Gregory Ronald Tintinger

Montelukast: More than a Cysteinyl Leukotriene Receptor Antagonist?

The prototype cysteinyl leukotriene receptor antagonist, montelukast, is generally considered to have a niche application in the therapy of exercise- and aspirin-induced asthma. It is also used as add-on therapy in patients whose asthma is poorly controlled with inhaled corticosteroid monotherapy, or with the combination of a long-acting β(2)-agonist and an inhaled corticosteroid. Recently, however, montelukast has been reported to possess secondary anti-inflammatory properties, apparently unrelated to conventional antagonism of cysteinyl leukotriene receptors. These novel activities enable montelukast to target eosinophils, monocytes, and, in particular, the corticosteroid-insensitive neutrophil, suggesting that this agent may have a broader spectrum of anti-inflammatory activities than originally thought. If so, montelukast is potentially useful in the chemotherapy of intermittent asthma, chronic obstructive pulmonary disease, cystic fibrosis, and viral bronchiolitis, which, to a large extent, involve airway epithelial cell/neutrophil interactions. The primary objective of this mini-review is to present evidence for the cysteinyl leukotrien–independent mechanisms of action of montelukast and their potential clinical relevance.

Cysteinyl leukotriene receptor-1 antagonists as modulators of innate immune cell function

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

Harmful interactions of non-essential heavy metals with cells of the innate immune system

In trace amounts, some heavy metals are essential for optimum health, while exposure to others, which are non-essential, presents the potential hazard of acute or chronic organ toxicity. Cadmium, mercury, lead, vanadium, platinum and palladium are commonly encountered, non-essential heavy metals which mediate their toxic activities by various mechanisms. All have the potential to interact with extracellular and intracellular protein sulfhydryls, rendering them not only potentially allergenic, but also predisposing to oxidative stress, while displacement of essential elements from their protein carriers may result in deficiency disorders. In addition, several of these metals, especially cadmium, palladium, platinum, and vanadium interact pro-oxidatively with the phagocytic cells of the innate immune system, potentiating the reactivity and toxicity of phagocyte-derived reactive oxygen species. This review is focused on the pro-oxidative/pro-inflammatory interactions of non-essential heavy metals with the cells of the innate immune system, a somewhat under-appreciated mechanism of metal induced toxicity.

Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4

The primary objective of this study was to probe the involvement of leukotriene B4 (LTB4) in itraconazole (0·1–5 µM)‐mediated inhibition of Ca2+ uptake by chemoattractant‐activated human neutrophils. Following exposure of the cells to platelet‐activating factor (PAF, 200 nM), LTB4 was measured by immunoassay, while neutrophil cytosolic Ca2+ concentrations were determined by a fura‐2/AM‐based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca2+ which was associated with increased generation of LTB4, both of which were attenuated significantly by itraconazole at 0·5 µM and higher. The inhibitory effect of the anti‐mycotic on Ca2+ uptake by PAF‐activated cells was mimicked by an LTB4 antibody, as well as by LY255283 (1 µM) and MK886 (0·5 µM), an antagonist of LTB4 receptors and an inhibitor of 5′‐lipoxygenase‐activating protein, respectively, while addition of itraconazole to purified 5′‐lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole‐mediated inhibition of LTB4 production and Ca2+ influx was also supported by the observation that pulsed addition of purified LTB4 to PAF‐activated neutrophils caused substantial restoration of Ca2+ uptake by cells treated with the anti‐mycotic. Taken together, these observations suggest that the potentially beneficial anti‐inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB4, with consequent attenuation of a secondary LTB4‐mediated wave of Ca2+ uptake by the cells.

Endogenous adenosine regulates neutrophil pro-inflammatory activities by cyclic AMP-dependent accelerated clearance of cytosolic calcium.

Abstract. Objective and design: To identify the involvement of adenosine in restoration of Ca2+ homeostasis to activated human neutrophils.¶Materials: Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers.¶Treatment: The cells were exposed to adenosine deaminase (ADA, 0.1–2 units/ml) for 10 min at 37°C prior to activation with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 μM).¶Methods: Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation in FMLP-activated neutrophils ± ADA were measured using spectrofluorimetric and radiometric procedures respectively, while intracellular cAMP and inositol triphosphate were measured by radioassay, and superoxide production and elastase release by, chemiluminescence and colourimetric methods respectively. Levels of statistical significance were calculated using the Mann-Whitney U-test and ANOVA.¶Results: Although FMLP-activated generation of inositol triphosphate and mobilisation of Ca2+ from neutrophil internal stores, as well as the magnitude of the subsequent efflux and store-operated influx of the cation were unaffected by ADA, there was a prolonged elevation in cytosolic Ca2+ in the presence of the enzyme, which was associated with failure to activate adenylate cyclase and with increased production of superoxide and release of elastase. These effects of ADA were attenuated by dibutyryl cAMP (4 mM), CGS 21680 (1 μM) and rolipram (0.5 μM), as well as by EGTA (10 mM).¶Conclusions: These results are compatible with a physiological role for adenosine in promoting deactivation of neutrophils, possibly by promoting cAMP-dependent clearance of Ca2+ from the cytosol of the cells by the endo-membrane Ca2+-ATPase.

Can the anti-inflammatory activities of β2-agonists be harnessed in the clinical setting?

Beta2-adrenoreceptor agonists (β2-agonists) are primarily bronchodilators, targeting airway smooth muscle and providing critical symptomatic relief in conditions such as bronchial asthma and chronic obstructive pulmonary disease. These agents also possess broad-spectrum, secondary, anti-inflammatory properties. These are mediated largely, though not exclusively, via interactions with adenylyl cyclase-coupled β2-adrenoreceptors on a range of immune and inflammatory cells involved in the immunopathogenesis of acute and chronic inflammatory disorders of the airways. The clinical relevance of the anti-inflammatory actions of β2-agonists, although often effective in the experimental setting, remains contentious. The primary objectives of the current review are: firstly, to assess the mechanisms, both molecular and cell-associated, that may limit the anti-inflammatory efficacy of β2-agonists; secondly, to evaluate pharmacological strategies, several of which are recent and innovative, that may overcome these limitations. These are preceded by a consideration of the various types of β2-agonists, their clinical applications, and spectrum of anti-inflammatory activities, particularly those involving adenosine 3′,5′-cyclic adenosine monophosphate-activated protein kinase-mediated clearance of cytosolic calcium, and altered gene expression in immune and inflammatory cells.

Pharmacological approaches to regulate neutrophil activity

Although indispensable in host defense against microbial pathogens, misdirected hyperacute and chronic activation of neutrophils presents the potential hazard of tissue damage, organ dysfunction, and carcinogenesis. In many clinical settings, particularly inflammatory disorders of the airways, over-reactivity of neutrophils is exacerbated by their relative resistance to conventional, pharmacological anti-inflammatory therapies, including, but not limited to, corticosteroids. Notwithstanding their sheer numbers, which can increase rapidly and dramatically during inflammatory responses, these cells are not only pre-programmed to release reactive oxygen species, proteinases, and eicosanoids/prostanoids immediately on exposure to pro-inflammatory stimuli but may also subsequently undergo the process of netosis, thereby enhancing and protracting their inflammatory potential. All of these mechanisms are likely to underpin the resistance of neutrophils to pharmacological control and have triggered the search for alternatives to corticosteroids. In addition to macrolides and adenosine 3′,5′-cyclic adenosine monophospate-elevating agents, more recent innovations in the control of neutrophilic inflammation include activators of histone deacetylases and antagonists of chemokine receptors, as well as monoclonal antibodies which target neutrophil-activating cytokines and their receptors. These and other neutrophil-targeted strategies represent the focus of the current review.

Comparison of the Effects of Selective and Non-selective Beta-adrenoreceptor Agonists on the Pro-inflammatory Activities of Human Neutrophils in Vitro

The objective of this study was to measure and compare the effects of 4 selective (fenoterol, formoterol, salbutamol, salmeterol) and 3 non-selective (epinephrine, norepinephrine, isoproterenol), β-adrenoreceptor agonists, at a fixed, final concentration of 1 μM, on intracellular cyclic AMP levels in human neutrophils in vitro and to relate alterations in these to the effects of the test agents on the production of superoxide and release of elastase following activation of the cells with the chemoattractant, FMLP. Intracellular cAMP was measured by radioimmunoassay, while superoxide and elastase were assayed using lucigenin-enhanced chemiluminescence and colorimetric procedures respectively. Of the 7 agents tested, fenoterol, formoterol, epinephrine and isoproterenol caused a substantial increase in neutrophil cAMP levels, which correlated well with the inhibitory effects of these agents on superoxide production and elastase release. The other agents were either inactive (salmeterol), or weakly active (norepinephrine, salbutamol). Pretreatment of neutrophils with the non-selective β-adrenoreceptor blockading agent, propranolol (2 μM), attenuated the cAMP-mediated, anti-inflammatory interactions of formoterol, epinephrine and isoproterenol with neutrophils, while atenolol, a selective β1-blockading agent, as well as α1- and α2-adrenoreceptor antagonists were ineffective. These findings demonstrate that some, but not all, currently used β-agonists suppress the proinflammatory activity of human neutrophils through interaction with β2-adrenoreceptors on these cells and activation of adenylyl cyclase. If operative in vivo, these anti-inflammatory properties may contribute, albeit in a secondary manner, to the therapeutic activity of fenoterol, formoterol, epinephrine and isoproterenol.

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 &mgr;M) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca2+ fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) (1 &mgr;M) in combination with cytochalasin B (CB; 3 &mgr;M). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca2+ fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca2+, as well as release of elastase and production of superoxide and LTB4, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

Beneficial and harmful interactions of antibiotics with microbial pathogens and the host innate immune system

In general antibiotics interact cooperatively with host defences, weakening and decreasing the virulence of microbial pathogens, thereby increasing vulnerability to phagocytosis and eradication by the intrinsic antimicrobial systems of the host. Antibiotics, however, also interact with host defences by several other mechanisms, some harmful, others beneficial. Harmful activities include exacerbation of potentially damaging inflammatory responses, a property of cell-wall targeted agents, which promotes the release of pro-inflammatory microbial cytotoxins and cell-wall components. On the other hand, inhibitors of bacterial protein synthesis, especially macrolides, possess beneficial anti-inflammatory/cytoprotective activities, which result from interference with the production of microbial virulence factors/cytotoxins. In addition to these pathogen-directed, anti-inflammatory activities, some classes of antimicrobial agent possess secondary anti-inflammatory properties, unrelated to their conventional antimicrobial activities, which target cells of the innate immune system, particularly neutrophils. This is a relatively uncommon, potentially beneficial property of antibiotics, which has been described for macrolides, imidazole anti-mycotics, fluoroquinolones, and tetracyclines. Although of largely unproven significance in the clinical setting, increasing awareness of the pro-inflammatory and anti-inflammatory properties of antibiotics may contribute to a more discerning and effective use of these agents.