Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c.
BACKGROUND
Histamine is known to elicit a negative feedback effect on anti-IgE and allergen-induced basophil activation. A series of experiments performed between 1981 and 1995 using a manual method showed biological activity of highly diluted histamine. Most of the experiments used histamine in the range 10(-30) (15C)-10(-36) M (18C). These results were confirmed by automated flow cytometry, but this method is based on the selection of basophils by anti-IgE and analysis of basophil activation by anti-CD 63, showing significant but relatively low inhibition (approximately 14%), insufficient to convince the scientific community of the reality of the phenomenon.


OBJECTIVE
We investigated if the use of CD 203c, a basophil specific, earlier marker than CD 63 of the activation cascade, increased the sensitivity of the method, testing two target histamine dilutions, 10(-4) (2C) and 10(-32) M (16C).


METHODS
Basophils, obtained from buffy coats, were pre-incubated with the histamine dilutions and activated by two agonists: anti-IgE and fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Basophil activation was stopped with EDTA. The cells were labelled with anti-IgE, anti-CD 13 and anti-CD 14 for basophil selection, and anti-CD 63 and anti-CD 203c for basophil activation. Results were expressed in up-regulation percentage for CD 63 or mean intensity of fluorescence (MFI) for CD 203c.


RESULTS
Histamine 10(-4) M (2C) and histamine 10(-32) M (16C) were capable of inhibiting both IgE-dependent (anti-IgE) and IgE-independent (fMLP) basophil activation. The percentage inhibition depended on the activation marker used. The highest inhibition for histamine dilution 16C was observed with CD 203c (38%, P<0.001), approximately half the inhibition observed with histamine 2C (73%).


CONCLUSIONS
These new flow cytometric protocols confirmed that high dilutions of histamine may inhibit basophil activation and that the inhibitory effect is not restricted to IgE-dependent activation. The use of CD 203c instead of CD 63 increased the magnitude of the response.
Sainte-Laudy
J
J
Laboratoire d'immunologie et d'allergologie, 5 boulevard du Montparnasse, 75006 Paris, France. jslaudy@wanadoo.fr
Belon
P
P
D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D001491:Basophils / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism; D005434:Flow Cytometry; D006632:Histamine / Q000494:pharmacology*; D006636:Histamine Release / Q000187:drug effects*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D015415:Biomarkers; C427944:ENPP3 protein, human; C026408:anti-IgE antibodies; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006632:Histamine; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases