key: cord-0002828-ykvkbegu
authors: Hasan, Djo; Blankman, Paul; Nieman, Gary F.
title: Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury
date: 2017-05-26
journal: Purinergic Signal
DOI: 10.1007/s11302-017-9564-5
sha: 2b94fed9c359d39e9cccab476f64077d5edc1f4e
doc_id: 2828
cord_uid: ykvkbegu

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-017-9564-5) contains supplementary material, which is available to authorized users.

Diffuse lung injury can be triggered by several experimental conditions, amongst others: Severe pulmonary infection or extrapulmonary infection leads to acute pulmonary distress syndrome (ARDS) [1] [2] [3] [4] [5] ; Mechanical ventilation with high inspiratory pressures and high tidal volumes of >30 ml/kg ideal body weight in animals with healthy lungs causes interstitial and alveolar oedema and diffuse lung injury (ventilation-induced lung injury (VILI)) [6] ; And mechanical ventilation in surfactant-deactivated animals causes VILI even with low tidal volume (LV T ) of 6 ml/kg [7] . On the other hand, experimental mechanical ventilation with airway pressure release ventilation (APRV) with relatively long inspiration time and very short pressure release period may protect the lungs [7] [8] [9] [10] [11] [12] [13] [14] .

It is clear that lung infection results in pro-inflammatory immune response. In addition, there is a general agreement that ventilation of inhomogeneous lung tissue and/or with a Electronic supplementary material The online version of this article (doi:10.1007/s11302-017-9564-5) contains supplementary material, which is available to authorized users. high tidal volume triggers a pro-inflammatory response [15] [16] [17] [18] [19] . The corollary to this is that reduction of tidal volume and return to homogeneous ventilation will reduce (but not prevent) this pro-inflammatory response [19] . However, the exact mechanism of ventilation-induced pro-inflammatory response that leads to VILI is still not fully understood [15] [16] [17] [18] . Moreover, the combination of all mechanical breath parameters (known as the mechanical breath profile (MBP) airway pressures, volumes, flow rates and the duration at both inspiration and expiration) applied with each breath, which injure or protect the lung, is still not known.

In this review, we have searched the literature thoroughly to identify the (missing) link between the MBP, alveolar mechanics (i.e. the dynamic change in alveolar size and shape during tidal ventilation) and the inflammatory response that causes VILI.

Conventional mechanical ventilator settings consist of a static and a dynamic component. The static component is a continuous pressure or stress applied during both the inspiration and expiration and results in a static volume change or static strain. Strain is defined as a change in volume normalised by the original volume. The static stress and strain are referred to as positive end-expiratory pressure (PEEP) and the change in end-expiratory lung volume (EELV) normalised by the functional residual capacity (FRC) of the lungs, respectively. If the applied PEEP is zero, the EELV equals the FRC. Thus, EELV equals FRC plus PEEP volume. The dynamic component is the strain (i.e. tidal volume (V T ) normalised by FRC or EELV) inflicted by the cyclic stress, i.e. the inspiratory pressure [20] .

Protti et al. determined the mean value of the global energy load calculated from the mean inspiratory capacity by means of whole-lung CT scan in 76 pigs with healthy lungs [6] . Mean inspiratory capacity was defined as the mean value of total lung capacity (TLC) measured at 45 cm H 2 O airway pressure minus the FRC measured at 0 cm H 2 O airway pressure. The global energy load during conventional mechanical ventilation comprised a static component due to lung volume caused by the applied PEEP and a dynamic component due to the applied inspiratory pressure. The lower and upper limit of the applied global energy load was set at the value of mean global energy load ±2SD (equivalent to an inspiratory capacity range of 30.9-59.7 ml/kg ideal bodyweight). Then, the animals were subjected to different levels of PEEP and inspiratory pressures. When the mean global energy load did not exceed its lower limit, lung damage was not observed and only 1 of the 29 experimental animals did not survive the experiment. In contrast, when the mean global energy load exceeded the lower limit of the global energy load, lung injury, pro-inflammatory cytokine production and death were observed in 26 of the 47 experimental animals. In these cases, lung damage was associated with high dynamic energy load rather than high static energy load [6] . In addition, the rate of the applied strain was associated with ventilation-induced lung oedema and lung injury [21] .

In surfactant-deactivated lungs cyclic lung volume change causes VILI even with low energy load

Remarkably, the threshold of the energy load above which lung damage occurred in healthy pig lungs was quite high (equivalent to a mean V T of >30 ml/kg ideal body weight) [6] , whereas a V T of 6 ml/kg is already injurious in the surfactant-deactivated lungs [11] . This difference is clearly illustrated in the following experiment. In anaesthetised mechanically ventilated pigs, normal subpleural alveoli were filmed in real time using in vivo video microscopy. This study discovered that there was very little change in alveolar size in healthy lungs when ventilated with LV T (6 ml/kg ideal body weight) or with HV T (12 or 15 ml/kg ideal body weight). In contrast, after surfactant deactivation with a detergent as a model of ARDS, the alveoli became unstable: Many alveoli collapsed at the end of the expiration, and some of these collapsed alveoli were recruited during inspiration irrespective of the magnitude of the tidal volume. Alveoli that remained open at the end of the expiration became over-distended during the inspiration resulting in an inhomogeneous ventilation pattern in the lungs [22] .

Bachofen and Schürch demonstrated with scanning electron micrographs that surfactant deactivation and consequently increased surface tension in the alveoli in detergent rinsed lungs increase the alveolar duct surface area at the expense of alveolar surface area as compared to normal lungs. The increased alveolar duct size is accompanied by a decrease in the alveolar diameter perpendicular to the axis of the alveolar ducts (alveolar wall compression) and by an increase in the alveolar diameter parallel to the axis of the alveolar ducts. Therefore, surfactant deactivation is accompanied by alveolar wall stretching [23] . This finding was confirmed in an experiment in anaesthetised rats by the group of Nieman. After en bloc excision of the lungs, one of the lungs was clamped and fixed in formalin at peak inspiration pressure and the other at end expiration pressure for histologic analysis. In this way, they were able to visualise the effect of surfactant deactivation (Tween-induced ARDS model) and mechanical ventilator settings on alveolar duct microstrain and the distribution of inspired air between the alveolar ducts and the alveoli (Fig. 1 ) [8] . Microstrain was calculated as the change in length of the alveolar ducts between inspiration and expiration normalised by their original length. As the increase in the alveolar duct size after surfactant deactivation is accompanied by alveolar wall stretching parallel to the axis of the alveolar ducts [23] , alveolar duct microstrain is likely to be proportional to the alveolar wall stretching and alveolar wall strain.

Four ventilation strategies were tested by this group [8] : LV T plus two levels of PEEP (5 and 16 cm H 2 O) and APRV with the expiratory termination set at either an end-expiratory flow (EEF) of 10% (APRV 10%) of the peak-expiratory flow (PEF) or an EEF of 75% of the PEF (APRV 75%) [24, 25] ; the higher the EEF/PEF ratio (i.e. 75% as compared with 10%), the shorter the expiratory duration (see Table 1 for the MBP of the set ventilator modes). Previous studies have shown that APRV 75% stabilises alveoli preventing alveolar collapse during expiration, whereas an APRV 10% extends the duration of expiration resulting in alveolar collapse [12, 24, 25] . This study demonstrated that APRV 10% caused high microstrain and redistribution of air towards the alveolar ducts as compared with APRV 75%, which resulted in almost normal distribution of air (Fig. 1) . The order of ventilation strategies that caused the most to the least redistribution of inspired air into alveolar ducts (inspiratory ratio C a /A a in Table 2 ) and microstrain was as follows: APRV 10%, PEEP 5, PEEP 16, APRV 75% and control (Table 2) . Obviously, the least injurious ventilator settings are those that mimic the microstrain and air redistribution levels in ventilated healthy lungs with intact surfactant function (control in Fig. 1 ). APRV 75% resulted in near normal microstrain and alveolar air distribution, which was very similar to that measured in normal lungs, even though APRV 75% was in a Tween-induced ARDS model (Fig. 1 , APRV 75% and Table 2 ). In this high surface tension ARDS model, the lower the PEEP (PEEP 5 vs PEEP 16) and the longer the expiratory duration (10 vs 75%), the worse the microstrain and alveolar air distribution (Fig. 1, Table 2 ). This research group hypothesised that the mechanism by which APRV 75% was so effective at normalizing microstrain and alveolar air distribution was as follows: The extended time at inspiration (achieved by applying continuous positive airway Fig. 1 Photomicrographs of rat lungs. After en bloc excision of the lungs, one of the lungs was clamped and fixed in formalin at peak inspiration pressure and the other at end expiration pressure for histologic analysis. The respiratory bronchiole, alveolar ducts and alveolar sacs are green; the alveoli are lilac; and the alveolar walls are magenta. Healthy lungs ventilated with controlled continuous mandatory ventilation (CMV) with low tidal volume (≤6 ml/kg ideal body weight) and 5 cm H 2 O PEEP (control). APRV: surfactant-deactivated lungs with intratracheal instillation of a detergent ventilated with T low being interrupted when the endexpiratory flow (EEF) reached 75% (APRV 75%) or 10% (APRV 10%) of the peak expiratory flow (PEF). (PEEP 5) Surfactant-deactivated lungs ventilated with the same settings as 'control' or with PEEP 16 cm H 2 O (PEEP 16) (see Table 1 for the mechanical breath profile data). Note that the highest alveolar duct surface area is reached during inspiration in APRV 10% followed by PEEP 5; the lowest alveolar duct surface area is observed in the control group. The highest alveolar stability (the smallest difference in alveolar duct surface area between inspiration and expiration) with the lowest microstrain is reached in healthy lungs (control) followed by APRV 75%. The lowest alveolar stability and the highest microstrain are seen in APRV 10% and PEEP 5. Microstrain is calculated as the change in length of the alveolar ducts between inspiration and expiration normalised by their original length. The difference between the control group and PEEP 5 is exclusively attributed to the surfactant function. APRV can either significantly increase (APRV 10%) or decrease (APRV 75%) the microstrain and the redistribution of air towards the alveolar ducts. Figure from Kollisch-Singule et al. [8] with permission pressure) recruited almost all alveoli, and the very short time at expiration prevented these newly recruited alveoli from collapsing. In contrast, APRV 10% resulted in the worst microstrain and air distribution since the high pressure and extended inspiratory duration opened alveoli but the excessively long expiratory duration allowed time for all of the newly opened alveoli to collapse ( Fig. 1 ) [8] . One mechanism of this lung protection was that APRV 75% preserved surfactant function, measured by increased concentrations of surfactant protein-A (SP-A) and SP-B in the bronchoalveolar lavage fluid, as compared to controlled CMV with a V T of 10 ml/kg ideal body weight [10] . Apparently, redistribution of air towards the alveolar ducts and the increase in microstrain in controlled CMV deplete pulmonary surfactant. This supports earlier works showing that mechanical ventilation can impair pulmonary surfactant function [26, 27] .

The protective effect of APRV 75% (APRV settings: P high 16-22 cm H 2 O, T high 4.5 s, P low 0 cm H 2 O) on lung tissue injury was also demonstrated in anaesthetised pigs with extrapulmonary ARDS. In contrast to LV T controlled CMV (V T 6 ml/kg ideal body weight, RR 55/min, I/E 1:2, FiO 2 21% PEEP 5 cm H 2 O and guided by the ARDSnet protocol [28]), APRV 75% was capable of preventing the development of DAD while maintaining lung elastance low, despite the fact that APRV 75% generated similar transpulmonary pressure compared to LV T [11] .

The characteristics of lung tissue damage in ARDS and VILI are pointing at the activation of the innate immune system

The histopathology of VILI is referred to as diffuse alveolar damage (DAD) and is very similar to the histopathology associated with experimental ARDS. Reportedly, in contrast to the animal models of ARDS [29, 30] , histopathology of DAD is found in only one third to half of patients with clinical diagnosis of ARDS [31] [32] [33] . ARDS is caused by multiple primary disease conditions such as sepsis, haemorrhagic shock and pneumonia, and these patients will require In non-infectious circumstances such as physiological mechanical deformation (stretch or compression during breathing or mechanical ventilation) of the AT I cells stimulates the P2X7Rs followed by the controlled release of ATP molecules through the P2X7R channel into the extracellular space ( Fig. 2) [54, 57].

Recently, using real-time luciferin-luciferase bioluminescence imaging coupled with simultaneous infrared differential interference contrast imaging, Furuya et al. were able to produce real-time imaging of inflation-induced ATP release in the ex vivo rat lung. They observed that lung inflation induces ATP release into alveolar spaces and into pulmonary blood capillaries [58] . In this way, extracellular purine nucleotides (such as ATP) play the key role in the extracellular signalling (purinergic signalling) of many biological processes and can activate purinergic receptors-for example, the P2Y2R-on the cell membranes [52] . The extracellular ATP originating from the AT I cells, released through the P2X7Rs by mechanical deformation, activates the P2Y2Rs on the surface of the AT II cells. The P2Y2R is a metabotropic G protein-coupled ATP receptor (GPCR). Activation of the P2Y2R by ATP results in the release of diacylglycerol (DAG) and inositol triphosphate (IP 3 ) into the cytoplasm. DAG activates the enzyme protein kinase C (PKC) that promotes the fusion of LBs with the cell membrane ( Fig. 2 ). Cytoplasmic IP 3 results in the release of intracellular Ca 2+ by stores that are sensitive to IP 3 and Ca 2+ entry from the extracellular space through several pathways (TRPV2 and STIM1/Orai1). The increased cytoplasmic Ca 2+ level also promotes the fusion of the LBs with the cell membrane of the AT II cells and creates a fusion The extracellular ATP activates the P2Y2Rs on the surface of the AT II cells in a paracrine manner. The G protein-coupled P2Y2 ATP receptor releases DAG and IP 3 into the cytoplasm. DAG release leads to the activation of PKC-dependent pathway of fusion of LBs with the cell membrane. IP 3 release results in the release of intracellular Ca 2+ by stores that are sensitive to IP 3 and Ca 2+ entry from the extracellular space through several pathways (TRPV2 and STIM1/Orai1). Increased cytoplasmic Ca 2+ level also promotes the fusion of the LBs with the cell membrane of the AT II cells. These two processes create a fusion pore causing the P2X4 ATP receptors in the membrane of the LBs to be exposed to extracellular ATP. Activation of these P2X4Rs by extracellular ATP strongly increases the local Ca 2+ concentration to a much higher level around the membrane of the fused vesicles (FACE). FACE promotes a significant expansion of the fusion pore resulting in surfactant release by the AT II cells (LBs unpacking) and is accompanied by the FACE-induced cations and water migration from the alveolar space to the subepithelial interstitium. Clearance of the ATP molecules from the extracellular space occurs through the stepwise conversion by ecto-enzymes or by soluble extracellular enzymes (CD39 and CD73) to adenosine (ADO). ADO is returned to the cytoplasm through ENTs or CNTs and converted by ADA to inosine in the cytoplasm or converted by soluble ADA in the extracellular space. AT I alveolar epithelial type I cell, AT II alveolar epithelial type II cell, ER endoplasmic reticulum, LB lamellar body, DAG diacylglycerol, PKC protein kinase C, IP 3 inositol triphosphate, IP 3 R inositol triphosphate receptor, a membrane bound glycoprotein complex functioning as a Ca 2+ channel sensitive to activation by inositol triphosphate, TRPV2 transient receptor potential cation channel subfamily V member 2, a nonselective cation channel, STIM1 stromal interaction molecule 1, a calcium sensor, Orai1 calcium release-activated calcium channel protein 1, a calcium selective ion channel, CD39 nucleoside triphosphate diphosphohydrolase 1 (NTPD1), NPP nucleotide pyrophosphatase/phosphodiesterase, CD73 5′-nucleotidase (5′-NT), ADA adenosine deaminase, ENTs equilibrative nucleoside transporters 1 and 2, CNTs concentrative nucleoside transporters 1 and 2 pore causing the P2X4Rs in the membrane of the LBs to be exposed to the extracellular ATP (Fig. 2) . Activation of these P2X4Rs by extracellular ATP strongly increases the local Ca 2+ concentration around the membrane of the fused vesicles to a much higher level (Fig. 2) . This process-referred to as 'fusion-activated' Ca 2+ entry (FACE)-promotes a significant expansion of the fusion pore resulting in surfactant release by the AT II cells (LBs unpacking). Thus, the AT I cells induce the unpacking of LBs by the AT II cells in a paracrine manner (Fig. 2) [54, 55, 59, 60]. In addition, at a higher degree of stretch of the AT II in vitro (probably comparable to a deep sigh), ATP molecules can be released to the extracellular space and induce LBs unpacking in an autocrine manner [57] .

FACE drives a trans-epithelial current of Na + and Ca 2+ from the alveolar lumen through the P2X4R in the fused LB membranes and the cytoplasm to the subepithelial interstitium. This is followed by a passive water resorption from the alveolar space to the subepithelial interstitial space. In other words, extracellular ATP induces extracellular excretion of surfactant to the alveolar space and reduces the quantity of fluid in the alveolar liquid lining maximizing the reduction of the surface tension of the alveoli (Fig. 2) [56].

The P2X7Rs that release the extracellular ATP molecules are not subject to a refractory period during mechanical deformation [57]. In rat glomerular mesangial cell culture, 72% of the cells show an increase in Ca 2+ levels during a 2-min stimulus with 100 μM ATP through the P2Y2Rs. In contrast to the p2X7Rs, the P2Y2Rs start to desensitise within 1 min, and the sensitivity to stimuli reaches its minimum within 2 to 4 min [61, 62] . After a recovery period of another 4 min, only 38% of the cells respond to a second stimulus. The response following the second stimulus is much weaker than the first stimulus [62] . The P2X4Rs become desensitised faster than the P2Y2Rs (within a few seconds after a stimulus with ATP), and the sensitivity to stimuli reaches its minimum after 30 to 60 s [63, 64] . Therefore, vigorous mechanical deformation of the AT I cells may cause excessive extracellular release of ATP but is unlikely to cause massive LBs unpacking.

Clearance of the ATP and ADP molecules from the extracellular space is performed through the stepwise conversion of extracellular ATP molecules by ecto-enzymes or by soluble extracellular enzymes to adenosine (Fig. 2) . These enzymes comprise nucleoside triphosphate diphosphohydrolase 1 (NTPD1 or CD39, conversion of ATP to ADP and ADP to AMP), nucleotide pyrophosphatase/phosphodiesterase (NPP, conversion of ATP to AMP) and 5′-nucleotidase (5′-NT or CD73, conversion of AMP to adenosine). Then, adenosine is converted by soluble extracellular adenosine deaminase (ADA) to inosine or is returned to the cytoplasm of the cells through the equilibrative nucleoside transporters (ENT1 and ENT2) and concentrative nucleoside transporters (CNT1 and CNT2). Inside the cells, adenosine is being further processed by ADA and purine nucleoside phosphorylase (PNP) to inosine and hypoxanthine, respectively, and by adenosine kinase (ADK) to AMP [43, 52, 53, 65] . Under normal conditions, extracellular levels of ATP are kept low by the controlled release of these nucleotides and by the clearance of these nucleotides by the soluble or ecto-enzymes CD39, NPP and CD73 (Fig. 2) [43, 52, 53].

There are several mechanisms to clear surfactant from the alveolar space: (1) a small fraction of the surfactant lipids is squeezed out during the respiratory cycles to the bronchiole [66] ; (2) Obviously, the loss of the alveolar epithelial cells in DAD results in reduced LB surfactant stores and reduced surfactant production. Moreover, surfactant production can be decreased by pulmonary infection [67] and by hyperoxia (ventilation with air with 98% oxygen content) [68] . Surfactant deactivation by extravasated serum proteins [69] and the conversion of the active to the non-active surfactant subfraction contribute to further surfactant impairment [27] . Loss of surfactant production is accompanied by the deficiency of SP-A and SP-D molecules. As discussed above, SP-A and SP-D are soluble PRRs; they are important components of the innate immunity for the recognition of PAMPs and several DAMPs [41, 42] . Therefore, deficiency of SP-A and SP-D weakens the immune defence against invading pathogens.

Prolonged induced excess of purinergic signalling leads to pro-inflammatory immune response, immunodepression and lung fibrosis

The effects of purinergic signalling through the binding of nucleotides (ATP, ADP, UTP and UDP) and nucleoside (adenosine) to different receptors on the innate and adaptive immune system are presented in Table 3 . This table clarifies the importance of purinergic signalling in the regulation of both pro-inflammatory and anti-inflammatory responses of the immune system and the fibrotic process in the tissues.

Although many ATP receptors play a role in the activation of the pro-inflammatory immune response (Table 3) , activated 49 AdorR2B knockout mice exposed to systemic bleomycin Substantial reduction of fibrosis and IL-6 production. This results the in amplification of the chemoattractant gradient sensing and the self-generated gradients.

[ [134] [135] [136] [137] [138] [139] [140] 52

Play an important role in the chemotactic navigation towards apoptotic cells [141] 53 Microglial cells and colonic epithelial cells Suppresses LPS-induced TNF-α production. [142, 143] 54 Anti-CD3-activated CD8 + CTLs Reduces the expression of mRNAs coding for granzyme B, perforin, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL). Diminishes Nalpha-CBZ-L-lysine thiobenzylester esterase activity (enzyme with cytotoxic activity). Reduces IL-2 sand IFN-γ release. [144] 55 AdoRA3 knockout mice exposed to intratracheal bleomycin Increases eosinophil numbers and selective upregulation of eosinophil-related chemokines and cytokines. But decreases eosinophil peroxidase activity in the BALF. [145] 56 P2X1R ATP Neutrophils and platelets Promotes thrombosis and fibrinogenesis: Keeps circulating neutrophils in quiescent state, recruit neutrophil to the injury site, activate adhered neutrophils and platelets [146] 57 P2X1R, P2X4R and P2X7 ATP Naïve T cells TCR stimulation results in the translocation of pannexin-1 hemichannels, P2X1Rs and P2X4Rs to the immune synapse.

While the P2X7Rs remain uniformly distributed, this process is required to induce calcium entry, NFAT and release of IL-2.

[147] [184, 191, 192] 92

Induce IFN-β release [193] 93 P2Y11R ATP Neutrophils Inhibits neutrophil apoptosis [194] 94 Neutrophils Enhances chemotactic response [195] 95 Neutrophils and moDCs Induces maturation of the granulocytic progenitors and monocyte differentiation [196, 197] 96 moDCs Inhibits migratory capacity [198] 97 moDCs Induces IL-8 release [199] 98 101 DCs Increases antigen endocytosis with subsequent enhancement of specific T cell activation [202] 102

Microglial cells Induces movement of juxta-vascular microglial processes to close the injured blood-brain barrier (BBB) and microglial activation [203, 204] 103 Microglial cells Promotes migratory, inflammatory (TNF-αand IL-6 release) responses [205] 104

T cells [206] 105 P2Y13R ADP > ATP

Inhibits ATP release [207] 106 P2Y14R UDP > UDP-glucose Neutrophils Enhances chemotactic response through IL-8 dependent manner [208, 209] In general, extracellular ATP activates the immune system, and extracellular adenosine exerts a depressive action on the immune system and possesses pro-fibrotic properties [70, 71] AdoR adenosine receptor, TNF-α tumour necrosis factor alpha, FcγR receptors belonging to the immunoglobulin superfamily, IFN-γ interferon gamma, IFN-β interferon beta, MAC-1 macrophage-1 antigen comprised CD11b (integrin P2X7Rs are important initiators of the pro-inflammatory response by the innate immunity [43, 53, 210] . P2X7R is an ATP receptor and an intrinsic cation channel, but here, the P2X7R serves as an ATP receptor initiating intracellular transduction. Furthermore, the P2X7Rs have a low affinity for ATP molecules. They are activated only when the concentration of extracellular ATP is quite high reaching millimolar concentrations [70, 211] , for example, during a severe inflammation leading to ARDS and/or vigorous mechanical deformation of the AT I cells (Fig. 3) . In these conditions with high levels of extracellular ATP, the P2X7R does not appear to exhibit desensitization [64, 215] . Consequently, high levels of extracellular ATP molecules continuously act as DAMPs and activate the immune system [43, 53, 70] . In case of massive release, ATP may saturate the ATP hydrolysis enzymes CD39 and CD73 causing persistent high ATP levels despite the conversion to adenosine. The resulting pro-inflammatory immune response causes damage to the lung tissue and ends in DAD. Interstitial and alveolar oedema are the result of capillary leakage due to the pro-inflammatory response in the early exudative stage of DAD [34, 36] .

Reportedly, another mechanism of the development of alveolar oedema in ARDS after influenza virus infection is proposed [216, 217] : influenza virus-induced disruption of the tight junctions between the alveolar epithelia cells and direct inhibition of amiloride-sensitive epithelial sodium channels (ENaCs) [217] . The devastation of the normal alveolar histology in DAD is such that the tight junctions between the epithelial cells are indeed disrupted. But massive ATP release by alveolar epithelial cells after H1N1 influenza A virus (IAV) [218, 219] , respiratory syncytial virus (RSV) infection [220, 221] and parainfluenza infection virus infection [222] as detected by increased ATP in bronchoalveolar lavage fluid (BALF) have also been reported. Consequently, activation of P2X7Rs in epithelial cells and macrophages after a viral infection results in a pro-inflammatory response of the innate immune system [151] (Table 3 , rows 63-66 and row 76) [162] [163] [164] 174] and lung tissue injury. Therefore, it is extremely difficult to assess whether the influenza-induced ENaC inhibition in the lung tissue plays a significant role in the development of alveolar oedema in ARDS with profound capillary leakage as part of the proinflammatory response and disrupted alveolar histology (DAD).

Mechanical barrier against invading pathogens, Sp-A and Sp-D (PRRs) production, innate and adaptive immunity against pathogens Adenosine converted from extracellular ATP Extracellular ATP

Up-regulated soluble enzymes and ecto-enzymes CD39, NPP and CD73 Pro-fibrotic proteins production (TGF-β, α-SMA, CTGF, fibronectin, bFGF, VEGF, insulin-like growth factor-1, etc.)

ATP level threshold for P2X7R activation Fig. 3 Putative model of local tissue purinergic signalling, pathogen barriers, adaptive immunity and pro-fibrotic proteins during ARDS and/ or VILI [70] . In the very early phase of ARDS and VILI, infection [43, 52, 53] and vigorous mechanical deformation of the alveoli by mechanical ventilation [212, 213] lead to the substantial increase of extracellular ATP. The ATP levels exceed the threshold for the activation of P2X7R and induce pro-inflammatory immune response [70, 211] . This causes capillary congestion and capillary leakage causing interstitial and alveolar oedema. CD39 expression is upregulated in severe sepsis [214] and after several hours of mechanical ventilation [212] . Consequently, extracellular levels of ATP gradually decrease to a certain extent and extracellular adenosine increases. In general, adenosine has potent anti-inflammatory properties. This may lead to immune paralysis against secondary specific infections. Moreover, lung tissue damage due to DAD is accompanied by the disruption of the physical barrier as a component of the innate immunity for the defence against invading pathogens and by decreased Sp-A and Sp-D levels that function as soluble pattern recognition receptors (PRRs) of the innate immune system. This renders the host susceptible to invading pathogens [42] . TGF-β expression is increased by the activation of P2X7Rs [175, 176] and activation of the adenosine receptor AdoRA2B [132] . AdoRA2B activation also increases the expression of TGF-β and other fibrotic factors such as alpha smooth muscle actin (α-SMA), connective tissue growth factor (CTGF or CCN2), IL-6, fibronectin, VEGF, CD206, arginase-1, hyaluronan, basic fibroblast growth factor (bFGF), insulin-like factor-1, etc. (Table 3 , rows 40, 44, 47 and 49) [128, 130, 132, 239] Conversion of extracellular ATP to adenosine with anti-inflammatory properties.

After several hours of in vitro mechanical deformation of Calu-3 cells (a human airway epithelial cell line that shows cAMP-dependent Cl − secretion [223] ), the messenger RNA (mRNA) levels of CD39 and CD73 are upregulated [212] . Moreover, CD39 expression is upregulated in severe infection such as sepsis [214] . This is probably the consequence of increased TGF-β release due to the activation P2X7Rs by extracellular ATP (Table 3 , row 77) [175, 176] and increased IFN-β [224] [225] [226] due to the activation of P2Y6Rs by extracellular ATP as presented in Table 3 row 92 [193] . Consequently, extracellular levels of ATP gradually decrease to a certain level, and extracellular adenosine increases. In general, adenosine has potent anti-inflammatory properties. Prolonged high levels of extracellular ATP lead to high levels of extracellular adenosine and to the secondary suppression of the innate and adaptive immune system (Fig. 3) . The suppressive effects of extracellular adenosine on the adaptive immune system are presented in Table 3 , rows 6-8 [81] [82] [83] [84] , 24-29 [106] [107] [108] [109] [110] [111] [112] , 37 [125] , 38 [126] and row 104 [206] . Note that adenosine promotes the differentiation of naïve T cells to CD4 + FoxP3 + Lag3 + Tregs [106] , increases the numbers of Tregs [112] , increases the expression of the co-inhibitory surface molecule CTLA-4 on Tregs [112] and upregulates the expression of CD39 and CD73 on Tregs [112] . Tregs are potent inhibitors of the activation and function of effector T cells and potent inhibitors of the maturation and function of DCs [227] . Moreover, Tregs reduce the survival of effector T cells [227] . This causes immune paralysis against secondary specific infections. Additionally, lung tissue damage due to DAD is accompanied by the disruption of the physical barrier, a component of the innate immunity for the defence against invading pathogens and by a decrease of certain soluble PRRs (SP-A and SP-D) production [41, 42].

Confirmation of the role of extracellular ATP in the pathogenesis of VILI is supplied by the following report by Matsuyama et al. [213] : Intratracheally ATP or UTP (100 and 200 mM) instilled mice showed a progressive increase in wet-to-dry (W/D) weight ratio of the lungs during the first 18 h, the extent of the increased W/D ratio was dose dependent. There was also a significant increase in the albumin permeability index. In comparison to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATP induced an increase in the gene expression (mRNA) of macrophage inflammatory protein-2 (MIP-2), TNF-α and IL-6 detected using real-time polymerase chain reaction (RT-PCR) within 60 min after the administration [213] . Note that the gene expression of IL-1β was not increased because ATP regulates the posttranscriptional activation of NLRP3 inflammasomes [152, 228, 229] , and this cannot be detected with the applied RT-PCR technique. There was a considerable rise in the number of neutrophil and macrophage counts in the BALF. These effects appeared in intratracheally ATP instilled mice and in mice mechanically ventilated with a V T of 40 ml/kg ideal body weight but not with a V T of 8 ml/kg. The ATP levels in BALF specimens in the high V T group were significantly increased when compared to the low V T group. In addition, the increased W/D weight ratio, albumin permeability and cytokine production (with the exception of TNF-α) in the intratracheal ATP group were mitigated by the administration of pyridoxal-5′-phosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS), a semi-selective antagonist of the purinergic receptor P2X and P2Y purinergic receptors [213] .

Increased extracellular ATP levels to micromolar concentrations and conversion of very high levels of extracellular ATP (millimolar concentrations) to adenosine improve survival Belete et al. applied five incremental steps of strain (0, 0.03, 0.06, 0.08, 0.10, 0.18, respectively) to experimental AT I cell monolayers [230] . Strain was defined as the proportion of radial length change of the monolayer during each stretch cycle. They found that the extent of the applied cell strain was proportional to the extracellular level of ATP and the extent of plasma membrane damage. Adding a small amount of ATP (10 μM) or ecto-enzyme inhibitor ARL 67156 (100 μM) decreased the proportion of the lethally wounded AT I cells. The addition of adenosine (5 μg/ml) did not provide increased cytoprotection. The addition of apyrase (20 U/ ml) or apyrase combined with ADA (5 U/ml) to increase the extracellular adenosine levels at the expense of the extracellular ATP levels caused an increase in the proportion of the lethally wounded AT I cells ruling out a potential adenosynergic cytoprotection pathway. Apparently, elevated extracellular ATP levels to micromolar concentrations facilitated the plasma membrane wound healing by increasing the fusion of the calcium-sensitive lysosomes with the plasma membrane at the wound site. ATP induced this process in the AT I cells in an autocrine manner through the P2Y2Rs activating the IP 3 and DAG/PKC pathways [230] . This process is similar to the paracrine ATP-induced fusion of the LBs with the cell membrane of the AT II cells [54, 55, 59].

Eckle et al. reported that the gene expression of the enzymes CD39 and CD73 that hydrolyse ATP to adenosine in the wild-type (WT) mice lungs was increased during mechanical ventilation with very high inspiratory pressure of 45 mbar (equivalent to 45.9 cm H 2 O) [212] . At this inspiratory pressure level, the lung volume reached TLC with a strain of 2.5 (strain was defined as the inspiratory volume divided by the functional residual capacity) [20, 231] . In CD39−/− and in CD73−/− knockout mice, diffuse lung injury was far more severe than in the WT mice [212] . Intraperitoneal administration of soluble CD39 in CD39−/− mice and soluble CD73 in CD73−/− mice attenuated the lung injury severity to the level of the WT mice [212] . In addition, intraperitoneal administration of soluble CD39 or CD73 in WT mice treated with the injurious mechanical ventilation increased the survival significantly, decreased the VILI score and increased the pulmonary adenosine levels [212] . As mentioned above, the very high strain levels cause a massive ATP release saturating the ATP hydrolysis enzymes CD39 and CD73 with persisting high ATP levels. The administered soluble CD39 and CD73 convert the excessive ATP molecules to adenosine causing the inflammatory ATP levels to drop and the anti-inflammatory adenosine levels to rise. In addition, adenosine attenuates the ventilator-induced capillary leakage through AdoRA2B receptor signalling ( Table 3 , row 43) [129] .

As stated earlier, the ATP hydrolysing enzymes CD39 and CD73 are upregulated during mechanical ventilation. One of the factors that leads to the upregulation of CD73 is the increased expression of IFN-β [224] [225] [226] by the activation of P2Y6Rs [193] . Reportedly, intravenous administration of IFN-β-1a reduces mortality in patients with ARDS treated with assisted mechanical ventilation significantly [232] .

On the other hand, when the extracellular ATP levels remain high due to continuous massive release of ATP, adenosine accumulates in the extracellular space due to the overload of the ADA enzyme and of the re-uptake process of adenosine through ENTs and CNTs (Fig. 2) . Prolonged high levels of extracellular adenosine result in immunosuppression, the Fig. 4 The summary of the physiological, pathophysiological and immunological consequences of controlled CMV with a V T ≤ 6 ml/kg ideal body weight (left) and of controlled CMV with extremely high V T (right) in healthy lungs (see text for explanation). The common cell signalling pathway for the release of surfactant by alveolar epithelial type 2 (AT II) cells and for the activation of the innate immunity (red arrows). Sequential processes related to mechanical ventilation (grey coloured text boxes). CMV continuous mandatory ventilation, V T tidal volume, APRV 10% airway pressure release ventilation with the expiration termination set at 10% of the peak-expiratory flow rate (PEFR), DAMPs danger-associated molecular patterns, DAD diffuse alveolar damage production of fibrotic proteins and finally end in fibrosis (Fig. 3) . Reportedly, adenosine-dependent fibrosis occurs in mice with ADA deficiency. In these mice, ADA suppletion leads to the resolution of the fibrosis [233, 234] . On top of the increased TGF-β expression by the activation of P2X7Rs (Table 3 , row 77) [175, 176] , activation of AdoRA2B increases the expression of TGF-β, α-SMA, connective tissue growth factor (CTGF or CCN2), IL-6 and fibronectin in fibroblasts. These proteins are known to be pro-fibrotic (Table 3 , row 47) (Fig. 3) [132] . One of the pro-fibrotic properties of TGF-β is the induction of epithelial-to-mesenchymal transition (EMT), a fundamental underlying pathogenic factor in lung fibrosis [235] . Presumably, treatment with IFN-β-1a, with soluble CD39 or with CD73, increases the adenosine levels. In turn, this probably leads to a significant decrease in extracellular ATP reducing the inflammation and the release of extracellular ATP by the lung tissue cells and the immune cells. For a proportion of patients [232] and experimental animals [212] , this may well be enough to halt the progression of DAD.

We summarise the physiological, pathophysiological and immunobiological consequences of controlled CMV, APRV 10% and APRV 75% in Figs. 4 and 5.

Diffuse lung tissue damage induced by systemic or local infection, trauma or haemorrhagic shock and mechanical ventilation all use a common cell signalling pathway to cause VILI. This pathway is as follows: AT I cells are the mechanosensors in the lung; the AT I cells secrete large quantities of ATP molecules after being infected or being vigorously stretched or compressed. This is followed by the paracrine stimulation of AT II cells by the extracellular ATP molecules leading to the unpacking of the LBs to the alveolar space. In infections, many infected immune cells and tissue cells (such as AT I and AT II cells) release ATP without requiring mechanical deformation of the cells.

Although increased extracellular ATP levels to micromolar concentrations may provide cytoprotection against stretched- Fig. 5 The summary of the physiological, pathophysiological and immunological consequences of controlled CMV with a V T ≤ 6 ml/kg ideal body weight or APRV 10% (left) and of APRV 75% (right) in infected lungs (see text for explanation). The common cell signalling pathway for the release of surfactant by alveolar epithelial type 2 (AT II) cells and for the activation of the innate immunity (red arrows). Sequential processes related to mechanical ventilation (grey coloured text boxes). CMV continuous mandatory ventilation, V T tidal volume, APRV 75% airway pressure release ventilation with the expiration termination set at 75% of the peak-expiratory flow rate (PEFR), APRV 10% APRV with the expiration termination set at 10% of the PEFR, DAMPs danger-associated molecular patterns, DAD diffuse alveolar damage induced plasma membrane damage by the facilitation of plasma membrane repair, very high levels (millimolar concentrations) of extracellular ATP activate the innate immune system by several other pathways: neutrophil recruitment and transmigration to the lung tissue, production of the proinflammatory cytokines IL-1β and IL-18, polarization of the macrophages towards M1 phenotype, etc. The resulting inflammation causes diffuse damage of the lung tissue and results in DAD. In addition, damage to the AT I and AT II cells (due to DAD) results in a decrease in surfactant production, extravasated serum proteins and the conversion of the active to the non-active surfactant subfraction ending in surfactant function impairment. The escalation of this process by ventilation (controlled CMVor APRV 10%) increases the extracellular ATP levels and surfactant impairment even further. Persistent release of extracellular ATP is followed by the upregulation of the ecto-enzymes (CD39 and CD73) decreasing extracellular ATP levels by conversion to adenosine. High levels of adenosine are associated with immune paralysis against invading pathogens and progressive lung fibrosis.

When the levels of the extracellular ATP are such that treatment with soluble CD39, CD73 or IFN-β is sufficient to reduce the pro-inflammatory response and restore the surfactant function, VILI development will be stopped and the lungs may recover. When the extent of lung damage is more severe and extensive with persistent high levels of extracellular ATP, conversion of extracellular ATP to adenosine does not lead to the recovery of the surfactant function but instead leads to very high levels of extracellular adenosine. In this case, VILI will progress to the next (fibrotic) stage (Fig. 3) .

Mechanical ventilation by means of controlled CMV or APRV 10% with relatively long expiration time results in cyclic recruitment of the surfactant-deactivated alveoli causing a redistribution of the inspired air volume from the non-recruited alveoli to the alveolar ducts. In addition, cyclic recruitment leads to an increase in the volume difference between inspiration and expiration in the alveolar ducts and therefore increases the microstrain. Increased microstrain is associated with the increase of mechanical deformation of the alveoli causing an increase in extracellular ATP release. This ends in a vicious circle of mechanical tissue damage → massive ATP release → increased inflammation → surfactant function impairment → exacerbating mechanical tissue damage → massive ATP release → etc. (Figs. 4 (right panel) and 5 (left panel) ).

The essence of the prevention of VILI is to escape this vicious circle by decreasing the extracellular levels of ATP in the lung tissue either by the administration of IFN-β [232] , soluble CD39 and soluble CD73 [212] or by continuously recruiting the alveoli until all are recruited and to keep the recruited alveoli open [8] . This ventilator strategy prevents the redistribution of inspired air towards the alveolar ducts, stabilises the alveoli, minimises the microstrain and limits the release of extracellular ATP by the cells, especially by the AT I cells. Consequently, the innate immune response is reduced, and the AT I, AT II cells and surfactant function are restored. Properly set, APRV 75% appeared to do just that by opening the lung and keeping it open, in experimental settings [7811] and in a clinical statistical analysis [9] (Fig. 5 (right panel) ). The rationale behind the lung tissue protective effect of the APRV 75%-induced recruitment and stabilization in surfactant-impaired ARDS model in animals is through understanding dynamic alveolar mechanics. Alveoli are not elastic (i.e. change size in a one to one relationship with the applied stress, which would be the V T ); rather, alveoli are a viscoelastic system. A viscoelastic system is represented best by the spring and dashpot [240] . In a viscoelastic system, there is a delay in the strain (i.e. change in alveolar size) following the applied stress, which in the case of the lung is the inspiratory pressure. At least two conditions have to be fulfilled to recruit the collapsed viscoelastic alveoli: (1) sufficiently high airway pressure of >30 cm H 2 O and (2) long enough duration of the pressure (varies between 2 s and several minutes for the individual alveolus due to the inhomogeneous nature of the collapsed alveoli) to allow the alveoli to be recruited [236, 237] . This is known as the creep phenomenon of the viscoelastic system [240] . Thus, by extending the time at inspiration (i.e. increasing the duration of the applied stress), we gradually 'nudge' alveoli open over time [236] . Fully recruiting the lung eliminates stress concentrators, a major VILI mechanism [238] . The lung is stabilised by a very short time at expiration. If the stress (i.e. inspiratory pressure) is released very quickly for a very short time, the viscoelastic alveoli will not have time to empty, maintaining a PEEP. Thus, the short expiratory duration stabilises the lung by two mechanisms: time and pressure. Using our understanding of dynamic alveolar mechanics and the fact that alveoli are viscoelastic, we use the MBP parameter of duration to open and stabilise the lung. Using a short expiratory duration stabilises alveoli and prevents the redistribution of the inspired air towards the alveolar ducts and prevents vigorous cyclic deformation of the alveoli [8] , which, in turn, prevents massive extracellular ATP release, massive short-term surfactant release, followed by surfactant function impairment, interstitial and alveolar oedema and activation of the innate immunity leading to the development or aggravation of DAD. This presumably prevents the loss of AT I cells, prevents the hyperplasia of AT II cells and restores the surfactant function, and the affected lung regions start to regain their normal compliance (increase their alveolar time constant). 

Acute respiratory distress syndrome

Acute respiratory distress syndrome induced by a swine 2009 H1N1 variant in mice

Acute respiratory distress syndrome induced by avian influenza A (H5N1) virus in mice

Acute lung injury induced by H9N2 virus in mice

Acute respiratory distress syndrome induced by H9N2 virus in mice

Lung anatomy, energy load, and ventilator-induced lung injury

Early airway pressure release ventilation prevents ARDS-a novel preventive approach to lung injury

Airway pressure release ventilation reduces conducting airway micro-strain in lung injury

Early application of airway pressure release ventilation may reduce mortality in high-risk trauma patients: a systematic review of observational trauma ARDS literature

Airway pressure release ventilation prevents ventilator-induced lung injury in normal lungs

The effects of airway pressure release ventilation on respiratory mechanics in extrapulmonary lung injury

Mechanical breath profile of airway pressure release ventilation: the effect on alveolar recruitment and microstrain in acute lung injury

Preemptive application of airway pressure release ventilation prevents development of acute respiratory distress syndrome in a rat traumatic hemorrhagic shock model

Preemptive mechanical ventilation can block progressive acute lung injury

What the concept of VILI has taught us about ARDS management

Impact of mechanical ventilation on the pathophysiology of progressive acute lung injury

Ventilator-induced lung injury

The Bbaby lung^became an adult

Inflammatory response to oxygen and endotoxin in newborn rat lung ventilated with low tidal volume

Lung stress and strain during mechanical ventilation: any difference between statics and dynamics? Crit C a r e M

Role of strain rate in the pathogenesis of ventilator-induced lung edema

Tidal volume increases do not affect alveolar mechanics in normal lung but cause alveolar overdistension and exacerbate alveolar instability after surfactant deactivation

Alveolar surface forces and lung architecture

Other approaches to open-lung ventilation: airway pressure release ventilation

Protein kinase C in ATP regulation of lung surfactant secretion in type II cells

Ca2+ response of rat mesangial cells to ATP analogues

Growth hormones reverse desensitization of P2Y(2) receptors in rat mesangial cells

Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate

Molecular physiology of P2X receptors

DAMPs from cell death to new life

Pulmonary surfactant in the airway physiology: a direct relaxing effect on the smooth muscle

Impaired surfactant production by alveolar epithelial cells in a SCID-hu lung mouse model of congenital human cytomegalovirus infection

Impaired surfactant phospholipid metabolism in hyperoxic mouse lungs

Influence of serum protein and albumin addition on the structure and activity of an exogenous pulmonary surfactant

Purinergic regulation of the immune system

Regulation of neutrophil function by adenosine

Purinergic signalling: its unpopular beginning, its acceptance and its exciting future

The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively

Adenosine promotes neutrophil chemotaxis

Phorbol ester-stimulated adherence of neutrophils to endothelial cells is reduced by adenosine A2 receptor agonists

Acting via A2 receptors, adenosine inhibits the production of tumor necrosis factor-alpha of endotoxin-stimulated human polymorphonuclear leukocytes

Fc gamma receptor-mediated functions in neutrophils are modulated by adenosine receptor occupancy. A1 receptors are stimulatory and A2 receptors are inhibitory

The role of cyclic AMP, calcium and filamentous actin in adenosine modulation of Fc receptor-mediated phagocytosis in human neutrophils

Adenosine inhibits actin dynamics in human neutrophils: evidence for the involvement of cAMP

Adenosine effectively restores endotoxin-induced inhibition of human neutrophil chemotaxis via A1 receptor-p38 pathway

Adenosine, through the A1 receptor, inhibits vesicular MHC class I cross-presentation by resting DC

Role of adenosine receptors in regulating chemotaxis and cytokine production of plasmacytoid dendritic cells

Phenotypic and functional characteristics of CD39high human regulatory B cells (Breg)

Ecto-5′-nucleotidase (CD73) attenuates allograft airway rejection through adenosine 2A receptor stimulation

Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Effect of adenosine on the expression of beta(2) integrins and L-selectin of human polymorphonuclear leukocytes in vitro

Acting via A2 receptors, adenosine inhibits the upregulation of Mac-1 (Cd11b/CD18) expression on FMLP-stimulated neutrophils

Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early antiinflammatory signal

Neutrophil A2A adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type IV phosphodiesterase inhibitor, rolipram

Immunomodulatory impact of the A2A adenosine receptor on the profile of chemokines produced by neutrophils

Adenosine A2 receptorinduced inhibition of leukotriene B4 synthesis in whole blood ex vivo

Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligandactivated human neutrophils

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4)

Adenosine, a potent natural suppressor of arachidonic acid release and leukotriene biosynthesis in human neutrophils

Cyclic AMP-mediated inhibition of 5-lipoxygenase translocation and leukotriene biosynthesis in human neutrophils

Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists

Accelerated resequestration of cytosolic calcium and suppression of the proinflammatory activities of human neutrophils by CGS 21680 in vitro

Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils

Apparent involvement of the A(2A) subtype adenosine receptor in the anti-inflammatory interactions of CGS 21680, cyclopentyladenosine, and IB-MECA with human neutrophils

Adenosine A2a receptor activation delays apoptosis in human neutrophils

Activation of adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome

Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma

Adenosine suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production by murine macrophages through a protein kinase A and exchange protein activated by cAMP-independent signaling pathway

Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells

A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells

Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function

A2A adenosine receptor (AR) activation inhibits proinflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence

Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptordependent and independent mechanisms

Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo

A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells

The development and immunosuppressive functions of CD4(+) CD25(+) FoxP3(+) regulatory T cells are under influence of the adenosine-A2A adenosine receptor pathway

Baicalin attenuates bleomycininduced pulmonary fibrosis via adenosine A2a receptor related TGF-beta1-induced ERK1/2 signaling pathway

Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages

Adenosine promotes alternative macrophage activation via A2A and A2B receptors

The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Ralpha) signaling

Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages

Regulation of macrophage function by adenosine

Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1

Adenosine suppresses activation of nuclear factor-kappaB selectively induced by tumor necrosis factor in different cell types

Netrin-1 dampens pulmonary inflammation during acute lung injury

Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

A role for the lowaffinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils

Adenosine augments IL-10 production by macrophages through an A2B receptor-mediated posttranscriptional mechanism

Adenosine receptors in regulation of dendritic cell differentiation and function

The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6

A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c(+)Gr-1(+) dendritic cell subset that promotes the Th17 response

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension

A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells

A2B adenosine receptors induce IL-19 from bronchial epithelial cells, resulting in TNF-alpha increase

Adenosine signaling increases proinflammatory and profibrotic mediators through activation of a functional adenosine 2B receptor in renal fibroblasts

Mast cell-mediated stimulation of angiogenesis: cooperative interaction between A2B and A3 adenosine receptors

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors

Selfgenerated chemoattractant gradients: attractant depletion extends the range and robustness of chemotaxis

Self-generated chemotactic gradients-cells steering themselves

Directional tissue migration through a selfgenerated chemokine gradient

Where to go: breaking the symmetry in cell motility

Cell migration: sinking in a gradient

Involvement of adenosine A3 receptors in the chemotactic navigation of macrophages towards apoptotic cells

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of PI 3-kinase/Akt and NF-kappaB activation in murine BV2 microglial cells

Activation of adenosine A3 receptor alleviates TNF-alpha-induced inflammation through inhibition of the NF-kappaB signaling pathway in human colonic epithelial cells

Adenosine acts through an A3 receptor to prevent the induction of murine anti-CD3-activated killer T cells

A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis

Purinergic control of inflammation and thrombosis: role of P2X1 receptors

Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse

ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells (article retracted in 2011 due to figure irregularities)

ATP release and autocrine signaling through P2X4 receptors regulate gammadelta T cell activation

P2X4 receptors control the fate and survival of activated microglia

Activation of P2X(7) receptor by ATP plays an important role in regulating inflammatory responses during acute viral infection

Molecular mechanisms regulating NLRP3 inflammasome activation

Activation and regulation of the inflammasomes

P2X4 receptor regulates P2X7 receptordependent IL-1beta and IL-18 release in mouse bone marrowderived dendritic cells

Silicainduced inflammasome activation in macrophages: role of ATP and P2X7 receptor

Neutrophil P2X7 receptors mediate NLRP3 inflammasomedependent IL-1beta secretion in response to ATP

Uric acid induces caspase-1 activation, IL-1beta secretion and P2X7 receptor dependent proliferation in primary human lymphocytes

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells

Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages

Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor

Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing

P2X7 receptors induce degranulation in human mast cells

P2X7 receptors regulate NKT cells in autoimmune hepatitis

Human P2X7 receptor activation induces the rapid shedding of CXCL16

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells

P2X7 receptor-dependent tuning of gut epithelial responses to infection

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism

Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R

Activation of P2X7 purinoceptor-stimulated TGF-beta 1 mRNA expression involves PKC/MAPK signalling pathway in a rat brain-derived type-2 astrocyte cell line, RBA-2

Adenosine 5′-triphosphate stimulates the increase of TGF-beta1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP-and thrombin-induced human platelet activation

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation

receptor regulates VCAM-1 membrane and soluble forms and eosinophil accumulation during lung inflammation

Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis

Akt activation is involved in P2Y12 receptor-mediated chemotaxis of microglia

P2Y6 receptor antagonist MRS2578 inhibits neutrophil activation and aggregated neutrophil extracellular trap formation induced by gout-associated monosodium urate crystals

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Responses of macrophages to the danger signals released from necrotic cells

UDP facilitates microglial phagocytosis through P2Y6 receptors

The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2

P2Y6 receptor-mediated microglial phagocytosis in radiation-induced brain injury

Protein interacting C-kinase 1 modulates surface expression of P2Y6 purinoreceptor, actin polymerization and phagocytosis in microglia

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

Human neutrophil peptides induce interleukin-8 production through the P2Y6 signaling pathway

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP-1-dependent manner in epithelial cells

Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-beta production

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor

P2Y11 purinoceptor mediates the ATPenhanced chemotactic response of rat neutrophils

Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells

ATP gradients inhibit the migratory capacity of specific human dendritic cell types: implications for P2Y11 receptor signaling

1-phenylene-carbonylimino-3,1-(4-methylphenylene)-car bonylimino))-bis(1,3-xylene-alpha,alpha′-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocytederived dendritic cells

Autocrine regulation of macrophage activation via exocytosis of ATP and activation of P2Y11 receptor

Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion

Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP

Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier

The P2Y12 receptor regulates microglial activation by extracellular nucleotides

expression and function in alternatively activated human microglia

P2Y12 receptors in primary microglia activate nuclear factor of activated T-cell signaling to induce C-C chemokine 3 expression

ADP acting on P2Y13 receptors is a negative feedback pathway for ATP release from human red blood cells

The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8

A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils

Purinergic signalling and immune cells

The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7)

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

CD39 improves survival in microbial sepsis by attenuating systemic inflammation

Allosteric modulation of ATP-gated P2X receptor channels

Pathogenesis of influenza-induced acute respiratory distress syndrome

Influenza virus inhibits ENaC and lung fluid clearance

Postinfection A77-1726 treatment improves cardiopulmonary function in H1N1 influenza-infected mice

Influenza a virus inhibits alveolar fluid clearance in BALB/c mice

Inhibition of airway Na+ transport by respiratory syncytial virus

Inhibition of Na+ transport in lung epithelial cells by respiratory syncytial virus infection

Acute effects of parainfluenza virus on epithelial electrolyte transport

Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl-secretion

Mechanism of action of IFN-beta in the treatment of multiple sclerosis: a special reference to CD73 and adenosine

IFN-beta regulates CD73 and adenosine expression at the blood-brain barrier

IFN-beta protects from vascular leakage via upregulation of CD73

How regulatory T cells work

Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome

The inflammasomes

The role of purinergic signaling on deformation induced injury and repair responses of alveolar epithelial cells

Lung stress and strain during mechanical ventilation: any safe threshold?

The effect of intravenous interferon-beta-1a (FP-1201) on lung CD73 expression and on acute respiratory distress syndrome mortality: an open-label study

Adenosine-dependent pulmonary fibrosis in adenosine deaminase-deficient mice

Partially adenosine deaminasedeficient mice develop pulmonary fibrosis in association with adenosine elevations

Epithelial-mesenchymal transition as a fundamental underlying pathogenic process in COPD airways: fibrosis, remodeling and cancer

The role of time and pressure on alveolar recruitment

Is time the missing component in protective ventilation strategies?

Non-lobar atelectasis generates inflammation and structural alveolar injury in the surrounding healthy tissue during mechanical ventilation

Distinct roles for the A2B adenosine receptor in acute and chronic stages of bleomycin-induced lung injury

Acknowledgments We are grateful to Prof. Dr. Diederik Gommers, chairman of the Department of Adult ICU of the University Hospital Erasmus MC Rotterdam (NL) for his invaluable comments during the preparation of the manuscript. We thank Philip van der Zee, MD, researcher at the Department of Adult ICU of the University Hospital