N. Takahashi

Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6

Cytokines are important mediators of the effects observed after the administration of endotoxin. One of them, tumor necrosis factor, is particularly important since it plays a cardinal role in two major endotoxin activities: its antitumor effect and its capacity to induce a systemic inflammatory response syndrome. TNF exerts its activity on a wide variety of target cells by the triggering of two distinct receptor types. TNF-R55 and TNF-R75. They induce distinct intracellular signals but can have cooperative effects. So, their differential triggering or modulation may have clinically relevant consequences. Based upon observations in the mouse, where hTNF does not interact with the TNF-R75 while mTNF triggers both receptor types, we propose that both receptors need to be triggered to obtain lethality after the administration of TNF. Since human TNF has retained antitumor activity, esp. in combination with IFN-gamma, TNF-mutants that are selective agonists for the TNF-R55 might have a broader therapeutic margin. One such human TNF mutant was already shown to be as effective as the wild-type hTNF in a xenograft model. However, several sensitizing agents may mimic TNF-R75 triggering and so make TNF-R55 triggering a lethal challenge. The fact that two such agents, RU38486 and IL-1 have similar effects regarding their kinetics and their capacity to sensitize for the lethality- and IL-6-inducing effect of hTNF may give a hint regarding the mechanism of the sensitizing effect.

Species specificity and involvement of other cytokines in endotoxic shock action of recombinant tumour necrosis factor in mice

We compared the effects of human rTNF and murine rTNF in murine models of toxicity, esp. the induction of endotoxic shock. As was the case for the antitumour activity, we found a marked difference in activity between these two TNFs. Only murine rTNF was able to cause lethality, while human rTNF needed the synergistic action of sensitizing agents to become lethal. Further experiments, such as the study of IL-6 induction by TNF in mice, allowed us to distinguish two types of TNF effects: those that can equally well be exerted by human rTNF and by murine rTNF (type I effects) and those that can only be exerted by murine rTNF (type II effects). Both types of effects, the “toxic” (a type I effect) and the sensitizing (a type II effect) are needed to produce a lethal outcome. Other cytokines such as IL-1 and IFN-γ, however, can also exert such a sensitizing effect and consequently lead to a fatal outcome when co-administered with human rTNF.

Mechanism of induction of tolerance to tumour necrosis factor (TNF): no involvement of modulators of TNF bioavailability or receptor binding.

The repetitive administration of low doses of hTNF to mice induces tolerance to the lethal effects of mTNF. The underlying mechanism is unknown. In this study we have investigated whether changes in bioavailability and receptor binding could account for the observed differences. To that end we compared the pharmacokinetics of mTNF, the antibody response to TNF, the levels of soluble TNF receptors and the receptor binding of TNF in tolerant and control mice. No differences in pharmacokinetic parameters were observed. An antibody response towards hTNF occurred but the antibodies did not neutralize the mTNF used as a challenge. Furthermore, tolerance failed to protect mice against lethality induced by TNF in the presence of galactosamine, where 100- to 1000-fold lower dose of TNF is required. Also, tolerance could be induced in athymic nude mice where the antibody response is absent. These results show that the mechanism of induction of tolerance is not due to an antibody response. No differences in levels of soluble receptors or receptor binding could be observed in tolerant vs control mice. We conclude that the induction of tolerance involves mechanisms operating at the post-receptor pathways.

Mechanisms of sensitization by infections towards tumour necrosis factor induced sirs

The outcome of a challenge with TNF is highly dependent on the condition of the host. Doses of TNF that otherwise cause only minimal toxicitiy can induce lethality in mice suffering from infections or carrying some tumours. Also the coadministration of sensitizing substances such as IL-1, LPS or GalN has similar effects and can decrease the lethal dose of hTNF 50to 100-fold. We now have studied the mechanism by which an infection with BCG causes such a sensitization. Using knock-out mice, neutralizing antibodies and recombinant cytokines, we could show that IL-12 is a necessary and sufficient intermediate, which causes the sensitization mainly through the induction of IFN-y in NK-cells, probably in synergism with endogenous TNF. As is the case for tumour-induced sensitization, which is mediated by other factors than IL-12/IFN-~, an administration of c~-LFA1(CD11a), shortly before the challenge can prevent the lethality while this is not the case for lethality induced by mTNF in unsensitized mice, so identifying a step specific for the sensitization process. Regarding the involvement of NO in the process of lethality, we observed that in all these models, methylene blue could provide a clear protection while NO-synthase inhibitors were unable to do so. However, while NO-synthase inhibitors are able to abolish the protection provided by methylene blue in the models in unsensitized or tumour-sensitized mice, so pointing at a guanylate cyclase independent effect of NO as an essential protective event, this was not the case in the infection-sensitization model. The cause of this paradox is that the NO-synthase inhibitors were unable to decrease the NG levels to the same amount as in the other models. These results may help to clarify the complex mechanisms involved in a model which might be much more relevant for the effects of TNF in sepsis than the administration of TNF to healthy mice.

Tumor Necrosis Factor: Mechanism of Action and its Potential for Anticancer Therapy

Tumor Necrosis Factor (TNF) is secreted by appropriately induced monocytes and also by some T-cells. The subunit of TNF is a 17 kDa polypeptide, while the native molecule corresponds to a trimer. The three-dimensional structure has been solved at 2.6 A resolution. Studies on mutants provided evidence for localization of receptor binding sites (three per trimer) in clefts between subunits (one site for each cleft). TNF (in combination with IFN-γ) is a promising anticancer agent, provided the therapeutic index can be improved. The antitumor activity can be enhanced by the presence of LiC1, but this combination is not without effects on normal cells; when TNF + LiCI was injected in the skin, a rapid, local inflammation was observed.

Tumour Necrosis Factor and Interleukin-6: Structure and Mechanism of Action of the Molecular, Cellular and in Vivo Level

Tumour necrosis factor (TNF) can be induced in experimental animals by injection of Bacillus Calmette-Guerin followed, after one or two weeks, by treatment with lipopolysaccharide (LPS); serum taken a few hours later contains a high concentration of TNF (Carswell et al., 1975). Isolated macrophages, e.g. obtained from placenta, can be activated with interferon-γ (LFN-γ) and 24 h later induced to produce TNF by treatment with LPS. Also monocytic cell lines, such as the human U-937 line or the murine PU-518 line, can be induced under proper conditions to produce TNF (Mannel et al., 1980; Fransen et al., 1985; Marmenout et al., 1985). We have cloned and expressed to a high specific activity in E. coli both the human TNF (hTNF) gene (Marmenout et al., 1985) and the murine TNF (mTNF) gene (Fransen et al., 1985). Also the sequence of the rabbit TNF gene has been reported (Ito et al., 1986). TNF obtained from various species is highly homologous (about 80%). The subunit of the mature hTNF is a 157 amino acids long polypeptide (156 amino acids for mTNF). The native protein is a nearly spherical, trimeric molecule, containing 45% β-structure and little or no α-helix (Wingfield et al., 1987). TNF, as its name implies, was originally recognized as a substance causing necrosis of tumours in experimental animals; this was usually demonstrated by means of a methyl-cholanthrene-induced sarcoma, and it may be noted that obtaining effective tumour regression requires a rather strict adherence to a defined treatment protocol. Remarkably (and almost by coincidence as it later turned out), TNF is also selectively toxic to some transformed cell lines. But in the presence of concomitant treatment with interferon (IFN), many more transformed and malignant cell lines become sensitive to the cytotoxic action of TNF (Williamson et al., 1983; Fransen et al., 1986b).