Cysteinyl leukotrienes induce nuclear factor κb activation and RANTES. cytokine production in a murine model of asthma.

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1 Cysteinyl leukotrienes induce nuclear factor κb activation and RANTES production in a murine model of asthma Tetsuya Kawano, MD, Hiroto Matsuse, MD, Yuki Kondo, MD, Ikuko Machida, MD, Sachiko Saeki, MD, Shinya Tomari, MD, Kazuko Mitsuta, MD, Yasushi Obase, MD, Chizu Fukushima, MD, Terufumi Shimoda, MD, and Shigeru Kohno, MD Nagasaki, Japan Background: It has been demonstrated that both cysteinyl leukotrienes (cyslts) and cytokines are involved in the pathophysiology of bronchial asthma. Nonetheless, the exact mechanism involved in the interaction between these 2 molecules has yet to be determined. Objective: The aim of the present study was to determine the effects of cyslts on allergic airway inflammation and allergen-specific cytokine production in a murine model of asthma. Methods: Four groups of BALB/c mice (control mice, Dermatophagoides farinae allergen sensitized mice, pranlukast cyslt receptor antagonist treated allergen-sensitized mice, and dexamethasone-treated allergen-sensitized mice) were examined. Results: Allergen-sensitized mice exhibited increased airway responsiveness and inflammation. Pranlukast-treated mice showed significant attenuation of these changes concomitant with reduction of T H 2 cytokine and IFN-γ production by isolated lung mononuclear cells (MNCs). A much stronger inhibition of all cytokines was noted in dexamethasone-treated mice. Pranlukast also significantly inhibited production of RANTES and activation of nuclear factor κb (NF-κB) in the isolated lung MNCs. Leukotriene D 4 stimulated isolated lung MNCs to produce RANTES but not any other cytokines and also activated NF-κB in these cells. Conclusions: Our results suggest that cyslts activate NF-κB and induce RANTES production from isolated lung MNCs, which in turn might cause migration of eosinophils and activated T lymphocytes into the airway. (J Allergy Clin Immunol 2003;112: ) Key words: Leukotrienes, cytokines, nuclear factor κb, RANTES, inflammation Bronchial asthma is a chronic airway inflammatory disease characterized by inflammatory cell infiltration, including eosinophils, CD4 + T cells, and mast cells. 1 Suppression of airway inflammation is the main strategy From the Second Department of Internal Medicine, Nagasaki University School of Medicine. Received for publication January 7, 2003; revised April 9, 2003; accepted for publication April 29, Reprint requests: Hiroto Matsuse, MD, Second Department of Internal Medicine, Nagasaki University School of Medicine, Sakamoto, Nagasaki , Japan Mosby, Inc. All rights reserved /2003 $ doi: /mai Abbreviations used AHR: Airway hyperresponsiveness CysLT: Cysteinyl leukotriene LTD 4 : Leukotriene D 4 MNC: Mononuclear cell NF-κB: Nuclear factor κb sraw: Specific airway resistance in the treatment of asthma. The airway inflammation of asthma is regulated by a complex network of various chemical mediators, cytokines, chemokines, adhesion molecules, and transcription factors, although the exact pathomechanism remains unknown. Naive CD4 + helper T (T H 0) cells differentiate into T H 1 (producing IFN-γ and IL-2) and T H 2 (producing IL-4, IL-5, and IL-13) phenotypes. 1 T H 1 and T H 2 cells counterbalance each other with production of their cytokines. 2 Imbalance between T H 1 and T H 2 cells is an important aspect of the pathophysiology of asthma, in which T H 2 cells are the dominant cells. In addition to their primary chemotactic effects on inflammatory cells, chemokines are also important for the immune response, causing migration of immune cells (eg, lymphocytes or dendritic cells) to lymphoid tissues. Among various chemokines, the CC chemokine family is primarily involved in allergic inflammation through their chemotactic effects on eosinophils and activated T cells. 3-5 Cell adhesion molecules are regarded as important selective contributors to the migration of eosinophils, basophils, and T cells into the inflamed tissue. 6,7 The expression of adhesion molecules is regulated by various inflammatory cytokines and mediators, 8,9 and the expression of the latter is regulated by transcription factors and DNA-binding proteins. In this context activation of nuclear factor κb (NF-κB) has been demonstrated in allergic airway inflammation. 10,11 Recently, many reports have demonstrated the clinical efficacy of cysteinyl leukotriene receptor 1 (cyslt1) antagonists in the treatment of asthma CysLTs cause bronchoconstriction, vascular hyperpermeability, and mucous hypersecretion and are thus important factors in the pathogenesis of asthma. 1,16-18 In addition, recent reports suggested a distinct immunologic effect of cyslts in asthma, modulation of allergen-specific 369

2 370 Kawano et al J ALLERGY CLIN IMMUNOL AUGUST 2003 cytokine production. For example, leukotriene D 4 (LTD 4 ) induced airway eosinophilia in the guinea pig is inhibited by IL-5 mab, 19 and montelukast, a cyslt1 antagonist, decreases the number of IL-5 expressing T cells in the airways of sensitized rats. 20 Furthermore, selective 5-lipoxygenase inhibitors and pranlukast, a cyslt1 antagonist, inhibit the eotaxin-induced airway hyperresponsiveness (AHR) and eosinophilia in IL-5 transgenic mice. 21 Pranlukast inhibits the production of mite allergen specific cytokines from peripheral blood mononuclear cells (MNCs) of asthmatic patients. 22 Nonetheless, the exact mechanism involved in the interaction between cyslts and allergen-specific cytokine production has yet to be determined. It is interesting to examine the interaction between biologically different molecules, cyslts, and cytokines. Thus the present study was designed to determine the effects of cyslts on allergic airway inflammation, cytokine and chemokine production, and signaling by using a mite allergen sensitized murine model of asthma. METHODS Animals and immunization protocol Four groups of 4-week-old female BALB/c mice (Charles River Japan, Inc, Yokohama, Japan) were housed at the Laboratory Animal Center for Biochemical Research, Nagasaki University School of Medicine. All mice were immunized twice intraperitoneally on days 1 and 14 with 0.5 mg per mouse of Dermatophagoides farinae (LG-5339; Cosmo Bio, Tokyo, Japan) precipitated in aluminum hydroxide. Then these mice were challenged intranasally with 50 µl of PBS (control group) or 50 µg/50 µl of D farinae allergen (allergen-sensitized group) on days 14, 16, and 18, as described previously. 23 Next, 0.5 mg per mouse of pranlukast (Ono Pharmaceutical Co, Osaka, Japan), a selective cysltsr1 antagonist, or 0.02 mg per mouse of dexamethasone (Sigma, St Louis, Mo) was injected subcutaneously in D farinae allergen sensitized and challenged allergen-sensitized mice from days 13 to 19. AHR was determined on day 20 in unrestrained mice by means of whole-body plethysmography. On day 21, all mice were killed by means of dislocation of the cervical vertebrae, and lung tissues were obtained from each group. The procedures were reviewed and approved by Nagasaki University School of Medicine Committee on Animal Research. All experiments were repeated at least 3 times. Semiquantification of pulmonary inflammation Hematoxylin and eosin stained lung sections were coded and evaluated at least twice in a blinded fashion by 3 different observers, as described previously. 23 The number of eosinophils was determined in 10 perivascular areas per section by using an oil immersion lens. These examined areas were selected randomly under a low power of magnification (4 ), at which leukocyte subtypes were hardly distinguishable. The mean diameters of the selected blood vessels and bronchioles were not significantly different among the groups. Interoperative and intraoperative variations were less than 10%. The results were expressed as the mean cell numbers of each group. Determination of AHR AHR was measured in unrestrained mice by means of wholebody plethysmography (PULMOS-I; M.I.P.S., Osaka, Japan). AHR was expressed as specific airway resistance (sraw), a calculated value that closely correlates with pulmonary resistance measured by using conventional 2-chamber plethysmography in ventilated animals. The 4 groups of mice were exposed for 5 minutes to nebulizer PBS and subsequently to increasing concentrations (6, 12, 25, and 50 mg/ml) of nebulizer methacholine (Sigma) in PBS by using an ultrasonic nebulizer (NE-U17; Omron, Kyoto, Japan). After each dose, recordings were taken for 3 minutes. The sraw values, measured during a 3-minute sequence, were averaged. In a different set of experiments, similar results were obtained in 3 independent experiments by using control and allergen-sensitized mice, suggesting the specificity and low variances of this method. Analysis of cytokines and chemokines Lung MNCs were prepared from 4 groups of mice. Lung tissues were chopped with sterile scissors and digested in a 37 C water bath for 2 hours in digestion buffer containing 1.5 mg/ml collagenase A (type IA; Boehringer Mannheim, Mannheim, Germany), 0.02 mg/ml DNase I (type I, Boehringer Mannheim), and 0.75 mg/ml hyaluronidase (type I, Sigma). Lung digestives were filtered by using a metal mesh to exclude cell pellets. Then filtered digestives were washed 3 times with RPMI 1640 (Gibco BRL, Rockville, Md) containing 10% FBS (Gibco BRL) and 1% penicillin-streptomycin (Gibco BRL), followed by a density gradient method to purify MNCs. These MNCs were cultured at a density of /200 µl per well in a 96-well plate in an incubator with a 95% O 2 /5% CO 2 gas mixture at 37 C for 48 hours. Four forms of stimulation were used: no stimulation (none), 100 µg/ml D farinae allergen, 2.5 µg/ml LTD 4 stimulation, and D farinae allergen plus LTD 4 stimulation by using the same concentration of D farinae and LTD 4. The concentration of LTD 4 was based on previous studies. 19,24 The concentrations of IFN-γ, IL-4, IL-5, and RANTES in the culture supernatants were determined by means of ELISA (Quantikine; R&D Systems Inc, Minneapolis, Minn) by using the procedure described in the respective instruction manual. The detection limits of IFN-γ, IL-4, IL-5, and RANTES assays were 2, 2, 7, and 2 pg/ml, respectively. Assay for transcription factor NF-κB activity NF-κB DNA-binding activity in lung MNCs from 4 groups of mice was measured by using 10 µg of nuclear extract and the ELISA-formatted transcription factor assay (Trans-AM; Active Motif, Carlsbad, Calif), according to the method provided by the manufacturer. In another set of experiments, lung MNCs were prepared from 4 groups of mice as mentioned above. These MNCs were cultured in the absence or presence of LTD 4 (2.5 µg/ml) for 4 hours, and NF-κB nuclear translocation was similarly determined. The incubation time was based on studies reported previously. 25 Absorbance was measured by using a spectrophotometer at 450 nm, with a reference wavelength of 655 nm. Statistical analysis Results are expressed as means ± SEM. Data were evaluated by using repeated-measures ANOVA with a Bonferroni multiple comparison test. A P value of less than.05 was considered significant. RESULTS CysLTs play an important role in AHR AHR to inhaled methacholine was measured by means of whole-body plethysmography (Fig 1). None of the groups showed any significant increase in sraw in response to PBS inhalation. Compared with the control group, the sraw of allergen-sensitized mice significantly increased after inhalation of 25 mg/ml methacholine.

3 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 2 Kawano et al 371 The response of pranlukast-treated mice to 25 mg/ml methacholine was not significantly different from that of control mice; however, a significant increase in sraw occurred in response to 50 mg/ml methacholine compared with that seen in control mice. The airways of allergen-sensitized mice were significantly more reactive than those of pranlukast-treated mice to 50 mg/ml methacholine. The AHR of dexamethasone-treated mice was comparable with that of control mice. Collectively, allergen-sensitized mice showed AHR to inhaled methacholine, which was significantly inhibited in pranlukasttreated mice and was completely inhibited in dexamethasone-treated mice. In vivo antagonism of cyslts attenuates allergen-induced airway inflammation Representative pathologic changes are shown in Fig 2. Allergen-sensitized mice exhibited goblet cell metaplasia and accumulation of eosinophils. The mean numbers of infiltrating eosinophils per 10 perivascular areas in pranlukast- and dexamethasone-treated mice were significantly lower than in allergen-sensitized mice (allergensensitized group, ± 12.3 cells; pranlukast, 83.2 ± 22.4 cells; dexamethasone, 5.2 ± 1.4 cells; P <.05 between allergen-sensitized and pranlukast-treated mice; P <.01 between allergen-sensitized and dexamethasonetreated mice; P <.01 between pranlukast-treated and dexamethasone-treated mice). Effects of cyslts on in vitro allergen-specific cytokine production Lung MNCs prepared from the 4 groups of mice were cultured with the medium alone, D farinae, LTD 4, or D farinae plus LTD 4. The concentrations of IFN-γ,IL-4, and IL-5 in the cultured supernatant were determined by means of ELISA (Table I). The levels of these cytokines in supernatants of MNCs cultured in medium only were less than the detection limits. IFN-γ, IL-4, and IL-5 production by lung MNCs of allergen-sensitized mice increased significantly in response to D farinae allergen stimulation. Production of these cytokines in response to D farinae was significantly less in pranlukast- and dexamethasone-treated mice, and the inhibition in dexamethasone-treated mice was more marked than that in pranlukast-treated mice. The levels of these cytokines produced by LTD 4 -stimulated MNCs were less than the detection limits. D farinae plus LTD 4 stimulation induced comparable amounts of cytokines compared with D farinae stimulation in all 4 groups (data not shown). CysLTs stimulate lung MNCs to release RANTES The concentration of RANTES in the cultured supernatant of lung MNCs was determined by means of ELISA. RANTES showed distinct results that differed from T H cytokines because it was released spontaneously by lung MNCs. Allergen-sensitized mice (798.9 ± 87.5 pg/ml) showed significantly (P <.01) higher production of RANTES compared with control mice (243.5 ± 28.9 FIG 1. Pranlukast and dexamethasone inhibit D farinae (Df) allergen-induced airway responsiveness to inhaled methacholine. Data are shown as means ± SEM of sraw (n = 12 mice for each group). *P <.05 and P <.01 versus control mice, P <.05 versus pranlukast-treated mice. pg/ml). Both pranlukast and dexamethasone significantly inhibited RANTES production (pranlukast, ± 48.9 pg/ml [P <.05]; dexamethasone, ± 14.9 pg/ml [P <.01]). Because significant differences in spontaneous release of RANTES were noted among the 4 groups, the spontaneous release was subtracted from the respective value at each stimulus in each group to estimate stimulus-induced RANTES production (Fig 3). Stimulation with D farinae, LTD 4, or both significantly enhanced RANTES production in allergen-sensitized mice. RANTES production caused by stimulation with D farinae, LTD 4, or both was significantly less in pranlukast-treated mice compared with that in allergen-sensitized mice. On the other hand, LTD 4 -induced RANTES production was not inhibited in dexamethasone-treated mice compared with that in allergen-sensitized mice. Considered together, these results indicate that LTD 4 stimulates lung MNCs to produce RANTES. LTD 4 shows an additional effect on D farinae allergen induced RANTES production from allergen-sensitized mice lungs. CysLT1 antagonist attenuates NF-κB p65 expression In preliminary experiments immunohistochemical staining for NF-κB p65 in the lung tissues demonstrated positive nuclear staining for NF-κB p65 in lung MNCs, probably representing alveolar macrophages, and the number of positively stained cells was significantly higher in allergen-sensitized mice than in control mice (data not shown). Thus a specific ELISA was performed to examine the activity of NF-κB p65 in lung MNCs. NFκB p65 activity in allergen-sensitized mice was significantly higher than in control mice. Pranlukast-treated mice showed inhibition of NF-κB p65 activation compared with allergen-sensitized mice, and dexamethasone treatment resulted in further inhibition (Fig 4).

4 372 Kawano et al J ALLERGY CLIN IMMUNOL AUGUST 2003 FIG 2. Pranlukast and dexamethasone attenuate D farinae (Df) allergen induced lung inflammation. Representative photomicrographs (400 ) of lung tissue samples of control (A), allergen-sensitized (B), pranlukasttreated (C), and dexamethasone-treated (D) groups (n = 12 for each) are shown. Allergen-sensitized mice show airway inflammatory changes, but significant inhibition of such changes are noted in pranlukast-treated mice. Dexamethasone-treated mice show almost complete inhibition of these inflammatory changes. FIG 3. RANTES concentrations in culture supernatants of isolated lung MNCs. The amount of spontaneous release was subtracted for the basal amount to adjust for the difference in spontaneous release between groups. Bars represent mean ± SEM values of 12 mice in each group. *P <.01 and P <.05 versus control mice, P <.05 and P <.01 versus allergen-sensitized mice. FIG 4. LTD 4 activates NF-κB p65 in isolated lung MNCs. Isolated lung MNCs prepared from 4 groups of mice were cultured in the absence (none) or presence (LT) of LTD 4. Bars represent mean ± SEM (n = 12 for each) values of OD450. *P <.05 versus none, P <.05 versus control mice, P <.05 versus control mice. LTD 4 activates NF-κB in isolated lung MNCs Because pranlukast inhibited NF-κB activity in isolated lung MNCs, we examined whether LTD 4 per se could activate NF-κB in lung MNCs (Fig 4). LTD 4 significantly activated NF-κB in control mice, and this effect was further enhanced in allergen-sensitized mice. LTD 4 failed to activate NF-κB in lung MNCs isolated from pranlukast-treated mice, but it significantly activated NF-κB in dexamethasone-treated mice. DISCUSSION In the present study in vivo antagonism of cyslts in a murine model of allergic asthma resulted in significant attenuation of AHR and allergic airway inflammation concomitant with inhibition of T H 2 cytokines and IFN-γ in lung tissue. Dexamethasone, a prototype of corticosteroid, inhibited cytokine production by lung MNCs to a much greater extent than pranlukast. IFN-γ represents T H 1-like cytokine but also has pro-

5 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 2 Kawano et al 373 TABLE I. Production of cytokines by lung MNCs* Group Stimulation IFN-γ (pg/ml) IL-4 (pg/ml) IL-5 (pg/ml) Control mice Medium <2.0 <2.0 <7.0 D farinae 6.9 ± ± ± 4.8 Allergen-sensitized mice Medium <2.0 <7.0 <7.0 D farinae ± ± ± Pranlukast-treated mice Medium <2.0 <7.0 <7.0 D farinae 67.5 ± ± ± 24.1 Dexamethasone-treated mice Medium <2.0 <7.0 <7.0 D farinae 6.9 ± ± ± 9.2 *Data are given as means ± SEM of 12 mice in each group. P <.01 and P <.05 versus control mice. P <.05 versus allergen-sensitized mice. inflammatory effects, including prolongation of eosinophil survival and increased expression of cell adhesion molecules on airway epithelial cells. 1,26,27 Thus in vivo antagonism of cyslts by pranlukast resulted in anti-inflammatory effects in the lung. In vivo antagonism of cyslts resulted in attenuation of allergen-specific cytokine production, whereas cyslts per se failed to stimulate MNCs to produce cytokines. This finding adds support to the previous results, showing that lymphocytes do not primarily express cyslt1. 28 Menard and Bissonnette 29 found that LTD 4 did not stimulate human alveolar macrophages to produce macrophage inflammatory protein 1α and TNF, but it primed macrophages for LPSstimulated cytokine production. It is also reported that LTD 4 augments epidermal growth factor induced human airway smooth muscle cell proliferation; however, LTD 4 per se does not cause such proliferation. 30 These reports and the present results suggest an indirect effect for cyslts on allergen-induced cytokine production. In contrast to cytokines, our in vitro studies indicated that cyslts stimulated lung MNCs to produce a significant amount of RANTES and showed additional effects on allergen-specific RANTES production from lung MNCs. RANTES, a CC chemokine, represents a crucial chemokine involved in allergic and autoimmune diseases because of its potential effect on migration of eosinophils and lymphocytes. 3-5 Our results showed that pranlukast but not dexamethasone inhibited LTD 4 -induced RANTES production, suggesting that lung MNCs produce RANTES by means of LTD 4 through cyslt1. In the present study alveolar macrophages, the primary lung cells that produce RANTES, 31 produced RANTES through cyslt1 because they express abundant amounts of cyslt1. 28 Equally important was that antagonism of cyslts resulted in inhibition of NF-κB and LTD 4 -activated NF-κB in lung MNCs. In the present study NF-κB activity was estimated by using an ELISA-formatted transcription factor assay. This method is highly reproducible, specific for NF-κB, and more sensitive than the regular radioactive gel shift. 32,33 In agreement with the present results, previous studies suggested that arachidonic acid metabolites, especially leukotrienes, act as second messengers in TNF-induced NF-κB activation because TNFinduced NF-κB activation is attenuated by leukotriene synthesis inhibitors. 34,35 The production of RANTES is regulated by NF-κB at a transcriptional level. Thus cyslts might be involved in RANTES production through regulation of NF-κB. Collectively, these results suggest that cyslts modulate infiltration of eosinophils and lymphocytes through RANTES production from alveolar macrophages in addition to their direct chemotactic effects on eosinophils. Thus RANTES-induced accumulation of eosinophils and activated lymphocytes might result in increased release of T H 2 cytokines. Interestingly, RANTES has a distinct immune effect that differs from those of other chemokines. RANTES shows biphasic T-cell signaling pathways in a dosedependent manner RANTES acts as a typical chemokine at nanomolar concentrations by binding to chemokine receptors (eg, CCR3), but it also triggers the protein tyrosine kinase mediated pathway to cause mitogen-like leukocyte activation by binding to glycosaminoglycans at micromolar concentrations. 37 The latter action to activate T cells is followed by many diverse effects, including IL-5 production from T cells in the absence of allergen. These mechanisms could be involved in the present results. In conclusion, cyslts might contribute to allergic airway inflammation in 2 ways. First, cyslts directly cause airway smooth muscle constriction, increased vascular permeability, and mucous hypersecretion. Second, cyslts activate NF-κB in lung tissue, which leads to RANTES production. CysLT-induced RANTES production could exacerbate airway inflammation. We thank Dr F. G. Issa for the careful reading and editing of the manuscript. 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