ARDS represents a stereotypic progress through several phases after pulmonary or extrapulmonary insults. At first, alveolar macrophages produce mediators that cause inflammatory cells to accumulate in the lungs and evoke lung tissue damage. Pathologic impairment of vascular permeability in the alveolar epithelial barrier and apoptosis and/or necrosis of type I and II alveolar cells could be the result of induced inflammation and associated release of pro-inflammatory mediators. Pulmonary oedema, surfactant inactivation, and the deposition of dead cells and debris along the alveoli including hyaline membranes are the result of these alterations, which reduce lung compliance and affect gas exchange in the lungs [2, 27]. Inflammatory mediators such as IL-1, IL-6, TNFα, and IL-18 regulate the inflammatory process that is generated by immune cells [28].
PDEs are enzymes catalyzing the metabolism of intracellular cAMP and cGMP that are expressed in a variety of cell types and respiratory diseases [29, 30]. Thus, increasing cAMP by inhibition of its degradation by PDEs PDE inhibitors play a role in airway smooth muscle relaxation and inhibition of cellular inflammation or other immune responses [31] and may also be helpful in treating severe respiratory diseases.
In this study, we focused on the effects of an intravenously administered non-selective PDE inhibitor aminophylline on the inflammatory response, pro-inflammatory cytokine production, oxidative damage and oedema formation, and ultimately on the respiration and gas exchange during the acute phase of experimental ARDS.
Lavage-induced imbalance in the alveoli and associated inflammation lead to deterioration of alveolar-capillary membrane integrity and influx of plasma proteins and activated inflammatory cells into the alveoli. This process affects the function of pulmonary surfactant and ventilation-perfusion mismatch, and thus respiration. Repeated lung lavage led to worsening of lung function parameters (P/F, OI, VEI, Cdyn, MAP, Cstat, Raw, SatO2, PaCO2) within minutes after the insult, what is consistent with the findings of other authors [32–34]. In untreated ARDS group, respiratory failure persisted until the end of the experiment probably due to surfactant dysfunction caused by interaction with leaked plasma proteins (albumin and fibrinogen) and/or inflammation [35]. In our study, the therapy with the PDE inhibitor aminophylline improved lung function parameters and gas exchange compared to ARDS animals. We observed a rapid improvement in OI and the P/F ratio within the first 30 minutes after the aminophylline administration, and this beneficial effect persisted until the end of the experiment. These findings are consistent with previous studies which have shown that nonselective PDE inhibitors (e.g., pentoxifylline, aminophylline) can improve lung function by effective enhancing oxygenation and ventilation parameters [36–39].
In ARDS treatment, it is essential to manage the systemic and also pulmonary inflammatory response. The early phase of ARDS is characterized by neutrophil-mediated inflammation, lung cell injury and apoptosis while neutrophil activation and burst in the lungs play a key role in the progression of ARDS. In our experimental model, increased levels of pro-inflammatory cytokines (IL-8, IL-6, IL-13, IL-18, TNFα, and IL-1β) were observed in the lung tissue of ARDS animals. These results are consistent with previous findings [40, 41]. However, the aminophylline therapy decreased the levels of the observed pro-inflammatory cytokines in the lung tissue compared to the ARDS group. This may be attributable to the fact that increased intracellular secondary messenger cAMP due to activation of adenylyl cyclase affects a broad spectrum of cellular functions; modulates transcription factor nuclear factor-kappa B (NF-κB) and expression of pro-inflammatory cytokines (e.g. IL-1, IL-6, IL-12, IL-13, and TNFα) and regulates expression of anti-inflammatory interleukins [17, 42–44]. In addition, significantly decreased level of anti-inflammatory cytokine IL-10 was found in the lung tissue of ARDS animals, likely due to an imbalance between anti-inflammatory response and serious inflammatory response in ARDS animals [45]. The administration of aminophylline significantly prevented the reduction in IL-10 levels in our study as well as in the study by Elaidy [46]. Elevated IL-10 signaling can inhibit pro-inflammatory cytokine production through direct targeting of immune effector types, but can also indirectly modulate immune function by preventing macrophage and dendritic cell maturation, thus limiting the host's co-stimulatory, antigen presentation, and chemokine secretion capacity of the host [47, 48].
The lung tissue injury may be additionally caused by the oxidation of proteins and lipids due to neutrophil overactivation, especially due to oxidative neutrophil burst. Proteinases, cationic polypeptides, cytokines, and free radicals of reactive oxygen and nitrogen species (RONS) are among the cytotoxic and immune cell activating agents released by neutrophils [49]. After lavage-induced lung injury, significantly increased levels of protein nitrosylation (3-nitrotyrosine, 3NT) and lipid peroxidation products (TBARS) were detected in the lung tissue. Similar oxidation-induced lung damage was confirmed in several studies, demonstrating increased levels of RONS in alveolar spaces during ARDS [50–52]. After aminophylline therapy, the levels of TBARS and 3NT in lung tissue decreased significantly compared to untreated ARDS animals, while, the total antioxidant capacity (TAC) increased significantly after the therapy. The anti-inflammatory and antioxidant effects of nonselective PDE inhibitors at different doses have been demonstrated in various models of injury models [46, 53, 54], since RONS production can be reduced due to achieving a high local concentration of aminophylline in the airways [55].
Other marker evaluated in this study is receptor for advanced glycation end products (RAGE). RAGE is a membrane receptor expressed in alveolar type (AT)-1 epithelial cells of the lung and a marker of epithelial injury [56]. RAGE controls a variety of cellular processes such as cell proliferation and migration, inflammation, apoptosis, and microtubule stabilization [57, 58]. Activation of RAGE plays a role in cell signaling and propagation of the pro-inflammatory response [59–62]. In our study, a significantly higher level of RAGE in ARDS animals was found which was associated with the severity of pulmonary physiological disturbances (P/F ratio and compliance). These results are consistent with the previous study, where RAGE levels correlated with oxygenation [63].
Deterioration of the lung endothelium was demonstrated by sphingosine-1-phosphate (S1P). S1P is highly expressed in the lung endothelium, where it promotes survival and barrier function [64, 65]. S1P’s role as a key regulator of endothelial barrier function is attributed to its signaling through S1P1 & S1P3 that activates downstream Rho GTPases and rearrangement of cytoskeleton [66]. Elevated concentrations of S1P are associated with barrier disruption, as it was observed in ARDS animals in our study. On the other hand, increases in cAMP by inhibition of PDE, e.g. by aminophylline, may improve endothelial barrier functions and support cell-cell junctions [67].
Damage of endothelial and epithelial cells by the above mentioned bioactive compounds, results to increased permeability across the alveolar-capillary membrane and formation of pulmonary oedema. Large numbers of activated neutrophils can damage the alveolar epithelium, probably by the release of toxic intracellular molecules that induce the dissolution of tight junctions [49]. The formation of alveolar oedema containing high molecular weight plasma proteins worsens the gas exchange and increases the risk of disordered repair after extensive alveolar epithelial injury [9, 68, 69]. In our study, the degree of lung oedema was calculated from a ratio of wet and dry lung weight (W/D). The ARDS group had a significantly higher W/D value compared to the control group, indicating increased accumulation of pulmonary fluid in the pulmonary interstitium. Furthermore, the ARDS group had a significantly higher level of total proteins in their BALF. Similar findings had been reported by other authors [33, 69]. In this study, aminophylline therapy decreased the formation of lung oedema and the protein content in BALF compared to the ARDS group. These results are also consistent with the previous studies [70–72].