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Mast cells play a key role in Th2 cytokine-dependent asthma model through production of adhesion molecules by liberation of TNF-α

Mast cells play a key role in Th2 cytokine-dependent asthma model through production of adhesion... tion, and airway hyperresponsiveness (Cohn et al., Mast cells are well recognized as key cells in allergic 2004). Airway inflammation in atopic asthma is reactions, such as asthma and allergic airway di- associated with a T-helper type 2 (Th2) immune response in which Th2 cell-derived cytokines are seases. However, the effects of mast cells and TNF-α thought to contribute to eosinophil recruitment, on T-helper type 2 (Th2) cytokine-dependent asthma mucus hypersecretion, and airway hyperrespon- are not clearly understood. Therefore, an aim of this siveness (Cohn et al., 2004; Woodruff et al., 2009; study was to investigate the role of mast cells on Th2 Kim et al., 2010). cytokine-dependent airway hyperresponsiveness and Mast cells are the main effector cells of espe- inflammation. We used genetically mast cell-defi- cially the early phase of the allergic reaction and W W-v v cient WBB6F1/J-Kit /Kit (W/W ), congenic normal immediated asthmatic response. Through antigen + + WBB6F1/J-Kit /Kit (+/+), and mast cell-reconstituted cross-linking to high affinity IgE receptors on the W/W mouse models of allergic asthma to investigate cell surface, mast cells discharge a group of the role of mast cells in Th2 cytokine-dependent asth- mediators (Galli and Tsai, 2008), including his- ma induced by ovalbumin (OVA). And we investigated tamine. Mast cell-derived lipid mediators and whether the intratracheal injection of TNF-α directly in- inflammatory cytokines, such as leukotrienes, IL-6, duce the expression of ICAM-1 and VCAM-1 in W/W and TNF-α, have also been reported to play an mice. This study, with OVA-sensitized and OVA-chal- important role in the late asthmatic response and airway hyperresponsiveness (Matsuoka et al., 2000; lenged mice, revealed the following typical histopatho- 36 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 Results Airway responsiveness to methacholine in mice after OVA challenge The dose curve of PenH responses to aerosolized methacholine of OVA-sensitized and -challenged mice shifted to the left, compared with that of saline-sensitized and -challenged mice (Figure 1). PenH responses to aerosolized methacholine in- creased significantly in OVA-sensitized and -cha- llenged +/+ mice and BMCMC→W/W mice, com - pared with those of saline-sensitized and -cha- v v llenged +/+ W/W , and BMCMC→W/W mice. Figure 1. Resistance of mice to methacholine after saline sensitization PenH responses in OVA-sensitized and -cha- and challenge or ovalbumin (OVA) sensitization and challenge in +/+, llenged W/W mice were significantly lower than v v W/W , and BMCMC→W/W mice. Data represent the mean ± SEM of 5 v those in OVA-sensitized and -challenged +/+ mice mice in each group. *P < 0.05, ***P < 0.001 versus W/W (OVA). at 50 mg/ml of methacholine inhalation. However, PenH responses in OVA-sensitized and -challeng- ed BMCMC→W/W mice were significantly higher Zimmermann et al., 2003; Antunez et al., 2006; at 50 mg/ml of methacholine inhalation than in Woodruff et al., 2009). TNF-α is a chemotactic v OVA-sensitized and -challenged W/W mice. PenH cytokine for granulocytes, including eosinophils responses to aerosolized methacholine in BMCMC and neutrophils (Sabatini et al., 2002; Chai et al., 2005; Yoshifuku et al., 2007), probably by up- regulation of cellular adhesion molecules, such as vascular cellular adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1, thus fa- cilitating migration of eosinophils and neutrophils. During this process, they also become primed for mediator secretion. Ultimately, this will lead to chronic inflammation and irreversible airway re- modeling, a key feature in bronchial asthma. How - ever the role of TNF-α in allergic inflammation is still controversial. Also, the relationship between TNF-α and adhesion molecules in asthma is not clear. Therefore, in this study, we investigated the possible roles of mast cells with regard to certain parameters, such as ICAM-1 and VCAM-1, of al- lergic inflammation in a Th2 cytokine-dependent asthma model using genetically mast cell-defici- W W-v v ent WBB6F1/J-Kit /Kit (W/W ), congenic normal + + WBB6F1/J-Kit /Kit (+/+), and mast cell-reconsti- v v tuted W/W (BMCMC→W/W ) mice. And we in- vestigated whether the intratracheal injection of TNF-α directly induce the expression of ICAM-1 and VCAM-1 in W/W mice. We found that mast cells are essential in development of Th2 cy- tokine-dependent asthma, because of the libera- tion of TNF-α, which activates and enhances pro- duction of ICAM-1 and VCAM-1. Figure 2. Infiltration of (A) inflammatory cells and (B) differential cellular components and total cells in bronchoalveolar lavage fluid after saline sensitization and challenge (Saline) or ovalbumin sensitization and chal- v v lenge (OVA) in +/+, W/W , and BMCMC→W/W mice. **P < 0.01 ver- v ## v sus W/W (OVA), P < 0.01 versus W/W (Saline). Role of mast cells in asthma 37 Figure 3. Histopathologic changes in lung tissues after saline sensitization and challenge (Saline) or ovalbumin v v sensitization and challenge (OVA) in +/+, W/W , and BMCMC→W/W mice. Lung tissues were stained with hema- toxylin and eosin (H-E) for inflammatory cells, congo red for eosinophils, and periodic acid-Schiff (PAS) for goblet cells and mucus. infiltration around bronchioles, mucus had ac- →W/W mice were restored to levels found in OVA-sensitized and -challenged +/+ mice. cumulated in the lumen of bronchioles, and hy- perplasia of goblet cells was observed at the epithelium of large airways after OVA sensitization Cellular changes in mice after OVA challenge and challenge, compared with those after saline Numbers of macrophages, lymphocytes, neutro- sensitization and challenge in +/+ mice. However, phils, eosinophils, and total cells in BAL fluids infiltration of eosinophils around bronchioles, ac- showed a significant increase at 48 h after OVA cumulation of mucus in the lumen of bronchioles, sensitization and challenge, compared with those and hyperplasia of goblet cells at the epithelium of after saline sensitization and challenge in +/+ mice large airways of OVA sensitized and challenged (Figure 2). However, the numbers of macrophages, W/W mice were significantly lower than in OVA- lymphocytes, neutrophils, eosinophils, and total sensitized and -challenged +/+ mice. On the other cells in BAL fluids of OVA-sensitized and -cha- hand, infiltration of eosinophils around bronchioles, llenged W/W mice were significantly lower than in accumulation of mucus in the lumen of bron- OVA-sensitized and -challenged +/+ mice. On the chioles, and hyperplasia of goblet cells at the other hand, numbers of macrophages, lympho- epithelium of large airways of BMCMC→W/W cytes, neutrophils, eosinophils, and total cells in mice after OVA sensitization and challenge were BAL fluids of BMCMC→W/W mice after OVA significantly higher than in OVA-sensitized and sensitization and challenge were significantly -challenged W/W mice. higher than in OVA-sensitized and -challenged W/W mice. Levels of Th2 cytokines of BAL fluids in mice after OVA challenge Histopathologic changes of lung challenged by OVA Enzyme immunoassays showed that levels of Histopathologic analyses revealed typical patho- TNF-α, IL-4, and IL-5 protein in BAL fluids were logic features of asthma in OVA-sensitized and significantly increased after OVA sensitization and -challenged mice (Figure 3). Lung tissues in mice challenge, compared with saline-sensitized and v v given OVA were characterized by gross alterations -challenged +/+ , W/W , and BMCMC→W/W mice in the structural integrity of the airway walls and (Figure 4). Levels of TNF-α, IL-4, and IL-5 protein parenchyma, epithelial cell shedding, microvascu- in BAL fluids in OVA-sensitized and -challenged lar leakage and extensive mucosal edema, in- W/W mice were similar to levels found in sa- creased tissue cellularity, and particulate exudated line-sensitized and -challenged W/W mice. How- in airway lumina and alveolar septa. Numerous ever, levels of TNF-α, IL-4, and IL-5 protein in BAL inflammatory cells, such as eosinophils, showed fluids in OVA-sensitized and -challenged BMCMC 38 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 teins in lung tissue increased significantly with OVA challenge, compared with those after saline cha- llenge in +/+ mice (Figure 5C). However, levels of ICAM-1 and VCAM-1proteins in lung tissue of OVA-sensitized and -challenged W/W mice were significantly lower than in lung tissue of OVA- sensitized and -challenged +/+ mice. On the other hand, in BMCMC→W/W mice, levels of ICAM-1 and VCAM-1 protein were restored to the level found in OVA-sensitized and -challenged +/+ mice (Figure 5C). Figure 4. Enzymatic immunoassay for TNF-α, IL-4, and IL-5 proteins in Effects of TNF-α on ICAM-1 & VCAM-1 protein levels bronchoalveolar lavage fluid after saline sensitization and challenge in W/W mice (Saline) or ovalbumin sensitization and challenge (OVA) in +/+, W/W , v v ## and BMCMC→W/W mice. ***P < 0.001 versus W/W (OVA), P < To investigate the effect of TNF-α on expression of 0.01 versus W/W (Saline). ICAM-1 and VCAM-1 proteins, we assessed the →W/W mice had almost reached the levels found in OVA-sensitized and -challenged +/+ mice. ICAM-1 & VCAM-1 expression in OVA challenged mice Two common and distinct pathways mediating mononuclear cell adhesion to pulmonary endo- thelium were investigated. Expression of endo- thelial ICAM-1 was observed in sections of blood vessels from lung of OVA-sensitized and -cha- llenged +/+ mice and BMCMC→W/W mice, whereas in saline-sensitized and -challenged +/+, v v , and BMCMC→W/W mice, ICAM-1 was W/W hardly detected in endothelial cells of blood vessels. Also, ICAM-1 was hardly detected in endothelial cells of blood vessels of OVA-sen- sitized and -challenged W/W mice (Figure 5A). In the contrast with ICAM-1, VCAM-1 expression was significantly heightened in vascular endothelium in blood vessels of OVA-sensitized and -challenged +/+ mice and BMCMC→W/W mice and was most pronounced in vessels with perivascular inflam- matory foci. Interestingly, VCAM-1 was also ex- pressed on smooth muscle cells of blood vessels and bronchioles (Figure 5B). Whereas in sa- line-sensitized and -challenged +/+, W/W , and BMCMC→W/W mice, VCAM-1 was hardly de- tected in endothelial cells of blood vessels. Also, VCAM-1 was hardly detected in endothelial cells of Figure 5. ICAM-1 and VCAM-1 proteins in lung tissues after saline sen- blood vessels of OVA-sensitized and -challenged sitization and challenge (Saline) or ovalbumin sensitization and challenge v v v v W/W mice. In BMCMC→W/W mice, expression of (OVA) in +/+, W/W , and BMCMC→W/W mice. Immunohistochemistry ICAM-1 and VCAM-1 in endothelial cells of blood of (A) ICAM-1 (arrows) and (B) VCAM-1 (arrows) in endothelial cells and smooth muscle cells of blood vessels (*) of the lungs after saline sensiti- vessels was restored to the levels found in OVA- zation and challenge or ovalbumin sensitization and challenge in +/+, sensitized and -challenged +/+ mice (Figure 5B). v v W/W and BMCMC→W/W mice. Br indicates bronchioles. Arrow heads Consistent with these results, western blot analysis show immunoreactive ICAM-1 and VCAM-1. (C) Western blotting of revealed that levels of ICAM-1 and VCAM-1 pro- ICAM-1and VCAM-1proteins in lung tissue. Role of mast cells in asthma 39 immunohistochemical analysis and western blot analysis of ICAM-1 and VCAM-1 proteins of lung tissue after intra tracheal injection of TNF-α in W/W mice. Immunohistochemical analysis re- vealed that expressions of ICAM-1 and VCAM-1 were observed of in sections of blood vessels from lung of TNF-α intra tracheal injected W/W mice. Localization of ICAM-1 and VCAM-1 expressions in endothelial layers around inflammation area and immunoreactive expression of VCAM-1 was more intensive than ICAM-1 (Figure 6A). Western blot analysis revealed that levels of ICAM-1 and VCAM-1 proteins were increased in a dose-depend- ent manner from lung of W/W mice (Figure 6B). Discussion Figure 6. Effects of TNF-α on ICAM-1 & VCAM-1 protein levels in W/W mice. (A) Immunohistochemistry of ICAM-1 and VCAM-1 (arrows). * in- In this study, we investigated the possible roles of dicates blood vessels of the lungs; Br, bronchioles. (B) Western blotting mast cells with regard to certain parameters of of ICAM-1 and VCAM-1 24 h after intra-tracheal injection of TNF-α in allergic inflammation in a Th2 cytokine-dependent W/W mouse. asthma model using genetically mast cell-deficient W/W mice, congenic normal +/+, and BMCMC release spasmogenic, vasoactive and proinflam- →W/W mouse models. Our results suggest that mast cells play a key role in a Th2 cytokine- matory mediators, which in turn can enhance dependent asthma model through production of airway smooth muscle contraction, vascular per- meability, and inflammatory cell (T cells, dendritic VCAM-1 and ICAM-1 by release of TNF-α. Asthma is perceived as a Th2 disease with a cell, neutrophils, eosinophils, and monocytes) re- cruitment at sites of antigen challenge (Bieder- particular profile of cytokine release, which is thought to include IL-4 and IL-5 (Woodruff et al., mann et al., 2000; Bradding et al., 2006; Suto et 2009). Increasing evidence indicates that other al., 2006; Kneilling et al., 2009). Chemokines and cytokines, which in mice are classically considered cytokines are also released and induce neutrophil to belong to Th1-type profiles, are also associated influx upon injection of cytokines, such as TNF-α (Ryan et al., 2007; Galli et al., 2008; Kawakami et with the inflammatory response that characterizes human asthma (Antunez et al., 2006). In the last al., 2009). On the other hand, locally released decade it has become evident that cytokines play a chemokines may activate resident cells to release pivotal role in the pathogenesis of asthma (Zi- cytokines. According to these reports, it is sug- mmermann et al., 2003; Antunez et al., 2006). The gested that recruitment of inflammatory cells, ac- tivation and cytokine release from resident cells role of antigen-induced TNF-α release and antigen stimulation of other cytokines is an important area are consequent on mast cell activation by OVA. However, the asthma model used in this manu- of study. In addition to TNF-α, cytokines including script is mast cell-dependent, so there did not IL-1β, IL-2, IL-3, IL-4, IL-5, granulocyte-macro- show the changes accompanied by a mast cell- phage-colony stimulating factor, and interferon-γ dependent exacerbation of airway inflammation to have also been implicated in development of the asthmatic inflammatory response (Thomas, 2001; OVA, and then did not induce cascade reactions Zimmermann et al., 2003; Antunez et al., 2006). such as, migration, local accumulation, and ac- tivation of proinflammatory cells and residential cell In this study, TNF-α showed an increase in BAL fluid after the second OVA challenge in +/+ mice, populations in the airway. So it is suggested that absence of mast cell result in a significant de- whereas OVA-sensitized and -challenged W/W mice showed a significant decrease in TNF-α crease in TNF-α, IL-4, and IL-5 levels. Our study levels (up to 75%) in their BAL fluid. In addition, provides evidence to show that cytokines play a pivotal role in the pathogenesis of asthma (Zi- TNF-α levels were significantly restored in BAL fluid in OVA-sensitized and -challenged BMCMC mmermann et al., 2003; Antunez et al., 2006). Evidence for expression of ICAM-1 and VCAM-1 →W/W mice. Also, other cytokines, IL-4 and IL-5, in smooth muscle cells in inflammatory diseases, showed a pattern similar to that of TNF-α. Mast cell activation by specific antigen induces the cells to such as arthrosclerosis and asthma, is mounting. 40 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 The exact roles of expression of such adhesion the direct effect of TNF-α on up-regulation of molecules are not fully understood; however, it has ICAM-1 and VCAM-1 after intra tracheal injection been suggested that expression facilitates trans- of TNF-α in W/W mice. Up-regulation of ICAM-1 migration and accumulation of leukocytes at in- and VCAM-1 increased in a dose manner of TNF-α flammatory sites. Increasing evidence has demon- in W/W mice. This data showed that TNF-α strated the importance of cytokine-inducible leu- induced adhesion molecules in the pulmonary kocyte-endothelial adhesion molecules in recruit- endothelium of asthma. ment and migration of leukocytes to sites of TNF-α is stored in granules and is known to be inflammation (Montefort and Holgate, 1991; Ando released during allergic responses from both mast et al., 2001; Chihara, 2005). Expression of both cells and macrophages via IgE-dependent me- ICAM-1 and VCAM-1 by smooth muscle cells in chanisms (Thomas, 2001; Zimmermann et al., response to several inflammatory factors within 2003; Antunez et al., 2006). This finding indicated airways has been reported in asthma animal that this cytokine will be coreleased with more models and allergic asthmatic patients (Gosset et extensively characterized preformed mast cell al., 1995; Lee et al., 2003). In our study, we found granule mediators, such as histamine, chymase, that expression of ICAM-1 and VCAM-1 in lung and tryptase. Mast cell cytokines are predomi- tissue was significantly increased in OVA-sen- nantly Th2 cytokines, which are critical to de- sitized and -challenged +/+ mice, compared with velopment and maintenance of the asthmatic levels in OVA-sensitized and -challenged W/W IgE-driven condition, and TNF-α has been shown mice and saline-sensitized and -challenged +/+ to induce airway hyperreactivity. In our study, we mice. However, expression levels of ICAM-1 and demonstrated that expression of ICAM-1, VCAM-1, VCAM-1 in lung tissue of OVA-sensitized and and protein in lung tissues was significantly increased after OVA sensitization and challenge of -challenged BMCMC→W/W mice increased to levels similar to those found in OVA-sensitized and +/+ mice, compared with levels of saline sen- -challenged +/+ mice and was most pronounced in sitization and challenge in+/+ mice or OVA sen- vessels with perivascular inflammatory foci. Inter- sitization and challenge in W/W mice. However, estingly, VCAM-1 was also expressed on smooth expression of ICAM-1, VCAM-1, and protein in muscle cells of blood vessels and bronchioles. lung tissue of OVA-sensitized and -challenged Therefore, our data suggest that ICAM-1 and BMCMC→W/W mice increased to levels similar to VCAM-1 may play an important role in Th2 those found in OVA-sensitized and -challenged +/+ cytokine-dependent asthma and that mast cells mice. Therefore, our results suggest that mast cell may have an important action in induction and cytokines, particularly TNF-α, are critical to de- expression of ICAM-1 and VCAM-1 in Th2 cy- velopment and maintenance of the asthma model tokine-dependent asthma. through production of ICAM-1 and VCAM-1. TNF-α, a cytokine that plays a role in many inflammatory diseases, is produced mainly by macrophages; however, monocytes, dendritic cells, Methods B cells, mast cells, and eosinophils are also known to be crucial in the pathogenesis of asthma. Also, Mice TNF-α is a chemotactic cytokine for granulocytes Genetically male mast cell-deficient mice W/W mice and (Sabatini et al., 2002; Chai et al., 2005; Yoshifuku their congenic littermates +/+ mice, 5 weeks of age, were et al., 2007), probably by up-regulation of cellular obtained from Japan SLC, Inc. W/W mice ordinarily con- adhesion molecules, such as E-selectin, ICAM-1 tain less than 1.0% of the number of dermal mast cells and VCAM-1, thus facilitating migration of eosi- present in the skin of congenic normal +/+ mice and have nophils and neutrophils. During this process, they no detectable mature mast cells in the respiratory system or other anatomic sites (Wolters et al., 2005; Galli and Tsai, also become primed for mediator secretion. Ul- 2008). timately, this will lead to chronic inflammation and irreversible airway remodeling, a key feature in bronchial asthma. The role of TNF-α in allergic Mast cell reconstitution inflammation is still controversial. TNF-α induction Selective reconstitution of mast cells in mast cell deficient of adhesion molecules in pulmonary endothelium is W/W mice was carried out according to the method important for eosinophil recruitment (Wong et al., described by Williams and Galli (2000), with slight modi- 2006); however, it has also been implicated in fications. Suspended bone marrow cells from +/+ mice negative modulation of eosinophil level, IL-5 pro- were cultured in WEHI-3 conditioned medium containing duction, and, consequently, airway responsiveness IL-3 for 4-5 weeks, at which time the cell populations were (Kanehiro et al., 2001). In this study, we revealed composed of> 95% immature mast cells, as assessed by Role of mast cells in asthma 41 staining with toluidine blue and Kimura staining. Five mi- Differential cell counts were determined with cytospin llion bone marrow-derived cultured mast cells (BMCMCs) (Centrifuge 5403; Eppendorf, Hamburg, Germany) prepa- were infused via the tail vein into each W/W mouse, and ration, followed by Diff Quik staining (Sysmex Co., Kobe, the recipients (BMCMC→W/W mice) were studied at least Japan). 16 wks later. There were 0.508± 0.09 mast cells/mm in lung tissue of BMCMC→W/W mice (vs none or 0.38± 2 v Histopathology and immunohistochemistry 0.035 mast cells/mm in lung tissue of W/W or +/+ mice, respectively). Histopathologic analysis of lung and immunohistochemical examination of ICAM-1 and VCAM-1 were performed as previously described (Vrugt et al., 2000). Animals were Immunization and challenge with OVA sacrificed with an overdose of ether at 48 h after the last v v OVA exposure and histologic specimens were collected. BMCMC→W/W and age-matched +/+, W/W mice were Using routine histologic procedures for light microscopic used in these experiments. Mice were sensitized and evaluation, tissue specimens were taken from the mid challenged with ovalbumin (OVA) (Sigma-Aldrich Co., St. zone of the left lung of the mice, and fixed in 10% formalin Louis, MO), as described previously, with some modifi- cations (Williams and Galli, 2000; Kim et al., 2007) and embedded in paraffin. Serial sections measuring 5 μm in thickness were then cut, and stained with hematoxylin (Supplemental Data Figure S1). In brief, mice were and eosin for inflammatory cells, congo red for eosinophils, immunized by intraperitoneal injection of 20 μg OVA and and periodic acid-Schiff for goblet cells and mucus. The saline in a total volume of 200 μl on days 0 and 14. Twenty others were stained with anti-ICAM-1 and anti-VCAM-1 eight days after the beginning of the sensitization period, antibodies. these mice received their first intranasal challenge of 30 μl of saline containing 200 μg of OVA. After 7 days, these mice were challenged for 30 min with an aerosol of 5% Measurements of Th2 cytokines in BAL fluids (wt/vol) OVA in saline using ultrasonic nebulization (NE-U12; Omron Crop., Tokoyo, Japan). BAL was performed and BAL fluid was collected for analysis of Th2 cytokines in BAL fluid, smears of BAL cells were prepared by cytospin (Centrifuge 5403; Eppendorf, Measurement of airway reactivity Hamburg, Germany). Levels of Th2 cytokines in super- natants of BAL fluids were quantified by mouse IL-4, IL-5, Twenty four hours after the final OVA challenge, airway and TNF-α BD ELISA sets (BD PharMingen). reactivity to aerosolized methacholine was measured using a whole body plethysmograph, as described by Ohtomo et al. (2009). In brief, unrestrained conscious mice were Western blot analysis placed in whole body plethysmographic chambers, and, after 5 min of stabilization, dose response curves to aero- Mice lungs were minced and sonicated in Pro-Prep buffer solized methacholine were generated. Increasing concen- (50 mg/ml) containing 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, trations of methacholine were aerosolized for 3 min each, 150 mM NaCl, and protease inhibitors using a tip-type data for lung resistance and dynamic compliance were sonicator (Branson sonifier, Branson ultrasonics, CT, 20% continuously monitored by using OCP 3000CPS-A soft- amplitude, 3 min), followed by centrifugation for 20 min at ware (allmedicus) and mean airway bronchoconstriction 1,000× g. Protein expression levels in cell lysates were readings, as assessed by enhanced respiratory pause analyzed by Western blot analysis, as previously described (PenH), were obtained over 10 min periods. PenH can be (Vrugt et al., 2000; Lee et al., 2003). Equal amounts of cell conceptualized as the phase shift of the thoracic flow and lysate were separated on an SDS polyacrylamide gel nasal flow curves; increased phase shift correlates with under reducing conditions, and were then transferred onto increased respiratory system resistance. PenH is cal- Protran nitrocellulose membranes (Schleicher & Schuell, culated by the formula PenH = (Te/RT - 1) × PEF/PIF, Keene, NH). Membranes were blocked for 2 h at room where Te is expiratory time, RT is relaxation time, PEF is temperature in 3% skim milk in TBS, followed by overnight peak expiratory flow, and PIF is peak inspiratory flow. incubation with anti-TNF-α (BD PharMingen), anti-ICAM-1 (BD PharMingen), and anti-VCAM-1 (Santa Cruz). Blots were washed for 15 min with three changes of transfer Collection and analysis of bronchoalveolar lavage buffer-0.05% Tween 20 solution, followed by 1 h incubation (BAL) fluid at room temperature with HRP-conjugated anti-mouse antibody or anti-rabbit IgG antibody. Blots were washed Forty eight hours after the final OVA challenge, BAL fluid was collected by cannulating the upper part of the trachea again for 15 min and finally developed in ECL Western detection reagents (Amersham-Pharmacia Biotech, Pis- and lavaging three times with 1 ml saline. The lavage fluid cataway, NJ). Signal intensities of specific bands were collected was centrifugated at 400× g for 5 min at 4 C, analyzed quantitatively using a Fluor-STM Imager (Bio- and the cells were separated from the fluid. The fluid was Rad, Muncher, Germay) and plotted as relative intensity. then centrifugated at 1,000× g for 2 min at 4 C for removal of cellular debris and then stored at -20 C until it was evaluated. Cells were resuspended in PBS containing Statistical analyses 1% FBS, and the total number of viable cells was de- termined by trypan blue exclusion using a hemocytometer. Unless otherwise specified, differences in response bet- 42 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 ween different subgroups were tested for statistical sig- Nat Rev Immunol 2008;8:478-86 nificance using an unpaired Student's t test (two tailed). P Gosset P, Tillie-Leblond I, Janin A, Marquette CH, Copin MC, < 0.05 was considered statistically significant. Unless Wallaert B, Tonnel AB. Expression of E-selectin, ICAM-1 and otherwise specified, all data are presented as mean± VCAM-1 on bronchial biopsies from allergic and non-allergic SEM. asthmatic patients. Int Arch Allergy Immunol 1995;106:69- Acknowlegements Kanehiro A, Lahn M, Makela MJ, Dakhama A, Fujita M, Joetham A, Mason RJ, Born W, Gelfand EW. Tumor necrosis 1. OH Chai was supported by a Korea Research Founda- factor-alpha negatively regulates airway hyperresponsive - tion Grant funded by the Korean Government (MOEHRD, ness through gamma-delta T cells. Am J Respir Crit Care Basic Research Promotion Fund; KRF-2007-331-E00009). Med 2001;164:2229-38 2. CH Song was supported by a Korea Research Founda- tion Grant funded by the Korean Government (KRF-2008- Kawakami T, Ando T, Kimura M, Wilson BS, Kawakami Y. 313-E00014). Mast cells in atopic dermatitis. Curr Opin Immunol 2009; 21:666-78 Kim YS, Ko HM, Kang NI, Song CH, Zhang X, Chung WC, Supplemental data Kim JH, Choi IH, Park YM, Kim GY, Im SY, Lee HK. Mast cells Supplemental Data include a figure and can be found with play a key role in the development of late airway hyper- this article online at http://e-emm.or.kr/article/article_files/ responsiveness through TNF-alpha in a murine model of SP-43-1-05.pdf. asthma. 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Induction of adhesion molecules upon the interaction J Immunol 2006;176:4102-12 between eosinophils and bronchial epithelial cells: in- volvement of p38 MAPK and NF-kappaB. Int Immuno- Thomas PS. Tumour necrosis factor-alpha: the role of this pharmacol 2006;6:1859-71 multifunctional cytokine in asthma. Immunol Cell Biol 2001; 79:132-40 Woodruff PG, Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A, Koth LL, Arron JR, Fahy JV. T-helper type Vrugt B, Wilson S, Bron A, Holgate ST, Djukanovic R, Aalbers 2-driven inflammation defines major subphenotypes of R. Bronchial angiogenesis in severe glucocorticoid-de- asthma. Am J Respir Crit Care Med 2009;180:388-95 pendent asthma. Eur Respir J 2000;15:1014-21 Yoshifuku K, Matsune S, Ohori J, Sagara Y, Fukuiwa T, Williams CM, Galli SJ. Mast cells can amplify airway reactivity Kurono Y. IL-4 and TNF-alpha increased the secretion of and features of chronic inflammation in an asthma model in eotaxin from cultured fibroblasts of nasal polyps with mice. J Exp Med 2000;192:455-62 eosinophil infiltration. Rhinology 2007;45:235-41 Wolters PJ, Mallen-St Clair J, Lewis CC, Villalta SA, Baluk Zimmermann N, Hershey GK, Foster PS, Rothenberg ME. P, Erle DJ, Caughey GH. Tissue-selective mast cell re- Chemokines in asthma: cooperative interaction between constitution and differential lung gene expression in mast chemokines and IL-13. J Allergy Clin Immunol 2003;111: cell-deficient Kit(W-sh)/Kit(W-sh) sash mice. Clin Exp Al- 227-42 lergy 2005;35:82-8 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Experimental & Molecular Medicine Springer Journals

Mast cells play a key role in Th2 cytokine-dependent asthma model through production of adhesion molecules by liberation of TNF-α

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Springer Journals
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Copyright © 2011 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Molecular Medicine; Medical Biochemistry; Stem Cells
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2092-6413
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10.3858/emm.2011.43.1.004
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Abstract

tion, and airway hyperresponsiveness (Cohn et al., Mast cells are well recognized as key cells in allergic 2004). Airway inflammation in atopic asthma is reactions, such as asthma and allergic airway di- associated with a T-helper type 2 (Th2) immune response in which Th2 cell-derived cytokines are seases. However, the effects of mast cells and TNF-α thought to contribute to eosinophil recruitment, on T-helper type 2 (Th2) cytokine-dependent asthma mucus hypersecretion, and airway hyperrespon- are not clearly understood. Therefore, an aim of this siveness (Cohn et al., 2004; Woodruff et al., 2009; study was to investigate the role of mast cells on Th2 Kim et al., 2010). cytokine-dependent airway hyperresponsiveness and Mast cells are the main effector cells of espe- inflammation. We used genetically mast cell-defi- cially the early phase of the allergic reaction and W W-v v cient WBB6F1/J-Kit /Kit (W/W ), congenic normal immediated asthmatic response. Through antigen + + WBB6F1/J-Kit /Kit (+/+), and mast cell-reconstituted cross-linking to high affinity IgE receptors on the W/W mouse models of allergic asthma to investigate cell surface, mast cells discharge a group of the role of mast cells in Th2 cytokine-dependent asth- mediators (Galli and Tsai, 2008), including his- ma induced by ovalbumin (OVA). And we investigated tamine. Mast cell-derived lipid mediators and whether the intratracheal injection of TNF-α directly in- inflammatory cytokines, such as leukotrienes, IL-6, duce the expression of ICAM-1 and VCAM-1 in W/W and TNF-α, have also been reported to play an mice. This study, with OVA-sensitized and OVA-chal- important role in the late asthmatic response and airway hyperresponsiveness (Matsuoka et al., 2000; lenged mice, revealed the following typical histopatho- 36 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 Results Airway responsiveness to methacholine in mice after OVA challenge The dose curve of PenH responses to aerosolized methacholine of OVA-sensitized and -challenged mice shifted to the left, compared with that of saline-sensitized and -challenged mice (Figure 1). PenH responses to aerosolized methacholine in- creased significantly in OVA-sensitized and -cha- llenged +/+ mice and BMCMC→W/W mice, com - pared with those of saline-sensitized and -cha- v v llenged +/+ W/W , and BMCMC→W/W mice. Figure 1. Resistance of mice to methacholine after saline sensitization PenH responses in OVA-sensitized and -cha- and challenge or ovalbumin (OVA) sensitization and challenge in +/+, llenged W/W mice were significantly lower than v v W/W , and BMCMC→W/W mice. Data represent the mean ± SEM of 5 v those in OVA-sensitized and -challenged +/+ mice mice in each group. *P < 0.05, ***P < 0.001 versus W/W (OVA). at 50 mg/ml of methacholine inhalation. However, PenH responses in OVA-sensitized and -challeng- ed BMCMC→W/W mice were significantly higher Zimmermann et al., 2003; Antunez et al., 2006; at 50 mg/ml of methacholine inhalation than in Woodruff et al., 2009). TNF-α is a chemotactic v OVA-sensitized and -challenged W/W mice. PenH cytokine for granulocytes, including eosinophils responses to aerosolized methacholine in BMCMC and neutrophils (Sabatini et al., 2002; Chai et al., 2005; Yoshifuku et al., 2007), probably by up- regulation of cellular adhesion molecules, such as vascular cellular adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1, thus fa- cilitating migration of eosinophils and neutrophils. During this process, they also become primed for mediator secretion. Ultimately, this will lead to chronic inflammation and irreversible airway re- modeling, a key feature in bronchial asthma. How - ever the role of TNF-α in allergic inflammation is still controversial. Also, the relationship between TNF-α and adhesion molecules in asthma is not clear. Therefore, in this study, we investigated the possible roles of mast cells with regard to certain parameters, such as ICAM-1 and VCAM-1, of al- lergic inflammation in a Th2 cytokine-dependent asthma model using genetically mast cell-defici- W W-v v ent WBB6F1/J-Kit /Kit (W/W ), congenic normal + + WBB6F1/J-Kit /Kit (+/+), and mast cell-reconsti- v v tuted W/W (BMCMC→W/W ) mice. And we in- vestigated whether the intratracheal injection of TNF-α directly induce the expression of ICAM-1 and VCAM-1 in W/W mice. We found that mast cells are essential in development of Th2 cy- tokine-dependent asthma, because of the libera- tion of TNF-α, which activates and enhances pro- duction of ICAM-1 and VCAM-1. Figure 2. Infiltration of (A) inflammatory cells and (B) differential cellular components and total cells in bronchoalveolar lavage fluid after saline sensitization and challenge (Saline) or ovalbumin sensitization and chal- v v lenge (OVA) in +/+, W/W , and BMCMC→W/W mice. **P < 0.01 ver- v ## v sus W/W (OVA), P < 0.01 versus W/W (Saline). Role of mast cells in asthma 37 Figure 3. Histopathologic changes in lung tissues after saline sensitization and challenge (Saline) or ovalbumin v v sensitization and challenge (OVA) in +/+, W/W , and BMCMC→W/W mice. Lung tissues were stained with hema- toxylin and eosin (H-E) for inflammatory cells, congo red for eosinophils, and periodic acid-Schiff (PAS) for goblet cells and mucus. infiltration around bronchioles, mucus had ac- →W/W mice were restored to levels found in OVA-sensitized and -challenged +/+ mice. cumulated in the lumen of bronchioles, and hy- perplasia of goblet cells was observed at the epithelium of large airways after OVA sensitization Cellular changes in mice after OVA challenge and challenge, compared with those after saline Numbers of macrophages, lymphocytes, neutro- sensitization and challenge in +/+ mice. However, phils, eosinophils, and total cells in BAL fluids infiltration of eosinophils around bronchioles, ac- showed a significant increase at 48 h after OVA cumulation of mucus in the lumen of bronchioles, sensitization and challenge, compared with those and hyperplasia of goblet cells at the epithelium of after saline sensitization and challenge in +/+ mice large airways of OVA sensitized and challenged (Figure 2). However, the numbers of macrophages, W/W mice were significantly lower than in OVA- lymphocytes, neutrophils, eosinophils, and total sensitized and -challenged +/+ mice. On the other cells in BAL fluids of OVA-sensitized and -cha- hand, infiltration of eosinophils around bronchioles, llenged W/W mice were significantly lower than in accumulation of mucus in the lumen of bron- OVA-sensitized and -challenged +/+ mice. On the chioles, and hyperplasia of goblet cells at the other hand, numbers of macrophages, lympho- epithelium of large airways of BMCMC→W/W cytes, neutrophils, eosinophils, and total cells in mice after OVA sensitization and challenge were BAL fluids of BMCMC→W/W mice after OVA significantly higher than in OVA-sensitized and sensitization and challenge were significantly -challenged W/W mice. higher than in OVA-sensitized and -challenged W/W mice. Levels of Th2 cytokines of BAL fluids in mice after OVA challenge Histopathologic changes of lung challenged by OVA Enzyme immunoassays showed that levels of Histopathologic analyses revealed typical patho- TNF-α, IL-4, and IL-5 protein in BAL fluids were logic features of asthma in OVA-sensitized and significantly increased after OVA sensitization and -challenged mice (Figure 3). Lung tissues in mice challenge, compared with saline-sensitized and v v given OVA were characterized by gross alterations -challenged +/+ , W/W , and BMCMC→W/W mice in the structural integrity of the airway walls and (Figure 4). Levels of TNF-α, IL-4, and IL-5 protein parenchyma, epithelial cell shedding, microvascu- in BAL fluids in OVA-sensitized and -challenged lar leakage and extensive mucosal edema, in- W/W mice were similar to levels found in sa- creased tissue cellularity, and particulate exudated line-sensitized and -challenged W/W mice. How- in airway lumina and alveolar septa. Numerous ever, levels of TNF-α, IL-4, and IL-5 protein in BAL inflammatory cells, such as eosinophils, showed fluids in OVA-sensitized and -challenged BMCMC 38 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 teins in lung tissue increased significantly with OVA challenge, compared with those after saline cha- llenge in +/+ mice (Figure 5C). However, levels of ICAM-1 and VCAM-1proteins in lung tissue of OVA-sensitized and -challenged W/W mice were significantly lower than in lung tissue of OVA- sensitized and -challenged +/+ mice. On the other hand, in BMCMC→W/W mice, levels of ICAM-1 and VCAM-1 protein were restored to the level found in OVA-sensitized and -challenged +/+ mice (Figure 5C). Figure 4. Enzymatic immunoassay for TNF-α, IL-4, and IL-5 proteins in Effects of TNF-α on ICAM-1 & VCAM-1 protein levels bronchoalveolar lavage fluid after saline sensitization and challenge in W/W mice (Saline) or ovalbumin sensitization and challenge (OVA) in +/+, W/W , v v ## and BMCMC→W/W mice. ***P < 0.001 versus W/W (OVA), P < To investigate the effect of TNF-α on expression of 0.01 versus W/W (Saline). ICAM-1 and VCAM-1 proteins, we assessed the →W/W mice had almost reached the levels found in OVA-sensitized and -challenged +/+ mice. ICAM-1 & VCAM-1 expression in OVA challenged mice Two common and distinct pathways mediating mononuclear cell adhesion to pulmonary endo- thelium were investigated. Expression of endo- thelial ICAM-1 was observed in sections of blood vessels from lung of OVA-sensitized and -cha- llenged +/+ mice and BMCMC→W/W mice, whereas in saline-sensitized and -challenged +/+, v v , and BMCMC→W/W mice, ICAM-1 was W/W hardly detected in endothelial cells of blood vessels. Also, ICAM-1 was hardly detected in endothelial cells of blood vessels of OVA-sen- sitized and -challenged W/W mice (Figure 5A). In the contrast with ICAM-1, VCAM-1 expression was significantly heightened in vascular endothelium in blood vessels of OVA-sensitized and -challenged +/+ mice and BMCMC→W/W mice and was most pronounced in vessels with perivascular inflam- matory foci. Interestingly, VCAM-1 was also ex- pressed on smooth muscle cells of blood vessels and bronchioles (Figure 5B). Whereas in sa- line-sensitized and -challenged +/+, W/W , and BMCMC→W/W mice, VCAM-1 was hardly de- tected in endothelial cells of blood vessels. Also, VCAM-1 was hardly detected in endothelial cells of Figure 5. ICAM-1 and VCAM-1 proteins in lung tissues after saline sen- blood vessels of OVA-sensitized and -challenged sitization and challenge (Saline) or ovalbumin sensitization and challenge v v v v W/W mice. In BMCMC→W/W mice, expression of (OVA) in +/+, W/W , and BMCMC→W/W mice. Immunohistochemistry ICAM-1 and VCAM-1 in endothelial cells of blood of (A) ICAM-1 (arrows) and (B) VCAM-1 (arrows) in endothelial cells and smooth muscle cells of blood vessels (*) of the lungs after saline sensiti- vessels was restored to the levels found in OVA- zation and challenge or ovalbumin sensitization and challenge in +/+, sensitized and -challenged +/+ mice (Figure 5B). v v W/W and BMCMC→W/W mice. Br indicates bronchioles. Arrow heads Consistent with these results, western blot analysis show immunoreactive ICAM-1 and VCAM-1. (C) Western blotting of revealed that levels of ICAM-1 and VCAM-1 pro- ICAM-1and VCAM-1proteins in lung tissue. Role of mast cells in asthma 39 immunohistochemical analysis and western blot analysis of ICAM-1 and VCAM-1 proteins of lung tissue after intra tracheal injection of TNF-α in W/W mice. Immunohistochemical analysis re- vealed that expressions of ICAM-1 and VCAM-1 were observed of in sections of blood vessels from lung of TNF-α intra tracheal injected W/W mice. Localization of ICAM-1 and VCAM-1 expressions in endothelial layers around inflammation area and immunoreactive expression of VCAM-1 was more intensive than ICAM-1 (Figure 6A). Western blot analysis revealed that levels of ICAM-1 and VCAM-1 proteins were increased in a dose-depend- ent manner from lung of W/W mice (Figure 6B). Discussion Figure 6. Effects of TNF-α on ICAM-1 & VCAM-1 protein levels in W/W mice. (A) Immunohistochemistry of ICAM-1 and VCAM-1 (arrows). * in- In this study, we investigated the possible roles of dicates blood vessels of the lungs; Br, bronchioles. (B) Western blotting mast cells with regard to certain parameters of of ICAM-1 and VCAM-1 24 h after intra-tracheal injection of TNF-α in allergic inflammation in a Th2 cytokine-dependent W/W mouse. asthma model using genetically mast cell-deficient W/W mice, congenic normal +/+, and BMCMC release spasmogenic, vasoactive and proinflam- →W/W mouse models. Our results suggest that mast cells play a key role in a Th2 cytokine- matory mediators, which in turn can enhance dependent asthma model through production of airway smooth muscle contraction, vascular per- meability, and inflammatory cell (T cells, dendritic VCAM-1 and ICAM-1 by release of TNF-α. Asthma is perceived as a Th2 disease with a cell, neutrophils, eosinophils, and monocytes) re- cruitment at sites of antigen challenge (Bieder- particular profile of cytokine release, which is thought to include IL-4 and IL-5 (Woodruff et al., mann et al., 2000; Bradding et al., 2006; Suto et 2009). Increasing evidence indicates that other al., 2006; Kneilling et al., 2009). Chemokines and cytokines, which in mice are classically considered cytokines are also released and induce neutrophil to belong to Th1-type profiles, are also associated influx upon injection of cytokines, such as TNF-α (Ryan et al., 2007; Galli et al., 2008; Kawakami et with the inflammatory response that characterizes human asthma (Antunez et al., 2006). In the last al., 2009). On the other hand, locally released decade it has become evident that cytokines play a chemokines may activate resident cells to release pivotal role in the pathogenesis of asthma (Zi- cytokines. According to these reports, it is sug- mmermann et al., 2003; Antunez et al., 2006). The gested that recruitment of inflammatory cells, ac- tivation and cytokine release from resident cells role of antigen-induced TNF-α release and antigen stimulation of other cytokines is an important area are consequent on mast cell activation by OVA. However, the asthma model used in this manu- of study. In addition to TNF-α, cytokines including script is mast cell-dependent, so there did not IL-1β, IL-2, IL-3, IL-4, IL-5, granulocyte-macro- show the changes accompanied by a mast cell- phage-colony stimulating factor, and interferon-γ dependent exacerbation of airway inflammation to have also been implicated in development of the asthmatic inflammatory response (Thomas, 2001; OVA, and then did not induce cascade reactions Zimmermann et al., 2003; Antunez et al., 2006). such as, migration, local accumulation, and ac- tivation of proinflammatory cells and residential cell In this study, TNF-α showed an increase in BAL fluid after the second OVA challenge in +/+ mice, populations in the airway. So it is suggested that absence of mast cell result in a significant de- whereas OVA-sensitized and -challenged W/W mice showed a significant decrease in TNF-α crease in TNF-α, IL-4, and IL-5 levels. Our study levels (up to 75%) in their BAL fluid. In addition, provides evidence to show that cytokines play a pivotal role in the pathogenesis of asthma (Zi- TNF-α levels were significantly restored in BAL fluid in OVA-sensitized and -challenged BMCMC mmermann et al., 2003; Antunez et al., 2006). Evidence for expression of ICAM-1 and VCAM-1 →W/W mice. Also, other cytokines, IL-4 and IL-5, in smooth muscle cells in inflammatory diseases, showed a pattern similar to that of TNF-α. Mast cell activation by specific antigen induces the cells to such as arthrosclerosis and asthma, is mounting. 40 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 The exact roles of expression of such adhesion the direct effect of TNF-α on up-regulation of molecules are not fully understood; however, it has ICAM-1 and VCAM-1 after intra tracheal injection been suggested that expression facilitates trans- of TNF-α in W/W mice. Up-regulation of ICAM-1 migration and accumulation of leukocytes at in- and VCAM-1 increased in a dose manner of TNF-α flammatory sites. Increasing evidence has demon- in W/W mice. This data showed that TNF-α strated the importance of cytokine-inducible leu- induced adhesion molecules in the pulmonary kocyte-endothelial adhesion molecules in recruit- endothelium of asthma. ment and migration of leukocytes to sites of TNF-α is stored in granules and is known to be inflammation (Montefort and Holgate, 1991; Ando released during allergic responses from both mast et al., 2001; Chihara, 2005). Expression of both cells and macrophages via IgE-dependent me- ICAM-1 and VCAM-1 by smooth muscle cells in chanisms (Thomas, 2001; Zimmermann et al., response to several inflammatory factors within 2003; Antunez et al., 2006). This finding indicated airways has been reported in asthma animal that this cytokine will be coreleased with more models and allergic asthmatic patients (Gosset et extensively characterized preformed mast cell al., 1995; Lee et al., 2003). In our study, we found granule mediators, such as histamine, chymase, that expression of ICAM-1 and VCAM-1 in lung and tryptase. Mast cell cytokines are predomi- tissue was significantly increased in OVA-sen- nantly Th2 cytokines, which are critical to de- sitized and -challenged +/+ mice, compared with velopment and maintenance of the asthmatic levels in OVA-sensitized and -challenged W/W IgE-driven condition, and TNF-α has been shown mice and saline-sensitized and -challenged +/+ to induce airway hyperreactivity. In our study, we mice. However, expression levels of ICAM-1 and demonstrated that expression of ICAM-1, VCAM-1, VCAM-1 in lung tissue of OVA-sensitized and and protein in lung tissues was significantly increased after OVA sensitization and challenge of -challenged BMCMC→W/W mice increased to levels similar to those found in OVA-sensitized and +/+ mice, compared with levels of saline sen- -challenged +/+ mice and was most pronounced in sitization and challenge in+/+ mice or OVA sen- vessels with perivascular inflammatory foci. Inter- sitization and challenge in W/W mice. However, estingly, VCAM-1 was also expressed on smooth expression of ICAM-1, VCAM-1, and protein in muscle cells of blood vessels and bronchioles. lung tissue of OVA-sensitized and -challenged Therefore, our data suggest that ICAM-1 and BMCMC→W/W mice increased to levels similar to VCAM-1 may play an important role in Th2 those found in OVA-sensitized and -challenged +/+ cytokine-dependent asthma and that mast cells mice. Therefore, our results suggest that mast cell may have an important action in induction and cytokines, particularly TNF-α, are critical to de- expression of ICAM-1 and VCAM-1 in Th2 cy- velopment and maintenance of the asthma model tokine-dependent asthma. through production of ICAM-1 and VCAM-1. TNF-α, a cytokine that plays a role in many inflammatory diseases, is produced mainly by macrophages; however, monocytes, dendritic cells, Methods B cells, mast cells, and eosinophils are also known to be crucial in the pathogenesis of asthma. Also, Mice TNF-α is a chemotactic cytokine for granulocytes Genetically male mast cell-deficient mice W/W mice and (Sabatini et al., 2002; Chai et al., 2005; Yoshifuku their congenic littermates +/+ mice, 5 weeks of age, were et al., 2007), probably by up-regulation of cellular obtained from Japan SLC, Inc. W/W mice ordinarily con- adhesion molecules, such as E-selectin, ICAM-1 tain less than 1.0% of the number of dermal mast cells and VCAM-1, thus facilitating migration of eosi- present in the skin of congenic normal +/+ mice and have nophils and neutrophils. During this process, they no detectable mature mast cells in the respiratory system or other anatomic sites (Wolters et al., 2005; Galli and Tsai, also become primed for mediator secretion. Ul- 2008). timately, this will lead to chronic inflammation and irreversible airway remodeling, a key feature in bronchial asthma. The role of TNF-α in allergic Mast cell reconstitution inflammation is still controversial. TNF-α induction Selective reconstitution of mast cells in mast cell deficient of adhesion molecules in pulmonary endothelium is W/W mice was carried out according to the method important for eosinophil recruitment (Wong et al., described by Williams and Galli (2000), with slight modi- 2006); however, it has also been implicated in fications. Suspended bone marrow cells from +/+ mice negative modulation of eosinophil level, IL-5 pro- were cultured in WEHI-3 conditioned medium containing duction, and, consequently, airway responsiveness IL-3 for 4-5 weeks, at which time the cell populations were (Kanehiro et al., 2001). In this study, we revealed composed of> 95% immature mast cells, as assessed by Role of mast cells in asthma 41 staining with toluidine blue and Kimura staining. Five mi- Differential cell counts were determined with cytospin llion bone marrow-derived cultured mast cells (BMCMCs) (Centrifuge 5403; Eppendorf, Hamburg, Germany) prepa- were infused via the tail vein into each W/W mouse, and ration, followed by Diff Quik staining (Sysmex Co., Kobe, the recipients (BMCMC→W/W mice) were studied at least Japan). 16 wks later. There were 0.508± 0.09 mast cells/mm in lung tissue of BMCMC→W/W mice (vs none or 0.38± 2 v Histopathology and immunohistochemistry 0.035 mast cells/mm in lung tissue of W/W or +/+ mice, respectively). Histopathologic analysis of lung and immunohistochemical examination of ICAM-1 and VCAM-1 were performed as previously described (Vrugt et al., 2000). Animals were Immunization and challenge with OVA sacrificed with an overdose of ether at 48 h after the last v v OVA exposure and histologic specimens were collected. BMCMC→W/W and age-matched +/+, W/W mice were Using routine histologic procedures for light microscopic used in these experiments. Mice were sensitized and evaluation, tissue specimens were taken from the mid challenged with ovalbumin (OVA) (Sigma-Aldrich Co., St. zone of the left lung of the mice, and fixed in 10% formalin Louis, MO), as described previously, with some modifi- cations (Williams and Galli, 2000; Kim et al., 2007) and embedded in paraffin. Serial sections measuring 5 μm in thickness were then cut, and stained with hematoxylin (Supplemental Data Figure S1). In brief, mice were and eosin for inflammatory cells, congo red for eosinophils, immunized by intraperitoneal injection of 20 μg OVA and and periodic acid-Schiff for goblet cells and mucus. The saline in a total volume of 200 μl on days 0 and 14. Twenty others were stained with anti-ICAM-1 and anti-VCAM-1 eight days after the beginning of the sensitization period, antibodies. these mice received their first intranasal challenge of 30 μl of saline containing 200 μg of OVA. After 7 days, these mice were challenged for 30 min with an aerosol of 5% Measurements of Th2 cytokines in BAL fluids (wt/vol) OVA in saline using ultrasonic nebulization (NE-U12; Omron Crop., Tokoyo, Japan). BAL was performed and BAL fluid was collected for analysis of Th2 cytokines in BAL fluid, smears of BAL cells were prepared by cytospin (Centrifuge 5403; Eppendorf, Measurement of airway reactivity Hamburg, Germany). Levels of Th2 cytokines in super- natants of BAL fluids were quantified by mouse IL-4, IL-5, Twenty four hours after the final OVA challenge, airway and TNF-α BD ELISA sets (BD PharMingen). reactivity to aerosolized methacholine was measured using a whole body plethysmograph, as described by Ohtomo et al. (2009). In brief, unrestrained conscious mice were Western blot analysis placed in whole body plethysmographic chambers, and, after 5 min of stabilization, dose response curves to aero- Mice lungs were minced and sonicated in Pro-Prep buffer solized methacholine were generated. Increasing concen- (50 mg/ml) containing 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, trations of methacholine were aerosolized for 3 min each, 150 mM NaCl, and protease inhibitors using a tip-type data for lung resistance and dynamic compliance were sonicator (Branson sonifier, Branson ultrasonics, CT, 20% continuously monitored by using OCP 3000CPS-A soft- amplitude, 3 min), followed by centrifugation for 20 min at ware (allmedicus) and mean airway bronchoconstriction 1,000× g. Protein expression levels in cell lysates were readings, as assessed by enhanced respiratory pause analyzed by Western blot analysis, as previously described (PenH), were obtained over 10 min periods. PenH can be (Vrugt et al., 2000; Lee et al., 2003). Equal amounts of cell conceptualized as the phase shift of the thoracic flow and lysate were separated on an SDS polyacrylamide gel nasal flow curves; increased phase shift correlates with under reducing conditions, and were then transferred onto increased respiratory system resistance. PenH is cal- Protran nitrocellulose membranes (Schleicher & Schuell, culated by the formula PenH = (Te/RT - 1) × PEF/PIF, Keene, NH). Membranes were blocked for 2 h at room where Te is expiratory time, RT is relaxation time, PEF is temperature in 3% skim milk in TBS, followed by overnight peak expiratory flow, and PIF is peak inspiratory flow. incubation with anti-TNF-α (BD PharMingen), anti-ICAM-1 (BD PharMingen), and anti-VCAM-1 (Santa Cruz). Blots were washed for 15 min with three changes of transfer Collection and analysis of bronchoalveolar lavage buffer-0.05% Tween 20 solution, followed by 1 h incubation (BAL) fluid at room temperature with HRP-conjugated anti-mouse antibody or anti-rabbit IgG antibody. Blots were washed Forty eight hours after the final OVA challenge, BAL fluid was collected by cannulating the upper part of the trachea again for 15 min and finally developed in ECL Western detection reagents (Amersham-Pharmacia Biotech, Pis- and lavaging three times with 1 ml saline. The lavage fluid cataway, NJ). Signal intensities of specific bands were collected was centrifugated at 400× g for 5 min at 4 C, analyzed quantitatively using a Fluor-STM Imager (Bio- and the cells were separated from the fluid. The fluid was Rad, Muncher, Germay) and plotted as relative intensity. then centrifugated at 1,000× g for 2 min at 4 C for removal of cellular debris and then stored at -20 C until it was evaluated. Cells were resuspended in PBS containing Statistical analyses 1% FBS, and the total number of viable cells was de- termined by trypan blue exclusion using a hemocytometer. Unless otherwise specified, differences in response bet- 42 Exp. Mol. Med. Vol. 43(1), 35-43, 2011 ween different subgroups were tested for statistical sig- Nat Rev Immunol 2008;8:478-86 nificance using an unpaired Student's t test (two tailed). P Gosset P, Tillie-Leblond I, Janin A, Marquette CH, Copin MC, < 0.05 was considered statistically significant. Unless Wallaert B, Tonnel AB. Expression of E-selectin, ICAM-1 and otherwise specified, all data are presented as mean± VCAM-1 on bronchial biopsies from allergic and non-allergic SEM. asthmatic patients. Int Arch Allergy Immunol 1995;106:69- Acknowlegements Kanehiro A, Lahn M, Makela MJ, Dakhama A, Fujita M, Joetham A, Mason RJ, Born W, Gelfand EW. Tumor necrosis 1. OH Chai was supported by a Korea Research Founda- factor-alpha negatively regulates airway hyperresponsive - tion Grant funded by the Korean Government (MOEHRD, ness through gamma-delta T cells. Am J Respir Crit Care Basic Research Promotion Fund; KRF-2007-331-E00009). Med 2001;164:2229-38 2. CH Song was supported by a Korea Research Founda- tion Grant funded by the Korean Government (KRF-2008- Kawakami T, Ando T, Kimura M, Wilson BS, Kawakami Y. 313-E00014). Mast cells in atopic dermatitis. Curr Opin Immunol 2009; 21:666-78 Kim YS, Ko HM, Kang NI, Song CH, Zhang X, Chung WC, Supplemental data Kim JH, Choi IH, Park YM, Kim GY, Im SY, Lee HK. Mast cells Supplemental Data include a figure and can be found with play a key role in the development of late airway hyper- this article online at http://e-emm.or.kr/article/article_files/ responsiveness through TNF-alpha in a murine model of SP-43-1-05.pdf. asthma. 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