I am looking over protocols for differentiation and activation of mo-DCs (analysing with flow citometry). They always use CD14 as exclusion marker (monocytes are CD14+, iDC and mDC are CD14-) for CDs.
In many protocols LPS is used as stimulus for activation. How does this activation even work if CD14 is necessary part of "LPS-receptor complex" (TLR4+MD+CD14)?
And why is this complex always presented on DCs (in books, articles) if it is not indeed there? Is CD14 actually differently expressed in vivo?
Thanks for any clarification,
Marija
LPS is potent immunogenic substance that's by is use as positive control for any expression study
These type of stimulations often take place in serum environment. Serum contains soluble CD14 molecules who can substitute for membrane bound CD14 molecules.
Do LPS stimulations in the presence or absence of serum and notice the difference :)
Little update from my research on this topic - if I understand it correctly - CD14 is absolutely necessary only for TRAM/TRIF part of TLR4 signalisation (because of it's involvement in endocytosis of TLR4 and LPS), TIRAF/MyD88 part of signalling, however, is possible even without CD14.
So even if CD14 would be entirely absent (and as your answers explain, this is often not the case), signal could still be transduced downstream.
Thanks all for the input!
Specificities of the TLRs are also influenced by various
non-TLR accessory molecules. This is best defined
for the TLR4 response to LPS. LPS first binds to soluble
LPS-binding protein in the blood or extracellular fluid,
and this complex serves to facilitate delivery of the LPS
to the surface of the responding cell. An extracellular
protein called MD2 (myeloid differentiation protein 2)
binds to the lipid A component of LPS, forming a complex
that then interacts with TLR4 and initiates signaling.
Another protein called CD14 is also required for
efficient LPS-induced signaling. CD14 is expressed by
most cells (except endothelial cells) as a soluble protein
or as a glycophosphatidylinositol-linked membrane protein. from Cellular and Molecular Immunology 8E
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