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Changes in Surface Immunoglobulin Isotypes on Purified Antigen-Binding Cells after Antigenic Stimulation¹

From the Department of Microbiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109; Experimental Pathology Department, Naval Medical Research Institute, Bethesda, Maryland 20014; Department of Microbiology and Immunology, School of Medicine, University of California Los Angeles, California 90024; and Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20014

Abstract

Antigen-binding cells (ABC) were isolated from 5-day immune and nonimmune mice via a two-step centrifugation procedure. Their surface immunoglobulins (sIg) were then stained with fluorescein-conjugated anti-Ig, anti-µ, and anti--specific reagents and analyzed on the fluorescence-activated cell sorter (FACS), or labeled by lactoperoxidase-catalyzed surface iodination and examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Nonimmune ABC, unfractionated and ABC-depleted lymphocytes had identical FACS profiles after anti-µ and anti-Ig staining. Surface radioiodination and SDS-PAGE analysis showed that these nonimmune ABC exhibited a greater amount of than µ on their surface with only a trace of . In contrast, immune ABC bore more µ than and 30 to 50% of these ABC also bore surface . Immune ABC also showed a reduced amount of total sIg compared to Fl-anti-Ig stained unfractionated or ABC-depleted lymphocytes.

Since the intensity of staining with Fl anti-µ did not change after immunization, the reduced total sIg and increased µ/ on the immune ABC were attributable to loss of sIgD after antigenic stimulation of the ABC. This antigen-induced loss of sIgD could be simulated on non-immune ABC and on unfractionated spleen cells by proteolytic removal of the sIgD.

Footnotes

¹ This work was supported by United States Public Health Service Grants CA12800 and CA1920, Naval Medical Research and Development Command Work Unit No. M0095.PN.001.1030, and Uniformed Services University of the Health Sciences Research No. CO8310. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, National Research Council Publication No. (NIH) 74-23.

² Recipient of Postdoctoral Fellowship from the Cancer Research Institute, Inc., New York, New York. Address reprint requests to James J. Kenny, Department of Microbiology, University of Michigan, School of Medicine, 6643 Medical Science Building II, Ann Arbor, Michigan 48109.