Extracellular ATP drives systemic inflammation, tissue damage and mortality.

Systemic inflammatory response syndromes (SIRS) may be caused by both infectious and sterile insults, such as trauma, ischemia-reperfusion or burns. They are characterized by early excessive inflammatory cytokine production and the endogenous release of several toxic and damaging molecules. These are necessary to fight and resolve the cause of SIRS, but often end up progressively damaging cells and tissues, leading to life-threatening multiple organ dysfunction syndrome (MODS). As inflammasome-dependent cytokines such as interleukin-1ß are critically involved in the development of MODS and death in SIRS, and ATP is an essential activator of inflammasomes in vitro, we decided to analyze the ability of ATP removal to prevent excessive tissue damage and mortality in a murine LPS-induced inflammation model. Our results indeed indicate an important pro-inflammatory role for extracellular ATP. However, the effect of ATP is not restricted to inflammasome activation at all. Removing extracellular ATP with systemic apyrase treatment not only prevented IL-1ß accumulation but also the production of inflammasome-independent cytokines such as TNF and IL-10. In addition, ATP removal also prevented systemic evidence of cellular disintegration, mitochondrial damage, apoptosis, intestinal barrier disruption and even mortality. Although blocking ATP receptors with the broad-spectrum P2 purinergic receptor antagonist suramin imitated certain beneficial effects of apyrase treatment, it could not prevent morbidity or mortality at all. We conclude that removal of systemic extracellular ATP could be a valuable strategy to dampen systemic inflammatory damage and toxicity in SIRS.

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.

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation.

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIF(Lps2) mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.

Dual role of endogenous nitric oxide in tumor necrosis factor shock: induced NO tempers oxidative stress.

Tumor necrosis factor (TNF) is involved in pathologies like septic shock, inflammatory bowel disease and rheumatoid arthritis. TNF and lipopolysaccharide can incite lethal shock, in which cardiovascular collapse is centrally orchestrated by the vasodilating free radical nitric oxide (NO). However, NO synthase (NOS) inhibition causes increased morbidity and/or mortality, suggesting a dual role for NO. To investigate the potential protective role of NO during TNF shock, we treated mice with TNF with or without NOS inhibition. Experiments in endothelial- NOS- and inducible NOS-deficient mice identified inducible NOS as the source of protective NO. Distinctive TNF-induced lipid peroxidation, especially in liver and kidney, was aggravated by NOS inhibition. In addition, various antioxidant treatments and a phospholipase A2 (PLA2) inhibitor prevented sensitization by NOS inhibition. Together, these in vivo results indicate that induced NO not only causes hemodynamic collapse, but is also essential for curbing TNF-induced oxidative stress, which appears to hinge on PLA2-dependent mechanisms.

Protection against TNF-induced lethal shock by soluble guanylate cyclase inhibition requires functional inducible nitric oxide synthase.

Hypotension and shock observed in sepsis, SIRS, and tumor necrosis factor (TNF) or cytokine-based cancer treatment are the consequence of excessive nitric oxide (NO) production and subsequent soluble guanylate cyclase (sGC)-mediated vascular smooth muscle relaxation. We demonstrate here that, while NO synthase (NOS) inhibitors exacerbated toxicity, inhibitors of sGC activation protected against TNF-induced lethality, bradycardia, and hypotension. Importantly, sGC inhibition did not interfere with the antitumor activity of TNF. Using NOS inhibitors or iNOS-deficient animals, we furthermore observed that no protection against TNF toxicity could be obtained in the absence of NO. These data imply that iNOS- (and not eNOS-) derived NO is an endogenous protective molecule indispensable to survive a TNF challenge and exerting this beneficial effect via sGC-independent mechanisms.

Coexistence of CD14-dependent and independent pathways for stimulation of human monocytes by gram-positive bacteria.

The cell wall is a key inflammatory agent of gram-positive bacteria. Possible receptors mediating cell wall-induced inflammation include CD14 and platelet-activating factor (PAF) receptor. To delineate the conditions under which these various receptors might be used, human monocytic THP-1 cells and heparinized whole human blood were stimulated with lipopolysaccharide (LPS), intact Streptococcus pneumoniae bacteria, or purified pneumococcal cell wall. THP-1 culture supernatant or cell-free plasma was analyzed for the presence of tumor necrosis factor, interleukin-1beta (IL-1beta), and IL-6. For the cultured monocytes, anti-CD14 inhibited induction of the inflammatory cytokines by the cell wall and LPS but not by intact pneumococcal bacteria. Despite the difference in CD-14 usage, the intracellular pathways induced by the three agents demonstrated similarities, as revealed in the presence of specific signal transduction inhibitors such as cholera toxin, pertussis toxin, and genistein. Cytokine production in whole human blood indicated that anti-CD14 failed to block responses to cell wall and intact pneumococci, whereas while LPS-induced responses were inhibited. PAF receptor antagonist had no effect under any conditions in both assays. These results indicate that although cell walls bind to both CD14 and PAF receptor, only CD14 appears to engender a cytokine response under restricted conditions. Furthermore, host cell responses to intact pneumococci are consistently independent of CD14 and PAF receptor.

Murine IL-12 is involved in Calmette-Guérin bacillus-induced sensitization and is by itself sufficient to sensitize mice to the lethal effects of human TNF1.

Human TNF, a specific agonist of murine (m)TNF-R55, is fairly harmless in healthy mice, but readily induces lethality in animals sensitized by a malignant tumor or a bacterial infection. We here report that IL-12 is both a necessary and sufficient mediator in Calmette-Guérin bacillus-induced sensitization to the lethal effects of hTNF. Evidence for this involvement is provided by the protection observed after anti-mIL-12 Ab treatment and by the fact that Calmette-Guérin bacillus-induced sensitization can be mimicked by repetitive mIL-12 administration. IL-12 exerts this effect by inducing IFN-gamma in NK cells. Tumor-induced sensitization, however, does not seem to involve mIL-12.

Involvement of IFN-gamma in Bacillus Calmette-Guérin-induced but not in tumor-induced sensitization to TNF-induced lethality.

In healthy mice, murine (m) TNF is fairly lethal, whereas human (h) TNF (a selective murine TNF-R55 agonist) is rather harmless. However, we and others observed that mice suffering from a bacterial infection, such as Bacillus Calmette-Guérin (BCG), or bearing i.m. some types of tumor, develop a hypersensitivity to the IL-6-inducing and lethal properties of hTNF. This is a cardinal problem as it severely limits the potential use of hTNF-R55-specific agonists for systemic treatment of human cancer. Using mice carrying a targeted disruption in the gene encoding the IFN-gamma receptor (IFN-gamma Ro/o), we here report that endogenous IFN-gamma plays a crucial role in the development of TNF hypersensitivity during BCG infection. Indeed, both the lethality and the IL-6 induced by hTNF were drastically reduced in IFN-gamma Ro/o mice as compared with control mice. These results demonstrate that the enhancement of TNF effects is at least an equally important mechanism by which IFN-gamma contributes to BCG-induced hypersensitivity as the previously described augmentation of TNF production. Experiments in athymic nude mice, either depleted of NK cells or not, revealed that the latter cell population is an important source of the sensitizing IFN-gamma during BCG infection. In contrast, IFN-gamma Ro/o mice were as susceptible as control mice to the sensitizing effects of tumors. mTNF, which interacts with both mTNF-R55 and mTNF-R75 and causes lethality on its own, is as toxic in IFN-gamma Ro/o mice as in wt control mice; this means that TNF-induced IFN-gamma does not play a role in mTNF-induced lethality.

Inhibition of and sensitization to the lethal effects of tumor necrosis factor.

The extension of the positive results obtained with tumor necrosis factor (TNF) in the locoregional treatment of cancer to systemic treatments requires the selective inhibition of its shock-inducing properties. In this paper, recent data regarding the mechanisms by which infections and tumors render mice extremely sensitive to the lethal effects of TNF as well as regarding the inhibition of the dose-limiting toxicities, hypotension and hepatotoxicity, are summarized. An interleukin-12 (IL-12) driven induction of interferon-gamma (IFN-gamma), probably in synergism with endogenous TNF, was found to mediate infection-induced sensitization. The sensitization induced by tumors develops independent of the IL-12/IFN-gamma axis but ultimately leads to a common step, which can be inhibited by alpha-CD11a and is specific for sensitization. Hypotension can be inhibited by methylene blue (MB), an inhibitor of the nitric oxide (NO)-induced activation of the cytosolic guanylate cyclase, without the indispensable protective properties of NO being affected. Finally, two acute phase proteins, alpha 1-acid-glycoprotein (AGP) and alpha 1-antitrypsin (AT), were able to protect against the TNF-induced liver failure. None of these three inhibitors seems to affect the antitumor effects of TNF.

Response of interleukin-6-deficient mice to tumor necrosis factor-induced metabolic changes and lethality.

Whether interleukin (IL)-6 contributes to tumor necrosis factor (TNF)-induced lethal shock or whether, on the contrary, it is part of a protective feedback system, remains unresolved. Here, we report experiments with IL-6 gene-disrupted mice (IL-6(0/0)). We have tested the susceptibility of these to TNF-induced metabolic changes and lethality in different models, and compared the results with those obtained with IL-6+/+ wild-type mice. We studied the response to TNF in three different models: (i) murine TNF administration; (ii) TNF in galactosamine (GalN)-sensitized mice; (iii) TNF in Bacillus Calmette-Guérin-sensitized mice. We observed no significant difference between the two types of mice in any of the three models. Furthermore, IL-6(0/0) mice could be equally well desensitized (by IL-1) to TNF/GalN-induced lethality and tolerized to TNF-induced shock as IL-6+/+ mice. We also observed that, in response to turpentine, TNF or IL-1, IL-6(0/0) mice produced significantly less acute phase proteins (APP) than IL-6+/+ mice. In IL-6(0/0) mice, less corticosterone was induced by TNF than in the control mice, while the response to adrenocorticotropic hormone was the same. The results indicate that IL-6 is not contributing in a major way to the pathogenesis leading to TNF-induced shock, and that neither IL-6 nor the APP studied are essential for a protective feedback system.

Receptor-selective mutants of tumour necrosis factor in the therapy of cancer: preclinical studies.

The use of TNF-mutants that are selective agonists of the TNF-R55 is one strategy that is being explored to broaden the therapeutic margin of TNF. Several problems still have to be overcome before they can be used in clinical trials. Regarding the sensitizing effect of some infections and some tumours, we identified IFN-gamma as a mediator in BCG- but not in tumour-induced sensitization. In both models, the vessel wall is most probably the key tissue as alpha-LFA-1 antibodies could protect against lethality. Studies in primates showed that an unexpected feature, namely, the longer half-life of such mutants, might interfere with this strategy. Recent observations also indicate that the mechanism of tolerance-induction, another way to separate antitumour and toxic effects of TNF, might reside in the functional ablation of the TNF-R75. Using IL-60/0 knockout mice, we could not find any causal role for IL-6 in TNF-mediated lethality, this in contrast to results obtained previously with neutralizing antibodies. Finally, we identified the acute phase protein alpha 1-acid glycoprotein as a protein with protective properties towards TNF-induced lethality and liver damage.

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.