The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptorinduced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-B pathway, but NF-B activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-B activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IB␣ mutant (IB␣-SR) or lack of IB kinase ␥ expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-B in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IB␣-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-B inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-B activation.Several years ago, p65/RelA knockout mice, which show an embryonic lethal phenotype due to extensive apoptosis of hepatocytes, gave a first clue that NF-B may have an important role in the inhibition of apoptosis (3). Subsequent studies revealed that inhibition of NF-B activation enhances the apoptotic effects of a variety of death inducers, like tumor necrosis factor (TNF), ionizing radiation, and chemotherapeutic agents (4,25,36,39,43), whereas pretreatment of cells with NF-B inducers, like interleukin 1 (IL-1), can confer resistance against the induction of apoptosis (12,19). On the other hand, NF-B activation can be inhibited by caspase-generated cleavage products of components of the NF-B signaling pathway that act as dominant-negative variants of their full-length parental forms (1,16,22,24,31). Hence, activation of the NF-B pathway and induction of apoptosis inhibit each other. This leads to a rapid amplification of the particular signaling pathway that is initially dominant. This regulatory circuit facilitates a clear decision between life and death at the cellular level in cells that are exposed to a complex pattern of distinct and possibly counteracting stimuli.In line with its antiapoptotic properties, NF-B regulates several genes encoding proteins with antiapoptotic properties, such as A20 (33), cIAP2 (8), TRAF1 (35, 40), Bfl-1/A1 (20, 45), IEX-1L (44), and Bcl-x L (7). Although ectopic overexpression of one or a combination of these proteins may efficiently prevent apoptosis induced by TNF, ionizing radiation, or chemotherapeutic agents, it is still questionable wheth...

Macrophages stand in the first line of defense against a variety of pathogens but are also involved in the maintenance of tissue homeostasis. To fulfill their functions macrophages sense a broad range of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs) by plasma membrane and intracellular pattern recognition receptors (PRRs). Intriguingly, the overwhelming majority of PPRs trigger the production of the pleiotropic cytokine tumor necrosis factor-alpha (TNF). TNF affects almost any type of cell including macrophages themselves. TNF promotes the inflammatory activity of macrophages but also controls macrophage survival and death. TNF exerts its activities by stimulation of two different types of receptors, TNF receptor-1 (TNFR1) and TNFR2, which are both expressed by macrophages. The two TNF receptor types trigger distinct and common signaling pathways that can work in an interconnected manner. Based on a brief general description of major TNF receptor-associated signaling pathways, we focus in this review on research of recent years that revealed insights into the molecular mechanisms how the TNFR1-TNFR2 signaling network controls the life and death balance of macrophages. In particular, we discuss how the TNFR1-TNFR2 signaling network is integrated into PRR signaling.

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFκB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFκB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFκB was activated by overexpressed FLIPL and FLIPS in a cell type–specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFκB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFκB target gene IL8. As NFκB signaling is inhibited during apoptosis, FasL-induced NFκB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFκB-related response.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.