Recombinant Human C-C motif chemokine 17 protein (CCL17) (Active)

What is the primary biological function of CCL17?

CCL17, also known as Thymus and Activation-Regulated Chemokine (TARC), primarily functions as a chemotactic cytokine that attracts immune cells to sites of inflammation or infection. It acts by binding to its receptor CCR4, facilitating the migration of T cells, particularly activated T cells. Beyond its established role in immune cell recruitment, CCL17 has emerged as a multifunctional protein involved in signal transmission in the brain, cardiac remodeling, and metabolic regulation. Recent research has revealed that CCL17 production is compartmentalized within the body, with expression predominantly found in activated Langerhans cells and mature dendritic cells located in various lymphoid and non-lymphoid organs . This compartmentalization suggests tissue-specific roles that extend beyond simple immune cell attraction.

Which cell types primarily express CCL17?

CCL17 is primarily expressed by dendritic cells (DCs), particularly mature myeloid-related DCs. Using fluorescence-based in vivo reporter systems, researchers have identified that CCL17 expression occurs in activated Langerhans cells and mature DCs located in various lymphoid and non-lymphoid organs . CCL17-expressing DCs predominantly belong to the CD11b+ subset. In the context of the brain, studies have shown that neurons in the hippocampus can produce CCL17, particularly when stimulated with substances that mimic bacterial infections . This neuronal expression pattern suggests CCL17 may have important functions in both immune and nervous systems. Importantly, CCL17 expression is differentially regulated across tissues - it can be induced in most peripheral lymphoid and non-lymphoid organs but is notably absent in the spleen, even during systemic bacterial infection .

How is CCL17 expression regulated in different tissues?

In neurons, CCL17 production can be stimulated by substances contained in bacterial cell membranes, suggesting a potential role in neuroinflammatory responses . Additionally, age-related and angiotensin II-induced cardiac stress significantly upregulates circulatory CCL17 levels in cardiac tissue . Research has also demonstrated that CCL17 levels increase with age, unlike the related chemokine CCL22, which does not show age-dependent expression patterns .

How does CCL17 influence neuronal signaling in the brain?

CCL17 plays an unexpected role in neuronal signaling beyond its established function in the immune system. Research has identified that CCL17 is produced by neurons in the hippocampus, a brain region critical for learning and memory formation . This localized expression suggests CCL17 may regulate neural functions related to cognition, orientation, and memory processing.

Experimental evidence indicates a potential link between CCL17 and autism spectrum disorders. Studies using animal models with defects in CCL17 receptor expression have demonstrated behavioral abnormalities, including impaired nest-building abilities compared to normally developed counterparts . These behavioral changes strongly suggest that CCL17 influences brain function and behavior through mechanisms that may involve modulating synaptic transmission or neuronal connectivity. The exact signaling pathways through which CCL17 affects neuronal function remain under investigation, but its expression pattern in hippocampal neurons points to a potential role in learning and memory processes.

What experimental approaches are recommended to study CCL17's neurological effects?

To effectively investigate CCL17's neurological functions, researchers should consider implementing the following methodological approaches:

• Genetic reporter systems: Utilize fluorescence-based in vivo reporter systems that couple CCL17 release with fluorescent protein production. This approach has been successfully employed by coupling the release of CCL17 with the production of fluorescent dyes that illuminate CCL17-producing cells, enabling visualization under microscopy .

• Bacterial membrane component stimulation: Simulate infection conditions using substances from bacterial cell membranes to enhance CCL17 production in neuronal tissue, making production sites clearly visible under microscopy .

• Behavioral assessment in knockout models: Evaluate CCL17 or CCR4 receptor knockout models using standardized behavioral tests that assess cognitive function, learning, memory, and social behaviors to identify potential neurological effects .

• Electrophysiological recordings: Implement patch-clamp techniques and field potential recordings to directly measure how CCL17 affects synaptic transmission and neuronal excitability in hippocampal slices.

• Neuronal culture systems: Establish primary neuronal cultures or organoids to examine CCL17's direct effects on neuronal development, axonal growth, and synapse formation using immunocytochemistry and live imaging techniques.

These approaches should be combined with rigorous controls and quantitative analysis to elucidate the precise mechanisms through which CCL17 influences neurological function.

What evidence supports CCL17 as a therapeutic target for heart failure?

Multiple lines of evidence support CCL17 as a promising therapeutic target for heart failure (HF), particularly age-related and angiotensin II-induced cardiac dysfunction:

• Increased expression in cardiac dysfunction: Analysis of left ventricular transcriptomes from the Gene Expression Omnibus database revealed significantly higher CCL17 expression in HF patients compared to non-HF controls. This finding was corroborated by elevated serum CCL17 levels in HF patients .

• Age-dependent increases: Population studies demonstrated that circulating CCL17 levels show an age-dependent increase, correlating with decline in cardiac function. In mouse models, CCL17 expression in serum also increased with age .

• Response to treatment: In a clinical study of 17 patients with acute decompensated HF, circulating CCL17 levels decreased following standard treatment as cardiac function recovered. This correlation between CCL17 reduction and symptom improvement suggests a functional relationship .

• Experimental validation: In knockout models, Ccl17-KO mice showed significant protection against aging and angiotensin II-induced cardiac hypertrophy and fibrosis. Furthermore, therapeutic administration of anti-CCL17 neutralizing antibodies markedly inhibited angiotensin II-induced pathological cardiac remodeling without affecting blood pressure or heart rate .

• Mechanistic understanding: CCL17 appears to promote cardiac dysfunction by recruiting T helper 2 (Th2) cells through binding to CCR4, disrupting T cell subset balance, and promoting the release of fibrotic factors (predominantly IL-4 and IL-17), ultimately resulting in cardiac hypertrophy, fibrosis, and subsequent heart failure .

These findings collectively establish CCL17 as a promising therapeutic target in age-related and angiotensin II-induced pathological cardiac conditions.

What methodological approaches are effective for measuring CCL17 in cardiovascular research?

For robust assessment of CCL17 in cardiovascular research, the following methodological approaches are recommended:

• Serum protein quantification:

  • ELISA or multiplex immunoassay platforms for precise quantification of circulating CCL17 levels

  • SOMAscan aptamer technology, which has successfully identified CCL17 as significantly associated with age in population studies

• Transcriptomic analysis:

  • RNA sequencing of cardiac tissue to evaluate CCL17 expression levels

  • Quantitative RT-PCR to measure CCL17 mRNA expression in different cardiac regions

• In vivo modeling:

  • Angiotensin II infusion models to induce pathological cardiac remodeling

  • Age-related cardiac dysfunction models to assess natural progression

  • Ccl17-knockout models to evaluate cardioprotective effects

• Therapeutic intervention assessment:

  • Anti-CCL17 neutralizing antibody administration

  • Monitoring of cardiac function via echocardiography

  • Histological analysis for fibrosis and hypertrophy using:

    • Masson's trichrome staining for fibrosis

    • Wheat germ agglutinin staining for cardiomyocyte area

    • Expression analysis of hypertrophy-associated genes (ANP, BNP, β-MHC)

• T cell analysis:

  • Flow cytometry to quantify T cell subpopulations

  • Analysis of CCR4 expression on cardiac-infiltrating immune cells

These methodologies have been validated in research demonstrating CCL17's role in cardiac pathology and can be employed to further elucidate mechanisms and therapeutic potential .

How is CCL17 associated with obesity and insulin resistance?

CCL17 shows significant associations with obesity and insulin resistance through multiple mechanisms:

• Elevated circulating levels in obesity: Clinical studies have demonstrated that plasma levels of CCL17 are significantly elevated in patients with morbid obesity (median 67.8 pg/mL, range 16.6–185.6 pg/ml) compared to control subjects (median 51.8 pg/mL, range 22.2–84.5 pg/mL, p=0.029) . This elevation suggests a potential role in obesity-associated inflammation.

• Correlation with metabolic parameters: Spearman correlation analysis has revealed positive correlations between circulating CCL17 levels and key metabolic parameters:

  • HOMA-IR index (r=0.233, p=0.042), indicating a relationship with insulin resistance

  • BMI (r=0.283, p=0.013), showing a direct association with degree of obesity

• Parallel elevation with CCL22: Similar to CCL17, its related chemokine CCL22 also shows elevated levels in morbidly obese patients. CCL22 displays even stronger correlations with HOMA-IR (r=0.38, p<0.001) and BMI (r=0.359, p=0.0016) .

• Tissue expression patterns: Research examining paired subcutaneous (SCAT) and visceral adipose tissue (VCAT) samples has shown differential expression patterns of CCL17 and its receptor CCR4 in adipose tissues of obese patients .

• Functional effects on leukocyte-endothelial interactions: Ex vivo studies have demonstrated that neutralization of CCR4 (the receptor for CCL17) affects leukocyte-endothelial cell interactions, suggesting a role in the inflammatory processes associated with obesity .

These findings suggest that CCL17 may serve as a biomarker for obesity-related metabolic dysregulation and potentially participate in the pathophysiological processes linking obesity with systemic inflammation and insulin resistance.

What experimental protocols are recommended for studying CCL17 in adipose tissue?

When investigating CCL17 in adipose tissue, the following experimental protocols are recommended based on current research methodologies:

• Tissue sampling and processing:

  • Obtain paired subcutaneous (SCAT) and visceral adipose tissue (VCAT) samples to account for depot-specific differences

  • Process samples immediately after collection to preserve RNA and protein integrity

  • Create multiple aliquots for different analytical approaches (RNA extraction, protein isolation, histological analysis)

• Gene expression analysis:

  • Extract RNA using specialized protocols optimized for adipose tissue (addressing high lipid content)

  • Perform RT-PCR to quantify CCL17 and CCR4 expression levels

  • Validate with droplet digital PCR for absolute quantification in samples with low expression levels

• Protein quantification:

  • Extract proteins using methods that address the high lipid content of adipose tissue

  • Perform western blot analysis to determine CCL17 and CCR4 protein levels

  • Use ELISA to quantify CCL17 secretion in adipose tissue explant cultures

• Tissue localization studies:

  • Conduct immunohistochemical analysis to determine the cellular localization of CCL17 and CCR4

  • Use fluorescence microscopy with cell-specific markers to identify which cells (adipocytes, macrophages, endothelial cells) express CCL17/CCR4

• Functional assays:

  • Establish ex vivo leukocyte-endothelial cell interaction assays using isolated cells from adipose tissue

  • Test CCR4 neutralization effects on leukocyte adhesion and migration

  • Measure cytokine/adipokine production in adipose tissue explants with and without CCL17 stimulation or CCR4 blockade

• Correlation with clinical parameters:

  • Collect comprehensive metabolic data including BMI, HOMA-IR, lipid profiles

  • Apply statistical methods such as Spearman correlation test to assess relationships between CCL17 levels and metabolic parameters

  • Use Kolmogorov-Smirnov test for normality distribution and appropriate parametric or non-parametric tests (Student's t-test or Mann-Whitney U test) for group comparisons

These protocols have been effectively employed in research demonstrating CCL17 upregulation in human obesity and can be adapted for different research questions related to CCL17's role in metabolic disorders.

How does CCL17 contribute to inflammatory responses and immune regulation?

CCL17 serves as a critical mediator in inflammatory responses and immune regulation through several mechanisms:

• Chemotactic activity: As a chemokine, CCL17 exhibits potent chemotactic properties that attract specific immune cell populations, particularly activated T cells, to sites of inflammation. CCL17 binds to the CCR4 receptor, which is expressed on various T cell subsets, facilitating their recruitment and localization within inflammatory sites .

• Dendritic cell-T cell interactions: CCL17 is predominantly produced by mature dendritic cells (DCs) and activated Langerhans cells, suggesting a role in orchestrating the interaction between antigen-presenting cells and T lymphocytes. This interaction is crucial for initiating and regulating adaptive immune responses .

• T cell subset modulation: Research indicates that CCL17 influences the balance and plasticity of T cell subsets, particularly promoting T helper 2 (Th2) cell recruitment through binding to CCR4. This preferential recruitment can shape the nature of the immune response, potentially biasing it toward allergic or anti-parasitic reactions .

• Contact hypersensitivity regulation: Studies using CCL17-knockout mice have demonstrated reduced contact hypersensitivity responses, indicating CCL17's role in mediating delayed-type hypersensitivity reactions, which are T cell-dependent inflammatory responses .

• Tissue-specific immune regulation: The compartmentalized expression of CCL17 in lymphoid and non-lymphoid organs, but notably absent in the spleen even during systemic infection, suggests a role in directing immune responses to specific anatomical locations where environmental antigenic stimulation is prevalent .

• Allograft rejection: CCL17 deficiency results in delayed allograft rejection, indicating its role in transplant-related immune responses and potentially in promoting T cell-mediated rejection of foreign tissues .

• Age-related inflammation: CCL17 levels increase with age, suggesting a potential contribution to age-associated chronic inflammation ("inflammaging"), which may link to age-related pathologies including cardiac dysfunction .

These multifaceted roles position CCL17 as a significant regulator of inflammatory processes, with importance in both physiological immune responses and pathological inflammation.

What methods are most effective for studying CCL17 in inflammatory disease models?

For investigating CCL17 in inflammatory disease models, the following methodological approaches are recommended based on established research:

• In vivo reporter systems:

  • Utilize fluorescence-based reporter mouse models with EGFP cassette insertion into the endogenous CCL17 locus

  • This approach enables tracking of CCL17-expressing cells in vivo under various inflammatory conditions

  • Coupling CCL17 release with fluorescent protein production allows visualization of production sites under microscopy

• Knockout/transgenic models:

  • Employ Ccl17-knockout mice to assess the functional relevance of CCL17 in specific inflammatory models

  • Use conditional knockout approaches for tissue-specific deletion of CCL17 or CCR4

  • Compare phenotypes in inflammatory challenges such as contact hypersensitivity and allograft transplantation models

• Toll-like receptor stimulation protocols:

  • Apply TLR ligands to upregulate CCL17 expression in dendritic cells and other potential sources

  • Monitor kinetics of CCL17 induction following various inflammatory stimuli

  • Analyze tissue-specific differences in CCL17 responsiveness to TLR stimulation

• Flow cytometry and cell sorting:

  • Characterize CCL17-expressing cells using multiparameter flow cytometry

  • Identify and isolate dendritic cell subsets based on CD11b and other markers

  • Analyze CCR4 expression on responding T cell populations

• Neutralization studies:

  • Administer anti-CCL17 neutralizing antibodies in vivo to block its function during inflammatory responses

  • Assess the timing-dependent effects of CCL17 neutralization on disease progression

  • Compare with CCR4 antagonism to distinguish receptor-specific effects

• Ex vivo functional assays:

  • Establish leukocyte-endothelial cell interaction assays to measure adhesion and migration

  • Perform T cell chemotaxis assays to quantify CCL17-directed migration

  • Use adoptive transfer experiments with labeled T cells to track their migration in vivo

• Histopathological analysis:

  • Conduct detailed immunohistochemical studies of inflamed tissues

  • Quantify immune cell infiltration in target organs

  • Correlate CCL17 expression with inflammatory markers and tissue damage parameters

These methodologies provide complementary approaches to dissect CCL17's role in inflammatory conditions and have been validated in research demonstrating its importance in various disease models .

What is the evidence for targeting CCL17 in treatment strategies?

Evidence supporting CCL17 as a therapeutic target spans multiple disease areas, with particularly strong data in the following conditions:

• Cardiac dysfunction and heart failure:

  • Ccl17-knockout mice show protection against both age-related and angiotensin II-induced cardiac hypertrophy and fibrosis

  • Therapeutic administration of anti-CCL17 neutralizing antibodies significantly attenuates angiotensin II-induced cardiac remodeling and dysfunction

  • The intervention did not affect blood pressure or heart rate, suggesting a direct cardioprotective effect rather than hemodynamic changes

  • The therapeutic benefit appears to involve modulation of T cell subset balance and reduction in fibrotic factors (IL-4 and IL-17)

• Allergic conditions:

  • High CCL17 levels are established diagnostic markers for allergic diseases including atopic eczema

  • CCL17 promotes Th2 cell recruitment and allergic inflammation

  • Blocking CCL17-CCR4 interactions could potentially reduce allergic symptoms

• Contact hypersensitivity and allograft rejection:

  • CCL17-knockout mice demonstrate reduced contact hypersensitivity responses

  • Absence of CCL17 leads to delayed allograft rejection, suggesting potential application in transplantation medicine

• Obesity and metabolic dysfunction:

  • Elevated CCL17 levels correlate with obesity, BMI, and insulin resistance

  • CCL17 may represent a therapeutic target for obesity-associated inflammation and metabolic disorders

• Neurological conditions:

  • Evidence of CCL17 production in hippocampal neurons suggests potential roles in neurological disorders

  • Behavioral abnormalities in animal models with CCL17 receptor expression defects point to possible applications in neurodevelopmental conditions like autism

The therapeutic potential is further supported by:

• Age-dependent increases in CCL17 that correlate with age-related diseases

• Mechanistic understanding of CCL17's role in promoting pathological inflammation through CCR4-mediated T cell recruitment

• Successful demonstration of antibody-based neutralization strategies in preclinical models

• Correlation between decreased CCL17 levels and clinical improvement in heart failure patients

These findings collectively establish CCL17 as a promising therapeutic target across multiple disease areas, with cardiac and inflammatory conditions showing the most advanced evidence base.

What are the optimal experimental conditions for recombinant CCL17 in cellular assays?

For optimal use of recombinant human CCL17 in cellular assays, the following experimental conditions are recommended based on established research protocols:

Protein Handling and Preparation:

• Store lyophilized recombinant CCL17 at -20°C to -80°C

• Reconstitute in sterile, ultrapure water or buffer (PBS with 0.1% BSA) at concentrations of 0.1-1.0 mg/ml

• Allow protein to completely dissolve before use (gentle agitation without vortexing)

• Prepare single-use aliquots to avoid freeze-thaw cycles

• For short-term storage (≤2 weeks), keep reconstituted protein at 4°C

• For long-term storage, maintain aliquots at -80°C

Concentration Ranges for Different Assay Types:

• Chemotaxis assays:

  • Effective concentration range: 10-100 ng/ml

  • Optimal concentration for T cell migration: ~50 ng/ml

  • Use freshly prepared chemokine solutions

  • Include positive controls (e.g., CXCL12/SDF-1α) and vehicle controls

• Cell stimulation experiments:

  • For dendritic cells and T cells: 10-200 ng/ml

  • Duration: 15 minutes for signaling studies, 4-24 hours for gene expression analysis

  • Pre-warm media and CCL17 solutions to 37°C before addition to cells

• Receptor binding studies:

  • Concentration range: 1-500 ng/ml for dose-response curves

  • Use freshly isolated primary cells or stable CCR4-expressing cell lines

  • Perform binding at 4°C to prevent receptor internalization

• Functional blocking experiments:

  • Pre-incubate CCL17 with neutralizing antibodies (typically 1:5 to 1:10 molar ratio)

  • Allow 30-minute incubation at room temperature before adding to cells

  • Include isotype control antibodies as negative controls

Cell Systems and Compatible Media:

• T cell assays: RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, antibiotics

• Dendritic cell assays: IMDM or RPMI with appropriate supplements

• Neuronal cultures: Neurobasal medium with B27 supplement for CCL17 effects on neurons

• Cardiomyocyte assays: Specialized cardiac cell media for testing CCL17 effects on cardiac cells

Quality Control Parameters:

• Verify protein activity using a reliable bioassay (e.g., chemotaxis of CCR4+ cells)

• Confirm protein purity by SDS-PAGE (>95% purity recommended)

• Test for endotoxin contamination, especially for immunological experiments (<0.1 EU/μg protein)

• Validate batch-to-batch consistency before conducting critical experiments

These conditions will help ensure reproducible results when working with recombinant human CCL17 across different experimental systems and research applications.

How do CCL17 and CCL22 differ functionally despite sharing the CCR4 receptor?

Despite both binding to the CCR4 receptor, CCL17 and CCL22 exhibit important functional differences that researchers should consider:

• Age-dependent expression patterns:

  • CCL17 shows significant age-dependent increases in expression

  • In contrast, CCL22 expression does not increase with age, suggesting different roles in age-related processes

• Receptor binding characteristics:

  • While both chemokines bind CCR4, they may interact with different domains or with different affinities

  • These binding differences can result in distinct downstream signaling patterns and cellular responses

• Disease associations:

  • In obesity, both CCL17 and CCL22 show elevated levels, but CCL22 demonstrates stronger correlations with metabolic parameters:

    • CCL22 shows stronger correlation with HOMA-IR (r=0.38) compared to CCL17 (r=0.233)

    • CCL22 exhibits stronger correlation with BMI (r=0.359) than CCL17 (r=0.283)

• Tissue-specific expression:

  • CCL17 expression is predominantly found in mature dendritic cells and shows tissue-specific regulation patterns

  • CCL17 cannot be induced in the spleen even during systemic infection, while such restriction has not been reported for CCL22

• Cell type specificity:

  • CCL17 is primarily produced by CD11b+ dendritic cells and Langerhans cells

  • CCL22 may be produced by a broader range of cell types, including macrophages and B cells

• Therapeutic implications:

  • Anti-CCL17 neutralizing antibodies have shown significant protective effects in cardiac dysfunction models

  • The specific targeting of CCL17 rather than general CCR4 blockade may provide more selective therapeutic effects with potentially fewer side effects

• Temporal dynamics:

  • The two chemokines may be produced with different kinetics during immune responses

  • This temporal regulation could create sequential gradients that orchestrate different phases of leukocyte recruitment

These functional differences highlight the importance of studying CCL17 and CCL22 individually, despite their shared receptor, and suggest potential advantages to targeting each chemokine specifically depending on the pathological context.

What are the major technical challenges in measuring CCL17 activity in complex biological samples?

Researchers face several significant technical challenges when measuring CCL17 activity in complex biological samples:

• Low physiological concentrations:

  • CCL17 typically circulates at picogram/ml levels (e.g., median 67.8 pg/ml in obese patients, 51.8 pg/ml in controls)

  • These low concentrations require highly sensitive detection methods with appropriate lower limits of quantification

• Sample matrix interference:

  • Biological fluids (serum, plasma, tissue homogenates) contain proteins, lipids, and other molecules that can interfere with assay performance

  • Specific challenges include:

    • Non-specific binding to other proteins

    • Presence of autoantibodies against chemokines

    • Complement activation in improperly handled samples

    • Hemolysis affecting assay readouts

• Protein degradation and stability issues:

  • CCL17 may undergo proteolytic degradation during sample collection and processing

  • Multiple freeze-thaw cycles can significantly reduce detectable chemokine levels

  • Some storage conditions may promote protein aggregation or adsorption to container surfaces

• Heterogeneous forms of CCL17:

  • Post-translational modifications can generate multiple forms with different activities

  • N-terminal processing by proteases can alter receptor binding affinity

  • Distinction between active and inactive forms requires functional rather than just immunological detection

• Cross-reactivity concerns:

  • Structural similarities between chemokines may lead to cross-reactivity in immunoassays

  • Particularly challenging is distinguishing between CCL17 and other CC chemokines in multiplex assays

• Receptor occupancy:

  • Endogenous CCL17 bound to CCR4 or to decoy receptors may not be detectable in standard assays

  • Acid dissociation protocols may be necessary to measure total rather than just free chemokine

• Standardization challenges:

  • Different recombinant standards used across commercial assays lead to variability

  • Lack of international reference standards for CCL17 complicates cross-study comparisons

• Biological activity assessment:

  • Immunoassays detect protein presence but not necessarily functional activity

  • Cell-based functional assays are more physiologically relevant but have higher variability and are more resource-intensive

To address these challenges, researchers should:

• Use standardized collection protocols with protease inhibitors

• Limit freeze-thaw cycles and use consistent storage conditions

• Incorporate appropriate controls to account for matrix effects

• Consider using multiple detection methods (e.g., both ELISA and bioassay)

• Validate assays specifically for each biological matrix of interest

• Include recovery experiments with known amounts of recombinant CCL17

These strategies can improve the reliability and reproducibility of CCL17 measurements in complex biological samples.