Recombinant Human C-C motif chemokine 23 protein (CCL23), partial (Active)

What is the molecular structure of CCL23 and how is it processed?

Recombinant Human CCL23 (also known as CK-Beta-8, MPIF-1, or SCYA23) is a small cytokine belonging to the CC chemokine family. The CCL23 gene encodes a 120 amino acid precursor protein with a 21 amino acid signal peptide that undergoes proteolytic cleavage to generate a 99 amino acid mature protein (amino acids 22-120) . Further N-terminal processing can produce a 75 amino acid peptide (amino acids 46-120) that exhibits significantly higher biological activity than the 99 amino acid variant . This differential processing is critical for researchers to consider when selecting recombinant CCL23 for experimental applications, as the truncated form may provide more robust biological responses in functional assays .

What are the primary biological functions of CCL23 in immune regulation?

CCL23 functions primarily as a chemotactic factor that binds with high affinity to the CCR1 receptor . Its biological activities include:

• Chemotactic recruitment of monocytes, dendritic cells, and osteoclast precursors to sites of inflammation

• Enhancement of angiogenesis in endothelial cells

• Inhibition of progenitor cell proliferation, affecting granulocyte and monocyte lineage formation

In experimental systems, recombinant CCL23 demonstrates a dose-dependent ability to chemoattract CCR1-expressing cells, with the truncated form (aa 46-137) showing ED50 values of 0.002-0.01 μg/mL for chemotaxis of CCR1-transfected cells . Researchers investigating immune cell trafficking should note that CCL23's effects may vary significantly depending on the specific cell type and inflammatory context.

How is CCL23 expression regulated at the transcriptional level?

CCL23 expression is primarily induced by the Th2 cytokines IL-4 and IL-13 in human peripheral blood monocytes through a STAT6-dependent mechanism . The transcriptional regulation involves:

• A canonical STAT6 binding site in the CCL23 promoter region that is essential for IL-4-inducible expression

• GM-CSF synergy with IL-4 (but not IL-13) in enhancing CCL23 expression

• A negatively acting cis-element downstream of the STAT6 binding site that modulates IL-4 inducibility

For researchers studying CCL23 regulation, it is important to note that tandem copies of the STAT6 site can confer cytokine responsiveness to a heterologous minimal promoter, and the negative regulatory element identified in the CCL23 promoter can function on other IL-4-inducible promoters . This understanding of transcriptional control provides avenues for experimental manipulation of CCL23 expression in research models.

What methodological approaches are recommended for measuring CCL23 levels in biological samples?

When quantifying CCL23 in research or clinical samples, researchers should consider the following approaches:

• Immunoassays (ELISA) for blood and cerebrospinal fluid (CSF) samples, which have been successfully used in clinical studies of neurodegeneration and cardiac arrest

• Quantitative antibody arrays for multiplex analysis of CCL23 alongside other chemokines, particularly useful for temporal dynamics studies

• Flow cytometry for cellular-level analysis of CCL23 production, especially in neutrophils and other immune cells

Methodological considerations should include:

• Sample processing and storage to preserve protein integrity

• Accounting for the different forms of CCL23 (99aa vs. 75aa variants)

• Including appropriate controls to normalize for biological variation

• Consideration of temporal dynamics, as CCL23 levels may change significantly over time in disease states

What is the significance of CCL23 as a biomarker in neurodegenerative conditions?

Recent research has identified CCL23 as a potential biomarker for progression from mild cognitive impairment (MCI) to Alzheimer's disease (AD) . Longitudinal studies have demonstrated:

• Predictive value of elevated CCL23 protein levels in both plasma (hazard ratio = 2.5, CI 95%: 1.2–5.3, p=0.02) and CSF (hazard ratio = 3.05, CI 95%: 1.02–5, p=0.04) for MCI to AD progression

• Association between the APOE ε4 allele and higher CCL23 levels (470.33 pg/mL vs. 377.94 pg/mL, p=0.01)

These findings support CCL23's role in neuroinflammatory processes during early AD stages. When designing studies to evaluate CCL23 as a biomarker, researchers should include longitudinal sampling, account for APOE genotype as a potential confounder, and consider combining CCL23 with other established biomarkers to improve predictive accuracy for disease progression.

How can researchers effectively study CCL23's role in acute cardiac conditions?

Recent research has revealed important insights into CCL23's role in cardiac arrest outcomes and post-resuscitation inflammation . Effective experimental approaches include:

• Temporal analysis of chemokine dynamics showing distinct patterns (CCL23 shows delayed elevation at 48 hours post-cardiac arrest compared to earlier increases in CCL2 and CCL4)

• Correlative analysis of CCL23 levels with clinical outcomes (higher CCL23 associated with increased neutrophil counts, elevated neuron-specific enolase, worse cerebral performance scores, and higher mortality)

• Combined human-mouse models where human neutrophils from cardiac arrest patients are injected into immunodeficient mice after cardiac arrest and CPR, showing increased CCL23 production in neutrophils infiltrating brain tissue compared to peripheral circulation

Researchers studying CCL23 in this context should consider the delayed temporal dynamics of CCL23 elevation and focus on tissue-specific analysis, particularly in neural tissues affected by ischemia-reperfusion injury.

What are the critical considerations when using recombinant CCL23 in cell-based assays?

When designing experiments with recombinant CCL23 protein, researchers should consider:

• Protein variant selection: The 75 amino acid residue peptide (aa 46-120) demonstrates significantly higher biological activity than the 99 amino acid variant

• Dose-response relationships: Effective doses for chemotaxis vary by cell type, with ED50 values ranging from 0.002-0.04 μg/mL depending on the specific assay

• Formulation considerations: Recombinant CCL23 is typically supplied as a lyophilized powder in buffer (e.g., 20mM PB, 250mM NaCl, pH 7.2) and requires proper reconstitution

• Storage conditions: Lyophilized proteins maintain stability for up to 12 months when properly stored

• Endotoxin levels: For cell-based assays, endotoxin contamination should be minimal (<1.0 EU per μg)

How do experimental models for studying CCL23 differ between inflammatory and neurodegenerative research?

Different research contexts require tailored experimental approaches for studying CCL23:

For inflammatory skin conditions (e.g., atopic dermatitis):

• Immunohistochemistry to assess CCL23-expressing cells in skin biopsies

• Correlation with IL-4/IL-13 expression and STAT6 activation

For neurodegenerative research:

• Longitudinal sampling of blood and CSF in MCI patients with clinical follow-up

• Integration with APOE genotyping and other AD biomarkers

For cardiac arrest and ischemia-reperfusion studies:

• Temporal profiling of multiple chemokines including CCL23

• Combined human sample analysis with mouse models of cardiac arrest/CPR

• Flow cytometric analysis of neutrophil CCL23 production in affected tissues

Each approach requires specific methodological considerations and appropriate controls to effectively interpret CCL23's role in the particular disease context.

What methodological challenges exist when studying the differential functions of full-length versus truncated CCL23?

Researchers investigating functional differences between full-length and truncated CCL23 face several methodological challenges:

• Production considerations: Ensuring consistent post-translational processing during recombinant protein production

• Activity verification: The 75aa variant (aa 46-120) demonstrates significantly higher activity in functional assays, requiring careful calibration of experimental doses

• Receptor binding studies: Differential binding affinities to CCR1 between variants may influence experimental outcomes

• In vivo relevance: Understanding which form predominates in specific physiological or pathological contexts

To address these challenges, researchers should:

• Use multiple functional readouts (chemotaxis, calcium flux, receptor internalization)

• Include both variants in parallel experiments with appropriate dose-response curves

• Consider the biological context where specific processing may occur

• Develop tools to specifically detect different CCL23 forms in biological samples

What are promising areas for future CCL23 research based on current findings?

Based on the integrated analysis of current literature, several promising research directions emerge:

• CCL23 as a biomarker for neurodegenerative progression:

  • Further validation in larger cohorts

  • Integration with imaging and other fluid biomarkers

  • Development of standardized assays for clinical application

• CCL23 in post-cardiac arrest neuroinflammation:

  • Mechanistic studies of delayed CCL23 elevation

  • Potential therapeutic targeting of CCL23/CCR1 pathway

  • Investigation of neutrophil-specific CCL23 production in brain inflammation

• STAT6-dependent regulation in Th2-dominant conditions:

  • Further characterization of the negative regulatory element

  • Investigation of epigenetic factors influencing CCL23 expression

  • Exploration of CCL23 in other Th2-mediated conditions beyond atopic dermatitis

These directions represent opportunities for researchers to make significant contributions to understanding CCL23's role in health and disease.