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Leukocyte Complement: Neoantigens of the Membrane Attack Complex on the Surface of Human Leukocytes Prepared from Defibrinated Blood^1,2,

From the Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Abstract

The neoantigenic determinants (neoAg) which have been identified in the human C5b-9 membranolytic C complex were detected here by the direct fluorescent antibody technique on the surface of 27 ± 11% of viable peripheral blood leukocytes (PBL). The cells were prepared from defibrinated blood by sedimentation on Ficoll-Hypaque. Specificity of the antisera was established by quantitative inhibition of the fluorescent staining reaction, and of agglutination of EAC1-7, by highly purified C5b-9 complex. No inhibition was observed with fresh normal human serum. The majority of the PBL with surface neoAg was found in the B lymphocyte subpopulation that failed to form rosettes with sheep erythrocytes. NeoAg on B lymphocytes was removed to differing degrees by trypsin, papain, or pepsin treatment, and by maintaining the cells at 4°C for 20 hr in serum-free medium. The individual components, C5, C6, C7, C8, and C9, were also detected on the surface of PBL. With differential fluorescent stains, C5 and neoAg as well as C8 and neoAg could be detected on the same cells. The results indicate that viable B lymphocytes prepared from defibrinated blood, have the components of the membrane attack complex of C on their surface. The concomitant occurrence of the neoAg indicates that these proteins are present at least in part in the form of the assembled terminal complex.

Footnotes

¹ This is publication Number 1400. This investigation was supported by United States Public Health Service Grants AI 07007 and HL 16411 from the National Institutes of Health.

² A preliminary report of these studies has appeared previously: Sundsmo, J. S., W. P. Kolb, and H. J. Müller-Eberhard. 1977. In Regulatory Mechanisms in Lymphocyte Activation. Edited by D. O. Lucas. Academic Press, New York. P. 359.

³ Dr. Sundsmo is supported by United States Public Health Service Training Grant HL 07195.

⁴ Dr. Kolb is the recipient of Research Career Development Award 5 KO4 CA 70841 from the United States Public Health Service.

⁵ Dr. Müller-Eberhard is the Cecil H. and Ida M. Green Investigator in Medical Research, Scripps Clinic and Research Foundation.