CD14 Human HEK

What is the structural basis for CD14's interaction with bacterial lipopolysaccharides?

The X-ray crystal structure of human soluble CD14 reveals a bent solenoid typical of leucine-rich repeat proteins with an amino-terminal pocket that specifically binds acylated ligands including LPS. This structural arrangement facilitates CD14's role in physically delivering lipidated microbial products to various Toll-like receptor signaling complexes, which subsequently induce intracellular proinflammatory signaling cascades upon ligand binding .

How do membrane-bound CD14 (mCD14) and soluble CD14 (sCD14) differ functionally?

Membrane CD14 is anchored to the cell surface and directly involved in LPS-induced monocyte activation. It binds LPS, and antibodies against CD14 block the effects of low-dose LPS . In contrast, soluble CD14 is released by cells and can mediate LPS responses in CD14-negative cells. Interestingly, human intestinal epithelial cells were found to not only express membrane-bound CD14 but also release soluble CD14, which may have important implications in shaping the interaction between the mucosal immune system and bacteria in the gut .

What methods are most effective for detecting CD14 expression in human cell lines?

Research indicates multiple complementary approaches for comprehensive CD14 detection:

• Flow cytometry using specific anti-human CD14 antibodies (such as MEM-15 or MEM-18)

• Western blotting for protein detection

• Reverse transcription-PCR for mRNA expression

• Immunofluorescence staining for cellular localization

These techniques were successfully employed to detect previously unreported CD14 expression in human intestinal epithelial cell lines (SW-480, HT-29, and Caco-2), which had traditionally been considered CD14-negative .

How can HEK293 cells be utilized to study CD14-ligand interactions?

HEK293 cells serve as an excellent model system when transfected with CD14 constructs. Researchers have successfully transfected HEK293 cells with HA-CD14 plasmids to study CD14-ligand interactions. This system allows for the observation of competitive binding between LPS and potential inhibitors. For example, in competition assays, biotinylated LPS bound to HA-CD14 can be immunoprecipitated with anti-HA beads and detected using HRP-conjugated streptavidin .

What experimental conditions are optimal for studying LPS-induced CD14 regulation?

When investigating LPS effects on CD14 expression, researchers should consider:

• Time course: No significant changes occur during the first 3 hours of LPS stimulation, but after 6-15 hours, LPS weakly reduces CD14 mRNA and mCD14 while transiently enhancing sCD14 release. Longer incubation (2 days) causes increases in CD14 mRNA (2-fold), mCD14 (2-fold), and sCD14 (1.5-fold) .

• Concentration range: The maximal effect on mCD14 and sCD14 is reached with ≥1 ng of LPS per ml.

• Controls: Polymyxin B blocks the LPS effect and serves as an important control.

• Cellular model selection: Different cell types (monocytes, macrophages, epithelial cells) may show distinct regulation patterns .

How does CD14 expression change during LPS tolerance and repeated exposure?

After an initial LPS challenge, cells develop tolerance characterized by abolished TNF-α production upon a second 4-hour challenge. Interestingly, this tolerance does not extend to CD14 regulation. In fact, LPS-induced increases in CD14 mRNA, mCD14, and sCD14 are stronger and appear earlier after a second LPS challenge, suggesting differential regulation of CD14 expression versus inflammatory cytokine production during endotoxin tolerance .

What mechanisms underlie CD14 transcriptional regulation in response to bacterial components?

CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents through complex signaling pathways. Beyond LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 μg/ml) cause similar increases in mCD14 expression. This regulation appears to be direct and not mediated by secondary cytokines, as anti-tumor necrosis factor alpha, anti-interleukin-6, anti-gamma interferon, and anti-LPS-binding protein do not block the LPS effect .

How do CD14 inhibitors affect downstream signaling pathways?

CD14 inhibitors like MHP1-AcN competitively bind to CD14 in the presence of LPS. At different doses, MHP1-AcN shows concentration-dependent inhibition:

• At 10 μg/ml of MHP1-AcN treatment, the amount of LPS bound to CD14 or LPS-induced IL-6 is reduced by approximately 50%

• At 100 μg/ml, approximately 90% reduction is observed

This competitive inhibition affects not only TLR4 signaling but potentially also TLR3 and TLR9 signaling associated with CD14 .

How do next-generation humanized mouse models improve CD14-dependent immune studies?

Current humanized mouse models, such as huNRG mice, have demonstrated human CD45+ immune cell reconstitution, including human CD4+ and CD8+ T cells and B cells. Next-generation humanized mice expressing HLA transgenes (huDRAG-A2) show improved engraftment of key immune cell types, including those that express CD14. These advanced models offer several advantages:

• HuDRAG-A2 mice have an increased frequency of certain immune cells (hCD4, hCD14) compared to huNRG mice

• Both models can be infected by HIV and show similar viral loads and characteristic CD4+ T cell depletion

• When infected with Mycobacterium tuberculosis, huDRAG-A2 mice develop granulomas with more classic human pathology

• These models are particularly valuable for co-infection studies and research that depends on immune response evaluation .

What are the therapeutic implications of targeting CD14 in inflammatory diseases?

CD14 has emerged as an important therapeutic target for inflammatory conditions, particularly sepsis-induced acute lung injury. Traditional approaches using CD14 antibodies have failed to consistently decrease inflammatory responses in patients with severe sepsis. Novel CD14 inhibitors like MHP1-AcN demonstrate promise by competitively binding to CD14 and preventing LPS binding. This inhibitory effect on CD14/TLR4 downstream signaling suggests potential applications in treating sepsis-induced acute lung injury and possibly other inflammatory disorders where bacterial components drive pathology .

What controls are essential when evaluating CD14 expression in cell lines?

When studying CD14 expression, several critical controls should be included:

• Isotype-matched antibody controls for flow cytometry (e.g., FITC- and PE-conjugated IgG1 antibodies)

• Known CD14-negative cell lines (such as K562 human erythroblastic cell line) to exclude nonspecific staining

• RT-PCR controls including samples without reverse transcriptase to check for genomic DNA contamination

• Multiple CD14-specific antibody clones (such as MEM-15, MEM-18, MoP9, MoP15, and MoS39) to confirm specificity .

How can differentiation state influence CD14 expression in experimental systems?

The differentiation state of cells can significantly impact CD14 expression levels. For example, when studying intestinal epithelial cells, researchers have used glucose deprivation to induce differentiation in HT-29 cells (HT-29 Glc−). After 20 days of cultivation in glucose-free medium, these cells exhibit altered growth characteristics and differentiation markers. This approach allows researchers to examine the relationship between cellular differentiation and CD14 expression, providing insights into how tissue maturation might affect CD14-mediated responses in vivo .