Studies on the Role of Acid Sphingomyelinase and Ceramide in the Regulation of Tumor Necrosis Factor α (TNFα)-converting Enzyme Activity and TNFα Secretion in Macrophages

Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor alpha (TNF alpha) secretion in macrophages. ASMase-deficient (asm(-/-)) mice and isolated peritoneal macrophages produce severalfold more TNF alpha than their wildtype (asm(+/+)) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNF alpha converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNF alpha) to an active 17-kDa form (soluble (s) TNF alpha). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNF alpha production. A substantial portion of the translated pro-TNF alpha was not processed to sTNF alpha; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2-3-fold higher in asm(-/-) macrophages as compared with asm(-/-) macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNF alpha-positive structures in the close vicinity of the plasma membrane in asm(-/-) but not in asm(+/+) macrophages. asm(-/-) cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm(-/-) cells a significant portion of pro-TNF alpha was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNF alpha.