Phospholipase A2 subclasses in acute respiratory distress syndrome

Phospholipases A₂ (PLA₂) catalyse the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids. In acute lung injury-acute respiratory distress syndrome (ALI-ARDS) several distinct isoenzymes appear in lung cells and fluid. Some are capable to trigger molecular events leading to enhanced inflammation and lung damage and others have a role in lung surfactant recycling preserving lung function: Secreted forms (groups sPLA₂-IIA, -V, -X) can directly hydrolyze surfactant phospholipids. Cytosolic PLA₂ (cPLA₂-IVA) requiring Ca²⁺ has a preference for arachidonate, the precursor of eicosanoids which participate in the inflammatory response in the lung. Ca²⁺-independent intracellular PLA₂s (iPLA₂) take part in surfactant phospholipids turnover within alveolar cells. Acidic Ca²⁺-independent PLA₂ (aiPLA₂), of lysosomal origin, has additionally antioxidant properties, (peroxiredoxin VI activity), and participates in the formation of dipalmitoyl-phosphatidylcholine in lung surfactant. PAF-AH degrades PAF, a potent mediator of inflammation, and oxidatively fragmented phospholipids but also leads to toxic metabolites. Therefore, the regulation of PLA₂ isoforms could be a valuable approach for ARDS treatment.     

Chemokines in acute respiratory distress syndrome

A characteristic feature of all inflammatory disorders is the excessive recruitment of leukocytes to the site of inflammation. The loss of control in trafficking these cells contributes to inflammatory diseases. Leukocyte recruitment is a well-orchestrated process that includes several protein families including the large cytokine subfamily of chemotactic cytokines, the chemokines. Chemokines and their receptors are involved in the pathogenesis of several diseases. Acute lung injury that clinically manifests as acute respiratory distress syndrome (ARDS) is caused by an uncontrolled systemic inflammatory response resulting from clinical events including major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Systemic inflammatory response syndrome involves activation of alveolar macrophages and sequestered neutrophils in the lung. The clinical hallmarks of ARDS are severe hypoxemia, diffuse bilateral pulmonary infiltrates, and normal intracardiac filling pressures. The magnitude and duration of the inflammatory process may ultimately determine the outcome in patients with ARDS. Recent evidence shows that activated leukocytes and chemokines play a key role in the pathogenesis of ARDS. The expanding number of antagonists of chemokine receptors for inflammatory disorders may hold promise for new medicines to combat ARDS.     

Secretory phospholipases A2 induce beta-glucuronidase release and IL-6 production from human lung macrophages

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of beta-glucuronidase (16.2 +/- 2.4% and 13.1 +/- 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of beta-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in beta-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on beta-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced beta-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.     

Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats

Sublethaladministration of lipopolysaccharide (LPS) renders rats tolerant tomultiple lethal stimuli. Tolerant macrophages exhibit differentialalterations in LPS-stimulated cytokine and inflammatory mediatorrelease. Increased cAMP levels stimulated byPGE₂ or prostacyclin(PGI₂) result in differentialeffects on LPS-induced cytokine release and protect against thepathophysiological changes of endotoxemia. In the present studies, wesought to determine whether PGE₂-and PGI₂-stimulated cAMP levelsare altered in tolerant macrophages. Incubation of macrophages withcicaprost or 11-deoxy-PGE₁ in thepresence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not resultin significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNAlevels were significantly increased in tolerant cells compared withcontrols, whereas[³H]PGE₂binding and PGE₂ EP4 receptor mRNAlevels were not significantly changed. These studies suggest that LPStolerance induces selective alterations in eicosanoid regulation ofcAMP formation.

     

Chemokine production by rat macrophages stimulated with streptolysin O from Streptococcus pyogenes

The contribution of streptolysin O (SLO) from Streptococcus pyogenes to neutrophil infiltration in inflammatory lesions was determined by production of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 and -3, and macrophage inflammatory protein (MIP)-1alpha by rat macrophages stimulated with SLO in culture. Active SLO induced the production of CINCs and MIP-1alpha in dose- and time-dependent manners. These inductions were ascertained by chemokine mRNA expression in macrophages. Streptolysin S was without effect. The SLO-cholesterol complex induced the chemokine production in proportion to the residual hemolytic activity of the complex. In addition, the effects of SLO on the chemokine production were confirmed by the injection of active SLO into the preformed air pouch on the back of rats. The infiltration of neutrophils into the pouch fluid (exudate) increased steadily with a lag phase of about 2 hr. The major chemokine found in exudates was MIP-1alpha but not CINCs. In this study, it became clear that active SLO, but not the inactive one, contributed to the production of MIP-1alpha and CINCs in the conditioned medium and in exudates.     

Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism.

Methods

Ten patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D₉-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D₉-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry.

Results

BALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D₉-choline infusion. The enrichment of methyl-D₉-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals.

Conclusions

This study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.     

Quantitative proteomic analysis by iTRAQ for identification of candidate biomarkers in plasma from acute respiratory distress syndrome patients

Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critical patients. Proteomic analysis of plasma from individuals with ARDS could elucidate new biomarkers for diagnosis and pathophysiology and identify potential ARDS treatment targets. In this study, we recruited 26 patients (15 controls, 11 ARDS). The ARDS group was subdivided into two groups depending on the type of injury: (1) direct lung injury (AD) and (2) indirect lung injury (AI). Using iTRAQ (isobaric tags for relative and absolute quantitation) analysis, we identified 2429 peptides representing 132 plasma proteins. Among these, 16 were differentially expressed in ARDS patients, including 11 overlapping proteins between the AI and AD group and 5 AI-specific proteins. Protein annotation revealed that lipid transport and complement activation were significantly enriched in the biological process category, and lipid transporter, transporter, and serine-type peptidase activities were significantly enriched in the molecular function category. IPA (Ingenuity Pathway Analysis) signaling pathways revealed that the overlapping proteins were involved in a variety of signaling pathways, including those underlying acute phase response; liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X (FXR)/RXR activation; clathrin-mediated endocytosis; atherosclerosis; interleukin (IL)-12; complement system; and cytokine, nitric oxide, and reactive oxygen species production in macrophages. We present the first proteomic analysis of ARDS plasma using the iTRAQ approach. Our data provide new biomarker candidates and shed light on potential pathological mechanisms underlying ARDS.     

Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.     

Constitutive activation of prosurvival signaling in alveolar mesenchymal cells isolated from patients with nonresolving acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by stereotypic host inflammatory and repair cellular responses; however, mechanisms regulating the resolution of ARDS are poorly understood. Here, we report the isolation and characterization of a novel population of mesenchymal cells from the alveolar space of ARDS patients via fiber-optic bronchoscopy with bronchoalveolar lavage (BAL). BAL was performed on 17 patients during the course of ARDS. Immunofluorescence staining and multiparameter flow cytometric analysis defined a population of alveolar mesenchymal cells (AMCs) that are CD45-/prolyl-4-hydroxylase+/alpha-smooth muscle actin+/-. AMCs proliferated in ex vivo cell culture for multiple passages; early passage (3-5) cells were subsequently analyzed in 13 patients. AMCs isolated from patients with persistent or nonresolving ARDS (ARDS-NR, n = 4) demonstrate enhanced constitutive activation of prosurvival signaling pathways involving PKB/Akt, FKHR, and BCL-2 family proteins compared with AMCs from patients with resolving ARDS (ARDS-R, n = 9). Exogenous transforming growth factor-beta1 markedly induces PKB/Akt activation in AMCs from ARDS-R. ARDS-NR cells are more resistant to serum deprivation-induced apoptosis compared with ARDS-R. This study identifies a novel population of mesenchymal cells that can be isolated from the alveolar spaces of ARDS patients. AMCs in patients with ARDS-NR acquire an activational profile characterized by enhanced prosurvival signaling and an antiapoptotic phenotype. These findings support the concept that apoptosis of mesenchymal cells may be an essential component of normal repair and resolution of ARDS and suggest that dysregulation of this process may contribute to persistent ARDS.     

Prognostic significance of elevated cardiac troponin-T levels in acute respiratory distress syndrome patients

Background

Elevated levels of biochemical markers of myocardial necrosis have been associated with worsened outcomes in Acute Respiratory Distress Syndrome (ARDS), but there are few prospective data on this relationship. We investigated elevated cardiac troponin T (cTnT) levels and their relationship with outcome in patients with ARDS.

Methods

A prospective cohort study of patients with ARDS was conducted at a tertiary-care academic medical center. Patients had blood taken within 48 hours of ARDS onset and assayed for cTnT. Patients were followed for the outcomes of 60-day mortality, number of organ failures, and days free of mechanical ventilation. Echocardiographic and electrocardiographic (ECG) data were analyzed for signs of myocardial ischemia, infarction, or other myocardial dysfunction.

Results

177 patients were enrolled, 70 of whom died (40%). 119 patients had detectable cTnT levels (67%). Median cTnT level was 0.03 ng/mL, IQR 0–0.10 ng/mL, and levels were higher among non-survivors (P = .008). Increasing cTnT level was significantly associated with increasing mortality (P = .008). The association between increasing cTnT level and mortality remained significant after adjustment in a multivariate model (HR_adj = 1.45, 95% CI 1.17–1.81, P = .001). Elevated cTnT level was also associated with increased number of organ failures (P = .002), decreased number of days free of mechanical ventilation (P = .03), echocardiographic wall motion abnormalities (P = 0.001), and severity of tricuspid regurgitation (P = .04). There was no association between ECG findings of myocardial ischemia or infarction and elevated cTnT.

Conclusions

Elevated cTnT levels are common in patients with ARDS, and are associated with worsened clinical outcomes and certain echocardiographic abnormalities. No association was seen between cTnT levels and ECG evidence of coronary ischemia.     

Surfactant alteration and replacement in acute respiratory distress syndrome

The acute respiratory distress syndrome (ARDS) is a frequent, life-threatening disease in which a marked increase in alveolar surface tension has been repeatedly observed. It is caused by factors including a lack of surface-active compounds, changes in the phospholipid, fatty acid, neutral lipid, and surfactant apoprotein composition, imbalance of the extracellular surfactant subtype distribution, inhibition of surfactant function by plasma protein leakage, incorporation of surfactant phospholipids and apoproteins into polymerizing fibrin, and damage/inhibition of surfactant compounds by inflammatory mediators. There is now good evidence that these surfactant abnormalities promote alveolar instability and collapse and, consequently, loss of compliance and the profound gas exchange abnormalities seen in ARDS. An acute improvement of gas exchange properties together with a far-reaching restoration of surfactant properties was encountered in recently performed pilot studies. Here we summarize what is known about the kind and severity of surfactant changes occuring in ARDS, the contribution of these changes to lung failure, and the role of surfactant administration for therapy of ARDS.     

早期肠内营养联合益生菌对急性呼吸窘迫综合征患者预后的影响

探讨早期肠内营养联合益生菌对急性呼吸窘迫综合征（ARDS）患者预后的影响,为该类患者的治疗提供参考。

回顾性分析2018年6月至2020年12月我院急诊ICU收治的86例中重度ARDS患者的临床资料,按随机数字法将患者分为观察组（n = 48例）和对照组（n = 38例）。观察组患者在早期肠内营养基础上联合益生菌治疗,对照组患者采用常规治疗+早期肠内营养进行治疗。统计两组患者性别、年龄、既往基础疾病、原发疾病（包括肺内和肺外）、APACHE Ⅱ评分、SOFA评分、氧合指数、机械通气天数、脱机拔管成功率、ICU以及总的住院天数、28 d病死率等情况。

观察组患者机械通气时间短于对照组[（10.34±2.16）d vs （14.63±3.27）d,P = 0.020]。观察组患者脱机拔管成功率高于对照组（70.83% vs 63.16%,P = 0.038）。观察组患者ICU住院时间短于对照组[（15.34±3.28）d vs （18.68±3.54）d,P = 0.030]。观察组患者28 d病死率低于对照组（22.92% vs 26.32%,P = 0.035）。相关性分析提示早期肠内营养联合益生菌与患者的机械通气天数（r = −0.489,P = 0.039）和病死率（r = −0.312,P = 0.042）均存在相关性。非条件Logistic回归分析显示早期肠内营养联合益生菌能够降低患者的死亡风险。

相比单一予以早期肠内营养,采用早期肠内营养联合益生菌治疗ARDS患者更有利于缩短患者机械通气时间,降低病死率,改善患者的预后。

     

Amyloid associated with elastin-staining laminar aggregates in the lungs of patients diagnosed with acute respiratory distress syndrome

Background

The heterogeneity of conditions underlying respiratory distress, whether classified clinically as acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS), has hampered efforts to identify and more successfully treat these patients. Examination of postmortem lungs among cases clinically diagnosed as ARDS identified a cohort that showed a consistent morphology at the light and electron microscope levels, and featured pathognomonic structures which we termed elastin-staining laminar structures (ELS).

Methods

Postmortem tissues were stained using the Verhoeff-Van Gieson procedure for elastic fibers, and with Congo red for examination under a polarizing microscope. Similar samples were examined by transmission EM.

Results

The pathognomonic ELS presented as ordered molecular aggregates when stained using the Verhoeff-van Gieson technique for elastic fibers. In several postmortem lungs, the ELS also displayed apple-green birefringence after staining with Congo red, suggesting the presence of amyloid. Remarkably, most of the postmortem lungs with ELS exhibited no significant acute inflammatory cellular response such as neutrophilic reaction, and little evidence of widespread edema except for focal intra-alveolar hemorrhage.

Conclusions

Postmortem lungs that exhibit the ELS constitute a morphologically-identifiable subgroup of ARDS cases. The ordered nature of the ELS, as indicated by both elastin and amyloid stains, together with little morphological evidence of inflammation or edema, suggests that this cohort of ARDS may represent another form of conformational disease. If this hypothesis is confirmed, it will require a new approach in the diagnosis and treatment of patients who exhibit this form of acute lung injury.     

Microarray analysis identifies IL-1 receptor type 2 as a novel candidate biomarker in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states.

Methods

In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis.

Results

Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS.

Conclusions

These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0190-x) contains supplementary material, which is available to authorized users.