In vitro detection of apoptotic stimuli by use of the HL-60 myeloid leukemic cell line.

The human histiocytic lymphoma line HL-60 has served as a model of myeloid cell differentiation and can be induced to differentiate along the neutrophil or monocytic lineage, depending on the external stimulus. The nondifferentiated cell line retains a premyeloid leukemic phenotype and is capable of anchorage-independent growth and proliferation. The role of apoptosis in the regulation of immunologic and inflammatory events associated with homeostasis and disease has been most intensively studied in lymphocytes. In the present study, nondifferentiated HL-60 has served as a model for studying myeloid cell apoptosis by investigating apoptotic changes induced by camptothecin, a DNA topoisomerase inhibitor, as well as physiologic stimuli, including ceramide analogs and a monoclonal antibody against the Fas antigen. Multiparameter flow cytometry was used to evaluate apoptosis by measuring changes in both side scatter and propidium iodide staining. The appearance of apoptotic cells was confirmed biochemically by measuring DNA endonuclease activity by both enzyme-linked immunosorbent assay quantitation and DNA ladder formation on agarose gels and morphologically with the detection of micronuclei by confocal laser microscopy. These studies demonstrate that HL-60 can serve as an in vitro model for the detection of physiologic and pharmacologic apoptotic stimuli and for understanding the early and late cellular changes associated with induction of the apoptotic program.

Neuroendocrine regulation of in vivo cytokine production and effects: I. In vivo regulatory networks involving the neuroendocrine system, interleukin-1 and tumor necrosis factor-alpha.

We have investigated the in vivo regulatory network involving the neuroendocrine system, interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF). Adrenalectomy or hypophysectomy shifted the sensitivity curve to lipopolysaccharide (LPS)-induced lethal shock as well as TNF- and IL-1-induced deaths. Serum levels of IL-1 or TNF were altered in adrenalectomized or hypophysectomized mice following in vivo stimulation with LPS when compared to appropriate sham-operated control mice. Exogenous administration of either IL-1 or TNF could induce increases in serum corticosterone in sham-operated mice. Finally, treatment of adrenalectomized mice with corticosterone or dexamethasone could inhibit the induction of serum IL-1 and TNF and modified the pattern of these cytokine-induced deaths. Dexamethasone was more effective in these conditions than the natural glucocorticoid, corticosterone. Taken together, these data provide in vivo evidence for a feedback system involving the neuroendocrine axis (hypothalamus, pituitary and adrenal glands) leading to corticosterone production and subsequent regulation and/or modulation of IL-1 or TNF levels or activity.

Differential regulation of lipopolysaccharide-induced interleukin 1 and tumor necrosis factor synthesis: effects of endogenous and exogenous glucocorticoids and the role of the pituitary-adrenal axis.

Intraperitoneal injection of a sublethal dose of lipopolysaccharide (LPS) into mice resulted in the appearance of tumor necrosis factor (TNF) in the serum within 45 min. Maximal serum TNF was detected by 1 h, and by 3-4 h TNF levels were no longer significantly above baseline. Injection of mice with an additional dose of LPS at 4 h resulted in no further increase in serum TNF. The in vivo kinetics of TNF appearance correlated with in vitro studies in which TNF mRNA was detected in murine peritoneal macrophages 30 min after LPS stimulation. The increase in serum TNF was not detected in mice treated with dexamethasone, 3 mg/kg, prior to LPS stimulation. The decrease in TNF correlated with the appearance of significant amounts of endogenous serum corticosterone which were maximal by 3 h. Further evidence for the role of endogenous steroids in the modulation of serum TNF levels was obtained in studies with adrenalectomized or hypophysectomized mice. Compared to sham-operated animals, serum TNF levels remain elevated 5 h post LPS stimulation in adrenalectomized or hypophysectomized mice. In contrast with the transient increase in TNF, serum IL 1 was maximal 4 h post LPS injection and remained elevated at 24 h. In vitro studies with primary cultures of human peripheral blood monocytes and human umbilical cord vein endothelial cells demonstrated that LPS-induced monocyte IL 1 levels were reduced approximately 5-fold by 10(-7) M dexamethasone while dexamethasone had only minimal effects on endothelial cell IL 1. Therefore, the in vitro data would suggest that the maintenance of elevated IL 1 levels coincident with the appearance of endogenous corticosteroids during LPS shock is related to the synthesis of IL 1 by both monocyte-macrophages and non-myeloid cell populations including endothelial cells.

Direct identification of the murine pluripotential stem cell using rabbit anti-mouse brain serum.

Previous studies have shown that antisera prepared in rabbits against mouse brain tissue (RAMBS) contain activity against the murine bone marrow colony-forming unit (CFU-s) or pluripotential hemotopoietic stem cell. In the present study, the F(ab')2 portion of RAMBS was examined for its potential efficacy in the identification of the mouse CFU-s when used in an indirect immunofluorescence-labeling technique. After separation of mouse bone marrow cells by a discontinuous bovine serum albumin density gradient, fluorescent cells were observed only in those bands which, by the splenic colony-forming assay, demonstrated CFU-s. Furthermore, the quantity of CFU-s demonstrated by the spleen colony-forming assay approximated the number of fluorescent cells observed in the corresponding band. It appears that the F(ab')2 portion of RAMBS used in an immunofluorescent assay may provide a method for the direct quantitation and identification of the CFU-s content of murine bone marrow.

Effect of a sulfhydryl inhibitor on in vitro bone marrow colonies (CFU-c).

A dose-related increase in the number of in vitro colony-forming units. CFU-c, was observed in mouse bone marrow cell suspensions following the administration of the sulfhydryl inhibitor, sodium iodoacetate. No effect on CFU-s was observed at the dosages and the periods selected for examination. Direct exposure of marrow cells in vitro to various concentrations of iodoacetate did not influence colony formation.