Characterization of lymphocytes in a patient with Wiskott-Aldrich syndrome: studies by fluorescence polarization.
Lymphocytes from a patient with Wiskott-Aldrich syndrome (WAS) were employed for a study of the intracellular viscosity and fluorescein permeability through the cell membrane by a fluorescence polarization spectrofluorometer, which was designed to calculate polarization value and permeable fluorescein intensity automatically. Fluorescein diacetate (FDA) was used as the indicator probe. The fluorogenic substrate is taken up by viable cells and converted to a fluorescent molecule, fluorescein, by intracellular esterase, where upon the fluorescein easily effluxes through the cell membrane. The response to stimulation with phytohemagglutinin (PHA) for 45 min led to a decreased polarization value (p-value) as compared to lymphocytes of healthy donors, and the fluorescein efflux through the cell membrane was greater than that of healthy donors. Fluorescein efflux from lymphocytes in the patient during 48 and 72 h incubation with or without PHA was markedly increased. In healthy donors, the degree of fluorescein permeability was not increased during the culture. These results indicate that intracellular viscosity of lymphocytes is altered in initial mitogenic stimulation, but that there was some abnormality in the fluorescein permeability properties through the cell membrane of lymphocytes in a patient with WAS.
Shiotsuki
K
K
Department of Immunology, Kurume University School of Medicine, Fukuoka, Japan.
Hirata
T
T
Ota
M
M
Kimura
K
K
Hiyoshi
Y
Y
Yamashita
F
F
Yokoyama
M M
MM
D000293:Adolescent; D002463:Cell Membrane Permeability; D005452:Fluoresceins; D005454:Fluorescence Polarization; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000502:physiology; D008213:Lymphocyte Activation / Q000502:physiology; D008214:Lymphocytes / Q000276:immunology / Q000502:physiology*; D008297:Male; D010835:Phytohemagglutinins / Q000494:pharmacology; D014783:Viscosity; D014923:Wiskott-Aldrich Syndrome / Q000097:blood*
D005452:Fluoresceins; D010835:Phytohemagglutinins; C018506:diacetylfluorescein