .

• Time course considerations: When studying cell recruitment, researchers should collect samples between 24-48 hours post-injection to capture peak recruitment effects.

• Cellular analysis: Single-cell RNA sequencing has been successfully employed to characterize the immune cell response to CCL24, allowing identification of distinct cell populations .

For in vitro applications, recombinant CCL24 has been used to:

• Induce hepatic stellate cell (HSC) proliferation, motility, and expression of profibrotic genes

• Promote cholangiocyte senescence and proliferation

• Stimulate M2-like macrophage polarization and proliferation

• Induce migration and myofibroblast-differentiation of dermal fibroblasts

What are the key differences between CCL24 and related chemokines?

CCL24 demonstrates distinct biological activities compared to related chemokines such as CCL11 (eotaxin-1). While both signal through the CCR3 receptor, they elicit different immune responses. In comparative studies:

• CCL24 primarily recruits neutrophils, monocytes, and NK cells, whereas CCL11 has a more limited recruitment profile .

• In animal models, CCL24 injection induced distinct changes in immune cell compartments that were not observed in CCL11-treated animals .

• CCL24 has stronger fibrotic effects on multiple cell types compared to some other chemokines, including:

  • Promoting hepatic stellate cell activation

  • Inducing dermal and cardiac fibroblast proliferation and activation

  • Stimulating cholangiocyte senescence and proliferation

When designing experiments to compare chemokine functions, researchers should consider these functional differences and include appropriate controls to distinguish CCL24-specific effects from general chemokine activities.

What are the methodological approaches for investigating CCL24-mediated fibrosis?

Investigating CCL24-mediated fibrosis requires a multi-faceted approach. Based on current literature, the following methodological framework is recommended:

In vitro models:

• Fibroblast activation assays: Treat dermal fibroblasts with recombinant CCL24 to assess:

  • Myofibroblast differentiation (α-SMA expression)

  • Collagen synthesis (procollagen I expression)

  • Migration capacity

  • Proliferation rate

• Hepatic stellate cell (HSC) studies: Evaluate CCL24-induced:

  • HSC proliferation

  • Expression of profibrotic genes (TIMP1, IL1β, α-SMA, procollagen I)

  • Motility assays

• Cholangiocyte response: Assess:

  • Senescence markers

  • Proliferation rates

  • Inflammatory cytokine production

In vivo models:
Multiple validated animal models are available to study CCL24-mediated fibrosis:

For intervention studies, CM-101 (anti-CCL24 monoclonal antibody) has been effectively used at doses of 0.5–10 mg/kg administered intraperitoneally, intravenously, or subcutaneously, typically twice weekly .

How can researchers quantify CCL24 expression and activity in experimental samples?

Multiple validated methods exist for quantifying CCL24 expression and activity:

• Protein quantification:

  • ELISA has been successfully employed to measure CCL24 levels in serum samples from patients with fibrotic diseases

  • Proximity extension assay (Olink®) for serum proteomics can detect CCL24 alongside other inflammatory markers

• Tissue expression:

  • Fluorescence immunohistochemistry can effectively visualize CCL24/CCR3 expression in tissue samples

  • Quantitative analysis of immunostaining can be performed to assess differences between experimental groups

• Functional assays:

  • Chemotaxis assays using primary monocytes or neutrophils can measure CCL24 activity

  • CCL24-induced cell proliferation assays (e.g., with fibroblasts or hepatic stellate cells)

  • Gene expression analysis of downstream targets using qPCR for profibrotic markers (TIMP1, IL1β, α-SMA, procollagen I)

• Pathway activation:

  • Ingenuity pathway analysis (IPA) has been used to identify dysregulated canonical biological pathways, upstream regulators, and toxicity functions associated with CCL24 levels

  • Protein expression data can be analyzed to determine average pathway expression per individual

When analyzing correlations between CCL24 levels and disease markers, Spearman's correlation has been effectively used, revealing strong correlations between neutrophil accumulation, fibrosis, and biliary hyperplasia (correlation coefficients of 0.70 and 0.78, respectively) .

What experimental approaches can distinguish CCL24-specific effects from general inflammatory responses?

To isolate CCL24-specific effects from broader inflammatory responses, researchers should consider these methodological approaches:

• Selective inhibition studies:

  • Use specific anti-CCL24 monoclonal antibodies (e.g., CM-101) at various concentrations (2.5-10 mg/kg in animal models)

  • Compare CCL24 blockade effects with inhibition of other inflammatory pathways

  • Use CCL24 knockout models when available

• Comparative chemokine studies:

  • Side-by-side comparison of CCL24 with CCL11 or other chemokines that share the CCR3 receptor

  • Assess differential immune cell recruitment patterns

• Receptor antagonism:

  • Use CCR3-specific antagonists to determine if effects are mediated specifically through this receptor

  • Compare with broader chemokine receptor blockade

• Cell-specific responses:

  • Isolate specific cell populations (HSCs, fibroblasts, cholangiocytes) and test responses to CCL24 stimulation

  • Perform RNA-seq to identify CCL24-specific transcriptional signatures

• Pathway analysis:

  • Use bioinformatics approaches to identify pathways specifically altered by CCL24

  • The ingenuity pathway analysis (IPA) has revealed that CCL24 is associated with specific canonical pathways including HSC activation, immune cell trafficking, and inflammation pathways

A validated experimental design would include appropriate controls:

• Vehicle controls (PBS)

• Isotype control antibodies when using anti-CCL24 antibodies

• Alternative chemokine controls (e.g., CCL11)

• Both disease and healthy controls in animal models

What are the correlations between CCL24 levels and disease progression markers in fibrotic conditions?

CCL24 levels correlate significantly with multiple disease progression markers in fibrotic conditions, providing valuable endpoints for experimental studies:

In Primary Sclerosing Cholangitis (PSC):

• Serum CCL24 levels correlate with enhanced liver fibrosis (ELF) scores

• High CCL24 levels correspond to upregulation of monocyte and neutrophil chemotaxis pathways

• Proteomic analysis shows patients with high CCL24 levels exhibit elevated expression of:

  • HSC activation pathway components

  • Immune cell trafficking pathways

  • Th1/Th2 activation pathways

In preclinical models:

• Neutrophil accumulation strongly correlates with:

  • Fibrosis grade (Spearman correlation of 0.70)

  • Biliary hyperplasia grade (Spearman correlation of 0.78)

• Liver inflammatory and fibrotic markers show high correlation with CCL24 levels, including:

  • Liver macrophage accumulation

  • Peribiliary neutrophil presence

  • Collagen deposition

In Systemic Sclerosis (SSc):

• CCL24 circulating levels are significantly elevated in SSc patients compared to healthy controls

• CCL24/CCR3 expression is strongly increased in SSc skin biopsies

• CCL24 levels correlate with disease severity markers including:

  • Dermal thickness

  • Skin collagen content

  • Immune cell infiltration

When designing experiments to investigate these correlations, researchers should collect paired samples for CCL24 quantification and disease markers, and apply appropriate statistical methods such as Spearman's correlation and multivariate analysis.

How should researchers design translational studies to move from rat CCL24 findings to human applications?

Translational studies require careful consideration of species differences and clinical relevance. The following framework is recommended:

• Cross-species comparative analysis:

  • Compare sequence homology and functional conservation between rat, mouse, and human CCL24

  • Validate key findings in multiple species models

  • Test recombinant human CCL24 in rat cell systems and vice versa to determine cross-reactivity

• Parallel human sample studies:

  • Collect matched human samples (serum and tissue biopsies) from patients with analogous conditions

  • Apply identical analytical techniques to human and rat samples

  • Validate the expression pattern of CCL24 and CCR3 across species

• Humanized models:

  • Consider using immunodeficient mice reconstituted with human immune cells

  • Test anti-human CCL24 antibodies in these models

• Translational biomarkers:

  • Identify biomarkers that correlate with CCL24 activity across species

  • Use multiple analytical platforms:

    • Proteomic analysis (e.g., Olink® proximity extension assay)

    • Transcriptomic profiling

    • Metabolomic analysis

• Therapeutic translation considerations:

  • Assess pharmaceutical properties of anti-CCL24 therapies:

    • Dosing based on allometric scaling

    • Pharmacokinetic/pharmacodynamic relationships

    • Safety profile across species

The existing clinical studies with CM-101 (anti-CCL24 mAb) provide a roadmap for successful translation:

These clinical studies demonstrate that findings from preclinical models can be successfully translated to human applications, with appropriate consideration of dosing, safety, and efficacy parameters.