A ceramide analog inhibits T cell proliferative response through inhibition of glycosphingolipid synthesis and enhancement of N,N-dimethylsphingosine synthesis

Abstract

The ceramide analogue 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol(PDMP) (particularly the D-threo isomer, D-PDMP) caused inhibition of cell growth in some types of cells, and this growth-inhibitory effect has been attributed to inhibition of UDP-Glc:Cer .beta.-Glc transferase, resulting in reduced glycolipid synthesis and increased free ceramide [Inokuch, J., and Radin, N. S. (1987) J. Lipid Res. 28, 565-571; Okada, Y., et al. (1988) FEBS Lett. 235, 25-29]. In view of increasing evidence that the T cell proliferative immune response is modulated by glycosphingolipids (GSLs), the reagent D-PDMP was used to evaluate the role of GSLs in this respect. Con A induced or PHA-induced mitogenesis of C3H/HeJ mouse splenocytes, as well as IL2-dependent CTLL cell growth, were strongly inhibited in a dose-dependent manner when cells were preincubated in the presence of 5-10 .mu.M D-PDMP, but not with its stereoisomer L-PDMP. Closely associated with this growth-inhibitory effect in the presence of D-PDMP, levels of essentially all GSLs, including GM3 and other gangliosides, were greatly reduced, whereas ceramide accumulated. Importantly, metabolically labeled radioactive bands, corresponding to free sphingosine and N-monomethylsphingosine, were found to be present in very small quantities (5-12%) relative to the band corresponding to N,N-dimethylsphingosine (DMS), which showed significant accumulation in D-PDMP-treated lymphocytes. The quantity of IL2 receptors and their affinity to IL2 on T cells did not change, but IL2-dependent tyrosine phosphorylation was greatly stimulated, following D-PDMP treatment. Thus, a decrease in ganglioside levels (including GM3) and increase in DMS may cooperate to enhance IL2-dependent tyrosine phosphorylation of several functionally unknown proteins (Mr 55K, 85K, and 100K), leading to inhibition of cell growth (possibly through exhaustion of intracellular ATP). Exogenous addition of subtoxic doses (1-5 .mu.M) of DMS greatly inhibited IL2-dependent CTLL cell proliferation. Thus, a cell growth regulatory mechanism operating via DMS level must be equally important as that operating via GM3 level, and quantities of these two molecules may cooperatively regulate transmembrane signaling.