Related Experiment Videos
Cell activation mediated by glycosylphosphatidylinositol-anchored or transmembrane forms of CD14
J Pugin1, V V Kravchenko, J D Lee
1Division of Medical Intensive Care, University Hospital, Geneva, Switzerland.
Abstract:
CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-kappaB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.
Insights
The glycosylphosphatidylinositol (GPI) anchor of CD14 is not essential for lipopolysaccharide (LPS)-induced myeloid cell activation. However, GPI anchoring may be crucial for other CD14 signaling functions, such as calcium mobilization upon antibody cross-linking.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- CD14 is a key receptor on myeloid cells for microbial pathogen components like lipopolysaccharide (LPS).
- CD14 is typically anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) tail, localizing it to specific membrane microdomains.
Purpose of the Study:
- To investigate the role of the GPI tail of CD14 in cellular signaling pathways.
- To compare the signaling capabilities of GPI-anchored CD14 versus transmembrane CD14.
Main Methods:
- Utilized the THP-1 promonocytic cell line engineered to express either GPI-anchored CD14 (THP1-wtCD14) or transmembrane CD14 (THP1-tmCD14).
- Assessed cell activation markers including protein tyrosine kinase phosphorylation, NF-kappaB activation, and cytokine production in response to LPS.
- Investigated calcium mobilization triggered by anti-CD14 antibody cross-linking.
- Employed pharmacologic inhibitors to elucidate intracellular signaling pathways involved in antibody-induced calcium signaling.
Main Results:
- Both GPI-anchored and transmembrane CD14 were expressed in THP-1 cells.
- LPS-induced myeloid cell activation (cytokine production, phosphorylation, NF-kappaB activation) occurred similarly for both forms of CD14.
- Anti-CD14 antibody cross-linking induced rapid calcium mobilization exclusively in cells expressing GPI-anchored CD14.
- Phospholipase C and protein tyrosine kinases were implicated in the antibody-induced calcium signaling pathway.
Conclusions:
- GPI anchoring and localization to membrane microdomains are dispensable for LPS-mediated myeloid cell activation via CD14.
- The GPI anchor of CD14 appears critical for distinct signaling events, exemplified by antibody-induced calcium mobilization.