Recombinant Mouse C-C motif chemokine 7 protein (Ccl7)

What is the molecular structure of Recombinant Mouse CCL7?

Recombinant Mouse CCL7 (also known as MARC or MCP-3) is a member of the beta subfamily of chemokines. The mature protein consists of 74 amino acid residues (position 24-97 of the precursor) with a molecular weight of approximately 8.5 kDa. The complete amino acid sequence is: QPDGPNASTC CYVKKQKIPK RNLKSYRRIT SSRCPWEAVI FKTKKGMEVC AEAHQKWVEE AIAYLDMKTP TPKP . This protein is derived from a 97-amino acid precursor that includes a 23-amino acid signal peptide that is cleaved to yield the mature form .

What are the primary biological functions of Mouse CCL7?

Mouse CCL7 functions primarily as a chemotactic factor that attracts monocytes and eosinophils, but not neutrophils. It serves as a potent chemoattractant for various immune cells, including monocytes and T-lymphocytes . Additionally, CCL7 enhances degranulation and calcium influx in mast cells when these cells are treated with IgE and antigen . The protein plays crucial roles in allergic responses, demonstrated by its requirement for maximal OVA-induced ocular anaphylaxis and mast cell recruitment in vivo .

How does Mouse CCL7 relate to human CCL7/MCP-3?

Mouse MARC (CCL7) is considered the murine homologue of human MCP-3 based on sequence comparisons. Despite some structural differences, they share similar functional properties as chemotactic agents. Mouse MARC is nearly identical to mouse FIC (differing by only one amino acid substitution), which is the product of a growth factor-activated gene .

How should Recombinant Mouse CCL7 be reconstituted and stored for maximum stability?

For optimal stability and activity, Recombinant Mouse CCL7 should be reconstituted at a concentration of 100 μg/mL in sterile, deionized water if using the lyophilized form . After reconstitution, the protein should be stored at -20°C to -80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles that can compromise protein integrity. Working aliquots can be stored at 4°C for up to one week. The protein in liquid form generally remains stable for up to 6 months at -20°C/-80°C, while the lyophilized powder form maintains stability for up to 12 months at the same temperature range .

What are the established bioassays for measuring CCL7 activity?

The biological activity of Recombinant Mouse CCL7 can be determined through multiple established bioassays:

• Chemotaxis bioassay using human monocytes: The biologically active concentration range is typically 100-300 ng/ml .

• Chemotaxis assay with BaF3 mouse pro-B cells transfected with human CCR2A: The ED50 for this effect is 0.5-2 μg/mL .

• Calcium influx assays in mast cells: CCL7 enhances calcium influx in mast cells treated with IgE and antigen, which can be measured using fluorescent calcium indicators .

• Degranulation assays: CCL7-enhanced degranulation in IgE-sensitized mast cells can be quantified by measuring the release of mediators such as β-hexosaminidase .

How can researchers generate CCL7-deficient models for studying its function?

Researchers can employ CCL7-deficient mouse models to study the protein's function in vivo. These models can be generated using gene targeting techniques such as CRISPR/Cas9 genome editing or traditional homologous recombination in embryonic stem cells. When conducting studies with CCL7-deficient mice, it is important to include appropriate wild-type controls to accurately assess phenotypic differences. These models have been successfully used to demonstrate CCL7's role in allergic conjunctivitis by comparing symptoms of experimental conjunctivitis between wild-type and CCL7-deficient mice .

What role does CCL7 play in allergy and hypersensitivity models?

CCL7 serves critical functions in allergy and hypersensitivity models, particularly in IgE-mediated responses:

• Ocular anaphylaxis: CCL7 is required for maximal OVA-induced ocular anaphylaxis. In experimental models, CCL7-deficient mice showed suppressed early-phase clinical symptoms in the conjunctiva after OVA challenge compared to wild-type mice .

• Mast cell recruitment and activation: The numbers of conjunctival mast cells in OVA-induced allergic responses were lower in CCL7-deficient mice than in wild-type mice, indicating that CCL7 is essential for mast cell recruitment to tissues during allergic reactions .

• Respiratory allergy: Pretreatment of mice with anti-CCL7 reduces inflammation and eosinophilia associated with respiratory allergic responses, suggesting CCL7 as a potential therapeutic target .

Which receptors does Mouse CCL7 interact with, and how do these interactions mediate cellular responses?

Mouse CCL7 interacts with multiple chemokine receptors, primarily CCR1, CCR2, and CCR3, each mediating distinct cellular responses:

Mouse CCR2 binds JE/MCP-1 with high affinity while showing a lower affinity for MARC/MCP-3 (CCL7) . The molecules necessary for ocular hypersensitivity reactions include CCR1 and CCR3, for which CCL7 serves as a ligand .

How does CCL7 interact with mast cells in allergic responses?

CCL7 interacts with mast cells through a dual mechanism in allergic responses:

• Direct activation: CCL7 synergistically enhances degranulation and calcium influx in mast cells treated with IgE and antigen. This enhancement occurs through receptor-mediated signaling pathways that amplify FcεRI-triggered activation .

• Chemotactic recruitment: CCL7 induces chemotaxis in mast cells, facilitating their recruitment to sites of inflammation. This was demonstrated in vivo where CCL7-deficient mice showed decreased mast cell numbers in conjunctival tissue during allergic responses .

Notably, mast cells both produce CCL7 and express receptors for it, suggesting an autocrine/paracrine regulatory mechanism. CCL7-deficient bone marrow-derived mast cells showed decreased degranulation following IgE and antigen treatment compared with wild-type cells, but there was no difference when cells were activated via IgE-independent pathways .

How can Recombinant Mouse CCL7 be utilized in tissue-specific inflammation studies?

Recombinant Mouse CCL7 can be employed in tissue-specific inflammation studies through several methodological approaches:

• Direct administration: Purified recombinant CCL7 can be administered locally to specific tissues to evaluate its pro-inflammatory effects and cellular recruitment capabilities. Dosage typically ranges from 0.1-2 μg/mL depending on the experimental model .

• Ex vivo tissue culture systems: Tissue explants can be treated with recombinant CCL7 to study its effects on cellular composition and inflammatory mediator production in a controlled environment that maintains tissue architecture.

• Adoptive transfer experiments: CCL7-responsive cells can be labeled and transferred to recipient mice, followed by local CCL7 administration to track cell recruitment patterns to specific tissues.

• Conditional expression systems: Tissue-specific promoters can drive CCL7 expression in transgenic models to evaluate the consequences of localized CCL7 production in particular organs or tissues.

Research has demonstrated that CCL7 expression is upregulated in conjunctival tissue during OVA-induced allergic responses and has been detected during murine experimental allergic encephalomyelitis in the spinal cord, making it relevant for studies in diverse tissue environments .

What are the optimal conditions for producing bioactive Recombinant Mouse CCL7 in expression systems?

Producing bioactive Recombinant Mouse CCL7 requires careful attention to expression systems and conditions:

E. coli-expressed mouse CCL7 has been successfully used in research applications and demonstrates full biological activity in chemotaxis assays using human monocytes at concentrations of 100-300 ng/ml . When utilizing E. coli expression systems, it's crucial to implement endotoxin removal procedures to ensure preparations contain less than 1.0 EU/μg as determined by the LAL method .

How do genetic variations in CCL7 or its receptors influence experimental outcomes?

Genetic variations in CCL7 or its receptors can significantly impact experimental outcomes and should be considered when designing studies:

• Strain-dependent variations: Different mouse strains may express variant forms of CCL7 or its receptors, potentially resulting in strain-specific responses to inflammatory stimuli. Researchers should specify and maintain consistency in the mouse strains used across experiments.

• Polymorphisms in receptor genes: Variations in CCR1, CCR2, and CCR3 genes can alter binding affinity, signaling efficiency, or receptor expression levels, affecting cellular responsiveness to CCL7. Genotyping experimental animals for relevant receptor polymorphisms may help explain variability in results.

• Expression regulation: Genetic variations in promoter regions can influence CCL7 expression levels in response to stimuli. For instance, CCL7 expression is glucocorticoid-attenuated in LPS-stimulated murine WEHI-3 cells and Swiss 3T3 cells, suggesting regulatory elements sensitive to steroid hormones .

When comparing results across studies, it's essential to account for these genetic factors. Additionally, when developing CCL7-deficient models, researchers should verify that the genetic background is matched to controls to avoid confounding variables.

How does Mouse CCL7 compare functionally with other CC chemokines in inflammatory responses?

Mouse CCL7 shares functional similarities with other CC chemokines but also demonstrates unique properties:

CCL7's broader receptor usage profile allows it to influence a wider range of immune cells compared to more selective CC chemokines. Unlike CCL2 (which primarily acts through CCR2), CCL7's ability to engage multiple receptors (CCR1, CCR2, and CCR3) enables it to simultaneously recruit and activate various cell types during inflammatory responses .

In allergic models, CCL7 stands out for its ability to enhance FcεRI-mediated mast cell activation, a property not prominently associated with other CC chemokines. This dual function in both cell recruitment and cellular activation enhancement makes CCL7 particularly relevant in studying allergic inflammation .

What are the current technical challenges in studying CCL7-mediated interactions in complex tissue environments?

Researchers face several technical challenges when studying CCL7-mediated interactions in complex tissue environments:

• Spatial and temporal dynamics: Conventional techniques often fail to capture the spatiotemporal dynamics of CCL7 gradients in tissues. Advanced approaches like intravital microscopy and optogenetic control of CCL7 expression can help address this limitation.

• Receptor redundancy: CCL7 binds multiple receptors (CCR1, CCR2, CCR3) that are also targeted by other chemokines, making it difficult to isolate CCL7-specific effects. Receptor-selective antagonists or cell-specific receptor knockout models are needed to deconvolute these complex interactions.

• Context-dependent functions: CCL7's effects can vary dramatically depending on the tissue microenvironment. For example, its role in mast cell recruitment and activation in allergic conjunctivitis may differ from its functions in other inflammatory contexts. Using tissue-specific conditional knockout models can help address this challenge.

• Detecting low-abundance interactions: Standard immunoassays may lack the sensitivity to detect physiologically relevant CCL7 concentrations in certain tissues. More sensitive techniques like proximity ligation assays or single-molecule imaging may be required.

• Translating in vitro findings: While in vitro studies show that CCL7 enhances degranulation and calcium influx in mast cells, translating these findings to complex in vivo environments requires careful experimental design and appropriate animal models .

How might CCL7-targeted interventions be developed for treating allergic and inflammatory conditions?

Based on current research, several CCL7-targeted intervention strategies show promise for treating allergic and inflammatory conditions:

• Neutralizing antibodies: Studies using anti-CCL7 antibodies have shown reduction in inflammation and eosinophilia associated with respiratory allergy, suggesting therapeutic potential. These antibodies can be further engineered for improved half-life and tissue penetration .

• Receptor antagonists: Small molecule or peptide antagonists that selectively block CCL7 interaction with its receptors (CCR1, CCR2, CCR3) represent a viable therapeutic approach. Selective targeting may allow for modulation of specific aspects of the inflammatory response while preserving beneficial immune functions.

• Gene silencing approaches: siRNA or antisense oligonucleotides targeting CCL7 mRNA could provide localized suppression of CCL7 production in affected tissues. Delivery systems such as lipid nanoparticles or conjugated antibodies can be used to target specific cell populations.

• Modulation of CCL7 expression: Since CCL7 expression is glucocorticoid-attenuated, targeted glucocorticoid receptor agonists could potentially reduce CCL7 levels in inflammatory conditions while minimizing systemic side effects .

• Combined interventions: Given CCL7's synergistic effect on mast cell activation with IgE/antigen stimulation, combining CCL7 inhibition with existing anti-allergic therapies may provide enhanced efficacy in conditions like allergic conjunctivitis or asthma .

Preliminary experimental evidence supports the development of these interventions, as demonstrated by the reduction in early-phase clinical symptoms in CCL7-deficient mice during OVA-induced allergic conjunctivitis and the decreased conjunctival mast cell numbers observed in these animals .