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Concentration-Dependent Noncysteinyl Type 1 -Mediated Inhibitory Activity of Antagonists This information is current as of September 25, 2021. Grzegorz Woszczek, Li-Yuan Chen, Sara Alsaaty, Sahrudaya Nagineni and James H. Shelhamer J Immunol published online 18 January 2010 http://www.jimmunol.org/content/early/2010/01/18/jimmun ol.0900071 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 18, 2010, doi:10.4049/jimmunol.0900071 The Journal of Immunology

Concentration-Dependent Noncysteinyl Leukotriene Type 1 Receptor-Mediated Inhibitory Activity of Leukotriene Receptor Antagonists

Grzegorz Woszczek,*,†,1 Li-Yuan Chen,*,1 Sara Alsaaty,* Sahrudaya Nagineni,* and James H. Shelhamer*

The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 re-

ceptor (CysLT1)-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT1 signaling in human to characterize CysLT1-dependent and -independent anti-inflammatory activity of two chemically

different, clinically relevant LTRAs ( and zafirlukast). Using receptor-desensitization experiments in monocytes and Downloaded from

CysLT1-transfected HEK293 cells and IL-10– and CysLT1 small interfering RNA-induced downregulation of CysLT1 expression, we showed that reported CysLT1 agonists and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were

required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D4, induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also

inhibited calcium ionophore-induced leukotriene ( and ) production, indicating 5- inhib- http://www.jimmunol.org/ itory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non–

CysLT1-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma. The Journal of Immunology, 2010, 184: 000–000.

eukotrienes have been implicated in the pathophysiology of and the final individual compound plasma concentrations obtained bronchial asthma. Drugs inhibiting leukotriene signaling, during treatment. L such as cycteinyl leukotriene receptor antagonists (LTRAs) All LTRAs act on the cysteinyl leukotriene type 1 receptor by guest on September 25, 2021 (montelukast, , and zafirlukast), were shown to be effective (CysLT1) and by competitive antagonism at this receptor are be- in treating this disease (1, 2). However, their use has been associated lieved to be responsible for the control of airway inflamma- with a significant degree of interpatient variability in response to tion, bronchoconstriction, and remodeling (7, 8). However, anti- treatment. In clinical trials, 20–50% of patients receiving LTRAs inflammatory activity of LTRAs independent of CysLT1 antagonism could be classified as responders, showing a great heterogeneity in was recently suggested. Montelukast reduced the expression of response to these drugs, which affects the effectiveness of treatment urokinase plasminogen activator receptor and the secretion of (3, 4). Much of that variability may be attributable to genetic varia- metalloproteinase-9 in human in vitro (9), and it in- tions; several studies reported that genetic polymorphisms in genes hibited TNF-a–mediated IL-8 expression in U937 cells through encoding key in the leukotriene pathway might influence the mechanisms distinct from CysLT1 antagonism (10). Interestingly, it response to LTRAs (5, 6). Another cause of variability may be as- was shown that montelukast may have a novel inhibitory effect on sociated with the pharmacokinetic characteristics of the drugs used 5-lipoxygenase (5-LO) activity (11) and transport of by the multidrug resistance ABCC4 (12), suggesting a broader mechanism of action for this drug. Non–CysLT1-related mechanisms of LTRA actions might present another level of vari- ability in the response to treatment in patients with asthma. Some of *Critical Care Medicine Department, Clinical Center, National Institutes of Health, these non–CysLT -related activities of LTRAs may be compound Bethesda, MD 20892; and †Medical Research Council and Asthma UK Centre in 1 Allergic Mechanisms of Asthma, King’s College London, London, United Kingdom specific or may depend on drug concentration or the presence of 1G.W. and L.-Y.C. contributed equally to this work. a particular inflammatory pathway in patients with asthma; there- Received for publication January 9, 2009. Accepted for publication December 16, fore clinically significant effects of treatment may be observed in 2009. some, but not all, treated subjects. Address correspondence and reprint requests to Dr. James H. Shelhamer, Critical We previously showed that CysLT1 is the predominantly expressed Care Medicine Department, Clinical Center, National Institutes of Health, Building leukotriene receptor in human elutriated monocytes and that leuko- 10 Room 2C145, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: jshel- [email protected] triene D4 (LTD4), acting through CysLT1, can induce the activation and of these cells (13). In the current study, we used this Abbreviations used in this paper: Con, control oligonucleotides; cysLT, cysteinyl leukotriene; CysLT1, cysteinyl leukotriene type 1 receptor; CysLT2, cysteinyl leuko- model of CysLT1 signaling in human monocytes to characterize the triene type 2 receptor; GPCR, G-protein coupled receptor; 5-LO, 5-lipoxygenase; CysLT1-dependent and -independent inhibitory activity of two LTB4, leukotriene B4;LTC4, leukotriene C4;LTD4, leukotriene D4; LTRA, cysLT receptor antagonist; Mon, montelukas; MRS2578, P2Y6 inhibitor; siRNA, small in- chemically different, clinically relevant LTRAs (montelukast and terfering RNA; Zaf, zafirlukast. zafirlukast) and to define the pathways of their inhibitory action.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900071 2 LTRAs AND NON–CysLT1-MEDIATED EFFECTS

Materials and Methods collected by centrifugation at 4˚C. Cells were lysed by freezing (three Materials times), followed by lipid purification using an solid phase extraction (C-18) cartridge (Cayman Chemical), according to the manufacturer’s

LTD4, montelukast, and zafirlukast (Cayman Chemical, Ann Arbor, MI), protocol. Purified samples were resuspended in immunoassay calcium ionophore A23187 (EMD Chemicals, Gibbstown, NJ), UDP, buffer and assayed directly. The concentration of leukotriene B4 (LTB4), P2Y6 inhibitor (MRS2578), DMSO (all from Sigma-Aldrich, St. Louis, leukotriene C4 (LTC4), and PGE2 produced spontaneously and after MO), and human rIL-10 (R&D Systems, Minneapolis, MN) were obtained stimulation was assayed by means of competitive enzyme immunosorbent from the manufacturers. assay (Cayman Chemical), according to the manufacturer’s protocol.

Cell culture Statistical analysis Human elutriated monocytes from healthy donors were obtained by an Data were analyzed by one-way ANOVA or paired and unpaired Student , institutional review board-approved protocol from the National Institutes of t tests, as appropriate. Differences were considered significant when p 0.05. Health Blood Bank (Bethesda, MD), resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 2 mmol/l L-glutamine Results (all from Invitrogen, Carlsbad, CA), and allowed to rest overnight before LTRAs inhibit LTD4- and UDP-induced calcium flux experiments at 37˚C in a humidified 5% CO2 incubator. Human embryonic kidney (HEK293) cells (American Type Culture It was suggested that CysLT1 has a dual cysteinyl leukotriene Collection, Manassas, VA) were cultured in DMEM medium (Invitrogen) (cysLT)/UDP specificity (14). To investigate a role for LTRAs in supplemented with 10% FBS. CysLT1-mediated reactions, the responsiveness of human mono- Calcium mobilization assay cytes to LTD4 and UDP was evaluated by intracellular calcium

flux measurement. As shown in Fig. 1, monocytes responded in Downloaded from Calcium mobilization experiments were conducted using a FLIPR Calcium 3 assaykit(MolecularDevices,Sunnyvale,CA),according tothemanufacturer’s a concentration-dependent manner to LTD4 and UDP. However, 5 instructions. Cells (2 3 10 cells/well) were plated into poly-L-lysine–coated the concentration of UDP that was most effective at inducing 2 96-well plates and incubated in RPMI 1640 supplemented with 10 mmol/l calcium flux was 3-log greater (10 4 mol/l) than the most effec- HEPES and FLIPR 3 assay reagent. After incubation for 1 h at 37˚C, fluo- 27 26 tive concentration of LTD4 (10 –10 mol/l). CysLT1 antago- rescence was measured every 4 s using the FlexStation (Molecular Devices). nists (montelukast and zafirlukast) were used to analyze whether HEK293 cells were grown in 75-ml flasks and transiently transfected with LTRAs similarly inhibit UDP and LTD signaling in monocytes. 4 http://www.jimmunol.org/ empty pcDNA 3.1 vector or CysLT1 expression vector (Missouri University of Science and Technology cDNA Resource Center, Rolla, MO) using Lipofect- Both antagonists inhibited (Fig. 2) LTD4- and UDP-induced cal- amine 2000 (Invitrogen) in serum-free medium (Opti-MEM I, Invitrogen), cium flux in monocytes in a concentration-dependent manner. transferred to poly-L-lysine–coated 96-well plates after 24 h, and used for cal- However, as only low nanomolar concentrations of montelukast cium mobilization experiments after an additional 24 h of incubation. and zafirlukast were required to fully inhibit LTD4-induced cal- 25 24 CysLT1, cysteinyl leukotriene type 2 receptor, and P2Y6 cium flux, much higher (10 –10 mol/l) concentrations of an- knockdown tagonists were required to inhibit UDP-induced calcium changes.

Silencer Select predesigned small interfering RNA (siRNA) (CysLT1:59-G- LTD4 and UDP signal through separate receptors in monocytes GAAAAGGCUGUCUACAUUtt-39; cysteinyl leukotriene type 2 receptor

To further characterize the responsiveness of human monocytes to by guest on September 25, 2021 [CysLT2]: 59-GCACAAUUGAAAACUUCAAtt-39; P2Y6:59-GAAGCUC- ACCAAAAACUAUtt-39) and Silencer Select Negative Control siRNA LTD4 and UDP, the submaximally effective concentrations of LTD4 27 24 (Ambion, Austin, TX) were used for CysLT1, CysLT2, and P2Y6 knock- (10 mol/l) and UDP (10 mol/l) were usedtoanalyze the patternof down experiments. Elutriated monocytes (5 3 106) were nucleofected with calcium signaling and desensitization. In monocytes, LTD induced m 4 4 g negative control or specific siRNA using a Human Nu- a rapid increase in intracellular calcium levels that relatively quickly cleofector kit (Amaxa, Cologne, Germany), according to the manu- facturer’s protocol. After 24 h, media were replaced, and cells were used decreased to baseline levels (Fig. 3A). UDP also rapidly increased for functional studies. intracellular calcium levels; however, these were sustained for only 10–20 s and then decreased to baseline. The different patterns of Real-time PCR calcium changes induced by UDP compared with LTD4 suggested Total RNAwas extracted from cells using QIA Shredder columns and RNeasy that those compounds could activate different receptors or signaling minikitandwastreatedwithDNase(Qiagen,Valencia,CA).mRNAexpression pathways. In agreement with the above observation, stimulation with for selected genes was measured using real-time PCR performed on an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA) using the following commercially available probe and primers sets (Applied Biosystems): CysLT1, Hs00272624_s1; CysLT2, Hs00252658_s1; and P2Y6, Hs00602548_m1. Reverse transcription and PCR were performed using an RT kit and TaqManUniversal PCR master mix (AppliedBiosystems),according to the manufacturer’s directions. Relative gene expression was normalized to GAPDH transcripts and calculated as a fold-change compared with control. ELISA

6 For IL-8 determination, monocytes (2 3 10 ) were stimulated with LTD4 (100 nmol/l) or UDP (100 mmol/l) for 2 and 6 h in the presence or absence of inhibitors. Supernatants were stored at 220˚C until analysis by com- mercial Quantikine ELISA (R&D Systems) for IL-8.

Determination of leukotriene B4, leukotriene C4, and PGE2 synthesis Monocytes (1 3 106) were resuspended in culture medium without FBS; preincubated with different concentrations of montelukast, zafirlukast, or FIGURE 1. LTD and UDP induce calcium mobilization in monocytes. vehicle control (DMSO); and stimulated with calcium ionophore A23187 4 (1 mmol/l) or DMSO for 30 min. Supernatants were collected by centri- Monocytes were prepared and stimulated with different concentrations of fugation and frozen at 280˚C for leukotriene measurement. For intra- LTD4 or UDP,and calcium releasewas measured, asindicated in Materials and cellular leukotriene determination, elutriated monocytes (2 3 106) were Methods. Data are presented as means 6 SD from three separate experiments, used, reactions were stopped by the addition of cold PBS, and cells were each performed in triplicate. The Journal of Immunology 3 Downloaded from

FIGURE 2. Montelukast and zafirlukast inhibit UDP- and LTD4-induced calcium mobilization in monocytes. Monocytes were stimulated with 100 mmol/l UDP (A, C) or 100 nmol/l LTD4 (B, D) in the presence of increasing concentrations of montelukast (A, B), zafirlukast (C, D), or vehicle control

(10 min pretreatment). Results are shown as the percentage of peak fluo- http://www.jimmunol.org/ rescence obtained in cells stimulated without inhibitors. The means 6 SD from three independent experiments are presented. FIGURE 3. Desensitization effects in LTD4- and UDP-stimulated mono- cytes and CysLT1-transfected HEK293 cells. Representative traces of calcium 27 LTD4 (10 mol/l) potently desensitized monocytes to subsequent flux induced by repeated exposure toLTD4 (100 nmol/l) and UDP (100 mmol/l) 27 stimulation with the same concentration of LTD4 (10 ) (as expected in monocytes (A)andCysLT1-transfected HEK293 cells (B). Data from one of for homologous desensitization), but it did not have any effect on three separate experiments, each with similar results, are shown. subsequent UDP exposure, suggesting again that UDP could act through a separate receptor (not CysLT1). When cells were stimulated dependent manner (Fig. 4). We previously reported that IL-10 by guest on September 25, 2021 with UDP, full desensitization of calcium signaling was observed for downregulates CysLT1 expression and signaling in human monocytes the subsequent UDP exposure (homologous desensitization) and (15). Monocytes were exposed to IL-10 (20 ng/ml) overnight to partial inhibition of signaling to subsequent LTD4 stimulation. To downregulate CysLT1, and calcium flux to LTD4 and UDP was determine whether LTD4 and UDP could activate CysLT1,HEK293 evaluated. IL-10 downregulated CysLT1 mRNA (measured by real- cells were transiently transfected with the human CysLT1 construct. time PCR) and inhibited LTD4-induced calcium flux, whereas UDP HEK293 cells did not express CysLT1 mRNA and, when transfected signaling was not significantly affected (Fig. 5A), suggesting that with empty pcDNA 3.1 vector, did not show calcium responses after UDP does not signal through CysLT1 in monocytes. To further con- 26 LTD4 stimulation up to 10 mol/l concentration (data not shown). In firm this observation, CysLT1 expression was specifically down- cells transfected with CysLT1 (Fig. 3B), LTD4 induced calcium flux regulated by siRNA, as previously described (15), and calcium flux with a pattern similar to that observed in monocytes, and full ho- was measured in response to different concentrations of LTD4 and mologous desensitization to LTD4 was present. However, empty UDP. As expected, in CysLT1 siRNA-treated cells, LTD4-induced vector and CysLT1-transfected HEK293 cells responded to UDP with calcium flux was significantly inhibited, in contrast to UDP-induced 24 potent calcium flux; the most effective concentration was 10 mol/l. signaling, which remained similar in CysLT1 siRNA-treated cells and When the desensitization experiments were carried out, a similar level control cells (Fig. 5B). Treatment of cells with siRNA directed at of homologous desensitization to UDP was observed in empty control CysLT2 reduced CysLT2 mRNA by 87%, but it did not reduce LTD4- and CysLT1-transfected cells. Interestingly, the same pattern of het- or UDP-induced calcium flux (data not shown). To analyze whether erologous desensitization as in monocytes was identified in CysLT1- UDP signals through P2Y6 in monocytes, P2Y6 was knocked down transfected cells: partial desensitization by UDP to subsequent LTD4 using siRNA, and the response to LTD4 and UDP was measured. stimulation and no desensitization by LTD4 to subsequent UDP ex- P2Y6 mRNAwas knocked down by 85% (four measurements). UDP- posure. All of these data suggested that UDP induced calcium flux induced calcium flux was inhibited in P2Y6 siRNA-treated cells, through a separate receptor, not CysLT1. It was shown that UDP can whereas LTD4-induced signaling was not affected (Fig. 6), showing induce calcium flux in several cell systems acting through nucleotide that P2Y6 may be one of the target receptors for UDP-induced sig- G-protein coupled receptors (GPCRs), with P2Y6 being the most naling in monocytes. selective for UDP. We confirmed the presence of P2Y6 mRNA in All of these data suggest that in human monocytes, LTD4 and monocytes, but the level of expression of P2Y6 mRNA in HEK293 UDP activate separate receptors, LTD4 induces calcium flux cells was very low (data not shown), suggesting that UDP could ac- through CysLT1 and UDP signals at least in part through P2Y6. tivate calcium flux through this or other nucleotide receptors in these cells. UDP (100 mM) induced a calcium flux in untransfected HEK LTRA inhibits UDP-induced IL-8 production cells that was not inhibited by the P2Y6 inhibitor MRS2578 (5 mM). It was reported that UDP mediates IL-8 production in monocytic When used at micromolar concentrations, MRS2578 inhibited UDP- THP-1 cells (16). To determine whether LTRAs may inhibit other and LTD4-induced calcium flux in monocytes in a concentration- UDP-induced functions, in addition to intracellular calcium flux, 4 LTRAs AND NON–CysLT1-MEDIATED EFFECTS Downloaded from

FIGURE 4. The effect of P2Y6 inhibitor MRS2578 on UDP- and LTD4- induced calcium flux in monocytes. Representative traces of calcium flux http://www.jimmunol.org/ induced by exposure to UDP (100 mmol/l) or LTD4 (100 nmol/L) in the presence of increasing concentrations of MRS2578 (MRS) or vehicle con- trol. Data from one of three separate experiments, each with similar results, are shown. FIGURE 5. Downregulation of CysLT1 did not affect UDP-inducedcalcium mobilization in monocytes. A, Monocytes were incubated with or without IL- monocytes were stimulated with LTD4 or UDP, and the synthesis 10 (20 ng/ml) overnight, and calcium release was measured in response to of IL-8 was measured as described in Materials and Methods. different concentrations of LTD4 and UDP, as described in Materials and UDP, but not LTD4, induced IL-8 production in a time-dependent Methods. B, Monocytes were nucleofected with CysLT1 siRNA or negative by guest on September 25, 2021 manner (Fig. 7A) that was significantly inhibited by the P2Y6 in- control oligonucleotides (Con), as described in Materials and Methods,cul- hibitor MRS2578 (10 mmol/l). Montelukast and zafirlukast also in- tured for 24 h, and calcium flux was measured in response to LTD4 or UDP.Data 6 hibited IL-8 production in a concentration-dependent manner (Fig. are means SD from three separate experiments, each performed in triplicate. 7B). These data further confirm that LTD4 and UDP signal differently in human monocytes and that LTRAs may significantly affect the as described in Materials and Methods. Calcium ionophore stimu- UDP-induced activation of these cells. It was recently reported that lation produced detectable intracellular levels of both leukotrienes C CysLT1 knockdown inhibited LTD4- and UDP-induced chemokine (Fig. 8 ), which were inhibited by pretreatment with montelukast and generation in human mast cells (17). In contrast, we did not ob- zafirlukast, indicating that LTRAs may significantly downregulate the serve significant changes in chemokine (IL-8) generation in CysLT1 production of leukotrienes in human monocytes. siRNA-treated monocytes compared with control siRNA-treated cells (data not shown). This suggests that UDP and cysLTs may Discussion signal differently in different types of cells (mast cells and mono- We presented evidence herein that so-called “selective CysLT1 antag- cytes), probably reflecting differences in GPCR expression. onists” montelukast and zafirlukast possess concentration-dependent non–CysLT1-mediated inhibitory activities in primary human mono- LTRAs inhibit leukotriene production cytes. Both drugs effectively inhibited LTD4-induced signaling at the To establish whether LTRAs may affect 5-LO activity and leukotriene nanomolar concentration range, in agreement with CysLT1 antagonist production, monocytes were stimulated with calcium ionophore activity reported in previous studies (18, 19). Interestingly, montelukast A23187, and synthesis of LTB4 and LTC4 was measured. Stimulated and zafirlukast, acting at low micromolar concentrations, also inhibited monocytes produced significant levels of both leukotrienes, which LTC4 and LTB4 synthesis, probably through a direct inhibition of 5-LO. were inhibited by pretreatment with montelukast (Fig. 8A) and za- It was suggested that montelukast may directly inhibit 5-LO activity, firlukast (Fig. 8B) in a concentration-dependent fashion. Both an- binding to an undefined allosteric site on 5-LO (11). In our study, tagonists (5 mmol/l) fully inhibited leukotriene production, montelukast and zafirlukast similarly inhibited leukotriene synthesis, suggesting that 5-LO could be directly affected by the treatment. To but not PGE2 generation, suggesting that this 5-LO inhibitory activity is exclude the possibility that LTRAs affect synthesis not specific for montelukast; it can be applied to other LTRAs as well. by A2,PGE2, as a product of the cyclooxygenase Thenextproinflammatorypathway,inhibitedinourstudybyboth pathway of arachidonic acid cascade, was measured in the same ex- compounds at higher micromolar concentrations, is UDP-induced periments. Neither montelukast nor zafirlukast (5 mol/l) inhibited signaling and IL-8 production. Extracellular nucleotides, such as UTP calcium ionophore-induced PGE2 generation (Fig. 8D). To further and UDP, were shown to activate phospholipase C and intracellular analyze whether LTRAs inhibit the production of leukotrienes and calcium mobilization in differentiated U937 cells through purine P2Y not transport out of the cell, monocytes were stimulated with calcium receptors; those effects were inhibited by micromolar concentrations ionophore, and intracellular levels of LTB4 and LTC4 were measured of montelukast and pranlukast (20). We validated these observations The Journal of Immunology 5 Downloaded from

FIGURE 6. Knockdown of P2Y6 did not affect LTD4-induced signaling in monocytes. Monocytes were nucleofected with P2Y6 siRNA or negative control oligonucleotides (control), as described in Materials and Methods, cultured for 24 h, and calcium flux was measured in response to different concentrations of http://www.jimmunol.org/

UDP (A, B)orLTD4 (C, D). Representative traces of calcium flux from one of three separate experiments, each with similar results, are shown. in a human primary cell system, and we showed that such an inhibition [calculated maximum plasma concentration .3and7mmol/l, re- of signaling may have functional consequences for cell activation, spectively (29)], the morning and evening peak expiratory flow rates measured at the level of UDP-induced IL-8 production. We report showed nearly twice the improvement compared to patients receiving herein that two chemically different LTRAs might possess, in addition lower doses, suggesting that higher doses of this drug could have an to CysLT1 antagonism, anti-inflammatory activities in primary human cells. The fact that these additional inhibitory activities are strongly related to the drug concentration might have potential clinical rele- by guest on September 25, 2021 vance. LTRAs, in contrast to many other drugs, are believed to require membrane transporters for their absorption after oral administration (21–23). In addition, other features, such as strong binding to plasma albumin (.99%) and extensive hepatic metabolism, suggest that the bioavailability of those drugs might be affected by pharmacogenetic and pharmacokinetic factors, resulting in variability of plasma drug concentration and response to treatment. Although there are no pub- lished population-based data demonstrating pharmacogenetic influence on LTRA plasma concentrations, case reports showed significant var- iations in montelukast plasma levels in healthy subjects (24). In addi- tion, it was recently shown that genetic variation of OATP2B1, a membrane transport protein, was associated with variable mon- telukast plasma concentrations and variable response to treatment in patients with asthma (25). The maximum plasma concentration after orally administered montelukast, 10 mg, ranged from 0.57 to 0.63 mmol/l in healthy female and male subjects, respectively (26); it reached 0.80 mmol/l following administration of a 10-mg chewable tablet in adults and a 5-mg chewable tablet in 9- to 14-y-old children (27). These concentrations are very close to the concentrations of montelukast and zafirlukast required for leukotriene synthesis in- hibition (1–5 mmol/l) in our study. Bearing in mind potential genetically driven variability in drug absorption and metabolism, it is plausible to consider that even after the administration of a standard dose of LTRAs FIGURE 7. Montelukast and zafirlukast inhibit UDP-induced IL-8 pro- in some patients, inhibition of the leukotriene synthesis pathway might duction in monocytes. A, Monocytes were preincubated with MRS2578 or vehicle control for 30 min and stimulated with LTD (100 nmol/l) or UDP be observed in addition to CysLT antagonism. The clinical relevance 4 1 (100 mmol/l). Supernatants were collected after 2 and 6 h, and IL-8 expression of additional inhibitory activity of LTRAs on 5-LO has not been ad- was measured. B, Monocytes were preincubated with different concentrations dressed. In a placebo-controlled, dose-ranging study of montelukast in of LTRAs, stimulated with UDP, and IL-8 expression was measured after 6 h. patients with asthma, doses from 10–200 mg/d were analyzed; no dose- Data are presented as the percentage of UDP-induced IL-8 production from response relationship for asthma symptom control and spirometry cells pretreated with vehicle control. Means 6 SD from three separate ex- was observed (28). However, in patients treated with 100 and 200 mg periments are presented. pp , 0.001, Student t test. 6 LTRAs AND NON–CysLT1-MEDIATED EFFECTS Downloaded from http://www.jimmunol.org/

FIGURE 8. Montelukast and zafirlukast inhibit leukotriene synthesis in monocytes. Monocytes were preincubated for 10 min with different concen- trations of montelukast (A) and zafirlukast (B) or vehicle control, stimulated with calcium ionophore A23187 (1 mmol/l), and levels of LTB4,LTC4, and PGE2 (D) released were measured. C, Cells were preincubated with 5 mmol/l montelukast (Mon) or zafirlukast (Zaf), and intracellular levels of LTB4 and LTC4 were measured as described in Materials and Methods. The means 6 SD from three donors, performed in triplicate, are shown. pp , 0.01; ppp , by guest on September 25, 2021 0.001 for A23187-stimulated cells with versus without inhibitor. additional impact on asthma treatment. The inhibition of UDP signaling act on the same GPCR. A similar observation was reported in dif- by LTRAs required higher micromolar concentrations (10–100 mmol/l) ferentiated U937 cells (32); CysLT1 desensitization and trafficking in our study. The maximum plasma concentration exceeded 25 mmol/l were differentially regulated by LTD4 and extracellular nucleotides, after the oral administration of 800 mg of montelukast, a dose that was including UDP. Capra et al. (32) showed that CysLT1 may be a target still well tolerated (29); however, the clinical relevance of such doses for extracellular nucleotide-induced heterologous desensitization, has not been tested. It was shown that LTRAs may also affect other non– a potential feedback mechanism ininflammation. The presence of this CysLT1-mediated pathways in human primary cells. Montelukast in- heterologous desensitization mechanism was demonstrated for the hibited protease activity (9), and pranlukast suppressed LPS- first time in human primary cells in our study. UDP signals effectively induced IL-6 production in PBMCs (30); both effects were observed at through calcium flux acting on P2Y6 in human cells (33). Because micromolar drug concentrations. All of these data strongly suggest that human monocytes express mRNA for CysLT1 and P2Y6,wedown- in addition to CysLT1 antagonism, other proinflammatory pathways regulated CysLT1 expression by IL-10 preincubation or specific may be affected by LTRA treatment and, depending on plasma drug CysLT1 siRNA to further support the observation that UDP does not concentration, variable responses to treatment may be expected. signal through CysLT1 in monocytes; we noted a specific decrease in It was suggested that CysLT1 expressed on human mast cells is also LTD4-induced signaling, without an effect on UDP-induced calcium a pyrimidinergic receptor and that it responds to UDP stimulation in mobilization. Knockdown of P2Y6 decreased UDP-induced calcium addition to cysLTs (14, 31). Inour study, human monocytes responded flux but did not affect LTD4-induced signaling. All of these data to LTD4 and UDP, but with characteristics suggesting that UDP acts suggest that UDP is not a CysLT1 agonist in human cells, but it may through separate receptors. First, 3-log greater concentrations (1024 signal through a different GPCR. In fact, a newly described cysLT mol/l) of UDP were required to submaximally activate calcium flux in receptor (GPR17) was shown to be a dual UDP/cysLT receptor (34). monocytes than reported in the study by Mellor et al. (14). Although We showed previously that GPR17 is not expressed in human mon- UDP signaling in monocytes was inhibited by LTRAs, much greater ocytes (13), excluding this receptor as a potential target for UDP in concentrations of LTRAs were needed. Almost every GPCR studied monocytes. Interestingly,the presence of GPR17 was detected inmast undergoes desensitization following agonist stimulation, an impor- cell line LAD2, an observation that might explain cysLT receptor- tant process that is crucial for turning off the receptor-mediated sig- related signaling induced by UDP in mast cells (35). It was also naling pathway. As expected, CysLT1 underwent LTD4-induced suggested that additional, not-yet-characterized cysLT receptors homologous desensitization in monocytes, but we did not observe might be expressed in mast cells (35). desensitization of UDP-induced calcium flux subsequent to LTD4 The described inhibitory activity of LTRAs on the 5-LO pathway treatment, a phenomenon that should be present when both agonists and extracellular nucleotide-induced signaling may be relevant for The Journal of Immunology 7

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