Recombinant Human C-C motif chemokine 8 protein (CCL8) (Active)

What is CCL8 and what are its primary biological functions?

CCL8, also known as monocyte chemotactic protein-2 (MCP-2), is a chemoattractive cytokine belonging to the CC chemokine sub-family. It plays pivotal roles in leukocyte chemotaxis, inflammatory response regulation, and has been implicated in HIV entry mechanisms. CCL8 functions primarily as a signaling protein that coordinates immune cell recruitment and activation during inflammatory responses. Its biological activity is mediated through interactions with multiple chemokine receptors, particularly CCR2 and CCR5, with additional evidence supporting interaction with CCR3 . The protein is notably involved in various immune-related pathologies and has emerged as a significant factor in severe respiratory conditions, including COVID-19-associated acute respiratory distress syndrome .

Which receptors does CCL8 interact with and what are their binding characteristics?

CCL8 has been experimentally confirmed to interact with multiple chemokine receptors:

What is the recommended protocol for soluble expression of functional CCL8 in E. coli?

For efficient production of soluble and functional CCL8 in E. coli, the following optimized protocol has been developed:

• Expression System Selection: E. coli has proven effective for recombinant CCL8 production, yielding approximately 1.5 mg protein per liter of culture .

• Expression Region: Focus on the 24-99aa region of the full-length mature protein for optimal expression .

• Purification Approach: Implement systematic optimization of expression conditions to maximize soluble protein yield and minimize inclusion body formation, which is a common challenge with chemokine expression .

• Quality Control Metrics:

  • Confirm >96% purity via SDS-PAGE and HPLC analysis

  • Ensure endotoxin levels remain below 1.0 EU/μg (determined by LAL method)

  • Verify biological activity through functional assays

Despite the structural similarities among chemokines, it's important to note that different optimal expression conditions may be required for various chemokines. For instance, while this protocol works well for CCL8, CCL11, and CCL24, it may not achieve comparable results for CCL5 (RANTES), which requires alternative approaches .

How should researchers verify the functional activity of recombinant CCL8?

Verification of CCL8 functionality should include multiple complementary approaches:

• In vitro Binding Assays:

  • Utilize Quartz Crystal Microbalance (QCM) to determine binding kinetics with known receptors (CCR2, CCR3, CCR5)

  • Measure association rate (ka) and dissociation rate (kd) constants to calculate the KD value

• Receptor Internalization Assays:

  • Expose cells expressing the target receptor (e.g., CCR3-EGFP) to 100 nM CCL8

  • Monitor receptor internalization via fluorescence microscopy over time (significant internalization typically observable within 1 hour)

  • Compare against established CCR3 agonists like CCL11 or CCL24 as positive controls

• Chemotaxis Tests:

  • Employ Transwell chamber assays using receptor-transfected cells (e.g., TRx-HEK293 cells stably expressing CCR3-EGFP)

  • Calculate chemotactic index by comparing migration toward CCL8 versus random migration

  • Benchmark against established chemokines with known activity profiles

These combined approaches provide comprehensive confirmation of functional activity and receptor specificity.

What role does CCL8 play in acute lung injury and how can its inhibition be therapeutically relevant?

CCL8 appears to play a permissive role in mediating cytokine induction and sustaining inflammation during acute lung injury. In LPS-induced lung injury models, CCL8 inhibition demonstrates significant therapeutic potential:

• Inflammatory Mechanism: While CCL8 expression itself is not necessarily enhanced following LPS administration, the enhanced expression of its receptors (particularly CCR2 and CCR5) appears to amplify CCL8 signaling effects .

• Cytokine Network Disruption: Inhibition of CCL8 activity disrupts the CCL8-TNFα network, leading to reduced TNFα expression and diminished pulmonary inflammation. This disruption also reduces the co-regulation between different chemokine receptors, effectively interrupting the coordinated pro-inflammatory response .

• Therapeutic Approach: A neutralizing antibody (1G3E5) against human CCL8 has shown promise in preventing and reducing pulmonary inflammation in experimental models. Unlike failed MCP-1 (CCL2) neutralizing strategies that resulted in elevated endogenous ligand levels, anti-CCL8 treatment with 1G3E5 actually reduced CCL8 levels in the lungs .

• Relevance to Human Disease: CCL8 has been specifically identified as significantly stimulated during acute respiratory distress syndrome in severely ill COVID-19 patients, highlighting its potential relevance as a therapeutic target in acute respiratory conditions .

How can researchers effectively design CCL8 inhibition studies in animal models?

When designing CCL8 inhibition studies, consider these methodological insights:

• Model Selection: Conventional laboratory mice (genus Mus) may not be ideal due to low homology between human and mouse CCL8 (only 71%). Instead, outbred deer mice (Peromyscus maniculatus) offer advantages:

  • Express CCL8 with higher similarity to human CCL8

  • Exhibit genetic diversity that mimics human populations

  • Allow experimentation in sibling pairs for paired statistical analysis

• Administration Route Considerations: Pharmacokinetic studies with anti-CCL8 antibodies indicate that intraperitoneal (IP) administration maintains higher sustained levels for longer periods compared to intravenous (IV) administration .

• Experimental Design Elements:

  • Include appropriate controls (e.g., LPS-only group, antibody-only group)

  • Utilize sibling pairs to account for genetic variation

  • Monitor not only CCL8 levels but also expression of receptors (CCR1, CCR2, CCR3, CCR5, CCR8)

  • Assess co-regulation patterns between chemokine receptors and pro-inflammatory cytokines

• Outcome Measurements:

  • Evaluate pulmonary inflammation markers

  • Measure pro-inflammatory cytokine levels (TNFα, IL1β, IL6)

  • Assess receptor expression via qPCR

  • Consider longer-term follow-up to evaluate lung histology and function

How does CCL8 binding to CCR3 compare with other ligands, and what implications does this have for research?

CCL8 binding to CCR3 has distinct characteristics compared to other ligands:

While CCL8 demonstrates binding affinity to CCR3 comparable to other natural ligands, its functional effects (chemotaxis, receptor internalization) are generally weaker than those of CCL11 and CCL24, but similar to CCL5 . This distinctive profile has important research implications:

• Differential Receptor Activation: CCL8 may induce different signaling patterns or receptor conformational changes compared to other CCR3 ligands.

• Biased Agonism Investigation: CCL8 could serve as a valuable tool for investigating biased agonism at CCR3, potentially activating specific signaling pathways while minimizing others.

• Antagonist Development: The unique binding characteristics of CCL8 could inform the design of novel CCR3 antagonists with improved specificity profiles .

• Experimental Controls: When investigating CCR3-mediated processes, researchers should consider the differential effects of various ligands and select appropriate positive controls based on the specific pathway or effect being studied .

What methodological approaches can resolve data inconsistencies in CCL8-receptor interaction studies?

The literature contains contradictory findings regarding CCL8 interactions with certain receptors, particularly CCR3. To resolve such inconsistencies, researchers should implement:

• Comprehensive Binding Characterization:

  • Employ multiple complementary binding assays (e.g., QCM, surface plasmon resonance)

  • Determine both equilibrium and kinetic binding parameters

  • Use recombinant receptors and native receptor-expressing cells

• Functional Validation Hierarchy:

  • Establish a hierarchy of functional assays from binding to cellular response

  • Assess receptor internalization under standardized conditions

  • Quantify downstream signaling activation (e.g., calcium flux, MAPK activation)

  • Measure chemotactic responses with appropriate controls

• Experimental Variables Control:

  • Standardize protein preparation methods

  • Verify protein activity before experiments

  • Control for receptor expression levels

  • Use receptor-specific antagonists as negative controls

  • Replicate experiments in multiple cell types

• Standardized Reporting:

  • Document complete experimental conditions

  • Report raw data alongside processed results

  • Specify exact protein constructs and expression systems

  • Disclose limitations of experimental approaches

By implementing these rigorous approaches, researchers can generate more consistent and reliable data regarding CCL8-receptor interactions, helping to resolve current discrepancies in the literature.

What are the common challenges in working with recombinant CCL8 and how can they be addressed?

When working with recombinant CCL8, researchers frequently encounter several challenges:

• Inclusion Body Formation:

  • Problem: CCL8, like many chemokines, tends to form inclusion bodies when overexpressed in E. coli

  • Solution: Optimize expression conditions systematically, including induction temperature, inducer concentration, and expression duration; consider using specialized E. coli strains designed for improved protein folding

• Protein Solubility:

  • Problem: Maintaining solubility post-purification can be challenging

  • Solution: Use appropriate buffer systems with stabilizing agents; store lyophilized protein and reconstitute only when needed; avoid repeated freeze-thaw cycles

• Biological Activity Verification:

  • Problem: Inconsistent activity results between batches or assays

  • Solution: Implement multiple complementary functional assays (binding, internalization, chemotaxis); use established positive controls (CCL11, CCL24) for comparison; verify protein concentration accurately prior to testing

• Receptor Specificity Concerns:

  • Problem: Conflicting reports regarding receptor interactions

  • Solution: Use receptor-specific antagonists as controls; verify receptor expression in test systems; implement dose-response studies with proper statistical analysis

• Endotoxin Contamination:

  • Problem: Bacterial expression systems can introduce endotoxin

  • Solution: Verify endotoxin levels remain below 1.0 EU/μg; implement additional purification steps if necessary; consider endotoxin-removal columns if working with sensitive cell types

What quality control parameters should be monitored when producing and using recombinant CCL8?

Rigorous quality control is essential for reliable CCL8 research. Key parameters include:

Documentation of these parameters for each batch is critical for experimental reproducibility and reliable data interpretation.