Use of lipid rafting for the analysis of human basophil activation by flow cytometry.
Inflamm Res
; 59 Suppl 2: S193-5, 2010 Mar.
Article
in En
| MEDLINE
| ID: mdl-20013029
ABSTRACT
BACKGROUND:
Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization.OBJECTIVES:
Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation.METHODS:
Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells.RESULTS:
Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium.CONCLUSIONS:
Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.
Full text:
1
Collection:
01-internacional
Health context:
3_ND
Database:
MEDLINE
Main subject:
Basophils
/
Membrane Microdomains
/
Macrophage Activation
Type of study:
Guideline
Limits:
Humans
Language:
En
Journal:
Inflamm Res
Year:
2010
Document type:
Article