CEACAM8 Human

What is CEACAM8 and where is it expressed in humans?

CEACAM8 (CD66b) is a glycosylphosphatidylinositol (GPI)-linked glycoprotein belonging to the carcinoembryonic antigen (CEA) family within the immunoglobulin superfamily. It is exclusively expressed by human granulocytes and is not found in other cell types . Structurally, CEACAM8 contains immunoglobulin-like domains that facilitate its interactions with other proteins, particularly CEACAM1. The protein is stored in specific vesicles within granulocytes and serves as a marker for exocytosis . An important evolutionary note is that no homolog for CEACAM8 has been identified in rodents, suggesting strong selection pressure during the evolution of the CEA family, possibly due to exposure to microorganisms or parasites .

How is CEACAM8 released from human granulocytes?

CEACAM8 is released from human granulocytes through a regulated secretion process. This release can be triggered by various stimuli, with strong evidence for two particular pathways:

• PMA-induced release: Phorbol-12-myristate-13-acetate (PMA) stimulation leads to significant release of CEACAM8 into the supernatant of human granulocytes, coinciding with granulocyte activation as evidenced by decreased L-selectin (CD62L) cell surface expression .

• TLR9-dependent release: Unmethylated CpG oligodeoxynucleotides (CpG-ODN) trigger CEACAM8 release through TLR9-dependent mechanisms .

The release process is inhibited by cytochalasin D, which blocks the production and transport of secretory vesicles, indicating that vesicular transport is essential for CEACAM8 secretion . This inhibition affects both PMA-induced and TLR9-triggered release of CEACAM8 .

How does soluble CEACAM8 interact with CEACAM1, and what are the functional consequences?

Soluble CEACAM8 released by granulocytes can bind to CEACAM1 expressed on various cell types, including human airway epithelium . This interaction has been demonstrated through cell-based ELISA experiments showing dose-dependent binding of recombinant CEACAM8-Fc to membrane-anchored CEACAM1 on stably transfected A549 cells .

The functional consequence of this interaction is the downregulation of TLR2-dependent inflammatory responses. When CEACAM8-Fc binds to CEACAM1 on human pulmonary epithelial cells (both NHBE and A549 cells), it significantly reduces TLR2-dependent (Pam3Cys-stimulated) release of pro-inflammatory cytokines IL-8 and IL-6 . This inhibitory effect appears to be specific to the CEACAM8-CEACAM1 interaction, as control experiments using rat CEACAM1-Fc (which does not bind to human CEACAM1) showed no such inhibitory effect .

The mechanism involves tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in CEACAM1's intracellular domain and recruitment of the phosphatase SHP-1, which negatively regulates TLR2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway .

What is the relationship between granulocyte count in bronchial lavage fluid and CEACAM8 detection?

Research has established a significant relationship between granulocyte count in human bronchial lavage fluid (BALF) and the detection of soluble CEACAM8. In a study of 165 patients undergoing bronchoscopy for various diagnostic or therapeutic purposes, individuals with high granulocyte counts (≥15% of total cells) were significantly more likely to have detectable levels of soluble CEACAM8 in their BALF compared to those with normal (0-3.9%) or moderately elevated granulocyte counts (4-14.9%) .

The odds ratios were particularly striking:

• High vs. normal granulocyte count: OR 10 (95% CI 3.9-25.6)

• High vs. moderately elevated granulocyte count: OR 6.3 (95% CI 2.0-20.5)

This data strongly suggests that the percentage of granulocytes in BALF serves as a significant predictor for the release of CEACAM8 by activated granulocytes, with potential implications for diagnosing and monitoring neutrophil-driven inflammatory conditions in the lungs .

How does CEACAM8 differ from CEACAM6 in expression and release patterns?

Both CEACAM8 and CEACAM6 are expressed in human granulocytes, but they exhibit different patterns of release in response to stimuli. While CEACAM8 release is significantly increased in response to TLR9 stimulation with unmethylated CpG-ODN, CEACAM6 release is not modulated by CpG-ODN-associated TLR9 activation .

This differential regulation suggests distinct biological roles for these two CEA family members in granulocyte function and immune responses. CEACAM6 has been described to interact with CEACAM1, similar to CEACAM8, but the functional consequences of this interaction appear to be regulated differently . The specific mechanisms governing CEACAM6 release from granulocytes remain less well characterized compared to CEACAM8.

What are the recommended methods for detecting soluble CEACAM8 in biological samples?

For detection of soluble CEACAM8 in biological samples such as cell culture supernatants or bronchial lavage fluid, enzyme-linked immunosorbent assay (ELISA) is the recommended approach . When establishing an ELISA protocol for CEACAM8 detection, researchers should consider:

• Sensitivity requirements: The detection limit of standard CEACAM8 ELISA is approximately 0.84 ng/ml, which appears adequate for most research applications involving activated granulocytes .

• Sample preparation: Careful collection and processing of samples is essential, with immediate centrifugation to remove cells and debris, followed by storage at -80°C until analysis to prevent protein degradation.

• Validation controls: Include appropriate positive controls (such as PMA-stimulated granulocyte supernatants) and negative controls to ensure assay specificity.

For cellular expression analysis, flow cytometry using specific anti-CEACAM8 antibodies is the method of choice, allowing for simultaneous assessment of activation markers such as CD62L .

How should researchers design experiments to study CEACAM8-CEACAM1 interactions?

When designing experiments to study CEACAM8-CEACAM1 interactions, researchers should consider the following methodological approaches:

• Cell-based ELISA: This approach can demonstrate binding of recombinant CEACAM8-Fc to membrane-anchored CEACAM1 in a dose-dependent manner. Comparison between CEACAM1-expressing cells and control transfectants is essential to confirm specificity .

• Functional assays: To assess the functional consequences of CEACAM8-CEACAM1 interaction, researchers can measure TLR2-dependent inflammatory responses (e.g., IL-8 and IL-6 release) in the presence or absence of recombinant CEACAM8-Fc .

• Controls: Important controls include:

  • Rat CEACAM1-Fc, which does not bind human CEACAM1 and serves as a negative control

  • CEACAM1-blocking antibodies (e.g., antibody 18/20) to reverse CEACAM8-Fc effects

  • Verification of protein purity and concentration by SDS-PAGE and Coomassie blue staining

• Molecular signaling analysis: Co-immunoprecipitation experiments can be used to investigate CEACAM1 phosphorylation status and recruitment of phosphatases like SHP-1 .

What are the best approaches for studying CEACAM8 release from human granulocytes?

To effectively study CEACAM8 release from human granulocytes, researchers should consider these methodological approaches:

• Granulocyte isolation and priming:

  • Isolate fresh PMNs (polymorphonuclear neutrophils) from human blood

  • Prime with GM-CSF (50 ng/ml for 90 min) before experimentation

  • Verify priming doesn't alter CD62L expression or IL-8 release

• Stimulation protocols:

  • PMA stimulation: 20 ng/ml for 1 hour is effective

  • TLR agonist stimulation: Test various TLR agonists including:

    • TLR2: Pam3Cys

    • TLR3: Poly:IC

    • TLR4: LPS

    • TLR5: Flagellin

    • TLR7/8: Resiquimod-848 (R848)

    • TLR9: Unmethylated CpG-ODN

• Inhibitor studies:

  • Cytochalasin D (0.5 μg/ml) to block vesicular transport

  • Pan-caspase inhibitor Z-VAD (10 μM)

  • Pan-MMP inhibitor GM-6001 (10 μM)

  • Cycloheximide (1 μg/ml) to block protein synthesis

• Analysis parameters:

  • Measure CEACAM8 release by ELISA

  • Assess granulocyte activation by CD62L expression (flow cytometry)

  • Evaluate cell viability using Annexin V/Propidium iodide staining

  • Monitor IL-8 secretion as a marker of activation

What considerations are important when working with recombinant CEACAM8 proteins?

When working with recombinant CEACAM8 proteins, researchers should consider several important factors:

• Protein characteristics:

  • Molecular weight discrepancies: While the calculated MW of CEACAM8 is approximately 35.1 kDa, due to glycosylation, the protein migrates as 55-66 kDa under reducing conditions on SDS-PAGE

  • Tags and fusion partners: Common tags include polyhistidine tags for purification and AvitagTM for biotinylation

• Storage and handling:

  • Long-term storage: Store in lyophilized state at -20°C or lower

  • Avoid repeated freeze-thaw cycles to maintain protein integrity

  • Follow reconstitution protocols provided in the Certificate of Analysis for optimal performance

• Quality control:

  • Verify purity (>95% by SDS-PAGE)

  • Confirm proper glycosylation status, which may affect binding properties

  • Validate activity through functional binding assays

• Experimental controls:

  • Include proper tag-only controls to rule out tag-mediated effects

  • Use species-specific controls (e.g., rat CEACAM1-Fc) that don't bind human CEACAM1

  • Verify equal amounts of different fusion proteins by SDS-PAGE and Coomassie blue staining before comparative experiments

What are the implications of CEACAM8-CEACAM1 interactions in respiratory infections?

The interaction between CEACAM8 released by granulocytes and CEACAM1 expressed on respiratory epithelium appears to downregulate TLR2-dependent inflammatory responses . This suggests a potential negative feedback mechanism in neutrophil-driven bacterial infections of the respiratory tract. Future research should explore:

• The role of this interaction in specific respiratory infections, particularly those characterized by neutrophilic inflammation

• Whether this mechanism represents a host protective response or could potentially be exploited by pathogens to evade immune clearance

• The balance between beneficial anti-inflammatory effects and potentially detrimental suppression of appropriate immune responses

• The therapeutic potential of modulating this pathway in conditions characterized by excessive neutrophilic inflammation in the lungs

How might the absence of CEACAM8 homologs in rodents impact translational research?

The absence of CEACAM8 homologs in rodents presents significant challenges for translational research in this field . Researchers should consider:

• Alternative model systems: Human cell culture systems, organoids, or humanized mouse models may be more appropriate for studying CEACAM8 biology

• Evolutionary implications: The apparent selection pressure on CEACAM8 during evolution suggests important roles in human-specific host-pathogen interactions that may not be captured in rodent models

• Comparative approaches: Studying the functional differences between human and rodent neutrophil biology may reveal important insights into species-specific immune mechanisms

• Technological solutions: Development of transgenic mice expressing human CEACAM8 could potentially address some translational limitations