MCP 4 Human, His

What is human MCP-4 and what are its primary biological functions?

MCP-4, also known as CCL13, is a C-C motif chemokine that functions as a proinflammatory mediator in the human immune system . It is induced by inflammatory proteins such as IL-1 and TNFα and serves as a ligand for three different G protein-coupled receptors: CCR2, CCR3, and CCR5 . The primary biological function of MCP-4 involves activating signaling pathways in various immune cells including monocytes, T lymphocytes, eosinophils, and basophils . This signaling is particularly associated with allergic responses and inflammatory conditions . MCP-4 also plays a significant role in promoting the recruitment and activation of human eosinophils in chronic inflammatory diseases .

What cell types express MCP-4 and under what conditions?

Human dermal fibroblasts have been demonstrated to express high levels of MCP-4 mRNA when stimulated with cytokines, suggesting they are a physiological source of this chemokine . Research has shown that MCP-4 mRNA is also present in cells collected in bronchoalveolar lavage of both asthmatic and non-asthmatic subjects . Additionally, MCP-4 is prominently expressed in human lung and heart tissues . The expression is typically upregulated during inflammatory conditions, particularly in response to stimulation by IL-1 and TNFα .

How can researchers accurately quantify MCP-4 in human samples?

Human MCP-4 can be quantified using a solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) specifically designed for this protein . The assay methodology involves:

• Using a microplate pre-coated with a target-specific antibody

• Adding samples, standards, or controls to the wells

• Formation of a sandwich complex with the addition of a detector antibody

• Addition of a substrate solution that reacts with the enzyme-antibody-target complex

• Measurement of signal intensity, which is directly proportional to MCP-4 concentration

This method can exclusively recognize both natural and recombinant human MCP-4 in serum, plasma, or cell culture medium . Each manufactured lot of ELISA kit should be quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency to ensure reliable quantification .

What techniques are optimal for studying MCP-4 signaling pathways?

Studying MCP-4 signaling pathways requires multiple complementary techniques. Research has demonstrated that MCP-4 induces production of reactive oxygen species and actin polymerization in human eosinophils . To investigate the signaling cascade activated by MCP-4, researchers can use specific enzyme inhibitors that interact with different components of the signal transduction pathway . Studies have revealed that Gi protein, protein kinase C, tyrosine kinase, and phosphatidylinositol-3-kinase are involved in MCP-4 signaling .

A systematic approach to studying MCP-4 signaling should include:

• Cell isolation and purification (e.g., eosinophils from human blood)

• Measurement of functional responses (e.g., respiratory burst, actin polymerization)

• Selective inhibition of signaling components using pharmacological inhibitors

• Assessment of downstream effects through protein phosphorylation studies

• Molecular approaches such as gene knockout or knockdown to confirm specific pathway components

How should researchers design experiments to study circadian variation of MCP-4?

Studies have investigated whether MCP-4 exhibits circadian variation in human plasma . To properly design such experiments, researchers should:

• Collect samples at multiple timepoints across a 24-hour period

• Ensure consistent collection protocols to minimize technical variability

• Control for potential confounding factors such as gender, body mass index (BMI), and age

• Use appropriate statistical methods for time-series analysis, such as mixed models for repeated measures with time as a within-subjects factor

• Consider analyzing MCP-4 in relation to other chemokines, particularly MCP-1

Research has shown that while individual MCP-4 levels might vary over time, the MCP-4/MCP-1 ratio remains relatively invariant over circadian time, making it a potentially valuable biomarker for conditions like PTSD .

What approaches can effectively address variability in MCP-4 measurements?

Variability in MCP-4 measurements can be addressed through several methodological approaches:

• Use of ratio-based measurements (e.g., MCP-4/MCP-1) which can normalize for individual variations and are independent of hemo-concentration effects

• Application of mixed statistical models that account for within-subject correlation without requiring complete data from every subject

• Inclusion of relevant covariates in statistical analyses (e.g., gender, BMI, age)

• Collection of samples at standardized times to control for potential circadian effects

• Establishment of appropriate reference ranges based on healthy control populations

The table below shows an example of how comprehensive data collection and proper covariate control can be structured:

How should researchers interpret the MCP-4/MCP-1 ratio in clinical studies?

The MCP-4/MCP-1 ratio has emerged as a potentially significant biomarker in certain conditions. Research has shown that in plasma, this bivariate ratio is approximately twofold elevated in PTSD patients compared to healthy controls . When interpreting this ratio:

• Consider that the ratio is invariant over circadian time, making it a reliable measure regardless of collection time

• Note that it appears to be independent of gender, body mass index, and the age at which trauma was suffered

• Recognize that while individual chemokines may show gender-specific differences, the ratio provides a gender-independent metric

• Understand that the statistical significance of the ratio (p-value) and its area under the curve (AUC) value are both important for assessing its biomarker potential

• Be aware that this ratio normalizes for potential hemo-concentration effects that might confound individual analyte measurements

What are the known gender differences in MCP-4 expression and function?

Research has revealed significant gender-specific differences in MCP-4 expression patterns and their relationship to certain conditions:

• MCP-4 has been found to significantly biomark PTSD in females but not necessarily in males

• By contrast, MCP-1 (with which MCP-4 is often studied in ratio) significantly biomarks PTSD in males

• These gender differences should be accounted for in study design and data interpretation

• When analyzing MCP-4 data, gender should be included as a covariate in statistical models

• Gender-stratified analyses may be necessary to properly interpret MCP-4 findings in mixed populations

How is MCP-4 implicated in inflammatory and allergic diseases?

MCP-4 plays significant roles in various inflammatory and allergic conditions:

• It promotes the recruitment and activation of human eosinophils in chronic inflammatory diseases

• MCP-4 mRNA has been detected in bronchoalveolar lavage cells from asthmatic subjects

• The interaction between dermal fibroblasts (which produce MCP-4) and human eosinophils may play an important role within the cytokine network in inflammatory skin conditions

• MCP-4 activates signaling in basophils, which is associated with allergic responses

• Its expression in human lung tissue suggests a role in respiratory inflammatory conditions

Researchers investigating these conditions should consider measuring MCP-4 levels and functional activity as part of their experimental design, particularly when studying allergic or inflammatory pathways.

What is the significance of MCP-4 in PTSD research?

MCP-4 has emerged as a potentially important biomarker in PTSD research:

• The MCP-4/MCP-1 ratio in plasma is approximately twofold elevated in PTSD patients compared to healthy controls

• This elevation is consistent across circadian time points, making it a robust potential biomarker

• MCP-4 alone significantly biomarks PTSD in females, showing gender-specific effects

• The ratio is independent of factors such as BMI and the age at which trauma occurred

• These findings suggest MCP-4 may be involved in the inflammatory aspects of PTSD pathophysiology

It remains to be determined whether these disease-specific differences in circadian expression for MCP-4 and other immune signaling molecules are biomarkers, surrogates, or drivers for PTSD .

How can researchers validate MCP-4 as a clinical biomarker?

To validate MCP-4 as a clinical biomarker, researchers should follow these methodological steps:

What are the optimal expression systems for producing recombinant human MCP-4?

While the search results don't specifically address expression systems for MCP-4 with histidine tags, general principles for chemokine production can be applied. Researchers should consider:

• Bacterial expression systems (E. coli) for high yield but potential issues with proper folding

• Mammalian expression systems (HEK293 or CHO cells) for proper post-translational modifications

• Yeast or insect cell systems as alternatives that balance yield and proper protein folding

• Codon optimization for the chosen expression system to enhance protein yield

• Appropriate signal peptides to ensure proper secretion of the recombinant protein

What purification strategies are most effective for histidine-tagged MCP-4?

For his-tagged MCP-4 purification, researchers should follow these methodological steps:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins as the primary purification step

• Buffer optimization to reduce non-specific binding while maintaining protein stability

• Secondary purification steps such as ion exchange or size exclusion chromatography to achieve high purity

• Endotoxin removal procedures for preparations intended for functional studies

• Quality control testing including SDS-PAGE, Western blotting, and functional assays to verify identity and activity