Recombinant Human C-C motif chemokine 28 protein (CCL28) (Active)

What is the basic structure of human CCL28?

Human CCL28 is a CC chemokine family member encoded by a cDNA that produces a 127 amino acid residue precursor protein. This precursor contains a 22 amino acid signal peptide that is cleaved to generate the 105 amino acid mature protein. The mature CCL28 protein shares significant homology with CCL27/CTACK, with human and mouse variants showing 83% amino acid identity in their mature regions . The antimicrobial activity of CCL28 is particularly dependent on positively charged amino acids at the C-terminus, especially amino acids 85-89, though other regions of the C-terminus may also contribute to this function .

Which receptors does CCL28 interact with?

CCL28 primarily binds to two receptors: CCR10 (also known as GPR2 orphan receptor) and CCR3. CCR10 is the main receptor, which is also shared with CCL27/CTACK . In neutrophils, CCL28 signaling occurs predominantly through CCR3 and to a lesser extent through CCR10. This differential receptor binding contributes to CCL28's varied functions in different immune contexts . Experimental blockade with receptor-specific antagonists (such as SB328437 for CCR3 and BI-6901 for CCR10) has demonstrated that CCL28's enhancement of neutrophil functions, including ROS production and NET formation, is primarily CCR3-dependent .

What are the primary physiological sources of CCL28?

CCL28 is primarily expressed by epithelial cells across various mucosal tissues. The highest expression levels are observed in normal and pathologic colon epithelial cells, with significant expression also detected in normal and asthmatic lung tissues . During infection, CCL28 expression increases at mucosal sites, with studies demonstrating approximately four-fold increases in CCL28 levels in feces during Salmonella infection and similar increases in serum during systemic infection . This upregulation suggests an important role in mucosal immunity and response to pathogens.

How should CCL28 be reconstituted for experimental use?

For optimal reconstitution of lyophilized CCL28, researchers should use sterile techniques and prepare the protein in a buffer appropriate for downstream applications. Typically, reconstitution in sterile PBS containing at least 0.1% human or bovine serum albumin is recommended to prevent protein adsorption to plastic or glass surfaces. For antimicrobial assays, CCL28 has been shown to be effective at concentrations of 1μM for Staphylococcus aureus and 0.5μM for Pseudomonas aeruginosa . Following reconstitution, the solution should be gently mixed rather than vortexed to prevent protein denaturation. Aliquoting is recommended to avoid repeated freeze-thaw cycles that could compromise protein activity.

What methods are recommended for validating CCL28 activity in experimental systems?

To validate CCL28 activity, researchers should employ multiple complementary assays. For chemotactic activity, transwell migration assays with CCR3+ or CCR10+ cells (such as CD4+ T cells, CD8+ T cells, or eosinophils) can be used, measuring cell migration in response to different CCL28 concentrations. Calcium flux assays provide a quantitative measure of receptor activation, demonstrating dose-dependent calcium release following CCL28 binding to its receptors . For antimicrobial activity, bacterial killing assays with organisms like Staphylococcus aureus or Pseudomonas aeruginosa can be performed, comparing wild-type CCL28 with mutant variants to identify critical functional domains . When studying CCL28's effects on hematopoietic stem cells, colony-forming unit assays and in vivo repopulation assays in immunodeficient mice are appropriate validation methods .

What controls should be included when studying CCL28's antimicrobial properties?

When investigating CCL28's antimicrobial properties, several controls are essential. First, include a negative control chemokine such as CCL5, which binds to CCR1, CCR3, and CCR5 but lacks antimicrobial activity . Second, incorporate commercially available CCL28 as a positive control to validate in-house produced proteins. Third, test multiple bacterial strains with different sensitivity profiles to CCL28 (e.g., Staphylococcus aureus and Pseudomonas aeruginosa), as they may respond differently to the protein . Finally, include truncated or mutated CCL28 variants, particularly those with modifications to the C-terminal region (amino acids 85-108), to dissect the structural requirements for antimicrobial activity. This comprehensive approach allows for robust validation of CCL28's antimicrobial effects.

How does CCL28 modulate neutrophil responses during mucosal infections?

CCL28 plays a multifaceted role in neutrophil responses during mucosal infections. It promotes neutrophil accumulation at infection sites, particularly in the gut during Salmonella infection and in the lung during Acinetobacter infection . Mechanistically, CCL28 enhances neutrophil antimicrobial functions through several pathways:

• Surface receptor modulation: Neutrophils from infected mucosal tissues express CCR3 and, to a lesser extent, CCR10. Unstimulated neutrophils harbor preformed intracellular CCR3 that rapidly mobilizes to the cell surface following phagocytosis or inflammatory stimuli .

• Antimicrobial activity enhancement: CCL28 stimulation boosts neutrophil killing capacity in a pathogen-specific manner, effectively enhancing the elimination of Salmonella but not Acinetobacter .

• ROS production: CCL28 significantly increases neutrophil production of reactive oxygen species, a key component of antimicrobial defense, primarily through CCR3-dependent mechanisms .

• NET formation: CCL28 enhances the formation of neutrophil extracellular traps (NETs) when neutrophils are co-stimulated with activated platelets. This process is predominantly CCR3-dependent, as demonstrated by the inhibitory effect of the CCR3 antagonist SB328437 .

What experimental models are suitable for studying CCL28 function in vivo?

Several experimental models have proven effective for studying CCL28 function in vivo. The streptomycin-treated C57BL/6 mouse model of colitis is well-established for investigating CCL28's role during Salmonella Typhimurium (STm) gastrointestinal infection . Ccl28-/- mouse models, generated through CRISPR/Cas9 technology targeting exons 1 and 3, provide valuable tools for loss-of-function studies . For these knockout models, proper genotyping using specific primers is essential:

• Forward primer: 5′-TCATATACAGCACCTCACTCTTGCCC-3′

• Reverse primer: 5′-GCCTCTCAAAGTCATGCCAGAGTC-3′

• He/Wt-Reverse primer: 5′-AGGGTGTGAGGTGTCCTTGATGC-3′

The expected product size for the wild-type allele is 466 bp and for the knockout allele is 700 bp .

For studying CCL28's role in hematopoiesis, xenotransplantation models using immunodeficient mice receiving human CD34+ cells cultured with CCL28 have demonstrated the chemokine's ability to support long-term repopulation potential . These diverse models allow researchers to investigate CCL28's functions across multiple biological contexts, from mucosal immunity to hematopoietic stem cell biology.

How does CCL28 influence outcomes in different infectious disease models?

The impact of CCL28 on infection outcomes varies dramatically depending on the pathogen and infection site. During Salmonella Typhimurium (STm) gut infection, CCL28 plays a protective role. Ccl28-/- mice show increased susceptibility to STm infection, with significantly higher bacterial loads in extraintestinal tissues (Peyer's patches, mesenteric lymph nodes, bone marrow, and spleen) by 3 days post-infection compared to wild-type mice . This suggests CCL28 helps contain the infection at the gut mucosa, limiting bacterial dissemination.

Conversely, in Acinetobacter lung infection, CCL28 contributes to pathology. Ccl28-/- mice demonstrate high resistance to otherwise lethal Acinetobacter infection . This divergent outcome likely stems from differences in:

• Pathogen susceptibility to neutrophil killing mechanisms

• Tissue-specific inflammatory responses

• Collateral damage from excessive neutrophil activation

These contrasting effects highlight the context-dependent nature of CCL28's role in host defense, where the same immunomodulatory function can be either beneficial or detrimental depending on the specific infection scenario.

Which structural domains of CCL28 are critical for its antimicrobial versus chemotactic functions?

Chimeric protein experiments have revealed that the antimicrobial C-terminus of CCL28 must interact with a compatible CC chemokine N-terminal domain to maintain full antimicrobial function . When the N-terminal region of CCL28 was replaced with the N-terminal region of another chemokine (either CCL27 or CCL5), the resulting chimeric proteins showed distinct differences in antimicrobial capacity, confirming the importance of appropriate N-terminal/C-terminal interactions .

In contrast, the chemotactic functions of CCL28 are likely more dependent on the N-terminal region and core structure that interact with CCR3 and CCR10 receptors, following the general pattern observed in other CC chemokines. This functional dichotomy allows CCL28 to serve multiple roles in mucosal immunity through distinct structural domains.

How can site-directed mutagenesis be used to explore CCL28 functional domains?

Site-directed mutagenesis represents a powerful approach for exploring CCL28 functional domains. Based on current knowledge of CCL28 structure-function relationships, researchers should consider the following strategic approach:

• C-terminal positive charge modifications: Systematically substitute positively charged amino acids (lysine, arginine) in the C-terminal region (particularly amino acids 85-89) with neutral amino acids to assess the contribution of each residue to antimicrobial activity .

• Domain-swapping experiments: Create chimeric proteins between CCL28 and related chemokines (e.g., CCL27, which shares the receptor CCR10) to identify domains critical for receptor binding specificity versus antimicrobial function .

• Receptor-binding domain mutations: Target the N-terminal region amino acids likely involved in CCR3 versus CCR10 binding to create receptor-specific variants.

• Disulfide bond mutations: Modify conserved cysteine residues to disrupt the tertiary structure and evaluate effects on both chemotactic and antimicrobial functions.

When conducting these experiments, researchers should utilize multiple functional assays to assess both antimicrobial activity (bacterial killing assays) and chemotactic functions (migration assays with CCR3+ and CCR10+ cells). This comprehensive mutagenesis approach will provide insights into the structural basis of CCL28's multifunctional nature.

What approaches can be used to study the differential binding of CCL28 to its receptors CCR3 and CCR10?

Investigating the differential binding of CCL28 to CCR3 and CCR10 requires a multifaceted approach:

• Competitive binding assays: Using radiolabeled or fluorescently labeled CCL28 and receptor-expressing cell lines, researchers can perform displacement assays with receptor-specific antibodies or known ligands (e.g., CCL27 for CCR10) to determine binding affinities and specificities.

• Surface plasmon resonance (SPR): This technique allows real-time measurement of CCL28 binding kinetics (association and dissociation rates) to purified CCR3 and CCR10 receptors or receptor-expressing cell membranes.

• Receptor antagonism studies: Using specific antagonists such as SB328437 for CCR3 and BI-6901 for CCR10, researchers can selectively block receptor-mediated functions to determine their relative contributions to CCL28's biological effects .

• Cellular calcium flux assays: These can quantify the receptor activation potency, as CCL28 causes calcium release in a dose-dependent manner through both CCR3 and CCR10 .

• CRISPR/Cas9-mediated receptor knockout: Generating cell lines with specific receptor deletions allows for clean evaluation of CCL28 signaling through individual receptors.

This comprehensive approach will provide insights into how CCL28 differentially engages its receptors in various cellular contexts, helping explain its diverse biological functions across different immune scenarios.

How does CCL28 influence hematopoietic stem and progenitor cell function?

CCL28 serves as a novel growth factor for hematopoietic stem and progenitor cells (HSPCs) with multiple beneficial effects. It directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins . When added to cultures of purified putative hematopoietic stem cells (HSCs), CCL28 significantly enhances their long-term repopulation potential in immunodeficient mice compared to equivalent numbers of fresh cells .

Mechanistically, CCL28 supports HSPCs by:

• Stimulating cell cycling, thereby promoting proliferation of primitive hematopoietic cells

• Inducing gene expression changes associated with enhanced cellular survival

• Preserving the functional integrity of cultured primitive hematopoietic cells in both in vitro and in vivo settings

These findings position CCL28 as a promising factor for ex vivo expansion of HSCs, addressing a significant challenge in the field of HSC transplantation where maintenance of stem cell properties during culture remains difficult. The discovery of CCL28's effects on HSPCs suggests it may have applications in improving hematopoietic cell transplantation protocols .

What methodologies are recommended for assessing CCL28's effects on stem cell populations?

To comprehensively evaluate CCL28's effects on stem cell populations, researchers should employ a multi-parameter assessment approach:

• Proliferation assays: Carboxyfluorescein succinimidyl ester (CFSE) labeling to track cell divisions or BrdU incorporation assays to identify actively cycling cells in CCL28-treated versus control HSPC cultures.

• Phenotypic analysis: Multi-color flow cytometry to assess changes in stem cell marker expression (CD34, CD38, CD90, CD45RA, CD49f) following CCL28 treatment.

• Colony-forming unit (CFU) assays: Methylcellulose-based colony assays to evaluate the functional progenitor cell content of cultured cells, with detailed analysis of colony types (CFU-GEMM, CFU-GM, BFU-E, etc.).

• Gene expression analysis: RNA-sequencing or qPCR to identify CCL28-induced changes in genes associated with self-renewal, survival, and differentiation pathways.

• In vivo repopulation assays: The gold standard for HSC function, involving transplantation of CCL28-treated human CD34+ cells into immunodeficient mice followed by long-term multilineage engraftment analysis .

• Limiting dilution analysis: To quantitatively determine the frequency of functional HSCs in CCL28-treated versus control cultures.

This comprehensive assessment will provide insights into both the magnitude and mechanisms of CCL28's effects on primitive hematopoietic cells, critical for potential therapeutic applications.

How can researchers distinguish between direct and indirect effects of CCL28 on hematopoietic cells?

Distinguishing between direct and indirect effects of CCL28 on hematopoietic cells requires sophisticated experimental approaches:

• Receptor expression analysis: Quantify CCR3 and CCR10 expression on purified hematopoietic stem and progenitor cell subpopulations using flow cytometry, RNA-seq, or qPCR to determine which cells can directly respond to CCL28.

• Serum-free, stroma-free culture systems: Test CCL28's effects on highly purified HSC populations in the absence of accessory cells that might produce secondary signals in response to CCL28.

• Receptor blocking experiments: Use CCR3- and CCR10-specific antagonists or blocking antibodies to determine if CCL28's effects on HSPCs are mediated directly through these receptors.

• Signaling pathway analysis: Assess the rapid activation of downstream signaling pathways (phosphorylation events) following CCL28 stimulation of purified HSPCs to confirm direct receptor engagement.

• Conditioned media experiments: Compare the effects of direct CCL28 addition to HSPCs versus conditioned media from CCL28-treated accessory cells (e.g., endothelial cells, mesenchymal stromal cells).

• Single-cell analysis: Perform single-cell RNA-seq or proteomics on CCL28-treated heterogeneous populations to identify cell-specific responses and potential paracrine signaling networks.

By implementing these approaches, researchers can delineate the direct effects of CCL28 on primitive hematopoietic cells from indirect effects mediated through accessory cells or secondary signaling molecules.