Recombinant Rhesus Macaque Eotaxin protein (CCL11)

What is Rhesus Macaque Eotaxin (CCL11) and how does it compare to human Eotaxin?

Rhesus Macaque Eotaxin (CCL11) is a chemokine that functions primarily in eosinophil recruitment through interaction with the CCR3 receptor. Similar to human eosinophils, rhesus macaque eosinophils express functional CCR3 receptors that respond to eotaxin stimulation . While the search results don't provide exact sequence homology between species, many cytokines exhibit high homology between rhesus macaques and humans, making them attractive translational models . The functional conservation is evidenced by preserved chemotactic responses of rhesus eosinophils to eotaxin .

What are the main biological functions of Eotaxin (CCL11) in immune responses?

Eotaxin (CCL11) serves multiple immunoregulatory functions beyond its primary role in eosinophil recruitment. Research shows that eotaxin has significant regulatory effects on acute inflammatory processes. In experimental models, eotaxin modulates NF-κB activation and influences the expression of neutrophil chemoattractants like MIP-2 and CINC . When eotaxin is blocked, NF-κB activation increases, along with elevated levels of MIP-2 and CINC, while IL-10 levels decrease . This regulatory role appears to limit neutrophil accumulation and vascular injury during acute inflammatory responses. Additionally, recent evidence suggests CCL11 may promote cellular senescence through oxidative stress mechanisms and DNA damage signaling pathways .

How is eotaxin expression regulated during inflammatory responses?

During inflammatory responses, eotaxin expression is dynamically regulated at both transcriptional and protein levels. In experimental models of acute lung inflammatory injury, eotaxin mRNA and protein are significantly upregulated during the inflammatory response . Western blot analysis revealed eotaxin protein expression in alveolar macrophages and alveolar epithelial cells, with levels increasing from barely detectable at baseline to significant expression at 2 hours (24.50 ± 4.96 pg/ml, p < 0.01) and further increasing at 4 hours (72.97 ± 8.86 pg/ml, p < 0.001) post-inflammation induction . Interestingly, eotaxin appears in both monomeric (~8 kDa) and dimeric (~16 kDa) forms, particularly at the 4-hour timepoint , suggesting post-translational regulation that may influence its biological activity.

What are the established protocols for producing and purifying recombinant rhesus macaque proteins?

While specific protocols for rhesus macaque eotaxin production aren't detailed in the search results, methodological approaches used for other rhesus cytokines provide a valuable framework. For rhesus IL-9 and IL-33, researchers first confirmed their endogenous expression and sequence identity before generating expression vectors . RT-PCR and Sanger sequencing were used to define the sequences, followed by recombinant expression . The biological activity of the resulting proteins was then validated through appropriate functional assays. For eotaxin specifically, a similar approach would likely involve sequence confirmation, optimal expression system selection, and functional validation through chemotaxis assays with rhesus eosinophils.

How can researchers validate the biological activity of recombinant rhesus macaque Eotaxin?

Biological activity validation should employ multiple complementary approaches:

• Chemotaxis assays: Using isolated rhesus eosinophils to assess migration in response to recombinant eotaxin at various concentrations. Preserved chemotaxis to eotaxin has been established as a functional test for rhesus eosinophils .

• Receptor binding assays: Confirming specific binding to CCR3 receptors on rhesus cells.

• Molecular signaling assays: Measuring downstream activation of signaling pathways known to be activated by CCR3 engagement.

• Immunomodulatory effects: Assessing eotaxin's ability to modulate inflammatory mediator production, similar to its documented ability to suppress MIP-2 production in alveolar macrophages when applied at concentrations of 100-200 ng/ml .

What concentrations of recombinant Eotaxin are effective in experimental systems?

Based on experimental data from studies with eotaxin in other systems, effective concentrations for in vitro studies typically range from 25-250 ng/ml . In studies examining eotaxin's regulatory effects on macrophage responses, significant suppression of IgG immune complex-induced MIP-2 production was observed at 100 ng/ml (reducing levels from 17,225 ± 727 to 12,818 ± 197 pg/ml, p < 0.05) and 200 ng/ml (reducing to 14,315 ± 701 pg/ml, p < 0.05) . These suppressive effects were time-dependent, being significant at 2 and 4 hours but not at 8 hours post-stimulation . For chemotaxis assays with rhesus eosinophils, similar concentration ranges would provide a reasonable starting point, with dose-response experiments recommended to determine optimal concentrations for specific applications.

What are the optimized methods for isolating functional rhesus macaque eosinophils?

Isolation of functional rhesus macaque eosinophils requires species-specific adaptations to standard protocols. The recommended procedure involves a two-step process:

• Ficoll sedimentation: To separate granulocytes from peripheral blood mononuclear cells.

• Immunomagnetic negative selection: Using anti-CD64 antibody to remove contaminating cells .

This approach has achieved >90% purity in some animals, although variability exists between individuals . Importantly, unlike human neutrophils, rhesus neutrophils do not express CD16, which is commonly used for eosinophil purification in human samples . This highlights the necessity of developing species-specific isolation protocols. The isolated eosinophils maintain their functional integrity, as evidenced by preserved chemotactic responses to eotaxin and absence of activation markers such as CD69 .

How can researchers assess the purity and viability of isolated rhesus macaque eosinophils?

Purity assessment of isolated rhesus eosinophils should employ both morphological and phenotypic approaches:

• Morphological examination: Counting at least 300 cells on cytospin slides stained with Diff-Quik to determine percentage purity .

• Flow cytometric analysis: Using eosinophil-specific markers for phenotypic confirmation .

Functional integrity and activation status can be evaluated by:

• Surface marker expression: Measuring CD69 expression as an activation marker (properly isolated cells should show minimal activation) .

• Functional assays: Confirming preserved chemotactic responses to eotaxin in standard chemotaxis assays .

• Viability assessment: Using standard viability dyes to ensure cells remain viable throughout isolation and experimental procedures.

What are the phenotypic differences between rhesus and human eosinophils that researchers should consider?

Understanding phenotypic differences between rhesus and human eosinophils is crucial for translational research. Multi-parameter flow cytometry comparisons have revealed important distinctions:

• Surface marker expression: Rhesus neutrophils lack CD16 expression, which is commonly used for human eosinophil purification , necessitating alternative isolation strategies.

• Shared markers: Like human eosinophils, rhesus eosinophils express EPX and functional CCR3 .

• Absent markers: Notably, Siglec-8 expression was not detected on rhesus eosinophils , which represents a significant difference from human eosinophils where Siglec-8 is an important surface receptor.

These differences highlight the importance of species-specific approaches when working with rhesus eosinophils and suggest potential limitations in cross-species extrapolation of certain regulatory mechanisms.

How can researchers effectively design chemotaxis assays using rhesus eosinophils and recombinant Eotaxin?

Designing effective chemotaxis assays with rhesus eosinophils requires careful consideration of multiple parameters:

• Isolation quality: Begin with highly purified, non-activated eosinophils (>90% purity) .

• Chamber selection: Standard chemotaxis chambers (Transwell or Boyden chamber) should be used with appropriate pore sizes (typically 3-5 μm).

• Concentration gradient: Establish a dose-response curve with eotaxin concentrations ranging from 10-500 ng/ml, based on effective concentrations observed in other experimental systems .

• Appropriate controls: Include negative controls (buffer alone) and positive controls (established chemoattractants).

• Incubation conditions: Optimize temperature (37°C), duration (typically 1-3 hours), and medium composition.

• Quantification methods: Use consistent cell counting methods for migrated cells, either manual counting or automated analysis.

What molecular mechanisms underlie Eotaxin-CCR3 signaling in primates and how can they be studied?

Advanced computational and experimental approaches have revealed several key signaling pathways activated by Eotaxin-CCR3 interaction:

• Oxidative stress pathways: CCL11 stimulation induces dose-dependent upregulation of cytochrome b alpha (CYBA) and beta (CYBB) chains, responsible for ROS formation .

• DNA damage signaling: High CCL11 activation frequencies induce H2A Histone Family Member X (H2AX) and tumor suppressor protein TP53 activation .

• Inflammatory mediator production: CCL11 leads to dose-dependent increases in IL6, a component of the senescence-associated secretory phenotype (SASP) .

These mechanisms can be studied using:

• PseudoCell computational modeling: An in silico approach to predict molecular interactions between proteins and metabolites in response to CCL11 stimulation .

• Flow cytometry: For detecting intracellular signaling molecules.

• ELISAs: To measure secreted inflammatory mediators.

• Western blotting: For analyzing protein expression and modification.

• Gene expression analysis: RT-PCR and RNA sequencing to examine transcriptional changes.

How does Eotaxin influence gene expression patterns in target cells?

Eotaxin exerts complex effects on gene expression in target cells, with distinct temporal patterns and target specificity:

• Selective suppression of chemokine genes: In alveolar macrophages stimulated with IgG immune complexes, eotaxin (100 ng/ml) selectively suppresses MIP-2 and CINC mRNA expression at 1 hour post-stimulation, but this effect is not observed at 2 hours . This suggests a transient, early-phase regulatory mechanism.

• Target specificity: Importantly, eotaxin does not affect the expression of other inflammatory mediators like IL-1β, TNF-α, MIP-1α, and MIP-1β , indicating pathway-specific regulation rather than global immunosuppression.

• Temporal dynamics: The suppressive effects of eotaxin on MIP-2 production are significant at 2 and 4 hours (p < 0.05) but not at 8 hours post-stimulation , highlighting the time-dependent nature of its regulatory effects.

These patterns suggest that eotaxin functions as a selective modulator of specific inflammatory pathways rather than a broad immunoregulatory agent.

How can recombinant rhesus macaque Eotaxin be used in disease models?

Recombinant rhesus macaque Eotaxin offers valuable applications in various disease models, particularly those involving allergic inflammation and eosinophil-mediated pathologies:

• Asthma models: Given eotaxin's critical role in eosinophil recruitment to asthmatic lungs, recombinant rhesus Eotaxin can be used to study pulmonary eosinophilia in rhesus models of allergic asthma.

• Anti-inflammatory studies: The regulatory effects of eotaxin on neutrophil-dependent inflammatory injury suggest applications in studying resolution mechanisms in acute inflammation models.

• Aging research: Recent evidence linking CCL11 to cellular senescence opens possibilities for studying age-related pathologies in rhesus models, which are valuable for aging research due to their longer lifespan and physiological similarity to humans.

• Therapeutic testing: Rhesus models can be used to test CCR3 antagonists or anti-eotaxin antibodies as potential therapeutics, with assessment of both efficacy and safety in a physiologically relevant primate system.

What are the key considerations when comparing findings between rhesus and human experimental systems?

When translating findings between rhesus and human systems, researchers should consider several important factors:

• Receptor homology: While CCR3 is functionally expressed on both human and rhesus eosinophils , potential differences in binding affinity or downstream signaling may exist.

• Missing markers: The absence of Siglec-8 on rhesus eosinophils represents a significant difference from human eosinophils and may impact certain regulatory pathways.

• Isolation methods: Different isolation protocols are required for rhesus eosinophils compared to human eosinophils due to the absence of CD16 on rhesus neutrophils .

• Dose responses: Optimal concentrations of eotaxin may differ between species, necessitating full dose-response curves rather than single-concentration experiments.

• Temporal dynamics: The time course of responses may vary between species, as seen with the transient nature of eotaxin's suppressive effects on chemokine production .

How does Eotaxin interact with the broader cytokine network in inflammatory responses?

Eotaxin functions within a complex cytokine network with multiple regulatory interactions:

• Differential regulation of chemokines: Eotaxin selectively suppresses production of neutrophil chemoattractants MIP-2 and CINC, without affecting other inflammatory mediators like TNF-α, IL-1β, MIP-1α, and MIP-1β . This selective suppression suggests pathway-specific regulation.

• Impact on anti-inflammatory cytokines: Blocking eotaxin in vivo causes decreased IL-10 levels , suggesting eotaxin may promote anti-inflammatory cytokine production.

• NF-κB pathway modulation: Eotaxin appears to inhibit NF-κB activation, as evidenced by enhanced NF-κB activation when eotaxin is blocked .

• Temporal regulation: The effects of eotaxin on other inflammatory mediators are time-dependent, with significant effects at 2 and 4 hours but not at 8 hours post-stimulation .

These interactions position eotaxin as a regulatory node in inflammatory networks, potentially limiting excessive neutrophil recruitment and promoting resolution.

What are common technical issues when working with recombinant Eotaxin and how can they be addressed?

Researchers working with recombinant Eotaxin should anticipate and address several technical challenges:

• Protein aggregation: Eotaxin may exist in both monomeric (~8 kDa) and dimeric (~16 kDa) forms . Solution: Optimize buffer conditions and use freshly prepared solutions; consider including low concentrations of carrier proteins.

• Variability in isolation purity: >90% purity in eosinophil isolation was achieved in only 2 of 6 animals in reported studies . Solution: Optimize isolation protocols for individual animals and rigorously assess purity before experiments.

• Activation during isolation: Inadvertent activation may affect functional responses. Solution: Monitor activation markers like CD69 and minimize processing time .

• Distinguishing specific from non-specific effects: Solution: Include blocking antibody controls; anti-eotaxin antibodies can abolish eotaxin's ability to inhibit MIP-2 release from macrophages .

• Donor variability: Individual differences between animals may affect responses. Solution: Use multiple donors and appropriate statistical analysis to account for biological variation.

How can researchers validate antibody cross-reactivity between human and rhesus macaque systems?

Validating antibody cross-reactivity is essential for accurate detection of rhesus proteins:

• Preabsorption controls: One effective approach is demonstrated in the literature where anti-mouse eotaxin antibody cross-reactivity with rat eotaxin was confirmed by preabsorbing the antibody with murine eotaxin (1 μg/ml), which eliminated the banding pattern for both murine eotaxin standard and rat BAL fluids in Western blots .

• Multiple detection methods: Use complementary methods such as Western blotting, ELISA, and flow cytometry to confirm consistent detection.

• Positive controls: Include recombinant proteins of both species as standards.

• Blocking peptides: Test specificity using blocking peptides corresponding to the antibody epitope.

• Sequence comparison: Analyze epitope regions for sequence conservation between species to predict cross-reactivity.

What controls are essential when measuring Eotaxin's biological effects in vitro?

Rigorous controls are critical for accurate interpretation of Eotaxin's biological effects:

• Antibody neutralization: Anti-eotaxin antibodies should abolish observed effects, as demonstrated in studies where "Anti-eotaxin coculture completely abolished the ability of eotaxin to inhibit IC-induced MIP-2 release from macrophages" .

• Dose-response relationships: Test multiple concentrations (e.g., 25, 50, 100, and 250 ng/ml) to establish concentration-dependent effects .

• Time course analysis: Assess effects at multiple timepoints, as eotaxin's effects can be transient (significant at 2 and 4 hours but not at 8 hours) .

• Receptor antagonists: Include CCR3 antagonists to confirm receptor-dependent mechanisms.

• Heat-inactivated controls: Use denatured protein to distinguish between specific biological activity and non-specific effects.

• Vehicle controls: Include appropriate buffer controls to account for potential effects of carriers or stabilizers.

How might single-cell analysis techniques advance our understanding of Eotaxin-CCR3 signaling in rhesus models?

Single-cell analysis techniques offer powerful approaches to unravel the complexity of Eotaxin-CCR3 signaling:

• Single-cell RNA sequencing: Could identify cell-specific transcriptional responses to eotaxin stimulation, revealing potential heterogeneity in CCR3 expression and downstream signaling across different immune cell populations.

• CyTOF (mass cytometry): Would allow simultaneous assessment of multiple phosphorylation events in signaling cascades activated by CCR3 engagement at the single-cell level.

• Imaging mass cytometry: Could visualize the spatial distribution of CCR3-expressing cells and their interactions within tissues.

• Live-cell imaging: With fluorescent reporters could track real-time signaling dynamics following eotaxin stimulation.

• Spatial transcriptomics: Could map the expression patterns of eotaxin, CCR3, and responsive genes within tissue microenvironments.

These approaches would provide unprecedented resolution of how eotaxin signaling varies across cell types and tissue contexts in rhesus models.

What are promising approaches for studying Eotaxin's role in cellular senescence in primate models?

Building on recent findings linking CCL11 to cellular senescence , several approaches could advance this line of investigation:

• Computational modeling: Expanding on PseudoCell approaches that have identified activation of DNA damage response pathways (H2AX, TP53) and oxidative stress mediators (CYBA, CYBB) in response to CCL11 .

• In vitro senescence models: Testing recombinant rhesus eotaxin on rhesus macaque cell lines to assess senescence markers, including:

  • Senescence-associated β-galactosidase activity

  • Expression of p16INK4a and p21

  • Formation of senescence-associated heterochromatin foci

  • Production of SASP components like IL-6

• Ex vivo tissue analysis: Examining correlations between tissue eotaxin levels and senescence markers in tissues from young versus aged rhesus macaques.

• Intervention studies: Testing whether blocking CCR3 signaling attenuates age-related accumulation of senescent cells in rhesus models.

How might systems biology approaches integrate Eotaxin signaling into broader inflammatory networks?

Systems biology approaches offer powerful frameworks for understanding eotaxin's role within complex inflammatory networks:

• Network modeling: Expanding on approaches like PseudoCell to model interactions between eotaxin and other inflammatory mediators, integrating the selective suppression of MIP-2 and CINC observed in experimental systems .

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to create comprehensive maps of cellular responses to eotaxin in different contexts.

• Temporal dynamics modeling: Capturing the time-dependent nature of eotaxin's effects, including the transient suppression of chemokine production observed at 2 and 4 hours but not 8 hours .

• Comparative systems analysis: Analyzing differences in network topologies between human and rhesus systems to identify conserved and divergent regulatory modules.

• Agent-based modeling: Simulating cellular behaviors in tissue microenvironments to predict emergent properties of eotaxin signaling in complex tissues.

These approaches would help position eotaxin within the broader context of immune regulation and identify key nodes for potential therapeutic intervention.

What are the key advantages of using rhesus macaque models for studying Eotaxin biology?

Rhesus macaque models offer several significant advantages for studying eotaxin biology compared to murine models:

• Evolutionary proximity: Rhesus macaques share closer evolutionary relationships with humans, potentially making findings more translatable to human biology and disease .

• Functional conservation: Rhesus eosinophils express functional CCR3 and demonstrate chemotaxis to eotaxin , confirming conservation of this signaling pathway.

• Comparable eosinophil biology: Despite some differences (like lack of Siglec-8), rhesus eosinophils share many functional characteristics with human eosinophils .

• Longer lifespan: Allows for studying chronic diseases and age-related phenomena that are difficult to model in short-lived rodents.

• Similar immune system complexity: The rhesus immune system more closely resembles human immunity in terms of receptor repertoire, cell subsets, and regulatory mechanisms.

What critical gaps remain in our understanding of rhesus macaque Eotaxin biology?

Despite advances, several important knowledge gaps deserve research attention:

• Complete structural characterization: Detailed sequence and structural comparisons between human and rhesus eotaxin are needed to understand potential functional differences.

• Tissue-specific expression patterns: Comprehensive mapping of eotaxin and CCR3 expression across different rhesus tissues would provide context for disease models.

• Developmental regulation: Understanding how eotaxin expression changes throughout development and aging in rhesus models.

• Role in homeostasis: Beyond inflammatory conditions, eotaxin's role in normal physiological processes in rhesus macaques requires exploration.

• Pharmacological modulation: Testing how existing and novel CCR3 antagonists affect rhesus eotaxin signaling could inform therapeutic development.

• Cross-reactivity of human reagents: Systematic evaluation of which human-targeted reagents effectively cross-react with rhesus systems would facilitate research.

How might emerging technologies advance translational research using recombinant rhesus macaque Eotaxin?

Emerging technologies offer exciting opportunities to advance eotaxin research in rhesus models:

• CRISPR/Cas9 gene editing: Could enable precise modification of eotaxin or CCR3 genes in rhesus cells to study structure-function relationships.

• Organoid technologies: Rhesus-derived organoids could provide physiologically relevant 3D systems for studying eotaxin's tissue-specific effects.

• Advanced imaging: Techniques like intravital multiphoton microscopy could visualize eosinophil recruitment and behaviors in live tissues in response to eotaxin.

• Protein engineering: Could generate modified forms of eotaxin with enhanced stability or reporter functions for mechanistic studies.

• Artificial intelligence approaches: Could identify patterns in complex datasets to generate novel hypotheses about eotaxin's roles in different physiological and pathological contexts.