Recombinant Mouse C-C motif chemokine 12 protein (Ccl12) (Active)

What is the molecular characterization of Mouse C-C motif chemokine 12 (CCL12)?

CCL12 is a small cytokine belonging to the CC chemokine family, characterized as a potent monocyte-active chemokine. It has a molecular weight of approximately 9.3-13.3 kDa depending on the expression system and tags used . The full-length protein contains 82-104 amino acids, with the mature form typically comprising amino acids 23-104. The amino acid sequence of the mature protein is GPDAVSTPVTCCYNVVKQKIHVRKLKSYRRITSSQCPREAVIFRTILDKEICADPKEKWVKNSINHLDKTSQTFILEPSCLG . Structurally, CCL12 shares characteristics with other CC chemokines but has specific binding properties that determine its unique functional profile in inflammatory and immune responses.

How does CCL12 differ from other monocyte chemoattractant proteins?

CCL12 is functionally distinct from other chemokines in its specificity for attracting eosinophils, monocytes, and lymphocytes, while not attracting neutrophils . Unlike other mouse chemokines, CCL12 appears to be uniquely involved in fibrocyte recruitment and fibrotic responses . An important distinction is that CCL12 is mouse-specific with no direct human ortholog, though it shows high homology to human CCL2 (MCP-1) . This creates important species-specific considerations for translational research. While multiple chemokines signal through CCR2, CCL12 appears to have a distinctive role in fibroproliferation that is not fully replicated by other ligands such as CCL2 in the mouse model, despite similar receptor binding profiles.

What is the relationship between mouse CCL12 and human chemokines?

Though often assumed that mouse CCL2 (JE) is the equivalent of human CCL2 (MCP-1), research indicates that mouse CCL12 actually has higher homology to human CCL2 than mouse CCL2 does . CCL12 is found only in mice and likely represents the functional correlate to human CCL2 in fibroproliferative responses. As stated in the research: "As murine CCL12 is homologous to human CCL2, we suggest that the pathobiology of murine CCL12 in fibroproliferation may correlate to human CCL2 biology" . This has significant implications for translational research, as findings regarding mouse CCL12 may be more relevant to human CCL2 biology than mouse CCL2 studies, particularly in fibrotic diseases.

What are the primary biological functions of CCL12?

CCL12 functions as a chemotactic factor that specifically attracts eosinophils, monocytes, and lymphocytes but not neutrophils . It signals through the CCR2 receptor to mediate these chemotactic effects . Beyond basic chemotaxis, CCL12 is involved in:

• Allergic inflammation responses

• Host response to pathogens

• Early stages of allergic lung inflammation

• Recruitment of fibrocytes to sites of tissue injury

• Promotion of fibroproliferative responses in lung injury models

These functions make CCL12 a critical factor in understanding inflammatory disease progression, particularly in fibrotic conditions of the lung.

How should recombinant CCL12 be stored and reconstituted for optimal activity?

Based on standard protocols for similar chemokines, recombinant CCL12 should be reconstituted at approximately 100 μg/mL in sterile deionized water . After reconstitution, the protein should be stored at -20°C to -80°C and multiple freeze-thaw cycles should be avoided to maintain biological activity . For long-term storage, the lyophilized form is more stable when kept at -20°C to -80°C. Many commercial preparations of recombinant CCL12 are supplied in Tris-based buffer with 50% glycerol or as lyophilized powder. For experiments requiring higher protein stability, formulations containing carrier proteins like BSA are recommended, while carrier-free versions should be used for applications where BSA might interfere with the assay system .

What are the optimal conditions for using CCL12 in cell migration assays?

When designing cell migration assays with CCL12, researchers should consider:

• Concentration range: Effective doses (ED₅₀) typically range from 0.1-2 μg/mL for chemotaxis assays, depending on the responsive cell type

• Cell types: Most responsive to CCL12 are:

  • Monocytes/macrophages

  • Eosinophils

  • Lymphocytes

  • Fibrocytes

• Assay format: Transwell migration systems (Boyden chamber) are commonly used, with CCL12 placed in the lower chamber as chemoattractant

• Positive controls: Include known chemoattractants like CCL2 for comparative analysis

• Receptor blocking: Include CCR2 antagonists in control wells to confirm specificity of migration response

For fibrocyte migration specifically, which is relevant to fibrosis research, CCL12 has been shown to be a more potent chemoattractant than CCL2 in mouse models, suggesting its crucial role in fibrocyte recruitment to sites of tissue injury .

How can CCL12 be used effectively in mouse models of inflammation and fibrosis?

When utilizing CCL12 in mouse models of inflammation and fibrosis, researchers should consider these methodological approaches:

• Administration routes:

  • Intratracheal for lung models

  • Intraperitoneal for systemic effects

  • Site-specific injection for localized inflammation

• Dosage considerations:

  • Typically 1-10 μg per mouse, depending on the model

  • Consider time-course experiments to determine optimal timing

• Neutralization studies:

  • Anti-CCL12 neutralizing antibodies can be administered to block endogenous CCL12 function

  • Research has shown that "neutralization of CCL12 in wild-type mice significantly protects from FITC-induced fibrosis, whereas neutralization of CCL2 was less effective"

• Genetic approaches:

  • Compare CCL2⁻/⁻ and CCR2⁻/⁻ mice to distinguish CCL12-specific effects

  • Consider adoptive transfer of CCR2-expressing fibrocytes to augment fibrotic responses

• Readouts:

  • Histological assessment of fibrosis (Masson's trichrome staining)

  • Quantification of fibrocyte recruitment using flow cytometry

  • Measurement of extracellular matrix protein production

  • Analysis of inflammatory cell infiltration

This experimental framework can help elucidate the specific contributions of CCL12 to inflammatory and fibrotic processes in vivo.

What is the role of CCL12 in fibrocyte recruitment and lung fibrosis?

CCL12 plays a crucial role in fibrocyte recruitment during lung fibrosis that appears distinct from other chemokines. Research has demonstrated that CCL12 is likely the principal CCR2 ligand responsible for driving fibroproliferation in mouse models . Studies comparing CCL2⁻/⁻ and CCR2⁻/⁻ mice revealed that CCL2⁻/⁻ mice could still recruit fibrocytes to FITC-injured airspaces, unlike CCR2⁻/⁻ mice, suggesting another CCR2 ligand (CCL12) was responsible .

Experimental evidence shows:

• Both CCL2 and CCL12 are chemotactic for fibrocytes in vitro

• Neutralization of CCL12 in wild-type mice significantly protected from FITC-induced fibrosis

• Neutralization of CCL2 was less effective than CCL12 neutralization

• Adoptive transfer of CCR2-expressing fibrocytes augmented FITC-induced fibrosis in both wild-type and CCR2⁻/⁻ mice

These findings collectively suggest that CCL12-mediated recruitment of fibrocytes is a critical pathogenic mechanism in lung fibrosis. The differential effects of CCL2 and CCL12 neutralization highlight the non-redundant functions of these chemokines despite sharing the same receptor, which has significant implications for therapeutic targeting strategies.

How can researchers address the species-specific differences when translating CCL12 findings to human disease models?

Addressing species differences between mouse and human chemokine biology presents a significant challenge. Since CCL12 exists only in mice, researchers must consider several approaches:

• Homology mapping:

  • Focus on the functional homology between mouse CCL12 and human CCL2, rather than assuming mouse CCL2 is the human equivalent

  • Compare signaling pathways activated by mouse CCL12 with those activated by human CCL2

• Receptor-focused approaches:

  • Study CCR2 signaling as the common factor between species

  • Examine downstream effects of receptor activation rather than focusing solely on the ligand

• Validation strategies:

  • Use humanized mouse models expressing human chemokine receptors

  • Confirm findings in human primary cells and tissue samples

  • Employ comparative proteomics to identify functional equivalents

• Translational considerations:

  • When designing therapeutics based on mouse CCL12 studies, target human CCL2 rather than assuming mouse CCL2 is the translational equivalent

  • As the research indicates: "the pathobiology of murine CCL12 in fibroproliferation may correlate to human CCL2 biology"

This careful consideration of species differences will improve the translational value of research findings from mouse models to human applications.

What methodological approaches can distinguish between CCL12 and other CCR2 ligands in experimental systems?

Distinguishing the specific contributions of CCL12 from other CCR2 ligands requires sophisticated experimental approaches:

• Selective neutralization experiments:

  • Compare the effects of neutralizing antibodies against CCL12 versus CCL2 and other CCR2 ligands

  • Use combination approaches to assess additive or synergistic effects

• Genetic approaches:

  • Utilize CCL12-specific knockout models (not just CCR2⁻/⁻)

  • Compare phenotypes with CCL2⁻/⁻ mice

  • Consider conditional tissue-specific knockouts to address developmental compensation

• Recombinant protein studies:

  • Perform side-by-side comparisons of recombinant CCL12 versus other CCR2 ligands

  • Create dose-response curves for different biological activities

  • Assess differential binding kinetics to CCR2

• Receptor binding competition assays:

  • Use labeled CCL12 and competing unlabeled chemokines to assess binding site overlap

  • Study receptor internalization and recycling patterns after exposure to different ligands

• Signaling pathway analysis:

  • Compare signaling cascades activated by different CCR2 ligands

  • Identify pathway-specific inhibitors to differentiate downstream effects

These approaches can help researchers distinguish the unique biological activities of CCL12 from other chemokines that signal through the same receptor.

What are the most effective methods for detecting and quantifying CCL12 in biological samples?

Several methods can be employed for detecting and quantifying CCL12 in biological samples, each with specific advantages:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Most common method for quantification in serum, tissue homogenates, or cell culture supernatants

  • Commercial kits typically have detection limits in the pg/mL range

  • Consider sandwich ELISA format for improved specificity

• Western Blot Analysis:

  • Useful for confirming protein presence and approximate molecular weight

  • Can distinguish between native and recombinant forms based on size differences

  • Limited quantification capabilities compared to ELISA

• Flow Cytometry:

  • For detecting cell-associated CCL12 or CCR2 expression

  • Can be combined with other markers for cell-specific expression patterns

  • Useful for analyzing receptor internalization after ligand binding

• Immunohistochemistry/Immunofluorescence:

  • Localizes CCL12 expression within tissue sections

  • Can reveal spatial relationships with target cells

  • Consider double staining with cell-type markers to identify producing cells

• Quantitative PCR:

  • Measures CCL12 gene expression rather than protein levels

  • Useful for kinetic studies of induction

  • Must be correlated with protein measurements

• Bioactivity assays:

  • Cell migration assays to measure functional activity

  • CCR2 receptor binding assays

  • Signal transduction assays (calcium flux, ERK phosphorylation)

Each method provides different information, and researchers should select techniques based on their specific research questions and sample types.

Why might recombinant CCL12 show reduced activity in certain experimental systems?

Several factors can contribute to reduced activity of recombinant CCL12 in experimental systems:

• Protein degradation:

  • Chemokines are susceptible to proteolytic degradation

  • Multiple freeze-thaw cycles can reduce activity

  • Improper storage conditions (temperature, buffer composition)

• Expression system effects:

  • E. coli-expressed proteins (like many recombinant CCL12 preparations) lack post-translational modifications

  • Different expression systems (E. coli vs. CHO cells) may yield proteins with varying activity levels

  • Tag presence can interfere with activity (His-tagged vs. tag-free preparations)

• Receptor desensitization:

  • Prolonged exposure to high concentrations can cause receptor internalization

  • Reduced surface expression of CCR2 on target cells

• Species-specific effects:

  • When testing across species, receptor compatibility issues may arise

  • Human cells may not respond optimally to mouse CCL12

• Matrix effects:

  • Components in biological samples may bind or inhibit CCL12

  • Presence of soluble receptor fragments or autoantibodies

• Technical considerations:

  • Using inappropriate reconstitution buffers

  • Improper handling causing protein aggregation

  • Adsorption to laboratory plasticware

To troubleshoot activity issues, researchers should:

• Use carrier proteins (like BSA) to stabilize dilute solutions

• Include protease inhibitors in experimental buffers

• Perform comparative activity assays with different lots/sources

• Validate receptor expression on target cells

What are the potential confounding factors when studying CCL12 in complex biological systems?

When investigating CCL12 in complex biological systems, researchers should be aware of several confounding factors:

• Redundancy in chemokine networks:

  • Multiple chemokines (CCL2, CCL7, CCL8, CCL13) bind to CCR2

  • Compensatory mechanisms may mask phenotypes in knockout models

  • Consider combinatorial approaches targeting multiple chemokines

• Differential receptor expression:

  • CCR2 expression varies by cell type, activation state, and disease condition

  • Receptor expression can be dynamically regulated during experiments

  • Cell-specific responses may complicate interpretation

• Context-dependent functions:

  • CCL12 may have different effects depending on microenvironment

  • Acute vs. chronic models may yield different results

  • Presence of other inflammatory mediators can alter CCL12 function

• Technical considerations:

  • Antibody cross-reactivity between chemokines

  • Sample collection timing can miss transient expression patterns

  • Detection methods may have different sensitivities

• Disease model variations:

  • Mouse strains have different baseline inflammatory responses

  • Genetic background affects chemokine production and response

  • Age and sex differences can impact chemokine biology

• Experimental design limitations:

  • Pharmacokinetics of recombinant proteins

  • Neutralizing antibody specificity and efficacy

  • Timing of interventions relative to disease progression

Addressing these factors requires comprehensive experimental designs with appropriate controls, time-course analyses, and validation across multiple model systems.

How does CCL12 contribute to disease models beyond lung inflammation and fibrosis?

While CCL12 has been extensively studied in lung inflammation and fibrosis, emerging research suggests broader roles in other disease models:

• Autoimmune disorders:

  • CCL12 may contribute to inflammatory cell recruitment in models of multiple sclerosis and rheumatoid arthritis

  • The chemokine's ability to attract lymphocytes suggests potential roles in adaptive immune responses

• Cancer biology:

  • Chemokines including CCL12 may influence tumor microenvironment

  • Similar to CXCL12, which has established roles in cancer biology , CCL12 might affect tumor cell migration and metastasis

  • Cancer-associated fibroblast recruitment may be CCL12-dependent in some models

• Metabolic disorders:

  • Adipose tissue inflammation often involves CCR2-mediated pathways

  • CCL12 may contribute to macrophage recruitment in models of obesity and insulin resistance

• Neuroinflammation:

  • Expression has been detected during murine experimental allergic encephalomyelitis in the spinal cord

  • May contribute to microglial activation and neuroinflammatory responses

• Cardiovascular disease:

  • Potential roles in atherosclerosis through monocyte recruitment

  • Cardiac fibrosis models may involve CCL12-dependent fibrocyte recruitment

These emerging areas represent promising research directions that extend our understanding of CCL12 biology beyond respiratory disease models.

What are the latest approaches for targeting CCL12-CCR2 interactions in therapeutic applications?

Research into targeting CCL12-CCR2 interactions has evolved to include several sophisticated approaches:

• Neutralizing antibodies:

  • Highly specific anti-CCL12 antibodies for selective blockade

  • Combined approaches targeting multiple CCR2 ligands simultaneously

  • Bispecific antibodies targeting both ligand and receptor

• Receptor antagonists:

  • Small molecule CCR2 antagonists that prevent ligand binding

  • Peptide-based inhibitors derived from CCL12 structure

  • Allosteric modulators that alter receptor conformation

• RNA-based therapeutics:

  • siRNA targeting CCL12 expression

  • Antisense oligonucleotides to reduce CCL12 production

  • mRNA approaches to modulate receptor expression

• Cell-based therapies:

  • Ex vivo manipulation of CCR2+ cells before adoptive transfer

  • Chimeric antigen receptor (CAR) approaches targeting CCR2+ pathogenic cells

  • Stem cell therapies to replace or supplement CCR2-expressing populations

• Targeted delivery systems:

  • Nanoparticle-based delivery of CCL12/CCR2 inhibitors

  • Tissue-specific targeting to reduce systemic effects

  • Controlled release formulations for sustained inhibition

When developing these therapeutic approaches, researchers must carefully consider the translation from mouse CCL12 biology to human disease, given the species differences discussed earlier . The differential roles of CCL12 versus other CCR2 ligands also suggest that selective targeting might offer advantages over receptor-level inhibition in certain contexts.

How can contradictory data on CCL12 function be reconciled in different experimental systems?

Reconciling contradictory findings on CCL12 function requires systematic analysis of experimental variables and biological context:

• Methodological standardization:

  • Establish consistent protocols for CCL12 production and characterization

  • Standardize functional assays (migration, binding, signaling)

  • Create reference standards for activity measurement

• Context-dependent analysis:

  • Compare acute vs. chronic experimental models

  • Analyze cell type-specific responses rather than global effects

  • Consider the influence of microenvironment on CCL12 function

• Comprehensive approach to receptor biology:

  • Examine receptor expression levels across experimental systems

  • Consider post-translational modifications of CCR2

  • Analyze receptor oligomerization and interaction with other receptors

• Integrated multi-omics:

  • Combine proteomics, transcriptomics, and metabolomics approaches

  • Map complete signaling networks rather than isolated pathways

  • Identify context-specific cofactors that modify CCL12 function

• Reproducibility initiatives:

  • Multi-laboratory validation of key findings

  • Pre-registration of experimental designs

  • Open data sharing to identify sources of variability

By systematically addressing these factors, researchers can develop more nuanced models of CCL12 biology that account for apparently contradictory observations and advance our understanding of this important chemokine's functions in health and disease.