Eotaxin 2 Mouse

What is the molecular structure of mouse Eotaxin-2 and how does it differ from human Eotaxin-2?

Mouse Eotaxin-2 (also named myeloid progenitor inhibitory factor-2 or MPIF-2) is a member of the CC chemokine subfamily designated as CCL24. Its cDNA encodes a 119 amino acid precursor protein that shares approximately 58% amino acid sequence identity with human Eotaxin-2 . The active protein exists in multiple isoforms, with the most common being a 93 amino acid form (Val27-Val119) and a slightly longer 96 amino acid form (Ile24-Val119) .

Functionally, the Val27-Val119 isoform has been demonstrated to be approximately 150-fold more potent than the Ile24-Val119 isoform as a chemoattractant for mouse cells expressing CCR3 . This significant difference in potency between closely related isoforms highlights the importance of precise amino-terminal processing in determining chemokine activity.

What is the tissue distribution pattern of Eotaxin-2 in normal mice, and how is its expression regulated?

This regulated expression pattern suggests that Eotaxin-2 plays important roles in both homeostasis and inflammatory responses, with tissue-specific expression mechanisms that can be activated during immune challenges.

How does mouse Eotaxin-2 functionally relate to other eosinophil chemoattractants?

Eotaxin-2 is functionally most closely related to Eotaxin/CCL11 and Eotaxin-3/CCL26 . Despite relatively low sequence homology among these three chemokines, they share the important functional property of being potent eosinophil chemoattractants. All three bind and activate the chemokine receptor CCR3, which is highly expressed on eosinophils .

What are the most effective methods for measuring mouse Eotaxin-2 levels in different biological samples?

The most widely used method for quantifying mouse Eotaxin-2 in biological samples is enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA kits are commercially available and optimized for measuring Eotaxin-2 in mouse serum, plasma, or cell culture medium . These assays use a target-specific antibody pre-coated in microplate wells that captures Eotaxin-2 from the sample, followed by detection with a second antibody that completes the sandwich .

For tissue expression analysis, immunohistochemistry using specific anti-Eotaxin-2 antibodies can localize the protein within tissue sections. At the mRNA level, quantitative PCR (qPCR) is commonly employed to measure Eotaxin-2 gene expression in various tissues or cell populations.

For functional analysis, in vitro chemotaxis assays using cells that express the CCR3 receptor (such as BaF3 mouse pro-B cells transfected with mouse CCR3) provide a reliable method to assess Eotaxin-2 bioactivity . The migration of these cells in response to Eotaxin-2 can be quantified using techniques such as Resazurin fluorescence measurement of cells that migrate through a membrane into a lower chamber .

How can researchers effectively neutralize Eotaxin-2 activity in mouse models?

Several approaches can be employed to neutralize Eotaxin-2 activity in mouse models:

• Neutralizing antibodies: Specific anti-Eotaxin-2 antibodies, such as the goat anti-mouse CCL24/Eotaxin-2 antibody, can effectively block Eotaxin-2-induced chemotaxis . In vitro studies have demonstrated that these antibodies can neutralize the chemotactic activity of Eotaxin-2 on CCR3-expressing cells in a dose-dependent manner .

• Receptor antagonists: CCR3 receptor antagonists can block Eotaxin-2 signaling by preventing its interaction with the receptor.

• Genetic approaches: Genetic knockout of Eotaxin-2 or conditional deletion in specific tissues can provide a powerful tool to study the role of this chemokine in various disease models.

• Custom-developed antibodies: Researchers have developed specific monoclonal antibodies directed against Eotaxin-2, such as the D8 antibody reported in one study, which was used to treat experimental autoimmune encephalomyelitis (EAE) mice and significantly decreased disease severity .

The choice of method depends on the specific research question, the disease model being studied, and whether acute or chronic neutralization is required.

What are the optimal experimental conditions for studying Eotaxin-2-mediated chemotaxis in vitro?

When designing in vitro chemotaxis assays to study Eotaxin-2 function, several key factors should be considered:

How does Eotaxin-2 contribute to asthma and respiratory inflammation models in mice?

Eotaxin-2 plays a critical role in asthma and respiratory inflammation models in mice, particularly through its function as an eosinophil chemoattractant. Studies have shown that:

• Transgenic models: Coexpression of IL-5 systemically and Eotaxin-2 locally in the lungs of transgenic mice creates an eosinophil-dependent model that closely resembles severe asthma in humans . This model demonstrates several key pathologies including:

  • Structural remodeling with epithelial desquamation

  • Mucus hypersecretion leading to airway obstruction

  • Subepithelial fibrosis

  • Airway smooth muscle hyperplasia

  • Exacerbated methacholine-induced airway hyperresponsiveness

• Mechanism of action: The pathologies observed in these models are accompanied by extensive eosinophil degranulation, similar to what occurs in human patients with severe asthma . Importantly, genetic ablation of eosinophils from this model abolished all pulmonary pathologies, confirming that these changes are a direct consequence of eosinophil effector functions .

• Pharmacological studies: Compounds that attenuate eosinophilic airway inflammation, such as alendronate, have been shown to suppress Th2 cytokines, Th17 cytokines, and Eotaxin-2 , further demonstrating the integral role of this chemokine in respiratory inflammation.

What role does Eotaxin-2 play in neuroinflammatory models such as experimental autoimmune encephalomyelitis (EAE)?

Eotaxin-2 has been implicated in neuroinflammatory conditions, particularly in experimental autoimmune encephalomyelitis (EAE), which serves as a mouse model for multiple sclerosis. Research has demonstrated that:

• Therapeutic targeting: Blocking the Eotaxin-2 pathway in EAE-induced mice using a specific monoclonal antibody (D8) significantly decreased the severity of EAE symptoms . This suggests that Eotaxin-2 contributes to the pathogenesis of neuro-inflammatory conditions.

• Mechanism: While the exact mechanisms are still being elucidated, Eotaxin-2 likely contributes to EAE pathology by recruiting inflammatory cells, particularly eosinophils, to the central nervous system.

• Biomarker potential: Changes in Eotaxin-2 levels may serve as biomarkers for disease progression or treatment response in neuroinflammatory conditions.

This research area represents an expanding field where Eotaxin-2 functions extend beyond respiratory inflammation to neuroinflammatory processes, highlighting the diverse roles of this chemokine in different organ systems.

How is Eotaxin-2 involved in parasitic infection models in mice?

Eotaxin-2 plays significant roles in the immune response to parasitic infections in mice. Research has revealed:

• Helminth immunity: Studies investigating immunity to Heligmosomoides polygyrus, a gastrointestinal nematode parasite, have shown that TPL-2 (a MAP3K) restricts Ccl24-dependent immunity to this parasite . This suggests that Eotaxin-2 contributes to protective immune responses against helminth infections.

• Tissue-specific expression: Given that Eotaxin-2 is constitutively expressed in the jejunum , it likely plays a role in maintaining mucosal immunity in the gastrointestinal tract, which is particularly relevant for defense against intestinal parasites.

• Eosinophil recruitment: Eotaxin-2's primary function as an eosinophil chemoattractant is especially relevant in parasitic infections, where eosinophils serve as important effector cells against many parasites, particularly helminths.

Understanding Eotaxin-2's role in parasitic infection models provides valuable insights into both basic immunology and potential therapeutic approaches for parasitic diseases.

What are the molecular mechanisms that differentiate the biological activities of Eotaxin-2 from other CCR3 ligands?

Despite sharing the CCR3 receptor with Eotaxin-1 (CCL11) and Eotaxin-3 (CCL26), Eotaxin-2 exhibits unique biological activities that distinguish it from these related chemokines. Advanced research questions in this area include:

• Receptor binding dynamics: How do the kinetics and thermodynamics of Eotaxin-2 binding to CCR3 differ from those of other eotaxins? Studies suggest that despite low sequence homology, these chemokines all bind the same receptor but may engage different binding sites or induce different receptor conformations.

• Signaling pathway specificity: Eotaxin-2 uniquely suppresses myeloid cell proliferation, a function not shared by Eotaxin-1 . This suggests activation of distinct downstream signaling pathways. Research should focus on identifying these pathways through phosphoproteomic analysis and signaling inhibitor studies.

• Structure-function relationships: The remarkable 150-fold difference in potency between the Val27-Val119 and Ile24-Val119 isoforms of mouse Eotaxin-2 highlights the critical importance of N-terminal processing in determining function. Structural biology approaches, including X-ray crystallography and NMR studies of receptor-ligand complexes, could elucidate how these subtle differences translate to major functional changes.

• Receptor oligomerization: Does Eotaxin-2 induce different patterns of CCR3 homo- or hetero-oligomerization compared to other ligands? Such differences could explain unique signaling outcomes.

How do genetic variations in mouse Eotaxin-2 affect its function in different mouse strains?

Genetic variations in mouse Eotaxin-2 across different strains may contribute to strain-specific differences in inflammatory responses and disease susceptibility. Advanced research in this area should address:

• Strain-specific polymorphisms: Comprehensive sequencing of the Eotaxin-2 gene across common laboratory mouse strains to identify coding and regulatory polymorphisms.

• Functional consequences: How do identified polymorphisms affect protein expression, stability, receptor binding affinity, and downstream signaling? In vitro expression systems with site-directed mutagenesis can help address these questions.

• Disease susceptibility: Do strain-specific variations in Eotaxin-2 correlate with differences in susceptibility to inflammatory diseases like asthma or EAE? Cross-strain disease model studies could illuminate these relationships.

• Epigenetic regulation: Beyond sequence variations, how do epigenetic differences in the Eotaxin-2 gene locus contribute to strain-specific expression patterns? Techniques like ChIP-seq for histone modifications and DNA methylation analysis are valuable for addressing this question.

What is the therapeutic potential of targeting Eotaxin-2 in complex inflammatory conditions?

Given its role in various inflammatory conditions, targeting Eotaxin-2 represents a promising therapeutic strategy that warrants advanced investigation:

• Selective targeting strategies: Development of highly specific Eotaxin-2 inhibitors that don't affect other CCR3 ligands could provide more precise control of inflammatory responses. Options include monoclonal antibodies, small molecule inhibitors, or aptamers.

• Dual-targeting approaches: Would simultaneous inhibition of Eotaxin-2 along with other inflammatory mediators (e.g., IL-5, IL-13) provide synergistic therapeutic benefits in conditions like severe asthma? Combination therapy studies in mouse models could address this question.

• Biomarker development: Can Eotaxin-2 levels serve as predictive biomarkers for treatment response? Studies correlating baseline or dynamic changes in Eotaxin-2 levels with therapeutic outcomes could help personalize treatment approaches.

• Tissue-specific targeting: Given Eotaxin-2's expression in multiple tissues, development of delivery systems that target specific tissues (e.g., lung-specific delivery for asthma) could enhance therapeutic efficacy while reducing systemic effects.

• Long-term consequences: What are the long-term immunological consequences of Eotaxin-2 inhibition? Chronic inhibition studies in mouse models could help identify potential compensatory mechanisms or unforeseen consequences of prolonged Eotaxin-2 blockade.

How should researchers optimize storage and handling of recombinant mouse Eotaxin-2 to maintain bioactivity?

Maintaining the bioactivity of recombinant mouse Eotaxin-2 is crucial for experimental reproducibility. Based on available research and standard practices for chemokines:

What are the key considerations when developing transgenic mouse models overexpressing Eotaxin-2?

Developing transgenic mouse models that overexpress Eotaxin-2 requires careful planning:

• Promoter selection: For constitutive systemic expression, promoters like the human β-actin promoter may be appropriate. For tissue-specific expression, promoters such as CC10 (for lung epithelial expression) have been successfully used in models where Eotaxin-2 was expressed locally in the lungs .

• Expression level control: Since chemokines can have dramatic effects even at low concentrations, incorporating inducible systems (e.g., tetracycline-responsive elements) allows for temporal control of expression and helps avoid developmental complications.

• Isoform selection: Given the significant functional differences between Eotaxin-2 isoforms, careful consideration should be given to which isoform is used. The Val27-Val119 isoform shows 150-fold higher activity than the Ile24-Val119 isoform in chemotaxis assays .

• Combination models: As demonstrated in research, combining Eotaxin-2 overexpression with other relevant factors (like IL-5) can create models that more closely mimic human disease conditions . Researchers should consider breeding strategies to generate such combination models.

• Phenotypic characterization: Comprehensive phenotyping should include baseline eosinophil counts in blood and tissues, assessment of lung function parameters (if respiratory phenotypes are expected), and histological evaluation of tissues where Eotaxin-2 is known to be active.