CCL28 Human

What is CCL28 and how was it initially identified?

CCL28 is a CC chemokine identified through TBLASTN searches of the Human Genome Systems and Genbank dbEst database using human chemokine consensus sequences. Human CCL28 cDNA encodes a 127 amino acid residue precursor protein with a 22 amino acid signal peptide that is cleaved to produce the 105 amino acid mature protein . It shares significant homology with CCL27/CTACK among CC chemokines . The discovery methodology involved:

• Computational analysis using consensus sequence alignment

• cDNA isolation and characterization

• Protein domain structure identification

• Receptor binding assays that identified CCR10 (and later CCR3) as its receptors

Human and mouse CCL28 are highly conserved, sharing 83% amino acid identity in their mature regions, suggesting evolutionary importance .

Where is CCL28 primarily expressed in human tissues?

CCL28 expression has been comprehensively mapped across human tissues. Analysis of the Body Index of Gene Expression (BIGE) database representing 130 tissues from human subjects revealed distinct expression patterns . Highest expression occurs in:

The high expression in salivary glands is particularly notable, and CCL28 is readily detectable in saliva from normal humans . This expression pattern supports its classification as a mucosal chemokine with roles in multiple barrier tissues.

What are the primary receptors for CCL28 and their distribution?

CCL28 signals through two main G-protein coupled receptors:

• CCR10 - The primary receptor, shared with CCL27/CTACK

• CCR3 - A secondary receptor that mediates some of CCL28's effects

Distribution methodology: Receptor expression can be detected through:

• Flow cytometry with receptor-specific antibodies

• RT-PCR for mRNA expression

• Immunohistochemistry of tissue sections

• Single-cell RNA sequencing

CCR10 is expressed on IgA-secreting B cells, specific T cell subsets, and some epithelial cells. CCR3 is notably expressed on neutrophils, particularly after stimulation with proinflammatory molecules, where it can be rapidly mobilized from intracellular stores to the cell surface upon activation .

How does CCL28 regulate neutrophil responses during bacterial infections?

Recent research using CCL28 knockout mouse models (Ccl28−/−) has revealed CCL28's previously unrecognized role in neutrophil regulation during mucosal infections . The methodology for investigating this function involves:

• Comparison of wild-type and Ccl28−/− mice during infection with:

  • Salmonella enterica serovar Typhimurium (gut infection model)

  • Multidrug-resistant Acinetobacter baumannii (lung infection model)

• Key findings:

  • CCL28 promotes neutrophil accumulation at infection sites

  • Neutrophils at infected mucosal sites express CCL28 receptors, particularly CCR3

  • In vitro, CCR3 is stored intracellularly in neutrophils and rapidly mobilized to the cell surface upon stimulation

  • CCL28 stimulation enhances neutrophil antimicrobial activity against Salmonella

  • CCL28 increases production of reactive oxygen species (ROS) and formation of neutrophil extracellular traps (NETs)

• Differential outcomes based on infection site:

  • In gut infections, CCL28-mediated neutrophil responses help control bacterial burden

  • In lung infections, CCL28 activity can exacerbate lethality despite similar bacterial loads

This dual role suggests context-specific functions of CCL28 in different mucosal tissues .

What methodologies are effective for studying CCL28 regulation in human epithelial cells?

The regulation of CCL28 expression in epithelial cells can be studied through several complementary approaches:

• In vivo human tissue analysis:

  • Comparing CCL28 expression in normal versus inflamed colon epithelium through immunohistochemistry and quantitative PCR

• Human intestinal xenograft models:

  • Transplanting human intestinal tissue into immunodeficient mice

  • Stimulating with proinflammatory cytokines (IL-1)

  • Measuring CCL28 mRNA and protein expression

• Cell culture systems:

  • Human colon epithelial cell lines exposed to:

    • Proinflammatory cytokines (IL-1)

    • Bacterial products (flagellin)

    • Metabolites from commensal bacteria (n-butyrate)

  • Analysis through qPCR, ELISA, immunoblotting

• Pathway inhibition studies:

  • Using pharmacological inhibitors of NF-κB to demonstrate pathway dependence

  • siRNA/shRNA knockdown of pathway components

These methodologies have revealed that CCL28 expression is significantly upregulated by IL-1, bacterial flagellin, and n-butyrate, with synergistic effects when cells are pretreated with n-butyrate before exposure to IL-1 or flagellin .

How do commensal bacteria influence CCL28 expression patterns in intestinal tissue?

The relationship between commensal bacteria and CCL28 expression has been investigated using germ-free animal models. Research methodologies include:

• Germ-free versus colonized comparison:

  • Using germ-free neonatal pig models

  • Monoassociating pigs with representative bacterial species:

    • Gram-negative bacteria (Escherichia coli)

    • Gram-positive bacteria (Lactobacillus fermentum)

  • Comparing with conventionalized animals (exposed to fresh adult fecal material)

• Quantitative analysis:

  • Tissue sampling along the small intestine at defined positions (5%, 25%, 50%, 75%)

  • RNA isolation and cDNA synthesis

  • Quantitative PCR with primer/probe sets specific for CCL28

  • Statistical analysis using Student's t-test and Wilcoxon Signed Rank Test

Results demonstrate that CCL28 expression levels are significantly higher in conventionalized and E. coli-colonized pigs compared to germ-free animals at specific intestinal locations. For example:

• At 5% small intestine location: conventionalized > E. coli > L. fermentum = germ-free

• At 75% small intestine location: E. coli > conventionalized > L. fermentum = germ-free

This suggests bacterial species-specific regulation of CCL28, with Gram-negative bacteria appearing more potent in inducing expression.

What is the role of CCL28 in inflammatory bowel conditions?

CCL28 expression is markedly increased in the epithelium of pathologically inflamed human colon compared to normal tissue . Research methodologies to investigate this association include:

• Human tissue biopsy studies:

  • Comparing CCL28 protein and mRNA levels in normal versus inflamed colon samples

  • Immunohistochemistry to localize expression to specific cell types

  • Correlation with disease severity indices

• Mechanistic studies:

  • In vitro models using intestinal epithelial cell lines

  • Stimulation with inflammatory mediators found in IBD (IL-1, TNF-α, bacterial products)

  • NF-κB pathway inhibition studies to determine signaling mechanisms

• Functional assays:

  • Chemotaxis assays to measure immune cell recruitment

  • Analysis of antimicrobial activity against intestinal pathogens

  • Assessment of epithelial barrier function through transepithelial resistance measurements

The evidence suggests CCL28 functions as an "inflammatory" chemokine in human colon epithelium, with expression attenuated by pharmacological inhibitors of NF-κB activation . This positions CCL28 as a potential biomarker and therapeutic target in inflammatory bowel conditions.

How does CCL28 contribute to mucosal IgA immunity and microbiome regulation?

CCL28 plays a crucial role in mucosal IgA immunity through several mechanisms:

• Recruitment of IgA-producing cells:

  • CCL28 mediates the recruitment of CCR10+ IgA plasmablasts to mucosal sites

  • Particularly important in mammary gland development during lactation

  • Enables transfer of maternal IgA to newborns through milk

• Impact on microbiome:

  • Studies in Ccl28−/− mice show reduced IgA production and altered microbiota in the colon

  • Research methodologies include:

    • 16S rRNA sequencing to profile microbial communities

    • Flow cytometry to quantify IgA-coated bacteria

    • ELISA measurement of secretory IgA levels

• Evolutionary significance:

  • The Ccl28 gene exists only in mammalian genomes, suggesting mammalian-specific functions related to lactation and offspring protection

  • Comparative genomics approaches can identify conserved regulatory elements

• Dual antimicrobial mechanisms:

  • Direct antimicrobial activity through a Histidine motif at the carboxy terminus (candidacidal activity)

  • Indirect antimicrobial effects through orchestration of adaptive immune responses

Research in this area typically combines immunological techniques (ELISA, flow cytometry) with molecular microbiology methods (16S sequencing, bacterial culture) to understand the complex interactions between CCL28, mucosal immunity, and microbiome composition.

What are the optimal methodologies for studying CCL28-receptor interactions?

Investigating CCL28 binding to its receptors (CCR10 and CCR3) requires specialized techniques:

• Radioligand binding assays:

  • Using 125I-labeled CCL28 to quantify receptor binding

  • Competition assays with unlabeled ligands to determine binding affinities

  • Scatchard analysis to determine receptor numbers and affinity constants

• Surface plasmon resonance (SPR):

  • Real-time measurement of CCL28-receptor binding kinetics

  • Determination of association and dissociation rates

  • Analysis of binding in the presence of potential inhibitors

• BRET/FRET-based assays:

  • Bioluminescence/fluorescence resonance energy transfer

  • Tagging CCL28 and receptors with appropriate donor/acceptor pairs

  • Allows real-time monitoring of binding in living cells

• Functional receptor assays:

  • Calcium flux measurement after receptor stimulation

  • β-arrestin recruitment assays

  • G-protein activation measured through GTPγS binding

  • Chemotaxis assays using Transwell or Boyden chamber systems

• Deep sequencing of chemokine libraries:

  • Creating variant libraries to identify key binding determinants

  • Selecting variants based on binding to "molecular casts" of active receptor states

  • Identifying structural elements that contribute to receptor specificity and signaling bias

These techniques have revealed that chemokine receptors like US28 (a viral homolog) can accommodate highly degenerate chemokine sequences and distinguish them through sensing the steric bulk of ligands rather than specific bonding chemistries . Similar principles may apply to CCL28-receptor interactions.

How can researchers effectively measure CCL28-mediated neutrophil functions?

To investigate CCL28's effects on neutrophil activity, several specialized methodologies are employed:

• Neutrophil isolation techniques:

  • Density gradient centrifugation of human blood

  • Magnetic-activated cell sorting (MACS) for high purity

  • Bone marrow extraction from wild-type and Ccl28−/− mice

• Receptor expression analysis:

  • Flow cytometry with anti-CCR3 and anti-CCR10 antibodies

  • Cell surface versus intracellular staining to detect receptor pools

  • Stimulation with proinflammatory molecules to track receptor mobilization

• Functional assays:

  • Reactive oxygen species (ROS) measurement using:

    • Chemiluminescence (lucigenin or luminol)

    • Fluorescent probes (DCF-DA, DHE)

  • Neutrophil extracellular trap (NET) quantification:

    • Fluorescent DNA stains

    • Immunofluorescence for NET-associated proteins (MPO, elastase)

    • DNase sensitivity assays

• Antimicrobial activity assessment:

  • Co-culture of CCL28-stimulated neutrophils with bacteria

  • CFU counting at different time points

  • Bacterial viability assays (e.g., live/dead staining)

  • Phagocytosis quantification through fluorescent particle uptake

• In vivo neutrophil tracking:

  • Adoptive transfer of labeled neutrophils

  • Intravital microscopy of mucosal tissues

  • Tissue digestion and flow cytometry to quantify neutrophil accumulation

These methodologies have revealed that CCL28 enhances neutrophil antimicrobial activity against pathogens like Salmonella, increases ROS production, and promotes NET formation, contributing to both infection control and potential tissue damage .

What are the key unresolved questions in CCL28 research?

Despite significant advances in understanding CCL28 biology, several important questions remain:

• Receptor specificity determinants:

  • What structural features of CCL28 determine preferential binding to CCR10 versus CCR3?

  • How does receptor binding translate to differential downstream signaling?

• Tissue-specific functions:

  • Why does CCL28 promote protective immunity in the gut but potentially harmful responses in the lung?

  • What tissue-specific cofactors modify CCL28 activity?

• Therapeutic potential:

  • Can CCL28 modulation be exploited for enhanced mucosal vaccine responses?

  • Would CCL28 blockade be beneficial in specific inflammatory conditions?

• Developmental biology:

  • What regulates CCL28 expression during mammary gland development?

  • How does maternal CCL28 activity influence neonatal immune development?

• Methodological challenges:

  • Development of specific antagonists for CCR10 versus CCR3

  • Improved animal models that better recapitulate human CCL28 functions

  • Systems for studying CCL28 in complex tissue environments

Addressing these questions will require interdisciplinary approaches combining structural biology, immunology, microbiology, and advanced imaging techniques.

How can contradictory findings in CCL28 research be reconciled?

Researchers studying CCL28 occasionally encounter seemingly contradictory findings. Methodological approaches to resolve these discrepancies include:

• Careful consideration of experimental systems:

  • Cell lines versus primary cells

  • Mouse models versus human tissues

  • In vitro versus in vivo observations

• Dose-response relationships:

  • Different concentrations of CCL28 may activate distinct pathways

  • High concentrations (1 μM) exhibit direct antimicrobial activity

  • Lower concentrations may primarily activate cellular responses

• Context-dependent functions:

  • CCL28 acts as both a homeostatic and inflammatory chemokine

  • Effects may differ based on:

    • Tissue microenvironment

    • Presence of commensal bacteria

    • Inflammatory state of the tissue

    • Expression levels of different receptor isoforms

• Methodological standardization:

  • Consistent protein preparation techniques

  • Validation of antibody specificity

  • Use of multiple complementary techniques to confirm findings

• Genetic background considerations:

  • Different mouse strains may show variable responses

  • Human population genetic variants affecting CCL28 or receptor function

By systematically addressing these factors, researchers can better understand the complex and context-dependent functions of CCL28 in health and disease.