Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury

Abstract

Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.

Figure 1. Increased Death Rate upon Oxygenation of Mice with Acute Lung Injury

Mice were IT injected with SEB and LPS to model polymicrobial infection and were exposed to 21% or 100% oxygen for 48–60 h. Determination of time-dependent survival curves was prohibited by considerations of unrelieved severe respiratory distress in NIH-approved animal care protocol, which required termination of experiments immediately after differences between groups became apparent. Major differences between groups occurred within 48–60 hours after IT injection of toxins, when the majority of oxygenated animals with inflamed lungs had died, while the nonoxygenated, obviously sick control mice with inflamed lungs were still alive.

Figure 2. Exacerbation of Inflammatory Lung Injury after Exposure of Mice to Different Concentrations of Oxygen

(A) Enhanced lung vascular permeability (left graph) and impairment of lung gas exchange (right graph) in mice breathing 100% O₂ upon induction of acute lung injury. Following IT injection of mice with SEB and LPS, animals breathed 21% or 100% oxygen. After 48 h, lung vascular permeability and lung gas exchange were determined by the amount of protein recovered by BAL or by measuring pO₂ values in arterial blood drawn, respectively, 15 min after return of mice to normal atmosphere. During this equilibration period, three out of seven mice previously exposed to 100% oxygen died, so that no arterial blood gas analyses could be performed, but BAL protein concentrations were determined immediately thereafter. (B) Increased lung vascular permeability (left graph) and impairment of lung gas exchange (right graph) in mice with acute lung injury even upon exposure to lower levels of oxygen (60%), which are considered clinically safe in humans. Experimental conditions were the same as in (A), except oxygen concentration was 60% instead of 100%.

Figure 3. Role of PMNs in the Pathogenesis of LPS-Induced Lung Injury

(A) Depletion of granulocytes attenuates the endotoxin induced rise in alveolocapillary permeability. Pretreatment of mice with anti-Gr-1 was followed by a significant decrease in the number of granulocytes (left graph) and a significant reduction of the total amount of protein (right graph) recovered by BAL 48 h after IT LPS injection. (B) The more granulocytes immigrated into the alveolar spaces, the higher the alveolocapillary permeability rose. Bivariate analysis according to Pearson revealed a statistically significant correlation (p < 0,001) between the number of PMNs and the amount of protein in the BAL fluid 48 h after IT LPS injection, suggesting that inflammatory lung injury after IT injection of LPS is mostly mediated by granulocytes.

Figure 4. Evidence for the Up-Regulation of Immunosuppressive A_2AR Expression on In Vivo-Activated Granulocytes Isolated from Inflamed Lungs

(A) The selective A_2AR agonist CGS21680 inhibited the fMLP-stimulated hydrogen peroxide production by granulocytes in blood of healthy mice to only a small degree, reflecting low levels of expression of A_2AR on naïve blood granulocytes. In contrast, granulocytes recovered by BAL from inflamed lungs 48 h after IT LPS injection were much more inhibited by CGS21680, demonstrating functional up-regulation of A_2AR on in vivo-activated cells. (B) CGS21680 induces cAMP accumulation in in vivo-activated granulocytes isolated from lungs 48 h after IT LPS injection. No effects of the A_2AR agonist were observed in naïve granulocytes obtained from bone marrow of healthy mice (left graph) or in in vivo-activated granulocytes recovered from inflamed lungs of A_2AR gene-deficient mice (right graph). The CGS21680-stimulated cAMP production observed in lung granulocytes obtained from wild-type mice could also be antagonized by the selective A_2AR antagonist ZM241385. Naïve bone marrow granulocytes were used for cAMP measurements, since it was impossible to isolate naïve cells from blood of healthy mice in sufficient numbers. (C) Higher levels of A_2AR-specific mRNA in in vivo-activated granulocytes. In parallel with the much stronger A_2AR agonist-induced inhibition of hydrogen peroxide production and accumulation of cAMP in in vivo-activated granulocytes, the relative levels of A_2AR-specific mRNA were much higher in in vivo-activated granulocytes obtained from inflamed lungs 48 h after IT LPS injection, as compared with naïve granulocytes isolated from the bone marrow of healthy mice (left graph). Up-regulation of A_2AR mRNA in in vivo-activated granulocytes was confirmed in another set of experimental animals breathing 21% oxygen, but was increased to a much lesser extent in animals subjected to 100% oxygen (right graph). Levels of A₁R mRNA did not change much in inflammatory lung granulocytes from animals breathing normal atmosphere, but were clearly increased in mice exposed to 100% O₂. In the two sets of experiments (left and right graphs), granulocytes were pooled from five and six mice per treatment, respectively. Taken together, the results demonstrate that granulocytes recovered from alveolar spaces of inflamed lungs did, indeed, up-regulate their A_2AR expression during these in vivo lung injury assays, thereby confirming and extending previous findings in other inflammation models [11, 53]

Figure 5. Evidence for the Critical Role of Immunosuppressive A_2AR in Lung Protection from Inflammatory Damage

(A) In A_2AR gene-deficient mice, number of PMNs (left graph) and amount of protein recovered (center graph) 48 h after IT LPS injection by BAL was significantly higher than in similarly treated wild-type control mice, reflecting increased lung damage in the absence of A_2AR. The arterial oxygen tension (right graph) was lower in A_2AR gene-deficient mice as compared with wild-type mice. (B) Pharmacologic inactivation of A_2AR leads to exacerbated inflammatory lung tissue damage and decreased lung funciton. After estimation of biologically relevant half-life of A_2AR antagonist ZM241385 (ZM) in vivo (unpublished data), the IT LPS-injected mice were administered ZM241385 at a dose of 10 mg/kg body weight every 6 h subcutaneously to ensure sufficient levels of the antagonist. This dosing regimen of the A_2AR antagonist caused significant more lung tissue damage, as reflected by increased number of PMNs (left graph) and protein levels (center graph) in the BAL fluid obtained after 48 h. In parallel experiments, the A_2AR antagonist decreased lung function (right graph) as compared to untreated wild-type mice, in agreement with results of experiments with A_2AR gene-deficient mice.

Figure 6. Hypoxia Down-Regulates Neutrophils and Protects Lung Tissue from Inflammatory Damage

(A) Exposure of IT LPS-injected mice to hypoxic (10%) oxygen levels for 48 h atmosphere leads to a significantly decreased accumulation of PMNs (left graph), production of LPS-triggered oxygen reactive metabolites in lungs (center graph), and improved lung gas exchange (right graph) compared to a control group of endotoxin-treated mice that were kept at ambient (21%) oxygen. To standardize conditions, the arterial blood samples were taken 15 min after return of the previously hypoxia-exposed animals to normal atmosphere. (B) Treatment by a shorter period of hypoxia attenuates PMN sequestration (left graph) and lung vascular permeability (right graph). Hypoxic treatment of mice even for only 24 h was sufficient to delay PMN sequestration and to diminish the increase in lung vascular permeability. (C) Histologic evidence for the hypoxic inhibition of pulmonary PMN sequestration. Quantitative analysis of lung slices by a pathologist blinded to the experimental design revealed inhibition of PMN sequestration in IT LPS-injected mice following 4-h exposure to hypoxia. Hypoxia not only attenuated PMN accumulation, but the lung tissue damage was also significantly decreased as assessed by the LIS (n = 9, mean ± standard deviation). The representative H&E-stained slices in the right two photomicrographs show less intravascular granulocyte sequestration, less thickening of the alveolocapillary membrane, and almost no granulocytes in the alveolar spaces as compared to IT endotoxin-injected animals breathing 21% O₂. These observations demonstrate that hypoxia also inhibited the transmigration of granulocytes from capillaries into the alveolar spaces.

Figure 7. Hypoxia and Extracellular Adenosine A_2AR Function in the Same Anti-Inflammatory, Lung Tissue-Protecting Pathway

(A) Effects of breathing hypoxic (10%) oxygen on arterial blood oxygen tension (left graph) and plasma adenosine concentration (right graph) in healthy wild-type mice. As a control, data are also shown for healthy mice breathing 21% and 100% oxygen. (B) No survival of A_2AR gene-deficient mice was observed in acute hypoxic lung injury. Wild-type and A_2AR gene-deficient mice were injected IT with LPS and exposed to hypoxia (10%). While the majority of wild-type mice survived, all of the A_2AR gene-deficient mice died, indicating that expression of A_2AR is required for survival of hypoxic lung inflammation; this experiment mimics the clinical situation in which lung inflammation increases to such severity that hypoxia occurs. (C) Significantly higher levels of pulmonary and systemic inflammatory cytokine production in hypoxic A_2AR-deficient mice. Observations of survival were supported by significantly higher BAL and serum (Se) levels of inflammatory cytokines in hypoxic A_2AR-deficient mice compared to hypoxic wild-type mice. Cytokines were determined 2 h after IT LPS injection, because A_2AR-deficient mice started to die soon after LPS administration and thus could not be used in comparative studies with wild-type control mice. The early mortality of A_2AR-deficient mice also did not allow the comparative determination of effects of hypoxia on other late markers of inflammation such as PMN accumulation, lung vascular permeability, and pulmonary gas exchange, which in wild-type mice need about 48 h to develop after IT endotoxin injection. (D) Degree of inflammation is independent from level of oxygen in A_2AR-deficient mice but not in wild-type mice. While BAL fluid TNF-α concentration determined 2 h after IT LPS injection was significantly suppressed in hypoxic wild-type mice compared to animals breathing 100% oxygen, hypoxia had no effect on TNF-α BAL concentrations in A_2AR gene-deficient mice, demonstrating that suppression of TNF-α formation by hypoxia is mediated through A_2AR signaling.

Figure 8. Intratracheal Injection of A_2AR Selective Agonist Mimics Protective Effects of Hypoxia

(A) IT injection of the A_2AR agonist CGS21680 into endotoxin-inflamed lungs provides protection similar to that observed in hypoxia-treated mice. Number of PMNs recovered after 48 h by BAL from endotoxin-injected animals that were kept at normal 21% oxygen atmosphere was significantly diminished by IT injections of CGS21680 compared to placebo-treated mice. Lung PMNs (left graph) from A_2AR agonist-treated animals also produced lower levels of reactive oxygen metabolites (H₂O₂; right graph). (B) Significantly decreased lung vascular permeability (protein in BAL; left graph) and improved lung gas exchange (p_aO₂; right graph) in endotoxin-injected mice after treatment with the A_2AR agonist CGS21680. (C) Histologic evidence for the lung tissue-protecting effects of A_2AR agonist during endotoxin- and oxygenation-induced lung damage. Quantitative analysis of lung histopathology by a pathologist blinded to the experimental design revealed inhibition of PMN sequestration in IT LPS-injected mice after treatment with the A_2AR-selective agonist CGS21680 for 48 h. The lung tissue damage was also significantly decreased as assessed by the LIS (n = 9, mean ± standard deviation). Representative H&E-stained slices in the right two photomicrographs show less intracapillary PMN sequestration and almost no intraalveolar accumulation of PMNs in CGS21680-treated mice. These CGS21680-induced changes are similar to those observed for the effects of hypoxia on endotoxin- injected animals (compare with Figure 6C).

Figure 9. IT Administration of A_2AR Agonist Protects from Increased Death Rate upon Oxygenation of Mice with Acute Lung Injury

Compensation for the oxygenation-associated loss of the hypoxia → adenosine → A_2AR signaling pathway by IT injection of CGS21680 significantly decreased the oxygen-exacerbated death rate in mice with acute lung injury induced by IT injection of SEB and LPS. For further explanation, see legend for Figure 1.