Published online before print June 3, 2004
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*CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Madrid, Spain; Investigation Unit, Industrial Farmacéutica Cantabria, Madrid, Spain; Department of Immunology, Imperial College, London, United Kingdom; Flow Cytometry Unit, Cancer Research Center and Department of Medicine, University of Salamanca, Spain; and ¶Immune System Diseases and Oncology Service, University Hospital "Príncipe de Asturias," Alcalá de Henares, Madrid, Spain
@ To whom correspondence should be addressed. E-mail: mams{at}tsai.es.
The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative fluorescein-activated cell sorter sorting. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg-positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.
Key Words: FACS • CD • annexin V • 7-amino actinomycin D • physiological • loss
