Anne Burke-Gaffney

Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4.

Expression of cell adhesion molecules (CAM) on the lung microvascular endothelium is believed to play a key role in the recruitment of leukocytes in pulmonary inflammation. Moreover, regulation of CAM expression may be an important mechanism through which this inflammation may be controlled. Experimental evidence has suggested that combined phosphodiesterase (PDE) 3 and 4 inhibitors increase cyclic AMP levels within cells greater than inhibition of either isoenzyme alone. In the present study we assessed the effect of combinations of rolipram (PDE4 inhibitor), ORG 9935 (PDE3 inhibitor) and salbutamol (β‐agonist) on CAM expression and neutrophil or eosinophil adhesion to human lung microvascular endothelial cells (HLMVEC). Tumour necrosis factor‐α (TNF‐α)‐induced intercellular adhesion molecule (ICAM)‐1, vascular cell adhesion molecule (VCAM)‐1 and E‐selectin expression were measured on HLMVEC monolayers at 6 h by a specific ELISA technique in the presence of different combinations of medium, rolipram, ORG 9935 and salbutamol. Rolipram in combination with salbutamol, but neither agent alone, inhibited TNF‐α‐induced E‐selectin expression, whilst ICAM‐1 and VCAM‐1 expression were not affected. ORG 9935 had no significant effect on CAM expression alone. However, in combination with rolipram a syngergistic inhibition of VCAM‐1 and E‐selectin, but not ICAM‐1, expression was observed. No further inhibition was seen in the additional presence of salbutamol. Neutrophil adhesion to TNF‐α‐stimulated (6 h) HLMVEC was mainly E‐selectin dependent in this model, as ENA2 an anti‐E‐selectin monoclonal antibody (mAb) abrogated neutrophil adhesion. Eosinophil adhesion was E‐selectin‐, ICAM‐1‐ and VCAM‐1‐dependent, as assessed by the inhibitory activity of ENA2 and the ability of a mAb to the ICAM‐1 ligand, CD18, and a mAb to the VCAM‐1 ligand, VLA4, to attenuate adhesion. Rolipram in the presence of salbutamol or ORG 9935 significantly inhibited neutrophil adherence to TNF‐α‐stimulated HLMVEC. Eosinophil adherence to monolayers was inhibited only when HLMVEC were activated in the presence of rolipram and ORG 9935. Collectively, the findings presented in this manuscript suggest that inhibition of PDE4 with appropriate activation of adenylate cyclase is sufficient to inhibit induction of E‐selectin expression on HLMVEC to a level that has functional consequences for neutrophil adhesion. In contrast, combined inhibition of PDE3 and 4 isoenzymes is necessary to inhibit VCAM‐1 and to have inhibitory effects on eosinophil adhesion to activated HLMVEC. Upregulation of ICAM‐1 expression on HLMVEC does not appear to be modulated by PDE3 and 4 inhibition. These data may have implications for the use of selective PDE4 inhibitors in lung inflammation.

Tumour necrosis factor‐α‐induced ICAM‐1 expression in human vascular endothelial and lung epithelial cells: modulation by tyrosine kinase inhibitors

1 Tumour necrosis factor‐α (TNFα) increases the expression of the adhesion molecule intercellular adhesion molecule‐1 (ICAM‐1) on cultured endothelial and epithelial cells and modulation of this may be important in controlling inflammation. Activation of tyrosine kinase(s) is known to be involved in the signal transduction pathways of many cytokines. In this study we have investigated the effects of the tyrosine kinase inhibitors, ST638, tyrphostin AG 1288 and genistein, on TNFα‐induced ICAM‐1 expression in human alveolar epithelial (A549) and vascular endothelial (EAhy926) cell lines and also normal human lung microvascular endothelial cells (HLMVEC). 2 ICAM‐1 expression on cultured cells was determined by a sensitive enzyme‐linked immunosorbant assay (ELISA). Endothelial or epithelial monolayers were exposed to increasing doses of TNF‐α (0.01–10 ng ml−1), in the presence or absence of either ST638 (3–100 μm), AG 1288 (3–100 μm) or genistein (100 μm) and ICAM‐1 expression was measured at 4 and 24 h. Control experiments examined the effect of ST638 on phorbol 12‐myristate 13‐acetate (PMA, 20 ng ml−1, 4 h)‐stimulated ICAM‐1 and compared it to that of a specific protein kinase C inhibitor, Ro31‐8220 (10 μm). Also, functional consequences of changes in ICAM‐1 expression were assessed by measuring adhesion of 111In‐labelled human neutrophils to EAhy926 endothelial and A549 epithelial monolayers treated with TNFα, in the presence or absence of ST638. 3 ST638 caused a concentration‐dependent reduction in TNFα‐ (0.1–10 ng ml−1‐induced ICAM‐1 on EAhy926 endothelial (at 4 h) and A549 epithelial monolayers (at 4 and 24 h). In contrast, ST638 caused a concentration‐dependent increase in TNFα‐ (0.1–10 ng ml−1‐induced ICAM‐1 on EAhy926 endothelial cells at 24 h. Similar effects were seen with AG 1288 or genistein. ST638 (100 μm) significantly (P < 0.01) inhibited ICAM‐1 expression on HLMVEC endothelial cells induced by 0.01 ng ml−1 TNFα at 4 or 24 h or 0.1 ng ml−1 at 4 h, but increased ICAM‐1 expression induced by 0.1 ng ml−1 TNFα at 24 h. ST638 did not significantly change the expression of PMA‐stimulated ICAM‐1 on either A549 epithelial, EAhy926 or HLMVEC endothelial cells. However, PMA‐induced ICAM‐1 expression was inhibited by Ro31‐8220. Also, treatment of epithelial or endothelial monolayers with TNFα and ST638 altered adhesion of human neutrophils to A549 epithelial or EAhy926 endothelial cells in a manner that corresponded to the alteration in ICAM‐1 expression. 4 These results show that tyrosine kinase inhibitors alter TNFα‐induced ICAM‐1 expression, but that the cell type, concentration of TNFα and time of exposure to this cytokine determine whether expression is decreased or increased by the inhibitor. An increased understanding of the signal transduction pathway(s) involved in TNFα‐induced ICAM‐1 expression on lung epithelial and vascular endothelial cells may be of potential therapeutic value in the treatment of inflammatory disease.

The RAGE axis in systemic inflammation, acute lung injury and myocardial dysfunction: an important therapeutic target?

BackgroundThe sepsis syndromes, frequently complicated by pulmonary and cardiac dysfunction, remain a major cause of death amongst the critically ill. Targeted therapies aimed at ameliorating the systemic inflammation that characterises the sepsis syndromes have largely yielded disappointing results in clinical trials. Whilst there are many potential reasons for lack of success of clinical trials, one possibility is that the pathways targeted, to date, are only modifiable very early in the course of the illness. More recent approaches have therefore attempted to identify pathways that could offer a wider therapeutic window, such as the receptor for advanced glycation end-products (RAGE) and its ligands.PurposeThe objectives of this study were to review the evidence supporting the role of the RAGE axis in systemic inflammation and associated acute lung injury and myocardial dysfunction, to explore some of the problems and conflicts that these RAGE studies have raised and to consider strategies by which they might be resolved.MethodsMEDLINE was searched (1990–2010) and relevant literature collected and reviewed.Results and conclusionRAGE is an inflammation-perpetuating receptor with a diverse range of ligands. Evidence supporting a role of the RAGE axis in the pathogenesis of systemic inflammation, ALI and myocardial dysfunction is compelling with numerous animal experiments showing the beneficial effects of inhibiting the RAGE axis. Despite a number of unanswered questions that need to be further addressed, the potential for inhibiting RAGE-mediated inflammation in humans undoubtedly exists.

Regulation of chemokine expression in atherosclerosis

(1) Chemokines play a central role in the pathogenesis of atherosclerosis, contributing to leukocyte recruitment, angiogenesis and also proliferation and migration of smooth muscle cells into atherosclerotic plaques. (2) Leukocytes and endothelial cells are an important source of chemokines, and many of the risk factors associated with atherosclerosis increase chemokine expression. There is now a body of evidence to suggest that interactions between cells such as leukocytes and endothelial cells amplify chemokine release, and this may contribute to sustained chemokine generation in inflammatory conditions. (3) This article summarises, briefly, what is currently known about chemokines release. A number of important pharmacological strategies used in the treatment of atherosclerosis inhibit chemokine release and the extent to which this may contribute to their therapeutic effect will be discussed. Understanding the mechanisms controlling chemokine expression is essential for the design of specific therapeutic interventions in atherosclerosis.

Sepsis since the discovery of Toll-like receptors: disease concepts and therapeutic opportunities.

Objective:Sepsis and its sequelae are the leading cause of death in critically ill patients. Discovery in the late 1990s of Toll-like receptors as primary sensors of microbial infection led to significant advances in understanding the pathogenesis of sepsis, including emerging differences between Gram-positive and Gram-negative infection and the potential for the manipulation of Toll-like receptors for the treatment of sepsis. This review describes these advances. Methods:Bibliographic search of the literature since 1999, with particular emphasis on the conceptual and therapeutic implications of Toll-like receptors for patients with systemic sepsis. Results and Conclusions:Toll-like receptors initiate the inflammatory processes that underlie the clinical response to infection and therefore represent an important putative target for therapeutic intervention.

Severe sepsis and Toll-like receptors

Severe sepsis dominates the mortality of non-cardiac intensive care units. The ingenious Toll-like receptor (TLR) system can recognise many infectious organisms through relatively few receptors to trigger pro-inflammatory and anti-inflammatory cytokine release. Further complexity arises from positive and negative signalling feedback loops. Severe sepsis may be a consequence of an inappropriately excessive response or inadequate endogenous negative feedback. Therapies targeting these pathways are currently being evaluated. Alternatively, in clinical scenarios such as compensatory anti-inflammatory response syndrome, chronic viral sepsis or inadequate vaccine function, TLR signalling may be inadequate. TLR agonists may augment the innate response and are being investigated.

Lest we forget the endothelial glycocalyx in sepsis

Sepsis is the third largest cause of death in industrialised countries, but treatment remains largely supportive and effective therapeutic interventions are urgently needed. Disruption and dysfunction of the microvascular endothelium leading directly or indirectly to multiple organ failure are now recognised to underpin the pathophysiology of sepsis. Biomarkers of endothelial activation may therefore assume an important role in guiding future research efforts. We suggest that integral to this approach is the investigation and evaluation of endothelial glycocalyx biomarkers, not only as indicators of the pathogenic process but also to inform the development of pharmacological and other therapies.

Cytokine-induced airway epithelial ICAM-1 upregulation: quantification by high-resolution scanning and transmission electron microscopy

The aim of this study was to investigate the variation of intercellular adhesion molecule (ICAM)-1 which occurs between individual airway epithelial cells of distinct phenotype. 16HBE14o- (16HBE) and BEAS-2B cell lines and human airway explants were cultured with medium alone or a mixture of tumour necrosis factor (TNF)-alpha (10 ng x mL(-1)) and interferon (IFN)-gamma (40 ng x mL(-1)) before being immunogold-labelled and examined quantitatively using sensitive high-resolution electron microscopic techniques. By enzyme-linked immunosorbent assay there was a 1.6-fold increase of ICAM-1 in the BEAS-2B cells following the cytokine mix which was not apparent in the 16HBE cells. However, high-resolution scanning electron microscopy demonstrated that an upregulation had occurred; median and ranges for gold particle number per 10 microm2 cell surface were: 7.9 (0-40) for nonstimulated and 19.1 (0-60 for stimulated) (p<0.01, Mann-Whitney U-test). The value for the nonstimulated BEAS-2B cells was 24.2 (0-60), 3-times higher that the constitutive expression in the 16HBE cells (p<0.01), whereas following stimulation, it was 68.5 (20-130) (p<0.01). Values for explant epithelial outgrowths were similar to the 16HBE cells. Immunohistochemistry of the explanted mucosa showed both constitutive and upregulated expression of epithelial ICAM-1 associated with basal and indeterminate cells rather than with ciliated or goblet cells. These results using high-resolution techniques indicate that there is marked cell-to-cell variation in cellular adhesion molecule expression and that it is the basal cells and less well differentiated (indeterminate) epithelial cells which are likely to play key roles in leukocyte retention via intercellular adhesion molecule-1.

PMX464, a thiol-reactive quinol and putative thioredoxin inhibitor, inhibits NF-κB-dependent proinflammatory activation of alveolar epithelial cells

Subtle changes in the intracellular reduction–oxidation (redox) state can modulate nuclear factor‐κB (NF‐κB) activity. Thioredoxin‐1 (Trx) is a small, ubiquitous, redox‐active thiol (‐SH) protein that, with thioredoxin reductase‐1 (TrxR), modifies the redox status of NF‐κB pathway components. PMX464 is a novel thiol‐reactive quinol thought to inhibit the Trx/TrxR system. The aim of this work was to investigate whether PMX464 inhibited NF‐κB‐mediated proinflammatory activation of human type II alveolar epithelial cells (A549).

TNF-α Potentiates C5a-Stimulated Eosinophil Adhesion to Human Bronchial Epithelial Cells: A Role for α5β1 Integrin

Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-α and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-α nor IL-5, a known eosinophil-priming agent. Priming caused activation of αMβ2 integrin; mAb against either the common β2 integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against β1 or α5, but not α4 integrin subunit, together with anti-β2 integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking α5β1 integrin increased αMβ2 integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of α5β1 integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for α5β1 integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-β2 integrin mAb, reduced adhesion to basal levels. In conclusion, α5β1 integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-α, potentiated C5a activation of αMβ2 integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for α5β1 integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.