Inhibitory Activity of Pranlukast and Montelukas Against Histamine

Showa Univ J Med Sci 21（2）, 77～84, June 2009

Original Inhibitory Activity of Pranlukast and Montelukas Against

Histamine Release and LTC4 Production from Human Basophils

1, 2 1 1 Satoshi HIBINO ）, Ryoko ITO ）, Taeru KITABAYASHI ）, 1 2 Kazuo ITAHASHI ） and Toshio NAKADATE ）

Abstract : Leukotriene receptor antagonists（LTRAs）are routinely used to treat bronchial asthma and are thought to act mostly by inhibiting leukotriene receptors. However, there is no preclinical or clinical evidence of the direct

effect of LTRAs on histamine release from and leukotriene（LT）C4 production by basophils. We used anti-IgE antibody（Ab）, FMLP, and C5a to induce

histamine release, and anti-IgE Ab and FMLP to stimulate LTC 4 production. Basophils were exposed to different concentrations of pranlukast and montelukast, and then to anti-IgE Ab, FMLP, and C5a. Culture supernatant histamine

and LTC 4 levels were measured by using a histamine ELISA kit and a LTC 4 EIA kit, respectively. Histamine release was expressed as a percentage of the total histamine content（％HR）induced by anti-IgE Ab, FMLP, or C5a. To evaluate the effects of pranlukast and montelukast on histamine release and

LTC 4 production, we calculated the percent inhibition of histamine release and

LTC 4 production, expressed as percent inhibition, at different concentrations of pranlukast and montelukast. Pranlukast significantly inhibited histamine release stimulated by FMLP and C5a, but had no effect on histamine release stimulated by anti-IgE Ab. By comparison, montelukast signi cantly inhibited histamine release stimulated by FMLP, C5a, and anti-IgE Ab, in a concentration-dependent manner. Both pranlukast and montelukast signi cantly

inhibited LTC 4 production stimulated by anti-IgE Ab and FMLP. Together, these ndings demonstrate that LTRAs have a direct inhibitory effect against

histamine release and LTC 4 production from basophils and provide new insight into the potential clinical application of LTRAs for the treatment of allergic disease.

Key words : basophil, leukotriene, leukotriene receptor antagonist, histamine

Introduction

Basophils play an important role in the late phase of allergic response by releasing proinflammatory mediators including histamine, cysteinyl leukotrienes（cysLTs）, and pros-

1）Department of Pediatrics, Showa University School of Medicine, 1‒5‒8, Hatanodai, Shinagawa-ku, Tokyo 142‒8666, Japan. 2）Department of Hygieiology, Showa University School of Medicine. 78 Satoshi HIBINO, et al

taglandins 1, 2）. Leukotriene receptor antagonists（LTRA）inducing pranlukast, montelukast, and zafirlukast are effective in the treatment of bronchial asthma because they improve forced expiratory volume in one second as well as night-time asthma symptom scores and other clinical parameters in asthmatic patients 3‒6）. LTRAs are thought to exert their pharmacological activity by inhibiting the leukotriene receptors（LTRs）. However, previous studies have suggested that LTRAs interact not only with the cysLT receptors（cysLTRs） but also with allergic inammatory cells in the absence of cysLTR mediation. Tohda et al（1999）demonstrated that pranlukast suppresses the production of interleukin（IL）-4, IL-5, and granulocyte macrophage colony-stimulating factor（GM-CSF）by peripheral blood mononuclear cells in vitro 7）. Furthermore, Ramires et al（2004）revealed that montelukast exerts inhibitory activity against 5-lipoxygenase activity in human mast cells（HMC-1 cells） and human polymorphonuclear leukocytes in vitro 8）. With respect to the inhibitory activity of LTRAs on basophils, Kobi et al（2005）have provided only indirect evidence that montelukast, taken orally, inhibits cysLT release from basophils in patients with bronchial asthma after 4 weeks of treatment 9）. The objective of the present in vitro study was to examine the direct effects of pranlukast

and montelukast on histamine release and LTC 4 production from human peripheral blood basophils.

Materials and Methods

We purchased rabbit anti-IgE antibody（Ab）（Dako, Glostrup, Denmark）, N-formyl- methionyl-leucyl-phenylalanine（FMLP）, human recombinant-C5a（C5a）, Histopaque 1.083,

C6H18N2O6S2・1.5 Na piperazine diethanesulfonic acid（PIPES）, human serum albumin（HSA）,

glucose, perchloric acid（PCA）, and alcian blue（C56H68Cl4CuN16S4）（Sigma, St. Louis, MO）, saline（Otsuka, Naruto, Japan）, ethyl-enediaminetetraacetic acid（EDTA）, NaCl, KCl,

MgCl2, CaCl2（Wako, Osaka, Japan）. Pranlukast and montelukast were supplied by Ono Pharmaceutical Co, Ltd. and Banyu Pharmaceutical Co, Ltd., respectively. PIPES-albumin- glucose（PAG）buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1％ glucose,

and 0.003％ HSA. PAGCM consisted of PAG supplemented with 1 mM CaCl2 and 1 mM

MgCl2. PAG-EDTA consisted of PAG supplemented with 4 mM EDTA.

Puri cation of basophils According to previously described methods 10, 11）, basophils were puri ed from peripheral blood taken from healthy volunteer donors（n＝10）who had given consent to enrolment in the present study. Saline at the same volume as the collected peripheral blood and 0.1 M EDTA-2 Na at one-tenth the volume of the blood were mixed with the blood and the mixture was then gently added to Histopaque 1.803. The mixture was centrifuged（300 g, 20 min）to isolate the layer of mononuclear cells including basophils. Mononuclear cells were suspended in PAGCM at a concentration of 5×105 basophils / mL. Basophils were counted Inhibitory activity of LTRAs on basophils 79

following alcian blue staining 12）.

Stimulation of basophils Anti-IgE Ab, FMLP, and C5a were used to induce histamine release, whereas and anti-

IgE Ab and FMLP were used to induce LTC4 production. Basophils were incubated at 37℃ for 15 minutes with different concentrations（1, 10, and 100 μM）of pranlukast and montelukast, followed by incubation with anti-IgE Ab（1 μg / mL）, FMLP（1 μM）, or C5a （300 ng / mL）at 37° C for 30 minutes. The concentration of used in this study was based on published data 10）, whereas the concentrations of anti-IgE Ab and C5a used in this study were based on determination of the optimal concentrations to induce histamine release.

Histamine and LTC4 measurements Reactions were terminated with ice-cold PAG-EDTA and the cells were pelleted by centrifugation at 4500 g for 30 s. Culture supernatant histamine levels were measured with a

histamine ELISA kit（Immunotech, Marseille, France）, and culture supernatant LTC4 levels

were measured with an LTC4 EIA kit（Cayman Chemical Co, Ann Arbor, AI）. Each mea- surement was performed in duplicate. Percentage of histamine release（％HR）induced with anti-IgE Ab, FMLP, snd C5a was calculated according to the following equation : ％HR＝

（C-C spon）/（C total -C spon）×100（％）, where C is the culture supernatant histamine level in

each stimulated blood sample, C spon is the culture supernatant histamine level in the control

sample that shows spontaneous histamine release, and C total is the total amount of histamine in basophils that is identi ed by using PCA to destroy cell membranes. To evaluate the direct effects of pranlukast and montelukast on histamine release, we estimated the percent

inhibition of histamine release and LTC4 production at different concentrations of pranlukast and montelukast. Blood samples collected from nonresponders, whose ％HR were not more

than 20％ or whose LTC4 production were not more than that of control samples were not used in the statistical analysis.

Statistical analysis Values are expressed as mean±SEM. Statistical analyses were performed by the paired Student’s t-test, and a value of P＜0.05 was considered to be statistically signi cant.

Results

Effects of pranlukast and montelukast on histamine release following stimulation by anti-IgE Ab, FMLP, or C5a The mean values（mean±SEM）of ％HR induced by anti-IgE Ab, FMLP, and C5a were 42±4.6％, 44±7.1％, and 25±1.6％, respectively（Table 1）. The effects of pranlukast on histamine release from basophils are shown in Fig. 1A. Pranlukast inhibited FMLP-induced histamine release in a concentration-dependent manner ; this effect was signi cant at 10 μM 80 Satoshi HIBINO, et al

Table 1 Histamine release from peripheral blood basophils

Histamine release（％） Inducer n Mean±SEM Anti-IgE Ab 4 42±4.6 FMLP 5 44±7.1 C5a 5 25±1.6

IgE, immunoglobulin E ; Ab, antibody ; FMLP, N-formyl- methionyl-leucyl-phenylalanine ; C5a, human recombinant C5a ; SEM, standard error of the mean

and 100 μM pranlukast with percent inhibition（mean±SEM）values of 55％±10％（P＜0.01） and 87％±5.1％（P＜0.001）, respectively. Pranlukast also signi cantly inhibited C5a-induced histamine release with percent inhibition（mean±SEM）values of 80％±6.8％ at 100 μM （P＜0.01）. By comparsion, pranlukast did not inhibit anti-IgE-induced histamine release. The effects of montelukast on histamine release from human basophils are shown in Fig. 1B. Montelukast signi cantly inhibited anti-IgE Ab-, C5a-, and FMLP-induced histamine release in a concentration-dependent manner. The percent inhibition of FMLP-induced histamine release by montelukast was 28％±9.4％ at 1 μM（P＜0.05）, 83％±8.8％ at 10 μM（P＜ 0.001）, and 88％±2.9％ at 100 μM（P＜0.001）. The percent inhibition of C5a-induced histamine release by montelukast was 78％±9.4％ at 10 μM（P＜0.01）and 79％±4.8％ at 100 μM（P＜0.001）. The percent inhibition of anti-IgE Ab-induced histamine release by montelukast was 71％±14％ at 10 μM（P＜0.05）and 81％±2.7％ at 100 μM（P＜0.001）.

Effects of pranlukast and montelukast on LTC4 production following stimulation by anti-IgE Ab or FMLP.

The mean amounts of LTC4 production（mean±SEM）induced by anti-IgE Ab and FMLP were 296±90.7 and 116±28.7（ng / 10 5 basophils）, respectively（Table 2）. The effects

of pranlukast on LTC4 production by human basophils are shown in Fig. 2A. Pranlukast

inhibited anti-IgE Ab-stimulated LTC4 production ; this effect was signi cant at 10 μM with percent inhibition calculated as 50％±31％（P＜0.01）. Pranlukast also inhibited FMLP- stimulated LTC4 production, but the percent inhibition was only signi cant at 100 μM with

percent inhibition values of 81％±9.0％（P＜0.001）. The effects of montelukast on LTC4

production are shown in Fig. 2B. The inhibition of anti-IgE Ab-stimulated LTC4 production by montelukast was signi cant at 10 μM and 100 μM with percent inhibition values of 55％ ±12％（P＜0.01）and 48％±16％（P＜0.05）, respectivery. Montelukast inhibited FMLP-

stimulated LTC4 production in a concentration-dependent manner with percent inhibition values of 55％±11％ at 1 μM（P＜0.01）; 87％±3.9％ at 10 μM（P＜0.001）; and 87％± 4.0％ at 100 μM（P＜0.001）. Inhibitory activity of LTRAs on basophils 81

Fig. 1. Effects of pranlukast A and montelukast B on histamine release from peripheral blood basophils（n＝4-5） Solid square, FMLP ; open square, C5a ; open triangle, anti-IgE antibody ; ＊＊＊P＜0.001 ; ＊＊P＜0.01 ; ＊, P＜0.05 The percent inhibition of histamine release at different concentrations of pranlukast A and montelukast B are shown. Histamine release was signi cantly inhibited with the exception that pranlukast did not inhibit anti-IgE antibody-induced histamine release. P values were calculated according to the paired Student’s t-test.

Discussion

The present study demonstrated that pranlukast signi cantly inhibited FMLP- and C5a- induced histamine release from basophils in a concentration-dependent manner as well as anti-IgE Ab- and FMLP- stimulated LTC4 production by basophils. The present study also revealed that montelukast signi cantly inhibited anti-IgE Ab-, FMLP-, and C5a-induced histamine release from basophils in a concentration-dependent manner as well as anti-IgE Ab- and FMLP-stimulated LTC4 production by basophils. Basophils play a central role in IgE- 82 Satoshi HIBINO, et al

Table 2 LTC4 production by peripheral blood basophils

5 LTC4（ng / 10 basophils） Stimulator n Mean±SEM Anti-IgE-Ab 8 296±90.7 FMLP 6 116±28.7

LTC4, leukotriene C4 ; IgE, immunoglobulin E ; Ab, antibody ; FMLP, N-formyl-methionyl- leucyl-phenylalanine ; SEM, standard error of the mean

Fig. 2. Effects of pranlukast A and montelukast B on LTC4 production by peripheral blood basophils（n＝6-8） Solid square, FMLP ; open triangle, anti-IgE antibody ; ＊＊＊P＜0.001 ; ＊＊P＜0.01 ; ＊P＜0.05 The percent inhibition of LTC4 production at different concentrations of pranlukast A

and montelukast B are shown. LTC4 production induced by FMLP and anti-IgE Ab was signi cantly inhibited by pranlukast and montelukast. P values were calculated according to the paired Student’s t-test. Inhibitory activity of LTRAs on basophils 83

mediated inammation and present elevated tissue concentrations in allergic patients. The cross-linking of the high-af nity IgE receptors by allergens leads to histamine release and

LTC4 production. Therefore, our results suggest that pranlukast and montelukast improve symptoms of allergic disease by their inhibitory activity in basophils. The peak plasma concentration of pranlukast and montelukast is about 1 μM for clini-

cal use. Our results suggest that inhibition of histamine release and LTC4 production is not signi cant at this concentration. There is only indirect evidence that oral montelukast inhibits cysLT release from basophils in patients with bronchial asthma 9）. There is no clear evidence that indicates a difference between the in vitro and in vivo effects of pranlukast and montelukast. If higher doses of pranlukast and montelukast are permitted, depending on safety, their clinical effects may be improved.

Gaurreau et al（2005）reported that basophils express the cysLTRs（cysLT1 and cysLT2） 13） and that part of the LTD4 receptors . FMLP and anti-IgE Ab induce the release of both

histamine and LTC4 by basophils. Therefore, it is possible that LTRAs do not directly inhibit the FMLP- and anti-IgE Ab-induced release of histamine but rather inhibit histamine

release through antagonist activity against the LTC4 receptors. However, the inhibition of

histamine release and LTC4 production demonstrated in the present study was not mediated by cysLTR antagonism because of the following facts : 1）our in vitro system did not use

leukotrienes ; and 2）LTC4 production by basophils occurred 1 minute after stimulation with FMLP. Histamine release from basophils is complete within 30 seconds of stimulation with 10） FMLP , whereas histamine release and LTC4 production by basophils are complete simul- taneously within 15 minutes of stimulation with anti-IgE Ab 14）. Therefore, it is unlikely that

LTC4 production stimulated by LTC4 itself induces histamine release through the cysLTRs. Both pranlukast and montelukast are LTRAs but show little difference in their inhibitory activity against basophils. Our study showed that montelukast inhibited histamine release that had been induced by anti-IgE Ab, FMLP, and C5a. By comparison, pranlukast did not inhibit histamine release induced by anti-IgE antibody but did inhibit histamine release induced by FMLP and C5a. The mechanism underlying histamine release from human basophils involves multiple signaling pathways. Anti-IgE Ab induces histamine release through FcεRI and FMLP, whereas C5a induces histamine release through G-protein 10, 11, 15, 16）. It is conceivable that montelukast inhibits both the FcεRI and G-protein signaling pathways and that pranlukast inhibits only the G-protein signaling pathway. Further studies are necessary to elucidate the mechanism underlying inhibition of histamine release by pranlukast and montelukast. A limited number of previous studies have demonstrated that LTRAs control allergic inammation by a mechanism that is different from cysLTR antagonism 7‒9）. However, there are no reports on the direct effects of LTRAs on basophils. Our in vitro study indicates that pranlukast and montelukast directly inhibit histamine

release and LTC4 production by human basophils. Essentially, LTRAs exert their inhibitory 84 Satoshi HIBINO, et al activity through cysLTR antagonism. A novel nding of the present study is the direct inhibition of leukotriene production by LTRAs. Histamine and LTs are potent mediators that cause allergic reaction. Therefore, the direct inhibitory activity of LTRAs against histamine release and LTC4 production by basophils provides new insight into the potential clinical use of LTRAs for the treatment of allergic disease.

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［Received December 8, 2008 : Accepted January 10, 2009］