Zusammenfassung
Humane Lymphocyten aus dem peripheren Blut wurden durch Zentrifugation im diskontinuierlichen Albumingradienten nach ihrer Dichte in verschiedene Populationen getrennt. Vor der Zentrifugation wurden die Lymphocyten von haftenden Zellen durch Filtration an Nylonwolle gereinigt. Für jeden Lymphocytenspender fand sich ein charakteristisches Verteilungsmuster. Der größte Teil der Zellen reicherte sich in den dichteren Zonen des Gradienten an (>1,068 g/cm3). Die getrennten Zellfraktionen wurden in vitro kultiviert und mit Phytohämagglutinin und Streptolysin 0 Antigen stimuliert. Eine Anreicherung der PHA-reaktiven Zellen fand sich in den Schichten geringerer Dichte (<1060 g/cm3). In den gleichen Zonen befanden sich die auf SLO Antigen optimal reagierenden Zellen. In den Zellschichten 1–5, den Zonen geringerer Dichte, ließen sich nie mehr als 12% aller Zellen gewinnen. Auch unstimulierte Lymphocyten aus den Zonen geringerer Dichte wiesen in allen Fällen einen höheren Einbau von Uridin und Thymidin auf als die entsprechenden Originalzellen.
Die Bedeutung dieser Befunde für die Anreicherung von antigenreaktiven Zellen und für den Mechanismus der Transformation von Lymphocyten in vitro wird diskutiert.
Summary
Human blood lymphocytes were separated by density centrifugation in a discontinuous albumin gradient. Prior to centrifugation the lymphocytes were purified by passage through a nylon wool column. A characteristic pattern of density distribution of lymphocytes was found for individual donors. The majority of cells was consistently recovered from the interfaces representing the regions of higher density of the gradient (>1,068 g/cm3). The separated cell fractions were cultured in vitro and stimulated by phytohemagglutinin (PHA) and streptolysin 0 antigen (SLO). Most of the PHA-reacting cells were found in the region of low density (<1060 g/cm3). The optimal response to SLO antigen was observed in cell cultures recovered from the same region of low density. Cell fractions 1–5 of the low density region comprised not more than 12% of the total cells. Unstimulated lymphocytes, too, from the low density region consistently showed a higher uptake of uridine and thymidine compared to the original lymphocyte suspension.
The significance of these findings is discussed with respect to enrichment of antigen-reactive cells and to the mechanism of transformation of lymphocytes in vitro.
Literatur
kabat, D., Attardi, G.: Synthesis of chicken hemoglobins during erythrocyte differentiation. Biochim. biophys. Acta (Amst.)132, 382 (1967).
Legge, D. G., Shortman, K.: The effect of pH on the volume, density and shape of erythrocytes and thymic lymphocytes. Brit. J. Hemat.14, 323 (1968).
Leif, R. C., Vinograd, J.: The distribution of buoyant density of human erythrocytes in bovine albumine solutions. Proc. nat. Acad. Sci. (Wash.)51, 520 (1968).
Shortman, K.: The separation of different cell classes from lymphoid organs. Aust. J. exp. Biol. med. Sci.46, 375 (1968).
—— Seligman, K.: The purification of erythroid cells by pH-induced density changes. J. Cell Biol.42, 783 (1969).
Raidt, D. J., Mishell, R. I., Dutton, R. W.: Cellular events in the immune response. Analysis and in vitro response of mouse spleen cell populations separated by differential flotation in albumin gradients. J. exp. Med.128, 681 (1968).
Meuwissen, H. J., Alten, P. A. van, Bach, F. H., Good, R. A.: Influence of thymus and bursa on in vitro lymphocyte function. Immunologic deficiency disease in man. U.S. Natl. Found. Original SeriesIV, 253, (1969).
Meuwissen, H. J., Alten, P. A. van, Good, R. A.: Decreased lymphoid cell multiplication in the post-thymektomy state. Transplantation7, 1 (1969).
Davies, A. J. S., Festenstein, H., Leuchars, E., Wallis, V. J., Doenhoff, M. J.: A thymic origin for some peripheral blood lymphocytes. Lancet1968 I, 183.
Pearmain, G., Lycette, R. R., Fitzgerald, P. H.: Tuberculininduced mitosis in peripheral blood leucocytes. Lancet1963 I, 637.
Mills, J., Harden, D.: Immunological significance of antigen induced lymphocyte transformation in vitro. Fed. Proc.25, 370 (1966).
Archdeacon, J. W., Glazebrock, G. W., Wise, W. C.: Simple separation of rabbit marrow myeloid cells from erythroid cells. Nature (Lond.)204, 996 (1964).
Miller, R. G., Phillips, R. A.: Separation of cells by velocity sedimentation. J. cell. comp. Physiol.73, 191 (1969).
Vallee, B. L., Hughes, W. L., Gibson, I. G.: A method for the separation of leukocytes from whole blood by flotation on serum albumin. Blood,1, 82 (1947), (Special Issue).
Rabinowitz, Y.: Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass column, including tissue culture observations. Blood23, 811 (1964).
Marshall, W. H., Valentine, F. T., Lawrence, H. S.: Cellular immunity in vitro. Clonal proliferation of antigenstimulated lymphocytes. J. excp. Med.130, 327 (1969).
Maini, R. N., Bryceson, A. D. M., Wolstencroft, R. A., Dumonde, D. C.: Lymphocyte mitogenic factor in man. Nature (Lond.)224, 43 (1969).
Dumonde, D. C., Wolstencroft, R. A., Panayi, G. S., Matthew, M., Morley, J., Howson, W. T.: Lymphokines: Non-antibody mediators of cellular immunity generated by lymphocyte activation. Nature (Lond.)224, 38 (1969).
David, I. R., Al-Askari, S., Lawrence, H. S., Thomas, L.: The in vitro desensitization of sensitive cells by trypsin. J. exp. Med.120, 1189 (1964).
Bloom, B. R., Bennet, B.: Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science153, 81 (1966).
Schild, H. O., Willoughby, D. A.: Possible pharmacological mediators of delayed hypersensitivity. Brit. med. Bull.23, 46 (1967).
Hannig, K., Zeiler, K.: Zur Auftrennung und Charakterisierung immunkompetenter Zellen mit Hilfe der trägerfreien Ablenkungselektrophorese. Hoppe-Seylers Z. physiol. Chem.350, 467 (1969).
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Riethmüller, G., Rieber, E.P. & Riethmüller, D. Untersuchungen zur Heterogenität humaner Lymphocyten: Trennung peripherer Lymphocyten im Albumindichtegradienten und Untersuchung der einzelnen Zellpopulationen auf ihre proliferative Reaktion in vitro. Klin Wochenschr 48, 1343–1349 (1970). https://doi.org/10.1007/BF01485460
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DOI: https://doi.org/10.1007/BF01485460