Recombinant Rat C-C motif chemokine protein (Ccl17) (Active)

How does the three-dimensional structure of Rat CCL17 influence its receptor binding capability?

Rat CCL17 adopts the canonical chemokine fold consisting of three β-strands and a C-terminal α-helix. The N-terminal region preceding the first cysteine residue is critical for receptor activation, while the N-loop region (following the CC motif) is essential for receptor binding specificity. The presence of two disulfide bonds (C11-C35 and C12-C51) stabilizes the tertiary structure, which is crucial for maintaining the spatial orientation of the receptor-binding domains. This precise structural arrangement enables specific interaction with CCR4 receptors on target cells, facilitating chemotaxis of CCR4-expressing cells at concentrations of 1.0-10 ng/ml .

What are the optimal storage and reconstitution conditions to maintain CCL17 bioactivity?

For optimal bioactivity maintenance:

Reconstitution protocol:

• Centrifuge the vial briefly before opening to collect contents at the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%)

• Aliquot to minimize freeze-thaw cycles

Storage conditions:

• Long-term storage: -20°C/-80°C in aliquots

• Working aliquots: 4°C for up to one week

• Avoid repeated freeze-thaw cycles which reduce activity

The reconstituted protein maintains stability for up to 6 months at -20°C/-80°C when properly aliquoted with glycerol as a cryoprotectant . The lyophilized form is shipped with buffer components from a 0.2 μm filtered PBS, pH 7.4, which provides optimal stability during reconstitution.

What are the validated methods for accurately assessing CCL17 bioactivity in vitro?

Bioactivity of recombinant Rat CCL17 can be assessed through multiple complementary approaches:

Primary method - Chemotaxis bioassay:

• Isolate human or rat T-lymphocytes using density gradient centrifugation

• Prepare serial dilutions of CCL17 (1.0-10 ng/ml) in chemotaxis buffer

• Place 25-50 μl of cell suspension (1-2×10⁶ cells/ml) in the upper chamber of a transwell system

• Add CCL17 dilutions to the lower chamber

• Incubate at 37°C with 5% CO₂ for 2-3 hours

• Count migrated cells using flow cytometry or hemocytometer

• Calculate chemotaxis index: (migrated cells with chemokine)/(migrated cells without chemokine)

Active CCL17 typically induces significant chemotaxis in the 1.0-10 ng/ml concentration range .

Secondary validation methods:

• Calcium flux assay in CCR4-expressing cells

• Phosphorylation of downstream signaling molecules (e.g., ERK1/2, AKT)

• Receptor internalization assay using CCR4-expressing cells

These complementary approaches provide comprehensive validation of functional activity beyond simple binding assays.

How can researchers distinguish between CCL17-mediated and other chemokine-mediated effects in complex in vivo models?

To distinguish CCL17-specific effects from other chemokine activities:

Genetic approaches:

• Use CCL17E/E knockout models, where the CCL17 gene is replaced with a fluorescent reporter (EGFP)

• Employ CCR4-deficient models to eliminate the primary receptor response

• Create conditional cell-specific CCL17 knockout models using Cre-loxP systems

Pharmacological approaches:

• Use CCR4-specific antagonists such as AZD2098 to block CCL17 signaling

• Apply neutralizing antibodies against CCL17 with validated specificity

• Administer competitive inhibitors that displace CCL17 from its receptor

Experimental design considerations:

• Include appropriate controls for chemokine redundancy analysis

• Perform dose-response studies to identify concentration-dependent effects

• Compare responses between wild-type and CCL17-deficient animals in parallel experiments

This multi-faceted approach allows researchers to attribute observed effects specifically to CCL17 activity rather than to other chemokines that may have overlapping functions.

What is the neurobiological mechanism by which CCL17 exerts neuroprotection in subarachnoid hemorrhage models?

CCL17 provides neuroprotection in subarachnoid hemorrhage (SAH) through a multi-step mechanism:

• Primary signaling axis: CCL17 binds to CCR4 receptors on microglia, activating the mTORC2 pathway

• Microglial polarization: This activation shifts microglia from pro-inflammatory (M1-like) to anti-inflammatory (M2-like) phenotypes

• Morphological changes: Treatment with recombinant CCL17 (rCCL17) reverses SAH-induced alterations in microglial morphology, restoring normal branching patterns, endpoint numbers, and process lengths

• Gene expression modification: rCCL17 increases expression of M2-like microglia-associated genes

• Neuronal protection: The polarized microglia secrete anti-inflammatory cytokines and growth factors that protect neurons from apoptosis

The neuroprotective effect is abolished by inhibiting either CCR4 (with AZD2098) or mTORC2 (with JR-AB2-011), confirming the specificity of this pathway . In rat SAH models, exogenous rCCL17 administration significantly reduces neuronal death and improves neurological function through this mechanism.

How should researchers design experiments to study the role of CCL17 in modulating microglial polarization after brain injury?

Experimental design for studying CCL17-mediated microglial polarization:

In vivo experimental approach:

• Create rat/mouse SAH model using established protocols

• Administer rCCL17 intracerebroventricularly at different time points (0, 3, 6, 12, 24 hours post-injury)

• Implement the following experimental groups:

  • Sham (surgery without SAH)

  • SAH + vehicle (PBS)

  • SAH + rCCL17

  • SAH + rCCL17 + CCR4 inhibitor (AZD2098)

  • SAH + rCCL17 + mTORC2 inhibitor (JR-AB2-011)

• Assess microglial morphology using:

  • Immunofluorescent staining with microglial markers (Iba1)

  • Morphological analysis with ImageJ plugins to quantify:

    • Number of branches

    • Number of endpoints

    • Process lengths

In vitro validation:

• Isolate primary microglia from rat/mouse brains

• Stimulate with:

  • rCCL17 alone

  • rCCL17 + CCR4 inhibitor

  • rCCL17 + mTORC2 inhibitor

• Analyze polarization markers via:

  • RT-qPCR for M1/M2 gene expression profiles

  • Flow cytometry for surface marker expression

  • Cytokine/chemokine secretion profiles via ELISA/multiplex assays

Key measurements:

• mRNA expression of M2-like markers

• Protein levels of phosphorylated Akt at Ser473 (mTORC2 activity indicator)

• Neuronal apoptosis (TUNEL assay)

• Neurological function (behavior tests)

• Brain edema (brain water content)

This comprehensive approach enables researchers to establish both the causality and the mechanistic pathway of CCL17's effects on microglial polarization following brain injury.

How is CCL17 expression regulated in different dendritic cell populations, and what methods can detect this expression?

CCL17 expression in dendritic cells (DCs) demonstrates complex compartmentalized regulation:

Cell type-specific expression:

• CCL17 is predominantly expressed in CD11b+ DCs

• Activated Langerhans cells show high expression

• Mature DCs in lymphoid and non-lymphoid organs express CCL17

• Expression is minimal in plasmacytoid DCs

Regulatory mechanisms:

• Toll-like receptor (TLR) stimulation: CCL17 expression is upregulated after stimulation with TLR ligands

• Cytokine environment: Th2 cytokines (IL-4, IL-13) enhance CCL17 production

• Maturation state: Expression increases with DC maturation

• Tissue microenvironment: Different expression patterns in skin, lymph nodes, and other tissues

Detection methods:

Researchers can utilize CCL17/EGFP reporter systems (CCL17E/+ or CCL17E/E) to visualize CCL17-expressing cells in vivo without disrupting tissue architecture , providing valuable insights into the dynamic regulation of CCL17 in different DC populations.

What are the optimal experimental protocols for studying CCL17-dependent T cell activation and migration?

Protocol for studying CCL17-mediated T cell responses:

T cell migration assay:

• Isolate CD4+ T cells from lymph nodes and spleen using MACS purification

• Pre-activate T cells with anti-CD3/CD28 for 48 hours (to upregulate CCR4)

• Prepare chemotaxis chambers:

  • Add rCCL17 (0.1-100 ng/ml) to lower chamber

  • Place 5×10⁵ activated T cells in upper chamber

• Incubate for 2-3 hours at 37°C

• Count migrated cells by flow cytometry

• Calculate chemotactic index for each concentration

T cell activation/proliferation assay:

• Generate bone marrow-derived or lymph node-derived DCs

• Pulse DCs with antigenic peptide (e.g., CRP peptide at 1 μg/ml)

• Co-culture 5×10³ DCs with MACS-purified CD4+ T cells

• Add rCCL17 at various concentrations (0-100 ng/ml)

• Measure proliferation by ³H-thymidine incorporation (add 1 μCi/well during last 17 hours of 72-hour culture)

• Alternatively, use CFSE dilution assay to track cell division

• Analyze T cell activation markers (CD25, CD69, CD44) by flow cytometry

• Measure cytokine production (IL-2, IFN-γ, IL-4) by ELISA

Controls and validation:

• Include CCR4 antagonist (AZD2098) to confirm specificity

• Compare responses between wild-type and CCR4-deficient T cells

• Use CCL17-neutralizing antibodies as additional controls

• Include other chemokines (CCL22) that share the CCR4 receptor to assess specificity

This comprehensive approach allows researchers to distinguish CCL17-specific effects on T cell behavior from those mediated by other chemokines or signaling pathways.

How can researchers effectively utilize CCL17 knockout and reporter models to investigate chemokine network redundancy?

Strategic approaches for investigating chemokine redundancy using CCL17 models:

Genetic model utilization:

• Compare phenotypes: Analyze CCL17E/E (homozygous knockout) vs. CCL17E/+ (heterozygous) vs. wild-type mice to identify dose-dependent effects

• Create compound knockouts: Generate CCL17/CCL22 double knockouts to assess redundancy within the CCR4 ligand family

• Cell-specific deletion: Use conditional knockouts (Cre-loxP system) targeting specific cell populations:

  • DC-specific deletion using CD11c-Cre

  • Neuronal deletion using Thy1-Cre

  • Microglial deletion using CX3CR1-CreER

Experimental approaches:

• Challenge models: Subject knockout models to various disease challenges:

  • Neuroinflammation (SAH, stroke models)

  • Allergic inflammation

  • Infectious disease models

• Compensatory mechanism analysis:

  • Measure expression of related chemokines (CCL22, CCL1, CCL8) in CCL17-deficient models

  • Analyze receptor expression changes (CCR4, CCR8) in the absence of CCL17

  • Profile transcriptome changes in target cells using RNA-seq

Reporter system applications:

• Dynamic visualization: Use CCL17/EGFP reporter mice to track spatiotemporal expression patterns in:

  • Development

  • Disease progression

  • Response to therapeutic interventions

• Cell isolation strategies: Sort EGFP+ cells from CCL17E/+ mice for detailed molecular profiling

• Compound reporter systems: Cross with other reporter strains to simultaneously track multiple components of chemokine networks

This multi-dimensional approach enables researchers to systematically map the redundant and non-redundant functions of CCL17 within complex chemokine networks, revealing both unique and shared roles across different physiological and pathological contexts.

What methodological considerations are important when investigating CCL17/CCR4/mTORC2 signaling in neurological disorders beyond subarachnoid hemorrhage?

Methodological framework for studying CCL17 signaling in diverse neurological contexts:

Experimental design considerations:

• Disease model selection:

  • Acute models: Traumatic brain injury, ischemic stroke, intracerebral hemorrhage

  • Chronic models: Alzheimer's disease, Parkinson's disease, multiple sclerosis

  • Developmental models: Autism spectrum disorders, schizophrenia

• Temporal profiling:

  • Establish baseline CCL17 expression in healthy brain regions

  • Map temporal expression changes after injury/disease onset (acute, subacute, chronic phases)

  • Correlate expression with disease progression markers

• Cell-specific analysis:

  • Identify CCL17-producing cells in different neurological conditions

  • Characterize CCR4 expression on target cell populations

  • Determine mTORC2 activation status across cell types

Technical approaches:

• In vivo signaling visualization:

  • Implement intravital microscopy with CCL17/EGFP reporter mice

  • Use phospho-specific antibodies (p-Akt Ser473) to track mTORC2 activation

  • Apply CLARITY or iDISCO techniques for whole-brain CCL17/CCR4 mapping

• Pharmacological interventions:

  • Design administration protocols for rCCL17 considering blood-brain barrier permeability

  • Implement CCR4 antagonist (AZD2098) and mTORC2 inhibitor (JR-AB2-011) at disease-appropriate timepoints

  • Consider combination with standard-of-care treatments

• Microglial-specific manipulations:

  • Implement microglial-specific Rictor knockdown via adenovirus-associated virus delivery

  • Use CX3CR1-CreER systems for inducible gene modification

  • Apply colony-stimulating factor 1 receptor inhibitors for microglial depletion/repopulation studies

Outcome measures:

• Neurological function assessment (behavior tests specific to each model)

• Neuroinflammatory profiling (cytokine/chemokine arrays)

• Cellular response characterization (microglial morphology and polarization)

• Neurodegeneration/neuroprotection markers (apoptosis, synaptic density)

• Brain water content and blood-brain barrier integrity

This comprehensive approach enables researchers to establish whether the CCL17/CCR4/mTORC2 pathway represents a conserved neuroprotective mechanism across different neurological disorders or has context-specific functions that vary by disease etiology and progression.

What are the common technical challenges when working with recombinant CCL17 in experimental systems, and how can they be overcome?

Common challenges and solutions when working with recombinant CCL17:

Additional technical considerations:

• Storage stability:

  • Minimize freeze-thaw cycles by creating single-use aliquots

  • Add glycerol (50% final concentration) as cryoprotectant

  • Monitor solution clarity before each use (cloudiness indicates aggregation)

• Species cross-reactivity issues:

  • Rat CCL17 may have different potency on human vs. rat cells

  • Validate species-specific activity before cross-species experiments

  • Consider using species-matched systems when possible

• Detection limitations:

  • Use antibodies validated specifically for rat CCL17

  • Confirm specificity with appropriate controls (CCL17-deficient samples)

  • Consider developing sandwich ELISA with capture/detection antibody pairs

These evidence-based solutions help researchers troubleshoot common technical challenges when working with recombinant rat CCL17, ensuring consistent and reliable experimental results.

How can researchers accurately quantify and differentiate between endogenous and exogenous CCL17 in experimental models?

Strategies for distinguishing endogenous from exogenous CCL17:

Protein modification approaches:

• Tagged recombinant proteins:

  • Use His-tagged CCL17 for exogenous administration

  • Detect via anti-His antibodies in Western blot or immunohistochemistry

  • Purify using Ni-NTA pull-down from tissue lysates

• Fluorescent labeling:

  • Conjugate recombinant CCL17 with fluorophores (e.g., Alexa Fluor dyes)

  • Track distribution via microscopy or flow cytometry

  • Note: Verify that labeling doesn't affect bioactivity

Genetic approaches:

• Species differences:

  • Administer human CCL17 in rat models

  • Use species-specific primers or antibodies for differential detection

  • Example primers for rat CCL17: pCCL17-US 5′-CAT GTG AAG AAG GCC ATC AGA TTG GTG-3′ and pCCL17-DS 5′-GAG GGA GGA AGG CTT TAT TCC GTT GC-3′

• Reporter systems:

  • Use CCL17E/E knockout/reporter mice that express EGFP instead of CCL17

  • Any detected CCL17 protein must be exogenous

  • EGFP fluorescence indicates cells that would normally produce CCL17

Analytical methods:

• Temporal discrimination:

  • Measure baseline CCL17 before exogenous administration

  • Track concentration changes with high temporal resolution

  • Account for potential endogenous upregulation due to experimental procedures

• Spatial distribution analysis:

  • Map normal tissue distribution of endogenous CCL17

  • Identify abnormal distribution patterns after exogenous administration

  • Use tissue microarrays for high-throughput analysis

• Mass spectrometry approaches:

  • Use isotope-labeled recombinant CCL17 for exogenous administration

  • Perform mass spectrometry to differentiate labeled vs. unlabeled protein

  • Enables precise quantification of both pools simultaneously

This comprehensive toolkit allows researchers to accurately track and distinguish exogenous CCL17 from endogenous production, enabling more precise interpretation of experimental results in complex biological systems.