Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.
Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.
Department of Clinical Immunology, Royal North Shore Hospital, St. Leonards, New South Wales, Australia. making@doh.health.nsw.gov.au
M A
MA
King
Q X
QX
Wu
G R
GR
Donovan
B A
BA
Baldo
D000485:Allergens / Q000633:toxicity*; D000818:Animals; D000862:Ant Venoms / Q000633:toxicity*; D001000:Ants; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004912:Erythrocytes / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019476:Insect Proteins / Q000138:chemical synthesis / Q000633:toxicity; D008214:Lymphocytes / Q000187:drug effects; D008555:Melitten / Q000633:toxicity; D010455:Peptides / Q000138:chemical synthesis / Q000633:toxicity*; D013696:Temperature
D000485:Allergens; D000862:Ant Venoms; D019476:Insect Proteins; D010455:Peptides; C115595:pilosulin 1; D008555:Melitten