D-galactosamine lethality model: scope and limitations.

D-Galactosamine (D-galN) is well established as sensitizing mice and other animals to the lethal effects of TNF, specifically, and by several orders of magnitude. Protection by anti-TNF neutralizing antibody is complete, as is (metabolically-based) protection by uridine. Sensitization occurs regardless of the origin of the released TNF, whether it is released from macrophages and/or T-cells. The same is true for the challenging agent which leads to the release of TNF, whether it is endotoxin, a superantigen, lipoprotein, bacterial DNA, or bacteria, either killed or proliferating. Most studies have utilized endotoxin as the challenging agent, and more than 70 agents have been reported to confer protection against LPS and/or TNF challenge in the model. The model has provided new insight regarding modes of protection, including from dexamethasone, which protects against challenge from LPS but not from challenge by TNF. The D-galN lethality model has also been used to test for synergistic behavior between different bacterial components, and to test for lethality when only small amounts of the challenging agent are available (lipid A chemistry).

Differential modulation of the induction of inflammatory mediators by antibiotics in mouse macrophages in response to viable Gram-positive and Gram-negative bacteria.

We have investigated effects of beta-lactam antibiotics on TNF-alpha, and iNOS production from mouse peritoneal macrophages following co-culture with Escherichia coli or Staphylococcus aureus bacteria. Ceftazidime and aztreonam enhanced TNF-alpha secretion from macrophages stimulated with E. coli; however, imipenem does not alter either the kinetics or magnitude of TNF-alpha in E. coli-treated macrophages. Similar treatments with S. aureus co-cultured with macrophages markedly altered profiles of TNF-alpha response characterized by apparent early TNF-alpha peak relative to untreated S. aureus. All antibiotics increased E. coli-induced iNOS expression as assessed by both mRNA and protein. These same antibiotics significantly reduced S. aureus-induced iNOS levels of RNA. Both ceftazidime and aztreonam enhanced LPS release from E. coli in comparison to low-level LPS release from imipenem-treated bacteria, consistent with observed differences in TNF-alpha release. Incubation of all three antibiotics with S. aureus similarly increased levels of the cell wall constituent protein A detected in supernatants at early time points indicating microbial lysis. In parallel, S. aureus culture supernatants from 2-h incubation with antibiotics enhanced TNF-alpha release. These results indicate that different cellular mechanisms contribute to antibiotic-mediated regulation of TNF-alpha and iNOS secretion in mouse macrophages in response to E. coli versus S. aureus.

Endogenous versus exogenous glucocorticoid responses to experimental bacterial sepsis.

Although lack of adrenals dramatically reduces resistance against sepsis generally, the value of glucocorticoid levels above those normally produced by stress remains controversial. An early and long-held concept is that glucocorticoid protection against lipopolysaccharides in animal models is important. Supporting this concept, C3H/HeJ mice, lacking Toll-like receptor-4 (TLR-4), and consequently, endotoxin hyporesponsive, have recently been shown to be resistant to glucocorticoid protection against live Escherichia coli. Effective antibiotic intervention, as an additional parameter and with concomitant administration of glucocorticoid, not only allows for expected antibiotic protection but also for glucocorticoid protection against E. coli or Staphylococcus aureus of mice sensitized to tumor necrosis factor alpha, regardless of the status of the TLR-4 receptor. TLRs, including but not limited to TLR-2, may be involved in glucocorticoid protective efficacy against Gram-positive and Gram-negative sepsis. Overlapping and possibly endotoxin-independent signaling may become important considerations.

Anomalous role of tumor necrosis factor alpha in experimental enterococcal infection.

The murine D-galactosamine (D-gal) model of tumor necrosis factor alpha (TNF-alpha) hypersensitization was used as an initial tool to investigate the potential contribution of TNF-alpha to lethal intraperitoneal (i.p.) infection with Enterococcus faecalis. D-gal sensitized mice to lethal E. faecalis infection, whereas dexamethasone and neutralizing anti-TNF-alpha antibody protected D-gal-treated, E. faecalis-infected mice, implicating TNF-alpha in the lethal response to E. faecalis infection in D-gal-treated mice. Circulating TNF-alpha was undetectable for at least 8 h following i.p. E. faecalis infection, although low peritoneal levels of TNF-alpha were detected within 3 h, suggesting that localized TNF-alpha production contributed to the lethal response to E. faecalis infection in D-gal-treated mice. Although i.p. E. faecalis infection failed to induce a detectable systemic TNF-alpha response, circulating Interleukin-6 (IL-6) was detected within 3 h of infection. IL-6 was also detected in the peritoneum within an hour of infection, prior to the appearance of peritoneal TNF-alpha. In striking contrast to in vivo results, E. faecalis induced a potent and rapid TNF-alpha response from both mouse peritoneal macrophages and the RAW 264.7 cell line in vitro. This led us to hypothesize that TNF-alpha production in response to E. faecalis infection is suppressed by IL-6 in vivo. In vitro experiments demonstrated a statistically significant, but modest, inhibitory effect of IL-6 on TNF-alpha production by RAW cells stimulated with E. faecalis. Collectively, these data indicate that acute, lethal E. faecalis infection appears to induce an unusual cytokine response that differs in character from that previously described for most other gram-positive and gram-negative bacteria.