Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.

Staphylococcal enterotoxin B induces toxic shock and is a major virulence factor of staphylococcal diseases. We examined the effects of systemic adenoviral infection on responses to staphylococcal enterotoxin B in a murine model. We found that adenoviral infection markedly increases the severity of liver injury following exposure to staphylococcal enterotoxin B without D-galactosamine sensitization. In adenovirus-infected mice, staphylococcal enterotoxin B triggered a more profound hypothermia and increased apoptosis in the liver. Consistent with these observations, we also found that adenoviral infection primed for an increased production of gamma interferon in vivo and in vitro following stimulation with staphylococcal enterotoxin B. Gammainterferon-knockout mice did not show increased sensitivity to staphylococcal enterotoxin B following adenoviral infection. These data suggest that a preexisting viral infection primes mice for subsequent staphylococcal enterotoxin B exposure, possibly via a gamma-interferon-mediated mechanism.Staphylococcal enterotoxin B (SEB) belongs to a large family of staphylococcal and streptococcal pyrogenic toxic superantigens and is a major virulence factor of staphylococcal diseases (7). SEB induces toxic shock syndrome and severe food poisoning in humans (7). Aerosolized SEB has also been shown to induce a fatal respiratory distress syndrome in nonhuman primates and is considered a potential threat to public health and safety (31).As a superantigen, SEB binds to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and to specific T-cell receptor (TCR) V␤ chains (29). This interaction results in activation of a large proportion of T cells (up to 30%) and massive release of proinflammatory cytokines (4). In laboratory mice, SEB binds to MHC class II molecules I-A and I-E and activates almost all T cells bearing TCR V␤3, -7, -8.1, -8.2, -8.3, and -17 (29). Mice are known to have natural resistance to the toxic effects of SEB due to the lower affinity of SEB for mouse MHC class II molecules than for human MHC class II molecules (29). Many studies have used D-galactosamine (D-Gal), a hepatocyte-specific inhibitor of transcription, to sensitize mice to the lethal effect of SEB (30, 32).

Airway epithelial cells are often the sites of targeted adenovirus vector delivery. Activation of the host inflammatory response and modulation of signal transduction pathways by adenovirus vectors have been previously documented, including activation of MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase). The effect of activation of these pathways by adenovirus vectors on cell survival has not been examined. Both the PI3-kinase/Akt and ERK/MAP kinase signaling pathways have been linked to cell survival. Akt has been found to play a role in cell survival and apoptosis through its downstream effects on apoptosis-related proteins. Constitutive activation of either PI3-kinase or Akt blocks apoptosis induced by c-Myc, UV radiation, transforming growth factor-beta, Fas, and respiratory syncytial virus infection. We examined the effect of adenovirus vector infection on activation of these prosurvival pathways and its downstream consequences. Airway epithelial cells were transduced with replication-deficient adenoviral vectors containing a nonspecific transgene, green fluorescent protein driven by the cytomegalovirus promoter, or an empty vector with no transgene. They were then exposed to the proapoptotic stimulus actinomycin D plus TNF-alpha, and evidence of apoptosis was evaluated. Compared with the cells treated with actinomycin/TNF alone, the adenovirus vector-infected cells had a 50% reduction in apoptosis. When we examined induction of the prosurvival pathways, ERK and AKT, in the viral vector-infected cells, we found that there was significant activation of both Akt and ERK.

Effects of adenoviral infection on in vivo responses to LPS mediated by TNF-α were evaluated in a murine model. Adenovirus-infected mice showed decreased mortality from fulminant hepatitis induced by administration of LPS or staphylococcal enterotoxin B in the presence of D-galactosamine. Importantly, TNF-α resistance genes within adenoviral E3 region were not required, because E1,E3-deleted vectors showed similar effects. Adenovirus-infected mice exhibited higher TNF-α levels after LPS stimulation, no difference in TNFR1 expression, and similar mortality from Fas-induced fulminant hepatitis. Decreased production of IL-6 and KC in response to exogenous TNF-α, in addition to protection from TNF-α, suggested that adenoviral infection results in TNF-α tolerance.