Use of lipid rafting for the analysis of human basophil activation by flow cytometry.
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.


OBJECTIVE
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.
Sainte-Laudy
Jean
J
Laboratoire d'Immunologie, Hôpital Universitaire Dupuytren, Limoges, France. jslaudy@wanadoo.fr
Ouk
Catherine
C
D017304:Annexin A5 / Q000494:pharmacology; D015703:Antigens, CD / Q000187:drug effects; D001491:Basophils / Q000502:physiology*; D002772:Cholera Toxin / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007073:Immunoglobulin E / Q000494:pharmacology; D066298:In Vitro Techniques; D008262:Macrophage Activation / Q000502:physiology*; D021962:Membrane Microdomains / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects; D054397:Receptors, CCR3 / Q000037:antagonists & inhibitors; D017455:Receptors, IgE / Q000187:drug effects; D060149:Tetraspanin 30
D017304:Annexin A5; D015703:Antigens, CD; C554586:CD63 protein, human; D010980:Platelet Membrane Glycoproteins; D054397:Receptors, CCR3; D017455:Receptors, IgE; D060149:Tetraspanin 30; D007073:Immunoglobulin E; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002772:Cholera Toxin