CD14 Human, CHO

What is CD14 and what is its role in the immune system?

CD14 is a glycolipid-anchored membrane glycoprotein primarily expressed on cells of myelomonocyte lineage including monocytes, macrophages, and some granulocytes. It functions as a pattern recognition receptor (PRR) that recognizes pathogen markers, specifically acting as a co-receptor for lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria. Through this interaction, CD14 supports initiation of the innate immune response during bacterial infection . CD14 exists in both membrane-anchored and soluble forms and is encoded by a gene on human chromosome 5q31.1, a region where several genes implicated in asthma pathogenesis are localized . Beyond bacterial recognition, CD14 has been found to contribute to immune responses to viral pathogens such as human respiratory syncytial virus (RSV), and may amplify inflammatory responses in severe cases of SARS-CoV-2 infection . Additionally, CD14 is an integral part of the mechanism by which macrophages interact with and engulf apoptotic cells .

Why are CHO cells commonly used for expressing human CD14?

CHO cells provide an ideal mammalian expression system for studying CD14 functions because they lack endogenous CD14 expression, allowing researchers to examine the specific roles of human CD14 when transfected into these cells. When transfected with human CD14 cDNA, CHO cells (CHO/CD14) gain the ability to respond to bacterial components like LPS and Group B Streptococcal cell wall preparations (GBS) by activating NF-κB, whereas control CHO/NEO cells show no such response . This clean background allows researchers to study CD14-dependent signaling pathways in isolation from other innate immune receptors that might be present in monocytes or macrophages. CHO cells are also advantageous because they grow well in culture, can be stably transfected, perform appropriate post-translational modifications, and have been extensively used for expressing various recombinant proteins, including anti-CD14 antibodies .

What differences exist between membrane-bound CD14 and soluble CD14?

Membrane-bound CD14 (mCD14) is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) link and is primarily expressed on myeloid cells. In contrast, soluble CD14 (sCD14) lacks this GPI anchor and circulates in the bloodstream at considerable concentrations ranging from 800-3200 ng/mL in serum . While mCD14 functions as a receptor on cell surfaces, sCD14 serves as a mediator that can transfer LPS to cells that lack mCD14, thereby enabling non-myeloid cells to respond to LPS . Additionally, sCD14 can combine with LPS binding protein (LBP) to enhance and potentiate cellular responses to both LPS and other bacterial components like GBS . This dual nature of CD14 allows for a more comprehensive immune surveillance system, capable of recognizing bacterial components in various physiological compartments. The soluble form can be measured in serum, plasma, and cell culture supernatants using quantitative ELISA techniques with high sensitivity and specificity .

How can I establish a stable CHO cell line expressing human CD14?

Establishing a stable CHO cell line expressing human CD14 requires a systematic approach involving transfection and selection. Begin by obtaining a mammalian expression vector containing the full-length human CD14 cDNA with an appropriate promoter for expression in CHO cells. Prepare CHO-K1 cells by culturing them in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS) in a 5% CO₂ humidified atmosphere at 37°C .

For transfection, various methods can be employed including lipofection, electroporation, or calcium phosphate precipitation. After transfection, select for stable integrants using G418 (typically at 1 mg/ml) or another appropriate selection marker included in your expression vector . Once colonies appear, isolate and expand individual clones and screen them for CD14 expression using flow cytometry with specific anti-CD14 antibodies.

Confirm functional expression by testing the cells' ability to respond to LPS with NF-κB activation using electrophoretic mobility shift assay (EMSA) or a reporter gene assay. Maintain the established CHO/CD14 cell line in Ham's F-12 medium supplemented with 10% FCS and continuous selection pressure (G418) to prevent loss of CD14 expression over time . Verify that your cell line is free from mycoplasma contamination using appropriate detection methods such as the MycoAlert™ Mycoplasma Detection kit .

What methods are most effective for quantifying CD14 expression in experimental systems?

Several complementary methods can be used for quantifying CD14 expression in research settings:

• Flow Cytometry: This is the gold standard for confirming surface expression of CD14 on cells. Using fluorescently labeled anti-CD14 antibodies, you can determine both the percentage of cells expressing CD14 and the relative expression level (mean fluorescence intensity) on a per-cell basis .

• ELISA: For soluble CD14 released into culture medium or present in biological fluids, quantitative ELISA kits such as the Human CD14 Quantikine ELISA Kit provide precise measurements. These assays typically have excellent sensitivity and can detect CD14 across a range of 250-16,000 pg/mL in various sample types including cell culture supernatants, serum, and plasma .

• Western Blotting: This technique can detect CD14 protein in cell lysates, providing information about total protein expression levels. It requires specific anti-CD14 antibodies and can be semi-quantitative when compared with known standards.

• Functional Assays: Testing the responsiveness of cells to LPS can indirectly confirm functional CD14 expression. For CHO/CD14 cells, measuring NF-κB activation following LPS stimulation provides a functional readout of CD14 expression and activity .

For comprehensive characterization, it's advisable to use multiple methods, as each provides different information about CD14 expression in your experimental system.

How should I design experiments to distinguish CD14-dependent from CD14-independent pathways?

To design experiments that effectively distinguish CD14-dependent from CD14-independent pathways, a systematic approach using paired cell lines and specific inhibitors is required:

• Cell Line Selection:

  • Use matched cell lines: CHO/NEO (control transfectants) and CHO/CD14 (expressing human CD14)

  • Consider additional transfectants expressing other receptors (e.g., CHO/CR3, CHO/CR4) for comparative analysis

• Stimulation Conditions:

  • Test various concentrations of LPS (10-1000 ng/ml) with and without LPS binding protein (LBP)

  • Include other bacterial components (e.g., Group B Streptococcal cell wall preparations)

  • Use serum-free conditions to control for exogenous soluble CD14

• Blocking Experiments:

  • Employ anti-CD14 blocking antibodies (e.g., mAb 3C10) to inhibit CD14-dependent responses

  • For studies in monocytes, include anti-CD18 antibodies to block leukocyte integrin contributions

• Readout Systems:

  • For signaling: Measure NF-κB activation using electrophoretic mobility shift assay (EMSA)

  • Include time-course experiments (15-180 minutes) to capture different activation kinetics

  • Analyze dose-response relationships for all stimuli

This approach has revealed important distinctions in receptor usage. For example, in human monocytes, LPS-induced TNF production is predominantly CD14-dependent but minimally affected by CD18 blockade, while GBS responses are inhibited by both anti-CD14 and anti-CD18 antibodies . Similarly, in CHO cell systems, while both LPS and GBS activate NF-κB in CHO/CD14 cells, LPS but not GBS can also weakly activate NF-κB in CHO/CR3 or CHO/CR4 cells, suggesting differential receptor usage .

How should I interpret differences in activation kinetics between bacterial stimuli in CD14 systems?

When analyzing kinetic differences between LPS and other bacterial components (such as Group B Streptococcal cell wall preparations, GBS) in CD14-transfected CHO cells, several key interpretation guidelines should be considered:

• Temporal Response Patterns:

  • LPS typically induces rapid NF-κB activation in CHO/CD14 cells, with detectable translocation observed within 15 minutes, reaching a plateau at 30-60 minutes, and declining by 180 minutes.

  • In contrast, GBS induces a delayed response, with NF-κB activation only becoming apparent after 60 minutes but persisting at similar levels throughout the 180-minute observation period .

  • These different kinetic profiles suggest distinct mechanisms of receptor engagement and signal transduction.

• Concentration Dependencies:

  • LPS shows activity at relatively low concentrations (as little as 10 ng/ml), with increased potency at 100-1000 ng/ml.

  • GBS typically requires 250-500 fold higher concentrations to achieve comparable NF-κB activation levels .

  • These concentration differences reflect varying affinities for CD14 or differences in the efficiency of CD14-mediated signal transduction.

• Co-receptor Requirements:

  • If responses are predominantly CD14-dependent (blocked by anti-CD14 antibodies) but minimally affected by blocking CD18, this suggests CD14 is the primary receptor.

  • If responses are inhibited by both anti-CD14 and anti-CD18 antibodies, this indicates a requirement for both CD14 and leukocyte integrins for optimal signaling .

These kinetic and concentration-dependent differences provide crucial insights into the molecular mechanisms of pathogen recognition and cellular activation, suggesting that while CD14 represents a common pattern recognition receptor for diverse bacterial components, the specific engagement dynamics and co-receptor requirements vary substantially based on the nature of the bacterial stimulus.

What are normal ranges for soluble CD14 in different sample types?

Understanding normal ranges of soluble CD14 (sCD14) in different sample types is essential for proper experimental design and data interpretation. Based on available data from studies of healthy individuals, here are the reference ranges:

Normal Ranges by Sample Type:

• Serum: 800-3200 ng/mL (mean: 1900 ng/mL)

• EDTA plasma: 1200-2600 ng/mL (mean: 1800 ng/mL)

• Heparin plasma: 1200-3100 ng/mL (mean: 1900 ng/mL)

• Citrate plasma: 1500-3000 ng/mL (mean: 2000 ng/mL)

Cell Culture Supernatants (PBMC):

• Unstimulated: ~5,070 pg/mL (day 1) increasing to ~9,457 pg/mL (day 3)

• PHA-stimulated: ~12,287 pg/mL (day 1) increasing to ~24,720 pg/mL (day 5)

When measuring sCD14 in experimental settings, several factors should be considered for proper normalization and interpretation:

• For clinical samples, compare values to the appropriate reference range for that specific sample type, noting that different anticoagulants can affect measured values.

• For cell culture experiments, normalize to cell number and include time-matched controls, as values change over the culture period even in unstimulated cells.

• For transfected cell lines, establish baseline production for your specific clone and perform time-course experiments to determine optimal sampling times.

• Account for assay variation (typical inter-assay CV% is 4.8-7.4%) and verify that sample values fall within the linear range of the assay .

By understanding these normal ranges and applying appropriate normalization strategies, you can meaningfully interpret sCD14 data across different experimental conditions.

How does CD14 interact with complement receptors in bacterial recognition?

CD14 and complement receptors (CR3/CR4) exhibit complex interactions in bacterial recognition systems, with both independent and collaborative roles in pathogen detection. Several lines of evidence demonstrate this relationship:

• Differential Receptor Usage by Bacterial Components:

  • In CHO transfectant systems, LPS can activate NF-κB through either CD14 or leukocyte integrins (CR3/CR4), although CD14-mediated responses are stronger and occur with faster kinetics.

  • In contrast, GBS (Group B Streptococcal cell wall preparations) signal NF-κB activation exclusively through CD14 in CHO cells, failing to activate CHO/CR3 or CHO/CR4 cells .

• Cooperative Signaling in Primary Cells:

  • In human monocytes, blocking experiments reveal that GBS-induced TNF production requires both CD14 and CD18 (the common β-chain of CR3/CR4) for optimal responses.

  • Anti-CD18 antibodies cause even greater suppression of GBS-mediated TNF release compared to anti-CD14 antibodies, indicating significant contribution from leukocyte integrins .

  • For LPS, CD14 appears to be the predominant receptor in monocytes, as anti-CD14 but not anti-CD18 antibodies potently inhibit cytokine production .

• Enhancement by Accessory Molecules:

  • LPS binding protein (LBP) significantly enhances both LPS and GBS interactions with CD14, facilitating their transfer to the receptor.

  • Soluble CD14 (sCD14) also increases responses to both stimuli, and the combination of sCD14 and LBP further potentiates these effects .

These findings suggest a model where CD14 and complement receptors can function both independently and cooperatively in recognizing different bacterial components, with the relative contribution of each receptor system varying based on the specific pathogen structure. This receptor cooperation likely enhances the sensitivity and specificity of innate immune recognition mechanisms.

How can CD14-expressing CHO cells be used to study pathogen recognition mechanisms?

CD14-expressing CHO cells provide a sophisticated system for studying pathogen recognition mechanisms, offering several methodological advantages:

• Controlled Receptor Environment:

  • CHO/CD14 cells express human CD14 without the complex receptor milieu found on primary immune cells

  • This allows precise delineation of CD14-dependent versus CD14-independent recognition pathways

  • Additional receptors can be co-expressed to reconstruct specific recognition complexes

• Applications for Comparative Studies:

  • Binding Studies: Compare direct binding of labeled bacterial components to CD14-expressing versus control cells

  • Signaling Pathway Analysis: Examine differences in NF-κB activation kinetics between stimuli (LPS shows rapid activation within 15 minutes, while GBS requires 60 minutes)

  • Concentration-Dependency Analysis: Compare dose-response relationships (LPS is active at 10-1000 ng/ml, while GBS requires 250-500 fold higher concentrations)

  • Co-receptor Requirements: Determine need for additional receptors by comparing responses in various CHO transfectants (CD14, CR3, CR4, or combinations)

• Enhancing and Inhibitory Factors:

  • Study how LBP enhances CD14-mediated recognition of both LPS and GBS

  • Investigate how anti-CD14 antibodies block recognition of different pathogen components

  • Explore how CD14 polymorphisms might alter pathogen recognition efficiency

• Viral Pathogen Interactions:

  • Investigate CD14's reported roles in respiratory syncytial virus (RSV) recognition

  • Explore potential contributions to SARS-CoV-2 inflammatory responses

This experimental system has revealed that while CD14 was initially characterized as a receptor for bacterial LPS, it plays broader roles in pathogen recognition. The differential kinetics, concentration dependencies, and co-receptor requirements observed between different pathogen components provide crucial insights into the versatility of CD14 in innate immune recognition and may help explain the varied inflammatory responses seen in different types of infections .

What approaches can be used to develop therapeutic anti-CD14 antibodies using CHO expression systems?

Developing therapeutic anti-CD14 antibodies using CHO expression systems involves a structured methodological approach:

• Design and Construction of Chimeric/Humanized Antibodies:

  • Start with a murine anti-CD14 antibody with known specificity and affinity

  • Create chimeric constructs by joining murine variable regions with human constant regions

  • For example, construct a fusion of single-chain fragment variable (scFv) with the Fc region (hinge, CH2, and CH3 domains) of human IgG1

  • Design expression vectors with appropriate regulatory elements for CHO cells and selection markers

• Expression and Selection in CHO Cells:

  • Transfect constructs into CHO cells using established methods

  • Select stable transfectants using appropriate selection agents (e.g., G418)

  • Screen for high-expressing clones

  • Optimize culture conditions for antibody production

• Functional Characterization:

  • Binding Specificity: Verify using flow cytometry that the chimeric antibody retains strong specific binding to CD14 (comparable to the parental murine antibody)

  • Functional Activity: Assess ability to block LPS binding to CD14

  • Inhibitory Potential: Measure capacity to inhibit LPS-induced inflammatory responses

• Therapeutic Potential Assessment:

  • Evaluate ability to block the LPS-induced systemic inflammatory response syndrome

  • Test efficacy in models of bacteremia or endotoxemia

  • Assess potential applications in inflammatory conditions where CD14 plays a key role

This approach has been successfully implemented to develop chimeric anti-CD14 antibodies like Hm2F9, which retained strong specific antigen-binding ability to CD14 with comparable activity to its parental murine antibody (99.07% vs. 98.86% positive cells) and maintained the ability to block LPS binding to CD14 (reducing positive cells from 98.37% to 1.35%) . Such antibodies could have therapeutic potential in conditions characterized by excessive CD14-mediated inflammatory responses, including sepsis and endotoxemia.

How does CD14 polymorphism impact disease susceptibility and therapeutic approaches?

CD14 polymorphisms have significant implications for disease susceptibility and therapeutic strategies:

• Genetic Variations and Disease Associations:

  • A functional single nucleotide polymorphism (SNP) in the CD14 promoter region affects expression levels and is associated with an increased risk of developing atopy

  • CD14 polymorphisms have been linked to susceptibility or resistance to various diseases including septic shock, asthma, and other inflammatory conditions

  • These genetic variations may influence the balance between protective immunity and pathological inflammation

• Mechanistic Implications:

  • Polymorphisms affecting CD14 expression levels can alter the threshold for activation by bacterial components like LPS

  • Differences in CD14 structure may impact binding affinity for various pathogen-associated molecular patterns

  • Variations in CD14 shedding might influence the ratio of membrane-bound to soluble CD14, affecting systemic responses to infection

• Research Approaches Using CHO Systems:

  • CHO expression systems can be used to compare different CD14 variants under identical conditions

  • Functional differences can be assessed by measuring:

    • Surface expression levels (flow cytometry)

    • LPS binding capacity (competitive binding assays)

    • Signal transduction efficiency (NF-κB activation)

    • Interaction with co-receptors

• Therapeutic Implications:

  • CD14 polymorphisms may affect responsiveness to anti-CD14 therapeutic antibodies

  • Genetic screening for CD14 variants could potentially inform personalized approaches to treating inflammatory conditions

  • Understanding how polymorphisms alter CD14 function may guide development of more targeted anti-inflammatory strategies

The location of the CD14 gene on human chromosome 5q31.1 places it in a region where several genes implicated in asthma pathogenesis are localized, highlighting its potential relevance to allergic and inflammatory conditions . The interactions of CD14 with other receptors, influenced by genetic polymorphisms, are important for normal signaling of LPS and host-pathogen interactions and affect susceptibility or resistance to various diseases.

What are common pitfalls in CD14-CHO cell experiments and how can they be overcome?

Researchers frequently encounter several challenges when working with CD14-expressing CHO cell systems. Here are common pitfalls and their methodological solutions:

• Variable Expression Levels Between Clones:

  • Problem: Different CHO/CD14 clones often show varying levels of CD14 expression, complicating experimental reproducibility.

  • Solutions:

    • Screen multiple clones by flow cytometry and select those with consistent expression

    • Regularly verify expression levels throughout culture periods

    • Cryopreserve early-passage cells to minimize drift

• Loss of Expression Over Time:

  • Problem: CHO/CD14 cells may gradually lose CD14 expression during extended culture.

  • Solutions:

    • Maintain constant selection pressure with appropriate antibiotics

    • Establish maximum passage numbers for experiments

    • Regularly monitor CD14 expression by flow cytometry

• Inconsistent Functional Responses:

  • Problem: Variable NF-κB activation or other functional readouts despite consistent CD14 expression.

  • Solutions:

    • Standardize LPS preparations (source, purification method)

    • Control for cell density and growth phase at time of stimulation

    • Ensure consistent timing in functional assays (especially important given the different kinetics of LPS vs. GBS responses)

    • Include LPS binding protein (LBP) to enhance and stabilize responses

• Technical Challenges in Measuring CD14:

  • Problem: Difficulty distinguishing specific CD14 staining from background or quantifying soluble CD14 accurately.

  • Solutions:

    • For flow cytometry: Use multiple anti-CD14 antibody clones targeting different epitopes

    • For ELISA: Be aware of the assay range (typically 250-16,000 pg/mL) and dilute samples appropriately

    • Account for inter-assay variation (CV% typically 4.8-7.4%)

• Contamination Issues:

  • Problem: Mycoplasma or endotoxin contamination affecting experimental outcomes.

  • Solutions:

    • Regularly test for mycoplasma using validated methods (e.g., MycoAlert™ Mycoplasma Detection kit)

    • Use endotoxin-free reagents and include polymyxin B controls where appropriate

By implementing these technical solutions, researchers can achieve more consistent and reliable results when working with CD14-expressing CHO cell systems, enhancing the reproducibility and validity of their findings.

How can I optimize LPS stimulation protocols for CD14-expressing CHO cells?

Optimizing LPS stimulation protocols for CD14-expressing CHO cells requires systematic attention to multiple experimental parameters:

• LPS Source and Quality:

  • Use ultrapure LPS preparations from reputable sources (E. coli O111:B4 is commonly used)

  • Aliquot LPS upon receipt to avoid freeze-thaw cycles

  • Store at -20°C or -80°C in endotoxin-free vessels

• Concentration Optimization:

  • Perform detailed dose-response experiments (10 ng/ml to 1000 ng/ml)

  • CHO/CD14 cells show detectable NF-κB activation at 10 ng/ml, with increasing responses at 100-1000 ng/ml

  • Establish EC50 values specific to your cell line and readout system

• LPS Binding Protein (LBP) Supplementation:

  • Include recombinant LBP to enhance responses

  • LBP markedly potentiates CD14-dependent responses to LPS by catalyzing LPS transfer to CD14

  • In published studies, LBP significantly enhances NF-κB activation caused by both LPS and GBS in CHO/CD14 cells

• Media Conditions:

  • Use serum-free conditions for defined stimulations (AIM medium has been successfully used)

  • Serum contains variable amounts of soluble CD14 and LBP that can confound results

• Cell Culture Parameters:

  • Standardize cell density, passage number, and growth phase

  • Use cells at 70-80% confluence

  • Verify CD14 expression levels by flow cytometry before experiments

• Timing and Kinetics:

  • Account for the specific kinetics of LPS responses

  • In CHO/CD14 cells, NF-κB activation is detectable at 15 minutes, peaks at 30-60 minutes, and declines by 180 minutes

  • Include multiple time points when establishing a new system

• Technical Controls:

  • Include CHO/NEO cells as negative controls for CD14-specific effects

  • Use anti-CD14 blocking antibodies (e.g., mAb 3C10) to confirm CD14 dependency

  • Include positive controls for your readout system

By methodically optimizing these parameters, researchers can achieve consistent and reproducible LPS stimulation in CD14-expressing CHO cells, establishing a reliable experimental system for studying CD14-dependent innate immune recognition mechanisms.

What quality control measures ensure reliable CD14 detection in research applications?

Implementing robust quality control measures for CD14 detection is essential for obtaining reliable and reproducible results in research applications:

• For Flow Cytometric Detection of CD14 Expression:

  • Use validated anti-CD14 antibody clones with defined epitope specificity

  • Include appropriate isotype controls matched for concentration and fluorophore

  • Run quantitative beads to convert mean fluorescence intensity to absolute values

  • Validate staining protocols on known CD14-positive cells (e.g., monocytes) before applying to transfected systems

  • Perform periodic quality control of the flow cytometer using standardized beads

• For ELISA-Based Detection of Soluble CD14:

  • Follow validated protocols like the Quantikine Human CD14 Immunoassay

  • Include standard curves with each assay run

  • Ensure samples fall within the linear range of the assay (typically 250-16,000 pg/mL)

  • Be aware of precision parameters: intra-assay CV% is typically 4.8-6.4%, while inter-assay CV% is 4.8-7.4%

  • Verify recovery rates in your specific sample matrices (cell culture media: 91%, serum: 98%, various plasma types: 96-102%)

• Functional Validation of CD14:

  • Confirm biological activity through LPS responsiveness

  • Demonstrate CD14-dependency using blocking antibodies

  • Include stimulation controls (e.g., compare LPS with GBS responses)

  • Verify enhancement by accessory molecules like LBP

• Sample Handling for Reliable Results:

  • Process samples consistently and without delay

  • For serum/plasma, use standardized collection and processing protocols

  • Avoid repeated freeze-thaw cycles of samples containing CD14

  • For cell culture supernatants, standardize collection times and storage conditions

• Reference Standards and Controls:

  • Maintain internal reference preparations for long-term studies

  • Include positive and negative control samples in each experimental run

  • Consider using commercially available reference materials when available

  • Document lot numbers and sources of all reagents