Antiinflammatory Effects of Genistein, a Tyrosine Kinase Inhibitor, on a Guinea Pig Model of Asthma

Departments of Pharmacology, Paediatrics, and Anatomy, Faculty of Medicine, National University of Singapore, Republic of Singapore

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Protein tyrosine kinase signaling cascade plays a pivotal role in the activation of inflammatory cells. The purpose of this study was to investigate the effects of genistein, a broad-spectrum protein tyrosine kinase inhibitor, on airway inflammation in an in vivo guinea pig model of asthma. Guinea pigs were actively sensitized by intraperitoneal injections of ovalbumin. Aerosolized ovalbumin induced acute bronchoconstriction in conscious animals in a dose-dependent manner. Genistein (15 mg/kg given intraperitoneally) markedly inhibited ovalbumin-induced, but not histamine- and methacholine-induced, acute bronchoconstriction. In addition, genistein significantly reduced ovalbumin-induced increases in total cell counts and eosinophils recovered in bronchoalveolar lavage fluid, airway eosinophilia, and eosinophil peroxidase activity in cell-free bronchoalveolar lavage fluid and markedly attenuated ovalbumin-induced airway hyperresponsiveness to inhaled methacholine. Immunoblot analysis of lung lysates isolated from genistein-pretreated animals showed that epidermal growth factor–induced tyrosine phosphorylation in lung tissues was inhibited by genistein. These results implicate that inhibition of tyrosine kinase signaling cascade may have therapeutic potential for allergic airway inflammation.

　

Key Words: inflammation • ovalbumin • airway hyperresponsiveness • bronchoalveolar lavage fluid • methacholine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Allergic asthma is a chronic airway inflammatory disease that manifests itself as recurrent reversible acute bronchoconstriction and airway hyperresponsiveness (AHR) (1, 2). The pathophysiology of airway inflammation encompasses eosinophilic infiltration, epithelial denudation, mucus hypersecretion, and airway remodeling. It is attributable to the coordinated and sustained activation of inflammatory cells, including mast cells, T helper 2 cells, B cells, macrophages and eosinophils, and synthesis of a variety of proinflammatory mediators. Acute bronchoconstriction is triggered off by the release of bronchoconstrictors, including histamine, cysteinyl-leukotrienes, and platelet-activating factor from mast cells upon allergen-induced cross-linking of immunoglobulin E–bound high-affinity Fc receptors (FcRI) (1, 2). Airway eosinophilia is induced by a combination of cytokines and chemokines such as interleukin (IL)-5, regulated upon activation, normal T cell expressed and secreted, and eotaxin, indicating a major role of T helper 2 cells in orchestrating the allergic airway inflammation. Pulmonary eosinophilia together with airway remodeling, altered neural control of airway tone, and airway epithelial desquamation may contribute to AHR in asthma (3, 4).

Cumulative evidence concurs with the fact that tyrosine kinase signaling cascade plays a pivotal role in the initiation of activation of various inflammatory cells important for the pathogenesis of allergic inflammation (5, 6). Tyrosine kinases can be broadly divided into receptor tyrosine kinase (7) and nonreceptor tyrosine kinase (8). Stimulation of nonreceptor tyrosine kinases is the earliest detectable signaling response upon immunoreceptor activation in mast cells, T and B cells, eosinophils, and macrophages. It is believed that src-family kinases (Lyn and Lck) and Syk/ZAP-70 are responsible for the initial activation of these cells (6, 9–11). Terminal differentiation, recruitment, and activation of eosinophils into the lungs are regulated by IL-5, which acts through stimulation of tyrosine kinases, including Lyn and Janus Kinase–Signal Transducer and Activator of Transcription (JAK-STAT) pathway (12, 13). There is increasing evidence that links chemokine-induced inflammatory cell chemotaxis, adhesion, and degranulation to chemokine receptor dimerization-induced JAK-STAT signaling pathway activation (14). These observations implicate that tyrosine kinase signaling cascade can be a central target for pharmacologic intervention for the control of airway inflammation. Indeed, several recent in vivo studies using selective Janus kinase 3 inhibitor, Syk antisense, or fgr-/- knockout showed that inhibition or genetic ablation of tyrosine kinases could attenuate lung eosinophilia and AHR (15–17). In contrast, genetic knockout of p59fyn, a member of the src tyrosine kinase family, has been reported to exacerbate pulmonary inflammation in an allergic mouse model, indicating that tyrosine kinases can be negative regulators of allergic airway inflammation as well (18).

Our laboratory has recently shown that inhibitors of the tyrosine kinase signaling cascade markedly abated ovalbumin (OVA)-induced anaphylactic contraction of guinea pig bronchi and release of histamine and peptidoleukotrienes from guinea pig lung fragments, and thrombin-induced guinea pig airway smooth muscle cell proliferation (19–22). The purpose of this study was to examine the potential antiinflammatory effects of genistein, a broad-spectrum tyrosine kinase inhibitor (23, 24), on a guinea pig model of asthma. Genistein markedly attenuated OVA-induced acute bronchoconstriction and AHR to inhaled methacholine in guinea pigs. In addition, genistein significantly reduced OVA-induced increases in total cell counts and eosinophils recovered in bronchoalveolar lavage fluid (BALF), eosinophil peroxidase activity in BALF, and airway eosinophilia. Our findings indicate that protein tyrosine kinase inhibitor may have therapeutic potential for the treatment of allergic airway inflammation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Animal Sensitization
Male Hartley guinea pigs were sensitized by intraperitoneal injections of 10-µg OVA and 100-mg Al(OH)₃ on Days 0 and 14. On Day 18, animals were challenged with OVA aerosol.

Bronchoconstriction
Bronchoconstriction was measured using a single-chamber whole-body plethysmograph (Buxco, Sharon, CT) based on the method of Hamelmann and colleagues (25). Genistein (1–15 mg/kg), daidzein (15 mg/kg), or dimethyl sulfoxide (DMSO) (1%) was injected intraperitoneally into the animals 45 minutes before OVA aerosol challenge. Guinea pigs were challenged for 3 minutes, and bronchoconstriction was recorded for an additional 5 minutes for each increasing dose of aerosolized OVA or saline aerosol. The highest Penh value obtained during each OVA challenge was expressed as a percentage of the basal Penh value obtained after saline challenge. To determine potential direct bronchodilatory effect of genistein, nonsensitized guinea pigs were challenged with aerosolized histamine (10–200 µg/ml) or methacholine (10–500 µg/ml) using the same protocol as OVA challenge.

Bronchoalveolar Lavage
Bronchoalveolar lavage was performed 24 hours after OVA challenge. Guinea pigs were anaesthetized by an intraperitoneal injection of pentobarbital sodium (37 mg/kg). Tracheotomy was performed, and a cannula was inserted into the trachea. Phosphate-buffered saline was instilled into the lungs, and BALF was collected. Total cell counts were performed using a hemocytometer. For cytologic examination, cytospin preparations were prepared, fixed, and stained in a modified Wright stain (26). A differential cell count was then performed.

Histologic Examination
After bronchoalveolar lavage, the lungs were inflated via trachea with 10% neutral formalin and immersed in the same fixative for at least 24 hours. Tissues were parafinized, and 8-µm sections were cut and stained with hematoxylin and eosin before examination under a light microscope.

Eosinophil Peroxidase Activity
The eosinophil peroxidase (EPO) activity in the cell-free BALF was measured according to the method of Strath and colleagues (27). Results were expressed as ng/ml peroxidase activity, excluding 3-amino-1,2,4-triazole-insensitive peroxidase activities.

Measurement of Airway Responsiveness
Guinea pigs were sensitized by intraperitoneal injections of 10-µg OVA and 100-mg Al(OH)₃ on Days 0 and 14. On Days 18, 19, and 20, animals were challenged with aerosolized OVA (10 mg/ml) or saline for 3 minutes daily to maximize airway inflammation. Genistein (15 mg/kg) or DMSO was given 45 minutes before each OVA aerosol challenge. On Day 21, guinea pig airway responsiveness to doubling concentrations of methacholine (0.00625–4 mg/ml) was measured. The minimum provocative concentration of methacholine at which Penh increased by 200% above the basal value of individual animals was calculated and expressed as a provocative concentration of methacholine aerosols causing a 200% increase from control level of airway responsiveness (mg/ml).

Western Blot Analysis
Western blot analysis for phosphotyrosine was conducted in lung parenchymal tissues isolated from guinea pigs with and without genistein pretreatment. The lung fragments (0.1 g) were exposed to epidermal growth factor (EGF) for 5, 10, and 15 minutes before being homogenized in ice-cold lysis buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. The membrane was probed with antiphosphotyrosine monoclonal antibody (PY-20), followed by alkaline phosphatase-conjugated secondary antibody, and visualized colorimetrically.

Statistical Analysis
All data are presented as means ± SEM. One-way analysis of variance followed by the Tukey test was used to determine significant differences between treatment groups. The critical level for significance was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Effects of Genistein on OVA-induced Bronchoconstriction
 shows the representative waveform of box pressure signal derived from a sensitized animal challenged with saline aerosol. Penh (enhanced pause) was used as the parameter of bronchoconstriction and defined as Penh = pause x peak expiratory pressure/peak inspiratory pressure (25). Penh reflects changes in box pressure signal from both peak inspiratory pressure and peak expiratory pressure in combination with the timing comparison between early and later expiration (pause). Penh value in the saline control group was 0.44 ± 0.03 (n = 8), with a value of less than 1 being considered as free of bronchoconstriction. Treatment with OVA aerosol (10 mg/ml) induced acute bronchoconstriction (Penh) in guinea pigs, as reflected by the changes in the waveform of the chamber pressure signals . An increase in Penh was accompanied with decreased respiratory rate and increased Pause (timing comparison of early and late expiration). Pretreatment of guinea pigs with 15 mg/kg of genistein effectively alleviated OVA-induced alterations of the waveform and Penh elevation . As compared with the Penh in saline control group (108.9 ± 5.7%), OVA dose dependently increased Penh in sensitized animals with 772.8 ± 166.3% (n = 8) and 1,117.3 ± 211.2% (n = 8) of basal Penh level at 3 and 10 mg/ml OVA, respectively  . Genistein dose dependently inhibited the OVA-induced acute bronchoconstriction . At 5 mg/kg, genistein markedly inhibited 1 mg/ml of OVA-induced bronchoconstriction from Penh of 217.9 ± 19.1% (DMSO) to 131.6 ± 12.7% (p < 0.05, n = 8). At 15 mg/kg, genistein significantly abated 10 mg/ml of OVA-induced bronchoconstriction from Penh of 891.3 ± 115.3% (DMSO) to 283.9 ± 49.9% (p < 0.05, n = 8). Daidzein, a structural analogue of genistein, which is devoid of tyrosine kinase inhibitory activity (23), failed to inhibit OVA-induced bronchoconstriction. To determine whether genistein is a direct bronchodilator, we examined the effects of genistein on aerosolized histamine- or methacholine-induced bronchoconstriction in guinea pigs. Genistein (15 mg/kg) did not show any major inhibition on bronchoconstriction induced by inhalational histamine (10–200 µg/ml) and methacholine (10–500 µg/ml)  .

fig.ommitted Figure 1. Representative tracings showing the changes in box pressure waveform after 3 minutes of (A) nebulization with saline aerosol, (B) 10 mg/ml of OVA aerosol, or (C) 10 mg/ml of OVA aerosol with 15 mg/kg of genistein pretreatment. Penh (enhanced pause) was used as the parameter of bronchoconstriction and defined as Penh = pause x peak expiratory pressure (PEP)/peak inspiratory pressure (PIP) (25). Penh reflects changes in chamber pressure signal from both peak inspiratory pressure and peak expiratory pressure in combination with the timing comparison between early and late expiration (pause); f indicates frequency of respiration (breaths per minute); PIF = peak inspiratory flow; PEF = peak expiratory flow.

 

fig.ommitted Figure 2. Effects of genistein on aerosolized OVA-induced acute bronchoconstriction in sensitized guinea pigs. (A) Inhalation of OVA aerosol-induced acute bronchoconstriction in guinea pigs in a dose-dependent manner (n = 8). (B) Effects of genistein (GEN) or daidzein (DZ) on OVA-induced bronchoconstriction in guinea pigs. Penh was expressed as a percentage of the basal value. *Significant difference from DMSO control, p < 0.05.

 

fig.ommitted Figure 3. Effects of genistein (GEN) on aerosolized (A) histamine- or (B) methacholine-induced acute bronchoconstriction in guinea pigs. Penh was expressed as a percentage of the basal value. *Significant difference from DMSO control, p < 0.05.

 
Effects of Genistein on OVA-induced Airway Inflammation
Bronchoalveolar lavage was performed 24 hours after OVA aerosol challenge, and total and differential cell counts in BALF were performed. OVA inhalation significantly increased total cell numbers from 9.9 ± 1.3 x 10⁶ per ml in saline challenge group to 37.2 ± 4.0 x 10⁶ per ml (p < 0.05)  . Eosinophils recovered in BALF were significantly increased from 1.6 ± 0.3 x 10⁶ per ml in the saline challenge group to 14.4 ± 2.1 x 10⁶ per ml (p < 0.05). The number of macrophages was also significantly increased 24 hours after OVA challenge, whereas neutrophil and lymphocyte counts were unchanged. Genistein (5 and 15 mg/kg) significantly reduced the total cell number recovered in BALF as compared with DMSO control, which was mainly due to a substantial reduction in eosinophils from 15.2 ± 2.2 x 10⁶ per ml in DMSO group to 7.0 ± 1.0 x 10⁶ per ml (p < 0.05) . In contrast, genistein did not affect OVA-induced elevation in the macrophage count. Daizdein did not show any effects on the OVA-induced increase in total cell counts or differential cell counts.

fig.ommitted   Figure 4. (A) Numbers of inflammatory cells recovered in BALF from guinea pigs 24 hours after saline aerosol or 10 mg/ml of OVA aerosol challenge. (B) Effects of genistein (GEN) or daidzein (DZ) on OVA-induced increase in inflammatory cell counts in BALF from guinea pigs 24 hours after 10 mg/ml of OVA challenge. Differential cell count was performed on a minimum of 500 cells to identify eosinophil (EOS), neutrophil (NEU), macrophage (MAC), and lymphocyte (LYM). *Significant difference from DMSO control, p < 0.05.

 
The OVA-induced increase in eosinophil count in BALF was also accompanied with a significant rise in EPO activity in the cell-free BALF. There was a sevenfold increase in EPO activity in OVA-challenged group as compared with the saline group  . Genistein dose dependently inhibited OVA-induced EPO activity in BALF, and at 15 mg/kg, it significantly inhibited the EPO activity from 3.0 ± 0.5 ng/ml in the DMSO control group to 1.7 ± 0.3 ng/ml (p < 0.05).

fig.ommitted   Figure 5. Effects of genistein on EPO activity recovered in cell-free BALF from guinea pigs 24 hours after 10 mg/ml of OVA aerosol challenge. The EPO activity was measured based on the principle of oxidation of o-phenylenediamine (OPD) by EPO in the presence of H2O2 (27). The OPD substrate solution was prepared from Sigma Fast OPD tablets (Sigma Chemical Co., St. Louis, MO). Each point represents the mean ± SEM of three to eight experiments. *Significant difference from DMSO control, p < 0.05.

 
Histologic examination of guinea pig airways revealed that 10 mg/ml of OVA aerosol challenge induced marked infiltration of inflammatory cells, especially eosinophils, into the highly vascularized lamina propria, beneath the pseudostratified respiratory epithelium. Similarly, in the smaller airways of the same group of guinea pigs, inflammatory exudates were observed in the intrapulmonary peribronchiolar connective tissues  . Pretreatment of guinea pigs with 15 mg/kg of genistein before OVA aerosol challenge effectively diminished the eosinophil-rich leukocyte infiltration as shown in .

fig.ommitted Figure 6. Histologic examination of inflammatory cell infiltration into the trachea (A–D, magnification x200) and bronchioles (E–H, magnification x400) from guinea pigs 24 hours after inhalation of saline aerosol (A, E), OVA aerosol (B, F), OVA aerosol plus DMSO (C, G), or OVA aerosol plus 15 mg/kg of genistein (D, H). In trachea from guinea pigs that had inhaled OVA aerosol, abundant cell infiltrates were primarily localized in the highly vascularized lamina propria beneath the pseudostratified respiratory epithelium. In the smaller airways, inflammatory exudates were observed in the intrapulmonary peribronchiolar tissues. The rectangular boxes in B and F were magnified to x1,000 to reveal OVA-induced eosinophilia in trachea (I) and in bronchioles (J). Black arrowheads indicate eosinophils, which are characterized by prominent bilobed nuclei and specific granules stained in pinkish red with eosin. Lung tissues were fixed, sectioned at an 8-µm thickness, and stained with hematoxylin and eosin.

 
Effects of Genistein on AHR
Sensitized guinea pigs challenged with 10 mg/ml of OVA aerosol for 3 minutes daily for 3 consecutive days developed AHR to inhaled methacholine, as shown by a marked decrease in the provocative concentration of methacholine aerosols causing a 200% increase from control level of airway responsiveness from 300 ± 60 µg/ml of methacholine in the saline group to 150 ± 30 µg/ml of methacholine in the OVA-challenged group (p < 0.05)  . Pretreatment with genistein (15 mg/kg) before each OVA aerosol challenge for 3 consecutive days dramatically prevented AHR to methacholine by restoring the provocative concentration of methacholine aerosols causing a 200% increase from control level of airway responsiveness from 110 ± 30 µg/ml of methacholine (DMSO) to 300 ± 60 µg/ml of methacholine (p < 0.05) .

fig.ommitted Figure 7. (A) Airway responsiveness of sensitized guinea pigs challenged with aerosolized saline or 10 mg/ml of OVA for 3 consecutive days in response to increasing doses of inhaled methacholine. (B) Effects of 15 mg/kg of genistein given 45 minutes before each OVA inhalation for 3 consecutive days on inhaled methacholine-induced airway responsiveness. AHR to methacholine was expressed as a provocative concentration of methacholine aerosols causing a 200% increase from control level of airway responsiveness. Each point represents mean ± SEM of four experiments. *Significant difference from control groups, p < 0.05.

 
Phosphotyrosine Immunoblot Analysis
To verify that the inhibitory effects of genistein on the guinea pig model of asthma were mediated by protein tyrosine kinase inhibition, we examined the effects of genistein on 100-nM EGF-induced protein tyrosine phosphorylation in lung fragments isolated from guinea pigs pretreated with DMSO or 15 mg/kg of genistein.  shows that EGF rapidly induced protein tyrosine phosphorylation of the EGF receptor in lung fragments within 5 minutes. Genistein effectively blocked EGF-induced EGF receptor tyrosine phosphorylation.

fig.ommitted Figure 8. Western blot analysis of EGF-induced protein tyrosine phosphorylation in lung fragments isolated from guinea pigs pretreated with DMSO or 15 mg/kg of genistein. Lung fragments were exposed to 100-nM EGF for 5, 10, and 15 minutes before homogenization in ice-cold lysis buffer. Proteins (10 µg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with antiphosphotyrosine antibody. EGF-stimulated A431 cell extract provided by the manufacturer was used as positive control for tyrosine-phosphorylated EGF receptor.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Genistein, an isoflavone compound, has been shown to be a broad-spectrum tyrosine kinase inhibitor that is capable of inhibiting EGF receptor kinase, pp60^v-src, and pp110^gag-fes with negligible effect against other serine/threonine kinases such as protein kinase A, protein kinase C, and phosphodiesterase. The inhibition is competitive at the ATP-binding domain of the kinase and is noncompetitive at the protein substrate site of the kinase (23, 24). Our present findings revealed that inhibition of protein tyrosine kinases by genistein could attenuate OVA-induced acute bronchoconstriction, pulmonary eosinophil infiltration, and AHR in sensitized guinea pigs.

Allergen-induced immediate acute bronchoconstriction is primarily due to mast cell degranulation upon cross-linking of immunoglobulin E–bound FcRI. Histamine and cysteinyl-leukotrienes are the two major mast cell-derived mediators contributing to the acute early-phase bronchoconstriction (1, 28). Cumulative evidence obtained from various cultured mast cells has shown that src-related kinase Lyn, Syk, and Btk are activated rapidly after FcRI aggregation (10). Inhibitors of protein tyrosine kinases, including genistein, lavendustin A, tyrphostin 47 (RG50864), and piceatannol, have been shown to block antigen-induced activation of tyrosine kinases and histamine release from mast cells (29, 30). Genistein, tyrphostin 47, and piceantannol have also been shown to attenuate OVA-induced bronchial contraction and prevent OVA-induced release of both histamine and peptidoleukotrienes from guinea pig lung fragments (19–21). In line with those findings from cell cultures and isolated lung tissues, this study showed that genistein significantly (p < 0.05) inhibited OVA-induced immediate acute bronchoconstriction in an in vivo guinea pig model of asthma in a dose-dependent manner (1–15 mg/kg). In contrast, 15 mg/kg of daidzein, an analogue of genistein that has no inhibitory activity against tyrosine kinases (23), failed to alter the OVA-induced acute bronchoconstriction. On the other hand, genistein did not produce any major inhibitory effect on direct histamine (10–200 µg/ml) or methacholine (10–500 µg/ml)-induced bronchoconstriction in guinea pigs, indicating that the reduction of OVA-induced acute early-phase bronchoconstriction by genistein was not due to direct bronchodilation. Indeed, our previous studies revealed that genistein, tyrphostin 47, and piceatannol did not show any inhibitory effects on direct histamine-, leukotriene D₄-, or KCl-induced bronchial contraction in vitro (20, 21). Our data implicate that suppression of OVA-induced immediate acute bronchoconstriction by genistein is likely due to mast cell stabilization via inhibition of protein tyrosine kinases.

On the other hand, inhaled allergen can activate T cell receptor on T helper 2 cells to trigger release of a vast array of cytokines such as IL-4, IL-5, and IL-13 and chemokines such as regulated upon activation, normal T cell expressed and secreted and eotaxin. These mediators can initiate and coordinate inflammatory responses leading ultimately to activation and accumulation of eosinophils and other leukocytes such as T helper 2 cells, macrophages, and neutrophils in the airways (1, 2). Our present findings showed that genistein, but not daidzein, significantly reduced total cell counts and eosinophils recovered in BALF 24 hours after OVA challenge of the guinea pigs. In line with these findings, histologic examination of trachea and bronchioles from genistein-pretreated animals revealed a substantial reduction in OVA-induced eosinophil infiltration as compared with the DMSO control group. It has been established that T cell receptor activation rapidly stimulates the activity of src-family kinase Lck and Syk for signal propagation and T cell activation (9). In cultured human T cells, genistein has been shown to inhibit T cell receptor activation-induced protein tyrosine phosphorylation and subsequent cytokine gene expression and cell proliferation (31–33). As such, the observed reduction in eosinophil infiltration into the guinea pig airways by genistein may be a result of blockade of T cell receptor activation via tyrosine kinase inhibition resulting in diminished release of proinflammatory cytokines.

Pulmonary eosinophilia has been considered as one of the major mechanisms for allergen-induced AHR. Eosinophil-derived major basic protein, eosinophil cationic protein, and EPO combined with H₂O₂ can induce AHR via bronchial epithelial damage (12, 13, 34). IL-5 plays a critical role in the growth and differentiation, recruitment, and activation of eosinophils in the airways (1–3). Direct administration of IL-5 into the airways induced pulmonary eosinophilia and AHR in subjects with asthma (35). IL-5 receptor activation in eosinophils rapidly triggers phosphorylation of Lyn, Syk, and JAK2 tyrosine kinases leading to various functional responses of eosinophils (12, 13). Genistein has been shown to inhibit IL-5–induced protein tyrosine phosphorylation and gene expression in eosinophils (36, 37). In addition, other tyrosine kinase inhibitors such as herbimycin A, erbstatin, and src-selective inhibitor have been shown to inhibit platelet-activating factor-induced eosinophil chemotaxis, immunoglobulin G–induced eosinophil degranulation, and CD11b/ CD18-mediated nicotinamide adenine dinucleotide phosphate oxidase activation in human adherent blood eosinophils (11, 38, 39). Our findings demonstrated that genistein (15 mg/kg) significantly inhibited EPO activity in the recovered BALF and OVA-induced AHR to inhaled methacholine, indicating a reduction in eosinophil migration and activation. The observed antiinflammatory effects of genistein may be mediated by inhibition of tyrosine kinase signaling cascade upon IL-5 receptor activation. Recent in vivo studies using selective inhibitor of JAK3 and Syk antisense also revealed that inhibition of tyrosine kinases could suppress airway eosinophilia and AHR (15, 16). Furthermore, Ezeamuzie and colleagues (40) reported that worthmannin, an inhibitor of phosphatidylinositol 3-kinase, a downstream signaling molecule of the tyrosine kinase signaling cascade, decreased EPO activity and AHR in a guinea pig model of asthma.

Allergen-induced acute bronchoconstriction and chronic airway inflammation is a multistage process that involves multiple cell types and a wide array of mediators. Protein tyrosine kinases have been shown to participate in many steps of the inflammatory process. Using tyrosine kinase inhibitor to modulate allergic airway inflammation is a logical therapeutic strategy. We report here for the first time that genistein effectively reduced OVA-induced acute bronchoconstriction, pulmonary eosinophilia, and AHR in an in vivo guinea pig model of asthma. These findings support a potential role for protein tyrosine kinase inhibitor in the treatment of asthma.

	Acknowledgments

 
The authors thank Peter W. Stengel of Lilly Research Laboratories for his expertise advice on the in vivo study of guinea pig bronchoconstriction.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

1. Busse WW, Lemanske RF Jr. Asthma. N Engl J Med 2001;344:350–362.

2. Maddox L, Schwartz DA. The pathophysiology of asthma. Annu Rev Med 2002;53:477–498.

3. Wills-Karp M. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu Rev Immunol 1999;17:255–281.

4. Kumar RK. Understanding airway wall remodeling in asthma: a basis for improvements in therapy? Pharmacol Ther 2001;91:93–104.

5. Wong WSF, Koh DSK. Advances in immunopharmacology of asthma. Biochem Pharmacol 2000;59:1323–1335.

6. Kinet JP. The high-affinity IgE receptor (FcRI): from physiology to pathology. Annu Rev Immunol 1999;17:931–972.

7. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–225.

8. Bolen JB, Brugge JS. Leukocyte protein tyrosine kinases: potential targets for drug discovery. Annu Rev Immunol 1997;15:371–404.

9. Latour S, Veillette A. Proximal protein tyrosine kinases in immunoreceptor signaling. Curr Opin Immunol 2001;13:299–306.

10. Reischl IG, Coward WR, Church MK. Molecular consequences of human mast cell activation following immunoglobulin E-high-affinity immunoglobulin E receptor (IgE-FcRI) interaction. Biochem Pharmacol 1999;58:1841–1850.

11. Kato M, Abraham RT, Kita H. Tyrosine phosphorylation is required for eosinophil degranulation induced by immobilized immunoglobulins. J Immunol 1995;155:357–366.

12. Adachi T, Alam R. The mechanism of IL-5 signal transduction. Am J Physiol 1998;275:C623–C633.

13. Stafford S, Lowell C, Sur S, Alam R. Lyn tyrosine kinase is important for IL-5-stimulated eosinophil differentiation. J Immunol 2002;168:1978–1983.

14. Mellado M, Rodriguez-Frade JM, Manes S, Martinez-AC. Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu Rev Immunol 2001;19:397–421.

15. Malaviya R, Chen CL, Navara C, Malaviya R, Liu XP, Keenan M, Waurzyniak B, Uckun FM. Treatment of allergic asthma by targeting Janus kinase 3-dependent leukotriene synthesis in mast cells with 4-(3',5'-dibromo-4'-hydrocyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P97). J Pharmacol Exp Ther 2000;295:912–926.

16. Stenton GR, Ulanova M, Dery RE, Merani S, Kim MK, Gilchrist M, Puttagunta L, Musat-Marcu S, James D, Schreiber AD, et al. Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase. J Immunol 2002;169:1028–1036.

17. Vicentini L, Mazzi P, Caveggion E, Continolo S, Fumagalli L, Lapinet-Vera JA, Lowell CA, Berton G. Fgr deficiency results in defective eosinophil recruitment to the lung during allergic airway inflammation. J Immunol 2002;168:6446–6454. Kudlacz EM, Andresen CJ, Salafia M, Whitney CA, Naclerio B, Changelian PS. Genetic ablation of the src kinase p59fynT exacerbates pulmonary inflammation in an allergic mouse model. Am J Respir Cell Mol Biol 2001;24:469–474.

19. Wong WSF, Koh DSK, Koh AHM, Ting WL, Wong PTH. Effects of tyrosine kinase inhibitors on antigen challenge of guinea pig lung in vitro. J Pharmacol Exp Ther 1997;283:131–137.

20. Tsang F, Wong WSF. Inhibitors of tyrosine kinase signaling cascade attenuated antigen challenge of guinea-pig airways in vitro. Am J Respir Crit Care Med 2000;162:126–133.

21. Seow CJ, Chue SC, Wong WSF. Piceatannol, a Syk-selective tyrosine kinase inhibitor, attenuated antigen challenge of guinea pig airways in vitro. Eur J Pharmacol 2002;443:189–196.

22. Tsang F, Choo HH, Dawe GS, Wong WSF. Inhibitors of the tyrosine kinase signaling cascade attenuated thrombin-induced guinea pig airway smooth muscle cell proliferation. Biochem Biophys Res Commun 2002;293:72–78.

23. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe SI, Itoh N, Shibuya M, Fukam Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;262:5592–5595.

24. Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995;267:1782–1788.

25. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766–775.

26. Hsiue TR, Lei HY, Hsieh AL, Chang HY, Chen CW. Time course of pharmacological modulation of peak eosinophic airway inflammation after mite challenge in guinea pigs: a therapeutic approach. Int Arch Allergy Immunol 1999;119:297–303.

27. Strath M, Warren DJ, Sanderson CJ. Detection of eosinophils using an eosinophil peroxidase assay: its use as an assay for eosinophil differentiation factors. J Immunol Methods 1985;83:209–215.

28. Roquet A, Dahlen B, Kumlin M, Ihre E, Anstren G, Binks S, Dahlen SE. Combined antagonism of leukotrienes and histamine produces predominant inhibition of allergen-induced early- and late-phase airway obstruction in asthmatics. Am J Respir Crit Care Med 1997;155:1856–1863.

29. Kawakami T, Inagaki N, Takei M, Fukamachi H, Coggeshall KM, Ishizaka K, Ishizaka T. Tyrosine phosphorylation is required for mast cell activation by FcRI cross-linking. J Immunol 1992;148:3513–3519.

30. Oliver JM, Burg DL, Wilson BS, McLaughlin JL, Geahlen RL. Inhibition of mast cell FcRI-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J Biol Chem 1994;269:29697–29703.

31. Mustelin T, Coggeshall KM, Isakov N, Altman A. T cell antigen receptor-mediated activation phospholipase C requires tyrosine phosphorylation. Science 1990;247:1584–1587.

32. Trevillyan JM, Lu YL, Atluru D, Phillips CA, Bjorndahl JM. Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase. J Immunol 1990;145:3223–3230.

33. Liu B, Hackshaw KV, Whisler RL. Calcium signals and protein tyrosine kinases are required for the induction of c-jun in Jurkat cells stimulated by the T cell-receptor complex and oxidative signals. J Interferon Cytokine Res 1996;16:77–90.

34. Jacoby DB, Costello RM, Fryer AD. Eosinophil recruitment to the airway nerves. J Allergy Clin Immunol 2001;107:211–218.

35. Shi HZ, Xiao CQ, Zhong D, Qin SM, Liu Y, Liang GR, Xu H, Chen YQ, Long XM, Xie ZF. Effect of inhaled interleukin-5 on airway hyperreactivity and eosinophilia in asthmatics. Am J Respir Crit Care Med 1998;157:204–209.

36. Hiraguri M, Miike S, Sano H, Kurasawa K, Saito Y, Iwamoto I. Granulocyte-macrophage colony-stimulating factor and IL-5 activate mitogen-activated protein kinase through Jak2 kinase and phosphatidylinositol 3-kinase in human eosinophils. J Allergy Clin Immunol 1997;100:S45–S51.

37. Horiuchi T, Weller PF. Expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5. Am J Respir Cell Mol Biol 1997;17:70–77.

38. El-Shazly A, Masuyama K, Samejima Y, Eura M, Ishikawa T. Modulation of normal human eosinophil chemotaxis in vitro by herbimycin A, erbstatin and pervanadate. Int Arch Allergy Immunol 1998;117:10–13.

39. Lynch OT, Giembycz MA, Barnes PJ, Hellewell PG, Lindsay MA. Outside-in signaling mechanisms underlying CD11b/CD18-mediated NADPH oxidase activation in human adherent blood eosinophils. Br J Pharmacol 1999;128:1149–1158.

40. Ezeamuzie CI, Sukumaran J, Philips E. Effect of wortmannin on human eosinophil responses in vitro and on bronchial inflammation and airway hyperresponsiveness in guinea pigs in vivo. Am J Respir Crit Care Med 2001;164:1633–1639.