Recombinant Human C-C motif chemokine 22 protein (CCL22) (Active)

What is the molecular structure of human CCL22 and how does it compare to other chemokines?

Human CCL22, also named Stimulated T cell Chemotactic Protein (STCP-1) or Macrophage-Derived Chemokine (MDC), is a CC chemokine with distinctive structural features. The human CCL22 gene encodes a 93-amino acid precursor protein containing a 24-amino acid signal peptide that is cleaved to yield a mature 69-amino acid protein with a molecular weight of approximately 8 kDa . A notable feature of CCL22 is its relatively low sequence homology with other CC chemokines, sharing less than 35% amino acid identity with other family members . This suggests unique structural properties that may contribute to its specific biological functions.

Unlike many other CC chemokines whose genes cluster on chromosome 17, the human CCL22 gene is located on chromosome 16, further highlighting its evolutionary divergence from other chemokine family members . This distinct genomic location may have implications for its regulation and function in various physiological contexts.

What are the primary cellular sources of CCL22 in normal and pathological conditions?

CCL22 expression exhibits a relatively restricted pattern compared to some other chemokines. In homeostatic conditions, CCL22 is primarily expressed by:

• Dendritic cells

• Macrophages

• Activated monocytes

• Tissue locations: thymus, lymph nodes, and appendix

In pathological conditions such as endometrial cancer, the cellular source of CCL22 becomes particularly important. Research has demonstrated that both immune cells and tumor cells can produce CCL22, with different prognostic implications based on the producing cell type and localization . Interestingly, while peripheral blood mononuclear cells (PBMCs) appear to be the main source of secreted CCL22 in coculture systems, endometrial cancer cells can also express CCL22 intracellularly upon interaction with immune cells .

This differential pattern of expression suggests context-dependent regulation of CCL22 that may contribute to its varied roles in health and disease.

How can researchers effectively measure CCL22 expression at both mRNA and protein levels?

For comprehensive analysis of CCL22 expression, researchers should employ multiple complementary techniques:

mRNA Expression Analysis:

• Quantitative RT-PCR remains the gold standard for CCL22 mRNA quantification, allowing for sensitive detection of expression changes in different experimental conditions .

• Important considerations include proper reference gene selection and primer design targeting conserved regions of the CCL22 transcript.

Protein Expression Analysis:

• ELISA: Human CCL22/MDC DuoSet ELISA systems effectively quantify secreted CCL22 in culture supernatants . For optimal analysis, standard curves should be created using four-parameter logistic regression (4PL).

• Western blotting: Allows for detection of different CCL22 isoforms.

• Immunohistochemistry (IHC): Particularly valuable for tissue localization studies, as seen in endometrial cancer research where CCL22 expression patterns in different cellular compartments had distinct prognostic implications .

• Flow cytometry: Enables single-cell analysis of intracellular CCL22 expression in heterogeneous cell populations.

For comprehensive characterization of CCL22 biology, researchers should consider analyzing both intracellular and secreted CCL22 levels, as discrepancies between these measurements may reveal important regulatory mechanisms affecting CCL22 secretion .

What experimental systems are most effective for studying CCL22 function and regulation?

Several experimental systems have proven valuable for investigating CCL22 biology:

Coculture Systems:
Transwell coculture systems have been particularly informative for studying CCL22 regulation in the context of tumor-immune cell interactions . Key methodological considerations include:

• Using 0.4-μm-pore transwell inserts to separate different cell fractions

• Appropriate cell densities (e.g., 2×10^5 tumor cells with 2×10^6 PBMCs for supernatant analysis)

• Incubation period of 48 hours for optimal detection of regulatory effects

• Inclusion of appropriate controls (monocultures of each cell type)

Cell Incubation with Conditioned Media:
To identify the cellular source of CCL22, researchers can incubate cell populations with cell-free supernatants from other cell types . This approach has revealed that in endometrial cancer models:

• PBMCs are the primary source of secreted CCL22

• Tumor cells may actually consume or degrade extracellular CCL22

• Tumor cells can increase intracellular CCL22 expression without corresponding secretion

In vivo Models:
For studying CCL22 function in complex disease contexts, genetic mouse models with CCL22 gene modifications provide valuable systems for analysis of CCL22's role in immune cell recruitment and disease progression.

How do genetic polymorphisms in the CCL22 gene affect its expression and disease associations?

Genetic variation in the CCL22 gene has been linked to altered susceptibility to inflammatory conditions, with particularly strong evidence in atopic dermatitis (AD) . Comprehensive resequencing of the CCL22 gene identified 39 single nucleotide polymorphisms (SNPs), with seven tag SNPs selected for association studies in Japanese populations .

A significant association was observed at rs4359426, with meta-analysis yielding a combined P-value of 9.6×10^-6 (OR: 0.74; 95% CI: 0.65-0.85) . Functional analysis revealed that the risk allele of rs4359426 contributed to higher expression levels of CCL22 mRNA .

Further electrophoretic mobility shift assays demonstrated allelic differences in nuclear protein binding, with the G allele of rs223821 (in absolute linkage disequilibrium with rs4359426) showing higher DNA-protein complex formation than the A allele . This suggests that these variants may influence CCL22 expression through altered transcription factor binding.

These findings indicate that CCL22 genetic variants likely contribute to disease susceptibility in a gain-of-function manner, with elevated CCL22 expression potentially driving pathogenic immune responses .

What is the relationship between CCL22 expression and regulatory T cell (Treg) recruitment in cancer?

The relationship between CCL22 and regulatory T cells (Tregs) represents a critical axis in tumor immunology. In endometrial cancer, significant positive correlations have been observed between the Treg marker FoxP3 and CCL22 expression, suggesting a mechanism by which tumors might recruit immunosuppressive Tregs .

Critically, the prognostic impact of CCL22 in endometrial cancer depends on its localization and producing cell type:

These seemingly contradictory findings are potentially explained by the observation that tumor cells may increase intracellular CCL22 expression without corresponding secretion . Since CCL22 can only function as a Treg-attracting chemokine when secreted extracellularly, impaired secretion may result in less Treg invasion and thus better outcomes .

This complex relationship suggests that both the cellular source and secretory status of CCL22 must be considered when evaluating its role in tumor progression and as a potential therapeutic target.

How does CCL22 secretion differ between cell types in coculture systems?

Coculture experiments have revealed fascinating dynamics in CCL22 production and secretion between different cell types. In endometrial cancer models:

• PBMCs were identified as the primary source of secreted CCL22 in coculture supernatants

• Endometrial cancer cell lines (Ishikawa+, RL95-2) showed minimal CCL22 secretion when cultured alone

• When tumor cells were cultured in cell-free supernatants from PBMCs, reduced CCL22 levels were observed, suggesting either consumption/uptake by tumor cells or enhanced degradation

• Coculture with PBMCs induced significant upregulation of CCL22 mRNA in the Ishikawa+ endometrial cancer cell line

• This was confirmed at the protein level, with ELISA analysis of tumor cell lysates showing significantly increased intracellular CCL22 levels after coculture

These findings highlight a critical distinction between CCL22 expression and secretion. While tumor cells can upregulate CCL22 intracellularly in response to immune cell interactions, they may be restricted in their ability to secrete this chemokine, with important implications for Treg recruitment and tumor progression .

How might CCL22 serve as a therapeutic target in cancer and inflammatory diseases?

The emerging evidence on CCL22's role in immune regulation suggests several potential therapeutic approaches:

In cancer immunotherapy, targeting the CCL22-Treg axis may help overcome immune evasion mechanisms. Since CCL22 appears to recruit immunosuppressive Tregs to the tumor microenvironment, strategies disrupting this pathway could enhance anti-tumor immunity . Potential approaches include:

• Neutralizing antibodies against CCL22

• Small molecule inhibitors of CCL22-CCR4 interaction

• Targeting cell-specific production of CCL22

The differential expression patterns and secretion dynamics of CCL22 in various cell types complicate therapeutic development. For instance, in endometrial cancer, the observation that tumor cells may upregulate intracellular CCL22 without corresponding secretion suggests that targeting CCL22 production versus secretion might have distinct outcomes .

Future therapeutic development will require careful consideration of these nuances to effectively modulate CCL22 activity in a context-specific manner.

What are the methodological considerations for using recombinant CCL22 in functional assays?

When designing experiments using recombinant human CCL22 protein, researchers should consider several key methodological factors:

Protein Source and Preparation:

• Recombinant human CCL22/MDC typically comprises amino acids Gly25-Gln93 of the native protein

• E. coli-derived recombinant systems are commonly used for production

• Proper reconstitution and storage are critical for maintaining activity:

  • Avoid repeated freeze-thaw cycles

  • Store at -20 to -70°C for long-term stability (up to 12 months)

  • After reconstitution, maintain at 2-8°C under sterile conditions for up to 1 month

Functional Assays:

• Migration assays: The effective concentration range for chemotactic activity is typically 0.5-3 ng/mL

• Consider the specific target cell types for functional studies (dendritic cells, activated T cells, NK cells)

• Include appropriate positive controls and dose-response analyses

Antibody Selection:
When selecting antibodies for CCL22 detection or neutralization experiments, consider:

• Clone specificity (e.g., Clone #57203 has been validated for applications including flow cytometry)

• Recognition of specific epitopes within the Gly25-Gln93 region

• Validation for specific applications (flow cytometry, neutralization, immunohistochemistry)

These methodological considerations ensure reliable and reproducible results when studying CCL22 function in various experimental systems.

What are the most promising avenues for future CCL22 research?

Several emerging areas of CCL22 research warrant further investigation:

• Detailed structural and functional analysis of CCL22 isoforms:
  The CD8+ T lymphocyte-derived CCL22 isoform with the amino-terminal sequence YGANM differs from the predicted mature CCL22 by two amino acid residues . The functional consequences of this difference remain undetermined and represent an important area for future investigation.

• Cell-type specific regulation of CCL22 secretion:
  The observation that tumor cells may increase intracellular CCL22 expression without corresponding secretion suggests complex regulatory mechanisms that warrant further study . Understanding these processes could reveal novel therapeutic targets.

• Direct cell-cell contact effects on CCL22 regulation:
  Current research using transwell systems has identified important regulatory mechanisms, but direct coculture studies without physical separation could provide additional insights into contact-dependent regulation of CCL22 .

• Comprehensive mapping of CCL22 genetic variants across populations:
  While significant associations have been identified in Japanese populations, broader studies across diverse ethnic groups would enhance our understanding of CCL22 genetic variation and its disease implications .

• Therapeutic targeting strategies for CCL22:
  Development of approaches that can selectively modulate CCL22 function in specific cellular contexts while minimizing off-target effects remains an important challenge for translational research.

These research directions hold promise for advancing our understanding of CCL22 biology and its therapeutic potential across various disease contexts.