Human monocytes cultured in vitro exert cytostatic activity against the non‐adherent human leukemic cell line K‐562. K‐562 cells in asynchronous growth were added as target cells to adherent mononuclear blood cells, either freshly isolated or cultured in vitro for 4 and 12 days. After 24 hours of co‐culture, the non‐adherent cells were gently removed and incubated further. Target cell DNA‐synthesis and cellular DNA content were examined by methyl‐3H‐thymidine incorporation and by flow cytofluorometry, respectively. Cell‐proliferation curves were obtained. Monocytes cultured in vitro exert a reversible cytostatic activity, which is a partial cell cycle block in the G0/G1 phase. Target cells exposed to freshly isolated monocytes also showed accumulation of cells in the G0/G1 phase. These target cells did not regain their normal proliferation rate before day 3 after removal from the monocyte cultures. This effect may, however, to some extent be due to loosely adherent mononuclear blood cells removed from the monocyte cultures together with the target cells.
Adherent monolayers of freshly isolated human peripheral blood mononuclear cells consist mostly (>90%) of monocytes. The initially adherent nonmonocytic cells detach during the first day of in vitro culture. Concomitant with cell detachment the cytotoxicity mediated by adherent monolayers is reduced. This is in part due to medium exchange with removal of initially adherent cells from the monolayers, cells that are potent mediators of cytostasis and cytolysis. The temporarily adherent cells consist of 10% monocytes, 35% T lymphocytes, 10% B lymphocytes, and 45% non-T, non-B lymphocytes. In addition, 45% of these cells stain positively with the monoclonal antibody OKT 10. The reduction in monolayer-mediated cytotoxicity from day 0 to day 1 of in vitro culture is less pronounced if the temporarily adherent cells are kept within the culture during the cytotoxic assay time. When the temporarily adherent cells are returned to adherent monocyte monolayers an additive effect on cytolysis is demonstrated with nonactivated, lymphokine-activated and α-interferon-activated effector cells, and in antibody-dependent cell-mediated cytotoxicity. The heterogenous population of temporarily adherent cells in freshly isolated monolayers of human mononuclear blood cells seems to be responsible for some of the cytotoxic effects regularly ascribed to freshly isolated human blood monocytes.
Monolayers of freshly isolated peripheral blood monocytes are contaminated with 5–10% non‐monocytic cells. These cells detach during the first day of in vitro culture, concomitant with a disappearance of the strong cytostatic and cytolytic activity of freshly isolated monocytes. The detached cells, mostly non‐monocytic cells, exerted cytostasis and cytolysis on the human natural killer (NK) sensitive cell line K‐562. The lytic activity was sensitive to trypsin treatment. The kinetics of cytolysis was NK‐like, with maximal 51Cr‐release within 4–8 hours of co‐culture, and with a high degree of methyl‐3H‐thymidine release during the first 24 hours of co‐culture. It is suggested that these cells may be a loosely adherent subpopulation of mononuclear cells with NK‐like properties.
The in vitro cytotoxicity of human monocytes was enhanced by leukocyte interferon (IFN‐a). In contrast to lymphokine activation which is obtained gradually under long‐term contact with monocytes, maximal activation of effector cells was obtained within 4 hours of culture with IFN‐a prior to medium exchange and target cell addition. The degree of activation was clearly dose‐dependent, reaching a plateau level of cytolysis at 400 U/ml of IFN‐a. In cytostasis no such plateau was demonstrable. IFN‐a enhanced the level of cytolysis mediated by human monocytes without inducing any change in the kinetics of lysis, regardless of whether IFN‐a was used for activation of monocytes prior to or simultaneously with the cytolysis assay. The direct effect of IFN‐a on K‐562 target cells was sparse. The incorporation of thymidine was modestly depressed in the presence of IFN‐a. Exposure of K‐562 cells to IFN‐a did not have any effect on the spontaneous isotope leakage from prelabelled target cells. The exposure of K‐562 cells to IFN‐a prior to the start of co‐culture with monocytes did not change the level of cytolysis reached, either with non‐activated or with IFN‐a activated monocytes, and irrespective of the in vitro age of the monocytes. The effect of IFN‐a thus seems to be mediated via the effector cell population and not via target cell modulation.
Human monocytes cultured in vitro exert cytostatic activity against neoplastic cell lines. The cytostatic effect was examinated on the nonadherent human leukemic cell line K-562. After 24 hours of co-culture between monocytes and K-562 cells (in a ratio of 10: l), the K-562 cells were removed and their DNA-synthesis and DNA-content were examinated by methyL3H-thymidine incorporation and flow cytofluorometry. Cell proliferation curves were obtained.A partial and reversible cell cycle block in the Go/G1-phase was observed. After removal of the target cells from the monocyte Mononuclear phagocytes may express important effects in host resistence against neoplasia (4,8). We have recently demonstrated development of strong cytostatic activity in human monocytes during in vztro differentiation (5,21), an activity that may be augmented by lymphokines released from BCGstimulated human lymphocytes (6,23). Target cells exposed to monocytes or to monocyte-released factors have been extensively studied (19,20,22). In some animal systems the cytostatic effect of mononuclear phagocytes has been reported to be an irreversible "lethal hit" (lo), while the effect seems to be reversible in other systems (14). Little is known, however, of the behavior of target cells previously exposed to human monocytes. This report presents the behavior of target cells separated from monolayers of human mononuclear phagocytes after co-culture for 24 hr. The behavior of the target cells was followed by registration of proliferation rate, by measuring the incorporation of meth~l-~H-thymidine (m-'HTdR), and by evaluating phase distributions in the cell cycle by flow cytofluorometry (FCM).I This work was supported by grants from the Norwegian Council for Science and Humanities, the Norwegian Cancer Society, and the Norwegian Society for Fighting Cancer.Presented at the Fifth International Symposium on Flow Cytometry, Rome Bracciano, December 2-5, 1980.Research Fellow of the Norwegian Council for Science and Humanities. monolayers, the target cells regained their normal proliferation rate during the following 1 to 3 days, with a maximal number of cells in S-phase at 7 to 9 hours after separation of target cells from the monocytes. The most marked effect was induced by monocytes cultured in vitro for 12 days, and with monocytes stimulated with lymphokine, i.e. supernatants from BCG-exposed lymphocyte cultures.Key words: Macrophages, cytostasis, proliferation, flow cytofluorometry, lymphokines. Materials and MethodsIsolation and culture of human monocytes: Monocytes were isolated by centrifugation (800 x g, 15 min) of defibrinated peripheral blood through Ficoll/Isopaque (sp.w. 1.077) (Lymphoprep., Nyegaard & Co., Oslo, Norway), according to the method of B0yum (2). The cells were washed twice with Hanks' balanced salt solution (HBSS).Cell concentration was adjusted to 6 X lo6 cells/ml in RPMI 1640 (Gibco, Biocult, Glasgow, Scotland), supplemented with 25% pooled human AB Rh' serum, 1 mM 1-glutamine, and 40 p g / d gentamicin (HS-M). In aliquots of 1.0...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.