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Evaluation of Guinea pig models of the acute phase of allergic rhinitis AL Suleimani, Yousuf Mohammed 2006

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Evaluation of Guinea pig Models of the Acute Phase of Allergic Rhinitis by Yousuf Mohammed A l Suleimani B . S c , Sultan Qaboos University, 2003  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF MASTER O F SCIENCE  in  T H E F A C U L T Y O F G R A D U A T E STUDD3S (Pharmacology & Therapeutics)  UNIVERSITY O F BRITISH C O L U M B I A A p r i l 2006 © Yousuf Mohammed A l Suleimani, 2006  Abstract Allergic rhinitis is an allergen-IgE complex mediated inflammation o f the nasal mucosa characterized by the symptoms o f sneezing, nasal itchiness, rhinorrhea, and nasal congestion. The economical and social impact o f allergic rhinitis is substantial. The effectiveness  o f currently available medications is limited.  Investigation o f more  effective medications with fewer side effects is essential. Therefore, this study was intended to establish a model o f allergic rhinitis i n guinea pigs that can be utilized for further investigation o f new medications. Furthermore, this study was also aimed to systematically evaluate the role o f some inflammatory mediators o f acute allergic reactions in guinea pigs in vivo. Male Dunkin Hartley guinea pigs were intranasally sensitized to, and challenged with, ovalbumin. Sneezing (SN) and nose rubbing (NR) were evaluated on day 21 post initiation o f sensitization dose. From day 23 after first sensitization, the animals were anaesthetized  with  intraperitoneal  pentobarbital  (30-35mg/kg).  The  trachea  was  cannulated i n both directions, caudally for measurement o f nasal airway pressure ( N A P ) using a ventilator flow method (8ml/beat, 72beats/min) and rostrally for measurement o f lung inflation pressure (LIP). Drugs were administered prior to ovalbumin challenge. S N and N R were evaluated for 30 minutes and N A P was evaluated within 30 minutes post challenge. Cellular infiltration (CI) was assessed from nasal lavage collected 60 minutes post challenge. Sensitized guinea pigs produced symptoms o f S N , N R and nasal blockade ( N B ) in addition to eosinophil infiltration following ovalbumin challenge. A first generation H I antihistamine, mepyramine, inhibited S N only, whereas later H I antihistamine, cetirizine,  ii  inhibited S N , N R and N B . Montelukast, a leukotriene D 4 receptor antagonist,  and  heparin prevented N B and CI. L - N A M E , a non specific nitric oxide synthase inhibitor, inhibited N B and stimulated neutrophil infiltration. In  non-sensitized  guinea  pigs,  histamine  and  acetylcholine  introduced  intravenously caused dose-dependent decreases i n N A P (by the action o f histamine on H I , M 2 and perhaps M 5 receptors, and acetylcholine on M l receptors) and increases in L I P (by the action o f histamine on H I receptors, and acetylcholine on M l receptors). In conclusion pathophysiological changes due to allergic rhinitis in guinea pigs resemble to some extent those in humans. The models reported here reflect  the  effectiveness o f some drugs currently used to treat allergic rhinitis. The models can be used in investigating new potential drugs for the treatment o f allergic rhinitis.  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List o f Tables  viii  List o f Figures  ix  Preface  i  Ackowledgements 1  xii xiii  Introduction  1  1.0 Allergic rhinitis 1.0.1 Human allergic rhinitis 1.0.2 Rhinitis in common laboratory animals 1.0.3 Rhinitis in guinea pigs 1.1 The structure and function o f the normal human nose compared with common laboratory animals 1.1.1 Anatomy and Histology 1.1.2 Vasculature 1.1.3 Innervation 1.1.4 Physiological function 1.1.5 Nose structure and function o f other species 1.2 Pathophysiology o f allergic rhinitis in various species, including human 1.2.1 Sensitization 1.2.2 Acute phase 1.2.3 Chronic phase 1.2.4 Inflammatory cells in allergic rhinitis 1.2.4.1 Mast cells 1.2.4.2 Eosinophils 1.2.4.3 Basophils 1.2.4.4 T-lymphocytes 1.2.4.5 Epithelial cells..... : 1.2.5 Inflammatory mediators in allergic rhinitis 1.2.5.1 Histamine 1.2.5.2 Eicosanoids : 1.2.5.2.1 Leukotrienes 1.2.5.2.2 Prostaglandins and Thromboxanes 1.2.5.3 Nitric oxide 1.2.5A Platelet activating factor 1.2.5.5 Cytokines 1.2.5.6 Chemokines.  iv  1 1 4 5 7 7 9 12 14 16 19 20 24 24 25 ..26 27 29 30 31 32 32 34 34 36 37 38 39 40  1.2.5.7 Kinins .1.2.5.8 Neuropeptides 1.2.6 Inflammatory mediators in animal models o f allergic rhinitis..... . 1.2.7 Pathophysiological events in allergic rhinitis 1.2.7.1 Neuronal events 1.2.7.2 Vascular events • 1.2.7.3 Glandular events 1.2.8 Nasal airway hyperresponsiveness 1.2.9 Overall summary 1.3 Drug targets in allergic rhinitis 1.3.1 Current registered drugs: limitations to their use 1.3.2 Possible novel targets for treating allergic rhinitis 1.3.2.1 Mediator inhibitors : 1.3.2.1.1 Antihistamines 1.3.2.1.2 Leukotrienes inhibitors 1.3.2.1.3 Prostaglandin receptor antagonists 1.3.2.1.4 Tryptase antagonists 1.3.2.1.5 Nitric oxide synthase inhibitors 1.3.2.2 Mast cells stabilizers , 1.3.2.2.1 Cromones 1.3.2.2.2 Protein kinase inhibitors 1.3.2.2.3 Ion channel blocking drugs 1.3.2.3 Inhibitors of neuronal pathways 1.3.2.4 Immunotherapy 1.3.2.4.1 Allergen-specific immunotherapy 1.3.2.4.2 Peptide-based immunotherapy 1.3.2.4.3 D N A immunotherapy 1.3.2.5 IgE targeting 1.3.2.6 Cytokines and Chemokines inhibitors 1.3.2.7 Adhesion molecules inhibitors 1.3.2.8 Selective phosphodiesterase 4 inhibitors 1.3.2.9 Heparin 1.3.2.10 Phototherapy 1.4 Methods for studying allergic rhinitis i n experimental animals 1.4.1 General overview 1.4.2 Techniques used to assess pathophysiological changes in allergic rhinitis in guinea pigs in vivo 1.4.2.1 Sneezing and Nose rubbing 1.4.2.2 Nasal airway pressure 1.4.2.3 Nasal secretions 1.4.2.4 Exudation 1.4.2.5 Cellular and Biochemical changes 1.5 Rationale and A i m s o f this project  41 42 43 46 46 49 51 52 53 54 54 57 57 57 58 58 59 59 59 59 60 61 61 62 62 63 63 64 65 66 67 67 68 69 69  2. Materials and Methods 2.0 Materials  76 76  v  69 70 70 72 72 73 74  2.1 Methods 2.1.1 Methods o f sensitization used i n the study 2.1.2 Experimental animals 2.1.3 Details o f methods and models used in this study 2.1.3.1 Sneezing and nose rubbing in conscious guinea pigs in response to allergen challenge 2.1.3.2 Measurement o f nasal airway pressure and forced inflation pressure in anaesthetized guinea pigs subjected to allergen challenge 2.1.3.3 Attempts to measure nasal secretions and exudation i n conscious and anaesthetized guinea pigs subjected to allergen challenge 2.1.3.4 Measurement o f leukocyte infiltration into nasal lavage fluid in anaesthetized guinea pigs subjected to allergen challenge 2.1.3.5 Experimental design and drugs used in the study 2.1.3.6 Data analyses 2.1.3.7 Experimental overview :  77 77 78 79  3. Results..' : 3.0 Assessment o f models 3.0.1 Effectiveness o f sensitization procedures used 3.0.2 Sneezing and nose rubbing responses to allergen challenge 3.0.3 Changes i n nasal airway pressure and lung inflation pressure as well as blood pressure in response to allergen challenge 3.0.4 Leukocyte infiltration responses to allergen challenge 3.0.5 Sensitization test 3.1 A n investigation o f the receptors involved i n mediating nose and lung responses to intravenous histamine and acetylcholine 3.1.1 Effect o f histamine and acetylcholine on nasal airway pressure 3.1.2 Effect o f histamine and acetylcholine on lung inflation pressure 3.1.3 A n investigation o f receptors involved in histamine and acetylcholine induced changes in nasal airway pressure and lung inflation pressure 3.2 Drug modification o f the responses to allergen challenge ; 3.2.1 Effect o f antihistamines (mepyramine and cetirizine) on acute allergic reactions in sensitized ovalbumin challenged guinea pigs 3.2.1.1 Sneezing and nose rubbing 3.2.1.1 Nasal airway pressure 3.2.2 Effect o f leukotriene D 4 receptor antagonist, montelukast, on acute allergic reactions in sensitized ovalbumin challenged guinea pigs 3.2.2.1 Sneezing and nose rubbing 3.2.2.2 Nasal airway pressure 3.2.2.3 Leukocyte infiltration 3.2.3 Effect o f N (omega)-nitro-L-arginine methyl ester ( L - N A M E ) on acute allergic reactions in sensitized ovalbumin challenged guinea pigs 3.2.3.1 Nasal airway pressure , , 3.2.3.2 Leukocyte infiltration.. 3.2.4 Effect o f heparin on acute allergic reactions in sensitized ovalbumin challenged guinea pigs  92 92 92 93  vi  79 79 82 82 84 88 89  95 96 97 98 98 99 101 111 Ill Ill Ill 113 113 115 116 117 117 118 120  3.2.4.1 Sneezing and nose rubbing 3.2.4.2 N a s a l airway pressure 3.2.4.3 Leukocyte infiltration 3.2.5 Effect o f dexamethasone on acute allergic reactions in sensitized ovalbumin challenged guinea pigs 3.2.5.1 Sneezing and nose rubbing 3.2.5.2 Nasal airway pressure 3.2.5.3 Leukocyte infiltration : 3.2.6 Overall summary  120 121 122  4. Discussion 4.0 Value o f the models in terms o f responses to allergen challenge and relevance to clinical rhinitis with special reference to effects of histamine and acetylcholine 4.1 Possible clinical relevance o f model responses to clinical and experimental drugs 4.1.1 Effect o f antihistamines on sneezing, nose rubbing and nasal airway pressure in sensitized ovalbumin challenged guinea pigs 4.1.2 Effect o f leukotriene D 4 receptor antagonist, montelukast on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs 4.1.3 Effect o f L - N A M E on nasal airway pressure and leukocyte infiltration i n sensitized ovalbumin challenged guinea pigs 4.1.4 Effect o f heparin on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration i n sensitized ovalbumin challenged guinea pigs 4.1.5 Effect o f dexamethasone on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs 4.2 Conclusions ;  130  5.  153  :  References  vii  123 123 124 126 128  130 134 134  138 141 144 147 151  List of Tables  Table 1.1  Role o f eosinophil cationic proteins in allergic airway disease  29  Table 1.2  Inflammatory cells and mediators in guinea pig allergic rhinitis.  44  Table 1.3  Inflammatory cells and mediators i n rat allergic rhinitis  45  Table 1.4  Inflammatory cells and mediators in mouse allergic rhinitis  45  Table 1.5  Inflammatory cells and mediators in dog allergic rhinitis  46  Table 1.6  Inflammatory cells and mediators in pig allergic rhinitis  46  Table 2.1  List o f drugs used in the experiments  85  Table 2.2 List o f drugs and doses used i n autacoids and neurotransmitters effect on nasal airway pressure and lung inflation pressure study  86  Table 2.3 receptors  89  Relative affinities o f different antagonists for muscarinic and histamine  Table 3.1 Acetylcholine effects on nasal airway pressure i n the presence o f different doses o f antagonists 106 Table 3.2 Histamine effects on nasal airway pressure in the presence o f different doses of antagonists 106 Table 3.3 Acetylcholine effects on lung inflation pressure in the presence o f different doses o f antagonists 110 Table 3.4 Histamine effects on lung inflation pressure in the presence o f different doses o f antagonists  110  Table 3.5 A summary o f drug modification o f the response to allergen challenge experiments  128  Table 3.6 Data summary o f changes in nasal airway pressure, lung inflation pressure, blood pressure and heart rate in various groups used i n this study 129  vin  List of Figures Figure 1.1  Schematic representation o f the different layers o f human nasal mucosa. 11  Figure 1.2 Schematic representation o f the endothelial and neuronal control o f blood flow in nasal blood vessels .'. 12 Figure 1.3  Schematic representation o f the innervation o f the nose  13  Figure 1.4 noses.  External and internal anatomical arrangement o f human and guinea pig  Figure 1.5  Schematic representation o f pathophysiology o f allergic rhinitis  23  Figure 2.1  The process o f sensitization in guinea pigs  78  Figure 2.2  General protocol for sneezing and nose rubbing experiments  79  16  Figure 2.3 Schematic representation o f surgical and technical connections for nasal airway pressure, lung inflation pressure and blood pressure measurement 81 Figure 2.4 General protocol for nasal airway pressure, lung inflation pressure and cellular infiltration experiments  81  Figure 2.5  Schematic representation o f a hempcytometer  83  Figure 2.6  Protocol for histamine and acetylcholine experiment  87  Figure 2.7 Schematic representation o f experimental protocol for histamine and acetylcholine experiment  87  Figure 3.1 Time dependent effects o f ovalbumin administration on sneezing frequency in guinea pigs measured during the sensitization period  92  Figure 3.2 Time dependent effect o f ovalbumin administration on nose rubbing frequency in guinea pigs measured during the sensitization period  93  Figure 3.3 Effect o f ovalbumin challenge on sneezing frequency in sensitized and non-sensitized guinea pigs evaluated on day 21 post first sensitization 94 Figure 3.4 Effect o f ovalbumin challenge on nose rubbing frequency in sensitized and non-sensitized guinea pigs evaluated on day 21 post first sensitization 94 Figure 3.5 Effect o f ovalbumin challenge on nasal airway pressure in anaesthetizaed (sensitized and non-sensitized) guinea pigs 23-28 days following initiation o f sensitization '. 95  IX  Figure 3.6 Systemic evaluation o f the effects o f ovalbumin challenge on nasal airway pressure, lung inflation pressure, and blood pressure 96 Figure 3.7 Effect o f ovalbumin challenge on cellular infiltration (total cell count) i n anaesthetized (sensitized and non-sensitized) guinea pigs in nasal lavage collected 60 minutes following challenge 97 Figure 3.8 Effect o f ovalbumin challenge on cellular infiltration (differential cell count) in anaesthetized (sensitized and non-sensitized) guinea pigs in nasal lavage collected 60 minutes following challenge  98  Figure 3.9 Effect o f ovalbumin lmg/kg administered intravenously in sensitized and non-sensitized guinea pigs on blood pressure, heart rate and lung inflation pressure. 99 Figure 3.10 Dose-response curves for the decrease in nasal airway pressure following intravenous administration o f histamine and acetylcholine in non-sensitized guinea pigs.. " 100 Figure 3.11 Dose-response curves for the increase in lung inflation pressure following intravenous administration o f histamine and acetylcholine i n non-sensitized guinea pigs.. 100 Figure 3.12 Dose-response curves for the decrease in nasal airway pressure following intravenous administration o f acetylcholine before and after antagonists in non-sensitized guinea pigs 103 Figure 3.13 Dose-response curves for the decrease in nasal airway pressure following intravenous administration o f histamine before and after antagonists in non-sensitized guinea pigs 105 Figure 3.14 Dose-response curves for the increase in lung inflation pressure following intravenous administration o f acetylcholine before and after antagonists in non-sensitized guinea pigs 107 Figure 3.15 Dose-response curves for the increase in lung inflation pressure following intravenous administration o f histamine before and after antagonists in non-sensitized guinea pigs 109 Figure 3.16 Effect o f antihistamines (mepyramine and cetirizine) (3mg/kg ip) on sneezing frequency  112  Figure 3.17 Effect o f antihistamines (mepyramine and cetirizine) (3mg/kg ip) on nose rubbing frequency 112  x  Figure 3.18 Effect o f antihistamines (mepyramine and cetirizine) (3mg/kg ip) on nasal airway pressure 113 Figure 3.19  Effect of montelukast lOmg/kg iv on sneezing frequency  114  Figure 3.20  Effect o f montelukast lOmg/kg iv on nose rubbing frequency  114  Figure 3.21  Effect o f montelukast lOmg/kg iv on nasal airway pressure  115  Figure 3.22  Effect o f montelukast lOmg/kg iv on cellular infiltration (total cell count). ;  116  Figure 3.23  Effect o f L - N A M E 1 Omg/kg iv on nasal airway pressure  117  Figure 3.24  Effect o f L - N A M E 1 Omg/kg iv on cellular infiltration (total cell count) 118  Figure 3.25 count)  Effect o f L - N A M E 1 Omg/kg iv on cellular infiltration (differential cell  Figure 3.26  Effect o f heparin 20mg/kg iv on sneezing frequency  120  Figure 3.27  Effect o f heparin 20mg/kg iv on nose rubbing frequency  121  Figure 3.28  Effect o f heparin 20mg/kg iv on nasal airway pressure  122  Figure 3.29  Effect of heparin 20mg/kg iv on cellular infiltration (total cell count)... 123  Figure 3.30  Effect o f dexamethasone 20mg/kg ip on sneezing frequency  124  Figure 3.31  Effect o f dexamethasone 20mg/kg ip on nose rubbing frequency  125  Figure 3.32  Effect o f dexamethasone 20mg/kg iv on nasal airway pressure  125  Figure 3.33 count)  Effect o f dexamethasone 20mg/kg iv on cellular infiltration (total cell  Figure 3.34 cell count)  Effect of dexamethasone 20mg/kg iv on cellular infiltration (differential 127  119  126  xi  Xll  Acknowledgements  I would  like to thank  Professor  M.J.A.  W a l k e r for his supervision,  guidance, support and valuable directions throughout m y study. I w o u l d l i k e also to express m y thanks to Y i n g D o n g for her technical and experimental support. A great appreciation is g i v e n to m y graduate committee members D r . Catherine C . Y . P a n g and D r . D a r r y l A . K n i g h t for their valuable ideas and suggestions. I also thank the undergraduate students M i c h e l l e S o h and D a v i d K o for their help during experiments. I o w e m y profound gratitude and special thanks to m y w i f e w h o has been very patient and a source o f emotional and m o r a l support throughout the course o f this research w o r k . M y graduate research training w o u l d not have been possible without the award o f the scholarship from Sultan Q a b o o s U n i v e r s i t y , O m a n . I w o u l d l i k e to express m y deepest thanks and appreciation to m y country O m a n , for p r o v i d i n g me w i t h this chance. M y experiments w o u l d not have been possible to conduct without the research fund from R h i n o p h a r m a L t d . I o w e the c o m p a n y thanks.  xiii  1  Introduction This thesis describes the evaluation o f models o f rhinitis in guinea pigs in terms  of the hallmarks o f rhinitis, namely sneezing, itching, nasal secretions, nasal obstruction and leukocyte infiltration. The following introduction covers the relevant  subjects  including: Rhinitis -its clinical and experimental characteristics; Anatomy o f the nose in humans and experimental animals; The relevant nasal physiology i n humans and experimental animals; The pathophysiology o f rhinitis-clinically and experimentally; Current drug treatment for rhinitis-clinical and experimental; Experimental models o f rhinitis  The thesis w i l l describe the various models that I have developed and used. Thereafter the results o f the initial studies into the nature o f the models w i l l be discussed, followed by a description o f the actions o f drugs currently used i n the clinic.  1.0  Allergic rhinitis  1.0.1  Human allergic rhinitis  Allergic rhinitis is defined as an abnormal inflammation o f the membrane lining the nose. It is characterized by nasal congestion, rhinorrhea, sneezing, itching o f the nose and/or postnasal drainage (Bousquet et al., 2001). Additionally, airway hypersensitivity ( A H R ) may develop, and loss o f the sense o f smell and an inability to taste may occur.  1  Moreover, some patients experience sleep disturbances, decreased emotional well-being and social functioning, headache,  and irritability. O n physical examination, nasal  obstruction often can be seen, with pale to bluish nasal mucosa, enlarged or boggy turbinates, clear nasal secretions, and pharyngeal cobblestoning (streaks o f lymphoid tissue). Other characteristic signs o f allergic rhinitis in children include allergic shiners (darkening o f the lower eyelids due the edematous nasal tissue that compresses the veins that drain the eyes, leading to pooling o f blood under the orbits) and the allergic crease (transverse skin line below the bridge o f the nose) that is caused by constant rubbing upwards from the palm o f the hand ("allergic salute"). Due to the chronic nasal airway obstruction, some children are chronic mouth breathers, which can lead to craniofacial abnormalities and orthodontic disturbances, such as palatal arching, increased facial length, and a flattened mid-face. M a n y patients do not show all these abnormalities, although they often may be sneezing and have rhinorrhea with mucosal edema (Nayak et al., 2001; Todd et al., 2005).  Although the onset o f allergic rhinitis may occur at any age, it is most common in children and at adolescence. There is a decrease in incidence with advancing age. The financial impact o f allergic rhinitis is significant. In 1996, the overall direct costs o f treating allergic rhinitis exceeded $3 billion, with an additional $4 billion spent to treat related comorbidities triggered or exacerbated by the disease. Allergic rhinitis is the most common atopic disorder in the United States. It affects about 24 million (8% o f the population) with an equal distribution between males and females. The prevalence o f allergic rhinitis varies by age: 32% o f patients are 17 years o f age or younger, 4 3 % are 18  2  to 44 years o f age, 17% are 45 to 64, and only 8% are 65 years o f age or older. The cost of treating allergic rhinitis plus indirect costs o f the disorder, such as lowered productivity and time lost from work or school are substantial. In the United States alone, the number o f lost workdays resulting from allergic rhinitis is estimated as approximately 3.5 million a year and the total direct health care cost o f treating allergic rhinitis is estimated at $3.4 biilion (Law et al., 2003; Holgate et al., 2003; Todd et al., 2005). Epidemiologically, up to 40% o f patients with allergic rhintis also have asthma, and up to 80% o f patients with asthma experience nasal symptoms. Furthermore, patients with allergic rhinitis are at three times the risk o f developing asthma compared with those without allergic rhinitis. In children who develop rhinitis within the first year o f life the chance o f developing asthma are twofold greater as compared with those who develop rhinitis later in life (Settipane et al., 1994; Wright et al., 1994).  Traditionally, allergic rhinitis is classified as seasonal or perennial, and as either mild, moderate, or severe. M i l d allergic rhinitis involves no sleep interruption, no impairment o f daily activities, and no troublesome symptoms. Moderate-to-severe allergic rhinitis involves one or more o f those factors. A newer classification system specifies that allergic rhinitis be characterized as intermittent, or persistent. Intermittent disease involves symptoms for fewer than 4 days per week, or for a duration o f fewer than 4 weeks. Persistent disease involves symptoms that occur more than 4 days per week and are present for longer than 4 weeks (Noble 1995; Bousquet et al., 2001). With seasonal rhinitis, the symptoms are periodic occuring in a temporal relationship to the presence o f seasonal allergens in individuals who are appropriately sensitized. Pollens  3  causing seasonal allergic rhinitis are tree pollen present in the springtime, grass pollen present in M a y through July, and weed pollen and mould spores which may produce symptoms i n late summer and autumn. Perennial disease, which is present all year round, relates to the presence o f a non-seasonal allergen. The allergens causing perennial rhinitis are frequently indoor aeroallergens the most commonly being allergens from mites (25%; Dermatophagoides pteronyssinus/farinae), animal antigens (15%; cats, dogs, rodents), fungal spores (10%; Alternaria, Cladosporium, Aspergillus, Penicillium), or exposate to antigens at the workplace (Platts-Milis et al., 1987; Raab et al., 1989; Howarth & Holgate 1990; Noble 1995). Allergic rhinitis should be differentially diagnosed from other respiratory allergic diseases. The two nasal conditions most commonly confused with allergic rhinitis are infectious rhinitis and perennial nonallergic rhinitis (vasomotor rhinitis). Infectious rhinitis is characterized by constitutional symptoms and purulent rhinorrhea. A nasal smear shows a preponderance o f neutrophils, whereas in allergic rhinitis, eosinophils predominate. Perennial nonallergic rhinitis is more frequent in women and is precipitated by such nonspecific factors as changes in temperature,  humidity, and barometric  pressure; strong odours; alcohol; and cigarette smoke. Nasal congestion frequently shifts from side to side and is often alleviated by exercise (Zeiger 1989).  1.0.2  Rhinitis in common laboratory animals  Allergic diseases are very uncommon in the animal world. There is no animal species that suffers spontaneously from allergic rhinitis (Szelenyi 2000). However, the  4  disease  can be induced in animals using different  strategies.  Although,  different  laboratory animal species have been used for the establishment o f animal models o f allergic rhinitis, using a variety o f antigens, there is no animal model that mimics all o f the symptoms o f allergic rhinitis. Therefore, only single symptoms are normally induced in a certain animal species. A m o n g the species, guinea pigs have recently gained more attention as the most suitable experimental model for in vivo studies o f pharmacological and pathophysiological aspects o f the acute and chronic phases o f allergic rhinitis. In addition, B A L B / c mice have been utilized mostly for immunological studies in allergic rhinitis (Saito et al., 2002; Murasugi et al., 2005). Furthermore, B r o w n Norway rats are also used for studying drugs effects on symptoms o f experimental rhinitis (Sugimoto et al., 2000b; Shimizu et al., 2000; F u et al., 2003). A m o n g other species, dogs and pigs have been occassionally used for studies o f mucus secretions and nasal congestion after allergen challenge (Revington et al., 1997; Szelenyi et al., 2000; M a l i s et al., 2001; Tiniakov et al., 2003). Since this thesis concerns studies in guinea pigs, rhinitis is considered in detail for this species.  1.0.3  Rhinitis in guinea pigs Conventionally, guinea pigs have been the species o f choice for the evaluation o f  chemical-related respiratory allergy, primarily because it is possible in this species to elicit and measure with relative ease challenged-induced inflammatory reactions that resemble i n some ways the acute clinical manifestations o f human allergic rhinitis. Dunken Hartley guinea pigs have been widely used to evaluate the actions o f drugs and  5  their therapeutic applicability in allergic rhinitis. The disease can be induced in guinea pigs using different allergens. The process o f induction o f allergy requires an initial sensitization dose o f allergen, followed by repeated booster doses, and finally a challenge dose. Symptoms and signs o f allergic rhinitis including sneezing, nose rubbing, rhinorrhea, vascular permeability (exudation) and nasal congestion.. Biochemical and cellular changes can be quantified and evaluated after a challenge dose. Ovalbumin and Japanese cedar pollen are the two common used allergens used to sensitize guinea pigs. The techniques used range from injection (intraperitoneal, with or without, adjuvant), bolus instillation (intranasal) or steady-state inhalation exposures over short or long periods o f time with different concentrations o f allergen and adjuvant. In the purely nasal route for sensitization, 4% lidocaine is insufflated over a period o f five minutes followed by intranasal doses o f allergen absorbed out in aluminum hydroxide, given daily for seven days (Mizutani et.al., 1999; Yamasaki et al., 2001; Nabe et al., 2001; Fukuda et al., 2003; Zhao et al., 2005). Alternatively, guinea pigs can be exposed to 1% aerosol ovalbumin twice for ten minutes, a week apart (Yamasaki et al., 1997). Moreover, ovalbumin absorbed in aluminum hydroxide can be injected peritoneally as an initial sensitization dose (Namimatsu et al., 1991; Narita et al., 1998; Fujita et al., 1999; Imai et al., 2000; M c l e o d 2002; Sakairi 2005). Guinea pigs can also be sensitized passively to ovalbumin by intravenous, subcutaneous, or intraperitoneal administration o f a n t i - O V A serum (Mizuno et al., 1991; Kaise et al., 1999; Kaise et al., 2001a). Following initial sensitization dose, guinea pigs are either exposed to repeated doses o f allergen boosters, over a period o f time, or directly challenged with allergen. The waiting period from first sensitization dose to first challenge dose varies between  6  studies, ranging between two weeks to more than four weeks. Conscious guinea pigs are challenged by the intranasal route using inhalation o f aerosolized allergen or by instillation o f micro milliliters o f allergen absorbed i n saline, whereas  anaesthetized  animals are challenged through either intranasal route or from the tracheal side toward the nasal cavity by infusion o f high volumes o f allergen (Albert et al., 1998; Mizutani et al., 1999; Sakairi 2005).  1.1 The structure and function of the normal human nose compared with those of common laboratory animals  1.1.1  Anatomy and Histology  The human external nose surrounds the nostrils and one-third o f the nasal cavity, which i n its entirety consists o f a 5-cm high and 10-cm long dual chambers. The total surface area o f both nasal cavities is about 150 c m , and total volume about 15 m l . 2  Approximately 1.5 c m from the nares is the narrowest portion o f the entire airway, the internal ostium (or nasal valve), with a cross-sectional area o f about 30 m m on each side. The nasal valve accounts for approximately 50% o f the total resistance to respiratory airflow from the nostril to the alveoli (Baroody 1997). Each o f the two nasal cavities is limited by the septal wall and the lateral wall, dominated by inferior, middle and superior turbinates. They are important for maintaining a slit-like cavity, and thereby facilitate humidification and temperature regulation o f the inspired air (Niels et al., 1998).  7  The nostrils are covered by skin, the anterior one-third o f the nasal cavity by a squamous and transitional epithelium. The upper part o f the cavity is covered by an olfactory epithelium and the remaining portion by a typical airway epithelium, which is ciliated, pseudostratified, and columnar. The latter consists o f four major cell types, basal cells, ciliated and non-ciliated columnar cells, and goblet cells. Basal cells, which are progenitors o f the other cell types, lie on the basement membrane and do not directly have contact with the airway lumen (Evans et al., 1988). Each o f the columnar cells, ciliated and non-ciliated cells, are covered by about 300 microvilli uniformly distributed over the entire apical surface. These short and slender fingerlike cytoplasmic expansions increase the surface area o f the epithelial cells, thus promoting exchange processes across the epithelium. The microvilli also prevent drying by retaining moisture essential for ciliary function. The cilia have a typical ultrastructure, each ciliated cell containing about 100 cilia, 0.3 um wide and 5 um in length (Halama et al., 1990). The anterior one-third o f the nasal cavity is non-ciliated. C i l i a start occurring just behind the front edge o f the inferior turbinate, and cover the posterior part o f the nasal cavity. The paranasal sinuses, are densely covered by cilia. The distribution pattern o f ciliated cells corresponds well with the distribution o f nasal airflow, thus the density o f ciliated cells is inversely proportional to the linear velocity o f inspiratory air in the nasal cavity (Cole 1982). Another cell type characteristic o f airway epithelium is the goblet cell. The majority o f goblet cells are located in the posterior part o f the nasal cavity with the concentration o f 4000-7000 cells per m m  2  mean  (Tos 1983). Goblet cells produce small  amounts o f viscous mucus that contributes only little to the total volume o f nasal  8  secretion. Little is known about release mechanisms for goblet cells, which i n contrast to the glands, are not under the control o f the parasympathetic nervous system.  The glands in the nose are o f two types: anterior serous glands and seromucous glands. There are only 100-150 anterior serous glands on each side o f the nose. Their long excretory ducts have large openings in the upper part o f the internal ostium, where small droplets o f watery secretion can be seen after stimulation o f the nasal mucosa. Secretions produced in the anterior part o f the nose are more watery, have a considerably lower viscoelasticity than secretions produced i n the posterior part o f the nose (Brofeldt et al., 1979). There are about 100,000 seromucous glands in the human nose, and this number appears to remain constant during life (Tos 1983). Thus infants have a secretory capacity comparable to that o f an adult. Since the ciliated surface, however, is much smaller in children, one can imagine that slight glandular hypersecretion may result in nasal discharge i n the child, but not in the adult. Figure 1.1 describes the arrangement o f nasal mucosa layers.  1.1.2  Vasculature  Blood from the ophthalmic and internal maxillary arteries feeds a huge network o f arterioles, venules, capillaries, capacitance vessels and shunt vessels. Together these supply and drain the nasal mucosa with a greater blood flow per volume o f tissue than the liver or brain (Grevers et al., 1996). The arterioles are conspicuous i n an absence o f an internal elastic membrane such that the endothelial basement membrane is continuous with the basement membrane system of the smooth muscle cell (Cauna 1970). The capillaries, just below the surface epithelium and surrounding the glands, are o f the  9  fenestrated type. Thus these capillaries are well suited for rapid movement o f fluid through the vascular wall allowing water to escape into the airway lumen, and for evaporation to take place so as to condition (humidify) inspired air (Cauna et al., 1969). Large venous cavernous sinusoids, mainly localized to the inferior turbinates,  are  characteristic o f the nasal mucous membrane. They are normally found in a semicontracted condition resulting from sympathetic nerve-mediated smooth muscle tone. The cavernous sinusoids are regarded as specialized vessels adapted to the functional demands o f the nasal airway with respect to heating and humidification o f inhaled air. When they distend with blood the mucosa w i l l swell and tend to block the airway lumen (Niels et al., 1998).  Furthermore, extravasation takes place through the walls o f  postcapillary venules during inflammation o f the mucosa, by the opening o f gaps in the intercellular junctions between endothelial cells (Cauna 1970).  Blood can bypass the capillary bed v i a arteriovenous anastomoses. The role o f the arteriovenous anastomoses is probably related to temperature and water control. A t least 50% of the blood flow in the nasal mucosa is normally shunted through arteriovenous anastomoses and total blood flow per cm o f tissue is greater i n the upper airway mucosa than in muscle, brain and liver (Anggard 1974; Drettner et al., 1974).  Nasal blood vessels are under endothelial and neuronal control (Riederer et al., 2002). A dual (endothelial and neuronal) control exists in arterioles whereas the control in the subendothelial muscular swellings o f the cushion veins appears to be mainly neuronal.  10  Non-ciliated  Venule  Arteriole  Figure 1.1: Schematic representation o f the layers o f human nasal mucosa. From Netter 2004.  The swelling o f the nasal mucosa is achieved by a simultaneous relaxation o f all smooth muscle cells, which leads to dilatation o f arteries as well as venous sinuses. The drainage o f the vascular bed is reduced by the venous muscular bolsters protruding into the lumen o f the venous sinuses. V i c e versa, a contraction o f all smooth muscle cells leads to a contraction o f the arteries and, consecutively, to a reduction o f blood supply. Simultaneously the muscular bolsters are rise out o f the lumen o f venous sinusoids allowing blood drainage to be increased hence nasal decongestion (Figure 1.2).  11  Arteriole  Capillary Postcapillary Venule  Venous Sinus  Smooth muscle  Figure 1.2: Schematic representation of different endothelial and neuronal control of blood flow in nasal blood vessels. N A : norepinephrine, N P Y : neuropeptide Y , VIP: vasoactive intestinal peptide, N O : nitric oxide, ACh: acetylcholine, SP: substance P, ET1: endothelin 1, C G R P : calcitonin gene related peptide. Adapted from Riederer et al., 2002.  1.1.3  Innervation  The nasal mucosa, including glands and blood vessels, are supplied by both afferent and efferent neurons (Figure 1.3). The afferent neuronal supply can be divided into two parts: the first, the olfactory nerve (cranial nerve I), projects into the olfactory mucosa, and conducts the sensation o f smell; the second, cranial nerve V , projects to the epithelium and detects perception o f airflow v i a A fibres, and noxious stimuli v i a unmyelinated C fibres and A a fibres. Activation o f these afferent nerves leads to local axonal and central reflexes (Baraniuk 1998).  12  nervous system  Superior cervical sympathetic ganglion Trigeminal ganglion  Sphenopalatine (parasympathetic ganglion)  _ (ZJ  0 5  Nasal congestion, Plasma exudation, Inflammation, Mucus secretion  Nasal congestion, Mucus secretion  eg  Nasal decongestion  Figure 1.3: Schematic representation of the innervation of the nose. N A N C : non adrenergic non cholinergic, C G R P : calcitonin gene related peptide, Sub-P: substance P, N K - A : neurokinin A , A C h : acetylcholine, VIP: vasoactive intestinal peptide, N O : nitiric oxide, N E P : norepinephrine, N P Y : neuropeptide Y .  13  There is a rich parasympathetic innervation to glands. Nervous stimulation o f glandular cholinoceptors causes marked hypersecretion and is often part o f a reflex arc. B l o o d vessels, have both sympathetic and parasympathetic innervation, but are controlled mainly by sympathetic fibres. A continuous release o f noradrenaline is postulated to keeps the sinusoids partly contracted since vasoconstrictor effect o f stimulation o f the alpha- adrenoceptor is more marked than vasodilatation resulting from stimulation o f the p -receptor (Niels et al., 1998). 2  The classical neurotransmitters, noradrenaline and acethylcholine, have in recent years been found to be accompanied by a number o f peptide neurotransmitters. These are secreted by afferent unmyelinated C fibres (substance P, calcitonin gene-related peptide ( C G R P ) , neurokinin A ( N K A ) , gastrin-releasing peptide), from efferent parasympathetic nerve endings (vasoactive intestinal peptide (VIP), peptide histidine methionine), and from efferent  sympathetic nerve endings (neuropeptide  Y ) (Uddman et al., 1987;  Lundblad 1990; Baroody 1997). Neuropeptides are capable o f generating local reflexes which causes an increase i n vascular permeability, plasma leakage, vasodilatation and subsequent tissue oedema (Baraniuk 1997).  1.1.4  Physiological function  Apart from being the first part o f the airways, the nose has two major functions; firstly, olfaction, and secondly, conditioning o f the inspired air for the lungs, by heating, humidifying and cleansing inhaled air. The normal nose is characterized by slit-like passages, which provide for efficient exchange o f heat arid moisture and the width o f these nasal cavities are actively regulated v i a the sympathetic innervation and tone in the  14  venous sinusoids. Nasal cycling is the cyclic alteration between the resistances on the two sides o f the nose. This changes from one side to the other at 2-4-h intervals. Eighty percent o f humans show this nasal cycle, and it has also been demonstrated in rat, rabbit, and pig. In addition, the nasal cycle is perceived by subjects with a deflected septum and by rhinitis patients (Niels et al., 1998). The nasal cycle seems to be predominantly vascular, and it is mediated via the nervous control o f the sinusoidal erectile tissue. Cutting the cervical sympathetic nerves, or blocking the sympathetic supply by local anesthesia, abolishes the nasal cycle in human and in lower animals (Widdicombe, 1986).  The nose is well suited to its air-conditioning function: (i) the slit-like shape o f the nasal cavity assures close contact between the inhaled air and the mucous membranes; (ii) the width o f the cavity can adapt rapidly to changing needs by alteration in sinusoid contraction; (iii) heat exchange is facilitated by the large amount o f arterial blood flowing in arteriovenous anastomoses, analogous to hot water in a radiator; (iv) the nasal mucosa has a high secretory capacity. Furthermore, the body saves about 100 m l o f water per day, due to condensation o f exhaled water in the anterior part o f the nose, which has a temperature 3-4°C lower than that o f the lungs. This water may contribute to rhinorrhea i n cold weather.  In addition the nose acts as a filter. Almost all particles larger than 10 um (e.g. pollen grains) are retained in the nose during breathing at rest, while most particles smaller than 2 um (mould spores) can bypass the nose. The nose also acts as a protective filter for water-soluble gases (sulphur dioxide, formaldehyde). Inhaled particles, trapped  15  in the nasal filter, are cleared from the nose within 30 m i n by mucociliary transport (Hilding et al., 1963; Andersen et al., 1974).  1.1.5  Nose structure and function of other species  In addition to the obviously wide range o f size and external shapes o f the nose between human and animals, there are also clear interspecies differences in the internal anatomy and physiology o f the nose (Figure 1.4).  Figure 1.4: External and internal anatomical arrangement of human and guinea pig noses. A and B indicate external appearance of human and guinea pig noses respectively. C and D show nasal cavities of human and guinea pig respectively. Composite from non-copywrite web sources.  16  The development o f the nasal cavity i n most mammals, excluding man and some higher apes (orangutan, chimpanzee, gorilla), is reflected in its primary function o f olfaction. Carnivores including dog and cat, and other species, such as rodents have complex nasal cavities with large areas for olfaction. O n the other hand, the fact that optimum temperature and humidity are necessary for the detection o f odor and normal function o f the lower respiratory tract, plus the characteristic rapid movements and breathing patterns o f these animals, has resulted i n the development o f a relatively large surface area for air conditioning. This air conditioning mechanism is especially apparent in desert animals, such as the camel, and in cold-water diving mammals such as seals. Thus, from a comparative viewpoint, humans have relatively simple noses with the primary function for breathing, while other mammals have more complex noses with primary function for olfaction. A s a result o f this distinction in primary function, the anatomy o f the nasal cavity i n relation to the oral cavity is arranged i n such a manner that, while man (and some higher apes) can breathe both nasally and oronasally, other mammals are generally obligatory nose breathers, due to the close apposition o f the epiglottis to the soft palate in such species (Proctor et al., 1983).  Despite the greater complexity and the variations in the shape and dimension o f the nasal cavity and its turbinates, the nasal airways o f most animals have characteristics similar to those i n humans. From nostrils, which are in line with the nasal fossae i n most instances, air must pass the vestibule and through the nasal valve into the main chamber (nasal cavity) that is divided into two rather symmetrical compartments. Posterior to the termination o f the nasal septum, the air passages then merge into one and travel downward through the nasopharyngeal meatus into the nasopharynx. Beyond their  17  similarity in airway anatomy, there are major structural differences between man and other mammals in the nostrils, vestibules, nasal septum, and the turbinates that can modify the course o f the air current. In some diving mammals the nostrils can be regulated to open and close, while those o f others are comma-shaped. The vestibule o f rats, mice and cats contains atrioturbinates that are effective baffle systems to deflect a large volume o f air and trap particulates and contain lateral nasal glands which are absent in man. The septum o f rats, mice, hamsters, and guinea pigs contains the so-called "septal window: so that i n some experiments, the two halves o f the chamber cannot be treated individually (Kelemen 1950; Kelemen 1953; Bang et al., 1959).  Histologically, the relative distribution o f squamous, respiratory, and olfactory epithelium from nostrils to nasopharyx is similar between man and other mammals although the nasal respiratory epithelium o f man appears to be more evenly covered by cilia, goblet cells and secretory acini than most animals (Negus 1959).  Underneath the respiratory epithelium, the lamina propria is rich in venous plexes. When these are altered, they can affect the thickness o f the respiratory mucosa and thus the width o f the airway. In general, there is very little difference between man and other mammals in the organization and the ultrastructure o f the vascular system within the nose (Dawes et al., 1953; V a n Diest et al., 1979). The choncae o f guinea pig nose has extensive network o f arterioles and venules. The arterioles and the venules are richly innervated, abounding in cholinergic nerve endings with peptidergic vesicles. The glands are located in the posterior portion and are composed o f acini, and intercalated  18  and striated duct. A c i n i as well as the intercalated duct have cholinergic nerve endings and vesicles, but also a probable peptidergic produce (Pastor et al., 1990).  In guinea pig nose, the distribution o f fibers containing vasoactive intestinal polypeptide (VIP) is dense i n the glandular tissue, but sparse around blood vessels and very sparse in the subepithelial layer. Fibers containing calcitonin gene-related peptide (CGRP)  are  densely distributed around blood  vessels, glandular tissue  and  the  subepithelial layer. A moderate number o f C G R P containing fibers were observed i n the intraepithelial layer. Fibers containing substance P and neurokinin A are sparsely distributed around blood vessels, glandular tissue and the subepithelial layer. In addition, a greater content o f both neurokinin A and substance P is located in the nasal concha than in the nasal septum (Su 1989).  1.2  Pathophysiology of allergic rhinitis in various species including human The allergic sensitization that characterizes allergic rhinitis has a strong genetic  component. The tendency to develop IgE/mast cell/TH2 lymphocyte immune responses is inherited in atopic patients. In addition, the hygiene hypothesis, first proposed by Strachan in 1989, explains the increasing prevalence o f atopic conditions like allergic rhinitis (Strachan et al., 1989). The idea arose from epidemiological observations suggesting an inverse correlation between family size and the prevalence o f allergic rhinitis. The hypothesis proposes that reduced contact with microbes, and diminished burden o f infectious disease at an early age, leads to weakened immunological drive in the T h l direction resulting in overactivity o f Th2 responsiveness. However, substantial  19  evidence suggests a negative relationship between infection and atopic diseases ( L i et al., 2003). Exposure to threshold concentrations o f dust mite fecal proteins, cockroach allergen, cat, dog, and other danders, pollen grains, or other allergens for prolonged periods o f time leads to the presentation o f the allergen by antigen presenting cells to C D 4 + T lymphocytes, which then release interleukin (IL)-3, IL-4, I L - 5 , and other T H 2 cytokines. These cytokines drive proinflammatory processes, such as IgE production, against these allergens through the mucosal infiltration and actions o f plasma cells, mast cells, and eosinophils. Once the patient has become sensitized to allergens, subsequent exposures trigger a cascade o f events that result in the symptoms o f allergic rhinitis. Allergic rhinitis is characterized by a two-phase allergic reaction: an initial sensitization phase where allergen exposure results in IgE formation as well as induction of the humoral response, and subsequent clinical disease after repeated antigen exposure. The clinical phase can also be further subdivided into early- and late-phase responses.  1.2.1  Sensitization The first step towards generation of a T helper lymphocyte response is the  recognition and uptake o f antigen by antigen-presenting cells (e.g. dendritic cells, macrophages, B cells) that have the capacity to digest the antigen into short peptides that associate with major histocompatibility complex ( M H C ) molecules and to provide costimulation for naive T cells (Lambrecht 2001). Dendritic cells have been identified as the most effective antigen presenting cells for inducing and regulating the primary immune response in vivo and in vitro (Banchereau et al., 2000). The mucosa o f the nose  20  is covered with an extensive network o f dendritic cells which reside in the para and intercellular channels surrounding the basal epithelial cells (Evans et al., 2000). There are three dominant mechanisms by which immature dendritic cells can uptake an antigen. First, antigenic material can be acquired v i a receptor-mediated endocytosis involving clathrin-coated pits. Immature dendritic cells express a plethora o f specialized cell receptors for patterns associated with foreign antigens, such as the C-type lectin .carbohydrate receptors (Mahnke et al., 1999; Valladeau et al., 2000; Geijtenbeek et al., 2000; A r i i z u m i et al., 2000; Cochand et al., 2000). Secondly, an antigen can be taken up by a constitutive macropinocytosis that involves the actin skeleton-driven engulfment of large amounts of fluid and solutes (approximately one cell volume/hour) by the ruffling membrane o f the dendritic cell followed by concentration o f soluble antigen in the endocytic compartment (de Baey et al., 2000). Thirdly, dendritic cells have been shown to phagocytose particulate antigens such as latex beads, and even whole bacteria, as well as apoptotic cells. This could be the dominant mechanism o f uptake o f particulate allergens (Banchereau et al., 2000). After being taken up by any o f the  above  mechanisms antigens accumulate i n the endocytic compartment, where they are loaded on newly synthesized and recycling M H C class II molecules. However, they may also be transported into the cytosol, where they become accessible to the class I antigen presentation pathway (Rodriguez et al., 1999; de Baey et al., 2000). Within the endocytic compartment,  antigen is cleaved into short immunogenic peptides  by proteolytic  enzymes. Antigen is loaded on M H C class II molecules in an acidic cellular compartment rich in newly synthesized M H C class II molecules, called the M I I C  compartment  (Nijman et al., 1995). Alternatively, immunogenic peptides can be loaded onto pre-  21  formed M H C II molecules that have been internalized into mildly acidic endosomal vesicles after being expressed on the cell-surface (Cella et al., 1997). In addition, antigen processing by proteases can occur extracellulary generating peptides that can be loaded onto empty cell surface-expressed M H C class II. Surprisingly, proteolysis o f antigen by immature dendritic cells can also occur extracellularly through secreted proteases, resulting in the generation o f peptides that can be loaded onto empty cell surfaceexpressed M H C class II (Santambrogio et al., 1999). Subsequently, dendritic cells migrate through submucosa and present the processed antigen to naive undifferentiated ^Helper  ( T H ) lymphocytes. Antigen-specific T cells bind the dendritic cell M H C class II-  peptide complex with C D 4 and this interaction, along with other cell-cell signals, triggers the T cells to differentiate into T 2 cells and activation o f B lymphocytes which produce H  antigen- specific IgE. IgE is the principal trigger for allergic rhinitis. IgE interacts with both F c s R l and the lower-affinity receptor FcsR2 (CD23). Differentiation o f B cells into IgE-secreting plasma cells requires at least two distinct signals in IL-4 (or IL-13) and C D 4 0 L on the surface o f T 2 cells with C D 4 0 , a co-stimulatory molecule on B cells which triggers H  isotype switching to IgE. IgE binds to the a-chain o f the tetrameric F c s R complex on mast cells, basophils, monocytes  and dendritic cells. The molecular  interactions  responsible for high-affinity binding are complex and involve several sites i n the C 3 E  domain o f IgE (Chang 2000). In its free form, IgE has a half life o f only a few days, however, when bound by FcsRs it is protected against degradation and can remain on the surface o f inflammatory cell for months (Brostoff et al, 1996). Circulating antigen-specific IgE binds (using Fc  22  region) to FceRI receptors on the surface o f nasal mast cells and basophils exposing the antigen-specific Fab region to the local environment, ready to be activated by further allergen exposure. The initial exposures and the process o f priming the inflammatory cells for response to antigen is referred to as sensitization. Re-exposure to the same allergen on a mucosal surface, results i n a coupling or cross-linking o f the IgE molecule that leads to cellular degranulation and the release o f inflammatory mediators, a process resulting in both an acute and a chronic phases (Figure 1.5). On-going allergic inflamation  Early phase response Sneezing Mast cell  IgE class switch  Nasal obstruction Histamine *•'" Leukotriena Prostaglandin  Rhinorrhea  Mast cell  Cytokines Chemokines A  Late phase response  Histamine Tryptase  P-selectin  RANTES GM-CSF Eotaxin  [  I iJ  I L _ 5  RANTES R>  V/f7>s Tcell  § 1 »VLA-4  IL-4/ IL-13/ TNF-a  PAF ECP LTB4 MBP  Eosinophil  ( _ • _ )  IL-4/IL-13/ TNF-a Histamine  Basophil VCAM1-1  VLA-4  C5D dDCED QD Figure 1.5: Schematic representation of pathophysiology of allergic rhinitis. A g : antigen, IgE: immunoglobulin E, IL: interlukin, TNF-a: tumor necrosis factor-alpha, P A F : platelet activating factor, ECP: eosinophil chemotactic protein, LTB4: leukotriene B4, G M - C S F : granulocyte macrophage colony stimulating factor, V C A M - 1 : vascular cell adhesion molecule 1, V L A - 4 : very late antigen 4, EP: epithelium, R A N T E S : regulated upon activation normal T cell expressed and secreted. After Pawankar 2001  23  1.2.2  Acute phase During periods o f continuous allergen exposure increasing numbers o f IgE-coated  mast cells traverse the epithelium, recognize the mucosally deposited allergen, and degranulate  (Naclerio 1991a).  Products  o f this  degranulation  include  preformed  mediators such as histamine, tryptase (mast cell specific marker), chymase ("connective tissue"-mast  cells only), kininogenase  (generates bradykinin), heparin, and other  enzymes. In addition, mast cells secrete several inflammatory mediators de novo (ie, one that are not preformed and stored i n mast cell granules) including prostaglandin D 2 and the sulfidopeptidyl leukotrienes ( L T ) L T C 4 , L T D 4 , and L T E 4 . These mediators cause blood vessels to leak and produce mucosal edema plus a watery rhinorrhea characteristic of allergic rhinitis. Glands secrete mucoglycoconjugates and antimicrobial compounds and dilate blood vessels to cause sinusoidal filling and a resulting occlusion and congestion o f nasal air passages. These mediators also stimulate sensory nerves, which convey the sensations o f nasal itch and congestion, and recruit systemic reflexes such as sneezing. The above responses develop within minutes o f allergen exposure and are termed the early phase, or "immediate," allergic response  (Mygind et al., 1993).  Sneezing, itching, and copious clear rhinorrhea are characteristic symptoms during the early phase o f allergic responses, although nasal congestion may also occur.  1.2.3  Chronic phase Mast cell-derived mediators released during early phase responses, as well as  mediators released by basophils during the late phase, are hypothesized to act on postcapillary endothelial cells to promote the expression o f vascular cell adhesion  24  molecule and E-selectin which facilitate the adhesion o f circulating leukocytes to the endothelial cells. Chemoattractant cytokines such as IL-5 promote the infiltration o f the mucosa with eosinophils, neutrophils, and basophils, T lymphocytes, and macrophages (Naclerio et al., 1985; Bascom 1988). During the 4- to 8-hour period after allergen exposure, these cells become activated and release inflammatory mediators which i n turn reactivate many o f the proinflammatory reactions o f the immediate response. This cellular-driven late inflammatory reaction is termed the "late phase response." This reaction is clinically indistinguishable from the immediate reaction, but congestion tends to predominate (Skoner et al., 1988). Eosinophil-derived mediators such as major basic protein, eosinophil cationic protein, and leukotrienes have been shown to damage the epithelium, leading ultimately to the clinical and histological picture o f chronic allergic disease. Subsets of the T-helper lymphocytes likely orchestrate the chronic inflammatory response to allergens. T H 2 lymphocytes promote the allergic response by releasing I L - 3 , IL-4, I L - 5 , and other cytokines that promote IgE production, eosinophil chemoattraction and survival in tissues, and mast cell recruitment (Durham et al., 1992). Cytokines released from T H 2 lymphocytes and other cells may circulate to the hypothalamus and result in the fatigue, malaise, irritability, and neurocognitive deficits that commonly are noted in patients with allergic rhinitis (Sim et al., 1995).  1.2.4  Inflammatory cells in allergic rhinitis One o f the hallmarks o f allergic diseases is an intense accumulation o f  inflammatory cells in tissue locations at specific mucosal surfaces. The presence o f an increased number o f mast cells, basophils, T cells, and particularly eosinophils, has been  25  detected i n nasal smears and biopsies from patients with allergic rhinitis. It has also been shown in response to certain mediators, that these inflammatory cells undergo local activation, releasing their own mediators, thereby contributing to the pathological features o f the disease.  1.2.4.1 Mast cells Mast cells are constitutive cells within the normal nasal mucosa and are the recognized key cells for type 1 hypersensitivity reactions. These cells can be subdivided into connective and mucosal phenotypes. Connective tissue mast cells express chymase, tryptase and T N F - a (Bradding et al., 1995). This cell population represents 85% o f the IL4 positive mast cells i n the nasal lamina propria. During allergen exposure, there is an increase in the proportion o f mast cells i n the epithelial cell layer (Juluisson et al., 1995). These cells which produce predominantly tryptase, without chymase, are called mucosal mast cells and are 15% o f the IL4 positive mast cells. In sensitized individuals, the nasal mucosa is full o f IgE-binding mast cells (Enerback et al., 1986). Mast cells have long been considered to primarily serve as important effector cells for acute IgE-associated allergic reactions. Mast cells in patients with allergic rhinitis produce Th2 type cytokines, induce IgE synthesis in B cells, and can autoactivate v i a the mast cell-IgE-FcsRI cascade. In addition, mast cells upregulate the production o f a variety o f cytokines/chemokines in epithelial cells and fibroblasts and induce the recruitment  o f basophils, T cells and eosinophils into sites o f allergic  inflammation as well as their own intraepithelial accumulation by upregulation o f adhesion molecules like V C A M - 1 and through the interactions o f nasal mast cells with the extracellular matrix proteins, and nasal epithelial cells. Thus, it is increasingly evident  26  that mast cells are not only important for the genesis o f the allergic reaction, but also contribute to the late-phase allergic reaction, and to on-going allergic inflammation (Yamagishi et al. 20G0; Pawankar 2005). . Activation o f mast cells can occur by antigen and immunoglobulin E (IgE) v i a the high-affinity receptor (FcsRI) for IgE. The liberation o f proteases, leukotrienes, lipid mediators, and histamine contribute to tissue inflammation and allow recruitment o f inflammatory cells to tissue. In addition, the synthesis and expression o f a plethora o f cytokines and chemokines (such as granulocyte-macrophage colony-stimulating factor [ G M - C S F ] , interleukin-1 [IL-1], I L - 3 , I L - 5 , tumor necrosis factor-alpha [TNF-alpha], and the chemokines IL-8, regulated upon activation normal T cell expressed and secreted [ R A N T E S ] , monocyte chemotactic protein-1 [ M C P - 1 ] , and eotaxin) by mast cells can influence leukocytes biology which has a great effect in allergic inflammation (Shakoory et al., 2004).  1.2.4.2 Eosinophils The eosinophil, a granular bi-lobed leukocyte readily stained by eosin (eosinophil = eosin + philein, to love in Greek), comprises approximately 2 to 5% o f granulocytes in a nonallergic person. Eosinophil progenitors are released from the bone marrow into the circulation and are chemically attracted to their site o f action by chemotactic factors. The development and maturation o f eosinophils can also occur i n situ in peripheral sites o f inflammation containing pre-existing increased tissue eosinophils (Adamko et al., 2004). Activated eosinophils play a role in allergy, asthma, parasitic diseases, granulomatous disorders, fibrotic conditions and several malignant tumors (Munitz et al., 2004).  27  Immunohistochemical  staining  o f nasal  mucosa  biopsies  has  show  that  eosinophils are evident within the submucosa and epithelium in symptomatic rhinitis (Bentley et al., 1992, Bradding et al., 1993). Eosinophils are mainly involved in the latephase reaction after infiltration from the peripheral blood into the tissue. Cytokines secreted by Th-2 cells account for recruiting and activating eosinophils in the nose. A m o n g them, IL-4 is considered to be pivotal since it up-regulates adhesion molecules selective for eosinophil recruitment  (Krouse et al., 2002; Ciprandi et al., 2004).  Eosinophils contain granules composed o f four basic proteins. The core of these granules is major basic protein ( M B P ) , while the matrix surrounding the core is composed o f eosinophil cationic protein (ECP), eosinophil-derived neurotoxin ( E D N ) and eosinophil peroxidase (EPO) (Gleich et al., 1994). The possible roles o f these proteins i n allergic airway disease are summarized in Table 1.1. The levels o f E C P , E P O and M B P are raised following antigen challenge in allergic rhinitis (Knani et al., 1992; Shin et al., 1994; Nishioka et al., 1995). Another mechanism by which eosinophils stimulate the late-phase allergic inflammation is by releasing arachidonic acid metabolites such as prostglandins and leukotrienes (Krouse et al., 2002; Saito et al., 2004). Additionally, it has been shown that human eosinophils express and synthesize a number o f cytokines, including G M C S F , I L - 6 , I L - l a , I L - 2 , I L - 3 , IL-4, I L 5 , I L - 8 , R A N T E S , and T N F - a (Moqbel et al., 1991; Hamid et al., 1992; Costa et al., 1993).  28  Protein MBP  ECP EDN EPO  Cell content  | Role in  allciL'ii  anw  i\  JIMMM.*  ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Histamine release from basophils and mast 9 cells Cytotoxic to epithelial cells Causes bronchoconstriction and induces hyperresponsiveness - Activates neutrophils histamine release from mast cells 5 cytotoxic to variety o f cells 3 Undefined 12 Cytotoxic to airway epithelium Causes bronchoconstriction Inactives leukotrienes  Table 1.1: Role of eosinophil cationic proteins in allergic airway disease. M B P : major basic protein, ECP: eosinophil cationic protein, E D N : eosinophil-derived neurotoxin, EPO: eosinophil peroxidise.  It is clear that eosinophils are major participants in the immunopathogenesis o f allergic inflammation since they are characteristically recruited to such sites to release their cationic proteins, cytokines, and lipid mediators. Thus, they contribute to damage and dysfunction o f other resident cell types and influence the inflammatory process.  1.2.4.3 Basophils Basophils are only present in very low numbers in peripheral blood, and are not found  in normal non-inflamed  tissues,  indicating they  are  recruited  to  sites o f  inflammation by mediators from other cell types. Basophils are evident in nasal smears i n allergic rhinitis (Okuda et al., 1985, Otsuka et al., 1985) and can be demonstrated to increase in rhinitic patients following nasal allergen challenge (Bascom et al., 1988). Evidence o f basophil infiltration into the nasal mucosa during allergen challenge is based on the mediator profile i n nasal secretions (Naclerio et al., 1985, Bascom et al., 1988).  29  Basophils, like mast cells, possess high affinity IgE receptors, and are derived from CD34-positive progenitor cells i n the bone marrow (Knapp 1990). When activated, basophils are prominent sources o f the inflammatory mediators found in allergic latephase reactions, such as histamine and L T C . 4  Basophils possess fewer, larger granules and differ from mast cells in that they contain less histamine. Following IgE-dependent activation, basophils only releases 2030% o f the histamine released from a comparable number o f mast cells (Cantells et al., 1987). Human basophils have also been shown to secrete cytokines, particularly IL4 and IL-13, when activated by IgE-dependent stimuli, modulating their response and the immune responses o f other cell types that participate in allergic rhinitis (Mac Glashan et a l , 1994, Schroeder et al., 1994, Schroeder et al., 1996).  1.2.4.4 T lymphocytes T lymphocytes have evolved to coordinate and amplify the effector functions o f antigen specific and non-specific inflammatory cells such as B cells and eosinophils. T lymphocytes have been divided into two distinct subtypes based upon their effector functions. C D 4 + T cells represent the T helper cells, which are important in the regulation o f antigen-driven receptors,  inflammatory processes.  V i a antigen-specific  C D 4 T cells are capable o f recognizing processed  T  cell  foreign antigen i n  association with M H C class II on specialized antigen-presenting cells (e.g. macrophages and dendritic cells). O n the other hand, C D 8 + T cells which represent T suppressor cells, drive the cell-mediated response, and respond to A P C presenting antigen i n conjunction with M H C class II molecule. The T lymphocyte represents a significant.non-structural  30  cell within the nasal mucosa. A n increase in the release o f these cells has been described in nasal biopsies specimens from rhinitic patients. These are generally C D 4 + T 2 cells H  displaying the activated phenotype (CD25+) (Varney et al., 1992; Calderon et al., 1994). C D 4 + T H 2 lymphocytes have been shown to play a crucial role i n the induction and maintenance o f chronic allergic inflammation. The presence o f T lymphocytes in allergic inflammation has  been well demonstrated. However, the major  reason  for  their  importance lies on the profile o f cytokines they express upon activation. Although individual T cells have the capacity to produce a wide range o f cytokines, a restricted profile o f cytokines is seen in chronic inflammatory diseases (Kelso 1995). A major feature o f allergic diseases is the high expression o f Th2-type cytokines. T lymphocytes o f the T-H2 subpopulation can generate I L - 3 , IL-4, I L - 5 , G M - C S F and T N F - a (Mossman et al., 1989). The Th2 phenotype is thought to influence subsequent T cell activation and IgE production by B cells i n addition to promoting the attraction, activation, growth, and differentiation o f specific leukocytes such as eosinophils. In this way, activated T cells can initiate and propagate allergic inflammation and participate directly in the events responsible for allergic diseases.  1.2.4.5 Epithelial cells  Besides being part o f nasal mucosa barrier, epithelial cells have the ability to generate pro-inflammatory cytokines and chemokines that can play an important role i n the genesis and persistence o f allergic rhinitis. Following exposure to allergen, in-vitro nasal epithelial cells from atopic individuals are able to release significantly greater amounts o f I L - l p , IL-8, G M - C S F , T N F - a and the chemokine 'regulated upon activation,  31  normal T cell expressed and secreted' ( R A N T E S ) , as compared with nasal epithelial cells from non-atopic individuals. Nasal epithelial cells o f atopic individuals, with a genetic predisposition to upper airway disease, appear to release increased amounts o f proinflammatory cytokines with natural exposure to allergen, augmenting the release o f these cytokines, thereby exacerbating the allergic response (Calderon et al., 1997).  In a preparation o f epithelial-cell-conditioned medium, human upper airway epithelial cells secrete G M - C S F , while epithelial cells from inflamed nasal tissue were shown to secrete larger amounts o f pro-inflammatory cytokines as compared with normal nasal epithelial cells (Ohtoshi et al., 1991). During inflammation, complement activation has been shown to occur upon the nasal epithelial cell membrane with the nasal epithelium being capable o f regulating this process. The integrity o f the nasal epithelium in inflammatory states is thought to depend on the maintenance o f an equilibrium between complement activation and cell membrane regulation o f this activation (Varsano etal., 1996).  Other cells types have also been shown to increase i n allergic rhinitis including neutrophils, macrophages,  dendritic cells, monocytes, B cells (Bachert et al., 1998)  although their crucial role in the responses to allergen challenge is controversial.  1.2.5  Inflammatory mediators in allergic rhinitis  1.2.5.1 Histamine Histamine plays a pivotal role in allergic inflammation. It is synthesized from L histidine by histidine decarboxylase. Histamine is released from the granules o f FcsRI+  32  cells (e.g., mast cells and basophils), after the cross-linking o f surface IgE by allergen or through mechanisms that are independent o f IgE (Enerback et al., 1986; Kaliner 1994;' H o warm  1995). Nasal challenge with histamine causes sneezing, pain, pruritus,  rhinorrhea and nasal blockade (Doyle et a l , 1990). Activation o f sensory neurones by histamine causes sneezing and pruritus (Mygind 1982) i n addition to activating a central reflex-mediated increase in nasal parasympathetic  activity (Hilberg et al., 1995).  Increased release o f parasympathetic mediators (e.g. acetylcholine) stimulates nasal submucosal glands which, together with the increase in vascular permeability, cause rhinorrhea (Baroody et al., 1994).  A l l four histamine receptors ( H I , H 2 , H3 and H4) have been found in the nasal mucosa by molecular biology studies (Nakaya et al., 2004), with higher expression o f H i and H2 i n atopics (Iriyoshi et al., 1996; Hirata et al., 1999).  Most o f the effects o f  histamine in allergic disease occur through H i receptors (Schmelz et al., 1997; Schneider et al., 2002; A k d i s et al., 2003), whereas cutaneous itch and nasal congestion may occur through both the H | - and H -receptors ( M c L e o d et a l , 1999; Sugimoto et al., 2004). 3  Histamine also activates H2-receptors on the smooth muscle cells surrounding nasal capacitance vessels. They mediate muscle relaxation, increase blood content, and thereby enlarge the volume o f nasal mucosa. In addition, histamine modulates function o f immune cells v i a H4-receptors (Riechelmann 2005). In addition to its role in the early allergic response to antigen, histamine acts as a stimulatory signal for the production o f cytokines and the expression o f cell-adhesion molecules and class II antigens, thereby contributing to the late allergic response (Fujikura et al., 2001; MacGlashan 2003).  33  Hi-receptors belong to the superfamily o f G-protein-coupled receptor. In addition, it has recently been shown that these receptors demonstrate agonist-independent signal transduction. Hi-antihistamines inhibit this constitutive signaling, probably by stabilizing an inactive conformation o f the Hi-histamine receptor and acting as inverse agonists (Bakker et al., 2002). V i a the Hi-receptor, histamine has proinflammatory activity and is involved i n the development o f several aspects o f antigen-specific immune response, including the maturation o f dendritic cells, and the modulation o f the balance o f type 1 helper ( T h l ) T cells and type 2 helper (Th2) T cells. Histamine may induce an increase in the proliferation o f T h l cells and in the production o f interferon gamma and may block humoral immune responses by means o f this mechanism. Histamine also induces the release o f proinflammatory cytokines and lysosomal enzymes from human macrophages and has the capacity to influence the activity o f basophils, eosinophils, and fibroblasts ( M a et al., 2002; A k d i s et a l , 2003).  1.2.5.2 Eicosanoids  Eicosanoids are proinflammatory mediators resulting from metabolic degradation of the arachidonic acid originating from membrane  phospholipids. They include  leukotrienes, prostaglandins and thromboxanes.  1.2.5.2.1  Leukotrienes  The name leukotriene comes from the words leukocyte and triene (a compound with three double bonds). What would be later named leukotriene C , "slow reaction smooth muscle-stimulating substance" (SRS) was originally described between 1938 and  34  1940 by Feldberg and Kellaway. The researchers isolated S R S from lung tissue after a prolonged period following exposure to snake venom and histamine (Feldberg et al., 1938; Kellaway et a l , 1940). Leukotrienes are generated by the action o f 5-lipoxygenase on arachidonic acid. They are released i n both the early and late phases following antigen challenge in subjects with seasonal allergic rhinitis, and during the early phase in perennial allergic rhinitis (Naclerio et al., 1985; De Graaf-in't V e l d et al., 1996). There are two classes o f leukotrienes: LTB4 and the peptidyl-cysteinyl leukotrienes (LTC4, LTD4 and LTE4). Leukotrienes have important mediator functions in the upper airways, with implications for the treatment o f allergic rhinitis. A t least two classes o f receptors exist for the cysteinyl leukotrienes and are termed c y s L T l and cysLT2 (Nicosia et al., 1999). The C y s L T l receptor is found in the human airway (including airway smooth muscle cells and airway macrophages), on other pro-inflammatory cells (including eosinophils and certain myeloid stem cells) and in nasal vascular beds. C y s L T s have been intimately related to the pathophysiology o f asthma and allergic rhinitis. Leukotrienes are synthesized by inflammatory cells known to play a key role i n allergic rhinitis (i.e. mast cells, eosinophils and basophils). Moreover, LTC4 and LTD4 are released at measurable concentrations in nasal secretions when nasal mucosa is exposed to allergen (Howarth et al., 2000). In one study, ragweed-sensitive patients challenged intranasally with pollen grains demonstrated a dose-dependent release o f L T C , LTD4 and L T E in nasal lavage, which was correlated with the familiar symptoms 4  4  of increased nasal airway pressure, sneezing, and mucous secretion (Creticos et al., 1984). Furthermore, LTC4, L T D  4  and LTE4 cause a long-lasting eosinophilic infiltration,  35  and have been associated with airway hyperresponsiveness in the lower airways in rats and in man (Christie et al., 1992; Wang et a l , 1993).  Nasal challenge with C y s L T s can reproduce the symptoms o f allergic rhinitis, the effects o f which can be inhibited by administration o f leukotriene receptor antagonists. In a study in normal subjects, topical delivery o f L T D 4 into the nose produced a significant dose-dependent increase in nasal mucosal blood flow and nasal airway pressure. Similar data have been seen in allergic subjects, whereby nasal provocation with LTD4 produced a marked increase in nasal airway pressure ( M c L e o d et al., 1988). In addition, pretreatment with pranlukast (a leukotriene receptor antagonist) inhibits the nasal mucosal swelling induced by topical administration o f L T D (Numata et al., 1999). 4  1.2.5.2.2  Prostaglandins and thromboxanes  Prostaglandins  are  proinflammatory  mediators  resulting  from  metabolic  degradation o f arachidonic acid originating from membrane phospholipids. The most important products o f enzyme cyclooxygenation o f arachidonic acid are prostaglandins D2, E 2 , F 2 a , thromboxane A 2 and prostacyclin (Raskovic et al., 1998). Prostaglandins PGD2 and PGE2 are detected at increased levels i n nasal lavage fluid following allergen challenge in subjects with seasonal allergic rhinitis (Sugimoto et al., 1994; Wagenmann et al., 1996), and perennial allergic rhinitis (Ramisi et al., 1991), but only in the early response, and not the late phase o f inflammation. However, inhibitors o f cyclooxygenase, the enzyme required for the synthesis o f prostaglandins, do not affect the response to antigen in. human nasal airways (Naclerio et al., 1985), suggesting a minimal role o f prostaglandins involvement in allergic rhinitis.  36  On the other hand, thromboxane A 2 induces vascular permeability, eosinophil infiltration in nasal mucosa and nasal congestion after antigen challenge in allergic patients. In addition, the level o f nasal thromboxane A 2 is also increased after antigen challenge in allergic individuals (Motobayashi et al., 2001).  1.2.5.3 Nitric oxide (NO)  N O is produced by the action o f N O synthase (NOS) on the substrate L-arginine. Different isoforms o f nitric oxide synthase exist: neuronal, inducible, and endothelial forms. There is evidence that N O S activity is increased in perennial allergic rhinitis (Garrelds et al., 1995) and in seasonal allergic rhinitis (Martin et al., 1996; Kharitonov et al., 1997). In mice with allergic rhinitis, the distribution o f the different nitric oxide synthases in nasal mucosa was examined. Neuronal and endothelial nitric oxide synthases were found on the surface epithelial and vascular endothelial cells, with no differences between allergic mice and the control group mice. However, the amount o f inducible nitric oxide synthase was elevated in allergic mice (Oh et al., 2003).  Nitric oxide may also have a role in the production o f cytokines necessary for eosinophil survival, such as IL-4 and IL-5 (Barnes et al., 1995). Interestingly, N O is thought to be the main mediator o f inhibitory non-cholinerigic non-adrenergic  (NANC)  transmission. Therefore, inhibition o f N O S could cause a reduction in the activity o f inhibitory N A N C nerves, thereby potentiating neurogenic inflammation mediated by excitatory N A N C nerves. In chronic allergy, excessive N O production causes airway hyperresponsiveness airways  through  via the formation o f the peroxynitrite free radical in guinea pigs inhibiting  cGMP  production  37  (Sadeghi-Hashjin  et  al.,  1996).  Furthermore, other N O metabolites, such as nitryl chloride, can be synthesised by neutrophils, inactivating endothelial cell angiotensin-converting enzyme (Eiserich et al., 1998). This enzyme is involved in the degradation o f kinins and possibly tachykinins in allergic rhinitis (Lurie et al., 1994; Chatelain et al., 1995). Thus inhibition o f this enzyme may influence nasal A H R by potentiating the action o f these mediators.  1.2.5.4 Platelet activating factor (PAF)  P A F is not preformed in storage granules, but produced from phospholipids mobilized from cell membranes by phospholipase A 2 in many cell types (e.g., basophils, neutrophils, monocytes, macrophages or endothelial cells). P A F produced by monocytes and polymorphonuclear leukocytes is secreted, whereas P A F synthesized by vascular endothelial cells activated by various physiologic agonists (e.g., thrombin, bradykinin, histamine, hydrogen peroxide, and leukotrienes C 4 and D4) is not released ( Sisson et al., 1987; Leirisalo-Repo 1994; Cuss 1999; K r u m p et a l , 1999) O f all the inflammatory mediators involved in allergic rhinitis, P A F is perhaps the most potent for inducing vascular leakage, an event that contributes to rhinorrhea and nasal  congestion  (Cuss  1999;  Oppenheimer  et  al.,  2002).  P A F has  potent  proinflammatory properties that have been implicated i n bronchial asthma (Naclerio et al., 1985). However, its role in allergic rhinitis is less well established. Several studies have been performed with P A F receptor antagonists in animal allergic rhinitis. In guinea pigs, CV-3988 blocked vascular permeability and decreased nasal airway pressure induced by topical application of P A F , whereas SM-10661 attenuated antigen-induced increase in late-phase nasal airway pressure (Honda et al., 2002). Another P A F  38  antagonist, A B T - 4 9 1 , inhibited both antigen- induced leakage and decreased airwayresistance in rats and guinea pigs (Bousquet 1998). In the clinic, instillation o f P A F into the nose induces many o f the symptoms o f rhinitis, such as an increase in nasal airway pressure, rhinorrhea, nasal neutrophil influx, and nasal hyperresponsiveness (Andersson et al., 1988; Leggieri et al., 1991; Miadonna et al., 1996). Both P A F and its metabolite (lyso-PAE) have been detected in the nasal fluids and plasma o f patients with rhinitis (Labrakis-Lazanas et al., 1988; Miadonna et al., 1989; Shirasaki et al., 1990).  1.2.5.5 Cytokines  Cytokines as intercellular messenger peptides, are released by a variety o f cells to influence the  activity o f other  cells. Three cytokines are o f importance  in the  development and regulation o f eosinophil function: the interleukins IL-3 and I L - 5 , and granulocyte-macrophage  colony-stimulating factor  (GM-CSF).  A l l three  prevent  apoptosis and prolong the survival o f eosinophils in vitro. In particular, IL-5 is essential for the differentiation o f progenitor cells into eosinophils (Sanderson et al., 1993).  Both IL-4 and IL-5 have been implicated in the development o f airway hyperresponsiveness (Hogan et al., 1997). In animals, IL-5 causes marked eosinophilia, eosinophil activation and airway hyperresponsiveness (Van Oosterhout et al., 1996). IL-4 regulates the activity o f C D 4 + T-lymphocytes, which release a range o f cytokines capable o f priming and activating eosinophils (Mauser et al., 1993). It also activates neutrophils (Howarth 1995). Furthermore, memory T-cells in the nasal mucosa o f patients with nasal allergy can produce IL-4 during allergen exposure. This could upregulate the inflammatory response (Boey et al., 1989). Patients with seasonal allergic  39  rhinitis or perennial allergic rhintis have a raised number o f C D 4 + T-cells (Hellquist et a l , 1992).  Following nasal allergen challenge in humans, levels o f IL-1 alpha, IL-lbeta, I L - 5 , IL-6, IL-8 and G M - C S F are elevated in nasal secretions (Bradding et al., 1993; Gosset et al., 1993; S i m et al., 1995). Eosinophils are potential sources o f these cytokines (Lantero et al., 1996). Moreover, epithelial cells isolated from allergic rhinitic patients showed increased immunostaining for G M - C S F , IL-8, the receptors for IL-1 and TNF-alpha (Galli et al., 1994), and also they release more IL-lbeta, IL-8, G M - C S F and TNF-alpha compared to epithelial cells from non-allergic subjects (Nonaka et al., 1996). Similar increases in IL-4-, I L - 5 - and GM-CSF-positive cells are observed in biopsies from the nasal mucosa o f atopic patients (Calderon et al., 1997). Both interferon-gamma and T N F alpha (and possibly other cytokines) cause an upregulation o f I C A M - 1 on human nasal epithelial cells (Durham et al., 1992) while IL-4 upregulates the expression o f V C A M - 1 . Both these adhesion molecule are upregulated in allergic rhinitis (Bradding et al., 1993).  1.2.5.6 Chemokines  Chemokines are cytokines that possess chemotactic activity. They are divided into groups depending on their chemical structure. The two main groups are C C chemokines, where two cysteine residues are adjacent to each other (e.g. R A N T E S , M I P 1 alpha, eotaxin) and C X C chemokines, in which the two cysteine residues are separated by a third amino acid (e.g. IL-8) (Barnes et al., 1998). The concentrations o f R A N T E S , MIP-1 alpha, eotaxin, and IL-8 detected in nasal lavage are raised following nasal allergen challenge in man (Sim et al., 1995; Rajakulasingam et al., 1997; Minshall et al.,  40  1997; Gosset et al., 1997).  Mucosal cells obtained from the noses o f subjects with  allergic rhinitis show increased expression o f m R N A for R A N T E S (Rajakulasingam et al., 1997), and eotaxin (Minshall et al., 1997).  It is now generally accepted that  R A N T E S and eotaxin are important i n IL-5-mediated eosinophilia, where the latter causes the mobilisation o f eosinophils into the circulation while the local release o f chemokines provides a 'homing' mechansim for the migration o f eosinophils into tissues (Barnes et al., 1998). Administration o f R A N T E S into the nasal airway o f subjects with allergic rhinitis causes an eosinophilia, but does not increase other inflammatory cells (Kuna et al., 1998). However, the same study also found that, after allergen challenge, administration o f R A N T E S also caused an influx o f basophils, neutrophils, lymphocytes and monocytes, as well as causing epithelial shedding, a response similar to that observed in nasal hyperresponsiveness. It is therefore likely that chemokines have an important role i n the recruitment o f inflammatory cells that is observed during the development o f nasal hyperresponsiveness.  1.2.5.7 Kinins  Kinins are proinflammatory peptides that mediate numerous vascular and pain responses to tissue injury. T w o pharmacologically distinct kinin receptor subtypes have been identified and characterized for these peptides, which are named B , and B2 (Regoli ' et al., 1977). The B2 receptor mediates the action o f bradykinin ( B K ) and lysyl-bradykinin ( L y s - B K ) , whereas the B i receptor mediates the action o f d e s - A r g - B K and Lys-des9  A r g - B K (Fredrik et al., 2005). 9  41  Recent investigations have found that airway hyperresponsiveness in the human nasal airway may be kinin dependent. Icatibant, a highly potent bradykinin B 2 receptor antagonist, prevents PAF-induced airway hyperresponsiveness, while P A F causes an increase i n the concentration o f kinins in nasal lavage fluid. Kinins are produced in both perennial allergic rhinitis, and seasonal allergic rhinitis. They could therefore contribute to airway hyperresponsiveness in allergic rhinitis (Turner et al., 1999). Bradykinin causes sensitisation o f C-fibres in the guinea pig trachea (Fox et al., 1996), and there is evidence that, in the human nose, enhanced responsiveness to bradykinin is mediated by neural reflexes (Riccio et al., 1996). Bradykinin can also release substance P and other neuropeptides from sensory nerve endings (Saria et al., 1988; Geppetti et al., 1990), so it may induce airway hyperresponsiveness by a neuropeptide-dependent mechanism. Alternatively, bradykinin can initiate the production o f the cytokines I L - 1 , IL-6 and IL-8 in vivo (Ferreira et al., 1993), and stimulate the release o f TNFalpha/beta and IL-1 from macrophages (Tiffany et al., 1989). In addition, it has been found that bradykinin increases the expression o f the C X C chemokine receptors C X C R 1 and C X C R 2 in patients with allergic rhinitis (Eddleston et al., 2003), and this may contribute to nasal airway hyperresponsiveness.  1.2.5.8 Neuropeptides  Neuropeptides are contained in, and released from a wide range o f nerves. Chemically distinct, they exhibit characteristic, patterns o f localization within the peripheral and central nervous system and possess the ability to cause a range o f diverse biological responses. The nerves that contain and release neuropeptides are primarily  42  unmyelinated sensory C-fibres and myelinated A8-fibres. Such nerves provide a dense innervation to most organs and tissues, in particular, blood vessels, where perivascular nerves often terminate i n close association with endothelial cells. Parasympathetic nerve endings contain vasoactive intestinal peptide (VIP), peptide histidine methionine, and efferent sympathetic nerve endings contain neuropeptide Y . Nasal sensory nerve fibres contain a number o f different peptides, including calcitonin gene-related peptide ( C G R P ) and the tachykinins substance P and neurokinin A ( N K - A ) . These neuropeptides, metabolised by the enzyme neutral endopeptidase (NEP), are released from sensory nerves which form part o f the non-adrenergic non-cholinergic ( N A N C ) nervous system, and are capable o f generating local reflexes which causes an increase i n vascular permeability, plasma leakage, vasodilatation and subsequent tissue oedema (Baraniuk 1997). Please see the section on neuronal events for details o f the role o f neuropeptides i n allergic rhinitis.  1.2.6  Inflammatory mediators in animal models of allergic rhinitis Laboratory animals have been used widely to study the pathophysiological  changes  occurring in allergic rhinitis using pharmacological, immunological  and  histopathological approaches. Rhinitis is induced in guinea pigs, rats, mice, dogs and pigs using different kinds o f allergens with various methods o f sensitization. The sensitized animals produce the different symptoms and signs o f allergic rhinitis, including sneezing, nasal itching, nasal congestion, rhinorrhea. Various pro-inflammatory mediators and cells have been found to be involved i n producing allergic rhinitis symptoms i n the animals, mimicking what has been found in humans. Tables 1.2-1.6 summaries inflammatory  43  mediators and cells, and their role in producing symptoms and pathophysiological changes i n allergic rhinitis in various experimental species.  Table 1.2: Inflammatory cells and mediators in guinea pig allergic rhinitis Mediators and cells  -  Role  *  Reference  histamine ( H I ) , C y s L T s  sneezing, nasal congestion  Mizutani et al., 2003  Tachykinin  Sneezing  Kaise et al., 2001a  sneezing, nose rubbing, histamine ( H I ) , p L T s  vascular permeability,  Kaise etal., 1998  nasal congestion platelet activating factor  nasal congestion  Albert et al., 1998  histamine ( H I )  nasal congestion, A H R  Mizutani et al., 1999  histamine  vascular permeability  Mizuno et al., 1991  nasal congestion  Kaise et al., 2001b  T X A 2 , histamine, neuropeptides vascular permeability, Yamasaki et al., 1997  T X A 2 , histamine nasal congestion exudation, nasal  Shizawa et al., 1997  Leukotrienes (B4, C4) congestion histamine ( H I )  Sneezing  Nabe etal.,2001  histamine ( H I )  nasal congestion  M c L e o d et al., 2002  CysLTs  nasal congestion  Fujitaetal., 1999  44  nasal congestion, edema, Imai et al., 2000  eosinophils, E P O epithelial disruption sneezing, nose rubbing, eosinophils, T X B 2 , p L T s , rhinorrhea, nasal  Zhao et al., 2005  e N O S , histamine ( H I ) congestion  thromboxane, histamine  nasal congestion  Sakairi et al., 2005  leukotriene D 4 , nitric oxide  nasal congestion  Mizutani et al., 2001  histamine and nitric oxide  nasal congestion  Bockman et al., 2002  T X A , pLTs  nasal congestion  Yamasaki et al., 2001  nasal congestion  Imai etal., 2001  2  constitutively produced nitric oxide  Table 1.3: Inflammatory cells and mediators in rat allergic rhinitis Mediators and cells  Role aiid effect  Reference  Cytokines  Sneezing, nose rubbing  Sugimoto et al., 2000a  histamine ( H I )  Sneezing, nose rubbing  Sugimoto et al., 2000b  CysLTs  mucus production  Shimizu et a l , 2000  P A F , histamine, serotonin, LTs  Vascular permeability  Albert etal., 1998  Table 1.4: Inflammatory cells and mediators in mouse allergic rhinitis Mediators and cells cytokines (IFN-gamma, I L - 2 , IL-4) .  Role and effect Sneezing, vascular permeability  45  Reference Murasugi et al., 2005  expression o f i N O S in nasal mucosa  O h et al., 2003  TNF-a  Sneezing, nore rubbing, increase expression o f adhesion molecules, eosinophils infiltration  Iwasaki et al., 2003  T X A 2 , leukotrienes  eosinophil infiltration  Kayasuga et al., 2003  IL-5  Eosinophilopoiesis  Saito et al., 2002  basophils, eosinophils, C D 4 + cells, IL4 and IL5 cells  Sneezing, nose rubbing, A H R  Saitoetal., 2001  Table 1.5: Inflammatory cells and mediators in dog allergic rhinitis Role and effect  Mediators and cells  • -'ReferenSf W'"  leukotriene B 4  neutrophilia, nasal secretion  Cardell et al., 2000  histamine  nasal congestion  Tiniakov et al., 2003  neuropeptide Y  nasal secretion, vasodilatation  Revington et al., 1997)  Table 1.6: Inflammatory cells and mediators in pig allergic rhinitis Mediators and cells  Reference  Role and effect  calcitonin gene related peptide  mediate bradykinin and histamine induced nasal congestion  Malis etal., 2001  histamine ( H I )  watery secretion  Szelenyi et al., 2000  1.2.7  Pathophysiological events in allergic rhinitis  1.2.7.1 Neuronal events Apart  from  sympathetic  and  parasympathetic  nerves  which  contain  norepinephrine and acetylcholine, respectively, a major role has been attributed to the  46  sensory nasal innervation (Baraniuk et al., 1991). The participation o f sensory airway nerves has been demonstrated to play a key role i n allergic rhinitis (Heppt et al., 2004). Nasal sensory nerve fibres contain a number o f different peptides, including calcitonin gene-related peptide ( C G R P ) and the tachykinins, substance P and neurokinin A ( N K A ) . These neuropeptides, metabolised by the enzyme neutral endopeptidase (NEP), are released from the sensory nerves that form part o f the non-adrenergic non-cholinergic ( N A N C ) nervous system, and are capable o f generating local reflexes which causes an increase in vascular permeability, plasma leakage, vasodilation and subsequent tissue oedema (Baraniuk 1997). This response is known as neurogenic inflammation, and is mediated by the tachykinin N K - 1 and N K - 2 receptors. In addition, eosinophils are capable o f producing vasoactive intestinal peptide (VIP) and substance P (Metwali et al., 1994). Increased levels o f substance P and vasoactive intestinal polypeptide (VIP) in nasal secretions o f patients  with allergic rhinitis were demonstrated  after  nasal  provocation (Mosimann et al., 1993). Together with other mediators such as neuropeptide Y (Groneberg et a l , 2004), calcitonin gene-related peptide ( G C R P ) (Springer et al., 2003), they may participate i n - pathophysiological mechanisms underlying allergic rhinitis. Furthermore, the inflammatory mediators released during the allergic response are able to sensitize and activate the sensory nerve endings by inhibiting neuronal afterhyperpolarization and increasing P K C phosphorylation o f neuronal ion channels respectively. Additionally, exposure o f nerve ending to cytotoxic proteins (e.g major basic protein and eosinophil cationic protein) and increases i n the expression o f receptors  47  on the neuronal membranes by cytokines (e.g. IL-1 [3 and T N F a ) may increase neuronal hyperexcitability (Christiansen et al., 2002).  Neurotrophins change the phenotype o f sensory and other types o f nerves. Nerve growth factor ( N G F ) is a potent trophic substance for nerves that can change their phenotype. In sensory nerves, especially C fibers, N G F appears to be the only active neurotrophin. It can be released by several types o f cells, including possibly mast cells. N G F can have acute effects that change neuroterminal function. N G F can also reach the nucleus through retrograde transportation up the body o f the nerve, producing signals that increase neuropeptide content in these nerves, and stimulate nerve growth. Evidence now exists that N G F is present i n the nasal fluids o f individuals with active chronic allergic rhinitis. Furthermore, N G F is acutely released upon nasal allergen challenge (Togias 2000; Sanico et al., 2000).  In allergic rhinitis, the most significant effects o f sensory neuron activation are itch and the recruited reflexes such as sneeze, the allergic "salute" and bilateral parasympathetic reflexes (Baraniuk et al., 2000; Casale et al., 2002). Upon being activated by different mediators, with histamine being the most prominent, the sensory neurons are depolarized and the depolarization propagates up neurons. In the central nervous system, trigeminal nociceptive neurons enter the pons through the sensory root, and turn caudally in the trigeminal spinal tract to terminate i n the pars caudalis o f the nucleus o f the spinal tract i n the lower medulla and upper three cervical segments o f the spinal cord. Pars caudalis interneurons cross the midline to enter the trigeminothalamic tract and terminate in the medial part o f the ventral posterior thalamic nucleus (arcuate or  48  semilunar nucleus). Pain and itch stimuli are received at the thalamic level. Connections between the afferent interneurons o f the nuclei o f the trigeminal spinal tract and the solitary tract  with  the  nucleus  ambiguus  establish the  sneezing reflex.  Similar  connections regulate parasympathetically mediated glandular secretion in the  nose  (superior salivatory nucleus and facial nerve) (Calliet 1992).  1.2.7.2 Vascular events Acetylcholine, catecholamines, various peptides and also nitric oxide participate in nasal vascular control (vasconstriction or vasodilatation) (Lund 1996). These bioactive molecules arise from both sensory and autonomic nerve fibers, and from neuroendocrine cells widely dispersed in the nasal mucosa. The adult human nasal mucosa exhibits dense nerve networks containing vasoactive intestinal peptide, neuropeptide Y , or its C terminal peptide, substance P, calcitonin gene-related peptide ( C G R P ) among others. Sympathetic fibers carry both norepinephrine and neuropeptide Y . Immunoreactivities for neuropeptide Y and C-terminal peptide show full colocalization and are mainly found in perivascular fibers. The subepithelial region contains a dense plexus o f substance P and CGRP-immunoreactive fibers, but these nerves also appear around blood vessels (Anggard et al., 1983; Lacroix et al., 1992; Hauser-Kronberger et al., 1993). Nasal congestion is a common symptom o f acute and chronic rhinitis. It is caused by swelling o f nasal blood vessels that expand so as to restrict or obstruct airflow through nasal passages (Broms 1982). During allergic reactions, a large number o f inflammatory and immunological mediators derived from leukocytes, plasma, and neurons  (e.g.  leukotrienes, kinins, histamine, neuropeptides, N O , A C h ) effect the nasal vasculature by acting on receptors found on different components o f blood vessels to cause either  .49  vasodilatation or vasoconstriction (Lung et al., 1984; Widdicombe 1986; Widdcombe 1990). In the inferior turbinate o f human nasal mucosa, arterioles and venous sinus are constricted by norepinephrine, neuropeptide Y and endothelin-1 resulting in a sense o f decongestion, and dilated by acetylcholine, vasoactive intestinal peptide, nitric oxide, calcitonon gene related peptide and substance P resulting in a sense o f congestion (Riederer et al., 2000). Changes in vascular innervation could be one o f the factors involved in the maintenance o f rhinitis. Nasal vascular hyperinnervation has been detected in patients with allergic rhinitis, as compared with non-allergic individuals (Figueroa et al., 1998). Plasma extravasation (vascular permeability or exudation) is unfiltered plasma containing albumin, antibodies and complement fractions (Bousquet et al., 1996). A n increase in vascular permeability occurs i n both naturally occurring seasonal allergic rhinitis, and in perennial allergic rhinitis (Wilson et al., 1998). Additionally, it has been shown that vascular permeability increases after histamine and bradykinin challenge in individuals with allergic rhinitis (Rajakulasingam et al., 1993).  In addition, these two  vasoactive substances have been shown to increase after allergen challenge (Baroody et al., 1994; Paul et al., 1994). Furthermore, histamine produces  concentration-dependent  nasal airway exudation o f bulk plasma i n subjects with seasonal rhinitis (Svensson et al., 1995). The role o f kinins in induction o f vascular permeability has also been proposed as nasal stimulation with histamine or L T C 4 results in an increase o f nasal vascular permeability and o f kinins concentration in the nasal lavage fluid in allergic rhinitis (Shirasaki et al., 1989).  50  The increase in vascular permeability is particularly marked i n postcapillary venules  where  the  opening  o f the  intercellular gaps together with  anatomical  fenestrations provides the plasma with an alternative route other than through blood vessels (Widdicombe 1997). The inflammatory mediators act on specific receptors on the blood vessels to cause vascular extravasation through either vasodilation, which causes increase  i n intravascular pressure especially post  capillary venules,  or  increase  interendothelial gaps, and eventually escape o f the exudate to the interstitial space and then to the nasal cavity. Beside nasal congestion and exudation, cellular infiltration also occurs in allergic rhinitis. The expression o f adhesion molecules on the endothelial cells induced by acute and chronic released inflammatory mediators enhances the extravasation o f different leukocytes to the site o f inflammation. The role o f the inflammatory cells in allergic rhinitis is described in inflammatory cells section above.  1.2.7.3 Glandular events Rhinorrhea (watery secretion) is one of the symptoms o f allergic rhinitis. Secretions from the nose come from three main sources: the epithelial goblet and serious cells, the submucosal seromucous glands, and the anterolateral deep glands in the nose (Widdicombe et al., 1982; Wells et al., 1986). In addition transudation may contaminate secreted mucus. Nasal mucus secretion is controlled predominantly by parasympathetic cholinergic nerves (Widdicombe 1990). In allergic rhinitis, neurotransmitters ( A C h , Substance P) and inflammatory mediators (histamine, bradykinin, leukotrienes) cause increases i n glandular secretions (Widdicome et al., 1982; Wells 1986; Knowles et al.,  51  1987). In the nasal mucosa o f human inferior turbinates, nerve fibers are found in the periglandular tissue around the acini, ducts and in the periglandular connective tissue. It has been found that V I P is in contact with acinus cells and C G R P is found in the connective tissue around glandular cells suggesting a role in controlling glandular secretions (Knipping et al., 2001).  1.2.8  Nasal Airway Hyperresponsiveness (AHR) Nasal hyperresponsiveness is a hallmark o f allergic rhinitis (Druce et al., 1985;  Mullins et al., 1989). Subjects with allergic rhinitis show an increased response to nasal challenge with a variety o f stimuli, including histamine, bradykinin (both o f which are released following allergen challenge), methacholine, tobacco smoke and  perfume  (Baraniuk 1997; V a n Wijk et al., 1999). A H R is associated with nasal congestion, and increased mucus production and oedema following allergen challenge, in both the upper and lower airways. It is usually associated with the late phase reaction, but can continue well beyond this stage. In fact, it is induced irrespective o f whether the late phase o f inflammation occurs (Togias et al., 1988). Most patients with allergic rhinitis, besides having chronic inflammation in their nasal mucosa that results from allergic reactions, also have chronic inflammation in the lower respiratory tract that can lead to A H R ( M a et al., 2000).  There are a number o f potential mechanisms by which A H R might occur: greater receptor activation due to increased mediators release after initial exposure to allergen; increased exposure o f receptors to any stimulus present (due to damage and destruction of epithelial and interstitial cells, and mucociliary clearance system by platelet activating  52  factor and cytotoxic proteins like major basic protein and eosinophil chemotactic protein); reduced the metabolism o f the mediators (due to loss o f epithelial function); increased receptor expression (e.g. methacholine causes more secretion in allergic subjects than i n non-allergic subjects); and alteration o f intracellular pathways (Laitinen et al., 1985; Devillier et a l , 1988; K o g a et al., 1992; White 1993; Teixeira et al., 1997).  1.2.9  Overall summary  In this introduction, the human nose was discussed in detail. Since most pre clinical studies i n determining human nasal physiology, histopathology and pathogenesis of allergic diseases are conducted using animal tissues, comparisons between human and experimental animals was discussed in detail to assess similarities and differences in terms o f structure and function o f the nose, and to evaluate the value o f such studies in applying and generalizing them to humans. There was extensive discussion o f the guinea pig since this thesis deals with guinea pig allergic rhinitis. There are similarities and differences in terms o f distribution and function o f different component o f the nose (histology, innervation, and vasculation). The pathogenesis o f allergic rhinitis is complex. It involves neuronal, vascular, and glandular events.  Mediators eliciting allergic  responses which are released by inflammatory cells (e.g. mast cells, eosinophils, lymphocytes, basophils and epithelial cells) act i n a synergistic way to produce allergic inflammation.  They  include  cytokines,  chemokines,  neuropeptides and kinins.  53  autacoids,  neurotransmitters,  1.3  Drug targets in allergic rhinitis  1.3.1  Current registered drugs: limitations to their use The two major classes o f drugs used to treat symptoms o f allergic rhinitis are oral  HI  antihistamines and intranasal corticosteroids. These agents may be used  as  monotherapy or in combination, depending on the predominant symptoms and the patient's response to therapy. Alternative agents, such as cromolyn sodium, may be appropriate  in  some  patients.  The  first-generation  antihistamines  include  brompheniramine, chlorpheniramine, and diphenhydramine. Although these drugs relieve the sneezing and rhinorrhea in allergic rhinitis, they cross the blood-brain barrier and are associated with marked drowsiness and impaired mental performance. The secondgeneration antihistamines produce no, or considerably less, sedation than the firstgeneration drugs. However, oral terfenadine  and astemizole produced potentially  dangerous (fatal) cardiac arrhythmias i n some patients (Day, 1999) and have been removed from the market. The  newer  second-generation  antihistamines  (acrivastine,  cetirizine,  fexofenadine, desloratadine, loratadine) are not so associated with such troubling side effects  (Kay, 2000). Fexofenadine,  loratadine, and  desloratadine  are  considered  nonsedating antihistamines. Acrivastine may be sedating in some patients. Cetirizine is considered low sedating. Oral antihistamines are also first-line therapy for allergic rhinitis in children (Dykewicz et al., 1998). These drugs have demonstrated efficacy in the relief o f the seasonal and perennial rhinitis symptoms o f sneezing, itching, and nasal discharge. They have  also been  found to reduce  ocular symptoms  conjunctivitis, which frequently occur i n conjunction with allergic rhinitis.  54  o f allergic  H I antihistamines are generally not considered effective for nasal congestion (Dykewicz et al., 1998). Therefore, in patients with this symptom, combination therapy with an oral antihistamine plus a decongestant may be helpful. Such combinations are available in convenient fixed-dose products that can be taken once daily. However, such decongestants as pseudoephedrine and phenylpropanolamine can have unwanted effects, such as insomnia, loss or stimulation o f appetite, and should be used with caution in patients with conditions such as arrhythmias or angina. Intranasal H I antihistamines, such as azelastine and levocabastine, are also firstline therapy for mild-to-moderate allergic rhinitis (Dykewicz et al., 1998). These topical antihistamines are administered twice daily, and have a rapid onset o f action. Both azelastine and levocabastine have been shown to improve symptoms in patients with seasonal or perennial allergic rhinitis (Bousquet et al., 2001), and they appear to have the potential to reduce nasal congestion. Intranasal corticosteroids are considered first-line treatment for more  severe  symptoms o f allergic rhinitis ( Dykewicz et al., 1998; Bousquet et al., 2001). The most effective medications for controlling allergic rhinitis are nasally inhaled corticosteroids including beclomethasone, (Corren  2000;  budesonide, flunisolide, mometasone,  Kaszuba et  al., 2001).  Corticosteroids target  and triamcinolone the  inflammatory  mechanisms. Thus, intranasal steroids are particularly effective in ameliorating nasal congestion, which is often the main complaint in chronic allergic rhinitis, but they also relieve the other symptoms o f rhinitis, such as rhinorrhea, sneezing, and nasal itching. Most are administered once or twice daily. Intranasal  corticosteroids should be  administered continuously. For optimal benefit therapy should begin before the onset o f  55  symptoms (for example, before pollen season). The onset o f action is slower with intranasal corticosteroids than with oral antihistamines. M a x i m u m benefit usually occurs over days or weeks. Systemic side effects appear to be minimal in adults receiving intranasal corticosteroids. Nose bleeding or irritation may occur with the use o f intranasal corticosteroids although these effects may diminish over time. In rare cases, septal perforations may develop. Intranasal cromolyn sodium has been shown to relieve allergic rhinitis symptoms in some patients. It needs to be initiated before the onset o f symptoms and does not improve symptoms once they occur. It may need to be administered up to four times daily. Immunotherapy may be appropriate i n patients who have severe symptoms whose symptoms are caused by allergens for which potent extracts are available, and who have not responded to pharmacotherapy  (Dykewicz et al., 1998). A g e and concomitant  illnesses are among the factors that determine whether immunotherapy is appropriate. For example, immunotherapy is rarely appropriate in preschool children, the elderly and in those  with  severe  pulmonary  or  cardiovascular  disease.  In  general,  effective  immunotherapy requires three to five years o f treatment. Due to redundancy, synergy and pleiotropism existing amongst the mediators o f allergic rhinitis, there are limitations to the currently, available drugs in terms o f their effectiveness. Antihistamines ( H I ) cause symptomatic relief only, and steroids do not provide acute relief o f symptoms and they are nonselective, unsuitable for some patients (especially in pediatric use), may cause nasal irritation, bleeding and in rare cases even systemic side effects. In addition, timing o f treatment is critical with anti IgE and  56  immunotherapy needs identification o f allergen and multiple injections. Moreover, anticholinergics are useful only in reducing rhinorrhea.  1.3.2  Possible novel targets for treating allergic rhinitis A n improved understanding o f the cellular and molecular mechanisms underlying  the pathogenesis o f allergic rhinitis has resulted i n the identification o f potential novel therapeutic strategies. In theory, the inhibition o f an upstream pathway i n the allergic cascade (for example, dendritic cells or T H 2 cells) is likely to make a greater clinical contribution, compared with the inhibition o f a single downstream mediator (Holgate et al.,2003).  1.3.2.1 Mediator inhibitors 1.3.2.1.1  Antihistamines  The new generation o f H i antihistamines show greatly improved efficacy and safety because they act as inverse agonists (stabilize the inactive conformation o f the receptor and drive the equilibrium away from the active conformation which leads to reduction o f the constitutive activity o f the receptor and inhibition o f basal activity) (Oppenheimer et al., 2002). A number o f antihistamines (e.g. fexofenadine) are also claimed to exert anti-inflammatory actions (Baroody et al 2000). The recent discovery o f the H4 receptor expressed on mast cells, basophils and eosinophils has generated renewed interest i n histamine because H4 receptors mediate C a  57  2 +  signalling and chemotaxis  (Hofstra et al., 2003). It is possible that selective H4 antagonists could have antiinflammatory actions in allergic disease.  1.3.2.1.2  , Leukotrienes inhibitors  Cysteinyl leukotrienes  (CystLTs) are released  from mast  cells, basophils,  eosinophils and macrophages and are particularly important i n causing nasal blockade (Higashi et al., 2003). Clinical trials o f montelukast and zafirlukast ( C y s t L T i receptor antagonists) i n allergic rhinitis have demonstrated that their effectiveness is overall less than that o f topical nasal corticosteroids (Pullerits et al., 2002; Philip et al., 2002; Topuz et al., 2003). The recent discovery o f both C y s t L T i and LT2 receptors on eosinophils, which differ i n their avidity for the leukotrienes L T D  4  and LTC4, indicates a potential  pro-inflammatory role for this mediator class that could be usefully inhibited by a dual antagonist (Evans 2002).  1.3.2.1.3  Prostaglandin receptor antagonists  A m o n g the prostaglandins, P G D , which is a mast-cell-derived eicosanoid, has 2  potent vasodilator properties mediated by the D P i receptor. DP] receptor  antagonists  offer promise for situations i n which nasal blockade is problematic.. A second PGD2 receptor, D P has been identified as a T 2 marker but is also expressed on eosinophils 2  H  and basophils where it serves a chemotactic function antagonism o f which could have anti-inflammatory effects (Arimura et al., 2001; Holgate et al., 2003; Sugimoto et al., 2003).  ,  58  1.3.2.1.4  Tryptase antagonists  Mast-cell granules contain high concentrations o f protease, tryptase. Tryptase exerts a range o f inflammatory responses that have been implicated i n chronic tissue injury and remodelling possibly involving coagulation Factor II receptor-like 1 found on epithelial cells, fibroblasts and smooth muscle. A number o f tryptase inhibitors have been described and some efficacy i n humans has been reported i n allergic models, including allergen challenge (Newhouse 2002).  1.3.2.1.5  Nitric oxide synthase (NOS) inhibitors  There is an increase i n level o f nitric oxide production i n allergic rhinitis. Nitric oxide causes vasodilation and glandular secretion (Baraniuk 1997). N O S inhibitors have been shown to reduce nasal blockade  in perennial  allergic rhinitis and plasma  extravasation i n seasonal allergic rhinitis (Dear et al., 1996). However, N O S inhibitors, when nasally administered to human subjects, caused the development o f upper airway hyperreactivity and significant eosinophilia (Turner et al., 2000).  1.3.2.2 Mast cell stabilizers 1.3.2.2.1  Cromones  Sodium cromoglycate, and its successor nedocromil sodium, are thought to act as mast cell stabilizers, but their precise mechanism(s) o f action is not known. They have been shown to be effective i n reducing immediate phase symptoms i n allergic rhinitis (Kunkel et a l , 1987).  59  1.3.2.2.2  Protein kinase inhibitors  Activation o f Syk kinase, a transducer o f signaling through the Fee receptor o f mast cells via the binding o f ligand to IgE bound to the IgE receptor leads to an array o f responses including degranulation and neosynthesis o f proinflammatory mediators. In a clinical study, inhibitor of Syk kinase ( R l 12) has been shown to be effective i n relieving the symptoms o f allergic rhinitis (Meltzer et al., 2005). Moreover, The finding that genistein, a potent inhibitor o f tyrosine kinase, has potent anti-inflammatory activity on the mast-cell-dependent early- and late-phase allergen-provoked inflammatory reaction i n the airways o f guinea pigs provides proof o f concept for selective inhibitors o f protein kinases linked to mast-cell activation (Duan et al., 2003). In addition, mast cells also express receptors that are able to inhibit IgEdependent degranulation through the activation o f immuno-receptor  tyrosine-based  inhibitory motifs (ITIMS). O n associating with the F c R l , inhibitory receptors, such as £  immunoglobulin-like transcripts (ILTs) and leukocyte immunoglobulin-like receptors (LIRs), are able to affect IgE signalling by triggering phosphorylation o f I T I M sequences on the gamma-chains o f F c R l . A t present, 13 L I R s are recognized, L I R s 1, 2, 3, 5 and 8 E  have inhibitory effects. L I R 5 (gp49A and gp49B) is expressed at a high level by mast cells (Katz et al., 1996). Although no natural ligands for these receptors have yet been identified, they offer targets at which to direct novel inhibitory agents. In the process o f their differentiation, survival and optimal secretion, mast cells are dependent on stem-cell factor (SCF) (Costa et al., 1996). In disorders such as mastocytosis, blockade o f S C F Src kinase activity by the selective inhibitor PP1 has a marked effect in suppressing mast-cell' proliferation (Tatton et al., 2003). Ablation o f  60  mast cells i n the nasal mucosa would clearly have a large benefit i n allergic rhinitis, r  where the mucosal mast-cell population markedly increases.  1.3.2.2.3  Ion channels blocking drugs  Selective blokers o f inwardly rectifying and Ca -activated K channels, and 2+  +  Ca -independent CI" channels linked to IgE-dependent activation, expressed i n mast 2+  cells may also offer promise for a new generation o f anti-allergic drugs (Duffy et al., 2001).  1.3.2.3 Inhibitors of neuronal pathways  Allergic rhinitis is characterized by local neural activity, such as itching, sneezing and reflex-mediated secretion (Baraniuk 1992; Barnes 2001). Although substance P induces eosinophilia i n allergic rhinitis (Fajac et al, 1995), inhibition o f its receptors (neurokinin 1 and 2) has so far proven disappointing when tested in clinical trials. For bradykinin, which is a potent releaser o f neuropeptides, efficacy o f a bradykinin B2 agonist has been reported  i n nasal allergen challenge (Austen et al., 1994), but  subsequent Phase III clinical trials proved disappointing. Other neuropeptides  also  provide interesting targets, including calcitonin-gene-related peptide ( C G R P ) i n chronic vasodilation (Uddman et al., 1999) and secretoneurin, which is present i n cholinergic, adrenergic  and sensory nerves (Korsgren et al., 2003), and which exerts a pro-  inflammatory effect on eosinophils (Dunzeridorfer et al.,T998). If suitable antagonists for these mediators are found then they are likely to be efficacious in the more chronic forms of allergic rhinitis in which nasal blockade dominates.  61  1.3.2.4 Immunotherapy  Certain strategies have been used in immunotherapy. These include the use o f allergen-specific immunotherapy,  allergen peptide-based  immunotherapy,  and D N A  immunotherapy.  1.3.2.4.1  Allergen-specific immunotherapy  The goal o f allergen-specific immunotherapy (ASIT) is to modulate the immune response to allergen and thereby reduce the symptoms o f allergic rhinitis., A S I T is administered as a series o f subcutaneous injections, or sublingually, o f highly purified airborne allergen(s) with a dose o f 6-24 ug to patients with allergic rhinitis who are specifically sensitized to identified allergen(s). A S I T is clinically effective i n reducing symptoms o f allergic rhinitis, as evidenced by the inhibition o f both the allergenprovoked early- and late-phase nasal responses and, i n children sensitized to a single allergen, a reduced risk o f the subsequent development o f sensitization to further allergens (Pajno et al., 2001; Mailing 2002; Moller et al., 2003). The mechanisms through which A S I T produces its beneficial clinical effects are becoming clear (Canonica et al., 2003). A S I T reduces clinical symptoms by inhibiting allergen-specific T 2 cells i n H  favour o f a T 1 response (immune deviation) (Walker et al., 2002), and inducing H  regulatory lymphocytes carrying the C D 4 and C D 2 5 antigens and C D 4 + C D 2 5 - T 3 H  (immune tolerance) ( M c H u g h et al., 2002). Specific immunotherapy also increases allergen-specific 'blocking' I g G l and IgG4 antibodies, a variable decline i n allergenspecific IgE, and reduces both the number and activation state o f mucosal mast cells, basophils  and eosinophils  (Ebner  1999).  62  Even  though  SIT is efficacious, its  administration can be associated with local and systemic allergic reactions, and so a variety o f strategies to reduce this, and to enhance efficacy, are being investigated.  1.3.2.4.2  Peptide-based immunotherapy  The rationale for using short peptides is to reduce the potential for allergic side effects while retaining the beneficial effect o f peptide epitopes recognized by T cells i n modifying their response to allergens, because peptides are unable to crosslink F c R l e  bound IgE on mast cells and basophils (Holgate et al., 2003). The safety and efficacy o f peptides has been taken advantage o f i n the treatment o f cat allergy using several overlapping peptides derived from chain 1 or 2 o f the major cat allergen Fel d l . Weekly subcutaneous immunization with 27-amino-acid peptides derived from Fel d l led to a reduction in rhinitis symptoms on exposure to cats (Norman et al., 1996).  1.3.2.4.3  D N A immunotherapy  Immunostimulatory D N A sequences containing C p G motifs are strong inducers of a T H I immune response to antigen, and have therefore been investigated i n the treatment o f T 2-mediated diseases such as allergic rhinitis and asthma (Horner et al., H  2001a). C p G D N A  inhibits T H 2 responses to antigen indirectly by influencing the  function o f cells o f the innate immune system, rather than exerting direct effects on T lymphocytes. Studies with TLR9-deficient mice have demonstrated that these receptors of the innate immune response are essential in mediating the immunostimulatory activity of C p G D N A , which is characterized by the production o f IL-12, IL-18, interferon  63  gamma, IL-6 and IL-10 (Roman et al., 1997; Hemmi et al., 2000). The cytokine environment  induced by C p G D N A is highly effective at reducing the levels o f  expression o f T H 2 cytokine receptors (for example, the IL-4 receptor) (Horner et al., 2001b). In a mouse model o f allergic rhinitis, C p G D N A administration prevented both the development o f nasal symptoms and eosinophilic inflammation (Hussain et al., 2002).  1.3.2.5 IgE targeting To avoid sensitization with foreign proteins, a humanized monoclonal antibody containing 95% human I g G l and 5% murine IgE-binding epitope has been constructed (Presta et al., 1993). This antibody recognizes IgE selectively, inhibits binding o f IgE to both F c R l and F c R 2 , and therefore fails to initiate mast-cell or basophil activation. B y e  e  this mechanism, omalizumab therapy is accompanied  by a marked  reduction in  inflammatory leukocytes and expression o f F c R l , which, i f not occupied by IgE, £  becomes internalized (Plewako et al., 2002). When administered as two-weekly, or onemonthly subcutaneous injections, omalizumab decreases circulating free IgE by >90% by forming small (1000 kDa), non-complement-fixing complexes that are eliminated by the reticuloendothelial system without causing side effects. In clinical trials o f seasonal allergic rhinitis, omalizumab has shown efficacy (Adelroth et al., 2000; Casale et al., 2001). Furthermore, in children with allergic rhinitis, a combination o f SIT with anti-IgE for 24 weeks was more efficacious than when either treatment was given alone (Kuehr et al., 2002).  64  1.3.2.6 Cytokines and chemokines inhibitors One o f the difficulties i n deciding which cytokines or chemokines to target the treatment o f allergic rhinitis is the large variety o f these that are expressed at sites o f allergic inflammation, as well as their overlapping functions. In allergic inflammation, research has focused particularly on individual T 2 cytokines (for example, IL-4, I L - 5 , H  IL-9 and IL-13) and chemokines that attract cells to sites o f allergic inflammation (Kay 2001).  . To date, there are no published studies o f cytokines antagonists i n humans with  allergic rhinitis. However, animal studies and cytokine challenges in humans help illustrate their effect i n nasal allergy. The therapeutic potential o f a recombinant soluble IL-4 receptor (Nuvance) as an IL-4 antagonist has shown improvement i n asthmatics (Borish et al., 2001). In addition, monoclonal anti-IL-4 antibodies inhibit IgE production in mice (Zhou et al., .1997). Allergen challenge increases the level o f expression o f IL-13 in the nasal mucosa in vivo, whereas, in vitro, IL-13 increases the number o f secretory cells in human nasal epithelial cells (Wills-Karp et al., 1998; Skowron et al., 2003). Targeting IL-13 has been investigated in allergic inflammation in mouse models o f asthma, and its antagonism inhibits the allergic inflammatory response in the lower airways (Wynn et al., 2003). The C - C chemokines, including eotaxin, R A N T E S , monocyte chemoattractant proteins 1 and 3, are particularly relevant to allergic inflammation, since increased levels of these chemokines are detected i n the nasal mucosa following allergen challenge and all interact with the C C R 3 receptor on eosinophils, basophils and mast cells (Terada et al., 2001). Activation o f C C R 3 receptors by application o f eotaxin to the nasal mucosa  65  induces an influx o f eosinophils (Gorski et al., 2002). Studies demonstrating that an 11amino-acid synthetic peptide inhibits nasal influx o f neutrophils and protein exudation induced by nasal challenge with IL-8 in normal subjects indicate the potential for inhibiting the function o f chemokines in the nasal mucosa (Cooper et al., 2001). Given the importance o f the issue, three chemokine receptors C C R 3 , C C R 4 and preferentially expressed by Th2 cells, mast cells or eosinophils therefore  CCR8  represent  therapeutic targets in allergy.  1.3.2.7 Adhesion molecules inhibitors Adhesion molecules expressed on leukocytes and endothelial cells are important for inflammatory cell recruitment during allergic inflammation. A t present, there are no published studies o f anti-adhesion therapy in allergic rhinitis. However, targeting o f these molecules on leukocyte or endothelial cell surfaces has been investigated as an approach to inhibiting allergic inflammation. A m o n g these molecules is endothelial P-selectin, which is highly expressed in the nasal mucosa and has been shown to stimulate eosinophil recruitment i n mouse models o f allergic inflammation (Symon et al., 1994). Subsequent to endothelial tethering, eosinophils firmly adhere to either I C A M - 1 or V C A M - 1 . Blockade o f these receptors in mouse allergic inflammation, and inhibition o f eosinophilic tissue recruitment in ICAM-1-deficient mice, results i n marked inhibition o f adhesion o f eosinophils to endothelium (Broide et al., 1998). Furthermore, eosinophils, basophils, monocytes and T cells, but not neutrophils, express high levels o f very late antigen-4 ( V L A - 4 ) , the ligand for V C A M - 1 (Jackson 2002). Binding o f V L A - 4 to the  66  CS-1 region o f fibronectin also induces eosinophil activation (Anwar et al., 1993), such that by targeting V L A - 4 , cell activation as well as-cell recruitment might be inhibited.  1.3.2.8 Selective phosphodiesterase 4 inhibitors  -  One promising development is the use o f selective phosphodiesterase 4 (PDE4) inhibitors which exert anti-inflammatory activity by blocking the hydrolysis o f cyclic 3 ' 5 ' - A M P i n lymphocytes, eosinophils, neutrophils and monocytes, thereby attenuating their release o f mediators and cytokines (Giembycz 2000). Although known to be effective i n the treatment o f asthma and chronic obstructive pulmonary disease, oral once-daily therapy with the P D E 4 inhibitor roflumilast i n patients with allergic rhinitis subjected to repeated allergen exposure proved to be efficacious, especially on nasal blockade (Sorbera et al., 2000; Schmidt et al., 2001).  1.3.2.9 Heparin Heparin, a straight-chain, highly sulfated glycosaminoglycan, is present in mast cells at high concentrations. Anti-inflammatory and anti-allergic properties o f heparin have been demonstrated i n several in vitro and in vivo studies (Matzner et al., 1984; Lider et al., 1990). Despite its effects on asthma, very little research has been undertaken over the past years to position heparin i n the treatment o f allergic rhinitis. In a clinical study, intranasal heparin significantly reduced symptom scores 10 m i n after antigen challenge. In addition, eosinophil influx i n airway mucosa,  and eosinophil cationic  protein  concentratons i n nasal lavage fluids were reduced (Vancheri et al., 2001). Moreover,  67  heparin, prevents  nasal  mucosa  mast  cell  degranulation  induced  by  adenosine  monophosphate (Zeng et al., 2004).  1.3.2.10  Phototherapy  Ultraviolet ( U V ) light has been shown to exert both local and  systemic  immunosuppression (Salo et a l , 2000; Duthie et al., 2000), and has been widely used for decades in the therapy o f various skin diseases.  The major mechanisms for U V  irradiation-induced immunosuppression involves induction of apoptosis in infiltrating T cells, reductions in the number o f Langerhans cells and their function, and induction o f immunosuppressive cytokines such as interleukin-10 in the skin (Garssen et al., 2001; Nghiem et al., 2002). In addition, U V irradiation inhibits histamine release from mast cells in vitro and in vivo (Gollhausen et al., 1985; Danno et al., 1988). Recently, the immunosuppressive action o f U V has attracted researchers to apply this technique i n allergic rhinitis. In a clinical study, intranasal irradiation with the 308nm xenon chloride (XeCl) ultraviolet-B laser and irradiation with a combination o f ultraviolet-B ( U V B ) , ultraviolet-A ( U V A ) and visible light (VIS) was effective in treating allergic rhinitis (Csoma et al., 2004; Koreck et al., 2005). Furthermore, intranasal therapy with 8methoxypsoralen (8-MOP) plus U V A light three times weekly for three weeks inhibited significantly the  symptoms  o f allergic rhinitis (sneezing, rhinorrhea, itching and  congestion) (Csoma et al., 2006). These results suggest that intranasal phototherapy is effective i n the treatment o f allergic rhinitis.  68  1.4  Methods for studying allergic rhinitis in experimental animals  1.4.1  General overview Rhinitis is induced in guinea pigs, rats, mice, dogs and pigs using different kinds  of allergens with various methods o f sensitization. The sensitized animals produce the different symptoms and signs o f allergic rhinitis including sneezing, nasal itching, nasal congestion and rhinorrhea following allergen provocation. Sneezing and nasal scratching can be assessed immediately after allergen challenge in sensitized guinea pigs, rats, and mice. Nasal secretions can be studied by weighing nasal secretions absorbed onto cotton swabs or filter papers in sensitized guinea pigs, dogs and pigs after allergen challenge. Nasal congestion can be assessed by measuring nasal resistance to air flow using forced air passed to nasal cavities; nasal passage space using acoustic rhinometry; and air flow using plethysmogram in guinea pigs, dogs and pigs. Cellular and biochemical changes can be quantified from nasal lavage collected from sensitized animals.  1.4.2 Techniques used to assess pathophysiological changes in allergic rhinitis in guinea pigs in vivo  After a challenge dose o f a specific allergen, sensitized guinea pigs produce the acute and the chronic signs and symptoms o f allergic rhinitis including sneezing, nose rubbing, rhinorrhea, and nasal congestion. In addition, there are cellular and biochemical changes. The way these changes are evaluated varies from one study to another. The following is a summary of the different techniques used for evaluation o f allergic rhinitis signs and symptoms, and quantification o f inflammatory changes in vivo i n guinea pigs.  69  1.4.2.1 Sneezing and Nose rubbing Sneezing and itching are typical, mostly histamine-mediated signs o f allergic rhinitis. Itching cannot be detected directly in guinea pigs. However, it may be related to nose scratching or rubbing combined with sneezing. Conscious guinea pigs are kept individually i n cages in a quiet environment and sneezing and nose rubbing are counted by direct observation or recorded using digital camera for later revision. In most cases, sneezing and nose rubbing are counted immediately after allergen provocation and evaluated for a period ranging from first ten minutes to one hour (Kaise et al., 1998; Yamasaki et al., 2001; Nabe et al., 2001; Mizutani et al., 2003; Fukuda et al., 2003; Zhao et al., 2005).  1.4.2.2 Nasal airway pressure Nasal blockade (decreased nasal patency) can occur as a result o f swelling o f the nasal mucosa due to vasodilatation o f cavernous tissue, and increase glandular secretions. The symptom o f congestion i n allergic rhinitics is biphasic i n time. It occurs during an acute phase as well as a late phase o f allergic rhinitis. In sensitized guinea pigs, both phases o f nasal,congestion have been detected, with the acute phase occurring during the first 30 minutes, and a later phase 4 to 6 hours after exposure to allergen. The degree o f patency o f the nasal cavity can be measured i n both conscious and anaesthetized guinea pigs. In conscious guinea pigs, a two chambered double-flow plethysmograph has been used to measure air flow through the nasal cavity. In this technique, a guinea pig is placed with its neck extending through the partition o f a two chambered box (Albert et al., 1998; Mizutani et al., 1999; Fujita et a l , 1999; Imai et al., 2000; Nabe et al., 2001;  70  Yamasaki et al., 2002; Mizutani et al., 2003; Fukuda et al., 2003). According to this technique, nasal air flow is inversely proportional to nasal blockade. Although the use o f plethysmograph has gained much attention, its use to measure nasal blockade has been criticized by Swedish researchers who consider that changes in resistance measured in the plethysmograph originate at or below the larynx (Finney et al., 1994). Recently, respiratory rate has been used in conscious guinea pigs to reflect resistance changes in the upper airway (Zhao et al., 2005). In anaesthetized guinea pigs, nasal blockade can be measured using different methods including a ventilator flow method (Mizuno et al., 1991; Yamasaki et al., 1997; Shizawa et al., 1997; Albert et al., 1998; Fukuda et al., 2003; Sakairi et al., 2005), forced oscillation method (Narita et a l , 1998; M c l e o d et a l , 2002), and acoustic rhinometry (Kaise et al., 1998; Kaise et al., 2001a). In a ventilator flow method, pulsatile air (4-10 ml/stroke and 50-70strokes/minute) is forced toward the nasal cavity from the tracheal side. A n y change in air flow resistance is reflected as a nasal patency change. In the forced oscillation method (know also as flow pressure method), one side o f nasal cavity is cannulated and the flow o f humidified air is restricted from cannulated side through the other side o f the nasal cavity and out the nostril. A n y change in nasal patency causes reduction i n air flow. Acoustic rhinometry is used to measure the volume o f the nasal cavity. In guinea pigs, the device can measure changes i n the nasal cavity within 2 c m from the nostrils. The volume o f the nasal cavity decreases when nasal blockade increases and vice versa.  71  1.4.2.3 Nasal secretions (Rhinorrhea) Rhinorrhea is a troublesome symptom o f allergic rhinitis. Watery secretions can be produced i n sensitized guinea pigs after allergen provocation although i n most cases its production is not enough for easy evaluation. The usual quantification o f nasal secretions i n most studies is gravimetrically. A piece o f cotton thread dyed with flourescein can be inserted into the anterior naris o f guinea pigs for one minute. The stretch o f color is proportional to fluid volume and to increase in weight o f a thread due to absorbed nasal secretions (Namimatsu et al., 1991). Alternatively, a pre weighed cottonwool or cotton swab can be used to absorb the secretions from the anterior naris (Fujita et al., 1999; Fukuda et al., 2003). The weight gained is proportional to nasal secretions. In addition, a pre weighed filter paper strip can be continuously inserted into the nares and secretions absorbed for a period o f 10 minutes. The increase i n paper weight is proportional to nasal secretions (Zhao et al., 2005).  1.4.2.4 Exudation Exudation occurs as a result o f increases i n vascular permeability. In sensitized guinea pigs, exudation can be quantified after allergen provocation. Different dyes have been used for this purpose, including Evan's blue (Mizuno et al., 1990; Mizutani et al., 1999; Mizutani et al., 2001), pontamine sky blue (Yamasaki et al., 1997; Kaise et al., 1998) and brilliant blue (Shizawa et al., 1997). A dye whose, at concentrations wich vary from one study to another (1-10%), is administered intravenously before  allergen  provocation i n anaesthetized guinea pigs. After allergen provocation, nasal cavities are perfused with saline at a rate o f 0.2-0.25ml/min for 10 to 20 minutes. Dye concentration  72  in the collected perfusate is quantified by spectrophotometer using absorbance at 620nm. Dye concentration is proportional to vascular permeability (exudation). Instead o f using dye, intravenously administered  I-labelled human serum albumin can be quantified  after recovery from nasal cavities, and can be used to reflect exudation o f serum proteins (Elovsson et al., 2005).  1.4.2.5 Cellular and Biochemical changes Allergic rhinitis basically results i n an inflammatory reaction in the nasal mucosa. This can be investigated by measurement o f mediators and inflammatory cells i n nasal lavages. In sensitized guinea pigs, cells and mediators o f inflammation can be detected in nasal lavage in both the acute and chronic phases o f allergic.rhinitis. Nasal lavage can be collected from anesthetized guinea pigs by perfusion o f nasal cavities with saline from the tracheal side (Yamasaki et al., 1997; Shizawa et al., 1997; Imai et al., 2000; Kaise et al., 2001a; Elovsson et al., 2005). Alternatively, saline can be instilled into one nostril and sucked out from the other nostril simultaneously by applying a negative pressure (Mizutani et al., 2001; Yamasaki et al., 2001; Yamasaki et al., 2002; Zhao et al., 2005). Recovered nasal lavage is centrifuged and the resultant supernant is used for quantitative measurement o f mediators (e.g. thromoxanes, leukotrienes, eosinophil peroxidase, N O ", N O ", histamine) using E L I S A or radioimmunoassay. Furthermore, nasal lavage can be assessed for total leukocyte count using hemocytometer or semiautomated haematoloty analyzer, and for differential cell count using cytospin followed by staining and observation under a microscope.  73  1.5  The rationale and aims of this project The prevalence o f allergic rhinitis is substantial and the financial and social  impact o f the disease is significant. The effectiveness o f currently available drugs for allergic rhinitis is limited. Therefore, the discovery o f more effective drugs with fewer side effects  is important.  In order to evaluate the  effectiveness  o f new  drugs,  experimental studies in animals with allergic rhinitis are required. Conventionally, guinea pigs have been the species of choice for the evaluation o f chemical-related respiratory allergy, primarily because it is possible i n this species to elicit and measure with relative ease challenged-induced inflammatory reactions that resemble i n some ways the acute clinical manifestations o f human allergic rhinitis. Thus, this project was aimed to: 1. establish comprehensive models o f allergic rhinitis in guinea pigs 2. investigate possible inflammatory mediators o f acute phase reactions of allergic rhinitis in guinea pigs in vivo.  To achieve this, the following experiments were conducted: 1. Systematic evaluation o f the models through the assessment o f the effect o f ovalbumin/saline on acute symptoms o f allergic rhinitis including sneezing, nose rubbing,  nasal airway pressure and cellular infiltration during the sensitization  period and on challenge days.  2. Evaluation o f the effect o f the autacoid histamine and the acetylcholine  on nasal  airway pressure  and  lung  inflation  neurotransmitter pressure  and  investigation of the receptors involved i n those responses.  3. Investigation o f the effect o f autacoids (histamine, leukotriene D 4 and nitric oxide) on sneezing, nose rubbing, nasal airway pressure and cellular infiltration  74  during the acute allergic reactions in sensitized ovalbumin challenged guinea pigs using different antagonists.  Assessment o f the anti-inflammatory activity o f heparin and dexamethasone by evaluation o f their effect on sneezing, nose rubbing, nasal airway pressure and cellular infiltration in sensitized ovalbumin challenged guinea pigs.  75  Materials and Methods 2.0  Materials  The materials used in the following studies are shown below: MATERIAL  SOURCE  Pharmacological agents 4-Diphenylacetaxy-N-methylpiperidine methiodide ( 4 - D A M P )  Sigma, Germany  Cetirizine  Fluka, Switzerland  Dexamethasone  Sigma, Germany  Heparin  Fisher Scientific, Canada  L-NAME  Sigma, Germany  Mepyramine  Sigma, U . S . A  Methoctramine  Sigma, Germany  Montelukast  Merck, Canada  Ovalbumin Grade V  Sigma, Germany  Pentobarbital sodium  B i m e d a - M T C , Canada  Pirenzepine  Sigma, Germany  Ranitidine  Sigma, Germany  Thioperamide  Sigma, Germany  Cell staining dyes Methylene blue  Sigma, Germany  Wright stain  Fluka, Germany  Solvents Dextros  Sigma, Germany  Dimethylsulfoxide ( D M S O )  Sigma, Germany  Polyethylene glycol ( P E G )  Fisher Scientific, U . S . A  Lactose  Pharmacy dept. U B C , Vancouver, Canada  Sodium chloride  Sigma, Germany  76  Equipment 5 minute epoxy syringe Glue  Henkel, Canada  Balance (mettler P C 4400)  Mettler instrument, Switzerland  Electron balance (mettler A E 2 6 0 )  Mettler instrument, Switzerland  Filter cards thin brown  Thermo-Electron Corporation, U K  Grass model 79D  Grass instrument C o . Quincy, mass, U . S . A  Hemocytometer  Bright Live, Reichert, U . S . A  I V Catheter 14G  Medex, U . S . A  Light microscope  Carsen, Canada  Micropipette  Gilson, France  Micropipette tips (l-200ul)  TipOne, Germany  Microscope cover glass  Fisher Scientific, Canada  Microscope slides  V W R international, Canada  Needle 23 G l  Becton Dickinson, U . S . A  Polyethylene tubing (PE50)  Becton Dickinson, U . S . A  Pressure transducer  Astromed, Canada  Shandon Cytospinl  Thermo Electron Corporation, U . S . A  U L T R A - N E B 99  The D e V I L B I S S C o . Canada  Ventilation pump  Harvard apparatus Limited  2.1  Methods  2.1.1  Methods of sensitization used in the study  The sensitization procedure used was first described by Yamasaki 1997. Guinea pigs were initially exposed to 1% ovalbumin in saline as a 1% aerosol twice for ten minutes, 7 days apart. The aerosol was generated by an ultrasonic nebulizer ( U L T R A -  77  N E B 99) and a ventilation pump set at 4ml/stroke, 70 strokes/min. O n days 14, 15 and 16, a booster o f 1% ovalbumin in saline was instilled intranasally at a volume o f 20ul/nostril/day into both nostrils. The instillation was performed using a micropipette (200ul capacity). O n day 21 guinea pigs were challenged with 2 % ovalbumin i n saline instilled intranasally at a volume o f 20ul/nostril to both nostrils. The sensitization process is shown in Figure 2.1.  1 Day  1 0  _._t I  1—  7  - |  14  _t,  15  1  1  16  21  _t._._t_._._t_.  I % OVA aerosol for 10 minutes •  |  I '  I % OVA booster instillation  __t |  |  20ul/nostril I  1  2% OVA challenge instillation  |  20ul/nostril I  I  I  OVA: ovalbum 1%: lOmg/kg 2%: 20mg/kg  *ure 2.1: The process o f sensitization in guinea pigs.  2.1.2  Experimental animals  Male Hartley guinea pigs were used for this project. They weighed 300-400 grams during the sensitization period and 400-600 grams at the time o f experiments. The animals were housed in an air-conditioned room at 23°C ± 2 and 55 ± 5% humidity with alternating 12 h light/dark cycles and were exposed to food and water ad libitum. The use of the animals for this project was approved by the U B C A n i m a l Care Committee.  78  2.1.3  Details of methods and models used in this study  2.1.3.1 Sneezing and nose rubbing in conscious guinea pigs in response to allergen challenge  O n day 21 post first sensitization, guinea pigs were challenged intranasally either with 2% ovalbumin, 20ul/nostril or 20ul saline/nostril. Drugs were administered before challenge. Sneezing and nose rubbing were observed and counted directly following nasal challenge and for 30 minutes thereafter (Figure 2.2).  3 i: Drug administration  2% ovalbumin challenge instillation 20ul/nostril Sneezing and nose rubbing count  Figure 2.2: General protocol for sneezing and nose rubbing experiments.  2.1.3.2 Measurement of nasal airway pressure and forced inflation pressure in anaesthetized guinea pigs subjected to allergen challenge Nasal airway pressure and lung inflation pressure were measured in anaesthetized guinea pigs. A s previously described (Mizuno et al., 1991) with some modification,  79  guinea pigs were anaesthetized with pentobarbital (35mg/kg) given intraperitoneally. Subsequently, both carotid artery and jugular vein were isolated and cannulated with polyethylene  tubing  (PE50) for  blood pressure  monitoring, and  drug delivery,  respectively. The trachea was isolated and cannulated with catheters (14G) in both directions, caudally for passive ventilation and lung inflation pressure measurement, and rostrally for nasal airway pressure. For nasal airway pressure measurements, the catheter was passed from the trachea, through the laryngopharynx and the oropharynx, toward the nasopharynx just close to the posterior nares. A ventilation pump was used to deliver a pulsatile air at a rate o f 8ml/stroke, 72times/min toward the nasal cavity, and lOml/kg, 72beats/min toward the lungs. To prevent air leakage during nasal resistance recording, the buccal (oral) cavity was filled with epoxy-soaked cotton and the oesophagus was ligated with surgical thread. Intranasal pressure and lower tracheal pressure were used as indexes o f nasal airway pressure and lung inflation pressure respectively, and were recorded v i a a port off the cannula using a pressure transducer and a Grass-polygraph (Figure 2.3). Following surgery, guinea pigs were kept for 15 minutes for obtaining baseline blood pressure, nasal airway pressure and lung inflation pressure measures. Then, nasal ventilation pump was disconnected and 0.5ml o f 2% ovalbumin challenge infused from the tracheal side toward the nasal cavity for 5 minutes. Thereafter, the nasal ventilation pump was reconnected and the animal was observed for 1 hour (Figure 2.4). This method was previously described by Sakairi 2005 with some modification.  80  Pressure  A i r flow Figure 2.3: Schematic representation of surgical and technical connections for nasal airway pressure, lung inflation pressure and blood pressure measurement.  Antigen challenge (perfusion of 0.5 ml of 2% ovalbumin to nasal cavity from the tracheal side)  p. ° 6 .2 5.  I1 1I  8 1  Figure 2.4: General protocol for nasal airway pressure, lung inflation pressure and cellular infiltration experiments.  81  2.1.3.3 Attempts to measure nasal secretions and exudation in conscious and anaesthetized guinea pigs subjected to allergen challenge.  Nasal watery secretions can be produced i n sensitized guinea pigs after allergen provocation although in most cases its production is not enough for easy evaluation. In an attempt to measure nasal secretions, a pre-weighed cotton swab was rubbed around the anterior naris o f sensitized guinea pigs 30-60 minutes after allergen challenge i n both conscious and anaesthetized guinea pigs. However the amount collected was not enough to be quantified and evaluated. W e also attempt to quantify exudation in sensitized guinea pigs. W e used Evan's blue and pontamine sky blue dyes. The dyes were administered intravenously i n anaesthetized guinea pigs. Thereafter, the guinea pigs were challenged with ovalbumin. After allergen provocation, nasal cavities were perfused with saline at a rate o f 0.20.25ml/min for 10 minutes. Dye concentration i n the collected perfusate was quantified by spectrophotometer using absorbance at 620nm. Dye concentration is proportional to vascular permeability (exudation). However the amount o f dye i n the perfusate was not enough to be quantified and evaluated. Therefore because o f these reasons nasal secretions and exudation were not evaluated i n this study.  2.1.3.4 Measurement of leukocyte infiltration into nasal lavage fluid in anaesthetized guinea pigs subjected to allergen challenge  Nasal cellular infiltration (extravasation) is a characteristic hallmark o f allergic rhinitis. Total and differential cell counts in nasal washings were used as indices o f  82  cellular  infiltration.  A s described before,  guinea pigs  were  anaesthetized  with  pentobarbital and subsequently challenged with ovalbumin. Nasal lavage was collected . from guinea pigs one hour post challenge as follows: nasal cavities were washed with 2ml o f pre-warmed saline infused from the tracheal side.  The recovered saline was  collected from the anterior nares. Total and differential cell counts were assessed immediately after sample collection. Total cell count was assessed using a standard hemocytometer. The ruled area o f hemocytometer consists o f one large square known as a type A square. The volume o f type A square is 0.1 m m (Figure 2.5). This area was used to determine the number o f 3  cells per milliliter o f nasal lavage.  Figure 2.5: Schematic representation of a hemocytometer.  After sampling, 50ul o f nasal lavage was mixed with 50(0.1 o f methylene blue (for cell staining and clarity o f counting). The two hemocytometer chambers were filled with  83  12ul o f the mixture per chamber. Leukocytes (white blood cells) were counted under light microscope at power 4 0 X . The mean o f the two chambers cells was consider as total cell count. To determine the number o f cells per milliliter, the following formula was used:  Number of cells/ml = total number of cell counted x dilution factor x 1000 (there are 1000 3  3  mm /ml) / total volume counted (0.1 mm )  To perform a differential cell count, lOOul o f nasal lavage was centrifuged using a cytospin (Shandon Cytospin 1) at a speed o f 1500 revolutions per minutes for 5 minutes. The sample was then stained with Wright stain solution for 5 minutes. White blood cell type (eosinophil, neutrophil, basophil, monocyte and lymphocyte) was determined based on morphological characteristics. The number o f each type o f cell was counted under 4 0 X power. Total number o f cells per three consistent fields observed was used to reflect the total number o f cells.  2.1.3.5 Experimental design and drugs used in the study The .experiments were performed in a randomized double blind fashion to minimize personal bias i n data analysis. Guinea pigs were divided into sensitized ovalbumin challenged groups (control), non-sensitized ovalbumin/saline challenged groups (control) and sensitized ovalbumin challenged pre-treated with drug groups. The actions o f drugs currently used clinically or experimentally i n the treatment of allergic rhinitis were assessed in terms o f their effectiveness in ameliorating sneezing,  84  nose rubbing, nasal congestion as well as inhibiting cellular infiltration in sensitized guinea pigs. The drugs used in this study were selected based on the previous findings which show their effectiveness in allergic rhinitis in human and animal models. Different drug classes were used including first and second generation antihistamines, leukotriene D4 inhibitor, nitric oxide synthases inhibitor, anti-inflammatory drugs like heparin and corticosteroids. Table 2.1 shows the drugs, their doses and the route o f administration used. Drugs were administered intraperitoneally, a 15 minute-period waiting prior to ovalbumin challenge. When they were administered intravenously, a 5 minute-period was used. The doses o f the antagonists used i n this study were chosen based on literature findings that such doses were effective in blocking their corresponding receptors.  Drug  DO.NC (nig  ku)  Rome o f administration  Mepyramine  3  Intraperitoneal  Cetirizine  3  Intraperitoneal  Montelukast  10  Intravenous  L-NAME  10  Intravenous  Heparin  20  Intravenous  Dexamethasone  20  Intraperitoneal (for sneezing), Intravenously (for nasal airway pressure)  Table 2.1: List of drugs used in the experiments.  The doses o f the  antagonists  for blocking the  effects  o f histamine and  acetylcholine on nasal airway pressure and lung inflation pressure study were chosen  85  based  on  literature  findings  that these doses were  effective  in blocking  their  corresponding receptors. A l l drugs were administered intravenously. See Table 2.2.  Agonists Histamine (2ug-l mg kg)  III 112  Antagonists Mep\ramine Ranitidine  10X dose (mg/kg) 1 0.6  i98l 1 hiopcramide  Ml Acetylcholine M 2 M3 (2ug-l mg/kg)  0.5 0.9 0.3 0.3  Pirenzepine Methoctramine 4-DAMP  M 1 - M 5 Atropine 0.9 Table 2.2: List of drugs and doses used in autacoids and neurotransmitters effect on nasal airway pressure and lung inflation pressure study. "10X" is a ten times dose since in this study three doses were used, X , 3X and 10X.  In the histamine and acetylcholine experiments, guinea pigs were divided into 7 groups (n=5 each) (Figure 2.6). A l l animals received acetylcholine and histamine (2ug1 mg/kg) intravenously as agonists. Three groups, each one o f them received three doses ( X , 3 X , 10X) o f one o f the muscarinic antagonists [ M l (pirenzepine) (0.09, 0.27 and 0.9mg/kg), M 2 (methoctramine) (0.03, 0.09 and 0.3mg/kg) and M 3 ( 4 - D A M P ) (0.03, 0.09 and 0.3mg/kg)]. One group received atropine at three different doses (0.09, 0.27 and 0.9mg/kg). Three other groups, each one o f them received three doses ( X , 3 X , 10X) o f one o f the histamine receptors antagonists [(HI (mepyramine) (0.1, 0.3 and 1 mg/kg), H 2 (ranitidine) (0.06, 0.18 and 0.6mg/kg) and H3 (fhioperamide) (0.05, 0.15 and 0.5mg/kg)]. Figure 2.7 shows the experimental design.  86  35 guinea  Muscarinic antagonists  Atropine (5)  Histamine receptors antagonist  Pirenzepine (5) Mepyramine (5)  Ranitide (5)  Methoctramine (5) Thioperamide (5)  4 - D A M P (5)  Figure 2.6: Protocol for histamine and acetylcholine experiment. Guinea pigs were divided into seven groups (n=5 per groug) based on the antagonist they received.  L&H Time  J  Tft Hi-  + ACh  +  +  L & H  L  J  L & H  L  W  Repeated  Antg (X)  Anl» <3X)  Vili? I I 0 \ l  UMCC  Ri'pi.ik'il l\\ l^c III 1 l.liulillll (Hik'l  His: histamine A C h : acetylcholine L & H : lower (25% of maximum response and higher doses (75% of maximum response) of His and A C h Antg: antagonist X : dose  Figure 2.7: Schematic representation of experimental protocol for histamine and acetylcholine experiment.  87  2.1.3.6 Data analyses The data from all the studies were analyzed using parametric statistical tests to determine the probability o f rejecting the null hypothesis. In all the studies, the null hypothesis stated that there was no difference between control and treated groups (i.e. all the means are equal). In studies where more than two groups were involved (e.g. nonsensitized control groups, sensitized control group and sensitized pretreated groups), one way analysis o f variance ( A N O V A ) was used to determine whether the variances were different or not, and i f they were different (p<0.05), a post-hoc test was used to compare between grougs. The post hoc-test used was Bonferroni multiple comparison test and i f p>0.05, a less powerful test such as the unpaired student t test was used to compare the means between two selected groups. In studies where two groups were involved and studied over a period o f time, a repeated measurement two way A N O V A was used. A probability, p, o f less than 0.05 was taken as significant. In each bar in the graphs, the error bar was a standard error o f mean. In experiments where we evaluated the model i n terms o f allergen responses we tried to use different forms o f transformation (e.g. log transformation) to analyze our data, however the outcome did not improve the analysis in terms o f the homogenecity o f variance, and although the statistical power after transformation was the same as before transformation, we decided to use untransformed raw data for statistical analysis. Dose ratio method was used to analyze the effectiveness o f the antagonists i n blocking the actions o f acetylcholine or histamine. For nasal airway pressure, ED1.5 A N A P (mmHg) was used to measure the potency for acetylcholine, and ED2 - A N A P (mmHg) for histamine. [ E D - A N A P (mmHg)] was the effective dose producing 1.5 or 2 X  88  m m H g decrease in nasal airway pressure, from pre-drug value. For lung inflation pressure (LIP), ED20+ALIP (mmHg) was used to measure antagonist  shifts  for  acetylcholine and histamine. [ED20+AIPP (mmHg)] is the dose producing a 20 m m H g increase in lung inflation pressure from pre-drug value. A l l the data i n the graphs represent mean ± S E M . The relative affinities o f the antagonists for muscarinic ( M l - 5 ) . and histamine ( H l - 3 ) receptors were tabulated (Table 2.3) using K B values from Leurs et al., 1995 and Bockman et al., 2001. These affinities were used to reflect the possible receptors involved in histamine and acetylcholine-induced changes in nasal airway pressure and lung inflation pressure.  Drug' ':'  .'  Receptor affinity  *  Atropine Pirenzepine Methoctramine  M l = M 2 ~ M 3 = M4 = M5 M l » M4 > M3 > M 2 ~ M5 M2 > M4 > M l > M5 » M3  4-DAMP Mepyramine Ranitidine Thioperamide  M3 = M5 = M l > M4 » M2 HI selective H2 » > HI H3 > HI ~ H2  = > » » >  equal in potency 2X-4X ratio of potencies 4X-10X ratio of potencies more than 10X ratio of potency  Table 2.3: Relative affinities of the antagonists used for muscarinic and histamine receptors  2.1.3.7 Experimental overview In this study different experiments were conducted in order to evaluate the model and drug modification o f responses to allergen challenge. A l l allergic responses, including sneezing, nose rubbing, nasal blockade, and leukocyte infiltration, were  89  evaluated during the acute phase o f allergic reactions. The following experiments were conducted: i . Assessment o f models The aim o f this experiment was to evaluate the effectiveness o f sensitization procedures used and to study the effects o f allergen challenge on sneezing, nose rubbing, nasal airway pressure, lung inflation pressure, blood pressure and leukocyte infiltration.  i i . A n investigation o f the receptors involved in mediating nose and lung responses to intravenous acetylcholine and histamine. During the process o f establishing the model, we explored the effects o f the autacoid histamine and the neurotransmitter acetylcholine on nasal airway pressure since they are important in allergic rhinitis. Surprisingly when they were administered intravenously, they tended to decrease nasal airway pressure while increasing lung inflation pressure. It is well known that histamine and acetylcholine cause vasodilatation. Applying this concept to the nasal blood vessels, they should be expected to increase nasal airway pressure (Powell et al., 1979; Feigl 1998). Some studies in human being (Tylor-Clark et al., 2005) and animals, namely, dogs (Ruslan et al., 2003) and guinea pigs (Mizutani et al., 1999) have shown that when histamine is given intranasally, nasal airway pressure increases. Other studies in guinea pigs indicated that histamine and acetylcholine introduced topically into the nasal mucosa may increase or decrease nasal airway pressure depending upon the dose (Lung et al., 1987; Lung et al., 1994). Histamine H I antagonists have been used widely to treat acute symptoms o f allergic  90  rhinitis (Dykewicz et a l , 1998). In guinea pigs oral histamine H I antagonists inhibit histamine-induced nasal congestion (Shigeru et al., 2003). Therefore this study was conducted in order to determine which receptors are involved in intravenous histamine and acetylcholine-induced decreases i n nasal airway pressure in anaesthetized  guinea pigs. Furthermore, the study was also aimed to  determine the receptors involved i n histamine and acetylcholine-provoked increases in lung inflation pressure.  i i i . Drug modification-of the response to allergen challenge The aim o f this study was to evaluate the effects o f therapeutically important drugs on allergen induced sneezing, nose rubbing, nasal airway pressure, and leukocyte infiltration in sensitized guinea pigs. To achieve this aim the following experiments were conducted: Effect o f antihistamines mepyramine and cetirizine on sneezing, nose rubbing, and nasal airway pressure. Effect o f montelukast on sneezing, nose rubbing, nasal airway pressure and cellular infiltration. Effect o f L - N A M E on nasal airway pressure and cellular infiltration. Effect o f heparin on sneezing, nose rubbing, nasal airway pressure and cellular infiltration. Effect o f dexamethasone on sneezing, nose rubbing, nasal airway pressure and cellular infiltration.  91  3.  Results  3.0  Assessment of models  3.0.1  Effectiveness of the sensitization procedures used During the process o f sensitization, the time effect o f ovalbumin administration  on the frequency o f sneezing and nose rubbing i n guinea pigs was evaluated. There was a frequency-dependent effect o f ovalbumin administration on number o f sneezes and nose rubbings (p<0.0001 two way A N O V A ) . The number o f sneezes increased from day 1 to day 16 in the ovalbumin group. N o significant effect o f saline aerosol/instillation on sneezing frequency was observed (Figure 3.1). The effect o f ovalbumin on sneezing frequency achieved significant levels (p<00.1) compare to saline group on days 15 and 16.  aerosol 10m in/nostril/day  instillation 20ul/nostril/day  Figure 3.1: Time dependent effect of ovalbumin administration on sneezing frequency in guinea pigs measured during the sensitization period. Days 1 and 7 guinea pigs received 2% aerosolized ovalbumin or saline for 10 minutes in both nostrils. Days 14-16 guinea pigs were boosted with intranasal instillation of 1% ovalbumin or saline 20ul /nostril in both nostrils/day. Sneezes were counted for 30 minutes immediately after aerosol/liquid exposure. * Significant increase in number of sneezes as compared to saline group (p<0.001, Bonferroni multiple comparison test). The data are the means of 10 guinea pigs. Vertical bars represent standard error of mean.  92  In comparison to saline, ovalbumin sensitization increased nose rubbing (p=0.002 two  way  A N O V A ) . The  significant levels were observed on days 14 (p<0.05),  15  (p<0.01) and 16 ( p O . 0 0 1 ) (Figure 3.2).  CZZl saline  o  1% ovalbumin  day 1  day 7  day 14  aerosol lOmin/nostril/day  day 15  day 16  instillation 20ul/nostril/day  Figure 3.2: Time dependent effect of ovalbumin administration on nose rubbing frequency in guinea pigs measured during the sensitization period. Days 1 and 7 guinea pigs received 2% aerosolized ovalbumin or saline for 10 minutes in both nostrils. Days 14-16 guinea pigs received booster doses as intranasal instillation of 1% ovalbumin or saline 20ul/nostril in both nostrils/day. Nose rubs were counted for 30 minutes immediately after aerosol/liquid exposure. * Significant increase in number of nose rubbing as compared to saline group (* p<0.05, ** p<0.01, *** pO.001, Bonferroni multiple comparison test). The data are the means of 10 guinea pigs. Vertical bars represent standard error of mean.  3.0.2  Sneezing and nose rubbing responses to allergen challenge Ovalbumin  challenge  in  sensitized  group  significantly  increased  sneezing  frequency (p<0.01) and number o f nasal rubs (p<0.001) as compared to non-sensitized groups (Figures 3.3 and 3.4).  93  control (non sensitized)  sensitized  Figure 3.3: Effect of ovalbumin challenge on sneezing frequency in sensitized and nonsensitized guinea pigs evaluated day 21 post first sensitization. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril in both nostrils and sneezes were counted for 30 minutes after challenge. * Significant increase in sneeze frequency as compared to non-sensitized ovalbumin challenged guinea pigs (* p<0.01, Bonferroni multiple comparison test). The data are the means of 5 guinea pigs. Vertical bars represent standard error of mean.  60  n  saline challenge  ova challenge  control (non sensitized)  ova challenge sensitized  Figure 3.4: Effect of ovalbumin challenge on nose rubbing frequency in sensitized and nonsensitized guinea pigs evaluated day 21 post first sensitization. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril to both nostrils and nose rubs were counted for 30 minutes after challenge. * Significant increase in nose rubbing frequency as compared to non-sensitized ovalbumin challenged guinea pigs (* p<0.001, Bonferroni multiple comparison test). The data are the means of 5 guinea pigs. Vertical bars represent standard error of mean.  94  3.0.3  Changes in nasal airway pressure and lung inflation pressure as well as blood pressure in response to allergen challenge  Ovalbumin challenge in sensitized group significantly increased nasal airway pressure (p<0.05) as compared to non-sensitized groups. N o significant change i n nasal airway pressure was observed in ovalbumin/saline non-sensitized groups (Figure 3.5).  O O, C  2 o .2 ^ | gf  5.5-1  4.5-1 3.52.5-  1 ova challenge  0.5-1  5  -0.5-1.5-  I  0  i  v a challenge  i  sensitized  saline challenge control (non sensitized)  -2.5' Figure 3.5: Effect of ovalbumin challenge on nasal airway pressure in anaesthetizaed (by pentobarbital) (sensitized and non-sensitized) guinea pigs 23-28 days following initiation of sensitization. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge values was considered as a change in nasal airway pressure. * Significant increase in nasal airway pressure as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are the means of 5 guinea pigs. Vertical bars represent standard error of mean.  Simultaneous recording o f lung inflation pressure and blood pressure besides nasal airway pressure recording showed no significant changes in these variables when compared between sensitized and non-sensitized groups. Intranasal ovalbumin did not cause significant changes in lung inflation pressure (Figure 3.6).  95  7.5-  nasal airway resistance i i lung inflation pressure CUD mean arterial pressure  5.04  2.5H  o.oH -2.5- JL  ova challenge ova challenge sensitized  saline challenge control (nonsensitized)  Figure 3.6: Systemic evaluation of effect of ovalbumin challenge on nasal airway pressure, lung inflation pressure, and blood pressure measured simultaneously after 2% ovalbumin or saline challenge at a volume of 0.5ml infused to the nasal cavities for 5 minutes in anaesthetized (by pentobarbital) sensitized and non-sensitized guinea pigs. The experiments were conducted 23-28 days following initiation of sensitization. The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.0.4  Leukocyte infiltration responses to allergen challenge Ovalbumin challenge in sensitized group significantly increased total cell  count  (p<0.01) as compared  ovalbumin/saline challenge  to non-sensitized groups. N o significant effect  in non-sensitized  groups  was  observed  (Figure  of  3.7).  Furthermore, ovalbumin significantly induced both eosinophil (p<0.01) and neutrophil (p<0.05) infiltration in sensitized guinea pigs compared with non-sensitized guinea pigs. N o significant effect o f ovalbumin was observed on other cell types (Figure 3.8).  96  40-i o  35 30H 25  u o  o  i  2015105 0J  [ saline challenge ova challenge  ova challenge  control (non sensitized)  sensitized  Figure 3.7: Effect of ovalbumin challenge on cellular infiltration (total cell count) in anaesthetized (sensitized and non-sensitized) guinea pigs evaluated from nasal lavage collected 60 minutes following 2% ovalbumin or saline challenge at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted using hemocytometer under light microscope and the value were expressed as number of cells per milliliter of nasal lavage. * Significant increase in total cell count as compared to non-sensitized ovalbumin challenged guinea pigs ( p O . O l , Bonferroni multiple comparison test), n = 5 per group. Vertical bars represent standard error of mean.  3.0.5  Sensitization test To test whether the guinea pigs were sensitized overall or not, at the end o f the  experiment, l m g / k g o f ovalbumin was administered intravenously. In sensitized guinea pigs anaphylactic shock occurred. It was characterized by an increase in blood pressure, heart rate and lung inflation pressure immediately (within one minute) after ovalbumin administration.  In  non-sensitized  guinea  pigs,  no  effect  of  lmg/kg  intravenously administered was observed as exemplified in Figure 3.9.  97  ovalbumin  150-i  <u  I eosinophil 1 neutrophil I basophil monocyte lymphocyte  d 3 X 12 o OJ  •* i o o H  3 rn aj </) O. CO  I H  O S_ O u o u a •2  <+-  50-|  1 0-J  , saline challenge  ova challenge  control (nonsensitized)  J  L  ova challenge sensitized  Figure 3.8: Effect of ovalbumin challenge on cellular infiltration (differential cell count) in anaesthetized (sensitized and non-sensitized) guinea pigs evaluated from nasal lavage collected 60 minutes following 2% ovalbumin or saline challenge at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted under light microscope at 40X power and total cells counted in three consistent fields were used to express the changes. * Significant increase in eosinophil number as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in neutrophils as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.1  An  investigation of the  receptors involved in  mediating nose and  lung  responses to intravenous acetylcholine and histamine in vivo  3.1.1  Effect of histamine and acetylcholine on nasal airway pressure  Both histamine and acetylcholine produced dose dependent decreases i n nasal airway pressure with histamine being more potent ( E D Q  98  6-AmmHg  =  1.8ug/kg for  Blood pressure  1 mg/kg ova iv  inmiiiiiliiiiiijiii miiiiiitnliiiiiiiiiiiliUi  IMMkkiimi  1 mg/kg ova iv  Ulltimill.illlllUlllllliimirlil.  ml  Heart rate Heart rate Lung inflation pressure Lung inflation pressure  •  Non-sensitized guinea pig  Sensitized guinea pig  Figure 3.9: Effect o f ovalbumin l m g / k g administered intravenously in sensitized and nonsensitized guinea pigs on blood pressure, heart rate and lung inflation pressure.  histamine and 6.3|ug/kg for acetylcholine) and probably with greater efficacy (Emax = 2 . 4 ± 0 . 2 m m H g for histamine and less than 1.5mmHg for acetylcholine) (Figure 3.10).  3.1.2  Effect of histamine and acetylcholine on lung inflation pressure Acetylcholine and histamine produced dose dependent increases in lung inflation  pressure, with histamine being more potent (ED50 = 5 ± l u g / k g for histamine and 36.3±lpg/kg  for acetylcholine) and  probably  with the  same efficacy  (Emax  2 5 . 4 ± 0 . 8 m m H g for histamine and 24.9±0.7mmHg for acetylcholine) (Figure 3.11).  99  =  Figure 3.10: Dose-response curves for the decrease in nasal airway pressure following intravenous administration of histamine and acetylcholine at doses of ug/kg in non-sensitized guinea pigs. The changes in nasal airway pressure are the difference between pre drug and after drug administration. The data are means of 35 guinea pigs. Vertical bars represent standard error of mean.  ine  |—i i imiq—I -1.0  -0.5  I IIIIIII—I  0.0  i  |—  0.5  n—I  1.0  I  IIIIIII—i  i  1.5  dose(log)  1—i i linn 2.0  2.5 ^  /  k  §  Figure 3.11: Dose-response curves for the increase in lung inflation pressure following intravenous administration of histamine and acetylcholine in non-sensitized guinea pigs. The changes in lung inflation pressure are the difference between pre drug and after drug administration. The locators ( E D ) , maximum effects (Emaxs) and hill slopes are shown on the graph. The data are mean responses from 35 guinea pigs. Vertical bars represent standard error of mean. 50s  100  3.1.3 A n investigation of receptors involved in histamine and acetylcholine induced changes in nasal airway pressure (NAP) and lung inflation pressure (LIP) In order to assess the effects o f supposedly selective antagonist on the responses to  acetylcholine  and  histamine,  the  following  procedures  were  used:  The initial sensitivity o f anaesthetized animals to histamine and acetylcholine were assessed in terms o f effects on N A P and L I P .  Two suitable test doses o f either agonist  were chosen from the data used to produce Figures 3.10 and 3.11.  After obtaining  control responses the first dose o f antagonist (x) was administered and the test doses were repeated. This was repeated after 3x and lOx the original dose o f antagonist. After the final dose o f antagonist an attempt was made to construct a full dose response curve to the particular agonist used. It was presumed that the effector concentration o f antagonist in the animal did not change significantly during the test procedure.  This procedure  resulted in seven groups, each o f 5 animals. The data in Figures 3.10 and 3.11 are the accumulated data from the control (no antagonist) data. Each o f the 7 groups received one o f the seven antagonists (four antimuscarinic and three antihistaminic). The effects of the antagonists were assessed in terms o f their ability to shift the responses to the two test doses o f each o f the two agonists to the left (for N A P , Figure 3.12 for acetylcholine and 3.13 for histamine) and to the right (for L I P , Figure 3.14 for acetylcholine and 3.15 for histamine) and to express a shift as a dose ratio. In terms o f the effects o f the various antagonists on acetylcholine and histamine responses Figs 3.12 and 3.13 showed that most antagonists failed to change responses to test doses o f agonists.  For acetylcholine there was some degree o f shift for atropine,  pirenzepine and D A M P but none for methoctramine and histamine antagonists (Figure 3.12). O n the other hand (Figure 3.13), for histamine the only antagonist that produced  101  any notable shift was mepryramine. In those cases where any shift occurred it was less than that expected for competitive antagonism (Tables 3.1 and 3.2) on the basis that increasing the antagonist dose from the initial X to 3 X and then to 10X would increase the dose ratio (i.e. shift the curves) by a factor o f 10 and 30 respectively for competitive antagonism. In the case o f the response o f the L I P to acetylcholine and histamine, a better degree o f antagonism was seen in some cases.  Thus, for acetylcholine, marked shifts  were seen with atropine, pirenzepine and D A M P  but were non competitive for  methoctramine and absent with the histamine antagonists (Figure 3.14). For histamine, marked shift was seen with mepyramine but absent with ranitidine, thioperamide and muscarinic antagonists (Figure 3.15). Tables 3.1-3.4 summaries results obtained from figures 3.12-3.15, respectively.  102  Dose (log ) 2  D  o  s  e  (i  o g 2  D  ACh ACh+DAMP(x)  O.OO-i  • ACh • ACh+meth(x) • ACh+meth(3x) ACh+meth(10x)  ao  X  i  )  ACh+DAMP(3x) ACh+DAMP(10x)  op -0.25-1 X -0.50H -0.75'  •  x=0.03mg/kg  x=0.03mg/kg  .00' 4  5  6  T (Mg/kg)  5  Dose (log )  6  (Mg/kg)  7 8  Dose (log )  2  2  J -0.21 -0.65 -i.o^  -1.4-  a  < x=0.06mg/kg  x=0.1mg/kg  -1.8-  1 5  ACh ACh+ranit(x) ACh+ranit(3x) ACh+ranit(10x)  O-i  •ACh ACh+mep(x) ACh+mep(3x) ACh+mep(10x)  -2-  1  1 (Mg/kg)  (Hg/kg)  6  Dose (log )  Dose (log )  2  2  103  (log ) 2  Figure 3.12: Dose-response curves for the decrease in nasal airway pressure following intravenous administration of acetylcholine before and after intravenous administration of an antagonist in nonsensitized guinea pigs. A , B , C, D, E, F ahd G show the effect of atropine (nonselective muscarininc antagonist), perinzepine ( M l antagonist), methoctramine (M2 antagonist), 4 - D A M P (M3 antagonist), mepyramine (HI antagonist), ranitidine (H2 antagonist) and thioperamide (H3 antagonist) respectively, on acetylcholine induced decreases in nasal airway pressure. The drugs were administered at three successive doses (X, 3X, and 10X). The X axis is log . N A P is nasal airway pressure. The data are means of 5 guinea pigs. Vertical bars represent standard error of mean. 2  104  O-l  his his+mep(x) his+mep(3x) his+mep(10x)  •  B  -H O.O-i  -2-\  —*— n^+ranit(x) —his+ranit(3x) —•— his+ranit(10x)  6fi  5 -3H  -2.5H  x=0.1 mg/kg -4J  i—i—r 1  2  ~l—I—I—I—I—l 4  3  5  6  7  8  9  l  10  5 I -5.0H  I (Mg/kg)  x=0.06mg/kg  t  -7.5-  11  1 (log )  1  r  2  3  4  Dose (bg )  2  2  60  his his+thio(x) his+thio(3x) his+thio(10x)  O.0i  5 -25H 5  his his+pire(x)  D  O.On  60  a  1 -2.5H x=O.05mg/kg  -5.0-1 1  2  3  < x=0.09mg/kg  -5.0-1  I (Mg%)  1 (Mg/kg)  T" 2  Dose (logj)  Dose (tag ) 2  O.O-i  his  his his+DAMP(x) his+DAMP(3x) his+DAMP(10x)  his+meth(x) his+meth(3x) his+meth(10x)  a -2.5H  < x=0.03mg/kg  -5.0-1  x=O.03mg/kg  1 (Mg/kg) 1  2  I  3  Dose (fog )  l(Mgfcg) 4  2 Dose (k)g )  2  2  Figure 3.13: Dose-response curves for the decrease in nasal airway pressure following intravenous administration of histamine before and after intravenous administration of an antagonist in non-sensitized guinea pigs. A , B , C, D , E, and F show the effect of mepyramine (HI antagonist) and ranitidine (H2 antagonist), thioperamide (H3 antagonist), atropine (nonselective muscarininc antagonist), perinzepine ( M l antagonist), methoctramine (M2 antagonist) and 4 - D A M P (M3 antagonist), respectively, on histamine induced decreases in nasal airway pressure. The drugs were administered at three successive doses (X, 3X, and 10X). The X axis is log . N A P is nasal airway pressure. The data are means of 5 guinea pigs. Vertical bars represent standard error of mean. 2  105  Antagonist Atropine  A C h - N A P fEDi.5 -ANAP(mmHg)! Summary o f actions o f antagonist in terms o f shifts o f dose-response curves A degree o f antagonist-induced shift (maximum dose ratio 7) but shift not increased with increasing doses o f antagonist  Pirenzepine  Methoctramine  Slight shift but not clearly related to dose o f antagonist N o antagonist-induced shifts seen  4-DAMP  Similar pattern o f responses to that seen with atropine  Mepyramine Ranitidine Thioperamide  N o antagonist-induced shifts seen N o antagonist-induced shifts seen N o antagonist-induced shifts seen  Type o f blockade Not obviously competitive Not competitive N o block Not obviously competitive N o block N o block N o block  Receptor involved Ml-5  Ml,4  No M 2 , M4 M3,M5, Ml No H NoH NoH  Table 3.1: A summary of Figure 3.12. Acetylcholine effects on nasal airway pressure in the presence of different doses of antagonists. Possible receptors blocked by the antagonists are shown based on type of blockade and relative affinities of the antagonists (refer to table 2.3).  Pirenzepine  His-NAP [ED -ANAP(mmHg)l Summary o f actions o f antagonist i n terms o f shifts o f dose-response curves N o antagonist-induced shifts seen  Methoctramine  Slight shift  4-DAMP  Slight shift (maximum dose ratio 2)  Mepyramine  V e r y marked shift by the all doses (dose ratio >100) but the effect o f larger doses o f antagonist could not be seen due to extend to blockade N o antagonist-induced shifts seen N o antagonist-induced shifts seen  2  Antagonist  Ranitidine Thioperamide  Type o f blockade N o block  Receptor involved  Not competitive Noncompetitive block  M2  No M l  M3,5,l  Possibly competitive  HI  N o block N o block  No H2 N o H3  Table 3.2: A summary of Figure 3.13. Histamine effects on nasal airway pressure in the presence of different doses of antagonists. Possible receptors blocked by the antagonists assessed based on the type of blockade and relative affinities of the antagonists (refer to table 2.3).  106  A LIP (mmHg) A LIP (mmHg)  o A LIP (mmHg)  A LIP (mmHg)  ?  Y  ?  Y  5  G •ACh ACh+thio(x) ACh+thio(3x) ACh+thio(10x)  m X § 20-1  1 1  cu  J 10H x=0.05mg/kg 4  5  —r (ng/kg)  Dose (log ) 2  Figure 3.14: Dose-response curves for the increase in lung inflation pressure following intravenous administration of acetylcholine before and after intravenous administration of an antagonist in nonsensitized guinea pigs. A , B, C, D, E, F and G show the effect of atropine (nonselective muscarininc antagonist), perinzepine ( M l antagonist), methoctramine (M2 antagonist), 4 - D A M P (M3 antagonist), mepyramine (HI antagonist), ranitidine (H2 antagonist) and thioperamide (H3 antagonist) respectively, on acetylcholine induced increases in lung inflation pressure. The drugs were administered at three successive doses (X, 3X, and 10X). The X axis is log and the values shown in X axis are anti-log . The data are means of 5 guinea pigs. Vertical bars represent standard error of mean. 2  2  108  30-i  - his • his+mep(x) • his+mep(3x) his+mep(10x)  B ^ 20ep X  nis+ranittx) his+ranit(3x) his+ranit(10x)  ioH —i  x=0.06mg/kg x=0. lmg/kg 0  1 2  3  7  4  8  9 10  (Mg/kg)  1  2  2  —•— his  ^ 30-i (mm  IOH  0. -J 10-  <  <  x=0.05mg/kg  x=0.09mg/kg  o-i  J  i  0  1  1  2  1  1 (Mg/kg) 4  r  3  n (Mg/kg) 1  Dose (log )  2  3  Dose (log )  2  2  •his • his+meth(x) his+meth(3x 'his+methilO  20-1 60  •—'  »— his —*— his+pire(x) —*— his+pire(3x) * his+pire(10x)  00 X  ftis+tliioflOx)  20-  D  20-1  —*— his+thio(x)  BO  s E  J (Mg/kg)  Dose (log )  Dose (log )  0  "]  2  his his+DAMP(x) his+DAMP(3x) his+DAMP(10x)  30-i  so  1  a  |20H  10H  CM  OH  J ioH  -1  x=€.03mg/kg  x=0.03mgkg  0-"  — I (Mg/kg)  1 (Mg/kg)  I  Dose (log )  Dose (log )  2  2  Figure 3.15: Dose-response curves for the increase in lung inflation pressure following intravenous administration of histamine before and after intravenous administration of an antagonist in non-sensitized guinea pigs. A , B, C, D, E, and F show the effect of mepyramine (HI antagonist) and ranitidine (H2 antagonist), thioperamide (H3 antagonist), perinzepine ( M l antagonist), methoctramine (M2 antagonist) and 4 - D A M P (M3 antagonist), respectively, on histamine induced decreases in nasal airway pressure. The drugs were administered at three successive doses (X, 3X, and 10X). The X axis is log and the values shown in X axis are anti-log . The data are means of 5 guinea pigs. Vertical bars represent standard error of mean. 2  2  109  ACh-LRP | E D  2 0  Receptor involved  +ALRP(mmHg)|  Antagonist Atropine  Pirenzepine Methoctramine 4-DAMP Mepyramine Ranitidine Thioperamide  Very marked shift by the all doses (dose ratio >10G) but the effect o f larger doses o f antagonist could not be seen due to extend to blockade Parallel (?) shift at lower doses o f antagonist (maximum dose ratio shift 5) N o antagonist-induced shifts seen  Possibly competitive  Ml-5  Possibly competitive N o block  Ml  Large shift (dose ratio >100) but competitive shift difficult to see N o antagonist-induced shifts seen N o antagonist-induced shifts seen N o antagonist-induced shifts seen  Possibly competitive N o block N o block N o block  No M 2 , 4 M3,5,l, No HI No H2 N o H3  Table 3.3: A summary of Figure 3.14. Acetylcholine effect on lung inflation pressure in the presence of different doses of antagonists. The assumed receptors involved is based on blocking property and relative affinities of the antagonists (refer to table 2.3).  His-LRP [ED +ALRP(mmHg)l Summary o f actions o f antagonist in terms o f shifts o f dose-response curves N o antagonist-induced shifts seen 20  Antagonist Pirenzepine Methoctramine 4-DAMP Mepyramine  Ranitidine Thioperamide  N o antagonist-induced shifts seen N o antagonist-induced shifts seen Very marked shift by the all doses (dose ratio >100) but the effect o f larger doses o f antagonist could not be seen due to extend to blockade N o antagonist-induced shifts seen N o antagonist-induced shifts seen  Type o f blockade N o block  Receptor involved  N o block N o block Possibly competitive  NoM NoM HI  N o block N o block  No H2 N o H3  NoM  Table 3.4: A summary of Figure 3.15. Histamine effects on lung inflation pressure in the presence of different doses of antagonists. Possible receptors blocked by the antagonists are stated based on blocking properties and relative affinities of the antagonists (refer to table 2.3).  110  3.2  Drug modification of responses to allergen challenge  3.2.1 Effect of antihistamines (mepyramine and cetirizine) on acute allergic reactions in sensitized ovalbumin challenged guinea pigs  3.2.1.1 Sneezing and nose rubbing Intranasal instillation of ovalbumin i n sensitized guinea pigs significantly induced sneezing and nose rubbing evaluated during the first 30 minutes following intranasal challenge as compared to non-sensitized guinea pigs. Pretreatment with mepyramine and cetirizine intraperitoneally administered at doses 3mg/kg 15 minutes prior to nasal challenge significantly ( p O . O l ) inhibited ovalbumin-induced sneezing in sensitized guinea pigs as compared to sensitized untreated guinea pigs (Figure 3.16). In addition, cetirizine at the same dose significantly (p<0.05) attenuated nose rubbing frequency. Mepyramine failed to inhibit ovalbumin-induced nose rubbing i n sensitized guinea pigs (Figure 3.17).  3.2.1.1 Nasal airway pressure Ovalbumin challenge in sensitized guinea pigs significantly caused increases in nasal airway pressure within 30 minutes post ovalbumin challenge as compared to nonsensitized guinea pigs. Cetirizine intraperitoneally administered at a dose o f 3mg/kg significantly (p<0.05) attenuated ovalbumin-provoked increases i n nasal airway pressure, whereas no effect o f mepyramine (3mg/kg) pretreatment on nasal airway pressure was observed (Figure 3.18).  Ill  lO.On  7.5H o  CO <u  5.0H  N <U <U  a  C/2  2.5-  0.0  J  ^aline challenge  0 V  control  a challenge,  control (nonsenskized)  pretreated with pretreated with mepyramine cetirizine  J  sensitized ova challenge Figure 3.16: Effect of antihistamines; mepyramine 3mg/kg and cetirizine 3mg/kg on sneezing frequency induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril in both nostrils and sneezes were counted for 30 minutes after challenge. Drugs were administered ip 15 minutes prior to challenge. * Significant inhibition of sneezing frequency as compared to sensitized untreated guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in sneezing frequncy as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  75  n  saline challenge ova challenge control control (nonsensitized) — 1  1  1  pretreated w ith pretreated with mepyramine cetirizine ——: ;—~ sensitized ova challenge  Figure 3.17: Effect of antihistamines; mepyramine 3mg/kg and cetirizine 3mg/kg on frequency of nose rubbing induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril in both nostrils and nose rubs were counted for 30 minutes after challenge. Drugs were administered intraperitoneally 15 prior to ovalbumin challenge. * Significant inhibition of nose rubbing frequency as compared to sensitized untreated guinea pigs (p<0.05, Bonferroni multiple comparison test). # Significant increase in nose rubbing frequncy as compared to nonsensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  112  4-1 3H •5 M  2H H OH  ova challenge  control ^  -H  pretreated with pretreated with mepyramine cetirizine  sensitized ova challenge saline challenge control (nonsensitized)  Figure 3.18: Effect of antihistamines; mepyramine 3mg/kg and cetirizine 3mg/kg on nasal airway pressure induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin, or saline, at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge was considered as a change in nasal airway pressure. Drugs were administered intraperitoneally 15 minutes prior to ovalbumin challenge. * Significant reduction of nasal airway pressure as compared to sensitized untreated guinea pigs (p<0.05, Bonferroni multiple comparison test). # Significant increase in nasal airway pressure as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.2.2  Effect of leukotriene D4 receptor antagonist, montelukast, on acute allergic  reactions in sensitized ovalbumin challenged guinea pigs  3.2.2.1 Sneezing and nose rubbing Ovalbumin challenge in sensitized guinea pigs induced sneezing and nose rubbing as compared to non-sensitized guinea pigs. Pretreatment with montelukast administered  lOmg/kg  intravenously 5 minutes prior to ovalbumin challenge in sensitized guinea  pigs failed to attenuate both sneezing and nose rubbing (Figures 3.19 and 3.20).  113  7.5  n  5.OH  o <u N <D  2.5H  C/3  o.o-J  saline challenge ova challenge I  control  l  control (nonsenskized)  L  pretreated with montelukast  sensitized ova challenge  Figure 3.19: Effect of montelukast 1 Omg/kg on sneezing frequency induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril in both nostrils and sneezes were counted for 30 minutes after challenge. Montelukast was administered intravenously 5 minutes prior to ovalbumin challenge. # Significant increase in sneezing frequncy as compared to nonsensitized ovalbumin challenged guinea pigs (p<0,05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  30  n  a o  20H  O  ioH  o  J  saline challenge ova challenge I  control  l  control (nonsensitized)  L  pretreated with montelukast  sensitized ova challenge  Figure 3.20: Effect of montelukast 1 Omg/kg on nose rubbing frequency induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received a challenge dose of 2% ovalbumin or saline 20ul/nostril in both nostrils and nose rubs were counted for 30 minutes after challenge. Montelukast was administered intravenously 5 minutes prior to ovalbumin challenge. # Significant increase in nose rubbing frequency as compared to nonsensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  114  3.2.2.2 Nasal airway pressure In sensitized guinea pigs, ovalbumin provocation produced significant increases i n nasal airway pressure as  compared to  non-sensitized guinea pigs.  Montelukast  1 Omg/kg administered intravenously 5 minutes prior to ovalbumin challenge significantly (p<0.01) alleviated increases in nasal airway pressure induced by ovalbumin i n sensitized guinea pigs (Figure 3.21).  4n O  OH /—S  1-  T  0-  n  S3  ,1-  ova challenge saline challenge I  5  pretreated with montelukast i sensitized ova challenge contro  I  control (nonsensitized)  Figure 3.21: Effect of montelukast 1 Omg/kg on nasal airway pressure induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge was considered as a change in nasal airway pressure. Montelukast was administered intravenously 5 minutes prior to ovalbumin challenge. N A P : nasal airway pressure. * Significant reduction of nasal airway pressure as compared to sensitized untreated guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in nasal airway pressure as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  115  3.2.2.3 Leukocyte infiltration Ovalbumin challenge i n sensitized guinea pigs induced cellular infiltration during first hour following nasal challenge. This was reflected by increases in total cell count o f nasal lavage collected one hour post ovalbumin challenge. N o significant change was observed i n non-sensitized guinea pigs. Pretreatment with montelukast 1 Omg/kg iv 5 minutes prior to ovalbumin challenge significantly (p<0.01) reduced total cell count as compared to sensitized ovalbumin challenged group (Figure 3.22).  saline challenge ova challenge ' '. control (nonsensitized)  pretreated with montelukast ——; ;— sensitized ova challenge Figure 3.22: Effect of montelukast lOmg/kg on cellular infiltration (total cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted using a hemocytometer as number of cells per milliliter of nasal lavage collected 60 minutes after challenge. Montelukast was administered intravenously 5 minutes prior to ovalbumin challenge. * Significant decrease in total cell count as compared to sensitized untreated guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in total cell count as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean. 1  116  control  3.2.3  Effect of  N  (omega)-nitro-L-arginine methyl ester ( L - N A M E )  on  acute  allergic reactions in sensitized ovalbumin challenged guinea pigs  3.2.3.1 Nasal airway pressure In sensitized guinea pigs, ovalbumin provocation produced significant increases in nasal airway pressure as compared to non-sensitized guinea pigs. L - N A M E 1 Omg/kg administered (pO.OOl)  intravenously  inhibited the  5  minutes  prior  to  ovalbumin  challenge  significantly  increase i n nasal airway pressure induced by ovalbumin in  sensitized guinea pigs (Figure 3.23).  10.07.55.02.5H 0.0 ova challenge -2.5-5.0-  control  saline challenge control (nonsensitized) pretreated with L-NAME  -7.5-  sensitized ova challenge  -10.0-  Figure 3.23: Effect of L - N A M E 1 Omg/kg on nasal airway pressure (NAP) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge was considered as a change in nasal airway pressure. L - N A M E was administered intravenously 5 minutes prior to ovalbumin challenge. * Significant reduction of nasal airway pressure as compared to sensitized untreated guinea pigs (pO.OOl, Bonferroni multiple comparison test). # Significant increase in nasal airway pressure as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean  117  3.2.3.2 Leukocyte infiltration Ovalbumin challenge i n sensitized guinea pigs induced cellular infiltration i n the first hour following nasal challenge. This was reflected by increases in total cell count o f nasal lavage collected one hour post ovalbumin challenge. N o significant change was observed  in  non-sensitized  guinea  pigs.  Pretreatment  with  L-NAME  1 Omg/kg  intravenously administered 5 minutes prior to ovalbumin challenge failed to reduce total cell count within one hour post ovalbumin challenge in sensitized guinea pigs (Figure 3.24).  75 C3 S3 ao  n  50H  TI-  cs  25u saline challenge ova challenge control (nonsensitized)  control  pretreated with L-NAME  sensitized ova challenge  Figure 3.24: Effect of L - N A M E lOmg/kg on cellular infiltration (total cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted using hemocytometer under light microscope and the value were expressed as number of cells per milliliter of nasal lavage collected 60 minutes after challenge. L - N A M E was administered intravenously 5 minutes prior to ovalbumin challenge. # Significant increase in total cell count as compared to nonsensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean  118  Ovalbumin challenge in sensitized guinea pigs significantly stimulated eosinophil infiltration within first hour after ovalbumin challenge as compared to non-sensitized guinea pigs. This was reflected by an increase in eosinophils i n nasal lavage sample detected by differential cell count. N o significant increase in neutrophils, basophils, monocytes and lymphocytes was detected when compared between sensitized and  non-  sensitized guinea pigs. Pretreatment with L - N A M E 1 Omg/kg intravenously administered 5 minutes prior to ovalbumin challenge significantly ( p O . 0 0 1 ) induced increases i n neutrophil infiltration in treated group as compared to untreated groups (Figure 3.25).  200-1  eosinophil neutrophil basophil monocyte lymphocyte  T3 «9 >< 150T3  u  O  8 o g. 100o O u  s-  3 Z  5<H  0-"  saline challenge  ova challenge  control (nonsensitized)  I* control  pretreated with L-NAME  i  sensitized ova challenge  Figure 3.25: Effect of L - N A M E lOmg/kg on cellular infiltration (differential cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted under light microscope at 40X power and total cells counted in three consistent fields were used to express the changes. L - N A M E was administered intravenously 5 minutes prior to ovalbumin challenge. * Significant increase in neutrophil number as compared to non-sensitized ovalbumin challenged guinea pigs (* pO.001, Bonferroni multiple comparison test). # Significant increase in eosinophil number as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  119  3.2.4 Effect of heparin on acute allergic reactions in sensitized ovalbumin challenged guinea pigs  3.2.4.1 Sneezing and nose rubbing Ovalbumin challenge in sensitized guinea pigs induced sneezing and nose rubbing as compared with non-sensitized. Pretreatment with heparin 20mg/kg administered intravenously 5 minutes prior to ovalbumin challenge failed to attenuate both sneezing and nose rubbing (Figures 3.26 and 3.27).  7.5  n  Figure 3.26: Effect of heparin 20mg/kg on sneezing frequency induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received ovalbumin, or saline, at 20ul/nostril in both nostrils. Sneezes were counted for 30 minutes after challenge. Heparin iv was administered 5 minutes prior to ovalbumin. # Significant increase in sneezing frequncy as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  120  3(H  #  I  saline challenge ova challenge  control  control (nonsensitized)  pretreated with heparin  sensitized ova challenge  Figure 3.27: Effect of heparin 20mg/kg iv on nose rubbing induced by 2% ovalbumin challenge in sensitized guinea pigs. Guinea pigs received ovalbumin, or saline, at 20ul/nostril in both, nostrils. Nose rubs were counted for 30 minutes after challenge. Heparin was administered iv 5 minutes prior to challenge. # Significant increase in nose rubbing frequency as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.2.4.2 Nasal airway pressure In sensitized guinea pigs, ovalbumin provocation produced significant increases in nasal airway pressure as compared with non-sensitized guinea pigs. Heparin 20mg/kg iv 5 minutes prior to ovalbumin challenge significantly (p<0.01) alleviated the increase in nasal airway pressure induced by ovalbumin challenge in sensitized guinea pigs (Figure 3.28).  121  5! I OH 5  ova challenge  control I  I  sensitized ova challenge  saline challenge J  pretreated with heparin  control (nonsensitized)  Figure 3.28: Effect of heparin 20mg/kg iv on nasal airway pressure (NAP) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin, or saline, at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge was considered as a change in nasal airway pressure. Heparin was administered 5 minutes prior to ovalbumin challenge. * Significant reduction of nasal airway pressure as compared to sensitized untreated guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in nasal airway pressure as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.2.4.3 Leukocyte infiltration Ovalbumin challenge in sensitized guinea pigs induced cellular infiltration during first hour following nasal challenge. This was reflected by increases in total cell counts in the nasal lavage collected one hour post ovalbumin challenge. N o significant change was observed in non-sensitized guinea pigs. Pretreatment with heparin 20mg/kg significantly (p<0.01) reduced total cell count as compared with sensitized ovalbumin challenged group (Figure 3.29)  122  I  I  i  control (nonsensitized)  heparin sensitized ova challenge  ,  Figure 3.29: Effect of heparin 20mg/kg iv on cellular infiltration (total cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted using hemocytometer under light microscope and the value were expressed as number of cells per milliliter of nasal lavage collected 60 minutes after challenge. Heparin was administered 5 minutes prior to ovalbumin challenge. * Significant decrease in total cell count as compared to sensitized untreated guinea pigs (p<0.01, Bonferroni multiple comparison test). # Significant increase in total cell count as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.01, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.2.5  Effect of dexamethasone on acute allergic reactions in sensitized ovalbumin  challenged guinea pigs  3.2.5.1 Sneezing and nose rubbing Intranasal instillation o f ovalbumin in sensitized guinea pigs significantly induced sneezing and  nose rubbing in the  first 30 minutes following  intranasal challenge.  Pretreatment with dexamethasone (20mg/kg ip) 15 minutes prior to nasal challenge significantly (p<0.001) inhibited ovalbumin-induced sneezing in sensitized guinea pigs as  123  compared  with  sensitized  non  treated  guinea  pigs  (Figure  3.30).  However,  dexamethasone failed to attenuate ovalbumin induced.nose rubbing (Figure 3.31).  10.0  n  #  7.5H o  5.0H 25H  I  0.0 saline challenge  *  J  i  ova challenge ;  control (nonsensitized)  control  i  I  pretreated with dexamethasone  sensitized ova challenge  Figure 3.30: Effect of dexamethasone 20mg/kg ip on number of sneezes induced by ovalbumin challenge in sensitized guinea pigs. Guinea pigs received ovalbumin, or saline, 20ul/nostril in both nostrils and sneezes were counted for 30 minutes after challenge. Dexamethasone administered 15 minutes prior to ovalbumin challenge. * Significant reduction in number of sneezes as compared to untreated sensitized guinea pigs (pO.001, Bonferroni multiple comparison test). # Significant increase in sneezing frequency as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  3.2.5.2 Nasal airway pressure Nasal airway pressure increased following  ovalbumin challenge in sensitized  guinea pigs. Dexamethasone 20mg/kg administered intravenously 5 minutes prior to ovalbumin challenge failed to prevent ovalbumin induced increases i n nasal airway pressure (Figure 3.32).  124  AO-i  •g.3<H o m CO  20H CO  £ 10J saline challenge ova challenge l  :  i  control  control (npnsensitized)  pretreated with dexamethasone  J  sensitized ova challenge  Figure 3.31: Effect of Dexamethasone 20mg/kg administered intraperitoneally on nose rubbing frequency induced by ovalbumin challenge in sensitized guinea pigs. Guinea pigs received ovalbumin, or saline, 20ul/nostril in both nostrils and nose rubs were counted for 30 minutes after challenge. Dexamethasone was administered 15 minutes prior to ovalbumin challenge. # Significant increase in nose rubbing frequency as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, unpaired student t-test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  I 2  5  ,  I ova challenge  J saline challenge  _ ,  control (nonsensitized)  control  pretreated with dexamethasone  §  sensitized ova challenge  Figure 3.32: Effect of dexamethasone 20mg/kg iv on nasal airway pressure (NAP) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with Ovalbumin, or saline, at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Nasal airway pressure was measured 30-35 minutes after challenge. The difference between pre-challenge and 30-35 minutes post-challenge was considered as a change in nasal airway pressure. Dexamethasone was administered 5 minutes prior to ovalbumin challenge. # Significant increase in nasal airway pressure as compared to nonsensitized ovalbumin challenged guinea pigs (p<0.05, Bonferroni multiple comparison test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  125  3.2.5.3 Leukocyte infiltration There was an increase in leukocyte infiltration following ovalbumin challenge in sensitized guinea pigs. This was reflected by increases in total cell count o f nasal lavage collected one hour post ovalbumin challenge. N o significant change was observed in non-sensitized guinea pigs. Pretreatment with dexamethasone (20mg/kg iv) 5 minutes prior to ovalbumin challenge failed to reduce total cell (Figure 3.33).  g  60H  saline challenge ova challenge control pretreated with I 1 dexamethasone control (nonsensitized) ' ——: ;—sensitized ova challenge Figure 3.33: Effect of dexamethasone 20mg/kg administered intravenously on cellular infiltration (total cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with ovalbumin, or saline, at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted using hemocytometer under light microscope and the value were expressed as number of cells per milliliter of nasal lavage collected 60 minutes after challenge. Dexamethasone was administered 5 minutes prior to ovalbumin challenge. # Significant increase in total cell count as compared to nonsensitized ovalbumin challenged guinea pigs (p<0.05, unpaired student t-test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean  Ovalbumin challenge in sensitized guinea pigs significantly stimulated eosinophil infiltration within first hour after ovalbumin challenge as compared to non-sensitized guinea pigs. This was reflected by an increase in eosinophils i n nasal lavage sample  126  detected by differential cell count. N o significant increase in neutrophils, basophils, monocytes and lymphocytes was detected when compared between sensitized and nonsensitized  guinea  pigs.  Pretreatment  with  dexamethasone  20mg/kg  intravenously  administered 5 minutes prior to ovalbumin challenge did not cause significant changes in differential cell count when compared with untreated groups although there is a none statistically significant increase in neutrophils (Figure 3.34).  150n  «5 X -a o 125H u  I eosinophil I neutrophil I basophil lymphocyte monocyte  ^  — o  100H  <~ 2 o b  75-\  JO  50H  c  £ <D Z  §  25H  ° saline challenge ova challenge control (nonsensitized)  control L  pretreated with dexamethasone  sensitized ova challenge  Figure 3.34: Effect of dexamethasone 20mg/kg intravenously administered on cellular infiltration (differential cell count) induced by ovalbumin challenge in anaesthetized, sensitized guinea pigs. Guinea pigs were challenged with 2% ovalbumin or saline at a volume of 0.5ml infused to the nasal cavities for 5 minutes. Cells were counted under light microscope at 40X power and total cells counted in three consistent fields were used to express the changes. Dexamethasone was administered intravenously 5 minutes prior to ovalbumin challenge. # Significant increase in eosinophil number as compared to non-sensitized ovalbumin challenged guinea pigs (p<0.05, unpaired student t-test). The data are means of 5 guinea pigs. Vertical bars represent standard error of mean.  127  3.2.6  Overall summary  Tables 3.5 and 3.6 summarise the findings o f the above conducted studies. Allergic responses ....  Possible target Drug  Mepyramine  Sneezing  Inhibited  Nose rubbing  N o effect  Nasal airway  Cellular  pressure  infiltraion  N o effect  Not done  mediator  Histamine ( H I ) Histamine  Cetirizine  Inhibited  Inhibited  Inhibited  (Hl)+  other inflammatory  Not done  mediators Montelukast  N o effect  N o effect  Inhibited  Inhibited induced  L-NAME  Not done  Not done  Inhibited  Leukotriene D 4 nasal  neutrophilia  Nitric oxide Inflammatory  Heparin  N o effect  N o effect  Inhibited  Inhibited  mediators Inflammatory  Dexamethasone  Inhibited  N o effect  N o effect  N o effect  mediators  Table 3.5: A summary of drug modification of the response to allergen challenge experiments.  128  (J roil}ililllll^^B o 3 -1  O  \ \ Xl'immllL-:  Al  irmniill'j)  AAlAPdnmllg)  \ I I U i h . min)  -o.y ± 0.5 0.1 ± 0 . 3  -0.1 -:  0.2 0.5 ± 1.0  2.5 -:. 0.9  .0 :l. 4.o  NOC  6.2 ± 3 . 5  -2.5 ± 6.6  SOC  3.3±0.9  -0.7 ± 0 . 6  4.7 ± 3 . 7  6.0 ± 6 . 0  NSC  a  Mepyramine  2.9 ± 0 . 4  Cetirizine  0.5 ± 0.4  Montelukast Heparine L-NAME  -4.0 ± 1.7" 4.7 ± 1.8  Dexamethasone  0.3 ± 0 . 9  7.8 ± 3 . 6  10.0 ± 9 . 1  2.3 ± 1.2  1.6 ± 1.3  -12.0 ± 4 . 1  0.2 ± 0 . 3 "  -0.1 ± 0 . 1  -1.5 ± 0 . 8  -0.5 ± 4 . 1  0.3±0.2  0.3 ± 0.5  1.6 ± 1.8  -3.3 ± 5 . 6  0.6 ± 0 . 4  2.5 ± 2 . 0  -3.0 ± 3 . 7  1.4 ± 0 . 9  10.9 ± 5 . 6  7.0 ± 7 . 5  b  b  Table 3.6: A data summary of changes in nasal airway pressure (NAP), lung inflation pressure (LIP), main arterial blood pressure ( M A P ) and heart rate (HR) in various control and treated guinea pigs measured 30 minutes post challenge. N S C : non-sensitized saline challenged, N O C : non-sensitized ovalbumin challenged, SOC: sensitized ovalbumin challenged. is statistical significant from The values represent mean ± S E M of n = 5. b  129  a  4  Discussion  4.0 Value of the models in terms of responses to allergen challenge and relevance to clinical rhinitis with special reference to effects of histamine and acetylcholine.  Guinea pigs have been the species o f choice for the evaluation o f chemical-related respiratory allergy, primarily because it is possible in this species to elicit and measure with relative ease challenged-induced inflammatory reactions that resemble in some ways the acute clinical manifestations o f human allergic rhinitis. In this study the procedures used to sensitized guinea pigs were effective. Sensitized guinea pigs produced acute allergic responses after allergen provocation characterized by sneezing, nose rubbing, increase in nasal airway pressure and eosinophil infiltration. In clinical situations, people who suffer from allergic rhinitis suffer from symptoms of sneezing, itching, nasal secretions,  nasal  congestion  in addition to eosinophil infiltration. The  currently  established model can be used to accurately compare effectiveness o f drugs currently used in clinic in addition to further investigation o f new drugs. In addition, this model shows no effect o f intranasal allergen challenge on lung inflation pressure which may indicate that intranasal allergen challenge specifically triggers nasal  inflammatory  responses without affecting those o f lower respiratory tract. Furthermore, sensitized guinea pigs produced anaphylactic shock immediately after intravenous administration o f l m g / k g ovalbumin. The response was characterized by rapid increase in blood pressure, heart rate and lung inflation pressure. This reflects systemic sensitization following topical nasal sensitization. Histamine is thought to be a main mediator in the acute inflammatory reaction o f allergic rhinitis (Passalacua et al., 2000). Histamine H I antagonists are commonly used  130  to treat the acute phase symptoms including sneezing, itchiness and nasal secretions (Dykewicz et al., 1998). However their action in ameliorating nasal congestion is poor (Nabe et al., 2001). Some studies have shown that histamine H I antagonists reduced nasal congestion (Dykewicz et al., 1998). Other studies on the other hand, concluded there was no effect o f H I antagonists in decreasing nasal airway pressure (congestion) (Nabe et al., 2001) although when histamine was applied topically into the nasal mucosa, it causes increases in nasal resistance (Mizutani et al., 1999; Ruslan et al., 2003; TylorClark et al, 2005). Other studies have shown that a combination o f H I and H 3 histamine receptor antagonists may reduce histamine-provoked increases in nasal airway pressure (Tylor-Clark et al., 2005). It also has been shown that, depending on the dose given intranasally, histamine may cause an increase or decrease in the nasal airway pressure (Lung etal., 1987). There are no published studied on the effect o f histamine on nasal airway pressure when it is given intravenously. In our study we have found that when histamine is administered intravenously to guinea pigs, it causes decreases in the nasal airway pressure in a dose-dependent fashion. Mepyramine, possibly competitively, antagonized histamine induced-decreases in nasal airway pressure, whereas no effect o f ranitidine and thioperamide were observed. This may suggest the involvement o f histamine H I receptors i n this action. Pirenzepine did not prevent histamine-induced decreases in nasal airway pressure. N o muscarinic M l receptors were involved in histamine action. However, methoctramine (to some extent selective on M 2 receptors but has very low affinity to M 3 receptors) and 4 - D A M P (same affinity on M l , 3, and 5 receptors) produced a slight shift in histamine action. This may suggest the involvement o f M 2 and  131  perhaps M 5 receptors. Taking all this into account, it may lead to the conclusion that histamine-induced decreases i n nasal airway pressure is due its action on H I , M 2 and perhaps M 5 receptors. The reason why topical application causes increases in nasal resistance, whereas intravenous application causes decreases in the nasal resistance, is not well understood. Results from previous norepinephrine  studies  transmission  have  shown that there is a local regulation o f  in nasal mucosa involving excitatory H I  receptors,  inhibitory H 2 receptors and inhibitory muscarinic receptors which can be facilitated by sensory stimulation with histamine. Histamine regulation may vary with a balance between the excitatory and inhibitory effects. Histamine i n the high concentrations seen in cases o f nasal allergy may shift this balance causing increased norepinephrine release. A t low concentrations, however, norepinephrine release is inhibited. Norepinephrine causes nasal vasoconstriction that lead to decrease in the nasal airway pressure (Kubo et al., 1989). Study i n rabbits showed that Hl-receptors mediate negative inotropic effects and vasoconstriction, whereas H2-receptors are responsible for positive inotropic and chronotropic effects and vasodilatation (Sakai 1980). Although anticholinergic drugs are known to decrease mucous secretion in allergic rhinitis (Lahny et al., 2002), few studies have shown their role in congestion. One study in dogs indicated that acetylcholine given intraarterially may increase or decrease nasal airway pressure, depending on the dose administered, probably v i a a dosedependent differential action on different components o f the nasal vascular bed (Lung et al., 1994). In our study we found that acetylcholine administered intravenously caused a decrease i n the nasal airway pressure in a dose-dependent manner. Methoctramine failed  132  to prevent acetylcholine-induced decreases in nasal airway pressure which may suggest that no muscarinic M 2 and M 4 receptors were involved, whereas pirenzepine and 4D A M P , non-competitively, slightly prevented acetylcholine action. Since 4 - D A M P acts equally on M l , M 3 and M 5 receptors, and pirenzepine acts maily on M l receptor, our best guess for the receptor involved in this action would be muscarinic M l receptors. None o f the antihistamines prevented acetylcholine-induced decreases i n nasal airway pressure. This may lead to conclusion that acetylcholine action to decrease nasal airway pressure does not involve histamine receptors. The main focus in our study was nasal airway pressure. However, we also studied the role o f muscarinic and histamine receptors on changes i n lung inflation pressure. Although we did not study muscarinic 4 and 5 receptors, the results were in general agreement with the literature. Lung inflation pressure increased in a dose-dependent fashion after acetylcholine and histamine were administered. Acetylcholine and histamine bind to receptors in airway smooth muscle cells causing increases in global intracellular calcium release. Calcium-calmodulin complex binds to myosin light chain resulting i n phosphorilation o f myocin light chain and eventually smooth muscle contraction, and thus  bronchoconstriction  which  leads  to  increase  in  lung  inflation  pressure.  Methoctramine did not prevent acetylcholine-induced increases in lung inflation pressure, hence no involvement o f M 2 and M 4 receptors. O n the other hand, pirenzepine (has higher affinity to M l receptors than other muscarinic receptors) and 4 - D A M P (shows equal affinity to M l , 3 and.5 receptors) presumably competitively blocked acetylcholineinduced increases in lung inflation pressure. N o effects o f antihistaminics were observed. Thus, acetylcholine acts mainly on M l receptors to cause increases i n lung inflation  133  pressure. Mepyramine, presumably competitively, blocked histamine induced-increases in lung inflation pressure. However, neither the muscarinic antagonist nor ranitidine and thioperamide blocked histamine effect. The most probably receptor involved i n the histamine action was H I .  4.1 Possible clinical relevance of model responses to clinical and experimental drugs  4.1.1 Effect of antihistamines on sneezing, nose rubbing and nasal airway pressure in sensitized ovalbumin challenged guinea pigs  Histamine,  a natural  body  constituent,  is  a  low-molecular-weight amine  synthesized from L-histidine exclusively by histidine decarboxylase, an enzyme that is expressed in cells throughout the body, including central nervous system neurons, gastricmucosa parietal cells, mast cells, and basophils (Schayer 1956; Fujikura et al., 2001; A k d i s et al., 2003). Histamine plays a pivotal role in allergic inflammation. Nasal challenge with histamine causes sneezing, pain, pruritus, rhinorrhea and nasal blockage (Doyle et al., 1990). In addition, histamine levels i n nasal lavages increase following allergen challenge (Naclerio et al., 1985). Histamine is one o f the major chemical mediators triggering the symptoms o f rhinitis, including sneezing, nasal itiching and rhinorrhea in allergic rhinitis patients, although it is not extensively involved in the increase in nasal airway pressure i n allergic rhinitis patients as shown by the limited effect o f antihistamines on nasal congestion (Skoher et al., 1994; Numata et al., 1999). Most o f the effects o f histamine in allergic disease occur through H i receptors (Schmelz et a l , 1997; Schneider et al., 2002; A k d i s et  134  al., 2003). HI-antihistamines act as inverse agonists that combine with and stabilize the inactive form o f the H I - receptor, shifting the equilibrium toward the inactive state (Bakker et al., 2001; Leurs et al., 2002). Hi-antihistamines (second  generation  antihistamines being mostly used currently for their minimal sedative effect), either administered orally or applied topically to mucosal surfaces, are the most commonly used first-line medications for both seasonal and perennial allergic rhinitis (Bousquet et al., 2001). W i t h the regard to their mechanism o f action, HI-receptors are coupled to phospholipase C , and their activation leads to formation o f inositol-1,4,5-trisphosphate (IP3) and diacylglycerols from phospholipids in the cell membrane; IP3 causes a rapid release o f calcium from the endoplasmic reticulum. Diacylglycerol and calcium activate protein kinase C , while calcium activates calcium/calmodulin-dependent protein kinases and phospholipase A 2 in the target cell to generate the characteristic response. Cetirizine and mepyramine are both highly selective for H I receptors as compared to H 2 , H3 (Bernheim et al., 1991; Leurs et al., 1995). In addition, cetirizine is highly selective for H I receptors as compared to serotonergic, adrenergic and muscarinic receptors (Gillard et al., 2003). In this study, mepyramine (first generation H l antihistamine) and cetirizine (second generation HI-antihistamine) significantly inhibited sneezing frequency during the acute phase o f allergic reactions in guinea pigs. In addition, cetirizine, but not mepyramine significantly, inhibited nose rubbing. These results support what has been postulated about the important role o f histamine in induction o f sneezing during allergic rhinitis and that antihistamines inhibit antigeninduced sneezing (Narita et al., 1997; Fujita et al., 1999; Yamasaki et al., 2001). Itch  135  (pruritus) is commonly defined in humans as an unpleasant sensation o f the superficial layers o f the skin (Shelley et al., 1957) provoking the desire to scratch (Ekblom 1995). It is a common clinical condition that can be associated with cutaneous (e.g. atopic eczema, contact dermatitis) or systemic (e.g. chronic renal failure) disease. Itch is difficult to study objectively in man and there are currently few reliable animal models o f itch. In addition, the role o f histamine in induction o f nose rubbing in guinea pig models o f allergic rhinitis has not been shown clearly, although some studies reported that antihistamines may inhibit nose rubbing (Zhao et al., 2005). Histamine acts on H l receptor on nerve endings, resulting in central neuronal reflexes, leading to sneezing and itching. Mepyramine failed to alleviate nasal scratching. It has been postulated that classical H i receptor antihistamines (e.g. mepyramine) are not effective in alleviating many chronic pruritic conditions (Greaves 1997). Furthermore, it has been shown in a murine model o f itching that histamine mediates this symptom through its action on histamine H 4 receptors (Bell et al., 2004). Therefore, histamine released after allergen provocation acts also on H 4 receptors mediating itching sensation suggesting why mepyramine failed to inhibit these responses. However, cetirizine attenuated nose rubbing, suggesting that this agent has additional pharmacological actions. In fact, cetirizine has a number o f antiallergic, anti-inflammatory properties that appear to be independent o f its HI-blockade activity (Assanasen et al., 2002), and these may account for its effectiveness in inhibition o f nose rubbing. Our results are consistence to what has been found in studies conducted in allergic rhinitic guinea pigs, i n that mepyramine ameliorates  sneezing  (Mizutani et al., 2003; Nabe et al., 2001; Narita et al., 1993). Additionally,  136  frequency clinical  studies have shown that cetirizine is effective in inhibition o f sneezing (Allegra et al., 1993; B a r o o d y e t a l . , 1989). In our study, cetirizine, but not mepyramine, significantly inhibited ovalbumininduced increases i n nasal airway pressure during the acute phase o f allergic rhinitis in guinea pigs. Preclinical studies in animal models o f allergic rhinitis have shown that histamine has a role in induction o f nasal airway pressure especially during the acute phase o f the disease (Mizutani et al., 1999; M c L e o d et al., 2002; Bockman et al., 2002 Mizutani et al., 2003). In addition, not all antihistamines alleviate the increase in nasal airway pressure induced by antigen in animal models o f rhinitis (Nabe et al., 2001; Mizutani et al., 2003; Sakairi et al., 2005). Mepyramine failed to alleviate the increase i n nasal airway pressure, mimicking what has been found i n previous studies i n guinea pig model o f allergic rhinitis (Terasawa et al., 1988; Narita et al., 1993; Mizutani et al., 1999; M c L e o d et al., 2002; Bockman et al., 2002; Mizutani et al., 2003). O n the other hand, cetirizine prevented nasal airway pressure increase after allergen provocation. Murata has shown that cetirizine inhibits antigen-induced nasal resistance in guinea pigs (Murata et al., 1997). The reason behind this contradiction i n antihistamine effects on nasal airway pressure is not well understood. It has been postulated that HI-antihistamines have multiple effects on the allergic inflammatory response. It is equally clear that these antiallergic effects are not uniformly shared among all drugs o f this class. Furthermore, data from in vitro,  in vivo, and ex vivo studies suggest that  second-generation  antihistamines  cetirizine) have a number  anti-inflammatory  (e.g.  o f antiallergic,  properties that appear to be independent o f their Hl-blockade activity (Assanasen et al.,  137  2002). Moreover, a number o f antihistamines  have been shown to relieve nasal  congestion, an effect possibly related to the combination o f the direct and potent antihistaminic effects and their well-established anti-inflammatory properties. This is especially true for one o f the best-investigated and widely used  second-generation  antihistamines, cetirizine, which is a 50/50 racemate mixture o f levocetirizine and dextrocetirizine. The predecessor o f levocetirizine has been shown to exhibit a number o f actions that determine its anti-inflammatory properties, including inhibition o f eosinophil accumulation, induced by pollen or platelet-activating factor ( P A F ) (Fadel et al., 1990; Walsh 2000), inhibition o f eosinophil migration to, and  infiltration, at the sites o f  allergic challenge (Fadel et al., 1990; Charlesworth et al., 1989), reduction o f I C A M - 1 (intercellular adhesion moleduce 1) expression (Fasce et al., 1996),  and  inhibition o f  monocytes and T lymphocytes (Jinquan et al., 1995). The potent inhibitory effect o f lovecetirizine on the H I receptor, histamine-induced inflammation, its ability to. control vascular dilation and plasma exudation, as well as direct evidence from nasal provocation tests, confirm the potential o f this novel antihistamine to control the early-phase reactions in the pathogenesis o f nasal congestion. Thus, it could be these additional anti inflammatory properties o f cetirizine behind its additional role i n nasal airway pressure alleviation, the property which is not observed i n mepyramine.  4.1.2 Effect of leukotriene D4 receptor antagonist, montelukast on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs  The cysteinyl. leukotrienes (cysLTs) produce their biological actions by binding and activating specific receptors located on the cell membranes o f target cells. T w o  138  subtypes o f c y s L T receptor have been pharmacologically characterized c y s L T i and cysLT2 (Coleman et al., 1995; Nicosia et al., 1999). Montelukast has twice potency than L T D 4 to c y s L T i receptor. Montelukast inhibits the binding o f L T D 4 to this receptor (Aharony 1998). In our study, montelukast significantly inhibited ovalbumin-induced increases in nasal airway pressure in guinea pigs during the acute phase o f allergic rhinitis. Furthermore,  montelukast  also  significantly inhibited cellular infiltration  (correlated with reduction i n total cell count) during the acute phase o f antigen-induced rhinitis. However the drug failed to alleviate sneezing and nose rubbing after allergen challenge. This is the first study to demonstrate the effect o f montelukast on such allergic symptoms using a guinea pig model o f antigen-induced rhinitis. In terms o f nasal airway pressure, our results are consistent with previous studies in guinea pig model o f allergic rhinitis. It has been demonstrated (using different c y s L T i receptor antagonists) that cysLTs (especially L T D 4 ) are significantly involved in the development o f nasal congestion (Shizawa et al., 1997; Kaise et al., 1998; Fujita et al., 1999; Mizutani et al., 2001; Yamasaki et a l , 2001; Mizutani et al., 2003). Cysteinyl L T s such as leukotriene C 4 , D and E (LTC4, L T D 4  4  4  and LTE4) are  released from various inflammatory cells including mast cells and eosinophils. These mediators are able to increase nasal blood flow (Bisgaard et al., 1986). A recent human study has shown that nasal provocation by L T D 4 can induce nasal obstruction, as indicated by a prolonged increase o f nasal airway pressure (Okuda et al., 1988). Furthermore, it has been demonstrated that nasal congestion i n the early-phase is accompanied by a significant increase o f cysteinyl L T s in nasal lavage fluid from patients with allergic rhinitis (Naclerio et al., 1991b). In addition, L T D 4 has been demonstrated to  139  dilate nasal blood vessels, which can be related to hyperproduction o f nitric oxide through cysteinyl LT1-receptor activation. These findings suggest the hypothesis that cysteinyl L T s play an important role in allergic rhinitis, especially in nasal obstruction due to edema o f the nasal mucosa membrane (Mizutani et al., 2001). Besides elleviating nasal congestion, montelukast reduces eosinophil infiltration (reduction o f the total cell count after allergen provocation) in the nasal mucosa as associated with the early phase (eosinophils are involved i n the acute phase o f allergic reactions in a guinea pig model o f rhinitis (Imai et al., 2000)) o f allergic rhinitis. This result is consistent with previous findings that LTD4 causes significant eosinophil infiltration in nasal mucosa which persists for up to 24 h after the topical challenge in guinea pigs (Fujita et al., 1997). Moreover, a recent report that cysteinyl L T s induces eosinophil infiltration in lower airway in human (Laitinen et al., 1993) and guinea pigs (Underwood et al., 1996) also supports the suggestion that cysteinyl L T s contribute to eosinophil infiltration. However, in spite o f its potent chemotactic activity (as low as 10~  10  M ) in human eosinophils (Spada et al., 1994), LTD4 up to 10 u M had no apparent  chemotactic activity in guinea pig eosinophils in vitro (Fujita et al., 1999). Therefore, its action is not attributable to direct chemotactic activity in guinea pig eosinophils. It is speculated that cells other than eosinophils produce eosinophil chemoatractant(s)  in  response to LTD4. Thromboxane A2 may be one o f the candidate mediators, since it can be released from tissues by LTD4 stimulation (Cheng et a l , 1990) and its involvement has been suggested in nasal eosinophil migration in guinea pigs (Narita et al., 1996). In contrast to the prevention o f nasal airway pressure increases, sneezing and nose rubbing in the early phase were not reduced by montelukast. In recent clinical studies,  140  montelukast prevents symptoms scores in patients with perennial allergic rhinitis (Patel et al., 2005) and seasonal allergic rhinitis (Chervinsky et al., 2004). However, our result is consistent with the findings that LTD4 does not induce sneezing in human (Okuda et al., 1988) and guinea pigs (Fujita et al., 1997b). Thus, it appears that cysteinyl L T s play a minor direct role i n sneezing and nose rubbing. Nevertheless, the possibility that cysteinyl L T s participate indirectly in sneezing and nasal itchings v i a an increase in hypersensitivity to specific and/or non-specific stimulus may not be totally excluded. Recently, it has been suggested that LTD4 enhances the responsiveness o f capsaicinsensitive afferent fibers i n the guinea pig airway (Undem, 1993). The results o f the present study support the involvement o f cysteinyl L T s i n allergic rhinitis, especially in oedema o f nasal membrane mucosa causing nasal obstruction. Cysteinyl L T receptor antagonists, such as montelukast, thus have therapeutic potential in the treatment o f allergic rhinitis.  4.1.3 Effect of L - N A M E on nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs  Nitric oxide, a free radical gas, is an endogenous  cell-signaling molecule  implicated i n a wide range o f physiological and pathophysiological events i n numerous cell types and processes, including the cardiovascular, immune and nervous systems. Endogenous nitric oxide is produced from the amino acid L-arginine by a family o f enzymes called nitric oxide synthases. There are three isoforms o f nitric oxide synthases (NOS); constitutive N O synthase which has two isoforms, neural N O S (NOS-1) and endothelial N O S (NOS-3), which have been reported to be expressed in nerve cells and  141  endothelial cells in arterioles, sinusoid vessels and capillary bed, respectively. Another isoform is inducible N O S (NOS-2), the expression o f which in epithelial cells, submucosal glands and inflammatory cells in the nasal mucosa o f allergic rhinitis patients is more marked than that o f subjects without nasal allergy (Hanazawa et al., 1993; Kawamoto et al., 1998). Nitric oxide (NO) is a powerful vasodilator that modulates systemic vascular tone (Rees et al., 1989). In addition, N O can cause tissue injury by contributing to the generation o f highly reactive oxygen radicals (Beckman et al., 1991). One study showed that a large amount o f N O . originating from the paranasal sinuses was continuously produced i n the nasal cavities o f healthy subjects (Lundberg et al., 1995). Furthermore, it has been reported that the N O concentration in exhaled air was elevated in patients with allergic rhinitis compared to that in normal subjects (Arnal et al. 1997; Kharitonov et al. 1997). NO  is known to cause marked vasodilatation by producing an increase in  intracellular cyclic G M P level, and to control systemic vascular tone (Imai et al., 2001). There is evidence that nitric oxide is involved in pathogenesis o f allergic rhinitis. H i g h levels o f nitric oxide have been detected in patients with allergic rhinitis (Arnal et al. 1997;  Kharitonov et al. 1997). Furthermore, nitric oxide causes vasodilatation and  glandular secretion (Baraniuk 1997). The contribution o f N O to the antigen-induced increase in specific airway resistance was evaluated using L - N A M E , a non-specific nitric' oxide synthase inhibitor. The nasal airway pressure elevations at the early phase induced by antigen challenge was suppressed by L - N A M E , given iv 30 minutes before the specific airway resistance measurement, and the suppression was greater than 95%. Our  142  results support previous finding that N O is involved in nasal airway pressure increase after allergen challenge in guinea pig models of allergic rhinitis (Imai et al., 2001; Mizutani et al., 2001; Bockman et al., 2002; Zhao et al., 2005). Furthermore, L - N A M E failed to attenuate eosinophil infiltration after allergen challenge, suggesting that this drug alleviates nasal congestion by a mechanism that might not involve eosinophil inhibition. The inhibitory action o f L - N A M E on the early nasal airway pressure increases is assumed to be mainly due to the direct suppression o f nasal vasodilatation. Kageyama et al.,  1997 reported that N O induces microvascular leakage as a result o f indirect  vasodilatation with direct endothelial contraction at the site o f leakage. Thus, it could be that L - N A M E inhibits the specific airway resistance increase by attenuating NO-induced plasma extravasation, i n addition to suppressing vasodilatation. The expression o f which o f N O S isoforms during allergic rhinitis is not very clear. One study has reported that endothelial N O S is expressed in sensitized guinea pig mucosal tissue (Zhao et al., 2005). In other study, it has been shown that constitutive N O S (both neuronal and endothelial N O S ) , but not inducible N O S , is involved in nitric oxide production after allergen provocation i n sensitized guinea pigs (Imai et al., 2001). In the current study, L - N A M E caused neutrophil infiltration. There is a growing body o f evidence that N O from both endogenous and exogenous sources limits leukocyte recruitment into normal and inflamed vessels. Inhibition o f endogenous N O with L N A M E promotes leukocyte adhesion in various vascular beds and species (Kubes et al., 1991;  Kubes et al., 1993; M a et al., 1993). Thus, endogenous N O is an important  homeostatic regulator o f leukocyte adhesion in postcapillary venules. Exposure o f  143  venular endothelium and neutrophils for 60 minutes to N O inhibitors (e.g. L - N A M E ) in vitro does not induce neutrophil-endothelial cell interactions ( N i u et al., 1994). This observation raises the possibility that some cell types were missing in the sample in vitro system. Mast cells are closely apposed to the vasculature and upon activation induce neutrophil-endothelium interactions (Gaboury et al., 1995). Inhibition o f N O synthesis causes mast cell degranulation in vivo, and this event could conceivably induce leukocyte recruitment. Indeed, L - N A M E was shown to cause stabilization o f mast cells and subsequent neutrophil adhesion (Kubes et al., 1993). Thus, the stimulation o f neutophil infiltration caused by L - N A M E may be as a result o f mast cell degranulation activated by L - N A M E and subsequent induction o f neutrophil adhesion and eventually infiltration. Despite o f this discrepancy, our results show that nitric oxide plays an important role in the pathogenesis o f allergic rhinitis. Through its powerful vasodilatory effect, nitric oxide may control the filling o f nasal capacitance vessels, thus determining nasal patency and mediating acute congestion accompanying allergen challenge.  4.1.4 Effect of heparin on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs  Heparin is a highly sulfated unbranched glycosaminoglycan. In addition to its well established anticoagulant properties, it exerts other actions including modulation o f various proteases (Schwartz et al., 1986), inhibition o f cell growth (Castellot et al., 1985), and attenuation o f inflammatory responses (Okajima et al., 2001). Heparin is now recognized to interact with a wide range o f proteins implicated in inflammatory responses. M a n y o f these heparin-binding proteins are central to the inflammatory  144  process, including cytokines, growth factors, adhesion molecules, cytotoxic peptides and tissue-destructive enzymes (Rose et al., 2004). Heparin has been proposed to have a regulatory role in limiting inflammation, i n part through its capability to bind such proteins,  thereby  limiting  cellular activation and  subsequent tissue  damage  and  remodelling (Page 1991). Heparin has particular relevance to allergy and inflammation in that it is found exclusively i n mast cells (Esko et al., 2002). Very little research has been undertaken over the past years to study heparin i n the treatment o f allergic rhinitis. This is the first study to show the protective effect o f heparin in animal models o f allergic rhinitis in vivo. Our results show that intravenously administered  heparin  significantly attenuates the increase in nasal airway pressure after allergen provocation in sensitized guinea pigs. In addition, pretreatment with intravenous heparin significantly reduced  leukocyte  influx  (predominantly  eosinophils)  60  minutes  after  allergen  challenge. O n the other hand, heparin failed to alleviate sneezing and nose rubbing after allergen challenge in sensitized guinea pigs. Mast cells and eosinophils are crucial for the development o f allergic rhinitis. Eosinophils are in fact able to produce a wide array o f proinflammatory cytokines and have the capacity to release cytotoxic proteins, including major basic protein, eosinophil-derived neurotoxin, and eosinophil cationic protein, which are potentially harmful for the integrity o f the nasal mucosa. In a clinical study, intranasal heparin significantly reduced symptom scores 10 m i n after antigen challenge. In addition, eosinophil influx in airway mucosa, and the amount o f eosinophil cationic protein in nasal lavage fluid were reduced (Vancheri et al., 2001). Moreover, heparin prevents nasal mucosa mast cell degranulation induced by adenosine (Zeng et al., 2004).  145  monophosphate  The mechanisms by which heparin inhibits eosinophil infiltration is not well understood. Heparin, through its capacity to prevent mast ceil mediator release (Page 2000), could indirectly produce a downregulation o f the expression o f a wide range o f adhesion molecules and therefore limit eosinophil migration into the nasal mucosa. Furthermore,  heparin  can  also  directly regulate  cellular diapedesis  through  the  endothelium by reducing the adherence o f leukocytes to endothelial cells, probably by increasing the negative charge o f the endothelial cell surface (Kanwar et al., 1996). Moreover, other mechanisms have been suggested to explain how heparin can modulate eosinophil recruitment and activation. Heparin can modulate the activity o f soluble mediators crucial to the promotion o f eosinophil migration. It has been reported that heparin might inactivate platelet activation factor, a cationic protein with a potent chemotactic activity for human eosinophils (Seeds et al., 1993). In this case, the ability o f heparin to bind platelet activating factor would result in a reduced cell influx into the tissue. Another study showed that heparin binds to I L - 5 , the major cytokine regulating eosinophil migration and activation (Lipscombe et al., 1998). In addition, it has been described that there is a possible interaction between eosinophil cationic protein and heparin, suggesting that heparin could neutralize the deleterious effects o f eosinophil chemotactic protein (Fredens et al., 1991). Thus, it could be that heparin ameliorates nasal airway pressure increases by mechanisms  involving  mast  cell  stabilization, eonosinophil  chemotactic  protein  neutralization, and reduction o f eosinophil recruitment. However, although heparin may prevent mast cell degranulation, it failed to attenuate antigen induced sneezing and nose rubbing in sensitized guinea pigs in this study, though heparin attenuates sneezing in  146  allergic rhinitic patients, suggesting that sneezing and itching mechanisms is more complex in guinea pigs. Taken together, these findings indicate that heparin may have beneficial effects in alleviating symptoms o f allergic rhinitis, but more studies are needed to confirm our observations and to define the characteristics o f the patients who would benefit most from such a therapeutic approach.  4.1.5 Effect of dexamethasone on sneezing, nose rubbing, nasal airway pressure and leukocyte infiltration in sensitized ovalbumin challenged guinea pigs Glucocorticoids are used successfully to suppress inflammation in chronic inflammatory diseases, such as asthma and allergic rhinitis (Carryer et al., 1950; Pipkorn et al., 1987). Corticosteroids act on mast cells, eosinophils, basophils, T-lymphocytes, neutrophils and Langerhans' cells to alter the release o f inflammatory mediators. This includes many o f the cytokines that promote inflammation as a result o f releasing histamine and attracting cells to infiltrate into affected areas. Corticosteroids also increase the  synthesis  o f lipocortin-1, which has  a potent  inhibitory effect  on  phospholipase A 2 . Corticosteroids produce pharmacological effects v i a an initial interaction with a selective cytosolic/nuclear receptor. This receptor, which is located near, or on, the surface o f the nuclear membrane  in the cytoplasmic space, recognizes the three  dimensional characteristics o f steroids. If the correct structure is present, the receptor w i l l bind. The receptor-drug complex in dimer form then translocates into the nucleus where it binds v i a a glucocorticoid responsive element to D N A , and with the involvement o f heat-shock proteins regulates the transcription o f specific genes to modify the synthesis  147  rate o f individual proteins. Additionally, when present as a monomer drug-receptor complex, the corticosteroids can also act to inhibit gene transcription o f inflammatory factors such as many o f the cytokines, adhesion molecules and various enzymes (Barnes 1998). Some investigators have suggested that the latter action o f corticosteroid-receptor monomers is the most important explanation o f their anti-inflammatory actions. Corticosteroids have been shown to be effective in alleviating chronic symptoms of allergic rhinitis. The role o f these agents in acute phase symptoms o f rhinitis has not been  studies  clearly. In this  study,  we  evaluated  the  effect  o f corticosteroid  dexamethasone on the acute allergic responses in sensitized guinea pigs. Pretreatment with intraperitoneal dexamethasone  significantly reduced sneezing frequency but not  nose rubbing frequency, evaluated immediately (for 30 minutues) after allergen challenge in sensitized guinea pigs. Intravenously administered dexamethasone failed to ameliorate nasal blockade induced by allergen challenge during the acute phase o f rhinitis. In addition, intravenously administered dexamethasone  had no apparent effect on the  cellular infiltration (no effect on total cell count and eosinophilia measured from nasal lavage fluid) during the acute phase ( l h r after allergen provocation) o f allergic rhinitis i n sensitized guinea pigs. In terms o f sneezing, this is the first study to show that, corticosteroids ameliorate sneezing in guinea pig model o f allergic rhinitis. However it has been demonstrated i n rat model o f rhinitis that corticosteroids reduce sneezing responses after allergen challenge (Sugimoto et al., 2000a). The mechanism by which dexamethasone reduces sneezing frequency is not well documented.  148  Corticosteroids reduce both nasal symptoms and histamine levels o f the early phase as well as the late phase reactions when the drug was applied topically in patients with allergic rhinitis (Pipkorn et al., 1987). In addition, it has been demonstrated that topical corticosteroids can reduce inflammation by inhibition o f mediators release from mast cells and basophils (Blackwell et al., 1980; Schleimer et al., 1981). Moreover, intranasal fluticasone propionate also reduced both histamine and tryptase in the mucosa of allergic patients (Meltzer et al., 1993). Furthermore, It has been postulated that dexamethasone  inhibits histamine H I receptor upregulation induced by allergen in  sensitized rats (Kitamura et al., 2004). Histamine is a main mediator that induces sneezing in allergic rhinitis through its action on H I receptors on nerve endings. Therefore, dexamethasone  reduces sneezing presumably by inhibiting H I  receptor  expression on nerve endings and decreasing histamine release from mast cells and eventually lowers sneezing frequency. The effect o f corticosteroids on nasal itching has not yet been studied in guinea pig model o f rhinitis. Our results show that dexamethasone has no effect on nose rubbing. Since dexamethasone inhibits H I receptor upregulation i n nasal mucosa and it fails to inhibit nose rubbing and mepyramine (refer to antihistamine section) failed to inhibit nose rubbing, it can be concluded that the mechanism o f nose rubbing in guinea pig is not simply involve H I receptor activation. Further studies are needed to explain the mechanisms o f corticosteroids in acute nasal responses (sneezing and nasal itching). Topical nasal corticosteroids are effective i n alleviating chronic nasal congestion in patients with allergic rhinitis (Craig et al., 2005; Trangsrud et al., 2002). In our study, intravenous  dexamethasone  failed to alleviate acute nasal congestion as well  149  as  eosinophil infiltration. In another study, it has been shown that dexamethasone has no inhibitory effect on nasal airway pressure elevation during the early phase but not during the late phase, i n a guinea pig model o f allergic rhinitis (Yamasaki et al., 2001). Corticosteroids inhibit upregulation o f cytokines and adhesion molecules, and suppress the actions o f immune cells during an allergic response (Barnes 1998). In chronic phase but not early phase o f allergic rhinitis, cytokines and adhesion molecules play major role in induction o f nasal congestion. This may explain why dexamethasone failed to inhibit the increase in nasal airway pressure and cellular infiltration in acute phase o f rhinitis. Furthermore, it has been demonstrated that allergen exposure may contribute to poor asthma  control  by  reducing  glucocotricoid receptor  ( G C R ) binding  affinity  in  mononuclear cells mediated through IL-2 and IL-4 (Nimmagadda et al., 1997). This may indicate that allergen exposure lowers the binding affinity o f G C R to corticosteroids and hence reduces their potency. Despite o f their beneficial prophylactic effect  in chronic allergic rhinitis,  glucocorticoids seems to play minimal role in acute phase responses.  150  4.2  Conclusions The method o f sensitization used in this study was effective. Sensitized guinea  pigs produced acute allergic responses after allergen provocation characterized  by  sneezing, nose rubbing, increase i n nasal airway pressure and eosinophil infiltration. In addition, no effect o f intranasal allergen challenge on lung inflation pressure was observed. Furthermore, sensitized guinea pigs produced anaphylactic shock immediately after administration o f 1 mg/kg ovalbumin intravenously. The response was characterized by rapid increase in blood pressure, heart rate and lung inflation pressure. Intravenous administration  o f histamine  and  acetylcholine  produced  dose  dependent decreases in nasal airway pressure and dose dependent increases in lung inflation pressure. W e found that histamine decreases nasal airway pressure by its action on histamine H I and muscarinic M 2 and perhaps M 5 receptors, whereas acetylcholine acts on muscarinic M l receptors to cause decrease i n nasal airway pressure. The increase in lung inflation pressure was due to the action o f histamine on histamine H I receptors, and acetylcholine on muscarinic M l receptors. Sneezing frequency  induced by allergen challenge was attenuated by pre-  treatment with mepyramine, cetirizine, and dexamethasone. 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