Recombinant Rat C-C motif chemokine protein (Ccl28) (Active)

What is the biological function of CCL28 in mucosal tissues?

CCL28 plays multiple critical roles in mucosal tissues:

• Mucosal immunity regulation: CCL28 is expressed constitutively in epithelial cells at mucosal sites, including salivary glands, mammary glands, colon, and respiratory tissues. It regulates IgA antibody-secreting cell (ASC) accumulation in mammary glands, which is crucial for the transfer of IgA antibodies from mother to infant .

• Chemotactic activity: The protein is chemotactic for resting CD4 and CD8 T-cells, eosinophils, and IgA antibody-secreting cells. Its biological activity in chemotaxis assays typically occurs in the concentration range of 5-50 ng/ml .

• Antimicrobial activity: CCL28 displays direct antimicrobial activity against various microorganisms, including Candida albicans, gram-negative, and gram-positive bacteria, particularly in low-salt conditions . This function is largely dependent on its C-terminal positively charged amino acids .

• Neutrophil regulation: Recent research has shown that CCL28 significantly contributes to neutrophil accumulation and activation in mucosal tissues during infection, modulating responses to pathogens like Salmonella and Acinetobacter .

How does CCL28 signal in target cells and what receptors does it engage?

CCL28 signals through two primary receptors:

• CCR10: This is the primary receptor through which CCL28 signals. CCR10 is expressed on T cells, B cells, and IgA-secreting plasma cells. The interaction of CCL28 with CCR10 is crucial for the homing of IgA-producing cells to mucosal tissues .

• CCR3: Initially thought to be absent on neutrophils, CCR3 has been detected on neutrophils from patients with chronic inflammation. CCL28 binding to CCR3 plays a significant role in neutrophil activation and recruitment, particularly during infection .

The signaling cascade includes:

• Calcium mobilization in a dose-dependent manner upon receptor binding

• Activation of Src, ERK, and eNOS signaling pathways

• CCL28-induced CCR10/eNOS interaction that has implications in angiogenesis

Experimental data shows that CCL28-dependent neutrophil responses, including reactive oxygen species (ROS) production and neutrophil extracellular trap (NET) formation, are primarily mediated through CCR3 rather than CCR10 .

What structural elements of CCL28 are responsible for its antimicrobial properties?

The antimicrobial activity of CCL28 depends on specific structural elements:

What are the proper storage and handling procedures for Recombinant Rat CCL28?

To maintain optimal activity of Recombinant Rat CCL28:

Before opening, it is recommended to briefly centrifuge the vial to bring contents to the bottom. Further dilutions should be made in appropriate buffered solutions .

How does CCL28 modulate neutrophil responses during different types of infections?

CCL28 modulates neutrophil responses differently depending on the site and type of infection:

• During intestinal Salmonella infection:

  • CCL28 is upregulated approximately 4-fold in feces of infected mice

  • CCL28 helps control Salmonella infection at the gut mucosa, reducing dissemination to other sites

  • CCL28-deficient mice show higher bacterial counts in extraintestinal tissues (Peyer's patches, mesenteric lymph nodes, bone marrow, spleen)

  • CCL28 promotes neutrophil accumulation in the gut, with ~20% of gut neutrophils expressing CCR3 and ~2% expressing CCR10

  • CCL28 stimulation enhances neutrophil bactericidal activity against Salmonella, with ~40% of the bacterial inoculum cleared compared to ~10% by unstimulated neutrophils

• During lung Acinetobacter infection:

  • CCL28 exacerbates lethality, with 75% of wild-type mice dying within 48 hours while 88% of CCL28-deficient mice survived

  • CCL28 modulates neutrophil accumulation in the lung, but fails to reduce pathogen burden

  • CCL28 stimulation does not enhance neutrophil bactericidal activity against Acinetobacter

  • The protective effect of CCL28 deficiency is associated with reduced neutrophil recruitment and pulmonary inflammation

• General mechanisms across infections:

  • CCL28 enhances neutrophil reactive oxygen species (ROS) production and neutrophil extracellular trap (NET) formation, primarily through CCR3

  • CCL28 promotes neutrophil chemotaxis, though not as potently as CXCL1

  • CCL28's effects can be context-dependent, with outcomes varying based on pathogen and infection site

What are the experimental considerations for measuring CCL28-mediated chemotaxis?

When designing and interpreting CCL28 chemotaxis assays:

• Concentration range: The biologically active concentration range for CCL28 in chemotaxis bioassays using human lymphocytes is typically 5.0-50 ng/mL. Researchers should titrate the protein in each testing system to obtain optimal results .

• Target cells: Different cell types show varying levels of responsiveness to CCL28:

  • Resting CD4+ and CD8+ T cells

  • Eosinophils

  • IgA antibody-secreting cells

  • Neutrophils (particularly those with upregulated CCR3 expression)

• Receptor considerations:

  • For neutrophil chemotaxis studies, consider that CCR3 expression may need to be upregulated with cytokine cocktails (GM-CSF + IFNγ + TNFɑ)

  • CCL28 promotes neutrophil chemotaxis, though not as potently as CXCL1

  • The relative contributions of CCR3 versus CCR10 can be studied using receptor antagonists (SB328437 for CCR3 and BI-6901 for CCR10)

• Comparative controls: Include both positive controls (like CXCL1 for neutrophil chemotaxis) and related chemokines (like CCL11/eotaxin, which also binds CCR3) .

• Calcium mobilization: Measuring calcium flux in a dose-dependent manner can provide additional confirmation of CCL28 activity .

How does CCL28's function differ between species, and what should researchers consider when working with rat CCL28?

Species-specific considerations for CCL28 research include:

• Sequence variations: Analysis of CCL28 across multiple species shows variations in crucial regions:

  • The consensus sequence for the essential 85-89 amino acid region is KRNSK

  • Human sequence in this region is RKNSN

  • Mouse/rat sequence variations in this region may contribute to differences in antimicrobial activity

  • Human CCL28 has been shown to have slightly higher antimicrobial activity than mouse CCL28

• Conservation of function: Despite sequence variations, core functions appear to be conserved across species:

  • Chemotactic activity for T-cells, eosinophils, and IgA-producing cells

  • Antimicrobial properties (though with varying potency)

  • Expression patterns in mucosal tissues

• Experimental validation: When working with rat CCL28:

  • Consider cross-species reactivity when selecting antibodies (many antibodies show reactivity with human, mouse, and rat CCL28)

  • Validate biological activity using species-appropriate target cells

  • Be aware that direct comparisons between human and rodent studies may be complicated by these sequence variations

What techniques can be used to study CCL28-induced neutrophil activation and antimicrobial functions?

Several methodological approaches can be employed:

• Neutrophil ROS production assays:

  • Use luminol-enhanced chemiluminescence to measure ROS production

  • Compare CCL28-stimulated versus unstimulated neutrophils

  • Include receptor antagonists (SB328437 for CCR3 and BI-6901 for CCR10) to determine receptor contribution

  • Time-course experiments show that CCL28 stimulation (50 nM) increases neutrophil ROS production within 30 minutes

• Neutrophil extracellular trap (NET) formation:

  • Incubate human neutrophils with activated platelets with or without CCL28

  • Stain with DNA dyes like DAPI and HELIX

  • Evaluate NET formation by fluorescence microscopy

  • Analyze DNA-MPO complexes as confirmation of NET formation

  • Include receptor antagonists to determine which receptor (CCR3 or CCR10) mediates the response

• Bacterial killing assays:

  • Incubate bacteria (e.g., Salmonella typhimurium) with bone marrow neutrophils with or without CCL28

  • Quantify bacterial killing after 2.5 hours of incubation

  • Compare CCL28 (50 nM) stimulation to other chemokines like CCL11 (50 nM)

  • Control for direct antimicrobial activity by testing CCL28 against bacteria without neutrophils

• Antimicrobial activity testing:

  • Test against various microorganisms (e.g., Staphylococcus aureus, Pseudomonas aeruginosa)

  • Use protein concentrations of 0.5-1μM depending on bacterial species sensitivity

  • Compare wild-type CCL28 with mutant proteins to identify essential structural elements

  • Consider testing in various salt concentrations, as antimicrobial activity is more pronounced in low-salt conditions

What is the role of CCL28 in mucosal immune regulation beyond antimicrobial activity?

CCL28 plays multiple roles in mucosal immunity:

• IgA antibody-secreting cell (ASC) regulation:

  • CCL28 is critical for regulating IgA ASC accumulation in mammary glands

  • This regulation is essential for the transfer of IgA antibodies from mother to infant

  • The chemotactic effect on IgA-producing cells helps establish mucosal immunity at various sites

• T-cell recruitment and distribution:

  • CCL28 attracts resting CD4+ and CD8+ T-cells to mucosal sites

  • This recruitment helps establish and maintain the mucosal T-cell pool

  • The interaction occurs primarily through the CCR10 receptor

• Neutrophil function modulation:

  • Beyond simple recruitment, CCL28 enhances neutrophil effector functions:

  • Increases reactive oxygen species (ROS) production

  • Promotes neutrophil extracellular trap (NET) formation

  • Enhances bacterial killing capacity against certain pathogens

  • These effects are primarily mediated through CCR3 rather than CCR10

• Context-dependent immune regulation:

  • CCL28's effects can be protective or detrimental depending on the infection site and pathogen

  • In Salmonella infection, CCL28-dependent neutrophil recruitment is protective

  • In Acinetobacter lung infection, CCL28-mediated neutrophil responses can be detrimental due to excessive inflammation

  • This context dependence parallels other immune components like CXCR2, which can be protective during Salmonella infection but harmful during Mycobacterium tuberculosis lung infection

How can researchers generate and validate CCL28 mutants to study structure-function relationships?

To investigate structure-function relationships of CCL28:

• Mutation strategy design:

  • Generate truncation mutants by creating progressive C-terminal deletions

  • Create targeted deletion mutants that remove specific functional regions (e.g., amino acids 85-89)

  • Design site-specific substitution mutants that change positively charged amino acids to neutral or negatively charged ones

  • Develop chimeric proteins that combine N-terminal regions of related chemokines with the C-terminus of CCL28

• Production methods:

  • Use PCR-based mutagenesis to generate desired mutations

  • Express mutant proteins in appropriate expression systems (typically E. coli)

  • Purify proteins using similar methods as wild-type CCL28

  • Verify purity by SDS-PAGE and HPLC analyses (>96% purity)

• Functional validation assays:

  • Antimicrobial testing: Compare wild-type and mutant CCL28 activity against model organisms like Staphylococcus aureus and Pseudomonas aeruginosa

  • Chemotaxis assays: Determine if mutations affect chemotactic activity for T-cells or other target cells

  • Receptor binding studies: Assess if mutations alter binding to CCR3 or CCR10

  • Neutrophil activation: Measure effects on neutrophil ROS production, NET formation, and bacterial killing

• Structural considerations:

  • Consider the potential impact of mutations on protein folding and stability

  • Evaluate if mutations in the C-terminal region affect interactions with the N-terminal domain

  • Assess how charge distribution alterations affect antimicrobial properties

What are the key experimental controls and considerations when studying CCL28 in vivo?

When designing in vivo studies with CCL28:

• Mouse model selection:

  • Use appropriate disease models (e.g., streptomycin-treated C57BL/6 mouse model for Salmonella colitis)

  • Consider CCL28 knockout mice (Ccl28−/−) to study loss-of-function effects

  • Include wild-type littermates as proper controls to minimize genetic background effects

• Infection route considerations:

  • CCL28's effects differ based on infection route - oral/gastrointestinal versus respiratory tract

  • For Salmonella studies, compare oral infection (which shows CCL28-dependent protection) with intraperitoneal infection (which bypasses gut mucosal defenses and shows no CCL28 effect)

  • For respiratory pathogens like Acinetobacter, intratracheal infection models are appropriate

• Timepoint selection:

  • Include multiple timepoints to capture the dynamics of infection and immune response

  • For Salmonella studies, examine 2-4 days post-infection

  • For Acinetobacter lung infection, monitor within 48 hours (acute phase) and through 10 days (resolution phase)

• Parameter measurements:

  • Quantify CCL28 levels in relevant biological samples (e.g., feces, serum, BAL fluid) by ELISA

  • Determine bacterial colony-forming units (CFU) in affected tissues and dissemination sites

  • Analyze neutrophil accumulation and activation state by flow cytometry

  • Assess receptor expression (CCR3, CCR10) on neutrophils from different sites (bone marrow, blood, infected tissue)

• Controls for direct antimicrobial activity:

  • Test CCL28's direct antimicrobial effects against the pathogen in vitro

  • Include wild-type pathogens and mutants more susceptible to antimicrobial peptides (e.g., ΔphoQ Salmonella)

  • Test at physiologically relevant CCL28 concentrations

  • Consider salt concentration effects, as antimicrobial activity is more pronounced in low-salt conditions