Nuclear Translocation of NF-κB in Lipopolysaccharide-Treated Macrophages Fails To Correspond to Endotoxicity: Evidence Suggesting a Requirement for a Gamma Interferon-Like Signal

Abstract

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-κB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-κB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, usingd-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations fromEscherichia, Salmonella,Klebsiella, Bacteroides,Pseudomonas, Neisseria, andRhodobacter species as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by >160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-α) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice, Rhodobacter capsulatus LPS stimulated RAW cell NF-κB-like DNA binding protein mobilization and TNF-α production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-κB activation nor TNF-α production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability of R. capsulatus LPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-γ) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-γ-like signal that accounts for enhanced toxicity. In sum, the activation of NF-κB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-κB alone as a signal transduction factor necessary for LPS-mediated toxicity.

Role of Nitric Oxide in Lipopolysaccharide-Induced Hepatic Injury ind-Galactosamine-Sensitized Mice as an Experimental Endotoxic Shock Model

“…Interleukin-1or gamma interferon-specific antibody did not block LPS-induced hepatic injury, which suggested that (i) TNF-␣ may be primarily responsible for hepatic injury and (ii) TNF-␣-induced NO production may participate in the development of hepatic injury. It was recently reported that the lethal dose of LPS in D-GalN-sensitized mice correlated with in vitro TNF-␣ and NO production in a macrophage cell line treated with the same LPS (6), which also supported our conclusions derived from the present study.…”

Comparison of the innate immune responses of porcine monocyte-derived dendritic cells and splenic dendritic cells stimulated with LPS

“…21,39,40 LPS has been reported to trigger mitogen-activated protein kinases, NF-κB, STAT3, IRFs and activator protein signal-transduction pathways by the activation of the TLR4 receptor complex (TLR4/MD2), which evokes the transcription and production of immune genes, including cytokines that are critical for activation of innate immunity. 41,42–44 In this study, we found that increasing TLR4 and MD2 expression was detected in both types of DCs, with higher phagocytic activity, coinciding with previous reports. 45 Similar to the other studies in mice and humans, we demonstrated that the TLR4/MD2 complex genes were up-regulated in porcine moDCs and SDCs in a time-dependent manner in response to LPS stimulation.…”

Development and Characterization of an Endotoxemia Model in Zebra Fish

“…Systemic inflammation is a hallmark of endotoxemia ( 2 , 35 ) where multiple receptors are activated and signals converge to activate the NF-κB family of transcription factors ( 36 , 37 ). To determine the degree of inflammation in the whole larvae, an NF-κB reporter zebra fish line, Tg(NF- κ B:GFP) ( 38 ), was used, where the transcription of the GFP reporter gene is activated by NF-κB.…”