Recombinant Human C-C motif chemokine 18 (CCL18) (Active)

What is CCL18 and what are its key molecular characteristics?

CCL18, also known as Alternative macrophage activation-associated CC chemokine 1 (AMAC1), CC chemokine PARC, Dendritic cell chemokine 1 (DC-CK1), Macrophage inflammatory protein 4 (MIP4), Pulmonary and activation-regulated chemokine (PARC), and Small-inducible cytokine A18 (SCYA18), is a member of the intercrine beta (chemokine CC) family . Recombinant human CCL18 typically encompasses amino acids 21-89 of the full protein sequence with a theoretical molecular weight of approximately 7.9 kDa . The protein contains multiple disulfide bonds, with the Cys-30/Cys-54 disulfide bond being particularly critical for its biological activity .

The amino acid sequence of recombinant human CCL18 is:
AQVGTNKELCCLVYTSWQIPQKFIVDYSETSPQCPKPGVILLTTKRGRQICADPNKKWVQKYISDLKLNA

How is recombinant CCL18 typically produced for research applications?

Recombinant human CCL18 is commonly expressed in bacterial systems, particularly Escherichia coli, to ensure high yield and purity for research applications . Production typically focuses on the active region (amino acids 21-89) without additional tags to prevent interference with protein function . Quality control standards for research-grade recombinant CCL18 generally include:

• Purity exceeding 95-97% as determined by SDS-PAGE

• Endotoxin levels below 1.0 EU/μg to prevent non-specific immune activation

• Functional validation through chemotaxis assays, with typical ED50 values of 1-5 μg/mL

Which cell types naturally produce CCL18 and under what conditions?

CCL18 is primarily secreted by activated macrophages and dendritic cells, making it an important immune-regulatory molecule . In vitro studies have demonstrated that CCL18 mRNA expression is specifically induced in alternatively activated macrophages (M2 phenotype) upon stimulation with Th2 cytokines including IL-4, IL-10, and IL-13 . Conversely, IFN-gamma, a Th1 cytokine, inhibits CCL18 expression, suggesting a regulatory balance between pro-inflammatory and anti-inflammatory signals .

CCL18 mRNA is also expressed by monocyte-derived dendritic cells generated through GM-CSF/IL-4 induction . Interestingly, epidermal Langerhans cells do not express CCL18, indicating tissue-specific regulation of this chemokine .

What is the tissue distribution pattern of CCL18 expression?

CCL18 shows a distinct tissue distribution pattern with particularly high expression in the lung and placenta . This tissue-specific expression profile suggests specialized roles for CCL18 in pulmonary immunity and maternal-fetal interactions. Normal serum and plasma levels of CCL18 in healthy individuals have been measured, with mean values of:

Table 1: Normal physiological levels of CCL18 in human biological fluids

What are the primary immune cells targeted by CCL18 and what responses does it elicit?

CCL18 functions as a highly selective chemotactic factor that attracts specific subsets of leukocytes but not others. Research has demonstrated that CCL18:

• Attracts naive T lymphocytes, CD4+ and CD8+ T cells

• May direct B-cell migration into B-cell follicles within lymph nodes

• Guides naive T-cells toward dendritic cells and activated macrophages in lymph nodes

• Does not attract monocytes or granulocytes (neutrophils), highlighting its selective action

The chemotactic activity of CCL18 on T cells is relatively similar between helper T cells and cytotoxic T cells, with naive T lymphocytes showing greater responsiveness than memory T lymphocytes . Additionally, CCL18 has been observed to bind to approximately 5-10% of skin-homing (CLA+) T cells, suggesting specialized roles in skin immunity .

What concentration range of CCL18 is typically required to elicit biological responses in vitro?

The effective concentration of CCL18 required to elicit biological responses varies depending on the experimental system and cell types involved. In published research, concentrations ranging from 0.1 ng/mL to 1000 ng/mL have been used . For chemotaxis assays, the typical ED50 (effective dose for 50% maximal response) has been reported as 1-5 μg/mL .

The variable responsiveness to CCL18 observed in different studies may be partly attributed to:

• Differences in CCL18 isoforms used (with varying amino- or carboxy-terminal processing)

• Cell culture conditions and duration (e.g., monocytes cultured for 3-4 days respond to CCL18 while freshly isolated monocytes do not)

• Differences in receptor expression levels on target cells

What are the validated methods for quantifying CCL18 in biological samples?

The most widely used method for quantifying CCL18 in biological samples is the enzyme-linked immunosorbent assay (ELISA). Commercial ELISA kits typically employ the quantitative sandwich enzyme immunoassay technique . The procedure involves:

• Capturing CCL18 from samples using pre-coated monoclonal antibodies specific for CCL18

• Detecting bound CCL18 with enzyme-linked monoclonal antibodies

• Developing color proportional to CCL18 concentration using substrate solutions

• Measuring optical density and calculating concentration based on standard curves

Performance characteristics of a typical CCL18 ELISA include:

Table 2: Performance metrics for CCL18 quantification by ELISA

How should biological samples be prepared for optimal CCL18 detection?

Sample preparation is critical for accurate CCL18 quantification. Based on validated protocols:

• Cell culture supernatants typically require minimal processing but may need dilution if CCL18 concentrations exceed the assay's upper limit

• Serum and plasma samples generally need dilution prior to assay due to naturally high CCL18 levels

• Samples should be centrifuged to remove particulates

• Lipemic, hemolyzed, or bacterially contaminated samples should be avoided as they may interfere with assay performance

The assay's specificity should be considered when analyzing complex biological samples, as no significant cross-reactivity or interference has been observed when tested against other chemokines at concentrations up to 500 ng/mL .

How does post-translational processing affect CCL18 biological activity?

The Cys-30/Cys-54 disulfide bond has been identified as particularly crucial for CCL18 activity . This highlights the importance of proper protein folding and disulfide bond formation during recombinant protein production for research applications.

What experimental approaches can be used to investigate CCL18-receptor interactions?

Investigating CCL18-receptor interactions remains challenging due to incomplete characterization of its receptors. Current experimental approaches include:

• Calcium mobilization assays: Measuring intracellular calcium flux in response to CCL18 stimulation to identify responsive cell populations

• Actin polymerization studies: Assessing cytoskeletal rearrangements following CCL18 binding as an indicator of receptor engagement

• Competitive binding assays: Using labeled CCL18 to determine binding specificity and potential receptor sharing with other chemokines

• Migration assays: Evaluating directional cell movement in response to CCL18 gradients (e.g., using Transwell chambers)

When investigating monocyte responses to CCL18, researchers should note that freshly isolated monocytes typically do not respond, while monocytes cultured for 3-4 days may exhibit calcium mobilization, directed migration, and actin polymerization in response to recombinant CCL18 .

How can CCL18 be employed in immunological research models?

CCL18 can be utilized in various immunological research models to investigate:

• T cell trafficking and activation: By using recombinant CCL18 in chemotaxis assays to study naive T cell migration and potential costimulatory effects

• Dendritic cell-T cell interactions: Through in vitro co-culture systems where CCL18 mediates cellular proximity and subsequent immune response development

• Alternative macrophage activation: As a marker and mediator of M2 macrophage function in tissue repair and immune regulation

• B cell follicle formation: To investigate the mechanisms of B cell organization within lymphoid tissues

When designing such experiments, researchers should consider:

• Using appropriate positive controls (other well-characterized chemokines)

• Including specificity controls (antibody neutralization of CCL18)

• Testing concentration-dependent effects (typically 0.1-1000 ng/mL range)

• Ensuring high purity of recombinant CCL18 (>95%) with minimal endotoxin contamination (<1.0 EU/μg)

What are common challenges when working with recombinant CCL18 in experimental systems?

Researchers working with recombinant CCL18 may encounter several technical challenges:

• Protein stability: Ensuring proper storage (-20°C to -80°C) and avoiding repeated freeze-thaw cycles that may compromise activity

• Solubility issues: Using appropriate buffers and carriers to maintain protein solubility without introducing experimental artifacts

• Isoform heterogeneity: Recognizing that different commercial preparations may contain varying mixtures of CCL18 isoforms with potentially different activities

• Receptor complexity: Accounting for incomplete understanding of CCL18 receptors when interpreting experimental results

• Species specificity: Acknowledging potential differences between human CCL18 and rodent models, as direct orthologs may not exist in common research animals

To address these challenges, researchers should:

• Validate each new lot of recombinant CCL18 using established functional assays

• Include appropriate positive and negative controls in experimental designs

• Consider multiple concentrations when testing CCL18 bioactivity (typical range: 0.1-1000 ng/mL)

• Document the exact source and catalog number of recombinant CCL18 in research publications

How can contradictory research findings on CCL18 function be reconciled?

Contradictory findings regarding CCL18 function in the scientific literature may result from:

• Methodological differences: Variations in recombinant protein production, purification methods, and activity assays

• Cellular context: Differences in activation state, culture conditions, and co-stimulatory signals present during experiments

• Isoform variations: Use of different CCL18 isoforms with varying NH2- or COOH-terminal processing

• Experimental timing: Differences in acute versus prolonged exposure to CCL18

• Concentration ranges: Use of significantly different CCL18 concentrations across studies

To reconcile contradictory findings, researchers should:

• Clearly specify experimental conditions, including CCL18 source, concentration, exposure time, and cell types

• Validate findings using multiple complementary approaches (e.g., both in vitro and in vivo models)

• Consider context-dependent effects of CCL18 that may vary based on microenvironment

• Examine potential receptor expression differences across experimental systems