Role of TNF alpha in bryostatin-induced inhibition of human hematopoiesis

In previous studies bryostatin has been shown to cause dose-dependent stimu latory or inhibitory effects on colony formation in acute myeloid leukemias . In this study we investigated the inhibitory effect of high dose bryostat in-1 (bryo-1) on normal human bone marrow mononuclear cells (BMNC) colony-f orming capacity. Preculturing BMNC for 24 h with 250 nln bryo-1 reduced col ony formation by 66 +/- 11% whereas this treatment did not reduce clonogeni c capacity of highly purified CD34(+) BMNC. When precultures with bryo-1 we re performed in the presence of several cytokine neutralizing antibodies ab rogation of the inhibitory effect could only be demonstrated by anti-TNF al pha. However, preculturing of BMNC or CD34(+) cells with a wide range of TN F alpha concentrations as well as TNF alpha neutralization in supernatant o f bryo-1-stimulated BMNC failed to affect the inhibitory effect on CD34(+) cells. Both indicate that TNF alpha was not the only factor responsible for the inhibitory effect. Depletion of CD14(+) cells from BMNC cultures showe d that upon bryo-1 exposure the monocytes served as the main source of TNF alpha but not as a source of the inhibitory cytokine(s): in CD14(+)-deplete d cultures the combination of exogenous added TNF alpha and bryo-1 resulted in an inhibition of colony formation comparable to that found in crude BMN C. In contrast, purified CD34(+) cultures were not directly affected by bry o-1 and TNF alpha. However, clonogenic growth of purified CD34(+) cells was inhibited if mononuclear cells were preincubated with TNF alpha alone for 24 h, and the supernatant of these cultures was used together with bryo-1. These results shaw that bryo-1-induced inhibition of clonogenic cell growth is not mediated by a direct effect of bryo-1 on CD34 cells but is a result of a process involving production of TNF alpha by CD14(+) cells upon bryo- 1: stimulation together with the induction of (a) secondary factor(s) by TN F alpha, which together with bryo-1 itself is inhibitory towards clonogenic cell growth.