Doing what I like.

I have spent my entire professional life at Harvard Medical School, beginning as a medical student. I have enjoyed each day of a diverse career in four medical subspecialties while following the same triad of preclinical areas of investigation-cysteinyl leukotrienes, mast cells, and complement-with occasional translational opportunities. I did not envision a career with a predominant preclinical component. Such a path simply evolved because I chose instinctively at multiple junctures to follow what proved to be propitious opportunities. My commentary notes some of the highlights for each area of interest and the mentors, collaborators, and trainees whose counsel has been immensely important at particular intervals or over an extended period.

The role of leukotrienes in allergic rhinitis.

OBJECTIVE: To review the role of cysteinyl leukotrienes (cysLTs) in allergic rhinitis and the scientific rationale for therapy with leukotriene receptor antagonists (LTRAs). DATA SOURCES: Relevant basic science and clinical articles were identified by a search of the PubMed database for articles published from 1984 to 2004 using the following keywords: allergic rhinitis; nose; immune response; allergen challenge; leukotrienes C, D, and E; cysteinyl leukotriene; cysteinyl leukotriene receptor; cytokine; leukocyte; montelukast; zafirlukast; and pranlukast. STUDY SELECTION: The authors' expert opinion was used to select studies for inclusion in this review. RESULTS: CysLTs are synthesized via 5-lipoxygenase metabolism of arachidonic acid by mast cells and basophils during the early-phase response to antigen and by eosinophils and macrophages during the late phase. The cysLT levels in nasal secretions are elevated after short-term allergen instillation and in allergy season in patients with allergic rhinitis. These lipid mediators act locally and systemically by interacting with receptors, particularly the cysLT1 receptor, on target cells. Evidence derived from topical application of cysLTs in the nose and from the effects of LTRAs indicates that cysLTs contribute to nasal mucous secretion, congestion, and inflammation. CysLTs promote allergic inflammation by enhancing immune responses and the production, adhesion, migration, and survival of inflammatory cells such as eosinophils. They also increase the generation of an array of other proinflammatory mediators, such as cytokines, which in turn increase the production of and receptors for cysLTs. Clinical trials have demonstrated that LTRAs have significant but modest efficacy as single agents but additive efficacy when used with other classes of agents. CONCLUSIONS: CysLTs fulfill the criteria for relevant mediators of allergic rhinitis via their diverse effects on immune, inflammatory, and local structural components of disease. By blocking the cysLT1 receptor responsible for most of these effects, LTRAs represent a useful approach to treatment of this important and prevalent disorder.

Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses.

The cysteinyl leukotrienes (cys-LTs) are a family of potent bioactive lipids that act through two structurally divergent G protein-coupled receptors, termed the CysLT(1) and CysLT(2) receptors. The cloning and characterization of these two receptors has not only reconciled findings of previous pharmacologic profiling studies of contractile tissues, but also has uncovered their expression on a wide array of circulating and tissue-dwelling leukocytes. With the development of receptor-selective reagents, as well as mice lacking critical biosynthetic enzymes, transporter proteins, and the CysLT(1) receptor, diverse functions of cys-LTs and their receptors in immune and inflammatory responses have been identified. We review cys-LT biosynthesis; the molecular biology and distribution of the CysLT(1) and CysLT(2) receptors; the functions of cys-LTs and their receptors in the recruitment and activation of effector leukocytes and induction of adaptive immunity; and the development of fibrosis and airway remodeling in animal models of lung injury and allergic inflammation.

Roles of cysteinyl leukotrienes in airway inflammation, smooth muscle function, and remodeling.

A new paradigm for asthma pathogenesis is presented in which exaggerated inflammation and remodeling in the airways are a consequence of abnormal injury and repair responses arising from a subject's susceptibility to components of the inhaled environment. An epithelial-mesenchymal trophic unit becomes activated to drive pathologic remodeling and smooth muscle proliferation through complex cytokine interactions. Histamine, prostanoids, and cysteinyl leukotrienes (CysLTs) are potent contractile agonists of airway smooth muscle (ASM). The CysLTs appear to play a central role in regulating human ASM motor tone and phenotypic alterations, manifested as hypertrophy and hyperplasia in chronic severe asthma. The CysLTs augment growth factor-induced ASM mitogenesis through activation of CysLT receptors. Although they mediate their contractile effects by increasing phosphoinositide turnover and inducing increased cytosolic calcium, new data suggest that part of the contractile effect may be independent of calcium mobilization. Prostaglandin E(2), the predominant eicosanoid product of the airway epithelium, is a potent inhibitor of mitogenesis, collagen synthesis, and mesenchymal cell chemotaxis and therefore can suppress inflammation and fibroblast activation. The capacity of the epithelium for CysLT synthesis is inversely related to its ability to make PGE(2). The ASM is capable of expressing both leukotriene-synthesizing enzymes and CysLT receptors, and cytokines upregulate the receptor expression. This may be an explanation for the CysLTs promoting airway hyperresponsiveness in asthma. The CysLTs play an important role in the airway remodeling seen in persistent asthma that includes increases of airway goblet cells, mucus, blood vessels, smooth muscle, myofibroblasts, and airway fibrosis. Evidence from a mouse model of asthma demonstrated that CysLT(1) receptor antagonists inhibit the airway remodeling processes, including eosinophil trafficking to the lungs, eosinophil degranulation, T(H)2 cytokine release, mucus gland hyperplasia, mucus hypersecretion, smooth muscle cell hyperplasia, collagen deposition, and lung fibrosis.

Cysteinyl leukotriene interactions with other mediators and with glucocorticosteroids during airway inflammation.

Unexpected aspects of the antiasthmatic efficacy of leukotriene modifiers and glucocorticosteroids have been observed. For both classes, the observed effects may be partially explainable on the basis of underrecognized interactions involving leukotrienes. This review examines the interactions between leukotrienes and other mediators of asthma. It details the effects of glucocorticosteroids on leukotriene synthesis and on leukocyte populations in asthmatic airways. Unexpected controller effects of the leukotriene modifiers may reflect the fact that leukotrienes and other mediators of asthma, such as T(H)2 cytokines, positively influence each other's generation. The ability of the leukotriene modifiers to disrupt such extensive interactions means that other relevant mediators are targeted indirectly by leukotriene blockade. Among asthma therapies, the glucocorticosteroids have numerous anti-inflammatory activities, but their effects may be unpredictable. Many processes involved in inflammation appear to escape modulation by glucocorticosteroids, including leukotriene synthesis, and leukotriene generation is among them. Understanding whether glucocorticosteroids reduce cysteinyl leukotriene levels in the airway is important in determining the clinical value of combining glucocorticosteroid therapy with leukotriene modifier therapy.

Effects of cysteinyl leukotrienes and leukotriene receptor antagonists on markers of inflammation.

The understanding that asthma pathophysiology includes an inflammatory component has spurred the more aggressive use of anti-inflammatory therapies and created a need for effective tools to measure inflammation. Biomarkers of airway inflammation proposed are obtained by methods that are direct but highly invasive (bronchial biopsy, bronchoalveolar lavage), moderately direct, and less invasive (indirect sputum, exhaled air, breath condensate) or indirect and least invasive (blood, urine). Several studies described in this review have implicated the cysteinyl leukotrienes (CysLTs) as inflammatory mediators in a wide range of diseases, implying that their biological activities reach far beyond acute bronchoconstriction, the activity traditionally ascribed to them. The validity of examining sputum for "biomarkers" has improved the understanding of asthma pathophysiology, optimization of asthma treatment and management, and investigation of the relation between CysLTs and airway inflammation in asthma. Nitric oxide is also a surrogate marker of asthma and reflects airway inflammation. The anti-inflammatory effects of the leukotriene receptor antagonists and the markers of their activity continue to grow.

Cysteinyl leukotrienes modulate angiotensin II constrictor effects on aortas from streptozotocin-induced diabetic rats.

Angiotensin II (Ang II) is a vasopressor peptide involved in the pathogenesis of cardiovascular diseases associated with diabetes mellitus. We have previously reported that the 5-lipoxygenase-derived products, particularly the cysteinyl leukotrienes (CysLTs), are involved in Ang II-induced contraction. In this study, we demonstrated that CysLTs contribute to the contraction elicited by Ang II in isolated aortas from streptozotocin-induced diabetic (SS) rats but not from insulin-treated diabetic rats, fructose-fed rats, or control rats. In an organ bath, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/L) reduced by 37.6+/-8.2% and 30.1+/-10.9% the Ang II-induced contractions in intact and endothelium-denuded aortic rings, respectively, from SS rats. In contrast, the CysLT(1) receptor antagonist (MK571, 1 micromol/L) or the dual CysLT(1)/CysLT(2) receptor antagonist (BAY-u9773, 0.1 micromol/L) did not affect Ang II-induced contraction. In addition, Ang II induced a 6.2+/-1.5-fold increase in CysLT release through the stimulation of the Ang II type 1 receptor. Furthermore, the urinary excretion of leukotriene E(4) was increased in SS rats (leukotriene E(4), 13.7+/-2.9 ng/24 h [SS rats, n=10] versus 1.5+/-0.5 ng/24 h [control rats, n=6]; P<0.0004). These data suggest the activation of the 5-lipoxygenase pathway in SS rats and the involvement of 5-lipoxygenase-derived products, particularly the CysLTs, in Ang II-induced contraction in aortas from SS rats through stimulation of CysLT receptors different from the well-characterized CysLT(1) or CysLT(2) receptor.

Cysteinyl leukotrienes are involved in angiotensin II-induced contraction of aorta from spontaneously hypertensive rats.

OBJECTIVE: Non specific lipoxygenase inhibitors have been reported to reduce the in vitro constrictor response and the in vivo pressor effect of angiotensin II in rats. The aim of this study was to assess the role of cysteinyl leukotrienes, in the vascular response to angiotensin II in spontaneously hypertensive rats (SHR). METHODS: Rings of thoracic aorta from SHR and normotensive Wistar-Kyoto rats (WKY) were compared in terms of contractile responses and release of cysteinyl leukotrienes in response to angiotensin II. RESULTS: Pretreatment with the specific 5-lipoxygenase inhibitor AA861 10 microM reduced the efficacy of angiotensin II in intact and endothelium-denuded aorta from SHR (% inhibition vs. control: 65+/-12.6% with endothelium (n=6), P<0.05; 43+/-7.2% without endothelium (n=6), P<0.05) but not in aorta from WKY. In addition, in aorta from SHR, the CysLT(1) receptor antagonist MK571 1 microM reduced by 55+/-6.1% (n=6, P<0.05) the contractile effects of angiotensin II in rings with endothelium but not in endothelium-denuded rings. Angiotensin II induced a 8.6+/-2.1-fold increase in cysteinyl leukotriene production in aorta rings from SHR with endothelium which was prevented by the AT(1) receptor antagonist losartan 1 microM but not by the AT(2) receptor antagonist PD123319 0.1 microM. In aorta rings from WKY, cysteinyl leukotriene production remained unchanged after exposition to angiotensin II. The cysteinyl leukotrienes (up to 0.1 microM) induced contractions in aorta rings from SHR but not from WKY. CONCLUSIONS: These data suggest that cysteinyl leukotrienes, acting at least in part on endothelial CysLT(1) receptors, are involved in the contractile response to angiotensin II in isolated aorta from SHR but not from WKY.

Leukotrienes in respiratory disease.

Arachidonic acid metabolism via 5-lipoxygenase gives rise to a group of biologically active lipids known as leukotrienes: leukotriene B(4), which is a potent activator of leukocyte chemotaxis, and cysteinyl leukotrienes (leukotriene C(4), D(4)and E(4)) which account for the spasmogenic activity previously described as slow-reacting substance of anaphylaxis. The biological actions of leukotrienes and the observations that leukotrienes are synthesised in the lung following antigen provocation and are elevated in asthma, stimulated considerable activity in the pharmaceutical industry to find drugs that modulate the synthesis or actions of leukotrienes. Three cysteinyl leukotriene antagonists (zafirlukast [Accolate], montelukast [Singulair] and pranlukast) and one 5-lipoxygenase inhibitor (zileuton) have received regulatory approval for the treatment of asthma. The clinical data obtained from using these drugs are generally consistent and complimentary. As a class the leukotriene modulators produce a rapid improvement in lung function after the first oral dose. Lung function improvements are maintained on chronic administration and are associated with reductions in a variety of asthma symptom scores. All of the available data are consistent with the hypothesis that all the leukotriene modulators exert their clinical benefit primarily through interference with cysteinyl leukotrienes. There are no compelling clinical data for an additional contribution by leukotriene B(4)in human asthma. In other respiratory conditions such as COPD, which are characterised by pronounced neutrophil infiltration, it may be that the chemotactic properties of leukotriene B(4)are more important and therefore evaluation of 5-lipoxygenase inhibitors in this condition is warranted. The introduction of the leukotriene modulators into clinical practice is the culmination of over 60 years of research since the initial discovery of the slow-reacting substances. The leukotriene modulators, and in particular the cysteinyl leukotriene antagonists, provide respiratory physicians with an oral therapeutic option and have set an efficacy standard which new oral anti-inflammatory approaches will have to beat.

Leukotrienes antagonist-inhibitor and the bronchial response to inhaled antigen in actively sensitized guinea-pigs.

The aim of the study was to estimate an eventual contribution of leukotrienes in respiratory mechanic changes observed in guinea-pig during bronchial anaphylaxis. Twenty-eight guinea-pigs, actively sensitized to ovalbumin, were anesthetized and curarized before being challenged, during controlled ventilation, with antigen administered as an aerosol. Antigen challenge was performed before and after pretreatment with nebulized FPL55712 and BW755C, respectively used as an antagonist of leukotrienes and as a cyclo-oxygenase/lipoxygenase inhibitor. The experiments were carried out during continuous recording of tracheal pressure (Ptr), airflow (V) and tidal volume (VT) signals variations evidencing respiratory asynchronism (AS) and allowing measurements of the changes in airway resistance (AR) and dynamic compliance (Cdyn) during all the challenge. Administration of nebulized ovalbumin was stopped at the onset of AS appearance chosen as the threshold of the antigen-induced bronchoconstriction. The results showed that separate or combined pretreatment with FPL55712 and BW755C did not significantly modify the threshold of the ovalbumin-induced bronchoconstriction in guinea-pigs. Nevertheless pretreatment with nebulized FPL55712 reduced significantly the intensity and the duration of the response of these animals to inhaled leukotriene D4 (LTD4). Moreover the response of guinea-pigs to inhaled LTD4, characterized by a starting decrease in Cdyn, appeared quite different from the response to the antigen starting by an abrupt rise in RA induced by a sudden bronchoconstriction. From these results, we concluded that leukotrienes seem not to be the main mediator of the bronchial anaphylaxis in guinea-pigs.

[Peptidoleukotriene receptor antagonists in asthma therapy].

Peptidoleukotrienes reproduce some of the functional and histological features of clinical asthma, such as potent bronchoconstriction, airway microvascular leakage, mucus hypersecretion, eosinophilic airway infiltration, in addition to inducing bronchial hypersensitivity. An important role for peptidoleukotrienes has been demonstrated in experimental challenge studies in asthmatic patients and in acute asthma attacks. Antagonists of peptidoleukotrienes can inhibit bronchoconstriction in response to inhaled allergen, exercise or inhaled cold air, and oral or inhaled aspirin. These compounds demonstrate beneficial effects in clinical studies with improvement in symptoms and lung function. Peptidoleukotriene antagonists may provide a new therapeutic approach for the treatment of asthma.

The Paul Kallós Memorial Lecture. From slow-reacting substance of anaphylaxis to leukotriene C4 synthase.

The hypothesis of 35 years ago that slowreacting substance of anaphylaxis would prove to be a pathobiologic mediator of reversible airway disease in the human has been validated. The multidisciplinary approach required to achieve this goal has been particularly prominent and consistent for the entire period in the program described in this presentation.

Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in the rat.

The purpose of the study was to investigate whether allergen-induced hyperresponsiveness to methacholine and an increase in airway smooth muscle (ASM) in Brown Norway (BN) rats could be mediated by LTD4, an important mediator of allergic airway responses. Male BN rats, 8 to 12 wk of age, were sensitized with ovalbumin (OA). Rats were exposed 2 wk later to aerosols of saline (n = 6), OA (n = 8), or OA after pretreatment with the LTD4 antagonist MK-571 (2 mg/kg intraperitoneally, n = 9), on six occasions at 5-day intervals. Airway responsiveness to methacholine (the concentration required to double pulmonary resistance, EC200 RL) was measured immediately before the first aerosol exposure and 2 days after the last exposure. ASM was quantitated by morphometry, and areas were standardized for size using the epithelial basement membrane length (BM). Following OA challenges EC200 RL decreased from 6.5 to 3.1 mg/ml (p < 0.05) but did not change significantly after saline or OA exposures in MK-571-pretreated animals. ASM/BM2 in the large airways was significantly greater, 3.41 +/- 0.19 x 10(-3), after OA compared with 2.35 +/- 0.22 x 10(-3) for saline exposures (p < 0.01). The ASM/BM2 after OA exposures but with MK-571 pretreatment (2.75 +/- 0.25 x 10(-3)) was intermediate in value. The results indicate that both the increase in airway responsiveness and the increase in ASM following repeated antigen exposures appear to be mediated predominantly by LTD4.

Selective inhibition by H7 and staurosporin of phorbol ester responses in U-937 cells.

The kinase inhibitors H7 and staurosporin dose-dependently stimulate adhesion U-937 cells to plastic but fail to inhibit the CD11b/CD18-dependent adhesion of U-937 cells induced by phorbol ester. The protein kinase C activity of U-937 cells, measured as phorbol ester-stimulated phosphorylation of pep epsilon in streptolysin-O permeabilized cells, is strongly inhibited by the kinase inhibitors. H7 and staurosporin efficiently overcome the inhibitory effect of phorbol-12,13-dibutyrate (PDBu) on leukotriene D4-induced increase in intracellular Ca2+. The results suggest that U-937 cell adhesion may be controlled by a protein kinase C isoform not sensitive to the inhibitors. In addition, the data indicate that selective pharmacological interference with different protein kinase C-mediated processes is achievable.

Leukotriene D4 is a mediator of proteinuria and glomerular hemodynamic abnormalities in passive Heymann nephritis.

We assessed the role of leukotrienes (LTs) in Munich-Wistar rats with passive Heymann nephritis (PHN), an animal model of human membranous nephropathy. 10 d after injection of anti-Fx1A antibody, urinary protein excretion rate (Upr) in PHN was significantly higher than that of control. Micropuncture studies demonstrated reduced single nephron plasma flow and glomerular filtration rates, increased transcapillary hydraulic pressure difference, pre- and postglomerular resistances, and decreased ultrafiltration coefficient in PHN rats. Glomerular LTB4 generation from PHN rats was increased. Administration of the 5-LO activating protein inhibitor MK886 for 10 d markedly blunted proteinuria and normalized glomerular hemodynamic abnormalities in PHN rats. An LTD4 receptor antagonist SK&F 104353 led to an immediate reduction in Upr and to reversal of glomerular hemodynamic impairment. Ia(+) cells/glomerulus were increased in PHN rats. In x-irradiated PHN rats, which developed glomerular macrophage depletion, augmented glomerular LT synthesis was abolished. Thus, in the autologous phase of PHN, LTD4 mediates glomerular hemodynamic abnormalities and a hemodynamic component of the accompanying proteinuria. The synthesis of LTD4 likely occurs directly from macrophages or from macrophage-derived LTA4, through LTC4 synthase in glomerular cells.

Effect of leukotrienes of renal water excretion.

To investigate the renal actions of leukotrienes (LT), we infused arachidonic acid into the renal artery of anesthetized dogs during systemic cyclooxygenase inhibition (with ibuprofen) alone or in combination with lipoxygenase inhibition or LTD4/LTE4 receptor antagonism. Renal arachidonic acid infusion following ibuprofen alone decreased urine osmolality (945 +/- 143 to 698 +/- 144 mosm/kg; p < 0.01) and increased urine flow rate (0.34 +/- 0.11 to 0.56 +/- 0.16; p < 0.05) without altering renal blood flow, glomerular filtration rate or sodium excretion. In separate groups, prior inhibition of lipoxygenase (propylgallate) or blockade of LTD4/LTE4 receptors (LY171883) prevented the changes in urine osmolality and urine flow rate. Intrarenal oleic acid infusion following ibuprofen had no effect on renal function. Analysis of the renal papillae at the end of the experiment indicated that interstitial osmolality and sodium, potassium and urea contents were the same in all groups, ruling out a decrease in papillary interstitial osmolality as the cause of the decrease in urine osmolality in the arachidonic acid-infused group. Our experiments suggest that renal LT can decrease urine osmolality and increase urine flow rate and may play a role in renal water excretion.