Lynne Armstrong

Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis

Toll‐like receptors (TLRs) are a recently described family of immune receptors involved in the recognition of pathogen‐associated molecular patterns (PAMPs). The central role of TLR‐2 and TLR‐4 in microbial responses suggests they may be implicated in the pathogenesis of human sepsis. We hypothesized that the incidence and outcome of sepsis would be influenced by the expression of TLR‐2 and TLR‐4 on monocytes. We have examined the expression of TLR‐2 and TLR‐4 mRNA and protein and their response to pro‐ and anti‐inflammatory agents on monocytes from subjects in the intensive therapy unit (ITU) with and without Gram‐negative, Gram‐positive or polymicrobial sepsis. We compared these data to ITU and healthy control subjects. TLR‐2 mRNA was significantly up‐regulated on monocytes from subjects with both Gram‐positive and Gram‐negative sepsis. Similarly, we detected increased levels of TLR‐2 protein on the surface of monocytes from sepsis subjects relative to ITU controls. TLR‐4 mRNA was increased in Gram‐positive subjects; however, there was no corresponding increase in TLR‐4 protein. Although TLR‐4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with lipopolysaccharide or interleukin‐10, this was not observed in monocytes obtained from sepsis and ITU control subjects, suggesting that septic and ITU control milieus may alter the immunoregulation of TLR‐4 mRNA expression on monocytes. TLR‐2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR‐2, and to a lesser extent TLR‐4 may be abnormal in human sepsis.

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.

Vascular Endothelial Growth Factor (VEGF) isoform expression and activity in human and murine lung injury

BackgroundThe properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in lung injury. Alternate spliced VEGF transcript generates several isoforms with potentially differing functions. The purpose of this study was to determine VEGF isoform expression and source in normal and ARDS subjects and investigate the expression and regulation of VEGF isoforms by human alveolar type 2 (ATII) cells.MethodsVEGF protein expression was assessed immunohistochemically in archival normal and ARDS human lung tissue. VEGF isoform mRNA expression was assessed in human and murine lung tissue. Purified ATII cells were cultured with proinflammatory cytokines prior to RNA extraction/cell supernatant sampling/proliferation assay.Measurements and Main ResultsVEGF was expressed on alveolar epithelium, vascular endothelium and alveolar macrophages in normal and ARDS human lung tissue. Increases in VEGF expression were detected in later ARDS in comparison to both normal subjects and early ARDS (p < 0.001). VEGF121, VEGF165 and VEGF189 isoform mRNA expression increased in later ARDS (p < 0.05). The ratio of soluble to cell-associated isoforms was lower in early ARDS than normal subjects and later ARDS and also in murine lung injury. ATII cells constitutionally produced VEGF165 and VEGF121 protein which was increased by LPS (p < 0.05). VEGF165 upregulated ATII cell proliferation (p < 0.001) that was inhibited by soluble VEGF receptor 1 (sflt) (p < 0.05).ConclusionThese data demonstrate that changes in VEGF isoform expression occur in ARDS which may be related to their production by and mitogenic effect on ATII cells; with potentially significant clinical consequences.

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 α. 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.

Inter-relationship between tumour necrosis factor-alpha (TNF-α) and TNF soluble receptors in pulmonary sarcoidosis

BACKGROUND The importance of tumour necrosis factor-alpha (TNF-α) in the pathogenesis of pulmonary sarcoidosis has remained uncertain because of the paucity of clinical features associated with excessive levels of this cytokine. Increased levels of soluble TNF receptors (TNF-R), which are known to inhibit TNF-α activity, were recently described in the lungs of subjects with sarcoidosis. We hypothesised that TNF-α bioactivity may be inhibited in sarcoidosis by the presence of TNF-R. A study was therefore undertaken to investigate for the first time the relationship between soluble receptors and TNF-α bioactivity in the lungs of subjects with sarcoidosis. METHODS Alveolar macrophages (AMs) from 16 subjects with histologically proven sarcoidosis and 13 healthy controls were cultured in the presence and absence of lipopolysaccharide (LPS). The subjects with sarcoidosis were grouped by radiological assessment into stage I (n = 6) and stage II/III (n = 10). The cell culture supernatants and bronchoalveolar lavage (BAL) fluid were assayed for TNF bioactivity using the WEHI 164 clone 13 assay. Immunoreactive (bound and free) TNF-α and free TNF-Rs (p55 and p75) were determined by ELISA. RESULTS Bioactive TNF-α was undetectable in the BAL fluid of all the subjects with sarcoidosis and most of the healthy controls. However, there was significantly more immunoreactive TNF-α in the BAL fluid from subjects with sarcoidosis than from the controls (median values 0.304 ng/ml and 0.004 ng/ml, respectively, 95% CI 0.076 to 0.455, p<0.001). The levels of both p55 and p75 in the BAL fluid were higher in both sarcoidosis groups than in the controls (p<0.0005 and p<0.001, respectively). In LPS stimulated AM supernatants reduced TNF-α bioactivity was seen in subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.333 ng/ml vs 1.362 ng/ml and 2.385 ng/ml, respectively, p<0.01). This contrasted with increased p55 levels in the AM supernatants derived from subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.449 ng/ml vs 0.058 ng/ml and 0.078 ng/ml, respectively, p<0.01). The levels of p75 were increased in unstimulated AM cultures in subjects with stage II/III disease compared with those with stage I disease and healthy controls (median 0.326 ng/ml vs 0.064 ng/ml and 0.102 ng/ml, p<0.05). CONCLUSIONS These results indicate that TNF-α bioactivity may be inhibited by increased soluble TNF-R in the lungs of subjects with sarcoidosis, and this inhibition may be greater in patients with stage I sarcoidosis than in those with stage II/III disease. This may represent a homeostatic mechanism which protects the lung from excessive TNF production characteristic of chronic inflammation.

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.

Tumour necrosis factor-α processing in interstitial lung disease: a potential role for exogenous proteinase-3

Tumour necrosis factor (TNF) blockade has become an important immunomodulatory therapy, particularly in patients refractory to conventional immunosuppression, but responses can be unpredictable. Understanding the complex biology of TNF processing may be key to predicting such responses and reduce unwanted side effects. TNF bioavailability is regulated partly by TNF‐α converting enzyme (TACE) cleavage; however, it can also be cleaved by proteinase‐3 (PR‐3). We have demonstrated this mechanism previously in healthy human alveolar macrophages (AM), leading us to hypothesize that PR‐3‐mediated TNF processing may be an important mechanism in inflammatory lung disease. Furthermore, this may be more apparent in diseases with a neutrophil component typical of usual interstitial pneumonia (UIP), compared with sarcoidosis, where lymphocytes predominate. We isolated AM from patients with UIP and sarcoidosis and healthy subjects. We found increased TACE expression on AM in sarcoidosis. In contrast, TACE was not increased in UIP; we found increased cleavage of glutathione S‐transferase‐proTNF) substrate, relative to both sarcoidosis and healthy controls. Furthermore, cleavage was subject to inhibition by serine protease inhibitor, rather than a TACE inhibitor BB‐3103. Cleavage was proportional to the number of neutrophils isolated from bronchoalveolar lavage, whereas there was an inverse relationship between neutrophils and BB‐3103 inhibition. There was also increased PR‐3 on the AM surface in UIP relative to healthy controls. These data provide evidence for PR‐3‐mediated cleavage in UIP, which may have implications for future therapeutic targeting of TACE.