MCP 5 Mouse

What is MCP-5 and how does it compare to human chemokines?

MCP-5 is a murine CC chemokine (also known as CCL12) that belongs to the larger family of chemokines controlling leukocyte recruitment during immune and inflammatory responses. Structurally, mouse MCP-5 shares 66% amino acid identity with human MCP-1, making it most closely related to this human chemokine rather than having a direct human ortholog named MCP-5 . The gene is located within the CC chemokine cluster on mouse chromosome 11 . MCP-5 functions as a potent chemoattractant specifically for peripheral blood monocytes, with minimal activity on eosinophils and no significant effect on neutrophils .

What are the structural specifications of recombinant MCP-5?

Recombinant mouse MCP-5 consists of amino acids Gly23-Gly104 (Accession # Q62401) . The full protein name is C-C motif chemokine 12, with alternative names including MCP-1-related chemokine, Monocyte chemoattractant protein 5, and Small-inducible cytokine A12 . Gene symbols include CCL12, MCP5, and SCYA12, with gene IDs 102641905 and 20293 .

How is MCP-5 expression regulated during inflammatory responses?

MCP-5 expression is significantly upregulated in response to various inflammatory stimuli. In experimental models, MCP-5 expression increases in the lungs of mice following aerosolized antigen challenge in sensitized animals, demonstrating its role in allergic inflammation . Additionally, MCP-5 expression is enhanced during host responses to pathogen infection, such as with Nippostrongylus brasiliensis .

Interestingly, the regulation of MCP-5 appears to be interconnected with other chemokines. Research using different MCP-1 knockout strategies has revealed complex regulatory relationships—in some models, the absence of MCP-1 leads to altered MCP-3 production, suggesting compensatory or regulatory mechanisms between MCP family members .

What are the optimal techniques for detecting MCP-5 in mouse samples?

For precise quantification, the RayBiotech Mouse MCP-5 ELISA offers a detection range of 0.5-100 pg/mL with high specificity, showing no cross-reactivity with other murine cytokines including 6Ckine, CTACK, Eotaxin, GCSF, GM-CSF, or other MCP family members . For multiplexing capabilities, the AimPlex system can detect MCP-5 alongside other analytes with sensitivity <10 pg/mL and a quantitation range from <20 pg/mL to >5,000 pg/mL .

What considerations should be made when analyzing MCP-5 in different experimental contexts?

When quantifying MCP-5, sample handling is critical. For serum/plasma samples, a 2-fold dilution is typically recommended for accurate quantification within the standard curve range . Intra-assay CV should be <10% and inter-assay CV <20% for reliable results .

When analyzing MCP-5 in complex biological systems, it's essential to consider potential confounding factors such as the presence of other chemokines with overlapping functions or binding to soluble receptors that might affect detection. Standardization of sample collection timing is crucial, as chemokine levels can fluctuate rapidly during inflammatory responses.

How can the chemotactic activity of MCP-5 be experimentally measured?

The gold standard for measuring MCP-5 chemotactic activity is the transwell migration assay. This can be performed using BaF3 mouse pro-B cells transfected with human CCR2A, which respond to MCP-5 in a dose-dependent manner. Migration can be quantified using cellular viability indicators such as Resazurin .

A typical experimental setup involves:

• Placing recombinant MCP-5 (typically at 0.2 μg/mL) in the lower chamber

• Adding CCR2-expressing cells to the upper chamber

• Measuring the number of cells that migrate through the membrane

• Quantifying migration using fluorescence or cell counting techniques

Calcium flux assays provide another approach to measuring MCP-5 activity, as MCP-5 induces calcium flux in peripheral blood mononuclear cells and CCR2-transfected cell lines, but not in purified murine eosinophils or neutrophils .

What methods can effectively neutralize MCP-5 activity in experimental systems?

MCP-5 activity can be specifically neutralized using monoclonal antibodies. For example, rat anti-mouse CCL12/MCP-5 monoclonal antibody (clone 147801) effectively neutralizes MCP-5-induced chemotaxis in a dose-dependent manner. The neutralization dose (ND₅₀) is typically 0.5-2.5 μg/mL in the presence of 0.2 μg/mL recombinant mouse MCP-5 .

For receptor-level inhibition, antagonists targeting CCR2 can block MCP-5 signaling. Genetic approaches using siRNA or CRISPR/Cas9 to knockdown MCP-5 or CCR2 expression provide alternative strategies for longer-term inhibition studies.

What role does MCP-5 play in inflammatory disease models?

MCP-5 has been implicated in various inflammatory conditions, particularly allergic inflammation. Studies have shown significant upregulation of MCP-5 expression in the lungs of mice following aerosolized antigen challenge, suggesting its involvement in allergic airway responses .

The presence of MCP-5 in atherosclerotic lesions has also been documented, suggesting a potential role in cardiovascular disease pathogenesis. In ApoE−/− mice, which develop atherosclerosis, MCP-induced protein 1 (MCPIP1) expression increases significantly in atherosclerotic plaques compared to healthy vessel walls, indicating a relationship between MCP signaling pathways and vascular inflammation .

How does MCP-5 contribute to host defense against pathogens?

MCP-5 expression is significantly upregulated during infection with parasites such as Nippostrongylus brasiliensis, indicating its role in host defense . By recruiting monocytes to infection sites, MCP-5 contributes to pathogen clearance and the orchestration of adaptive immune responses.

Interestingly, research on Borrelia burgdorferi (the causative agent of Lyme disease) has identified a methyl-accepting chemotaxis protein also abbreviated as MCP5, which is essential for mammalian infection and tick transmission. This bacterial MCP5 helps spirochetes evade host immune defenses . Though this is a different protein than mouse CCL12/MCP-5, it highlights the importance of chemotactic proteins in host-pathogen interactions.

How does MCP-5 interact with other chemokines in inflammatory networks?

MCP-5 functions within a complex network of chemokines that orchestrate inflammatory responses. Research in MCP-1 knockout models has revealed interesting regulatory relationships between MCP family members. Different gene deletion strategies for MCP-1 have produced varying effects on MCP-3 and MCP-5 expression:

• In a mouse model where MCP-1 was deleted by removing a 2.3-kb DNA fragment (MCP-1Δ/Δ), the production of MCP-3 was significantly increased

• Conversely, in mouse models where MCP-1 was disrupted by inserting a neo-gene cassette, decreased production of MCP-3 was observed

These findings suggest compensatory regulatory mechanisms between different MCP family members that must be considered when studying any individual chemokine.

What receptor interactions does MCP-5 engage in and how do they compare to other chemokines?

MCP-5 primarily signals through the CCR2 receptor, inducing calcium flux in cells expressing this receptor. Studies have shown that MCP-5 can induce calcium flux in human embryonic kidney (HEK)-293 cells transfected with either human or murine CCR2, but not in cells transfected with CCR1, CCR3, or CCR5 .

This receptor specificity distinguishes MCP-5 from other chemokines with broader receptor binding profiles and helps explain its selective activity on monocytes rather than other leukocytes. The specificity for CCR2 is consistent with MCP-5's functional similarity to human MCP-1, which also signals predominantly through CCR2.

What approaches can be used to genetically manipulate MCP-5 in mouse models?

Several genetic approaches can be employed to study MCP-5 function:

• Conventional knockout: Complete deletion of the MCP-5/CCL12 gene

• Conditional knockout: Cell- or tissue-specific deletion using Cre/loxP systems

• Reporter gene knockin: Replacing or tagging MCP-5 with reporter constructs for expression monitoring

• Point mutations: Introducing specific amino acid changes to study structure-function relationships

• CRISPR/Cas9: For precise genetic modifications of the MCP-5 gene or its regulatory regions

The selection of approach depends on the specific research question and whether developmental versus acute effects of MCP-5 deficiency need to be distinguished.

What are critical considerations when interpreting data from MCP-5 genetic models?

When interpreting data from MCP-5 genetic models, researchers should consider:

• Potential compensatory mechanisms: Deletion of MCP-5 may lead to upregulation of other chemokines with similar functions

• Genetic background effects: The phenotypic manifestation of MCP-5 deficiency may vary depending on mouse strain

• Gene deletion strategy: As seen with MCP-1 models, different deletion approaches (complete deletion versus insertion of selection markers) can have varying effects on neighboring genes

• Developmental versus acute roles: Constitutive knockout models cannot distinguish between developmental requirements and functions in adult tissues

• Specificity of phenotypes: Effects attributed to MCP-5 deletion should be confirmed with complementary approaches such as antibody neutralization or receptor antagonism

Research has demonstrated that different knockout strategies for chemokine family members can lead to varying effects on the expression of other chemokines in the cluster, highlighting the importance of understanding the specific genetic modification when interpreting results .

What are the optimal storage and handling conditions for MCP-5 reagents?

For antibodies against mouse MCP-5, recommended storage conditions are 2-8°C in the dark . Lyophilized proteins should typically be reconstituted at 0.5 mg/mL in sterile PBS . For lyophilized standards containing MCP-5, proper reconstitution and aliquoting for single use is recommended to maintain protein activity.

When working with sodium azide-containing reagents (often used as preservatives), safety precautions must be followed as sodium azide can form explosive compounds with heavy metals found in plumbing .

How can researchers ensure reproducible results when working with MCP-5?

To ensure reproducibility in MCP-5 research:

• Use validated antibodies with documented specificity (e.g., antibodies showing no cross-reactivity with other mouse cytokines)

• Include appropriate positive and negative controls in all assays

• Standardize sample collection, processing, and storage protocols

• Verify protein activity through functional assays such as chemotaxis or calcium flux

• When comparing results across studies, consider differences in recombinant protein sources, detection methods, and experimental models

For neutralization experiments, titrate antibodies to determine the optimal concentration, as the typical ND₅₀ of 0.5-2.5 μg/mL may vary depending on the specific experimental conditions .

How do findings from mouse MCP-5 studies translate to human disease?

While mouse MCP-5 shares highest structural homology with human MCP-1 (66% amino acid identity) rather than having a direct human ortholog called MCP-5 , functional studies suggest similar roles in monocyte recruitment and inflammation. This structural and functional similarity makes mouse MCP-5 research potentially relevant to human diseases involving MCP-1-mediated monocyte recruitment.

What are emerging therapeutic approaches targeting MCP-5 or its signaling pathways?

While direct MCP-5 targeting therapies are still in experimental stages, several approaches targeting chemokine networks have therapeutic potential:

• Monoclonal antibodies against MCP-5 or its receptor CCR2

• Small molecule inhibitors of CCR2 signaling

• RNA interference approaches to downregulate MCP-5 or CCR2 expression

• Modulation of pathways that regulate MCP-5 expression

The effectiveness of these approaches depends on the specific disease context and the relative contribution of MCP-5 versus other chemokines in the pathological process. The potential for compensatory upregulation of other chemokines following MCP-5 inhibition must be carefully evaluated.