Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c

J Sainte-Laudy; P Belon

doi:10.1016/j.homp.2005.10.002

Homeopathy, Table of Contents

Homeopathy 2006; 95(01): 3-8
DOI: 10.1016/j.homp.2005.10.002

Original Paper

Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c

J Sainte-Laudy

¹Laboratoire d’immunologie et d’allergologie, 5 boulevard du Montparnasse, 75006 Paris, France

,

P Belon

²Laboratoires Boiron, 20 rue de la Libération, 69110 Lyon, France

› Author Affiliations
Subject Editor:

Abstract

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 histermine 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%).

Conclusion: 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.

Keywords

high dilutions - histamine - basophil activation - flow cytometry - CD 63 - CD 203c

Full Text

References

References
1 Sainte-Laudy J., Sambucy J.L., Belon P. Biological activity of ultra low doses: I/effect of ultra low doses of histamine on human basophil degranulation triggered by D Pteronyssinus extract. in: Ultra Low Doses. 1991. London: Taylor & Francis Ltd.; 127-138.
2 Sainte-Laudy J, Belon P, Halpern G. Homeopathy: effect of histaminum on ‘in vitro’ basophil degranulation. Abstract, ICACI 1992, p 338.
3 Sainte-Laudy J., Sambucy J.L., Belon P. Biological activity of ultra low doses: II/efffect of ultra low doses of histamine on human basophil degranulation triggered by anti-IgE. in: Ultra Low Doses. 1991. London: Taylor & Francis Ltd.;
4 Lichtenstein L.M., Gillespie E. The effects of the H1 and H2 antihistamines on “allergic” histamine release and its inhibition by histamine. J Pharmacol Exp Ther 1975; 192: 441-450.
5 Masini E., Blandina P., Brunelleschi S., Mannaioni P.F. Evidence for H2-receptor-mediated inhibition of histamine release from isolated rat mast cells. Agents Actions 1982; 12: 85-88.
6 Belon P., Cumps J., Ennis M., Mannaioni P.F., Sainte-Laudy J., Roberfroid M., Wiegant F.A. Inhibition of human basophil degranulation by successive histamine dilutions: results of a European multi-centre trial. Inflamm Res 1999; 48 (Suppl. 01) S17-S18.
7 Davenas E., Beauvais F., Amara J., Oberbaum M., Robinzon B., Miadonna A., Tedeschi A., Pomeranz B., Fortner P., Belon P. et al. Human basophil degranulation triggered by very dilute antiserum against IgE. Nature 1988; 333: 816-818.
8 Ovelgönne J.H., Bol A.W.J.M., Hop W.C.J., Van Wijk R. Mechanical agitation of very dilute antiserum against IgE has no effect on basophil staining properties. Experientia 1992; 48: 504-508.
9 Hirst S.J., Hayes N.A., Burridge J., Pearce F.L., Foreman J.C. Human basophil degranulation is not triggered by very dilute antiserum against human IgE. Nature 1993; 366: 525-527.
10 Sainte-Laudy J., Belon P. Analysis of immunosuppressive activity of serial dilutions of histamine on human basophil activation by flow cytometry. Inflamm Res 1996; 45 (Suppl. 01) S33-S34.
11 Knol E.F., Mul F.P.J., Jansen H., Calafat J., Roos D. Monitoring human basophil activation via CD 63 monoclonal antibody 435. J Allergy Clin Immunol 1991; 88: 328-338.
12 Sainte-Laudy J., Sabbah A., Vallon C., Guerin J.C. Analysis of anti-IgE and allergen induced human basophil activation by flow cytometry. Comparison with histamine release. Inflamm Res 1998; 47: 401-408.
13 Sainte-Laudy J., Sabbah A., Drouet M. et al. Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release. Clin Exp Allergy 2000; 30: 1166-1171.
14 Moneret-Vautrin D.A., Sainte-Laudy J., Kanny G. et al. Human basophil activation measured by CD 63 expression and LTC4 release in IgE-mediated food allergy. Ann Allergy Asthma Immunol 1999; 82: 33-40.
15 Belon P., Cumps J., Ennis M., Mannaioni P.F., Robertfroid M., Sainte-Laudy J., Wiegant F.A.C. Histamine dilutions modulate basophil activation. Inflamm Res 2004; 53: 181-188.
16 Hennersdorf F., Florian S., Jakob A., Baumgartner K., Sonneck K., Nordheim A., Biedermann T., Valent P., Buhring H.J. Identification of CD 13, CD 107a, and CD 164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of ige-dependent upregulation. Cell Res 2005; 15 (05) 325-335.
17 Binder M., Fierlbeck G., King T.P. et al. Idividual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/phosphodiesterase (CD 203c) in sensitised patients. Int Arch Allergy Immunol 2002; 129: 160-168.
18 Buhring H.J., Simmons P.J., Pudney M. et al. The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors. Blood 1999; 94: 2343-2356.
19 Sainte-Laudy J., Belon P. Application of flow cytometry to the analysis of the immunosuppressive effect of histamine dilutions on human basophil activation: effect of cimetidine. Inflamm Res 1997; 46 (Suppl. 01) S27-S28.
20 Brown V., Ennis M. Flow-cytometric analysis of basophil activation: inhibition by histamine at conventional and homeopathic concentrations. Inflamm Res 2001; 50 (Suppl. 02) S47-S48.
21 Lorenz I., Schneider E.M., Stolz P. et al. Sensitive flow cytometric method to test basophil activation influenced by homeopathic histamine dilutions. Forsch Komplementarmed Klass Naturheilkd 2003; 10: 316-324.
22 Aguejouf O., Malfatti E., Belon P., Doutremepuich C. Time related neutralization of two doses of acetyl salicylic acid. Thromb Res 2000; 100: 317-323.