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Studies on the Mechanism of Lymphocyte-Mediated Cytolysis

II. The Use of Various Target Cell Markers to Study Cytolytic Events¹

From the Department of Medicine of the Johns Hopkins University and the O'Neill Memorial Laboratories of The Good Samaritan Hospital, Baltimore, Maryland

Abstract

The lysis of DBA/2 mastocytoma cells by splenic lymphocytes from C57BL mice obtained 10 days after alloantigenic stimulation has been investigated by following the release of various target cell markers. Although the events initiating the cytolytic pathway occurred very rapidly, the rupture of the cell membranes took place some considerable time later, following a period of gradual, progressive increases in permeability. Thus, when the efflux of low molecular weight indicators (e.g., ATP, ⁸⁶Rb) from target cells was measured, increases in membrane permeability could be demonstrated within 20 min of the addition of sensitized lymphocytes. Target cell indicators which became associated with intracellular components, however, (⁵¹Cr, ³H-thymidine-DNA) could not be demonstrated extracellularly unless considerably longer periods of incubation were used. The rate of appearance of these indicators was inversely related to their effective molecular size.

Although the rate of efflux of different indicators varied considerably, the number of effector lymphocytes necessary to initiate specific release was comparable, and events leading to release of label were in all cases similar. Thus, all indicators were released in a linear manner both as a function of time and as a function of the number of effector cells present. Similarly, in the absence of divalent cations, none of the indicators were specifically released.

Measurement of the ATP released after incubation of cytolytically active lymphocytes with target cells indicated that both interacting cell types undergo membrane permeability changes as the result of their interactions, although in the case of the lymphocyte this increase was apparently of a nonprogressive nature.

Footnotes

¹ This work was supported by Grant AI-10280 and a Research Career Development Award from the National Institute of Allergy and Infectious Diseases. This is communication 50 from the O'Neill Memorial Laboratories.

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