Activity of pulmonary edema fluid interleukin-8 bound to alpha(2)-macroglobulin in patients with acute lung injury

The formation of alpha(2)-macroglobulin (alpha(2)-M)/interleukin-8 (IL-8) complexes may influence the biological activity of IL-8 and the quantitative assessment of IL-8 activity. Therefore, in this study, concentrations of free IL-8 and IL-8 complexes with alpha(2)-M were measured in pulmonary edema fluid samples from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and compared with control patients with hydrostatic pulmonary edema. Patients with ALI/ARDS had significantly higher concentrations of alpha(2)-M (P < 0.01) as well as alpha(2)-M/IL-8 complexes (P < 0.05). Because a substantial amount of IL-8 is complexed to alpha(2)-M, standard assays of free IL-8 may significantly underestimate the concentration of biologically active IL-8 in the distal air spaces of patients with ALI/ARDS. Furthermore, IL-8 bound to alpha(2)-M retained its biological activity, and this fraction of IL-8 was protected from proteolytic degradation. Thus complex formation may modulate the acute inflammatory process in the lung.     

Anti-IL-8 autoantibody:IL-8 immune complexes suppress spontaneous apoptosis of neutrophils

Our previous studies demonstrated that a significant fraction of interleukin-8 (IL-8) in lung fluids from patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) is associated with anti-IL-8 autoantibodies (anti-IL-8:IL-8 immune complexes). Neutrophils have been implicated in the pathogenesis of ALI/ARDS, and moreover, it is well-established that apoptosis of neutrophils is delayed in patients with ALI/ARDS. The aim of this study was, therefore, to examine the role of anti-IL-8:IL-8 immune complexes in modulating spontaneous apoptosis of normal human neutrophils. Apoptosis was assessed by evaluating morphological changes, measuring enzymatic activity of caspase-3, and determining the extent of DNA degradation. We found that samples containing anti-IL-8:IL-8 immune complexes but not samples from which these complexes were removed inhibited neutrophil apoptosis. Furthermore, the former samples or effectively anti-IL-8:IL-8 complexes induced an increase in the level of antiapoptotic protein, Bcl-X(L). In contrast, levels of proapoptotic proteins Bax and Bak were decreased in the same conditions. Activity of both caspase-3 and caspase-9 was also suppressed by anti-IL-8:IL-8 complex-containing samples. Finally, we established that IgG receptor, FcgammaRIIa, mediates antiapoptotic activity of anti-IL-8:IL-8 complexes and that the key components of the FcgammaRIIa signaling pathway, Src, Syk, PI3 kinase, and ERK, may be involved in regulation of neutrophil apoptosis by the complexes. These studies demonstrate for the first time that anti-IL-8:IL-8 immune complexes have the ability to prolong neutrophil life.     

Association of IL-8-251A/T polymorphism with incidence of Acute Respiratory Distress Syndrome (ARDS) and IL-8 synthesis after multiple trauma

INTRODUCTION: Interleukin-8 (IL-8) is regarded as one of the most important mediators in the pathogenesis of Adult Respiratory Distress Syndrome (ARDS). However, knowledge regarding the influence of genetic variations within the IL-8 gene either on the development of ARDS or on IL-8 production in the traumatic setting is sparse. PATIENTS AND METHODS: In this prospective cohort study, patients were included if the following criteria were fulfilled: Injury Severity Score (ISS) >16, age 18-60 years and a survival >48 h after injury. Systemic IL-8 concentrations and the polymorphisms (IL-8-251A/T) were determined. Patients were separated according to the development of ARDS (group +ARDS vs. group -ARDS) and the genotypes of the IL-8-251A/T polymorphism (genotypes A/A, A/T and T/T). RESULTS: Group +ARDS demonstrated significantly higher IL-8 plasma concentrations from day 3 until the end of the observation period compared to group -ARDS. In addition, duration of mechanical ventilation and length of stay in the ICU were significantly longer in this group. Furthermore, a significant association between the IL-8-251A allele and IL-8 production (day 4-8) was observed. Genotype A/A showed a significantly longer duration of mechanical ventilation compared to genotype T/T. A trend towards an association between the IL-8-251A allele and an increased incidence of posttraumatic ARDS was observed (p=0.08). CONCLUSION: This data reaffirms a central role of IL-8 in the pathogenesis of ARDS. Furthermore, it points towards a genetic predisposition for posttraumatic IL-8 synthesis which might also be associated with the development of posttraumatic ARDS.     

High concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome

alpha-Defensins, antimicrobial peptides localized in neutrophils, participate in tissue damage through their cytotoxic effects in neutrophil-mediated pulmonary diseases. Neutrophils play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). We measured alpha-defensins levels in plasma and bronchoalveolar lavage fluid (BALF) of ARDS patients to assess the kinetics of alpha-defensins in ARDS. Plasma alpha-defensins levels were higher in ARDS patients than in control subjects, and BALF levels were also higher in ARDS patients than in control subjects. In ARDS, BALF alpha-defensins levels correlated with those of interleukin (IL)-8, and plasma alpha-defensins levels also correlated with Lung Injury Score. Peripheral neutrophil alpha-defensins contents were higher in ARDS than the control. IL-8 dose-dependently stimulated alpha-defensins release from cultured neutrophils and these levels were higher in ARDS than the control. Reverse-phase high performance liquid chromatography showed high plasma levels of pro-defensins, precursors of alpha-defensins from the bone marrow in ARDS, although alpha-defensins in peripheral and BALF neutrophils were mature type. In conclusion, high plasma alpha-defensins in ARDS patients result from the release of pro-defensins from bone marrow, rather than mature alpha-defensins from neutrophils that accumulate in the alveolar space. The alpha-defensins contents of peripheral neutrophils in ARDS are higher and easier to release than control.     

Bacteria-specific neutrophil dysfunction associated with interferon-stimulated gene expression in the acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a poorly understood condition with greater than 30% mortality. Massive recruitment of neutrophils to the lung occurs in the initial stages of the ARDS. Significant variability in the severity and duration of ARDS-associated pulmonary inflammation could be linked to heterogeneity in the inflammatory capacity of neutrophils. Interferon-stimulated genes (ISGs) are a broad gene family induced by Type I interferons. While ISGs are central to anti-viral immunity, the potential exists for these genes to evoke extensive modification in cellular response in other clinical settings. In this prospective study, we sought to determine if ISG expression in circulating neutrophils from ARDS patients is associated with changes in neutrophil function. Circulating neutrophil RNA was isolated, and hierarchical clustering ranked patients' expression of three ISGs. Neutrophil response to pathogenic bacteria was compared between normal and high ISG-expressing neutrophils. High neutrophil ISG expression was found in 25 of 95 (26%) of ARDS patients and was associated with reduced migration toward interleukin-8, and altered responses to Staphylococcus aureus, but not Pseudomonas aeruginosa, which included decreased p38 MAP kinase phosphorylation, superoxide anion release, interleukin-8 release, and a shift from necrotic to apoptotic cell death. These alterations in response were reflected in a decreased capacity to kill S. aureus, but not P. aeruginosa. Therefore, the ISG expression signature is associated with an altered circulating neutrophil response phenotype in ARDS that may predispose a large subgroup of patients to increased risk of specific bacterial infections.     

Green tea polyphenol blocks h(2)o(2)-induced interleukin-8 production from human alveolar epithelial cells

Reactive oxygen species (ROS) play crucial roles in ischemia-reperfusion (IR) injury of lung transplants. Reactive oxygen species may stimulate the production of neutrophil chemotactic factors such as interleukin-8 (IL-8), from alveolar epithelial cells, causing recruitment and activation of neutrophils in the reperfused tissue. Green tea polyphenol has potent anti-oxidative activities and anti-inflammatory effects by decreasing cytokine production. In the present study, we found that green tea polyphenol significantly inhibited IL-8 production induced by hydrogen peroxide (H(2)O(2)) in human lung alveolar epithelial cells (A549 line). It has been shown that mitogen activated protein kinases, such as Jun N-terminal kinase (JNK), p38 and p44/42, could mediate IL-8 production from a variety of cell types. We further investigated the effect of green tea polyphenol on these protein kinases, and demonstrated that H(2)O(2)-induced phosphorylation of JNK and p38 but not p44/42 was inhibited by green tea polyphenol in A549 cells. We speculate that green tea polyphenol may inhibit H(2)O(2)-induced IL-8 production from A549 cells through inactivation of JNK and p38.     

Bronchoalveolar lavage fluids of ventilated patients with acute lung injury activate NF-kappaB in alveolar epithelial cell line: role of reactive oxygen/nitrogen species and cytokines.

In human alveolar epithelial cell line, we investigated the binding activity of NF-kappaB induced by the bronchoalveolar lavage fluids (BALs) from ventilated patients with acute lung injury (ALI), in correlation with the concentrations of inflammatory cytokines, RNOS, and the severity of the ALI. In BALs obtained in 67 patients (16 bronchopneumonia, 14 infected ARDS, 20 ARDS, and 17 ALI patients without bronchopneumonia and no ARDS), we measured endotoxin, IL-1beta, IL-8, and nitrated proteins (NTP), the activity of myeloperoxidase, and the capacity to activate the NF-kappaB in alveolar A549 cells by electrophoretic mobility shift and supershift assays. The neutrophil counts and mean IL-1beta, IL-8, myeloperoxidase, and NTP values were increased in bronchopneumonia and infected ARDS groups compared to ARDS and ALI without bronchopneumonia and no ARDS groups (P<0.001). The number of neutrophils was correlated to those of IL-1beta, IL-8, myeloperoxidase, NTP, and endotoxin in all groups (P<0.0001). NF-kappaB activity was induced in alveolar like cells by BALs in all groups, was higher in bronchopneumonia and infected ARDS groups (P<0.02), and was correlated to IL-1beta (P=0.0002), IL-8 (P=0.02), NTP (P=0.014), myeloperoxidase (P=0.016), and neutrophil counts (P=0.003). BALs of bronchopneumonia and infected ARDS patients had increased inflammatory mediators (compared to ARDS and ALI without bronchopneumonia and no ARDS patients) that correlated to neutrophil counts and to the NF-kappaB-binding activity. These mediators and NF-kappaB activation may induce an amplification of inflammatory phenomena. By in vitro studies, we confirmed that NO-derived species (10(-6) to 10(-5)M peroxynitrite and 10(-5)M nitrites) and myeloperoxidase (at concentration equivalent to that found in BALs) can participate in the NF-kappaB activation.     

Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway.

Acute ethanol (EtOH) intoxication has been identified as a risk factor for infectious complications in trauma and burn victims. However, the mechanism of this immune dysfunction has yet to be elucidated. The monocyte/macrophage production of cytokines, in particular IL-8 and TNF-alpha, is critical in the regulation of the acute inflammatory response to infectious challenge. IL-8 is a potent chemoattractant and activator of neutrophils. TNF-alpha, a proinflammatory cytokine, initiates expression of endothelial cell surface adhesion molecules and neutrophil migration. p38, a member of the mitogen-activated protein kinases, plays an important role in mediating intracellular signal transduction in endotoxin-induced inflammatory responses. We examined the effects of LPS and ethanol on p38 activation and the corresponding IL-8 and TNF-alpha production in human mononuclear cells. LPS-induced IL-8 and TNF-alpha production was inhibited in a similar pattern by pretreatment with either EtOH or SB202190 (1 microM), a specific inhibitor of p38 kinase. Western blot analysis, using a dual phospho-specific p38 mitogen-activated protein kinase Ab, demonstrated that EtOH pretreatment inhibited LPS-induced p38 activation. These results demonstrate that alcohol suppresses the normal host immune inflammatory response to LPS. This dysregulation appears to be mediated in part via inhibition of p38 activation. Inhibition of IL-8 and TNF-alpha production by acute EtOH intoxication may inhibit inflammatory focused neutrophil migration and activation and may be a mechanism explaining the increased risk of trauma- and burn-related infections.     

Disease-specific dynamic biomarkers selected by integrating inflammatory mediators with clinical informatics in ARDS patients with severe pneumonia

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome that occurs as a result of various risk factors, including either direct or indirect lung injury, and systemic inflammation triggered also by severe pneumonia (SP). SP-ARDS-associated morbidity and mortality remains high also due to the lack of disease-specific biomarkers. The present study aimed at identifying disease-specific biomarkers in SP or SP-ARDS by integrating proteomic profiles of inflammatory mediators with clinical informatics. Plasma was sampled from the healthy as controls or patients with SP infected with bacteria or infection-associated SP-ARDS on the day of admission, day 3, and day 7. About 15 or 52 cytokines showed significant difference between SP and SP-ARDS patients with controls or 13 between SP-ARDS with SP alone and controls, including bone morphogenetic protein-15 (BMP-15), chemokine (C-X-C motif) ligand 16 (CXCL16), chemokine (C-X-C motif) receptor 3 (CXCR3), interleukin-6 (IL-6), protein NOV homolog (NOV/CCN3), glypican 3, insulin-like growth factor binding protein 4 (IGFBP-4), IL-5, IL-5 R alpha, IL-22 BP, leptin, MIP-1d, and orexin B with a significant correlation with Digital Evaluation Score System (DESS) scores. ARDS patients with overexpressed IL-6, CXCL16, or IGFBP-4 had significantly longer hospital stay and higher incidence of secondary infection. We also found higher levels of those mediators were associated with poor survival rates in patients with lung cancer and involved in the process of the epithelial mesenchymal transition of alveolar epithelial cells. Our preliminary study suggested that integration of proteomic profiles with clinical informatics as part of clinical bioinformatics is important to validate and optimize disease-specific and disease-staged biomarkers.     

Proinflammatory activity of anti-IL-8 autoantibody:IL-8 complexes in alveolar edema fluid from patients with acute lung injury

A significant fraction of IL-8 in lung fluids from patients with the acute lung injury (ALI) is associated with anti-IL-8 autoantibodies (anti-IL-8:IL-8 complexes), and lung fluid concentrations of these complexes correlate with development and outcome of ALI. In this study, we examined whether anti-IL-8:IL-8 complexes exhibit proinflammatory activity in vitro. These complexes were purified from pulmonary edema fluid samples obtained from patients with ALI. First, we found that IL-8 bound to the autoantibody retained its ability to trigger chemotaxis of neutrophils, whereas control antibody did not have significant chemotactic activity. Next, we examined the ability of anti-IL-8:IL-8 complexes to induce neutrophil activation, i.e., neutrophil respiratory burst and degranulation. Anti-IL-8:IL-8 complexes triggered superoxide and myeloperoxidase release from human neutrophils, and in contrast, the control antibody had no effect. We also demonstrated that IgG receptor, FcgammaRIIa, is the receptor involved in cellular activation mediated by these complexes. Blockade of FcgammaRIIa completely reverses activity of the complexes with the exception of chemotaxis. Both FcgammaRIIa and IL-8 receptors mediate chemotactic activity of anti-IL-8:IL-8 complexes, with FcgammaRIIa being, however, a predominant receptor. Furthermore, activity of the complexes is partially dependent on the activation of the mitogen-activated protein kinases, i.e., ERK and p38, important components of the FcgammaRIIa signaling cascade. Anti-IL-8:IL-8 complexes may therefore be involved in the pathogenesis of lung inflammation in clinical acute lung injury.     

Regulation of hepatocyte growth factor secretion by fibroblasts in patients with acute lung injury

The mechanisms of pulmonary repair in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are poorly known. Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are key factors involved in alveolar epithelial repair, present in the bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS. The role of BALF mediators in their production remains to be determined. We evaluated the overall effect of BALF from 52 patients (27 ventilated patients with ALI/ARDS, 10 ventilated patients without ALI, and 15 nonventilated control patients) on HGF and KGF synthesis by lung fibroblasts. Fibroblasts were cultured in the presence of BALF. HGF and KGF protein secretion was measured using ELISA, and mRNA expression was evaluated using quantitative real-time RT-PCR. Only BALF from ALI/ARDS patients upregulated both HGF and KGF mRNA expression and protein synthesis (+271 and +146% for HGF and KGF, respectively). BALF-induced HGF synthesis from ALI/ARDS patients was higher than that from ventilated patients without ALI (P < 0.05). HGF secretion was correlated with BALF IL-1beta levels (rho = 0.62, P < 0.001) and BALF IL-1beta/IL-1 receptor antagonist ratio (rho = 0.54, P < 0.007) in the ALI/ARDS group. An anti-IL-1beta antibody partially (>50%) inhibited the BALF-induced HGF and PGE(2) secretion, whereas NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, completely inhibited it. Anti-IL-1beta antibodies as well as NS-398 reversed the COX-2 upregulation induced by BALF. Therefore, IL-1beta is a main BALF mediator involved in HGF secretion, which is mediated through a PGE(2)/COX-2-dependent mechanism. BALF mediators may participate in vivo in the production of HGF and KGF by lung fibroblasts during ALI/ARDS.     

Non-Invasive monitoring of diaphragmatic timing by means of surface contact sensors: An experimental study in dogs

Background

The predictive role of many cytokines has not been well defined in Acute Respiratory Distress Syndrome (ARDS).

Methods

We measured prospectively IL-4, IL-6, IL-6 receptor, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid (BALF) in 59 patients who were admitted to ICU in order to identify predictive factors for the course and outcome of ARDS. The patients were divided into three groups: those fulfilling the criteria for ARDS (n = 20, group A), those at risk for ARDS and developed ARDS within 48 hours (n = 12, group B), and those at risk for ARDS but never developed ARDS (n = 27, group C).

Results

An excellent negative predictive value for ARDS development was found for IL-6 in BALF and serum (100% and 95%, respectively). IL-8 in BALF and IL-8 and IL-10 serum levels were higher in non-survivors in all studied groups, and were associated with a high negative predictive value. A significant correlation was found between IL-8 and APACHE score (r = 0.60, p < 0.0001). Similarly, IL-6 and IL-6r were highly correlated with PaO2/FiO2 (r = -0.27, p < 0.05 and r = -0.55, p < 0.0001, respectively).

Conclusions

BALF and serum levels of the studied cytokines on admission may provide valuable information for ARDS development in patients at risk, and outcome in patients either in ARDS or in at risk for ARDS.     

The role of the neutrophil in inflammatory diseases of the lung

Under certain circumstances, the neutrophil has been implicated in causing disease by damaging normal host tissue. This may occur in the adult respiratory distress syndrome (ARDS). The neutrophil has been implicated since a) substances that activate neutrophils are produced in association with the predisposing risks that lead to ARDS; b) activated neutrophils migrate into the alveolar spaces and their toxic products can be found in lung lavage fluid and in the breath of patients with ARDS; and c) the magnitude of the physiologic alterations correlate with the number of neutrophils in the alveolar space. Additionally, the neutrophils may be primed by substances which are released by activated platelets within the confines of the lung. Both platelet adenine nucleotides and the platelet-derived extracellular matrix protein (ECM), thrombospondin, can prime the neutrophil for subsequent O2- generation following activation of the cells with the chemotactic peptide, F-met-leu-phe (FMLP). Furthermore, neutrophils can be primed or O2- generation by the basement membrane ECM protein, laminin. Since neutrophils express receptors for both laminin and thrombospondin, these constituents may serve to modulate neutrophil behavior for subsequent oxidative metabolism and contribute to exacerbating pulmonary disease.     

Inhibition of human neutrophil IL-8 production by hydrogen peroxide and dysregulation in chronic granulomatous disease

The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemoattractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H(2)O(2) scavenger) or diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Addition of hydrogen peroxide or an H(2)O(2)-generating system suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism.     

Increased interleukin (IL)-8 and decreased IL-17 production in chronic obstructive pulmonary disease (COPD) provoked by cigarette smoke

Recently, involvement of IL-17 in development of COPD has been noticed. Unlike IL-8, the role of IL-17 in COPD remains controversial. In order to further understand mechanisms in cigarette smoke (CS) induced COPD, we investigated IL-17 and IL-8 levels in different stages of COPD patients, and time courses of IL-17 and IL-8 release in CS induced COPD rats. A total of 73 elderly patients with COPD and 31 healthy volunteers were recruited in the study. IL-17 and IL-8 levels in the sputum and plasma were measured, and number of differential cells was counted. A newly developed CS induced rat COPD model was employed to study time courses of IL-17 and IL-8 release and nucleated cell accumulation. The results showed that IL-8 levels in the sputum of severe COPD patients were elevated by 16.5-fold, but IL-17 levels were reduced by 4.8-fold. While IL-8 correlated with neutrophils, IL-17 correlated with monocytes and lymphocytes. Similarly, level of IL-8 was increased, but IL-17 was decreased in the bronchoalveolar lavage fluid (BALF) of CS rats. Time course study showed that increased IL-8 production in the BALF initiated at 6 weeks, but decreased IL-17 production started at 10 weeks following CS exposure. In conclusion, increased IL-8 level in COPD patients appears mainly secreted from neutrophils and macrophages, whereas decreased IL-17 level seems resulted from reduced number of monocytes or damaged epithelial cells. Increased IL-8 (a proinflammatory cytokine) secretion and decreased IL-17 (a protective cytokine of airways) release can both contribute to development of COPD.     

Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8 interleukin-8 - fMLP fMet-Leu-Phe - MBP myelin basic protein - ERK extracellular signal regulated kinase - MAP2 microtubule-associated protein 2 - PK-A cAMP dependent protein kinase - PKI protein kinase inhibitor - PMSF phenyl-methanesulfonyl fluoride - PVDF poly-vinylidene difluoride - HBSF Hank's buffered salt solution - DAB 3,3-diaminobenzidine tetrahydrochloride - PNPP p-nitrophenyl-phosphate - HSA human serum albumin - EGTA [ethylenebis (oxyethylenenitrilo)]tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.     

Bronchoalveolar fluid and plasma inflammatory biomarkers in contemporary ARDS patients

Purpose: Bronchoalveolar fluid (BALF) and plasma biomarkers are often endpoints in early phase randomized trials (RCTs) in acute respiratory distress syndrome (ARDS). With ARDS mortality decreasing, we analyzed baseline biomarkers in samples from contemporary ARDS patients participating in a prior RCT and compared these to historical controls.

Materials and methods: Ninety ARDS adult patients enrolled in the parent trial. BALF and blood were collected at baseline, day 4?±?1, and day 8?±?1. Interleukins-8/-6/-1β/-1 receptor antagonist/-10; granulocyte colony stimulating factor; monocyte chemotactic protein-1; tumour necrosis factor-α; surfactant protein-D; von Willebrand factor; leukotriene B₄; receptor for advanced glycosylation end products; soluble Fas ligand; and neutrophil counts were measured.

Results: Compared to historical measurements, our values were generally substantially lower, despite our participants being similar to historical controls. For example, our BALF IL-8 and plasma IL-6 were notably lower than in a 1999 RCT of low tidal volume ventilation and a 2007 biomarker study, respectively.

Conclusions: Baseline biomarker levels in current ARDS patients are substantially lower than 6–20?years before collection of these samples. These findings, whether from ICU care changes resulting in less inflammation or from variation in assay techniques over time, have important implications for design of future RCTs with biomarkers as endpoints.