Extracellular ATP drives systemic inflammation, tissue damage and mortality

Abstract

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-1β 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-1β 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.

Figure 1

Apyrase dose dependently protects against LPS-induced shock. (a, b) Mice were injected i.v. with 175 μg (8 mg/kg) Sigma LPS, and pre-treated i.p. with 5, 10 or 15 U apyrase 30 min before. ****P<0.0001, ***P<0.001, compared with LPS controls, n=6. (c, d) Mice were injected i.v. with 175 μg Sigma LPS, and treated i.p. with 15 U apyrase 30 min before or after LPS challenge. ****P<0.0001, ***P<0.001, compared with LPS controls, n=6

Figure 2

Apyrase inhibits systemic TNF, IL-1 and IL-10, but not IL-6 production. (a, c) Systemic IL-1 and TNF induction determined by bioassay (n=4). **P<0.01, ***P<0.001, compared with LPS alone. (b, d, e) Serum IL-1β, TNF and IL-10 levels 2 h after LPS challenge, determined by Bio-Plex (n=3). (f) IL-6 concentrations in serum, assayed via Bio-Plex. *P<0.05, ***P<0.001, ****P<0.0001, compared with LPS alone

Figure 3

Apyrase prevents inflammation and tissue damage. (a) CD45 staining quantification in liver sections 6 h after LPS challenge, n=3–7. (b) Hexosaminidase in serum indicates cellular disintegration. Plotted is the relative increase compared with PBS-treated animals, n=7. (c, d) Mitochondrial complex I and II activity in liver tissue 6 h after LPS injection, n=3–4. (e) TUNEL staining of apoptotic cell death in the jejunum, n=4-6. (f) AB/PAS staining was used to visualize mucin-positive goblet cells in the jejunum, n=6–9. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with LPS (a, b) or PBS controls (c–f), or as indicated by horizontal lines

Figure 4

Suramin reduces inflammation and necrotic damage, but offers no survival benefit. (a–c) Suramin pre-treatment at −30 min reduces systemic TNF (2 h), CD45 staining in the liver (6 h) and systemic hexosaminidase activity (6 h), n=3. (d) Suramin does not reduce systemic IL-1β levels when given 30 min before or after LPS, rather contrary (2 h), n=3. (e) In contrast to apyrase, suramin does not prevent LPS-induced mortality. When given as a post-treatment (+ 30 min), suramin even accelerates mortality (e), correlated with increased TNF production (f, 2 h), earlier evidence of cellular damage (g, 2 h) and enhanced myeloperoxidase (MPO) accumulation in the liver (h, 6 h). *P<0.05, **P<0.01, ****P<0.0001, compared with PBS controls (except for e), with LPS (only for e), or as indicated by horizontal lines