Vitamin D-binding protein contributes to COPD by activation of alveolar macrophages.

BACKGROUND: Vitamin D-binding protein (DBP) genetic polymorphisms have been associated with chronic obstructive pulmonary disease (COPD). DBP has an indirect role in macrophage activation; thus it was hypothesised that DBP is present in the airway and contributes to lung disease by this mechanism. METHODS: 471 PiZZ subjects with α1-antitrypsin deficiency (AATD) were genotyped for tag single nucleotide polymorphisms (SNPs) covering the DBP gene (GC), together with known functional variants, prior to seeking association with COPD phenotypes. 140 subjects with usual COPD and 480 controls were available for replication. Vitamin D and DBP levels were measured by tandem mass spectrometry and ELISA, respectively, in serum and DBP in the sol phase of sputum in a subset of 60 patients. Concentrations were related to phenotype and to alveolar macrophage activation. RESULTS: rs2070741 was associated with airway bacterial colonisation (p=0.04) and bronchiectasis (p=0.01), as was rs7041 (p=0.03) which also influenced vitamin D concentrations (p=0.01). The GC2 variant predisposed to bronchiectasis in AATD (p=0.04) and protected against COPD (p=0.05); the latter association was replicated in usual COPD versus controls (p=0.04). Circulating DBP related inversely to forced expiratory volume in 1 s (FEV(1)) (p=0.02), in direct contrast to vitamin D, where deficiency related to low FEV(1) (p=0.04). Sol DBP related directly to alveolar macrophage activation (p=0.004). CONCLUSIONS: The genetic association of DBP with COPD may be mediated by effects on macrophage activation, since DBP relates to FEV(1), and affects macrophage activation. Vitamin D effects may be independent of this, relating more strongly to innate immunity.

Metalloproteinases in idiopathic pulmonary fibrosis.

In this article, we outline the current state of knowledge about the balance between collagen production and degradation in idiopathic pulmonary fibrosis (IPF). The dysregulated action of metalloproteinases implicated in IPF may play a central role in IPF pathogenesis. Inhibiting metalloproteinases in IPF may, therefore, have therapeutic potential, but our knowledge of their pathophysiological role is held back by limited animal models and the lack of specific inhibitors.

Neutrophil chemotaxis in granulomatosis with polyangiitis (Wegener's) and idiopathic pulmonary fibrosis.

The presence of antineutrophil cytoplasmic antibodies in granulomatosis with polyangiitis (Wegener's) (GPA) implicates the neutrophil as a key effector cell. Previous studies have reported elevated neutrophil counts in the lung, although the determinants of neutrophil chemotaxis in the GPA lung are unknown. Bronchoalveolar lavage fluid (BALF) cell counts, myeloperoxidase (MPO) and chemokines were measured in 27 patients with GPA, 20 disease controls with idiopathic pulmonary fibrosis (IPF) and six healthy controls. CXC chemokine ligand (CXCL)8, interleukin (IL)-1ß, epithelial neutrophil-activating protein 78, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor were measured by ELISA. The neutrophil chemotactic potential of BALF was investigated using the under-agarose method, and specific antibodies were used to examine the role of CXCL8 and IL-1ß. GPA BALF had an increased neutrophil percentage, and elevated MPO, CXCL8 and G-CSF concentrations compared with healthy controls. Chemotaxis of control neutrophils towards BALF from patients with active (p=0.006) and remission (p=0.077) GPA, and IPF (p=0.001) patients was increased compared with normal controls. BALF-induced chemotaxis correlated with BALF IL-1ß (r=0.761, p=0.001) and CXCL8 (r=0.640, p=0.012) in GPA, and was inhibited by anti-CXCL8 (85%; p<0.001) and anti-IL-1ß (69%; p<0.001). Our study confirms a neutrophilia and pro-inflammatory alveolar milieu that persists in clinical remission. CXCL8 and IL-1ß appear to play important roles in the neutrophil chemotactic response to BALF.

To What Extent do Social Determinants of Health Modulate Presentation, ITU Admission and Outcomes among Patients with SARS-COV-2 Infection? An Exploration of Household Overcrowding, Air Pollution, Housing Quality, Ethnicity, Comorbidities and Frailty.

Background: Internationally, researchers have called for evidence to support tackling health inequalities during the severe acute respiratory syndrome coronavirus 2 (COVID19) pandemic. Despite the 2020 Marmot review highlighting growing health gaps between wealthy and deprived areas, studies have not explored social determinants of health (ethnicity, frailty, comorbidities, household overcrowding, housing quality, air pollution) as modulators of presentation, intensive care unit (ITU) admissions and outcomes among COVID19 patients. There is an urgent need for studies examining social determinants of health including socioenvironmental risk factors in urban areas to inform the national and international landscape. Methods: An in-depth retrospective cohort study of 408 hospitalized COVID19 patients admitted to the Queen Elizabeth Hospital, Birmingham was conducted. Quantitative data analyses including a two-step cluster analysis were applied to explore the role of social determinants of health as modulators of presentation, ITU admission and outcomes. Results: Patients admitted from highest Living Environment deprivation indices were at increased risk of presenting with multi-lobar pneumonia and, in turn, ITU admission whilst patients admitted from highest Barriers to Housing and Services (BHS) deprivation Indies were at increased risk of ITU admission. Black, Asian and Minority Ethnic (BAME) patients were more likely, than Caucasians, to be admitted from regions of highest Living Environment and BHS deprivation, present with multi-lobar pneumonia and require ITU admission. Conclusion: Household overcrowding deprivation and presentation with multi-lobar pneumonia are potential modulators of ITU admission. Air pollution and housing quality deprivation are potential modulators of presentation with multi-lobar pneumonia. BAME patients are demographically at increased risk of exposure to household overcrowding, air pollution and housing quality deprivation, are more likely to present with multi-lobar pneumonia and require ITU admission. Irrespective of deprivation, consideration of the Charlson Comorbidity Score and the Clinical Frailty Score supports clinicians in stratifying high risk patients.

The vitamin D axis in the lung: a key role for vitamin D-binding protein.

There has been much recent interest in the role of the vitamin D axis in lung disease, which includes vitamin D, vitamin D receptor (VDR) and vitamin D-binding protein (VDBP; also known as Gc-globulin). VDBP is a serum protein which has immunomodulatory functions relevant in the lung, predominantly relating to macrophage activation and neutrophil chemotaxis. Variations within its gene are also associated with airways disease, implying a role for the protein product in pathogenesis. Thus far the majority of evidence relates to chronic obstructive pulmonary disease (COPD), but is scant in other airways diseases, such as asthma and bronchiectasis. VDBP also acts as a scavenger protein to clear extracellular G-actin released from necrotic cells, which may be of relevance in severe lung infections and acute lung injury. Vitamin D protects against the development of cancer and tuberculosis, although optimal levels are unknown. The majority of circulating vitamin D is bound to VDBP, and its uptake into cells occurs in both bound and unbound forms, which suggests the role of VDBP warrants further study in these conditions as well. This article reviews the evidence of the role VDBP and its gene (GC) in a range of lung diseases, including asthma, COPD and tuberculosis.

Pulmonary infection in Wegener granulomatosis and idiopathic pulmonary fibrosis.

RATIONALE: Wegener granulomatosis (WG) has previously been associated with increased nasal carriage of Staphylococcus aureus, but no studies have investigated the occurrence of pathogen growth in the lower airways. OBJECTIVES: To culture bronchoalveolar lavage fluid (BALF) from patients with WG, patients with idiopathic pulmonary fibrosis (IPF) and normal controls. METHODS: 33 patients with WG, 22 with IPF and 8 normal controls underwent bronchoscopy and bronchoalveolar lavage. Quantitative culture established bacterial levels in the lower airways. Culture experiments were designed to investigate whether BALF is a supportive environment for S aureus growth. BALF cytokines were measured by ELISA. RESULTS: Pathogens were commonly grown from BALF of patients with WG and those with IPF. S aureus was particularly associated with patients with WG both in relapse and in remission. BALF levels of interleukin 1 receptor antagonist (IL1ra) were statistically significantly elevated in those patients who grew a pathogen from lavage fluid. BALF from patients with WG and IPF stimulated S aureus growth compared with normal lavage fluid. CONCLUSIONS: Pathogens are more commonly isolated from BALF from patients with WG than from that of patients with IPF or normal controls, and with a different culture profile. IL1ra was associated with pathogen growth in WG and IPF. WG BALF is a trophic environment for S aureus growth. Pulmonologists treating patients with acute or relapsing WG should consider bronchoscopic microbiological sampling and consider antibiotics with antistaphylococcal activity.

Soluble endostatin is a novel inhibitor of epithelial repair in idiopathic pulmonary fibrosis.

BACKGROUND AND AIM: Aberrant angiogenesis and defective epithelial repair are key features of idiopathic pulmonary fibrosis (IPF). Endostatin is an antiangiogenic peptide with known effects on endothelial cells. This study aimed to establish the levels of endostatin in the bronchoalveolar lavage fluid (BALF) in IPF and to investigate its actions on distal lung epithelial cells (DLEC) and primary type II cells. METHODS: 20 patients with IPF and 10 controls underwent BAL. Endostatin was measured by ELISA. BALF cytokines and matrix metalloproteinase (MMP)-3 were measured by Luminex array. Primary DLEC monolayers were wounded and treated with endostatin. Apoptosis and cell viability were assessed. RESULTS: Endostatin was elevated in the BALF and plasma of patients with IPF compared with normal controls. There was a negative correlation between endostatin, forced vital capacity and gas transfer. Endostatin correlated with a number of proinflammatory cytokines and MMP3. Physiological endostatin doses inhibited DLEC wound repair by 44% in an effect that was partially FasL and caspase dependent. Endostatin increased apoptosis rates by 8% and reduced their viability by 34%. Similar effects of endostatin were seen in primary type II cells in terms of inhibition of wound repair and proliferation. CONCLUSIONS: Elevated BALF endostatin levels correlated with a number of elevated cytokines, MMP3 and lung function in IPF. Endostatin is a novel inhibitor of DLEC wound repair, inducing apoptosis and reducing cell viability in a FasL and caspase dependent manner. Endostatin may play a role in aberrant epithelial repair in IPF.

MMP expression and abnormal lung permeability are important determinants of outcome in IPF.

Matrix metalloproteinases (MMPs) degrade all of the extracellular matrix components of the intersititium and may play a role in abnormal alveolar permeability, which is a feature of idiopathic pulmonary fibrosis (IPF). The aims of the present study were to evaluate MMP protein levels in patients with IPF and determine any relationship to treatment and markers of permeability. In total, 20 patients with IPF and eight normal controls underwent bronchoalveolar lavage. MMP, tissue inhibitor of metalloproteinase, and vascular endothelial growth factor (VEGF) levels were related to clinical outcome and protein permeability index. MMP-3, -7, -8 and -9 were elevated in IPF lavage fluid and levels remained high despite treatment. Levels of MMP-3, -7, -8 and -9, VEGF and protein permeability index were higher in those who died early during follow-up. VEGF, and MMP-8 and -9 levels were higher in those with a rapidly declining lung function over 1 yr. Levels of MMP-3, -7, -8 and -9 correlated with an increased permeability index. Matrix metalloproteinase levels were elevated in idiopathic pulmonary fibrosis patients and were not modulated by current standard treatment. Matrix metalloproteinase production through an interaction with the known vascular permogen, vascular endothelial growth factor, was potentially associated with abnormal capillary permeability and may have potentiated the neo-angiogenesis seen in idiopathic pulmonary fibrosis. The changes were greatest in those who died or progressed during follow-up, suggesting that drugs targeting vascular endothelial growth factor or matrix metalloproteinase activity warrant assessment as novel therapy for idiopathic pulmonary fibrosis.

In vivo and in vitro effects of salbutamol on alveolar epithelial repair in acute lung injury.

BACKGROUND: Acute lung injury is an important cause of respiratory failure in the critically ill patient. It is caused by damage to the alveolar barrier with subsequent alveolar flooding leading to the development of refractory hypoxaemia. beta Agonists stimulate alveolar fluid clearance in animal models of lung injury. In a clinical trial (BALTI-1), intravenous beta agonists reduced extravascular lung water, an effect that took 72 h in contrast with what animal studies suggest. One possible explanation for the delay in change in extravascular lung water is the time required for salbutamol to stimulate alveolar epithelial repair. OBJECTIVE: To investigate whether salbutamol can stimulate alveolar epithelial repair in vivo and in vitro. RESULTS: Intravenous salbutamol reduced measures of alveolar-capillary permeability in patients with acute respiratory distress syndrome (ARDS). In vitro, salbutamol stimulated both wound repair, and spreading and proliferation of A549 cells and distal lung epithelial cells. Lung lavage fluid from patients treated with salbutamol enhanced wound repair responses compared with placebo treated patients in vitro by an interleukin 1beta dependent mechanism. CONCLUSIONS: Our in vivo and in vitro work suggests that salbutamol may stimulate epithelial repair--potentially a pharmacological first in ARDS. Clearly establishing the mechanisms and pathways responsible for this is important for the future, and may allow identification of novel therapeutic targets to promote alveolar epithelial repair in humans with ARDS.

Churg-Strauss syndrome and leukotriene antagonist use: a respiratory perspective.

BACKGROUND: Churg-Strauss syndrome (CSS) is a rare granulomatous small vessel vasculitis that occurs against a background of longstanding asthma. Leukotriene antagonists (LTAs) are used in the management of asthma and may facilitate a reduction in steroid dosage. Reports of the development of CSS in patients with asthma following the initiation of LTA therapy suggest either a causal association or an unmasking of latent CSS as steroid doses fall. We have undertaken a systematic review to establish whether evidence of a drug induced syndrome exists. METHODS: Systematic review searching Medline from database inception to August 2007 to identify cases with a possible association between LTAs and CSS. Hill's criteria of causation were used to assess strength of causality. RESULTS: 62 cases in which CSS developed after the introduction of LTA therapy were identified. Patients were divided into three groups: group 1 had received no previous steroid therapy; group 2 had been treated with oral and/or inhaled corticosteroids, but had no change in steroid therapy following LTA introduction; and group 3 had a clear reduction in steroid therapy following introduction of LTA therapy. The majority of patients from each group exhibited a clear temporal relationship between initiation of LTA and development of CSS, with no evidence of pre-existing disease. CONCLUSIONS: Currently available evidence suggests an association between LTA and CSS that may be causal.

Statins and sepsis.

Severe sepsis and septic shock is common and frequently fatal. Over the last few years, the primary treatments demonstrated to improve outcome from several major clinical trials have finally emerged. However, translating these recent therapeutic advances to routine clinical practice has proven controversial, and new approaches of additional strategies are continued to be developed. Given their pleiotropic effects related to many pathophysiological determinants of sepsis, statin therapy could be the next step in the search for adjuvant therapy. A future challenge may be to test both the efficacy and the safety by large randomized controlled clinical trials ascertaining the effects of statins administered at the onset of sepsis and in patients with severe sepsis or septic shock admitted into intensive care units.

Neutrophil Extracellular Trap Production in Patients with Colorectal Cancer In Vitro.

PURPOSE: Neutrophil Extracellular Traps (NETs) are extracellular neutrophil derived DNA webs which have been implicated in cancer progression and in the development of metastases. NETs production in patients with colorectal cancer was investigated to elucidate their role and prognostic significance. METHODS: Systemic neutrophils were isolated from consecutive patients with colorectal cancer and from age-matched healthy volunteers. Neutrophils were stimulated to produce NETs which were quantified by a measure of the fluorescence of the extracellular DNA. The impact of cancer location, tumour stage, and patient outcomes (complications, length of stay, and mortality) on NET production was investigated. RESULTS: Quantification of NET formation was performed in patients with colorectal cancer (n = 45) and in well-matched healthy individuals (n = 20). Significant increases in NETs production in response to no stimulant (9,735 AFU versus 11347 AFU, p = 0.0209), IL-8 (8,644 AFU versus 11,915 AFU, p = 0.0032), and LPS (10,576 AFU versus 12,473 AFU, p = 0.0428) were identified in patients with colorectal cancer. A significant increase in NETs production in response to fMLP was detected in patients who developed significant postoperative complications (11,760 AFU versus 18,340 AFU, p = 0.0242) and who had a prolonged hospital recovery (9,008 AFU versus 12,530 AFU, p = 0.0476). An increase in NETs production was also observed in patients who died, but this did not reach statistical significance. Cancer location and tumour stage did not appear to affect preoperative NETs production. CONCLUSIONS: Patients with colorectal cancer have significantly increased NETs production in vitro when compared to healthy volunteers, possibly implicating them in cancer development. Adverse patient outcomes were associated with increased preoperative NETs production, which highlights them as potential therapeutic targets.

The balance between collagen synthesis and degradation in diffuse lung disease.

Diffuse interstitial lung disease (DLD) is characterised by varying degrees of inflammation and fibrosis that result in derangement of the gas-exchanging units of the lung. A hallmark of these diseases is the abnormal deposition of collagen, which is a prime determinant of clinical course. This review considers the current information concerning collagen turnover in diffuse fibrotic lung disease. Considerable experimental evidence implicates both increased collagen production and reduced degradation in these diseases. Reduced degradation may result from both reduced production of collagenases and increased inhibition by tissue inhibitors. The known effects upon collagen turnover of novel therapeutic agents for pulmonary fibrosis (pirfenidone and interferon gamma) are discussed.

Beta-Agonist Lung injury TrIal-2 (BALTI-2): a multicentre, randomised, double-blind, placebo-controlled trial and economic evaluation of intravenous infusion of salbutamol versus placebo in patients with acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a major cause of mortality in intensive care patients and lacks effective treatments. A previous randomised controlled Phase II trial suggested that an intravenous (i.v.) infusion of salbutamol may be beneficial, as it reduced extravascular lung water and plateau airway pressure. The Beta-Agonist Lung injury TrIal-2 (BALTI-2) was initiated to evaluate the effects of this intervention on mortality in patients with ARDS. OBJECTIVES: To evaluate whether or not, in patients with ARDS, an i.v. infusion of salbutamol given at 15 µg/kg ideal body weight (IBW)/hour for up to 7 days, compared with a placebo (0.9% sodium chloride) infusion, reduces 28-day all-cause mortality and other clinical outcomes. To evaluate salbutamol's clinical effectiveness and its cost-effectiveness in subgroups of patients. DESIGN: A multicentre, randomised, placebo-controlled trial. SETTING: Forty-six intensive care units (ICUs) in the UK. PARTICIPANTS: Patients were eligible if they (1) were intubated and mechanically ventilated patients in participating ICUs; (2) were within 72 hours of onset of ARDS; (3) fulfilled American-European Consensus Conference definition for ARDS {acute-onset, severe hypoxaemic respiratory failure [partial pressure of oxygen in arterial blood/fraction of inspired oxygen ≤ 26.7 kPa (200 mmHg)] and bilateral infiltrates on the chest radiograph in the absence of clinical evidence of left atrial hypertension}; and (4) were aged ≥ 16 years. INTERVENTIONS: Intravenous infusion of salbutamol (15 µg/kg IBW/hour) or placebo (0.9% saline) for up to 7 days. MAIN OUTCOME MEASURES: All-cause mortality 28 days after randomisation, mortality at (first) discharge from ICU, mortality at (first) discharge from hospital, number of ventilator-free days, number of organ failure-free days, mortality at 12 months post randomisation, side effects (tachycardia/new arrhythmia/lactic acidosis) sufficient to stop treatment with trial drug, health-related quality of life (European Quality of Life-5 Dimensions and Short Form questionnaire-12 items at 6 and 12 months after randomisation), length of stay in critical care unit and length of stay in hospital. RESULTS: Forty-six ICUs recruited patients to the trial. A total of 326 patients were randomised; 162 were allocated to salbutamol and 164 to placebo. One patient in each group withdrew consent. Recruitment was stopped after the second interim analysis because of safety concerns. Salbutamol increased 28-day mortality: 55 (34%) of 161 patients died in the salbutamol group compared with 38 (23%) of 163 in the placebo group (risk ratio 1.47, 95% confidence interval 1.03 to 2.08). CONCLUSIONS: Treatment with i.v. salbutamol early in the course of ARDS was poorly tolerated, is unlikely to be beneficial and could worsen outcomes. Further trials of ß-agonists in patients with ARDS are unlikely to be conducted. Some questions remain, such as whether or not there may be benefit at a different dose or in specific populations, but any studies investigating these would require a very strong rationale. TRIAL REGISTRATION: Current Controlled Trials ISRCTN38366450. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury.

BACKGROUND: Intravenous salbutamol (albuterol) reduces lung water in patients with the acute respiratory distress syndrome (ARDS). Experimental data show that it also reduces pulmonary neutrophil accumulation or activation and inflammation in ARDS. AIM: To investigate the effects of salbutamol on neutrophil function. METHODS: The in vitro effects of salbutamol on neutrophil function were determined. Blood and bronchoalveolar lavage (BAL) fluid were collected from 35 patients with acute lung injury (ALI)/ARDS, 14 patients at risk from ARDS and 7 ventilated controls at baseline and after 4 days' treatment with placebo or salbutamol (ALI/ARDS group). Alveolar-capillary permeability was measured in vivo by thermodilution (PiCCO). Neutrophil activation, adhesion molecule expression and inflammatory cytokines were measured. RESULTS: In vitro, physiological concentrations of salbutamol had no effect on neutrophil chemotaxis, viability or apoptosis. Patients with ALI/ARDS showed increased neutrophil activation and adhesion molecule expression compared with at risk-patients and ventilated controls. There were associations between alveolar-capillary permeability and BAL myeloperoxidase (r = 0.4, p = 0.038) and BAL interleukin 8 (r = 0.38, p = 0.033). In patients with ALI/ARDS, salbutamol increased numbers of circulating neutrophils but had no effect on alveolar neutrophils. CONCLUSION: At the onset of ALI/ARDS, there is increased neutrophil recruitment and activation. Physiological concentrations of salbutamol did not alter neutrophil chemotaxis, viability or apoptosis in vitro. In vivo, salbutamol increased circulating neutrophils, but had no effect on alveolar neutrophils or on neutrophil activation. These data suggest that the beneficial effects of salbutamol in reducing lung water are unrelated to modulation of neutrophil-dependent inflammatory pathways.

Regulation of vascular endothelial growth factor bioactivity in patients with acute lung injury.

BACKGROUND: Reduced bioactive vascular endothelial growth factor (VEGF) has been demonstrated in several inflammatory lung conditions including the acute respiratory distress syndrome (ARDS). sVEGFR-1, a soluble form of VEGF-1 receptor, is a potent natural inhibitor of VEGF. We hypothesised that sVEGFR-1 plays an important role in the regulation of the bioactivity of VEGF within the lung in patients with ARDS. METHODS: Forty one patients with ARDS, 12 at risk of developing ARDS, and 16 normal controls were studied. Bioactive VEGF, total VEGF, and sVEGFR-1 were measured by ELISA in plasma and bronchoalveolar lavage (BAL) fluid. Reverse transcriptase polymerase chain reaction for sVEGFR-1 was performed on BAL cells. RESULTS: sVEGFR-1 was detectable in the BAL fluid of 48% (20/41) of patients with early ARDS (1.4-54.8 ng/ml epithelial lining fluid (ELF)) compared with 8% (1/12) at risk patients (p = 0.017) and none of the normal controls (p = 0.002). By day 4 sVEGFR-1 was detectable in only 2/18 ARDS patients (p = 0.008). Patients with detectable sVEGFR-1 had lower ELF median (IQR) levels of bioactive VEGF than those without detectable sVEGFR-1 (1415.2 (474.9-3192) pg/ml v 4761 (1349-7596.6) pg/ml, median difference 3346 pg/ml (95% CI 305.1 to 14711.9), p = 0.016), but there was no difference in total VEGF levels. BAL cells expressed mRNA for sVEGFR-1 and produced sVEGFR-1 protein which increased following incubation with tumour necrosis factor alpha. CONCLUSION: This study shows for the first time the presence of sVEGFR-1 in the BAL fluid of patients with ARDS. This may explain the presence of reduced bioactive VEGF in patients early in the course of ARDS.

Drug-induced antisynthetase syndrome.

We present a case of antisynthetase syndrome whose predominant feature was fibrosing alveolitis and which may have been drug-induced. This responded well to steroids and cyclophosphamide.

Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions.

BACKGROUND: Investigation and management of pleural effusions is an important clinical problem yet the pathogenesis of pleural fluid accumulation is poorly understood. Vascular endothelial growth factor (VEGF) is a potent inducer of capillary permeability that is produced by both malignant and inflammatory cells. A study was undertaken to determine whether VEGF has a potential pathogenic role in the development of pleural effusions and whether VEGF receptors are present on human pleural mesothelial cells. METHODS: Normal and inflamed pleura were examined immunohistochemically for the presence of FLT-1 (the fms-like tyrosine kinase receptor of VEGF). VEGF levels were measured by ELISA in 78 consecutive patients presenting with undiagnosed unilateral pleural effusions and the levels were correlated with the aetiology of the effusions. RESULTS: Immunohistochemical staining of normal and diseased pleura demonstrated the presence of the FLT-1 VEGF receptor on human mesothelial cells. Median VEGF levels were 2500 pg/ml in the malignant group and 305 pg/ml in the non-malignant group (median difference 1397.5 pg/ml (95% CI 851 to 2693), p<0.005). Median VEGF levels varied according to tumour histology. VEGF levels were also significantly raised compared with transudates (median 36.5 pg/ml) in empyema (4651 pg/ml (95% CI 833 to 10 000), p<0.001) and parainfectious effusions (360 pg/ml (95% CI 46 to 597), p<0.005). CONCLUSIONS: This first report of VEGF receptors on pleural mesothelial cells has indicated a potential mechanism for the biological activity of VEGF on pleural tissue. VEGF levels are raised in the majority of exudative effusions, implying a pathogenic role for this molecule in the development of pleural effusions.

Increased expression of functionally active membrane-associated tumor necrosis factor in acute respiratory distress syndrome.

Membrane-associated tumor necrosis factor (mTNF) has recently been shown to induce inflammatory cellular responses previously attributed to the soluble form. The present study measures for the first time the expression and function of mTNF on the surface of alveolar macrophages (AMs) to determine whether it is associated with the development of acute respiratory distress syndrome (ARDS). TNF expression was determined by flow cytometry, and the function of mTNF on the surface of AMs was determined by an in vitro cytotoxicity assay. Tumor necrosis factor (TNF)-alpha bioactivity was measured by bioassay. Soluble TNF receptor (TNFR) protein and messenger RNA (mRNA) expression were measured by enzyme-linked immunosorbent assay and reverse transcriptase/polymerase chain reaction, respectively. Increased detection of mTNF was observed on the surface of AMs derived from subjects with ARDS (mean percentage increase in fluorescence 22.30 +/- 3.50% for subjects with ARDS compared with 7.09 +/- 1.70% for At Risk subjects [P < 0.003]). mTNF cytotoxicity in the bioassay positively correlated with the mTNF expression determined by flow cytometry (r(2) = 0.97). Although there was increased mTNF expression and cytotoxic function in ARDS, there was no significant increase in soluble TNF expression in the bronchoalveolar lavage fluid or the AM supernatants. Lower levels of CD120b-soluble TNFR were detected in the AM supernatants derived from subjects with ARDS compared with At Risk (mean 0.264 +/- 0.058 versus 0.593 +/- 0.143 ng/ml, respectively [P < 0.05]). By contrast, there was increased CD120b mRNA expression in AMs derived from subjects with ARDS (P < 0.03), suggesting that increased surface expression of this receptor may be important in mediating the signal of mTNF. These data demonstrate for the first time the presence of functionally active mTNF on the surface of AMs in ARDS and highlight a potential mechanism for TNF-mediated lung injury.