Eotaxin 3 Human
Description
Introduction
Eotaxin-3 (CCL26) is a human CC chemokine encoded by the CCL26 gene on chromosome 7q11.2 . It belongs to the eotaxin family, which includes eotaxin-1 (CCL11) and eotaxin-2 (CCL24), and is primarily recognized for its role in eosinophil recruitment during allergic inflammation . Unlike other eotaxins, Eotaxin-3 exhibits unique regulatory properties, acting as both a chemoattractant and a natural antagonist for multiple chemokine receptors, suggesting a broader immunomodulatory role .
Molecular Structure and Production
-
Amino Acid Composition: Eotaxin-3 is synthesized as a 94-amino acid precursor protein with a 23- or 26-residue signal peptide. The mature protein consists of 68–71 amino acids, depending on the expression system .
-
Recombinant Forms: Produced in Escherichia coli and insect cells, recombinant Eotaxin-3 retains chemotactic activity for eosinophils and CCR3-transfected cells .
-
Structural Epitopes: Two distinct structural epitopes enable interactions with CCR1, CCR2, CCR3, and CCR5, explaining its dual agonist/antagonist functions .
Receptor Interactions
Repulsive Guidance
Eotaxin-3 drives monocytes away from its gradient, synergizing with MCP-1 to amplify repulsion. This mechanism is dependent on CCR2 and pertussis toxin-sensitive G proteins .
Eosinophil Recruitment in Disease
-
Allergic Inflammation: Elevated in asthma, atopic dermatitis (AD), and eosinophilic esophagitis (EoE). Serum levels correlate with disease severity (e.g., AD: mild = 27.6 pg/ml, severe = 62.2 pg/ml) .
-
Mucosal Eosinophilia: Plasma Eotaxin-3 >78.8 pg/ml predicts mucosal eosinophil infiltration (94.4 ± 12.9 vs. 47.5 ± 7.9 cells/HPF, p <0.001) and recurrence post-surgery .
Regulatory Functions
-
Cytokine Synergy: IL-13 induces Eotaxin-3 secretion in nasal fibroblasts (p <0.05) .
-
Genetic Polymorphisms: A single-nucleotide polymorphism in CCL26 is linked to EoE susceptibility .
Antagonistic Activity
Eotaxin-3 inhibits CCR1/CCR2/CCR5-mediated responses:
Biomarker Potential
Assays and Detection
Product Specs
Recombinant Human Eotaxin-3, produced in E. coli, is a single, non-glycosylated polypeptide chain consisting of 71 amino acids. It has a molecular weight of 8.4 kDa. The purification of Eotaxin-3 is achieved using proprietary chromatographic techniques.
Sterile Filtered White lyophilized (freeze-dried) powder.
Lyophilized from a 0.2 μm filtered concentrated solution in phosphate-buffered saline (PBS) at pH 7.4.
To reconstitute the lyophilized Eotaxin-3, it is recommended to dissolve it in sterile 18 MΩ-cm H2O at a concentration of at least 100 µg/ml. This solution can then be further diluted in other aqueous solutions as needed.
Lyophilized Eotaxin-3, while stable at room temperature for up to 3 weeks, should be stored desiccated at a temperature below -18°C. Upon reconstitution, CCL26 should be stored at 4°C for 2-7 days. For long-term storage, it is recommended to store it at a temperature below -18°C. To ensure optimal stability during long-term storage, the addition of a carrier protein (0.1% HSA or BSA) is recommended. It is important to avoid repeated freeze-thaw cycles.
The purity of Eotaxin-3 is determined using the following methods: (a) Analysis by RP-HPLC and (b) Analysis by SDS-PAGE. The purity is greater than 97.0%.
The biological activity of Eotaxin-3 is assessed based on its ability to chemoattract BaF3 mouse pro-B cells transfected with mouse CCR3. The ED50 for this effect typically falls within the range of 0.1-1.0 μg/ml.
TRGSDISKTC CFQYSHKPLP WTWVRSYEFT SNSCSQRAVI FTTKRGKKVC THPRKKWVQK YISLLKTPKQ L.
Q&A
What is Eotaxin-3 and what is its role in human biology?
Eotaxin-3 is particularly notable for its expression patterns in vascular endothelial cells and dermal fibroblasts after stimulation with IL-4 and IL-13, as well as in bronchial tissue following allergen challenge . It plays an essential role in the transendothelial migration of eosinophils, suggesting its importance in inflammatory cell trafficking during immune responses .
How does Eotaxin-3 differ from other eotaxin family members?
While all three eotaxin family members (Eotaxin-1, Eotaxin-2, and Eotaxin-3) signal through CCR3, they demonstrate distinct expression profiles and functional properties:
-
Receptor interactions: Unlike other eotaxins, Eotaxin-3 binds to CCR2 on monocytes and acts as a natural antagonist for this receptor .
-
Efficacy profile: Studies comparing chemotactic response of human eosinophils show that "eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4" . For actin polymerization, the activity ranking is "eotaxin-3 = eotaxin = eotaxin-2" .
-
Reactive oxygen species release: For the release of toxic reactive oxygen species, the ranking is "eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic protein-4 = RANTES" .
-
Unique repulsive property: Eotaxin-3 uniquely demonstrates the ability to repel monocytes (fugetaxis), promoting active movement away from its gradient - a property not observed with Eotaxin-1 despite its CCR2 antagonism .
What are the primary cellular targets of Eotaxin-3?
Eotaxin-3 primarily targets cells expressing the CCR3 receptor, which includes:
Additionally, Eotaxin-3 interacts with monocytes through their CCR2 receptors, though it acts as an antagonist rather than a typical agonist at this receptor . This interaction contributes to the complex role of Eotaxin-3 in orchestrating inflammatory responses by affecting multiple cell types simultaneously.
What assays are available for measuring human Eotaxin-3 levels?
Researchers can measure human Eotaxin-3 using immunoassay techniques such as ELISA. The Quantikine Human CCL26/Eotaxin-3 Immunoassay is a solid-phase ELISA designed specifically for this purpose, with the following characteristics:
-
Assay duration: 4.5 hours
-
Sample types: Cell culture supernatants, serum, and plasma
-
Assay basis: Utilizes E. coli-expressed recombinant human Eotaxin-3 and antibodies raised against it
Performance metrics:
| Parameter | Details |
|---|---|
| Sensitivity (MDD) | 0.87-5.2 pg/mL (range across 39 assays) |
| Intra-assay precision CV% | 3.3-5.6% |
| Inter-assay precision CV% | 4.4-7.5% |
| Recovery in cell culture media | 100% (92-112% range) |
| Recovery in serum | 91% (86-98% range) |
| Recovery in EDTA plasma | 92% (85-101% range) |
| Recovery in heparin plasma | 92% (86-100% range) |
Table 1: Performance characteristics of Eotaxin-3 immunoassay
The assay demonstrates good linearity with serial dilutions of samples containing high concentrations of human Eotaxin-3 .
What experimental systems are most effective for studying Eotaxin-3 function?
Several experimental systems have proven valuable for investigating Eotaxin-3 function:
-
Chemotaxis assays: Modified Boyden chamber assays allow measurement of both positive chemotaxis toward Eotaxin-3 and negative chemotaxis (fugetaxis) away from Eotaxin-3 . Checkerboard studies can assess directional versus random migration.
-
CCR receptor-transfected cell lines: These allow examination of receptor-specific responses, though they may lack additional interaction molecules required for some biological responses like cell repulsion .
-
Actin polymerization assays: Used to assess cytoskeletal rearrangements induced by Eotaxin-3 .
-
Calcium mobilization assays: These detect intracellular calcium transients in response to receptor activation .
-
ERK phosphorylation assays: Used to assess activation of the MAP kinase pathway .
-
Transendothelial migration models: Critical for studying the role of Eotaxin-3 in facilitating eosinophil migration across vascular barriers .
How does Eotaxin-3 function as both a CCR3 agonist and CCR2 antagonist?
Eotaxin-3 demonstrates a complex pharmacological profile through its interactions with multiple chemokine receptors:
-
CCR3 agonism: Eotaxin-3 binds CCR3 and triggers full activation, inducing chemotaxis, calcium mobilization, and cytoskeletal rearrangements in eosinophils, basophils, and Th2 lymphocytes .
-
CCR2 antagonism: Unlike its effect on CCR3, Eotaxin-3 binds to CCR2 on monocytes but does not trigger typical activation responses. Instead, it inhibits MCP-1–mediated responses, functioning as a natural antagonist . Specifically, Eotaxin-3:
This dual functionality represents an important mechanism for fine-tuning cellular responses at inflammatory sites where multiple chemokines are present .
What is the mechanism behind Eotaxin-3's repulsive effect on monocytes?
Eotaxin-3 demonstrates the unusual property of actively repelling monocytes (a process sometimes called fugetaxis or chemorepulsion), which represents a significant finding in chemokine biology:
-
Concentration-dependent effect: High concentrations of Eotaxin-3 in the upper well of a chemotaxis chamber (creating an oppositely oriented gradient) induce statistically significant monocyte migration away from the Eotaxin-3 source .
-
Specificity: This repellent property appears unique to Eotaxin-3. Neither Eotaxin-1 (despite also being a CCR2 antagonist) nor the CCR2 agonist MCP-1 exhibit this property .
-
Receptor dependency: The repulsive effect is largely abolished when cells are pretreated with MCP-1 or CCR2 antagonists, indicating that CCR2 availability is necessary for this response .
-
G-protein involvement: Like MCP-1–mediated migration, the repulsion is sensitive to Bordetella pertussis toxin, indicating the involvement of Gi protein–coupled receptors .
-
Synergistic effects: When applying a reverse gradient of Eotaxin-3 together with an optimal concentration of MCP-1 in the lower well, researchers detected a significant increase in migration efficacy. This suggests that "the synergistic effect of opposing gradients of MCP-1 and Eotaxin-3 is stronger than the inhibitory potential of Eotaxin-3 on MCP-1–mediated migration" .
Checkerboard studies have shown that monocytes migrate away from Eotaxin-3 in a dose-dependent fashion, with a repulsive index of 9.12 compared to MCP-1's chemotactic index of 26.33 .
What is the role of Eotaxin-3 in allergic inflammation and asthma?
Eotaxin-3 appears to play a significant role in allergic inflammation, particularly in asthma:
-
Temporal expression pattern: Data suggest that Eotaxin-3 may be responsible for the continuing recruitment of eosinophils to asthmatic airways following allergen challenge .
-
Cellular expression: Eotaxin-3 is expressed in vascular endothelial cells and dermal fibroblasts after stimulation with IL-4 and IL-13, which are typical Th2 cytokines associated with allergic responses .
-
Bronchial expression: Eotaxin-3 is expressed in bronchial tissue following allergen challenge , suggesting a direct role in pulmonary allergic responses.
-
Eosinophil recruitment: Eotaxin-3 is as efficient as Eotaxin-1 in attracting eosinophils and inducing shape changes in basophils, supporting its role as an important effector chemokine in allergic conditions .
-
Endothelial transmigration: Eotaxin-3 is essential for the transendothelial migration of eosinophils , a critical step in the development of tissue eosinophilia characteristic of allergic inflammation.
The coordinated expression of Eotaxin-3 along with its regulatory effects on both eosinophil and monocyte recruitment suggests a complex role in orchestrating the cellular components of allergic inflammatory responses.
How do the unique properties of Eotaxin-3 contribute to leukocyte trafficking?
Eotaxin-3's complex functionality provides a sophisticated mechanism for coordinating leukocyte positioning during inflammatory responses:
-
Bidirectional guidance: Eotaxin-3 can simultaneously attract CCR3-expressing cells (eosinophils, basophils, Th2 lymphocytes) while repelling CCR2-expressing monocytes , allowing for selective cell positioning.
-
Synergistic guidance mechanisms: The repulsive effect on monocytes is amplified when combined with an MCP-1 gradient in the opposite direction, suggesting that "2 chemokine gradients that are oriented in opposite directions could cooperate in efficiently driving out monocytes from blood vessels into tissue" .
-
Barrier crossing regulation: Eotaxin-3 expressed by vascular endothelial cells plays an essential role in facilitating eosinophil transmigration across the endothelial barrier , a crucial step in leukocyte recruitment to inflammatory sites.
-
Temporal coordination: The expression of Eotaxin-3 following allergen challenge in asthmatic airways suggests it may be responsible for sustained, late-phase recruitment of eosinophils , indicating a role in the temporal regulation of inflammatory responses.
What research questions remain unanswered regarding Eotaxin-3?
Several important questions remain to be fully addressed in Eotaxin-3 research:
-
Molecular mechanisms of repulsion: The precise signaling pathways that mediate Eotaxin-3's repulsive effect on monocytes remain incompletely understood. Research using transfected cells expressing CCR2 failed to detect F-actin formation or active movement away induced by Eotaxin-3, suggesting that "either expression of a single receptor type is not sufficient to mediate cell repulsion or that the used transfected cell lines lack additional interaction molecules that are required for reverse migration" .
-
Physiological significance: While Eotaxin-3 is constitutively expressed in the reproductive system and the heart , the significance of this expression remains to be clarified.
-
Therapeutic targeting: Given Eotaxin-3's role in allergic inflammation, research into specific antagonists or modulators might offer therapeutic benefits, but this area remains underexplored.
-
Integration with other chemokines: The complex interactions between Eotaxin-3 and other chemokines in inflammatory microenvironments require further investigation to understand the combinatorial effects on cell positioning and function.
-
Quantitative considerations: The concentration thresholds at which Eotaxin-3 switches from weak attraction to repulsion of monocytes, and how these concentrations relate to physiological levels in different tissues, remain important questions.
What are the recommended controls for Eotaxin-3 functional assays?
When designing experiments to study Eotaxin-3 function, researchers should consider the following controls:
-
Receptor antagonist controls: Using CCR2 antagonists and MCP-1 (as a competitive ligand) to verify CCR2-dependent effects of Eotaxin-3 on monocytes .
-
G-protein inhibitors: Bordetella pertussis toxin can be used to verify the involvement of Gi protein-coupled receptor signaling .
-
Gradient controls: Checkerboard assays with varying concentrations of Eotaxin-3 in upper and lower chambers to distinguish directed migration from random movement .
-
Related chemokines: Include other eotaxins (Eotaxin-1, Eotaxin-2) and other CC chemokines (MCP-1, MCP-3, MCP-4, RANTES) as comparative controls .
-
Assay sensitivity controls: When measuring Eotaxin-3 levels, include samples spiked with known quantities for recovery assessment (cell culture media: 92-112%, serum: 86-98%, plasma: 85-101%) .
What are the critical variables affecting Eotaxin-3 measurement accuracy?
When measuring Eotaxin-3 in biological samples, several factors can affect accuracy:
-
Assay sensitivity: The minimum detectable dose ranges from 0.87-5.2 pg/mL across different assays , so researchers should be aware of this variability.
-
Intra-assay and inter-assay precision: Variation coefficients range from 3.3-5.6% within assays and 4.4-7.5% between assays , requiring appropriate replication.
-
Sample type differences: Recovery rates vary slightly between sample types (cell culture media, serum, EDTA plasma, heparin plasma) , so matrix-matched calibration may be important.
-
Dilution linearity: Samples with high Eotaxin-3 concentrations should demonstrate linear results when serially diluted , which should be verified.
-
Standard curve preparation: A log/log curve-fit is recommended for data analysis, with the possibility of linearizing data by plotting the log of concentrations versus log of optical density values .
By addressing these critical variables, researchers can improve the reliability and reproducibility of their Eotaxin-3 measurements.
Product Science Overview
Eotaxin-3, also known as CCL26 or SCYA26, is a member of the CC chemokine family. Chemokines are small cytokines that play a crucial role in immune responses by directing the migration of immune cells to sites of inflammation or injury. Eotaxin-3 is particularly known for its role in attracting eosinophils, a type of white blood cell involved in allergic reactions and asthma.
Eotaxin-3 is constitutively expressed in various tissues, including the heart and ovary . It is also expressed in endothelial cells, which line blood vessels, and can be upregulated by cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13) . Eotaxin-3 binds to the CC chemokine receptor 3 (CCR3), which is highly expressed on eosinophils, basophils, and Th2 lymphocytes . This interaction induces chemotaxis, the directed movement of cells towards the chemokine source, and plays a significant role in allergic inflammation .
Recombinant Eotaxin-3 is typically produced in Escherichia coli (E. coli) using recombinant DNA technology . The protein is expressed as a single, non-glycosylated polypeptide chain and purified using high-performance liquid chromatography (HPLC) and other chromatographic techniques . The final product is lyophilized and can be reconstituted in sterile phosphate-buffered saline (PBS) for use in various applications .
The synthetic route for producing recombinant Eotaxin-3 involves cloning the gene encoding Eotaxin-3 into an expression vector, which is then introduced into E. coli cells . The bacteria are cultured, and the recombinant protein is expressed and accumulated within the cells. The cells are then lysed, and the protein is purified from the cell lysate using chromatographic methods .
The biological activity of recombinant Eotaxin-3 is measured by its ability to chemoattract cells expressing CCR3 . The effective dose (ED50) for this activity is typically in the range of 0.3-1.5 µg/mL . The protein is stable when stored at -20 to -70°C and should be reconstituted in sterile PBS containing at least 0.1% human or bovine serum albumin to maintain its stability .
Industrial production of recombinant Eotaxin-3 involves large-scale fermentation of E. coli cultures expressing the protein . The process includes several steps: fermentation, cell lysis, protein purification, and lyophilization. The purified protein is subjected to rigorous quality control tests, including SDS-PAGE to confirm purity and endotoxin testing to ensure safety .
