Recombinant Rat Monocyte differentiation antigen CD14 (Cd14)

What is rat CD14 and how does it function in the immune system?

Rat CD14 is a 53-55 kDa glycophosphatidylinositol (GPI)-linked glycoprotein belonging to the leucine-rich glycoprotein repeat superfamily of cell-surface proteins. It functions primarily as:

• A coreceptor for bacterial lipopolysaccharide (LPS) that partners with LPS-binding protein (LBP)

• A mediator that delivers LPS to the LY96/TLR4 complex, initiating innate immune responses

• A molecule that acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation and cytokine secretion

• A coreceptor for TLR2:TLR6 heterodimer responding to diacylated lipopeptides and TLR2:TLR1 heterodimer for triacylated lipopeptides

In rats, monocytes are typically defined by reactivity with mAbs ED1 and ED9, as specific antibodies against rat CD14 have not been as widely available as for mouse or human CD14 . The expression of CD14 on rat monocytes is inferred from functional studies and gene expression analysis rather than direct antibody detection in many studies.

How does soluble CD14 (sCD14) differ from membrane-bound CD14 in rat models?

Soluble CD14 (sCD14) represents a significant fraction of CD14 in circulation and differs from membrane-bound CD14 in several important ways:

Research shows that sCD14 can increase the rate at which cell-bound LPS is released from monocyte surfaces and binds to plasma lipoproteins, which significantly reduces monocytes' ability to produce cytokines in response to LPS . Furthermore, P2X7 receptor activation contributes to CD14 release in extracellular vesicles, maintaining optimal CD14 levels during sepsis .

What expression systems are recommended for producing recombinant rat CD14?

Several expression systems have been successfully used for producing recombinant rat CD14, each with specific considerations:

When expressing rat CD14 in insect cells, researchers should note that complex oligosaccharide synthesis does not occur as it does in mammalian cells, although the pentasaccharide core common to N-glycosylation is synthesized . The impact of glycosylation on function should be considered when selecting an expression system.

How can researchers verify the functional activity of recombinant rat CD14?

Verification of recombinant rat CD14 functionality should include multiple assays:

• LPS binding assays: Measure direct binding of CD14 to LPS or lipid A coated on microtiter wells

  • Essential controls: BSA as negative binding control, mannose competition to demonstrate specificity

• CD14 binding to TLR4 complex components: Assess interaction with TLR4/MD-2 complex

  • Methods: ELISA-based binding, co-immunoprecipitation, surface plasmon resonance

• Functional cell-based assays: Measure biological responses in relevant cell types

  • Parameters to assess: NF-κB activation, cytokine production (TNF-α, IL-6)

  • Key consideration: Use CD14-deficient cells reconstituted with recombinant rat CD14 to demonstrate specificity

• Ligand blotting analysis: Confirm binding specificity to LPS and related molecules

  • Technique: Transfer CD14 to PVDF membrane and probe with labeled ligands

How do experimental conditions affect rat CD14-LPS interactions?

Experimental conditions significantly impact rat CD14-LPS interactions and should be carefully controlled:

It's important to note that rat mannose-binding protein A (MBP-A) can bind CD14 in a concentration-dependent manner, and this binding is not inhibited by excess mannose or EDTA . This interaction should be considered when designing experiments with rat CD14.

What are the key differences between rat CD14 and human/mouse CD14 for cross-species studies?

Understanding species differences is crucial for translational research:

How does recombinant rat CD14 modulate inflammation in experimental models?

Recombinant rat CD14 affects inflammation through multiple mechanisms:

• LPS neutralization: Soluble recombinant CD14 can sequester LPS in circulation, limiting monocyte binding and reducing inflammatory responses

  • In mice expressing high levels of soluble CD14, LPS was retained in circulation and animals were protected from LPS-induced lethality

• Modulation of cytokine production: CD14 can both enhance and inhibit inflammatory cytokine production depending on context

  • When used to deplete CD14⁺ monocytes, LPS-induced TNF production was reduced by 28% while Pam3Cys-induced TNF was reduced by 64%

• Inflammatory monocyte regulation: In rats, CD14 expression distinguishes inflammatory monocyte populations

  • Monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 upregulate CD14 expression and show enhanced responsiveness to endotoxin

• Insulin sensitivity effects: Studies show CD14 modulates inflammation-driven insulin resistance

  • Recombinant human soluble CD14 (rh-sCD14) led to increased insulin action in several models, affecting the expression of thousands of genes in adipose tissue, particularly those related to lipid metabolism

What methodological approaches can detect CD14⁺ monocyte subpopulations in rat samples?

Identifying CD14⁺ monocyte subpopulations in rats requires specialized techniques:

• Flow cytometry panels for rat monocyte subsets:

  • Primary markers: CD14, CD16, CD43, CD11b/c, MHC class II (OX-6)

  • Technical note: While rat monocytes are defined by reactivity with mAbs ED1 and ED9, CD14 expression is often inferred rather than directly measured due to antibody limitations

  • Example gating strategy: CD14⁺CD16⁺DR⁺⁺ cells represent proinflammatory monocytes (~10% of all monocytes)

• Isolation of rat monocyte subpopulations:

  • Perfusion technique: To obtain comprehensive samples including cells from the marginal pool, perfuse the extrapulmonary circulation with cold PBS/EDTA

  • Density gradient separation: Use Histopaque 1077 for initial PBMC isolation

  • Magnetic bead separation: Based on CD14 and/or CD16 expression

• Characterization of monocyte activation state:

  • CD14⁺⁺CD16⁻DR⁺ cells represent classical monocytes

  • CD14⁺CD16⁺DR⁺⁺ cells represent proinflammatory monocytes with higher TNF production capacity

  • After LPS stimulation, the median fluorescence intensity for TNF protein was 3-fold higher in CD14⁺CD16⁺ proinflammatory monocytes compared to classical monocytes

How can researchers generate CD14-expressing dendritic cells from rat monocytes?

Generating CD14-expressing dendritic cells from rat monocytes involves several approaches:

• Conventional differentiation protocol:

  • Isolate rat monocytes from peripheral blood using standard techniques

  • Culture with GM-CSF (20-40 ng/ml) and IL-4 (20 ng/ml) for 7 days

  • Monitor CD14 expression throughout differentiation process

• CD14-ML-DC generation (adapted from human protocols):

  • Introduce lentiviral vectors expressing cMYC and BMI1 to expand monocytes

  • For improved efficiency, include BCL2 or LYL1 along with cMYC and BMI1

  • Add IL-4 to differentiate expanded CD14-ML cells into functional DCs (CD14-ML-DC)

  • For antigen-expressing DCs, introduce lentiviral antigen-expression vectors and culture for 2 weeks for drug-selection and expansion

• Verification of DC phenotype:

  • Flow cytometry for surface markers: CD14, CD11c, MHC II (high), CD103 (OX-62)

  • Functional assays: T cell stimulation capacity, cytokine production profile

  • Note: Unlike mice, rat macrophages often express high levels of MHC II, making distinction from DCs more challenging

What are the critical experimental controls when using recombinant rat CD14 in neutralization studies?

When conducting neutralization studies with recombinant rat CD14, the following controls are essential:

• Specificity controls:

  • Isotype-matched control proteins at equivalent concentrations

  • CD14-depleted samples to confirm specificity of observed effects

  • Dose-response curves to establish concentration-dependent effects

• Functional validation controls:

  • Positive control: Known CD14-dependent stimuli (e.g., smooth LPS)

  • Negative control: CD14-independent stimuli (some studies suggest MPLA can stimulate some responses in a CD14-independent manner)

  • Comparison of effects on CD14-knockout cells vs. wild-type cells

• Cross-species considerations:

  • When using antibodies against rat CD14, verify specificity using rat vs. mouse/human cells

  • For recombinant proteins from different species, establish comparative dose-response relationships

• Technical controls for neutralization:

  • Heat-inactivated recombinant CD14 to control for non-specific protein effects

  • Pre-absorption controls with target ligands

  • Timecourse studies to determine optimal neutralization conditions

When depleting sCD14 from serum, researchers should confirm that LBP levels remain unchanged, as this could affect interpretation of results. In one study, LBP levels after sCD14 depletion were 97 ± 6% of precolumn levels .

How does glycosylation affect rat CD14 function in experimental systems?

Glycosylation significantly impacts rat CD14 function through several mechanisms:

• Effects on protein stability and folding:

  • N-linked glycosylation contributes to proper folding and stability

  • Deglycosylated CD14 can maintain some binding capacity to certain partners, as demonstrated with rat MBP-A which bound deglycosylated CD14

• Influence on ligand binding properties:

  • Glycosylation patterns affect the interaction with various LPS chemotypes

  • Expression system selection impacts glycosylation: insect cells provide basic N-glycosylation while mammalian systems (especially CHO cells) provide more complex and physiologically relevant patterns

• Experimental considerations:

  • When comparing recombinant rat CD14 from different expression systems, researchers should be aware that functional differences may arise from glycosylation differences rather than protein sequence issues

  • For studies requiring physiologically relevant glycosylation, mammalian expression systems are preferred

What are the recommended methods for studying CD14-mediated signaling in rat models?

Several approaches are valuable for investigating CD14-mediated signaling in rat models:

• Gene expression analysis:

  • qPCR for key inflammatory genes (IL-6, TNF-α, IL-1β)

  • RNA-Seq to capture broader transcriptional changes

  • In recombinant human sCD14 treatment of high-fat diet mice, 3,479 genes showed significantly different expression in adipose tissue

• Protein-level signaling detection:

  • Western blotting for phosphorylated signaling intermediates (NF-κB, MAP kinases)

  • Flow cytometry for phospho-proteins in specific cell populations

  • Multiplex cytokine assays for secreted factors

• Functional readouts:

  • Cytokine production (TNF-α, IL-6) measured by ELISA

  • Phagocytosis assays

  • Migration/chemotaxis assays

• In vivo approaches:

  • Cecal ligation and puncture (CLP) model with CD14 manipulation

  • Administration of recombinant CD14 to modulate inflammatory responses

  • Extracellular vesicle isolation and characterization for CD14 content

When studying the impact of CD14 on insulin sensitivity, researchers should consider using techniques such as euglycemic hyperinsulinemic clamp, which has been successfully employed to demonstrate that recombinant human sCD14 leads to increased insulin action in various mouse models .