UBC Theses and Dissertations

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UBC Theses and Dissertations

Investigations into the development and mechanism of tolerance induced by the chronic antigen challenge… Andrew, Deborah Karen 1983

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INVESTIGATIONS INTO THE DEVELOPMENT AND MECHANISM OF TOLERANCE INDUCED BY THE CHRONIC ANTIGEN CHALLENGE OF S E N S I T I Z E D GUINEA PIGS by DEBORAH KAREN ANDREW B . S c , The U n i v e r s i t y o f B r i t i s h C o l u i m b i a , 1977 A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE S T U D I E S ( D e p a r t m e n t o f P a t h o l o g y ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA O c t . 1983 ( c ) D e b o r a h K a r e n Andrew, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f PATHOLOGY  The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 6. 1983 - i i -ABSTRACT P r e v i o u s s t u d i e s h a v e shown t h a t g u i n e a p i g s w h i c h h a v e b e e n s e n s i t i z e d t o o v a l b u m i n (OA) become u n r e a c t i v e t o OA a e r o s o l c h a l l e n g e a f t e r m u l t i p l e n o n l e t h a l e x p o s u r e s . The p r e s e n t s t u d i e s were u n d e r t a k e n t o d e f i n e t h e m e c h a n i s m ( s ) o f t h i s n o n r e a c t i v i t y , o r " t o l e r a n c e " . T o l e r a n c e was i n d u c e d i n p r e v i o u s l y s e n s i t i z e d g u i n e a p i g s by c h a l l e n g i n g w i t h OA a e r o s o l one h o u r / d a y , f i v e d a y s / w e e k f o r s i x weeks. R e a c t i v i t y was a s s e s s e d v i s u a l l y and by l u n g m e c h a n i c s . Maximum b r o n c h o c o n s t r i c t i v e r e s p o n s e s were o b s e r v e d i n t h e f i r s t two weeks o f a e r o s o l c h a l l e n g e , w i t h r e s p o n s e s d r o p p i n g o f f s t e a d i l y t h e r e a f t e r u n t i l v i r t u a l l y i n a p p a r e n t a f t e r f i v e weeks o f c h r o n i c ' c h a l l e n g e . L u n g m e c h a n i c s s t u d i e s p r o v i d e d an o b j e c t i v e means o f • a s s e s s i n g r e a c t i v i t y . D y n a m i c c o m p l i a n c e ( C d y n ) a n d p u l m o n a r y r e s i s t a n c e ( R L ) d a t a c o n f i r m e d i n i t i a l s t r o n g a n a p h y l a c t i c r e a c t i o n s i n t h e e a r l y weeks o f c h r o n i c c h a l l e n g e w h i c h s t e a d i l y d e c l i n e d o v e r t h e f o l l o w i n g f i v e weeks. To i n v e s t i g a t e t h e s t a b i l i t y o f t o l e r a n c e i n t h e a b s e n c e o f r e g u l a r a e r o s o l c h a l l e n g e , g r o u p s o f g u i n e a p i g s were w i t h h e l d f r o m c h a l l e n g e f o r 4, 6, 8, 10, and 24 day p e r i o d s . No p r o g r e s s i v e l o s s o f t o l e r a n c e o c c u r r e d when a e r o s o l c h a l l e n g e s were w i t h h e l d f o r 4 t o 24 day p e r i o d s . I n v i t r o c o n t r a c t i l e r e s p o n s e s o f a i r w a y s m o o t h m u s c l e f r o m s e n s i t i z e d a n d t o l e r a n t g u i n e a p i g s r e v e a l e d c o m p a r a b l e h i s t a m i n e r e s p o n s e s i n t h e two g r o u p s b u t a s i g n i f i c a n t l y - i i i -d e c r e a s e d r e s p o n s e t o OA f r o m t o l e r a n t a n i m a l s . T o t a l l u n g h i s t a m i n e c o n t e n t was s i g n i f i c a n t l y g r e a t e r i n t h e t o l e r a n t a n i m a l s ( 5 . 7 4 +_ 0.76 jug h i s t a m i n e / g wet l u n g ) t h a n i n c o n t r o l ( 3 . 0 5 +_ 0.33) o r s e n s i t i z e d a n i m a l s ( 3 . 3 0 +_ 0 . 3 8 ) . Serum f r o m t o l e r a n t a n i m a l s showed a r e d u c e d a r e a o f b l u e i n g c o m p a r e d w i t h s e r u m f r o m s e n s i t i z e d a n i m a l s by a s i x h o u r p a s s i v e c u t a n e o u s a n a p h y l a x i s s e n s i t i z a t i o n . L y m p h o c y t e s t r a n s f e r r e d f r o m t o l e r a n t g u i n e a p i g s t o s u b s e q u e n t l y s e n s i t i z e d a n i m a l s d i d n o t v i s i b l y r e d u c e an i n i t i a l r e p o n s e t o OA a e r o s o l c h a l l e n g e . A n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e f r o m c h o p p e d l u n g p r e p a r a t i o n s was s i g n i f i c a n t l y l e s s i n t o l e r a n t a n i m a l s ( 0 . 3 4 _+ 0.27 j /ug h i s t a m i n e r e l e a s e d / g t i s s u e , 1.0 _+ 0.7 % h i s t a m i n e r e l e a s e d ) t h a n s e n s i t i z e d a n i m a l s ( 1 . 2 _+ 0 . 3 j u g h i s t a m i n e r e l e a s e d / g t i s s u e , 5.1 +_ -9 1.4 % h i s t a m i n e r e l e a s e d ) a t a low ( 1 . 1 x 10 M) OA c o n c e n t r a t i o n . I t i s c o n c l u d e d t h a t a n t i g e n - i n d u c e d m e d i a t o r r e l e a s e o r a n t i b o d y s y n t h e s i s may be i n h i b i t e d i n t o l e r a n t a n i m a l s . - i v -TABLE OF CONTENTS Page T i t l e P a g e i A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s V L i s t o f F i g u r e s v i A c k n o w l e d g e m e n t s v i i L i s t o f A b b r e v i a t i o n s v i i i G l o s s a r y o f Terms X C h a p t e r 1. I n t r o d u c t i o n 1 C h a p t e r 2. M e t h o d s a n d M a t e r i a l s 28 C h a p t e r 3. D a t a A n a l y s i s 38 C h a p t e r 4. R e s u l t s 41 C h a p t e r 5. D i s c u s s i o n 66 R e f e r e n c e s 84 L I S T OF TABLES T a b l e 4.1. Lung M e c h a n i c s A s s e s s m e n t o f T o l e r a n c e D e v e l o p m e n t . T a b l e 4.2. L u n g M e c h a n i c s A s s e s s m e n t o f E f f e c t s o f L a y - O f f s on T o l e r a n c e . T a b l e 4.3 T o t a l L u n g P a r e n c h y m a l H i s t a m i n e . T a b l e 4.4 S i x H o u r PCA C o m p a r i s o n o f S e n s i t i z e d a nd T o l e r a n t S e r a . T a b l e 4.5 V i s u a l G r a d e A s s e s s m e n t o f L y m p h o c y t e T r a n s f e r E f f e c t on R e s p o n s e t o OA C h a l l e n g e . T a b l e 4.6 O v a l b u m i n - I n d u c e d H i s t a m i n e R e l e a s e f r o m C h o p p e d G u i n e a P i g L u n g . - v i -L I S T OF FIGURES Page F i g u r e 2.1. Summary o f E x p e r i m e n t a l D e s i g n . 27 F i g u r e 4.1. V i s u a l G r a d e A s s e s s m e n t o f t h e D e v e l o p m e n t 42 o f T o l e r a n c e . F i g u r e 4.2. L u n g M e c h a n i c s C d y n A s s e s s m e n t o f t h e 43 D e v e l o p m e n t o f T o l e r a n c e . F i g u r e 4.3. L u n g M e c h a n i c s RL A s s e s s m e n t o f t h e 44 D e v e l o p m e n t o f T o l e r a n c e . F i g u r e 4.4. V i s u a l G r a d e A s s e s s m e n t o f t h e E f f e c t o f 4 7 L a y - O f f s on T o l e r a n c e . F i g u r e 4.5. D o s e - R e s p o n s e T r a c i n g s a n d D r u g 4 9 P r o t o c o l f o r I n V i t r o S t u d y o f A i r w a y Smooth M u s c l e . F i g u r e 4.6. I n V i t r o T i s s u e R e s p o n s e s t o H i s t a m i n e 50 and C a r b a c h o l . F i g u r e 4.7. I n V i t r o T i s s u e R e s p o n s e s t o O v a l b u m i n . 51 F i g u r e 4.8. I n V i t r o T i s s u e H i s t a m i n e R e s p o n s e as a 52 P e r c e n t a g e o f Maximum H i s t a m i n e R e s p o n s e . F i g u r e 4.9. I n V i t r o T i s s u e O v a l b u m i n R e s p o n s e a s a 53 P e r c e n t a g e o f Maximum C a r b a c h o l R e s p o n s e . F i g u r e 4.10. I n V i t r o H i s t a m i n e G r o u p R e s p o n s e s a s a 55 P e r c e n t a g e o f Maximum T i s s u e R e s p o n s e s . F i g u r e 4.11. I n V i t r o O v a l b u m i n G r o u p R e s p o n s e s a s a 56 P e r c e n t a g e o f Maximum T i s s u e R e s p o n s e s . F i g u r e 4.12. A r e a s o f B l u e i n g I n d u c e d b y PCA C o m p a r i n g 59 S e n s i t i z e d a n d T o l e r a n t S e r a . F i g u r e 4.13. O v a l b u m i n - I n d u c e d P e r c e n t a g e H i s t a m i n e R e l e a s e 65 f r o m C h o p p e d G u i n e a P i g L u n g . F i g u r e 5.1. G u i n e a P i g R e s p o n s e s D i s t r i b u t i o n A s s e s s e d 6 7 by V i s u a l G r a d e . - v i i -ACKNOWLEDGEMENTS I w o u l d l i k e f i r s t a n d f o r e m o s t t o t h a n k my s u p e r v i s o r , D r . James C. Hogg f o r h i s u n e n d i n g s u p p o r t , e n c o u r a g e m e n t w i t h o u t p r e s s u r e , a nd g u i d a n c e a l o n g t h e ' f o c u s s e d ' p a t h . F o r t h e i r h e l p f u l s u g g e s t i o n s a n d comments I t h a n k my c o m m i t t e e members, D r . Hogg, D r . J . L e v y and D r . R. P e a r c e a n d members o f t h e P u l m o n a r y R e s e a r c h L a b , D r . P.D. P a r e a n d D r . R. R. S c h e l l e n b e r g . Many t h a n k s t o B a r r y W i g gs f o r t h e s t a t i s t i c a l a n a l y s e s a n d t o C a r i n P i h l f o r t h e p h o t o g r a p h y o f t h e f i g u r e s . A l s o , t h a n k s t o M r s . P a t B e r n o e a n d M r s . Anne B i s h o p f o r t h e i r p a t i e n c e and g u i d a n c e i n t h e u s e o f t h e Wang word p r o c e s s o r . And f i n a l l y , many t h a n k s t o my f a m i l y , f o r t h e i r c o n t i n u i n g e n c o u r a g e m e n t o f a l l my e n d e a v o u r s . I a l s o g r a t e f u l l y a c k n o w l e d g e t h e s u p p o r t o f a M e d i c a l R e s e a r c h C o u n c i l S t u d e n t s h i p t h r o u g h o u t t h i s work. L I S T OF ABBREVIATIONS A b — a n t i b o d y A C D — a c i d c i t r a t e d e x t r o s e A N O V A — a n a l y s i s o f v a r i a n c e B r — b r o n c h i B S A — b o v i n e s e r u m a l b u m i n C — c o n t r o l c A M P — c y c l i c a d e n o s i n e 3', 5'-monophosphate C d y n — d y n a m i c c o m p l i a n c e c G M P — c y c l i c g u a n o s i n e 3', 5'-monophosphate C F A — c o m p l e t e F r e u n d ' s a d j u v a n t C M I — c e l l - m e d i a t e d i m m u n i t y C o n A — C o n c a n a v a l i n A E C F - A — e o s i n o p h i l c h e m o t a c t i c f a c t o r o f a n a p h y l a x i s H — h e a v y c h a i n p o r t i o n o f I g I g — i m m u n o g l o b u l i n K L H — k e y h o l e l i m p e t h a e m o c y a n i n L — l i g h t c h a i n p o r t i o n o f I g L T - - l o w e r t r a c h e a M — m o l a r ( m o l e s / l i t r e ) M I F — m i g r a t i o n i n h i b i t i o n f a c t o r ( o f m a c r o p h a g e s ) m i n . — m i n u t e M L C — m i x e d l y m p h o c y t e c u l t u r e M. W t . — m o l e c u l a r w e i g h t O A — o v a l b u m i n P A F — p l a t e l e t a g g r e g a t i n g f a c t o r P a r — p a r e n c h y m a P B S — p h o s p h a t e b u f f e r e d s a l i n e P C A ~ p a s s i v e c u t a n e o u s a n a p h y l a x i s P G — p r o s t a g l a n d i n P L ~ t r a n s p u l m o n a r y p r e s s u r e R L — p u l m o n a r y r e s i s t a n c e S — s e n s i t i z e d s e c . — s e c o n d S F A — s u p p r e s s i v e f a c t o r o f a l l e r g y SRBC---sheep r e d b l o o d c e l l S R S - A — s l o w - r e a c t i n g s u b s t a n c e o f a n a p h y l a x i s T — t o l e r a n t T . V . — t i d a l v o l u m e U T — u p p e r t r a c h e a - X -GLOSSARY OF TERMS Antibody—a protein synthesized in response to antigen and which has the ability to bind to that antigen. Antigen—a substance which stimulates an immune response when present in an animal. B-cells—B lymphocytes, bone marrow-derived cells which are plasma c e l l precursors. Control—guinea pigs not sensitized, never challenged. Compliance—elasticity of the lung. Fc receptor—a receptor present on lymphocytes, eosinophils and mast cells which binds the Fc portion of immunoglobulins. Immunoglobulin—a glycoprotein consisting of 2 identical heavy (H) and 2 identical light (L) chains which functions as an antibody. Divided into classes A, D, E, G, and M. In vitro—outside the body. In vivo—within the body. Lay-offs—withholding of aerosol challenge. Pulmonary resistance—resistance of airways to airflow. Sensitized—guinea pigs immunized two weeks previously, never challenged. Suppressor T - c e l l s — a subset of T lymphocytes which inhibits other effector T-cell immune reactions and B-cell antibody synthesis. T- c e l l s — T lymphocytes, thymus-derived cell s , involved in cell-mediated immune reactions. Tolerant—guinea pigs immunized, and challenged until non-reactive. - 1 -CHAPTER 1. INTRODUCTION Asthma i s a c o n d i t i o n w h i c h a f f e c t s a p p r o x i m a t e l y 3 % o f t h e N o r t h A m e r i c a n p o p u l a t i o n ( 1 ) , b u t i s most p r e v a l e n t as a c h r o n i c c o n d i t i o n i n c h i l d r e n . C o m p l e t e u n d e r s t a n d i n g o f t h e c a u s e o r c a u s e s o f a s t h m a and t h e way i n w h i c h t h e s e a l t e r s t r u c t u r e and f u n c t i o n i s l a c k i n g , s o a s t r i c t e t i o l o g i c a l d e f i n i t i o n o f a s t h m a i s n o t p o s s i b l e . I n d i v i d u a l s h a v e d i f f e r e n t s t i m u l i w h i c h t r i g g e r t h e i r a s t h m a t i c e p i s o d e s . The s t i m u l u s o f a s t h m a may be an a l l e r g e n , e x e r c i s e , i n f e c t i o n , a c h e m i c a l compound, o r a p s y c h o g e n i c e v e n t . W h a t e v e r t h e i n i t i a t i o n , i t c u l m i n a t e s i n r e s p i r a t o r y d i s t r e s s o f v a r y i n g i n t e n s i t y . T h i s v a r i a b i l i t y i n d y s p n e a i s t h e b a s i s f o r t h e f u n c t i o n a l d e f i n i t i o n o f a s t h m a ( 2 ) . T h i s d i s o r d e r o f f u n c t i o n r e s u l t s f r o m v a r i a t i o n s i n r e s i s t a n c e t o g a s f l o w i n n a r r o w e d p e r i p h e r a l a i r w a y s . S p o n t a n e o u s v a r i a b i l i t y i n d y s p n e a w i t h i n t e r v a l s o f f r e e d o m f r o m d y s p n e a a n d s i g n i f i c a n t d e c r e a s e o f d y s p n e a b y b r o n c h o d i l a t o r s o r c o r t i c o s t e r o i d s a r e i m p o r t a n t f e a t u r e s i n d e f i n i n g a s t h m a . A n a t o m i c a l l y , t h e r e a r e c h a r a c t e r i s t i c p a t h o l o g i c c h a n g e s i n t h e a i r w a y s o f a s t h m a t i c s ( 3 , 4 ) . A g a i n , t h e s e a r e o f v a r y i n g d e g r e e s o f s e v e r i t y d e p e n d i n g upon t h e p a t i e n t . B r o n c h i a l p l u g s composed o f mucus, s e r o u s a n d c e l l u l a r e l e m e n t s a r e f o u n d . H y p e r p l a s i a a n d h y p e r t r o p h y o f s u b m u c o s a l g l a n d s and g o b l e t c e l l s r e s u l t s i n i n c r e a s e d mucus s e c r e t i o n . S u b m u c o s a l i n f l a m m a t i o n w i t h d i l a t e d c a p i l l a r i e s c o n t r i b u t e t o i n t e r s t i t i a l oedema and t h e s e r o u s component o f b r o n c h i a l p l u g s . E o s i n o p h i l i c i n f i l t r a t e s a r e commonly f o u n d i n a s t h m a , a s i s - 2 -sloughing of the mucosal layer. These bronchial epithelial cells and eosinophils comprise the cellular portion of the bronchial plugs. Hyperplasia and hypertrophy of bronchial smooth muscle and increased f i b r i l l a r collagen adjacent to the basement membrane are further morphologic abnormalities. Theories of the pathogenesis of asthma are numerous, and only those relevant to allergic asthma w i l l be discussed (3, 4, 5). Whatever the other contributing factors, i t i s now agreed that the primary event i s exposure of the individual to an antigen which i s processed by the immune system as an allergen. This induces the production of IgE antibodies which are specific for the allergen, and which bind to mast cells in the interstitium and airway lumen of the lungs. Upon subsequent exposure to the allergen, binding of the allergen to the mast cell-bound IgE causes degranulation of the mast c e l l with release of i t s chemical mediators. Some of these mediators are histamine, slow-reacting substance of anaphylaxis (SRS-A), eosinophil chemotactic factor (ECF-A), platelet aggregating factor (PAF) and prostaglandins (PG). The total effects of these mediators are not completely understood, but the immediate effects appear to be stimulation of i r r i t a n t receptors leading to rapid shallow breathing via a vagus nerve reflex (6). Increased epithelial permeability by loosening of tight junctions (7) allows the entry of antigen and mediators to the submucosal region, where greater numbers of mast cells are present. Interaction of antigen with IgE bound to i n t e r s t i t i a l mast cells provokes release of larger quantities of mediators. These - 3 -m e d i a t o r s c a n r e a d i l y i n t e r a c t w i t h s m o o t h m u s c l e c e l l s , n e r v e e n d i n g s a n d c a p i l l a r i e s , p r o d u c i n g d i r e c t a n d i n d i r e c t b r o n c h o c o n s t r i c t i o n a n d i n t e r s t i t i a l oedema and i n f l a m m a t i o n f r o m l e a k y c a p i l l a r i e s . V a r i o u s h y p o t h e s e s o f t h e p a t h o g e n e s i s o f a l l e r g i c a s t h m a h a v e b e e n i n v e s t i g a t e d i n s t u d i e s i n a n i m a l s and man. I t h a s b e e n f o u n d t h a t t h e a i r w a y s o f a s t h m a t i c s a r e e x t r e m e l y s e n s i t i v e t o p h y s i c a l , c h e m i c a l , and p h a r m a c o l o g i c s t i m u l i ( A , 8 ) . T h e a b n o r m a l i t y w h i c h g i v e s r i s e t o t h i s b r o n c h i a l h y p e r r e a c t i v i t y i s s t i l l u n c e r t a i n , a l t h o u g h s e v e r a l t h e o r i e s a r e b e i n g i n v e s t i g a t e d . T h e r e i s s u p p o r t f o r a number o f h y p o t h e s e s , b u t none o f them a l o n e a p p e a r s t o a c c o u n t f o r a l l c a s e s . S i n c e t h e s e p r o v o k i n g s t i m u l i a r e a n t i g e n - i n d e p e n d e n t a n o n - i m m u n o l o g i c m e c h a n i s m c a n a l s o be r e s p o n s i b l e f o r b r o n c h o -c o n s t r i c t i o n . Some p o s s i b l e c a u s e s o f b r o n c h i a l h y p e r r e a c t i v i t y a r e a b n o r m a l p a r a s y m p a t h e t i c , s y m p a t h e t i c o r n o n - a d r e n e r g i c i n n e r v a t i o n , i n c r e a s e d e p i t h e l i a l p e r m e a b i l i t y , i n c r e a s e d s m o o t h m u s c l e r e s p o n s i v e n e s s , o r c h r o n i c a l l y d e c r e a s e d b a s e l i n e a i r w a y c a l i b r e . The m a i n n e r v o u s c o n t r o l o f a i r w a y s m o o t h m u s c l e d e p e n d s upon a b a l a n c e b e t w e e n t h e b r o n c h o c o n s t r i c t i v e s y m p a t h e t i c ( a l p h a - a d r e n e r g i c ) a n d p a r a s y m p a t h e t i c v a g a l a c t i v i t y a n d t h e b r o n c h o d i l a t i v e s y m p a t h e t i c b e t a - a d r e n e r g i c and n o n - a d r e n e r g i c i n h i b i t o r y a c t i v i t i e s ( 9 ) . E v i d e n c e i s a v a i l a b l e t h a t v a g a l e f f e r e n t a c t i v i t y p r o d u c e s a m i l d d e g r e e o f r e s t i n g t o n e i n a i r w a y s m o o t h m u s c l e ( 8 ) . S t u d i e s i n d o g s ( 1 0 ) and g u i n e a p i g s (6) h a v e shown t h a t i n r e s p o n s e t o a n t i g e n o r h i s t a m i n e t h e v a g u s m e d i a t e s a r e f l e x b r o n c h o c o n s t r i c t i o n . E x p e r i m e n t s w i t h b o v i n e - 4 -l u n g s h a v e shown t h a t t r a c h e a l m u s c l e m u s c a r i n i c b i n d i n g s i t e s a r e 3 7 - f o l d g r e a t e r t h a n i n t h e l u n g p e r i p h e r y , w h i l e b e t a - r e c e p t o r s a r e 8 - f o l d g r e a t e r i n t h e p e r i p h e r y t h a n i n t h e t r a c h e a l m u s c l e ( 1 1 ) . V a g a l s t i m u l a t i o n c o u l d t h u s be e x p e c t e d t o p r o d u c e a m a r k e d r e d u c t i o n i n t h e t r a c h e a l a i r w a y d i a m e t e r , and a l t h o u g h t h i s h a s b e e n r e p o r t e d b y J a c k s o n e t a l ( 1 2 ) a p r e f e r e n t i a l d e c r e a s e i n t r a c h e a l c r o s s - s e c t i o n was n o t o b s e r v e d , w i t h d i s t a l a i r w a y c r o s s - s e c t i o n s a l s o b e i n g r e d u c e d . W o o l c o c k e t a l (13) h a v e r e p o r t e d d i f f e r e n c e s i n c e n t r a l a n d p e r i p h e r a l r e s i s t a n c e i n d o g s r e c e i v i n g v a g a l s t i m u l a t i o n . An a b s o l u t e i n c r e a s e i n c e n t r a l a n d p e r i p h e r a l r e s i s t a n c e u pon s t i m u l a t i o n o c c u r r e d i n a l l d o g s . However, i n d i v i d u a l d i f f e r e n c e s b e t w e e n d o g s were o b s e r v e d . I n some, t h e i n c r e a s e i n c e n t r a l r e s i s t a n c e was g r e a t e r t h a n t h e i n c r e a s e i n p e r i p h e r a l r e s i s t a n c e , and i n o t h e r s , t h e r e v e r s e was o b s e r v e d . C o n s i d e r a t i o n o f v a r i o u s p o s s i b i l i t i e s t o a c c o u n t f o r t h e phenomenon s u g g e s t e d t h a t t h e most l i k e l y e x p l a n a t i o n was t h a t t h e d i s t r i b u t i o n o f v a g a l f i b r e s a l o n g t h e t r a c h e o b r o n c h i a l t r e e v a r i e s among d o g s . I r r i t a n t r e c e p t o r s l y i n g i n t h e b r o n c h i a l e p i t h e l i u m a p p e a r s e n s i t i v e t o t h e s e v a g a l i n f l u e n c e s a n d o t h e r s t i m u l i ( 6 , 8, 1 0 ) . I t h a s b e e n s u g g e s t e d ( 1 4 ) t h a t i n c r e a s e d v a g a l n e r v o u s a c t i v i t y i s r e s p o n s i b l e f o r i n c r e a s e d a i r w a y s m o o t h m u s c l e t o n e i n t h e a s t h m a t i c s t a t e . O t h e r p o s s i b l e d e f e c t s i n a i r w a y s i n n e r v a t i o n a r e i n t h e b r o n c h o d i l a t i o n m e c h a n i s m s . Th e s y m p a t h e t i c b e t a - a d r e n e r g i c s y s t e m h a s b e e n o b s e r v e d i n a n i m a l s ( 8 , 15-19) a l t h o u g h no d e f i n i t e c o n t r i b u t i o n i n humans h a s b e e n e s t a b l i s h e d ( 8 , 2 0 ) . A s t u d y o f g u i n e a p i g - 5 -e x p e r i m e n t a l a s t h m a ( 1 6 ) h a s f o u n d t h a t t h e a l p h a - a d r e n e r g i c r e c e p t o r s a r e s i g n i f i c a n t l y i n c r e a s e d i n c h r o n i c a l l y a n t i g e n c h a l l e n g e d a n i m a l s . T h i s i s s u p p o r t i v e o f a t h e o r y p u t f o r w a r d by S z e n t i v a n y i ( 2 1 ) s u g g e s t i n g t h a t i n c r e a s e d a l p h a - a d r e n e r g i c a c t i v i t y c o m b i n e d w i t h p a r t i a l b e t a - a d r e n e r g i c b l o c k a d e i s a c a u s e o f some f o r m s o f a s t h m a . Human s t u d i e s ( 4 , 8) h a v e a l s o p r o v i d e d s u p p o r t f o r t h i s t h e o r y . A n o n - a d r e n e r g i c i n h i b i t o r y s y s t e m h a s b e e n w e l l d o c u m e n t e d i n a n i m a l s ( 8 , 22-24) a n d humans ( 8 , 18, 2 0 ) . V a s o a c t i v e I n t e s t i n a l P e p t i d e h a s b e e n c i t e d a s a p o s s i b l e t r a n s m i t t e r o f n o n - a d r e n e r g i c r e l a x a t i o n i n g u i n e a p i g a i r w a y s ( 2 4 ) , a n d t h i s n o n - a d r e n e r g i c i n h i b i t o r y i n n e r v a t i o n h a s b e e n f o u n d t o r u n i n t h e v a g i a n d r e c u r r e n t l a r y n g e a l n e r v e s ( 2 2 ) . I t h a s b e e n h y p o t h e s i z e d t h a t a d e f e c t i n t h i s n o n - a d r e n e r g i c i n h i b i t o r y s y s t e m w o u l d r e s u l t i n e x c e s s i v e b r o n c h o c o n s t r i c t i o n l e a d i n g t o b r o n c h i a l h y p e r r e a c t i v i t y ( 8 , 2 0 ) . A l t h o u g h e v i d e n c e h a s b e e n p r e s e n t e d f o r a b n o r m a l f u n c t i o n o f a u t o n o m i c r e g u l a t i o n i n b r o n c h i a l h y p e r r e a c t i v i t y , o t h e r f a c t o r s may be i n v o l v e d w h i c h a r e p r i m a r y o r p r o m o t i n g f o r t h i s d e f e c t . One o f t h e s e may be e p i t h e l i a l damage r e s u l t i n g i n i n c r e a s e d p e r m e a b i l i t y o f t h e m u c o s a l b a r r i e r . The i r r i t a n t r e c e p t o r s a n d n e r v e e n d i n g s p r e s e n t w i t h i n t h e e p i t h e l i u m w o u l d t h e n be e x p o s e d t o s t i m u l i w h i c h may p r o d u c e h y p e r r e a c t i v i t y e i t h e r t h r o u g h r e p e a t e d s t i m u l a t i o n o r t h r o u g h a " s e n s i t i z a t i o n " o f t h e n e r v e e n d i n g s ( 3 , 4, 8 ) . S t u d i e s h a v e shown t h a t when c h a l l e n g e d w i t h b r o n c h o c o n s t r i c t o r s s u b j e c t s w i t h u p p e r r e s p i r a t o r y t r a c t i n f e c t i o n s h a d i n c r e a s e d b r o n c h i a l r e a c t i v i t y when c o m p a r e d w i t h n o r m a l c o n t r o l s ( 2 5 ) . - 6 -A i r w a y e p i t h e l i a l damage i s an a b n o r m a l i t y d u r i n g r e s p i r a t o r y v i r a l i n f e c t i o n s , a n d r e s p i r a t o r y i n f e c t i o n i s one o f t h e s t i m u l i o f a s t h m a . T h a t e p i t h e l i a l damage i s p r e s e n t i n a s t h m a and may c o n t r i b u t e t o h y p e r r e a c t i v e a i r w a y s i s c o n c e i v a b l e i n v i e w o f t h e f a c t t h a t damaged mucosa i s a n o r m a l p a t h o l o g i c a l f i n d i n g i n a s t h m a ( 3 ) . E o s i n o p h i l i a i s a l s o a common f i n d i n g i n a s t h m a a n d a r e c e n t s t u d y by F r i g a s and o t h e r s ( 2 6 ) d e m o n s t r a t e s t h a t t h e m a j o r b a s i c p r o t e i n w h i c h i s a c o m p o n e n t o f e o s i n o p h i l s i s s e v e r e l y c y t o t o x i c t o b r o n c h i a l e p i t h e l i u m c a u s i n g c i l i a r y damage, oedema, and d i s r u p t i o n o f t h e e p i t h e l i u m . T h i s may be a m a j o r c o n t r i b u t o r t o t h e damage o b s e r v e d i n a s t h m a t i c b r o n c h i a l e p i t h e l i u m . F c r e c e p t o r s f o r I g E h a v e b e e n d e t e c t e d on human e o s i n o p h i l s , a n d t h e number o f r e c e p t o r s were f o u n d t o " i n c r e a s e when t h e e o s i n o p h i l s were p r e i n c u b a t e d w i t h ECF-A ( 2 7 ) . T h i s s i t u a t i o n may o c c u r i n an a l l e r g i c i n d i v i d u a l , b u t t h e i m p l i c a t i o n s o f e o s i n o p h i l I g E - a n t i g e n b i n d i n g a r e u n c l e a r . A n o t h e r c o n t r i b u t i o n t o a i r w a y e p i t h e l i a l damage may be made b y m a c r o p h a g e s . T h e y h a v e b e e n shown t o b i n d I g E a n d may p l a y a r o l e i n t h e p a t h o l o g y o f a s t h m a by i n d u c i n g t i s s u e damage ( 2 8 ) . The a n a p h y l a t o x i n s C 5 a a n d C 3 a a r e a l s o c a p a b l e o f i n d u c i n g c o n t r a c t i o n s o f l u n g a i r w a y s and p a r e n c h y m a , a n d may w e l l be p r e s e n t i n t h e a l l e r g i c r e a c t i o n ( 2 9 ) . A n o t h e r c o n t r i b u t i o n t o b r o n c h i a l h y p e r r e a c t i v i t y may be made by a l t e r a t i o n s i n t h e s m o o t h m u s c l e . I t i s w e l l e s t a b l i s h e d t h a t t h e q u a n t i t y o f s m o o t h m u s c l e i s i n c r e a s e d i n a s t h m a , and an i n c r e a s e d amount o f m u s c l e i s l i k e l y t o c a u s e a l a r g e r n a r r o w i n g o f a i r w a y s ( 8 ) . H owever, t h i s m e c h a n i s m i s u n l i k e l y t o be t o t a l l y r e s p o n s i b l e f o r - 7 -b r o n c h i a l h y p e r r e a c t i v i t y s i n c e t h e mass o f a i r w a y s m o o t h m u s c l e i n humans d u r i n g r e s p i r a t o r y i n f e c t i o n s o r e x p o s u r e t o c h e m i c a l s t i m u l i w o u l d n o t be i n c r e a s e d . A n o t h e r c h a n g e i n t h e s m o o t h m u s c l e may be a c h a n g e i n i t s i n t r i n s i c c o n t r a c t i l e p r o p e r t i e s i n r e s p o n s e t o i o n s o r a g o n i s t s . T h e m e c h a n i s m o f t h i s may r e s i d e i n t h e s e n s i t i v i t y o f s m o o t h m u s c l e t o c a l c i u m i o n s . M a r t o r a n a ( 3 0 ) h a s shown t h a t g u i n e a p i g s s e n s i t i z e d t o o v a l b u m i n h a v e c a l c i u m i o n - b i n d i n g c h a r a c t e r i s t i c s w h i c h a r e m a r k e d l y d i f f e r e n t f r o m n o r m a l . The a i r w a y h y p e r r e a c t i v i t y s e e n p o s t - a n a p h y l a c t i c a l l y may t h e n be due t o an i n c r e a s e d s e n s i t i v i t y o f t h e s m o o t h m u s c l e c e l l s t o e x t r a c e l l u l a r c a l c i u m i o n s ( 3 1 ) . T r a c h e a l s m o o t h m u s c l e p r e p a r a t i o n s f r o m o v a l b u m i n - a l l e r g i c d o g s h a v e b e e n shown t o be h y p e r s e n s i t i v e a n d h y p e r r e a c t i v e i n r e s p o n s e t o h i s t a m i n e when c o m p a r e d w i t h l i t t e r m a t e c o n t r o l s ( 3 2 ) . I n c o n t r a s t t o t h i s s t u d y on d o g s , S o u h r a d a d i d n o t f i n d t h a t t h e r e a c t i v i t y o f t r a c h e a l s m o o t h m u s c l e f r o m e x p e r i m e n t a l a s t h m a t i c g u i n e a p i g s was s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f c o n t r o l t r a c h e a l p r e p a r a t i o n s ( 3 3 ) . However, S o u h r a d a h a s d e m o n s t r a t e d w i t h s e n s i t i z e d g u i n e a p i g s m o o t h m u s c l e i n v i t r o t h a t s p o n t a n e o u s m e c h a n i c a l a c t i v i t y i s i n c r e a s e d ( 3 4 ) and s u g g e s t s t h a t t h i s i s due t o an i n c r e a s e d r e s t i n g membrane p o t e n t i a l o f t h e s m o o t h m u s c l e c e l l s ( 3 5 ) . The e x t e n t o f t h e c o n t r i b u t i o n o f a l t e r e d a i r w a y s m o o t h m u s c l e p r o p e r t i e s on b r o n c h i a l h y p e r r e a c t i v i t y r e m a i n s u n c e r t a i n . The p r e s e n c e o f m u c o s a l oedema, s m o o t h m u s c l e h y p e r p l a s i a a n d h y p e r t r o p h y , and m u c o s a l f o l d i n g may a l l c o n t r i b u t e t o a n a r r o w i n g o f t h e b a s e l i n e a i r w a y c a l i b r e . A i r w a y f l o w r e s i s t a n c e i s a common i n - 8 -v i v o p a r a m e t e r f o r m e a s u r i n g b r o n c h o c o n s t r i c t i o n , a n d r e s i s t a n c e i s i n v e r s e l y p r o p o r t i o n a l t o t h e f o u r t h , power o f t h e r a d i u s when a i r w a y f l o w i s l a m i n a r . T h e r e f o r e , when a l r e a d y n a r r o w a i r w a y s a r e f u r t h e r d e c r e a s e d b y b r o n c h o c o n s t r i c t i o n t h e r e i s a g r e a t e r c h a n g e i n a i r w a y r e s i s t a n c e t h a n t h e r e w o u l d be i f w i d e r a i r w a y s c o n t r a c t e d t h e same amount. B e n s o n ( 3 6 ) h a s s u g g e s t e d t h a t n a r r o w i n g o f a i r w a y s i s r e s p o n s i b l e f o r b r o n c h i a l h y p e r r e a c t i v i t y . However, b r o n c h i a l h y p e r r e a c t i v i t y i s o b s e r v e d e v e n when n a r r o w i n g o f t h e b a s e l i n e a i r w a y c a l i b r e i s a b s e n t ( 7 ) . A l t h o u g h i t may c o n t r i b u t e t o h y p e r r e a c t i v i t y when i t i s p r e s e n t , i t i s c l e a r l y n o t a p r i m a r y m e c h a n i s m . I m p a i r e d m u c o c i l i a r y f u n c t i o n i s a l s o i n v o l v e d i n a s t h m a . Some c h a r a c t e r i s t i c s o f t h i s h a v e a l r e a d y b e e n d e s c r i b e d , s u c h a s h y p e r t r o p h y a nd h y p e r p l a s i a o f s u b m u c o s a l g l a n d s and g o b l e t c e l l s a n d i n c r e a s e d mucus i n t h e a i r w a y s . T h e r e a r e a number o f f a c t o r s w h i c h may c o n t r i b u t e t o a b n o r m a l h a n d l i n g o f r e s p i r a t o r y s e c r e t i o n s . One o f t h e s e i s t h e a f o r e m e n t i o n e d f a c t t h a t t h e e p i t h e l i a l l a y e r i s o f t e n damaged. C i l i a w h i c h a r e a b s e n t o r h a v e u n c o o r d i n a t e d o r r e d u c e d b e a t i n g p a t t e r n s w o u l d be l e s s a b l e t o c l e a r mucus f r o m t h e a i r w a y s . T h e r e i s a l s o e v i d e n c e t h a t t h e mucus i n a s t h m a t i c s i s b i o c h e m i c a l l y a b n o r m a l ( 3 7 ) . I n c r e a s e d c o n c e n t r a t i o n s o f u n u s u a l p o l y s a c c h a r i d e s , s e r u m p r o t e i n s w h i c h c r o s s b i n d , a nd s e c r e t o r y IgA h a v e b e e n f o u n d i n t h e s p u t u m o f a s t h m a t i c p a t i e n t s . I n c r e a s e d c a l c i u m i o n s a n d s e r u m p r o t e i n s h a v e a l s o b e e n r e p o r t e d i n t h e s o l p h a s e o f s p u t u m . The g r e a t e r v i s c o s i t y o f s p u t u m w h i c h h a s b e e n o b s e r v e d i n a s t h m a t i c s may w e l l be due t o i t s a l t e r e d c o m p o s i t i o n , e s p e c i a l l y t h e i n c r e a s e d - 9 -glycoproteins. It has been, found that when viscosity is increased, the rate of transport on cil i a t e d epithelia is decreased. Increased mucus volume and viscosity, and decreased transport provide the conditions for mucus stasis. The fibrin content of mucus increases, and appears as lamina when mucus stagnates in the airways. This too, increases the viscosity of the secretions. Secretion from submucosal glands is regulated at least in part by parasympathetic and sympathetic innervation (38). In vitro studies with cat, dog, ferret, and human trachea have shown that vagus nerve stimulation and alpha-adrenergic agonists stimulate secretion from submucosal glands. If increased activity of the vagus nerve and the alpha-adrenergic system is a part of the pathogenesis of asthma, this may account for the increased mucus secretion. When the c i l i a r y function is impaired, this excess mucus cannot be cleared, and mucus plugs form in the narrowed airways. Although the studies investigating airway hyperreactivity and mucociliary dysfunction have contributed much to our knowledge of these components of the pathology of bronchial asthma, there i s s t i l l uncertainty about the basic mechanism(s) of asthma. Additional experiments have been performed using animal models to investigate the contributions of the immune system, functions of cells and mediators, and the pulmonary pathophysiology of the disease. Some general animal models of the asthmatic state have been described by Patterson and Kelly (39). As previously stated, the handling of an antigen by the immune system to produce an antibody which w i l l bind to mast cells is an - 10 -essential part of allergic asthma. Whether or not the production of these antibodies is the main pathogenesis of asthma is uncertain. The components of the immune system which are thought to be involved in the production of reaginic antibodies w i l l now be discussed. The f i r s t consideration is the route by which the antigen i s introduced into the body. Entry via the mucosal surfaces of the respiratory tract or the intestinal tract w i l l allow "sensitization" to the antigen to occur. Evidence has been derived from studies in guinea pigs and mice that indicate that alveolar and peritoneal macrophages are involved in antigen processing and presentation to T-lymphocytes (40-44). Antigen entering via the respiratory tract may be processed by alveolar macrophages, or i t may be processed by gut-associated lymphoid tissue. A study by Willoughby (45) has shown that rabbits exposed to BSA aerosol for 90 min. have 72 % of the antigen in the stomach immediately after the aerosol exposure. Within the intestinal tract there are two routes by which antigens may be absorbed. One is via the epithelium overlying the lymph nodules, and the other i s via the columnar epithelium covering the v i l l i (46). Horseradish peroxidase placed in the lumen of a mouse intestine can be observed in vesicles passing through the "membranous" epithelial cells which separate lymphocytes from gut lumen. Later the horseradish peroxidase is detected in the space between the epithelial cells and lymphocytes, and finally within lymphocytic vesicles. Antigen absorption across columnar epithelium may also occur. Mice and rats given radio-labelled proteins intragastrically have been shown to absorb varying amounts of - l i -the protein in i t s native state. Distribution of antigens to lymphoid tissue throughout the body may then occur. Interactions between macrophages, suppressor and helper T-cells and B-cells are required for the ultimate formation of antibodies. Some evidence is available which suggests that a deficiency of suppressor T-cells allows hyperproduction of IgE, and a resulting atopic state (47). The production of antibodies is the result of an intricate shuffling and joining of segments of DNA and RNA within developing B lymphocytes (48, 49). An antibody (Ab) molecule is composed of two identical heavy (H) and two identical light (L) polypeptide chains, each consisting of sequences of polypeptides producing regions termed "variable" and "constant". There are two types of L chains, kappa (K) and lambda (A), and five classes of H chains, alpha (<*), epsilon (&), delta (o), gamma (if) and mu (A). Within each type of L or H chain the constant region of the polypeptide varies l i t t l e from Ab to Ab. However, the variable regions vary greatly, and confer 6 8 antigen-specificity upon the Ab. In the range of 10-10 unique antibodies may be produced by an animal. The usual practice of one gene giving rise to one molecule does not operate in this situation. Rather, heterogeneity is produced by the random selection of one variable sequence from among a large number of possible genes. Although the exact number is not yet known, there is probably in the range of one hundred to two hundred variable gene sequences for H chain synthesis and one hundred to two hundred variable gene sequences for L chain synthesis. During development of a B-cell, the 'chosen' variable - 12 -gene i s l i n k e d t o t h e one c o n s t a n t gene p r e s e n t i n t h e genome. F u r t h e r v a r i a b i l i t y i s i n t r o d u c e d by t h e c h o i c e o f o n e o f s e v e r a l J g e n e s , a s e g m e n t w h i c h j o i n s t h e v a r i a b l e and c o n s t a n t r e g i o n s i n L and H c h a i n s . H eavy c h a i n s h a v e a n o t h e r c o m p o n e n t o f v a r i a b i l i t y , t h e D o r d i v e r s i t y g e n e s , w h i c h p r o d u c e one o f t h e t h r e e h y p e r v a r i a b l e r e g i o n s o f t h e h e a v y c h a i n . I n a d d i t i o n t o t h e c h o i c e o f d i f f e r e n t v a r i a b l e , J and D g e n e s , r e c o m b i n a t i o n i s f l e x i b l e o v e r a r a n g e o f s e v e r a l n u c l e o t i d e s f o r j o i n i n g v a r i a b l e a n d D s e g m e n t s a n d D a n d J s e g m e n t s . F u r t h e r v a r i a b i l i t y i s a v a i l a b l e f r o m H and L c h a i n p a i r i n g . A n o t h e r m e c h a n i s m f o r p r o m o t i n g Ab d i v e r s i t y i s t h a t o f s i n g l e n u c l e o t i d e m u t a t i o n s d u r i n g t h e c o u r s e o f B l y m p h o c y t e m a t u r a t i o n . The a r r a n g e m e n t o f g e n e s a l o n g t h e DNA m o l e c u l e i s s u c h t h a t t h e v a r i a b l e g e n e s a r e f o l l o w e d by t h e D, J and c o n s t a n t g e n e s . The g e n e s w h i c h c o d e f o r t h e c o n s t a n t r e g i o n s o f e a c h o f t h e c l a s s e s o f h e a v y c h a i n s a r e a r r a n g e d i n t h e o r d e r mu, d e l t a , gamma, e p s i l o n and a l p h a a l o n g t h e DNA m o l e c u l e . H e n c e , when t r a n s c r i p t i o n o c c u r s , mu a n d d e l t a a r e t h e f i r s t g e n e s t r a n s c r i b e d and t r a n s l a t e d . C o m b i n a t i o n w i t h s u b s e q u e n t l y s y n t h e s i z e d l i g h t c h a i n m o l e c u l e s f o r m s c o m p l e t e IgM a n d IgD m o l e c u l e s . The i n i t i a l t r a n s c r i p t i o n o f t h e s e g e n e s i n c l u d e s i n f o r m a t i o n w h i c h p r o v i d e s a h y d r o p h o b i c e n d t o t h e s e m o l e c u l e s , t h u s e n s u r i n g t h a t t h e f i r s t IgM and IgD w i l l be membrane-bound. By t h i s s t a g e , t h e n , r e a r r a n g e m e n t a n d h y p e r m u t a t i o n o f m u l t i p l e g e r m l i n e g e n e s i n t h e d e v e l o p i n g B l y m p h o c y t e h a v e g i v e n r i s e t o a B l y m p h o c y t e w i t h membrane-bound IgM a n d IgD, b o t h h a v i n g t h e same v a r i a b l e r e g i o n s a n d s o t h e r e f o r e s p e c i f i c f o r t h e same a n t i g e n . - 13 -Upon b i n d i n g o f t h e s p e c i f i c a n t i g e n , t h e B l y m p h o c y t e i s s t i m u l a t e d t o c o n t i n u e i t s d e v e l o p m e n t t o a f u l l y A b - p r o d u c i n g p l a s m a c e l l . T h e membrane-bound Ab a r e l o s t , and IgM, IgG, I g E o r IgA may b e s y n t h e s i z e d a n d s e c r e t e d . T h i s c o n s t i t u t e s a 'heavy c h a i n c l a s s s w i t c h ' and may be a c c o m p l i s h e d by s e v e r a l m e c h a n i s m s . One o f t h e s e i s r e a r r a n g e m e n t o f t h e DNA s e q u e n c e s by means o f a s i m i l a r ' s w i t c h i n g s i g n a l ' b e t w e e n t h e c o n s t a n t g e n e s and t h e v a r i a b l e r e g i o n . T h e t r a n s c r i b e d RNA i s t h e n s p l i c e d t o make a mRNA e n c o d i n g a gamma, e p s i l o n , o r a l p h a r e g i o n . A n o t h e r p o s s i b i l i t y i s t h a t t h e r e a r e c l a s s - s p e c i f i c ' s w i t c h i n g p r o t e i n s ' (50) w h i c h r e c o g n i z e t h e s p e c i f i c ' s w i t c h i n g s i g n a l ' a r e a s and b i n d t o them, t h e r e b y j u x t a p o s i n g t h e a p p r o p r i a t e r e g i o n s a n d a l l o w i n g DNA r e c o m b i n a t i o n . F r o m t h i s s e r i e s o f e v e n t s e a c h c l a s s o f Ab may be s y n t h e s i z e d by i t s own c l o n e o f p l a s m a c e l l s w h i c h d e v e l o p s f r o m t h e i n i t i a l B l y m p h o c y t e . IgM and IgG w h i c h c i r c u l a t e i n b l o o d p l a s m a , IgA w h i c h a s s o c i a t e s w i t h s u r f a c e - l i n i n g c e l l s , a n d I g E , w h i c h b i n d s t o a l l e r g e n i c c e l l s may a l l be p r o d u c e d , u n d o u b t e d l y s u b j e c t t o c o n t r o l by o t h e r m o d i f y i n g f a c t o r s . Humans h a v e b e e n f o u n d t o s y n t h e s i z e a s h o r t - t e r m s e n s i t i z i n g a n t i b o d y ( I g G S - T S ) i n a d d i t i o n t o t h e commonly d e s c r i b e d I g E ( 5 1 ) . Numerous a n i m a l m o d e l s h a v e b e e n u s e d t o s t u d y t h e e f f e c t s o f s e n s i t i z a t i o n on a n t i b o d y l e v e l s . G u i n e a p i g s , a commonly u s e d a n i m a l m o d e l , h a v e b e e n shown t o s y n t h e s i z e I g G j g , a r a p i d l y s e n s i t i z i n g ( 4 - 6 h o u r s ) , s h o r t - l i v e d ( 3 - 8 d a y s ) , h e a t - s t a b l e a n t i b o d y ( 5 2 , 5 3 ) . O t h e r s t u d i e s h a v e d e t e c t e d t h e p r e s e n c e o f I g E i n a d d i t i o n t o IgG, - 14 -(54-57). IgE is heat-labile, has a long sensitizing time (48 hours or longer), and persists at least 2-4 weeks in the skin. Rats sensitized with egg albumin and Bordetella pertussis (58, 59) have been shown to produce IgE specific for egg albumin. The assessment of antibody levels is most often done by passive cutaneous anaphylaxis (PCA) experiments (52-58, 60, 61). This involves injecting serum from a sensitized animal intracutaneously into the back of a non-sensitized animal. Antigen and an indicator dye are later injected intravenously. At the sites of serum injection a reaction i s seen by a patch of dye, the size of which is proportional to the amount of antibody in the serum from the sensitized animal. In vitro precipitin tests are another means of determining antibody levels (53, 60, 61). IgE can also be measured by a radioimmunosorbent method. This allows measurement of nanogram levels of IgE and enables detection of small differences in total versus specific IgE antibody levels (53, 62). Much information is now available on the biochemical changes which precede degranulation of mast cell s . Degranulation is commonly assessed via histamine release. The f i r s t requirement of degranulation is for cross-linking of the IgE receptors in the c e l l membrane (63-71). These receptors are composed of a 50,000 M. Wt. glycoprotein alpha-component and a 30,000 M. Wt. beta-component in a 1:1 ratio (72, 73). Divalent antigen performs this role in vivo, but anti-IgE antibody, chemically dimerized IgE, anti-receptor antibody, or Concanavalin A (cross-links IgE by binding to Fc region) have been successsfully used in vitro. Cross-linking of the IgE receptors leads - 15 -t o a c t i v a t i o n o f a m e t h y l t r a n s f e r a s e a n d a d e n y l a t e c y c l a s e . An e a r l y r i s e i n cAMP i s p r o d u c e d w h i c h i n t u r n a c t i v a t e s two p r o t e i n k i n a s e i s o e n z y m e s ( 6 9 ) t h e f u n c t i o n o f w h i c h i s u n c e r t a i n . O t h e r b i o c h e m i c a l s t e p s i n c l u d e d e c a r b o x y l a t i o n o f p h o s p h a t i d y l s e r i n e t o f o r m p h o s p h a t i d y l e t h a n o l a m i n e w h i c h i s m e t h y l a t e d b y a m a g n e s i u m - d e p e n d e n t m e t h y l t r a n s f e r a s e 1 t o p r o d u c e an N-monomethyl d e r i v a t i v e . P h o s p h a t i d y l c h o l i n e i s t h e n p r o d u c e d by t h e a c t i o n o f a s e c o n d m e t h y l t r a n s f e r a s e on p h o s p h a t i d y l e t h a n o l a m i n e . P h o s p h a t i d y l c h o l i n e i s a s u b s t r a t e f o r membrane p h o s p h o l i p a s e A. T h e r e s u l t i n g l y s o p h o s p h a t i d y l c h o l i n e a p p e a r s r e s p o n s i b l e f o r o p e n i n g c h a n n e l s f o r c a l c i u m i n f l u x . I n c r e a s e d i n t r a c e l l u l a r c a l c i u m i s a r e q u i r e m e n t f o r d e g r a n u l a t i o n . The p r o d u c t i o n o f ATP i s a l s o a r e q u i r e m e n t a s i s an a d e q u a t e e n d o g e n o u s l e v e l o f a s c o r b i c a c i d ( 7 4 ) . The r e l a t i o n s h i p b e t w e e n h i s t a m i n e r e l e a s e a n d cAMP, cGMP, a n d P G E 2 l e v e l s h a s b e e n s t u d i e d by a number o f i n v e s t i g a t o r s . C o n c l u s i v e a n s w e r s a r e n o t y e t a v a i l a b l e . T he o b s e r v a t i o n was made t h a t d u r i n g t h e a n a p h y l a c t i c r e l e a s e o f h i s t a m i n e i n t r a c e l l u l a r cAMP l e v e l s f e l l , w h i l e cGMP l e v e l s r o s e , p r o d u c i n g a d e c r e a s e i n t h e cAMP/cGMP r a t i o . The e f f e c t s o f t h e s e two c y c l i c n u c l e o t i d e s t h u s a p p e a r s t o be o p p o s i t e , a n d i s p a r t o f t h e " Y i n - Y a n g " t h e o r y p r o p o s e d b y G o l d b e r g ( 7 5 ) . I t h a s b e e n t h e o r i z e d t h a t a f a l l i n i n t r a c e l l u l a r cAMP i s an i n i t i a t i n g e v e n t f o r h i s t a m i n e r e l e a s e , s i n c e cAMP l e v e l s d e c r e a s e d u r i n g h i s t a m i n e r e l e a s e , a n d m a i n t e n a n c e o f h i g h cAMP p r e v e n t s t h e r e l e a s e o f h i s t a m i n e ( 6 3 , 64) by p r e v e n t i n g t h e o p e n i n g o f c a l c i u m c h a n n e l s . The i n a c t i v a t i o n o f c a l c i u m c h a n n e l s may t h e n o c c u r as cAMP l e v e l s i n c r e a s e . However, i t - 16 -h a s r e c e n t l y b e e n r e p o r t e d t h a t a s h o r t - l i v e d i n c r e a s e i n cAMP p r e c e d e s t h e f a l l i n cAMP ( 6 4 , 6 9 , 7 6 ) . A s t u d y u s i n g a n t i g e n - c h a l l e n g e d g u i n e a p i g l u n g s l i c e s , m i n c e d l u n g , and p e r f u s e d l u n g h a s shown t h a t t h e cAMP a n d cGMP l e v e l s c h a n g e i n p a r a l l e l ( 7 6 ) . B o t h n u c l e o t i d e s e x h i b i t e d an i n i t i a l i n c r e a s e , f o l l o w e d by a d e c r e a s e , t h e n a g r e a t e r and more p r o l o n g e d i n c r e a s e . The b a s i c e v e n t , h o w e v e r , d o e s n o t seem t o b e c h a n g e s i n n u c l e o t i d e l e v e l s . R a t s t u d i e s by Okabe and o t h e r s ( 7 7 ) h a v e shown t h a t a f a l l i n PGEg p r e c e d e s t h e d e c r e a s e i n cAMP/cGMP r a t i o . I n f u s i o n o f P G E 2 e l e v a t e d t h e cAMP l e v e l , t h e cAMP/cGMP r a t i o , a nd i n h i b i t e d t h e a n t i g e n - i n d u c e d e l e v a t i o n o f p l a s m a h i s t a m i n e . The f i n d i n g t h a t p r o s t a g l a n d i n (PG) i n f u s i o n p r e v e n t s r e l e a s e o f h i s t a m i n e f r o m t h e l u n g h a s b e e n r e p o r t e d by o t h e r s ( 7 8 , 7 9 ) . W h a t e v e r t h e p r e c i s e b i o c h e m i c a l s e q u e n c e o f e v e n t s w h i c h r e s u l t i n d e g r a n u l a t i o n , a c t i v a t i o n o f t h e c y t o s k e l e t a l e l e m e n t s o f t h e c e l l ( 7 1 ) , p o s s i b l y v i a c o n t r a c t i o n o f m i c r o f i l a m e n t s ( 6 3 ) , i s a n e c e s s a r y s t e p i n g r a n u l e r e l e a s e . A l t h o u g h h i s t a m i n e h a s b e e n most o f t e n c i t e d a s t h e m e d i a t o r r e l e a s e d by mast c e l l d e g r a n u l a t i o n , i t i s by no means t h e o n l y o n e p r e s e n t . A v a r i e t y o f m e d i a t o r s , some s t o r e d p r e - f o r m e d , a n d some s y n t h e s i z e d de n o v o i n r e s p o n s e t o s t i m u l a t i o n a r e r e l e a s e d u p o n d e g r a n u l a t i o n . F u n c t i o n s o f t h e s e m e d i a t o r s r a n g e f r o m b r o n c h o s p a s t i c and v a s o a c t i v e a c t i v i t y t o c h e m o t a c t i c , e n z y m a t i c , and s t r u c t u r a l a c t i v i t i e s ( 7 1 ) . H i s t a m i n e i s s t o r e d p r e - f o r m e d a n d when r e l e a s e d a c t s r a p i d l y u p on H^ r e c e p t o r s i n s m o o t h m u s c l e o f l a r g e a n d s m a l l a i r w a y s t o g i v e - 17 -a short-lasting bronchoconstriction. Stimulation of i r r i t a n t receptors causes a reflex neural bronchoconstriction (71, 80). Partial disconnection of vascular endothelium produces vasodilation and increased vascular permeability leading to oedema and inflammation. The receptor on which histamine acts has been reported as (71), H 2 (80), and H-^  and H 2 (81). The action of histamine at an receptor produces an increase in cellular cGMP, while action at an H 2 receptor produces increased cAMP. This latter action decreases IgE-mediated histamine release (71) and mediates relaxation (82-85). Histamine has also been shown to relax trachea by stimulation of adrenergic receptors (63). Prostaglandin increases are observed after histamine stimulation, but i t is unknown whether this is a direct action of histamine, or a reflex from bronchoconstriction (71, 80). Histamine causes a release of catecholamines by direct action on the adrenal medulla (80, 86-88). The epinephrine released influences the recovery from histamine, apparently by the beta-effects of the epinephrine (87). In addition, non-adrenal adrenergic factors have been shown to modify responses to histamine (68). The effect of histamine is short-lived. It appears within 30 sec. of antigen challenge, and has reached i t s maximum within 2 min. (80). The inactivation of histamine occurs by oxidative deamination from histaminase, or methylation plus oxidative deamination from histaminase plus histamine N-methyltransferase (89). Slow-reacting substance of anaphylaxis (SRS-A), now frequently referred to as the leukotrienes C and D have been assigned a major role - 18 -i n g u i n e a p i g l u n g a n a p h y l a x i s by s t u d i e s e m p l o y i n g t h e SRS-A r e c e p t o r a n t a g o n i s t , FPL 55712 ( 9 0 - 9 2 ) . Upon s t i m u l a t i o n o f t h e m a s t c e l l , SRS-A i s s y n t h e s i z e d de n o v o . I t a p p e a r s w i t h i n 30 s e c . o f a n t i g e n c h a l l e n g e , b u t o n l y a t a l o w c o n c e n t r a t i o n . R e l e a s e o f SRS-A c a n s t i l l be d e t e c t e d 30 m i n . a f t e r c h a l l e n g e ( 7 1 , 80) and i t s r e l e a s e may be i n h i b i t e d by l e u k o t r i e n e ( 9 3 ) . The t y p e o f b r o n c h o c o n s t r i c t i o n i t i n d u c e s i s s l o w , l o n g - l a s t i n g , and c o n t r i b u t e s t o t h e l a t e r , p r o l o n g e d a i r w a y a n t i g e n r e s p o n s e (94, 95). V a s o d i l a t i o n i s a l s o o b s e r v e d w i t h SRS-A s t i m u l a t i o n ( 7 1 ) , a s w e l l a s p r o s t a g l a n d i n r e l e a s e ( 7 1 , 8 0 ) . SRS-A i s a c i d l a b i l e , a l k a l i n e s t a b l e , a n d s t a b l e t o h e a t i n g a t 6 0 ° C f o r 60 m i n . ( 9 6 ) . I t i s f o r m e d f r o m a r a c h i d o n i c a c i d v i a t h e l i p o x y g e n a s e p a t h w a y (97, 98). The c o m p l e t e s t r u c t u r e o f t h e SRS-A r e l e a s e d f r o m an i m m u n o l o g i c a l l y c h a l l e n g e d g u i n e a p i g h a s b e e n d e t e r m i n e d . I t i s an u n s a t u r a t e d s u l p h u r - c o n t a i n i n g l i p i d o f 3 80-400 M. Wt. ( 9 6 , 97) w h i c h a p p e a r s t o h a v e t h e same p r o p e r t i e s f r o m man, g u i n e a p i g , a n d r a t ( 9 9 ) . The s m o o t h m u s c l e c o n t r a c t i o n s i n d u c e d by SRS-A r e q u i r e an e x t r a c e l l u l a r s o u r c e o f c a l c i u m , and a p p e a r t o h a v e a s i m i l a r c o n t r a c t i o n m e c h a n i s m t o t h a t o f h i s t a m i n e a n d a c e t y l c h o l i n e , a l t h o u g h s p e c i f i c SRS-A r e c e p t o r s a r e p r e s e n t on t h e s m o o t h m u s c l e ( 1 0 0 ) . I n g u i n e a p i g s t h e a c t i o n o f SRS-A on p e r i p h e r a l t i s s u e s h a s b e e n f o u n d t o be s i g n i f i c a n t l y g r e a t e r t h a n t h e e f f e c t on c e n t r a l a i r w a y s ( 7 1 , 80, 1 0 1 ) , b u t i n humans c e n t r a l a n d p e r i p h e r a l a i r w a y s a p p e a r t o r e s p o n d t o SRS-A t o t h e same d e g r e e ( 1 0 2 ) . SRS-A d o e s n ' t h a v e a p o w e r f u l d i r e c t c o n t r a c t i l e e f f e c t o n t h e l u n g , b u t may i n d i r e c t l y p r o d u c e b r o n c h o c o n s t r i c t i o n . A l t h o u g h SRS-A i s most o f t e n - 19 -c o n s i d e r e d i n t e r m s o f i t s r e l e a s e f r o m a n t i g e n - s t i m u l a t e d mast c e l l s , o t h e r c e l l s a p p e a r t o p r o d u c e SRS-A, a n d may b e r e q u i r e d f o r m a s t c e l l SRS-A p r o d u c t i o n ( 7 1 , 103, 1 0 4 ) . F u r t h e r m o r e , SRS-A may b e r e l e a s e d b y n o n - i m m u n o l o g i c a l s t i m u l i f r o m o t h e r t i s s u e s , s u g g e s t i n g a b r o a d e r p h y s i o l o g i c a l r o l e t h a n j u s t a n a p h y l a x i s ( 7 7 ) . T h e i n a c t i v a t i o n o f SRS-A i s a c c o m p l i s h e d b y a r y l s u l p h a t a s e s A a n d B w h i c h a r e s t o r e d p r e - f o r m e d i n mast c e l l s ( 7 1 ) . P r o s t a g l a n d i n s , a p r o d u c t o f a r a c h i d o n i c a c i d o x i d a t i o n ( 7 1 ) a r e a l s o p r o d u c e d upon a n a p h y l a x i s . PGE2 a n d P G F 2 o < a r e t h e m a j o r PG's w h i c h a r e p r o d u c e d i n t h e l u n g ( 8 0 , 105, 1 0 6 ) . T h e s e PG's h a v e o p p o s i n g a c t i o n s , a n d a r e p r o d u c e d p r e f e r e n t i a l l y i n d i f f e r e n t p a r t s o f t h e l u n g . PGE^ a n d i t s 1 5 - k e t o m e t a b o l i t e r e l a x e s b r o n c h i a l s m o o t h m u s c l e and i s a v a s o d i l a t o r i n a v a r i e t y o f v a s c u l a r b e d s ( 8 0 , 1 0 7 ) . P G E 2 i s p r o d u c e d p r e f e r e n t i a l l y i n t h e a i r w a y s i n humans, a n d i s a l s o p r o d u c e d by g u i n e a p i g a i r w a y s . B e t a - a d r e n o c e p t o r a c t i v a t i o n h a s b e e n shown t o i n d u c e P G E 2 g e n e r a t i o n i n g u i n e a p i g t r a c h e a ( 1 0 8 ) . P G F 2 o < and i t s 1 5 - k e t o m e t a b o l i t e c o n t r a c t b r o n c h i a l s m o o t h m u s c l e a n d a r e p r o d u c e d p r i m a r i l y by p a r e n c h y m a ( 8 0 , 105, 1 0 6 ) . T h e s t i m u l u s , s o u r c e a n d f u n c t i o n o f PG p r o d u c t i o n i s a s y e t u n c e r t a i n . The r e l e a s e o f PG's i s u n r e l a t e d t o t h e a n t i g e n d o s e , a nd i t h a s b e e n v a r i o u s l y h y p o t h e s i z e d t h a t h i s t a m i n e ( H ^ s t i m u l a t i o n ) , b r o n c h o c o n s t r i c t i o n , o r t i s s u e damage a r e t h e i n i t i a t i n g f a c t o r s f o r PG p r o d u c t i o n ( 8 0 , 105, 1 0 6 ) . P a r e n c h y m a l p r o d u c t i o n o f P G F 2 o t d o e s n o t a p p e a r t o b e d e p e n d e n t u p o n h i s t a m i n e o r b r o n c h o c o n s t r i c t i v e s t i m u l i ( 1 0 5 ) . T h e m a j o r s o u r c e o f PGF^ i n p a r e n c h y m a m a y n o t b e t h e m a s t c e l l > b u t - 20 -p e r h a p s a n o t h e r c e l l t y p e more common i n p a r e n c h y m a t h a n i n b r o n c h i ( 1 0 5 ) . P r o s t a g l a n d i n s do n o t a p p e a r t o make a s i g n i f i c a n t d i r e c t c o n t r i b u t i o n t o t h e b r o n c h o c o n s t r i c t i o n a s p e c t o f a n a p h y l a x i s , a l t h o u g h i t h a s b e e n s u g g e s t e d t h a t t h e m e t a b o l i t e s o f P C s may s e n s i t i z e b r o n c h i a l s m o o t h m u s c l e t o t h e a c t i o n o f o t h e r m e d i a t o r s o f a n a p h y l a x i s ( 8 0 ) . A l t e r n a t i v e l y , P C s may be i n v o l v e d i n a f e e d - b a c k m e c h a n i s m w h e r e b y h i s t a m i n e , SRS-A and k i n i n s s t i m u l a t e PG r e l e a s e , t h e n P C s i n h i b i t f u r t h e r m e d i a t o r r e l e a s e . PGE a n d P G F 2 o c h a v e b e e n shown t o i n c r e a s e cAMP l e v e l s i n r a t p e r i t o n e a l mast c e l l s a nd human l u n g f r a g m e n t s . V i a t h e i r d i f f e r e n t i a l r e l e a s e , P G E 2 a n d P G F 2 o < may m a i n t a i n v e n t i l a t i o n / p e r f u s i o n r a t i o s i n t h e l u n g ( 8 0 ) . The m e t a b o l i z a t i o n o f p r o s t a g l a n d i n s r e l e a s e d d u r i n g a n a p h y l a x i s o c c u r s b e f o r e t h e P C s r e a c h t h e p u l m o n a r y c i r c u l a t i o n . T h e r a t e - l i m i t i n g enzyme i s 15-OH PG d e h y d r o g e n a s e ( 8 0 ) . K i n i n o g e n l e v e l s h a v e b e e n o b s e r v e d t o d e c r e a s e d u r i n g a n a p h y l a x i s , t h u s s u g g e s t i n g t h a t b r a d y k i n i n i s r e l e a s e d . I n d u c t i o n o f t a c h y p h y l a x i s t o b r a d y k i n i n p r o d u c e s a d e c r e a s e d b r o n c h o c o n s t r i c t i o n , t h u s p r o v i d i n g f u r t h e r e v i d e n c e f o r t h e r e l e a s e o f b r a d y k i n i n d u r i n g a n a p h y l a x i s . T h e s m a l l e r a i r w a y s a p p e a r t o be s e l e c t i v e l y n a r r o w e d by b r a d y k i n i n , c a u s i n g a d e c r e a s e i n l u n g c o m p l i a n c e ( 8 0 ) . A n o t h e r e f f e c t i s v a s o d i l a t i o n , w h i c h r e s u l t s i n a d r o p i n t h e s y s t e m i c b l o o d p r e s s u r e . However, t h e r e l e a s e o f c a t e c h o l a m i n e s r a p i d l y r e v e r s e s t h e i n i t i a l h y p o t e n s i o n . A g a i n , k i n i n s may be r e q u i r e d f o r t h e r e l e a s e o f p r o s t a g l a n d i n s o r o t h e r m e d i a t o r s . P e p t i d a s e s i n t h e b l o o d m e t a b o l i z e t h e k i n i n s , p r o b a b l y m o s t l y i n t h e p u l m o n a r y c i r c u l a t i o n ( 8 0 ) . - 21 -A mediator chemotactic for eosinophils, ECF-A, is stored pre-formed in mast cells and is released dose-dependently in a time course similar to that of SRS-A. Its release is modulated by cAMP and cGMP in the same way as histamine and SRS-A. The 400 M. Wt., acidic dipeptide structure attracts eosinophils, and deactivates them to further migration. Eosinophils contain arylsulphatase, which inactivates SRS-A. This suggests the presence of a feed-back mechanism to limit the effect of SRS-A (71, 80). Another mast c e l l mediator which acts on another c e l l type is platelet aggregating factor (PAF). The l i p i d - l i k e PAF has been shown to promote relaxation of guinea pig trachea (109) as well as inducing the release of serotonin from platelets. This monoamine is stored pre-formed in platelets and has been shown to contract smooth muscle in some non-human systems. Serotonin also increases vascular permeability. Heparin, a structural proteoglycan, is stored pre-formed in mast c e l l s . It functions as an anti-coagulant by interacting with human anti-thrombin III. A variety of enzymes are also present in mast cel l s , some of which appear to diminish the effects of other anaphylactic mediators (71). Chymase, stored pre-formed, is a protease with alpha-chymotrypsin-like activity, and has been shown to inhibit IgE-dependent inflammation in rabbit skin. Arylsulphatases A and B hydrolyze SRS-A and are released from pre-formed stores. Although there are other mediators and factors present in mast cell s , those whose functions are best understood have been described here. The summation of the actions of the mediators of anaphylaxis give rise to - 22 -c h a r a c t e r i s t i c c h a n g e s i n t h e p u l m o n a r y p h y s i o l o g y . T h e s e w i l l be d i s c u s s e d n e x t . I n v e s t i g a t i o n o f t h e e f f e c t s on p u l m o n a r y p h y s i o l o g y d u r i n g a n a p h y l a x i s a r e o f t e n s t u d i e d w i t h l u n g m e c h a n i c s . The p a r a m e t e r s most commonly r e p o r t e d a r e p u l m o n a r y r e s i s t a n c e (RL) a n d d y n a m i c c o m p l i a n c e ( C d y n ) . RL i s a m e a s u r e o f t h e r e s i s t a n c e o f a i r w a y s t o a i r f l o w , a n d i s m o s t l y d e r i v e d f r o m l a r g e a i r w a y s b r o n c h o c o n s t r i c t i o n . Cdyn i s a m e a s u r e o f t h e e l a s t i c i t y o f t h e l u n g , and i n d i c a t e s p e r i p h e r a l a i r w a y i n v o l v e m e n t . Numerous i n v e s t i g a t o r s h a v e f o u n d t h a t d u r i n g a n a p h y l a x i s RL i n c r e a s e s and C d yn d e c r e a s e s ( 1 1 0 - 1 1 5 ) . The i n i t i a l e x t e r n a l l y v i s i b l e r e s p o n s e t o a n t i g e n c h a l l e n g e i s an i n c r e a s e d f r e q u e n c y o f b r e a t h i n g w i t h a d e c r e a s e d t i d a l v o l u m e ( 1 1 4 ) . From t h i s p h a s e , l a b o u r e d b r e a t h i n g w i t h m a r k ed r e t r a c t i o n o f t h e t h o r a c i c w a l l f o l l o w s ( 1 1 1 ) . T h e b r o n c h o c o n s t r i c t i o n p r o d u c e s o t h e r c h a n g e s i n l u n g f u n c t i o n p a r a m e t e r s . The f u n c t i o n a l r e s i d u a l c a p a c i t y a n d r e s i d u a l v o l u m e b o t h i n c r e a s e due t o a i r t r a p p i n g w i t h i n t h e l u n g s ( 1 1 1 ) . A n o t h e r s t u d y o f p u l m o n a r y m e c h a n i c s i n g u i n e a p i g s was d e s i g n e d t o a l l o w c o m p a r i s o n w i t h t h e m e a s u r e m e n t s r o u t i n e l y u s e d i n s t u d i e s o f human a s t h m a . Upon e x p o s u r e t o a n t i g e n , s e n s i t i z e d g u i n e a p i g s e x h i b i t e d a d e c r e a s e i n t h e p e ak e x p i r a t o r y f l o w r a t e , t h e m a x i m a l m i d - e x p i r a t o r y f l o w r a t e , t h e f o r c e d v i t a l c a p a c i t y , a n d t h e f o r c e d e x p i r a t o r y v o l u m e i n 0.1 s e c . T h i s i s s i m i l a r t o t h e a c u t e r e s p o n s e i n a s t h m a t i c humans e x c e p t t h a t t h e f o r c e d e x p i r a t o r y v o l u m e o v e r 1.0 s e c . i s a s s e s s e d i n humans, s i n c e humans h a v e a l o n g e r d u r a t i o n o f e x p i r a t i o n . F u r t h e r m o r e , t h e f o r c e d e x p i r a t o r y v o l u m e i n 0.1 s e c . a s m e a s u r e d by t h i s g r o u p was n o t - 23 -p r e c e d e d b y a m a x i m a l i n s p i r a t i o n t o t o t a l l u n g c a p a c i t y a s i s t h e u s u a l m ethod ( 1 1 6 ) . An e x p e r i m e n t i n d o g s h a s shown t h a t d u r i n g a n a p h y l a x i s t h e r e i s a d e c r e a s e i n t h e a r t e r i a l p 0 2 , pH, a n d h e a r t r a t e ( 1 1 3 ) . I t c a n be s e e n , t h e n , t h a t t h e m e d i a t o r s o f a n a p h y l a x i s h a v e e x t e n s i v e d e t r i m e n t a l i n f l u e n c e s on t h e e f f i c i e n c y o f g a s e x c h a n g e i n t h e l u n g s and t h a t many s t u d i e s h a v e b e e n u n d e r t a k e n t o e x p a n d o u r u n d e r s t a n d i n g o f t h e p a t h o g e n e s i s o f a s t h m a . A v a r i e t y o f a n i m a l m o d e l s h a v e b e e n d e s c r i b e d . Among t h e s e a r e d o g s ( 1 0 , 1 1 7 ) , r a b b i t s ( 4 5 , 6 1 ) , r a t s ( 5 8 , 1 1 8 ) , m i c e ( 1 1 9 - 1 2 1 ) , monkeys ( 3 9 , 122, 123) and c a l v e s ( 1 2 4 ) . The most commonly u s e d a n i m a l i n s t u d i e s o f a n a p h y l a x i s , h o w e v e r , i s t h e g u i n e a p i g ( 3 4 , 92, 111, 115, 116, 1 2 5 ) . T h e r e a r e a number o f s i m i l a r i t i e s b e t w e e n g u i n e a p i g s a n d humans w h i c h h a s made them t h e a n i m a l o f c h o i c e . The f i r s t i s t h a t t h e l u n g i s t h e m a j o r " s h o c k " o r g a n a n d s o u r c e o f m e d i a t o r s d u r i n g a n a p h y l a x i s i n b o t h humans a n d g u i n e a p i g s ( 8 0 ) . F u r t h e r m o r e , t h e m e c h a n i c a l p r o p e r t i e s o f t h e l u n g s i n g u i n e a p i g e x p e r i m e n t a l a s t h m a a r e s i m i l a r t o t h e m e c h a n i c s i n human b r o n c h i a l a s t h m a , b o t h s h o w i n g a d e c r e a s e i n Cdyn a nd an i n c r e a s e i n RL d u r i n g a n a p h y l a x i s ( 8 1 ) . I n a n o t h e r s t u d y i n g u i n e a p i g s u n d e r g o i n g a n a p h y l a x i s t h e p u l m o n a r y m e c h a n i c s were e v a l u a t e d i n s p o n t a n e o u s l y b r e a t h i n g , a n a e s t h e t i z e d g u i n e a p i g s f o r c e d b y e x t e r n a l p r e s s u r e t o p e r f o r m a n i n s p i r a t o r y / e x p i r a t o r y m a n e u v r e . T h i s a l l o w s c o m p a r i s o n o f r e s u l t s w i t h t h o s e f r o m human s t u d i e s . The r e s p i r a t o r y r a t e i n c r e a s e d , t h e peak e x p i r a t o r y f l o w r a t e a n d maximum m i d - e x p i r a t o r y f l o w r a t e f e l l 20 %, f o r c e d v i t a l c a p a c i t y f e l l 50 %, f o r c e d e x p i r a t o r y v o l u m e i n 0.1 s e c f e l l 40 %, a n d - 24 -t h e u s e o f a m i n o p h y l l i n e r e v e r s e d t h e s e r e s p o n s e s . T h e s e a r e s i m i l a r t o r e s u l t s f r o m a c u t e a s t h m a t i c humans ( 9 0 ) . The p r e d o m i n a n t a n t i b o d y m e d i a t o r o f a n a p h y l a x i s i n humans i s I g E , w i t h some a p p e a r a n c e o f an IgG ( 5 1 ) , w h i l e i n g u i n e a p i g s t h e r e a r e I g G ^ a n d I g E - l i k e a n t i b o d i e s i n v o l v e d ( 5 7 ) . The SRS-A p r o d u c e d by a n t i g e n c h a l l e n g e o f p a s s i v e l y s e n s i t i z e d human l u n g a n d a c t i v e l y s e n s i t i z e d g u i n e a p i g l u n g w e r e f o u n d t o be p h a r m a c o l o g i c a l l y , c h e m i c a l l y , and c h r o m a t o g r a p h i c a l l y i n d i s t i n g u i s h a b l e ( 1 0 4 ) . The r e s p o n s e o f b o t h t i s s u e t y p e s t o a n t i g e n - i n d u c e d m e d i a t o r s i s an i m m e d i a t e , s h o r t - l i v e d c o n t r a c t i o n , f o l l o w e d b y a s u s t a i n e d c o n t r a c t i o n . A n t a g o n i s m o f t h i s a i r w a y s m o o t h m u s c l e c o n t r a c t i o n w i t h d i p h e n h y d r a m i n e , a n a n t a g o n i s t a n d FPL 55712, an SRS-A a n t a g o n i s t i n h i b i t e d t h e i n i t i a l f a s t c o n t r a c t i o n and s u b s e q u e n t s l o w c o n t r a c t i o n i n b o t h g u i n e a p i g s a n d humans ( 9 9 ) . O t h e r m e d i a t o r s a r e known t o be r e l e a s e d upon a n t i g e n s t i m u l a t i o n , s u c h a s p r o s t a g l a n d i n s . B o t h human a n d g u i n e a p i g p e r i p h e r a l l u n g t i s s u e s r e s p o n d t o H-^  s t i m u l a t i o n b y p r o d u c i n g P G F 2 o < and PGE. H 2 s t i m u l a t i o n , a n d s t i m u l a t i o n o f p a r e n c h y m a l t i s s u e w i t h KC1 o r c a r b a c h o l d i d n o t p r o v o k e PG g e n e r a t i o n . S t i m u l a t i o n o f a i r w a y s m o o t h m u s c l e c o n t r a c t i o n w i t h h i s t a m i n e , KC1, and c a r b a c h o l c a u s e d t h e g e n e r a t i o n o f PGE i n humans, a n d PGE a n d p G F 2 o ( i n g u i n e a p i g s ( 1 0 6 ) . So, w h i l e t h e r e a r e some d i f f e r e n c e s a p p a r e n t i n t h e g u i n e a p i g and human s y s t e m s , t h e b a s i c s i m i l a r i t i e s c o u p l e d w i t h t h e e a s e o f s e n s i t i z a t i o n a n d e a s e o f h a n d l i n g g u i n e a p i g s h a s made them a p r a c t i c a l a n d p o p u l a r a n i m a l m o d e l f o r t h e s t u d y o f human a l l e r g i c a s t h m a . - 25 -Many a p p r o a c h e s h a v e b e e n u s e d by d i f f e r e n t i n v e s t i g a t o r s t o p r o d u c e an a n a p h y l a c t i c a l l y r e s p o n s i v e g u i n e a p i g , w i t h v a r i a t i o n s i n t h e r e g i m e s o f a n t i g e n i m m u n i z a t i o n , c h a l l e n g e , and s e n s i t i v i t y a s s e s s m e n t . N a r e s d u s t i n g w i t h r a g w e e d p o l l e n o r a d i s t i l l a t e o f r a g w e e d p o l l e n h a s b e e n u s e d t o s e n s i t i z e g u i n e a p i g s ( 1 2 6 ) , a s h a v e i n t r a m u s c u l a r i n j e c t i o n s o f 5 % (w/v) o v a l b u m i n ( 1 1 4 , 1 2 7 ) . The most common s e n s i t i z a t i o n p r o c e d u r e i s an i n t r a p e r i t o n e a l i n j e c t i o n o f a n t i g e n , m o s t o f t e n o v a l b u m i n , w i t h o r w i t h o u t a n a d j u v a n t ( 5 7 , 110, 111, 128, 1 2 9 ) . P a s s i v e s e n s i t i z a t i o n w i t h h o m o l o g o u s a n t i b o d y i s a l s o a s u c c e s s f u l method ( 1 1 5 ) . C h a l l e n g e v i a an a n t i g e n a e r o s o l i s common ( 1 1 0 - 1 1 2 , 114-116, 126-129) a l t h o u g h i n t r a v e n o u s o r s u b c u t a n e o u s c h a l l e n g e s may a l s o be u s e d ( 5 7 ) . The a s s e s s m e n t o f t h e l e v e l o f a n a p h y l a c t i c r e s p o n s e t o a n t i g e n e x p o s u r e h a s b e e n m e a s u r e d as t h e l e n g t h o f t i m e b e f o r e o n s e t o f d y s p n e a ( m i c r o s h o c k t i m e ) ( 1 2 6 - 1 2 8 ) c h a n g e s i n C d yn and RL ( 5 7 , 110-112, 114, 1 1 5 ) , PCA r e a c t i o n s ( 5 7 , 126, 130) a n d i n v i t r o r e s p o n s e s o f t i s s u e t o a n t i g e n a n d d r u g s ( 1 2 9 ) . S t u d i e s w h i c h h a v e i n v o l v e d r e p e a t e d c h a l l e n g e s o f g u i n e a p i g s o v e r a p e r i o d o f t i m e h a v e shown t h a t t h e h i g h l e v e l o f a n a p h y l a c t i c r e a c t i v i t y o b s e r v e d on i n i t i a l a n t i g e n e x p o s u r e s d i m i n i s h e s w i t h t h e c o n t i n u a t i o n o f a n t i g e n e x p o s u r e ( 3 4 , 111, 115, 1 2 6 -128, 1 3 0 ) . T h i s phenomenon h a s b e e n e x p l o r e d by a number o f i n v e s t i g a t o r s , b u t no s a t i s f a c t o r y e x p l a n a t i o n o f t h i s a p p a r e n t i n d u c t i o n o f t o l e r a n c e i n t h e g u i n e a p i g h a s b e e n f o u n d . The s t u d y o f t h i s p r o p e r t y o f t o l e r a n c e d e v e l o p m e n t i s o f i n t e r e s t b e c a u s e o f a p o s s i b l e a p p l i c a t i o n t o t h e human s i t u a t i o n . - 26 -A l t h o u g h t h e c h a r a c t e r i s t i c s o f human a s t h m a h a v e b e e n w e l l d e s c r i b e d , t h e p a t h o g e n e s i s i s u n c e r t a i n . T h e s e o b s e r v a t i o n s o f g u i n e a p i g t o l e r a n c e i n d u c t i o n l e a d t o t h e h y p o t h e s i s t h a t human a s t h m a a r i s e s due t o a f a i l u r e o f t h e m e c h a n i s m w h i c h o p e r a t e s i n g u i n e a p i g s w h i c h become t o l e r a n t . The a p p r o a c h t o t h i s p r o b l e m was t o u s e g u i n e a p i g s s e n s i t i z e d w i t h o v a l b u m i n , w h i c h i s an i n e x p e n s i v e , e f f e c t i v e a n t i g e n and h e a t - k i l l e d B o r d e t e l l a p e r t u s s i s a s an a d j u v a n t s i n c e i t h a s b e e n shown i n r a t s t o p r e f e r e n t i a l l y s t i m u l a t e t h e s y n t h e s i s o f r e a g i n i c a n t i b o d i e s ( 1 2 0 , 131, 1 3 2 ) , p e r h a p s b y e n h a n c i n g t h e I g E - b i n d i n g a b i l i t y o f l y m p h o c y t e s ( 1 3 3 ) . The g u i n e a p i g s r e c e i v e d d a i l y a e r o s o l c h a l l e n g e s t o m i m i c t h e s i t u a t i o n o f c h r o n i c human e x p o s u r e . E x p e r i m e n t s were t h e n c o n d u c t e d t o c o m p a r e t h e r e s p o n s e s o f a n i m a l s no l o n g e r r e s p o n s i v e i n v i v o t o a e r o s o l c h a l l e n g e — t h e " t o l e r a n t " g u i n e a p i g s , w i t h t h o s e o f s e n s i t i z e d , r e s p o n s i v e g u i n e a p i g s . A summary o f t h e e x p e r i m e n t a l d e s i g n ( F i g . 2.1) i s g i v e n t o o r i e n t t h e r e a d e r t o t h e o v e r a l l p l a n a nd s e q u e n c e o f s t u d i e s . F i g u r e 2.1. Summary o f E x p e r i m e n t a l D e s i g n - 27 -1 mg/ml OA a e r o s o l R e a c t i v i t y A s s e s s m e n t : a ) V i s u a l G r a d e b ) L u n g M e c h a n i c s 1 h r / d a y 5 d a y s / w e e k 6 weeks A e r o s o l C h a l l e n g e L a y - O f f s : A, 6, 8, 10, 24 d a y s i n t r a p e r i t o n e a l I ml o f 1 mg/ml OA & 1 m l o f B. p e r t u s s i s I n V i t r o Smooth M u s c l e S t u d y T o t a l H i s t a m i n e A s s a y P a s s i v e C u t a n e o u s A n a p h y l a x i s L y m p h o c y t e s t r a n s f e r r e d f r o m T o l e r a n t a n d C o n t r o l a n i m a l s t o s u b s e q u e n t l y s e n s i t i z e d a n i m a l s A n t i g e n - I n d u c e d H i s t a m i n e R e l e a s e f r o m C h o p p e d L u n g - 28 -CHAPTER 2. METHODS AND MATERIALS P a r t 2.1: D e v e l o p m e n t a n d A s s e s s m e n t o f T o l e r a n c e 2.1.1) S e n s i t i z a t i o n T h i r t y 250 g m a l e a n d f e m a l e H a r t l e y s t r a i n g u i n e a p i g s ( o u t b r e d a l b i n o s ) were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 1 mg o v a l b u m i n (OA) ( 4 5 , 0 0 0 M. Wt.) ( G r a d e I I I , S i g m a C h e m i c a l C o . ) i n 1 ml s a l i n e a n d 1 ml h e a t - k i l l e d B o r d e t e l l a p e r t u s s i s v a c c i n e (HKP) ( C o n n a u g h t L a b o r a t o r y ) . 2.1.2) C h a l l e n g e P r o c e d u r e Two weeks a f t e r s e n s i t i z a t i o n t h e a n i m a l s were p l a c e d i n a P l e x i g l a s s a e r o s o l i z a t i o n chamber and e x p o s e d t o a s o l u t i o n o f 2.2 x 10" 5 M OA i n s a l i n e n e b u l i z e d a t a f l o w r a t e o f 5 1 c o m p r e s s e d a i r / m i n . f r o m a H u d s o n d i s p o s a b l e n e b u l i z e r . T h e a e r o s o l c h a l l e n g e was p e r f o r m e d f o r one h o u r e a c h d a y , f i v e d a y s / w e e k , f o r s i x w e e k s . 2.1.3) A s s e s s m e n t o f R e a c t i v i t y 2.1.3.1) V i s u a l G r a d e W i t h e v e r y e x p o s u r e t o t h e a e r o s o l t h e b r o n c h o c o n s t r i c t i v e r e s p o n s e o f e a c h g u i n e a p i g was g r a d e d . T h i s was b a s e d on a v i s u a l - 29 -a s s e s s m e n t o f t h e r a t e a n d d e p t h o f b r e a t h i n g and t h e d e g r e e o f c y a n o s i s . When no r e s p o n s e was a p p a r e n t a g r a d e o f z e r o was a s s i g n e d . R a p i d s h a l l o w b r e a t h i n g r e c e i v e d a g r a d e o f o n e , s l o w l a b o u r e d b r e a t h s w i t h c y a n o s i s r e c e i v e d two, a n d a s e v e r e b r o n c h o c o n s t r i c t i v e r e s p o n s e l e a d i n g t o c o l l a p s e and r e s c u e o f t h e a n i m a l r e c e i v e d a g r a d e o f t h r e e . 2.1.3.2) L u n g M e c h a n i c s A s s e s s m e n t D u r i n g e a c h o f t h e s i x weeks o f c h r o n i c a e r o s o l e x p o s u r e , s i x t o e i g h t a n i m a l s f r o m t h e g r o u p o f 30 g u i n e a p i g s were r a n d o m l y c h o s e n f o r l u n g m e c h a n i c s m e a s u r e m e n t s . T h e s e a n i m a l s were n o t c h a l l e n g e d i n t h e a e r o s o l i z a t i o n chamber p r i o r t o t h e s e s t u d i e s , b u t were a n a e s t h e t i z e d w i t h 20 mg/kg o f s o d i u m p e n t o b a r b i t a l i n t r a p e r i t o n e a l l y a n d p l a c e d p r o n e i n a p r e s s u r e s e n s i t i v e g u i n e a p i g body p l e t h y s m o g r a p h . A p r e s s u r e t r a n s d u c e r ( V a l i d y n e MPA5 + 2 cm H 2 0 ) d e t e c t e d c h a n g e s i n box p r e s s u r e and y i e l d e d a v o l u m e s i g n a l ( T . V . ) . A s a l i n e - f i l l e d e s o p h a g e a l c a t h e t e r w i t h m u l t i p l e s i d e h o l e s m e a s u r e d p l e u r a l p r e s s u r e , and a d i f f e r e n t i a l p r e s s u r e t r a n s d u c e r ( H e w l e t t P a c k a r d 267BC) w h i c h c o m p a r e s p l e u r a l a n d a t m o s p h e r i c p r e s s u r e p r o d u c e d a t r a n s p u l m o n a r y p r e s s u r e r e a d i n g ( P L ) . The t i d a l v o l u m e and t r a n s p u l m o n a r y p r e s s u r e w ere r e c o r d e d by a t e m p e r a t u r e - s e n s i t i v e r e c o r d e r ( H e w l e t t - P a c k a r d 7 7 5 8 B ) , w h i c h a l s o d i f f e r e n t i a t e d t i d a l v o l u m e t o p r o d u c e a f l o w s i g n a l . From t h e s i m u l t a n e o u s r e a d i n g s o f t i d a l v o l u m e , f l o w a n d t r a n s p u l m o n a r y p r e s s u r e c a l c u l a t i o n s o f p u l m o n a r y r e s i s t a n c e ( R L ) a n d d y n a m i c c o m p l i a n c e ( C d y n ) w e r e made ( 1 3 4 ) . A t p o i n t s o f z e r o f l o w t h e v o l u m e c h a n g e ( T . V . ) was d i v i d e d by PL t o d e t e r m i n e C d y n . A t p o i n t s o f - 30 -e q u a l v o l u m e PL was d i v i d e d by t h e f l o w t o d e t e r m i n e RL. A e r o s o l s o f OA w e r e a d m i n i s t e r e d v i a a f u n n e l - l i k e a p p a r a t u s t h a t d i r e c t e d t h e s p r a y a r o u n d t h e h e a d o f t h e g u i n e a p i g . A e r o s o l s were -12 g i v e n i n 1 0 - f o l d i n c r e a s e s o f c o n c e n t r a t i o n r a n g i n g f r o m 10 t o 1 0 ~ ^ M OA. M e a s u r e m e n t s w e r e t a k e n a f t e r a c o n t r o l s a l i n e a e r o s o l , t h e n one m i n u t e and f o u r m i n u t e s a f t e r t h e s t a r t o f e a c h 30 s e c . OA a e r o s o l . D o s e - r e s p o n s e c u r v e s o f RL a n d Cdyn v e r s u s n e b u l i z e d c o n c e n t r a t i o n were c o n s t r u c t e d . 2.1.4) I n v e s t i g a t i o n o f t h e S t a b i l i t y o f T o l e r a n c e F o l l o w i n g t h e s i x weeks o f c h r o n i c OA e x p o s u r e t h e a n i m a l s were d i v i d e d r a n d o m l y i n t o g r o u p s a n d w i t h h e l d f r o m OA c h a l l e n g e f o r a v a r y i n g number o f d a y s ( L a y - o f f s ) t o i n v e s t i g a t e t h e s t a b i l i t y o f t o l e r a n c e when t h e a n i m a l s a r e n o t c o n t i n u a l l y e x p o s e d t o t h e a n t i g e n . One g r o u p o f f o u r a n i m a l s s e r v e d a s c o n t r o l s a n d w e r e m a i n t a i n e d on t h e f i v e d a y s / w e e k s c h e d u l e , w h i l e o t h e r g r o u p s o f f o u r , f i v e , f o u r , f i v e and f o u r a n i m a l s were n o t a e r o s o l i z e d f o r 4, 6, 8, 10, a n d 24 d a y s r e s p e c t i v e l y . A f t e r t h e p e r i o d o f n o n - e x p o s u r e t h e r e s p o n s e s were a s s e s s e d by b o t h t h e v i s u a l g r a d i n g s y s t e m a n d by RL and Cdyn d e t e r m i n e d f r o m l u n g m e c h a n i c s . - 31 -P a r t 2.2: I n v e s t i g a t i o n s o f t h e M e c h a n i s m o f T o l e r a n c e 2.2.1) I n V i t r o A i r w a y Smooth M u s c l e S t u d i e s To t e s t f o r i n v i t r o a n t i g e n r e s p o n s i v e n e s s , t e n o f t h e 30 a n i m a l s w h i c h h a d b e e n c h r o n i c a l l y e x p o s e d t o a n t i g e n ( T ) w e r e c o m p a r e d w i t h t e n s e n s i t i z e d w e i g h t - m a t c h e d a n i m a l s ( S ) and t e n n o n - s e n s i t i z e d w e i g h t - m a t c h e d c o n t r o l s ( C ) . The a n i m a l s were a n a e s t h e t i z e d w i t h 60 mg/kg s o d i u m p e n t o b a r b i t a l i n t r a p e r i t o n e a l l y a n d e x s a n g u i n a t e d by c a r d i a c p u n c t u r e . The s e r u m was s e p a r a t e d a n d s t o r e d a t - 2 0 ° C f o r l a t e r c o m p a r i s o n o f a n t i b o d y l e v e l s . T h e l u n g s a n d t r a c h e a w e r e r a p i d l y r e m o v e d a n d p l a c e d i n o x y g e n a t e d K r e b s 1 s o l u t i o n ( N a C l , 95 mM; KC1 4.7 mM; M g S 0 4 , 2.3 mM; C a C l 2 , 2.5 mM; K H 2 P 0 4 , 1.2 mM; NaHCO-j, 25 mM; a n d a n h y d r o u s d e x t r o s e 12 mM). A s t r i p o f p a r e n c h y m a ( P a r ) was r e m o v e d f r o m t h e p e r i p h e r y o f t h e r i g h t l u n g f o r t h e s m o o t h m u s c l e s t u d y , a n d t h e r e m a i n d e r o f t h e r i g h t l u n g f r o z e n a t - 2 0 ° C f o r a l a t e r h i s t a m i n e a s s a y . A l l e x d e s s c o n n e c t i v e t i s s u e a n d f a t w e r e r e m o v e d f r o m t h e t r a c h e a a n d b r o n c h i . The t r a c h e a was c u t i n h a l f t o f o r m u p p e r t r a c h e a l ( UT) a n d l o w e r t r a c h e a l ( L T ) s e g m e n t s , a n d t h e b r o n c h i ( B r ) d i s s e c t e d f r e e o f l u n g a n d s e p a r a t e d f r o m t h e t r a c h e a . T h e s e t r a c h e a l s e g m e n t s and t h e b r o n c h i were s p i r a l l e d by p l a c i n g a t h i n w i r e r o d t h r o u g h t h e lumen a n d c u t t i n g a t an a n g l e w h i l e g e n t l y p u l l i n g on one e n d o f t h e t i s s u e ( 1 3 5 ) . The two t r a c h e a l s p i r a l s , b r o n c h i a l s p i r a l a n d p a r e n c h y m a l s t r i p w e r e p l a c e d i n 20 ml o r g a n b a t h s - 32 -c o n t a i n i n g K r e b s ' s o l u t i o n c o n t i n u o u s l y b u b b l e d w i t h 95 % 0^/5 % C 0 2 ( v / v ) . The t r a c h e a l a n d b r o n c h i a l s t r i p s w ere s t r e t c h e d b y a p p l y i n g an i n i t i a l w e i g h t o f 2 g and t h e p a r e n c h y m a s t r e t c h e d w i t h a n i n i t i a l w e i g h t o f 0.6 g. F o l l o w i n g a n e q u i l i b r a t i o n p e r i o d o f a p p r o x i m a t e l y 60 m i n . , c u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o h i s t a m i n e d i h y d r o c h l o r i d e ( F i s h e r S c i e n t i f i c ) w e r e p e r f o r m e d . 0.1 ml a l i q u o t s o f a g o n i s t were a d d e d t o t h e b a t h s t o g i v e f i n a l h i s t a m i n e b a s e b a t h c o n c e n t r a t i o n s o f 5 x 1 0 " 7 M, 5.5 x 1 0 " 6 M, a n d 5.5 x 1 0 " 5 M. F o l l o w i n g t h o r o u g h w a s h i n g t o r e t u r n t h e t i s s u e s t o t h e i r b a s e l i n e s t a t e o f c o n t r a c t i o n , t h e t i s s u e s were c h a l l e n g e d w i t h 1 0 - f o l d -12 i n c r e a s e s i n c o n c e n t r a t i o n s o f OA r a n g i n g f r o m 1 . 1 x 1 0 M t o 1.2 x 1 0 ~ 7 M. L a s t l y , a l a r g e e x c e s s o f c a r b a c h o l ( 2 . 2 x 1 0 " ^ M) ( C a r b a m y l c h o l i n e c h l o r i d e , S i g m a C h e m i c a l C o . ) , a c h o l i n o m i m e t i c , was a d d e d t o e l i c i t t h e maximum t i s s u e c o n t r a c t i o n . C o n t r a c t i o n s were m e a s u r e d u s i n g f o r c e - d i s p l a c e m e n t t r a n s d u c e r s ( G r a s s M o d e l F T 0 3 C ) c a l i b r a t e d t o r e f l e c t d e v e l o p e d f o r c e i n g/9.8 m/sec . A f o u r c h a n n e l p o l y g r a p h (Beckman D y n o g r a p h R511 A) r e c o r d e d t h e raw d a t a . C o n t r a c t i l e r e s p o n s e s o f t h e t i s s u e s were a n a l y z e d b y p l o t t i n g t i s s u e r e s p o n s e s i n grams v e r s u s l o g H i s t a m i n e a n d v e r s u s l o g OA a n d by p l o t t i n g t h e p e r c e n t a g e o f t h e maximum h i s t a m i n e o r c a r b a c h o l r e s p o n s e v e r s u s l o g H i s t a m i n e a n d l o g OA. - 33 -2.2.2) T o t a l P a r e n c h y m a l H i s t a m i n e A s s a y T h e t h i r t y l u n g s s t o r e d a t - 2 0 ° C were t h a w e d a n d two 0.5 g s a m p l e s t a k e n f r o m e a c h o f t h e l u n g s . T h e s a m p l e s were m i n c e d f i n e l y w i t h s c i s s o r s a n d p l a c e d i n 10 ml t e s t t u b e s w i t h 2 ml P I P E S b u f f e r ( 2 5 mM P I P E S , 120 mM N a C l , 5 mM KC1, pH 7 . 4 ) . A f t e r b o i l i n g f o r 10 m i n . i n a w a t e r b a t h t h e t i s s u e was p e l l e t e d by c e n t r i f u g i n g 15 m i n . a t 1300 x g (Beckman T J - 6 c e n t r i f u g e , TH-4 r o t o r ) t h e n 1 ml a l i q u o t s o f t h e s u p e r n a t a n t w e r e r e m o v e d a n d f r o z e n a t - 2 0 ° C . The f o l l o w i n g d a y t h e h i s t a m i n e c o n t e n t s o f t h e s a m p l e s were a s s a y e d f l u o r o m e t r i c a l l y ( A m i n c o Bowman SPF 124) a s p r e v i o u s l y d e s c r i b e d ( 7 0 ) u s i n g t h e m i c r o - m o d i f i c a t i o n o f May e t a l ( 1 3 6 ) o f t h e method d e s c r i b e d by S h o r e e t a l ( 1 3 7 ) . R e s u l t s were e x p r e s s e d a s j u g h i s t a m i n e / g t i s s u e . 2.2.3) P a s s i v e C u t a n e o u s A n a p h y l a x i s (PCA) 2.2.3.1) S i x h o u r s e n s i t i z a t i o n PCA's were p e r f o r m e d a c c o r d i n g t o t h e m ethod o f O v a r y ( 1 3 8 ) . B r i e f l y , s i x f e m a l e H a r t l e y g u i n e a p i g s w e i g h i n g a p p r o x i m a t e l y 300 g e a c h were l i g h t l y a n a e s t h e t i z e d p r i o r t o b e i n g s h a v e d w i t h an O s t e r s m a l l a n i m a l c l i p p e r (# 40 r a z o r ) . S e r a s t o r e d f r o m t h e t o l e r a n t , s e n s i t i z e d a n d c o n t r o l a n i m a l s u s e d i n t h e a i r w a y s m o o t h m u s c l e s t u d y w ere u s e d . S e r a f r o m f o u r a n i m a l s i n e a c h o f t h e t h r e e g r o u p s were p o o l e d , t h e n d i l u t i o n s o f 1/50, 1/100, 1/200, a n d 1/400 i n s a l i n e w ere - 34 -made on t h e t o l e r a n t and s e n s i t i z e d s e r a . Two h o u r s a f t e r s h a v i n g 0.1 ml i n j e c t i o n s o f t h e d i l u t i o n s o f t o l e r a n t a n d s e n s i t i z e d s e r a , s a l i n e , and u n d i l u t e d c o n t r o l s e r u m were g i v e n , m a k i n g a t o t a l o f 10 d o r s a l i n t r a c u t a n e o u s i n j e c t i o n s p e r a n i m a l . A f t e r t h e s i x h o u r s e n s i t i z a t i o n p e r i o d 13 mg/kg i n t r a p e r i t o n e a l s o d i u m p e n t o b a r b i t a l and s u b c u t a n e o u s x y l o c a i n e ( A s t r a C h e m i c a l s L t d . ) w e r e a d m i n i s t e r e d . One ml o f a s o l u t i o n o f 10 mg/ml E v a n s b l u e dye ( J . T. B a k e r C h e m i c a l Co.) a n d 2.2 x 1 0 " ^ M OA i n s a l i n e was i n j e c t e d v i a a c a r o t i d c a n n u l a t i o n . A f t e r 45 m i n . t h e g u i n e a p i g s were k i l l e d w i t h an o v e r d o s e o f s o d i u m p e n t o b a r b i t a l a n d t h e s k i n r e m o v e d , s t r e t c h e d a n d c o v e r e d w i t h b o r a x . T w e n t y h o u r s l a t e r t h e b o r a x was b r u s h e d away and t h e l o n g a n d s h o r t a x e s o f t h e b l u e e l l i p s e s m e a s u r e d w i t h v e r n i e r c a l i p e r s t o a l l o w c a l c u l a t i o n o f t h e a r e a ( i f a b ) o f e a c h o f t h e b l u e e l l i p s e s . 2.2.3.2) F o u r d a y a n d s e v e n d a y s e n s i t i z a t i o n s F o u r and s e v e n day s e n s i t i z a t i o n s were done by t h e m ethod o f W atanabe a n d O v a r y (55) a n d P a r i s h ( 5 4 ) . S e v e n d a y s b e f o r e t h e OA c h a l l e n g e f o u r 300 g f e m a l e H a r t l e y g u i n e a p i g s were i n j e c t e d i n t r a c u t a n e o u s l y on one s i d e o f t h e b a c k w i t h u n h e a t e d , s e n s i t i z e d a n d t o l e r a n t s e r a (same p o o l e d s e r a a s f o r s i x h o u r s e n s i t i z a t i o n ) a t d i l u t i o n s o f 1/2, 1/4, a n d 1/8. F o u r d a y s b e f o r e t h e OA c h a l l e n g e h e a t e d (56°C f o r 30 m i n . ) s e n s i t i z e d a n d t o l e r a n t s e r a w e r e i n j e c t e d on t h e o t h e r s i d e o f t h e b a c k i n d i l u t i o n s o f 1/50, 1/100, and 1/200. The r e m a i n d e r o f t h e e x p e r i m e n t i s a s d e s c r i b e d f o r t h e s i x h o u r s e n s i t i z a t i o n . 2.2.4) L y m p h o c y t e T r a n s f e r s - 35 -F o u r t o l e r a n t a n d f o u r n o n - s e n s i t i z e d ( C o n t r o l ) S t r a i n 13 g u i n e a p i g s ( i n b r e d t o a l l o w c e l l t r a n s f e r s b e t w e e n a n i m a l s ) w e i g h i n g a p p r o x i m a t e l y 1 k g were k i l l e d w i t h 100 mg/kg s o d i u m p e n t o b a r b i t a l i n t r a p e r i t o n e a l l y . A m o d i f i c a t i o n o f t h e t e c h n i q u e d e s c r i b e d by F o r d ( 1 3 9 ) was e m p l o y e d . T h e s p l e e n , t h r e e t o f o u r h i l a r l y m p h n o d e s a n d t h r e e t o f o u r c e r v i c a l lymph n o d e s were r e m o v e d and p l a c e d i n room t e m p e r a t u r e , pH 7.3, 0.73 % (w/v) N a C l / P h o s p h a t e b u f f e r e d s a l i n e / a c i d c i t r a t e d e x t r o s e (PBS c o m p o s i t i o n : N a ^ P O ^ 7 H 2 0 , 3.21 mM; KFLjPO^, 6.0 mM; ACD c o m p o s i t i o n : a n h y d r o u s c i t r i c a c i d , 6.5 mM; s o d i u m c i t r a t e , 7.7 mM; a n h y d r o u s d e x t r o s e , 10 mM). E x c e s s f a t and c o n n e c t i v e t i s s u e were r e m o v e d a n d t h e s p l e e n a n d lymph n o d e s t h o r o u g h l y r i n s e d b e f o r e b e i n g f i n e l y m i n c e d w i t h s c i s s o r s . T h e t i s s u e s w e r e g e n t l y p r e s s e d t h r o u g h a f i n e mesh s i e v e (W. S. T y l e r T y p e 304, 100jjm g r i d ) and t h e s p l e e n a n d l y m p h node c e l l s c o l l e c t e d i n N a C l/PBS/ACD. The 40 ml o f s p l e e n c e l l s u s p e n s i o n were d i v i d e d among 8 p l a s t i c c o n i c a l c e n t r i f u g e t u b e s , e a c h w i t h 30 ml d i s t i l l e d H 2 o t o l y s e t h e RBC. A f t e r m i x i n g f o r 30 s e c . t h e t o n i c i t y was r e c o n s t i t u t e d w i t h 15 ml o f pH 7.3, 2.5 % (w/v) NaCl/PBS/ACD. The p e l l e t f r o m a 10 m i n . , 2 2 ° C 200 x g (Beckman T J - 6 , TH-4 r o t o r ) c e n t r i f u g a t i o n was r e s u s p e n d e d i n 20 ml 0.73 % NaCl/PBS/ACD. E a c h o f t h e 20 ml l y m p h node a n d s p l e e n c e l l s u s p e n s i o n s were l a y e r e d o n t o 8 ml o f F i c o l l - P a q u e ( P h a r m a c i a F i n e C h e m i c a l s ) a n d c e n t r i f u g e d 30 m i n . a t 2 2 ° C a t 4 0 0 x g . The u p p e r c l e a r l a y e r and p e l l e t were d i s c a r d e d and t h e m i d d l e w h i t e - 36 -f l u f f y l a y e r washed t w i c e i n 0.73 % (w/v) NaCl/PBS/ACD, t h e n r e s u s p e n d e d i n 1 ml s a l i n e . C e l l s were c o u n t e d i n a h a e m o c y t o m e t e r u s i n g 2 % ( v / v ) a c e t i c a c i d / m e t h y l e n e b l u e a n d v i a b i l i t y a s s e s s e d u s i n g T r y p a n b l u e e x c l u s i o n . C e l l v i a b i l i t y was a p p r o x i m a t e l y 94 %. T o t a l c e l l s i n j e c t e d r a n g e d f r o m 0.57-2.2 x 10 c e l l s . E a c h o f t h e e i g h t S t r a i n 13 r e c i p i e n t s w ere i n j e c t e d i n t r a p e r i t o n e a l l y ( 1 4 0 ) w i t h t h e c o m b i n e d s p l e e n a n d l y m p h node c e l l s f r o m one d o n o r . A p p r o x i m a t e l y 24 h o u r s l a t e r t h e g u i n e a p i g s were s e n s i t i z e d w i t h OA a s p r e v i o u s l y d e s c r i b e d , t h e n a e r o s o l c h a l l e n g e d two weeks l a t e r . 2.2.5) I n V i t r o H i s t a m i n e R e l e a s e E i g h t s e n s i t i z e d , c h r o n i c a l l y c h a l l e n g e d S t r a i n 13 g u i n e a p i g s ( T ) , e i g h t g u i n e a p i g s s e n s i t i z e d 14 d a y s p r i o r t o t h e s t u d y b u t n e v e r c h a l l e n g e d ( S ) a n d two n o n - s e n s i t i z e d , n o n - c h a l l e n g e d c o n t r o l ( C ) g u i n e a p i g s were u s e d i n t h i s e x p e r i m e n t . The a n i m a l s were h e a v i l y a n a e s t h e t i z e d w i t h a p p r o x i m a t e l y 100 mg/kg s o d i u m p e n t o b a r b i t a l i n t r a p e r i t o n e a l l y , t h e n e x s a n g u i n a t e d by c a r d i a c p u n c t u r e . The i n v i t r o h i s t a m i n e r e l e a s e m e t h o d o l o g y o f A u s t e n a n d B r o c k l e h u r s t was f o l l o w e d ( 1 4 1 ) . T h e l u n g s a n d h e a r t were r e m o v e d en b l o c , a n d t h e l u n g s f l u s h e d w i t h 3 7 ° C T y r o d e s s o l u t i o n ( N a C l , 13.7 mM; C a C l 2 - 2 H 2 0 , 0.14 mM; KC1, 0.27 mM; M g C l 2 ' 6 H 2 0 , 0.10 mM; N a H 2 P 0 4 ' H 2 0 , 0.04 mM; N a H C 0 3 , 1.2 mM; a n h y d r o u s d e x t r o s e , 0.56 mM) v i a t h e p u l m o n a r y a r t e r y u n t i l t h e b l o o d was c o m p l e t e l y - 37 -c l e a r e d . Any p o r t i o n s o f l u n g s t i l l l e f t w i t h b l o o d w e r e r e m o v e d . T h e l u n g p a r e n c h y m a was f i n e l y m i n c e d , t h e n washed w i t h 2 2 ° C T y r o d e s s o l u t i o n v i a s u c t i o n f i l t r a t i o n . E i g h t t i s s u e s a m p l e s o f 0.5 g were o b t a i n e d f r o m e a c h l u n g a n d p l a c e d i n 10 ml g l a s s t e s t t u b e s . Two ml o f T y r o d e s s o l u t i o n f o r c o n t r o l t u b e s and T y r o d e s / O A i n a p p r o p r i a t e c o n c e n t r a t i o n s w e r e a d d e d t o t h e d u p l i c a t e t u b e s . F o u r t o l e r a n t g u i n e a p i g a nd 4 s e n s i t i z e d g u i n e a p i g l u n g s a m p l e s r e c e i v e d OA i n c o n c e n t r a t i o n s o f 2.2 x 1 0 " 9 M, 1.1 x 1 0 " 8 M, a n d 1.1 x 1 0 " 7 M. T h e r e m a i n i n g 4 t o l e r a n t , 4 s e n s i t i z e d , a n d 2 c o n t r o l a n i m a l s r e c e i v e d OA i n c o n c e n t r a t i o n s o f 1.1 x 1 0 " 6 M, 1.1 x 1 0 ~ 5 M a n d 1.1 x 1 0 " 4 M. The t i s s u e s were i n c u b a t e d w i t h a n t i g e n f o r 15 m i n . a t 3 7 ° C w i t h i n t e r m i t t e n t s w i r l i n g . The s u p e r n a t a n t f r o m a 15 min., 4 ° C 1300 xg (Beckman T J - 6 , TH-4 r o t o r , T J - R r e f r i g e r a t i o n u n i t ) was r e m o v e d and s t o r e d a t - 2 0 ° C . The t i s s u e p e l l e t was r e s u s p e n d e d i n 2 ml T y r o d e s s o l u t i o n and b o i l e d 10 m i n . F o l l o w i n g a c e n t r i f u g a t i o n a s p r e v i o u s l y d e s c r i b e d , 1 ml a l i q u o t s o f t h e s u p e r n a t a n t were r e m o v e d a n d s t o r e d a t - 2 0 ° C . The h i s t a m i n e a s s a y was c a r r i e d o u t a s d e s c r i b e d i n t h e T o t a l P a r e n c h y m a l H i s t a m i n e A s s a y s e c t i o n . H i s t a m i n e r e l e a s e d f r o m t u b e s c o n t a i n i n g o n l y T y r o d e s s o l u t i o n was s u b t r a c t e d b e f o r e c a l c u l a t i o n o f h i s t a m i n e r e l e a s e i n r e s p o n s e t o OA. T h e r e s u l t s w e r e e x p r e s s e d a s p e r c e n t a g e h i s t a m i n e r e l e a s e v e r s u s OA c o n c e n t r a t i o n . - 38 -CHAPTER 3. DATA ANALYSIS I n t h e a n a l y s i s o f t h e l u n g m e c h a n i c s d a t a a one-way a n a l y s i s o f v a r i a n c e (ANOVA) ( 1 4 2 , 143) was p e r f o r m e d on Cdyn and RL u s i n g weeks as a g r o u p i n g f a c t o r . T h i s a n a l y s i s was p e r f o r m e d a t e a c h OA c o n c e n t r a t i o n . T h i s method was c h o s e n s i n c e i t was d e s i r a b l e t o f i n d d i f f e r e n c e s b e t w e e n an RL o r Cdyn r e s p o n s e b e t w e e n e x p e r i m e n t a l weeks a t a g i v e n OA c o n c e n t r a t i o n . The RL and C d y n v a l u e s f o r e a c h a n i m a l w e r e a c t u a l l y a p e r c e n t a g e o f a c o n t r o l m e a s u r e m e n t made on e a c h a n i m a l . E x p r e s s i o n o f r e s u l t s i n t h i s way a l l o w s r e m o v a l o f some i n t e r - a n i m a l v a r i a t i o n . The c o n t r o l m e a s u r e m e n t was r e c o r d e d e a c h week p r i o r t o an OA d o s e . Upon c o m p l e t i o n o f t h e ANOVA a s i g n i f i c a n t d i f f e r e n c e b e t w e e n some g r o u p s was i n d i c a t e d . A D u n c a n ' s m u l t i p l e r a n g e t e s t ( 1 4 4 ) was t h e n u s e d t o d e t e r m i n e t h e weeks w h i c h w e r e d i f f e r e n t . The a e r o s o l l a y - o f f s a s s e s s e d by a v i s u a l g r a d e w e r e c o m p a r e d u s i n g an u n p a i r e d T - t e s t ( 1 4 4 ) . T h e a e r o s o l c h a l l e n g e l a y - o f f d a t a f r o m l u n g m e c h a n i c s was a n a l y z e d u s i n g D u n n e t t ' s T - t e s t ( 1 4 4 ) s i n c e t h i s t e s t c o m p a r e s s e v e r a l g r o u p s t o a c o n t r o l . I n t h i s e x p e r i m e n t t h e day 43 Cdyn and RL s e r v e d a s c o n t r o l s w h i l e t h e d a y s s i n c e l a y - o f f w e r e c o m p a r e d t o them. F o r t h e i n v i t r o a i r w a y s m o o t h m u s c l e s t u d i e s a one-way a n a l y s i s o f v a r i a n c e was p e r f o r m e d on t h e mean h i s t a m i n e r e s p o n s e a s a p e r c e n t a g e o f t h e maximum r e s p o n s e u s i n g c o n t r o l , s e n s i t i z e d a n d t o l e r a n t a n i m a l s a s t h e g r o u p i n g f a c t o r . F o r t h i s a n a l y s i s UT, L T , a n d - 39 -B r were g r o u p e d t o g e t h e r a n d a n a l y z e d . T h i s was done s i n c e p r i o r a n a l y s i s by one-way ANOVA a n d O r t h o g o n a l C o n t r a s t s y i e l d e d no d i f f e r e n c e b e t w e e n t h e s e t i s s u e t y p e s f o r a g i v e n g r o u p o r c o n c e n t r a t i o n . Upon c o m p l e t i o n o f t h e ANOVA, D u n c a n ' s m u l t i p l e r a n g e t e s t was a g a i n u s e d t o d e t e r m i n e w h i c h g r o u p s were d i f f e r e n t . T h e a b o v e a n a l y s i s was r e p e a t e d f o r P a r e n c h y m a d a t a . T h e a n a l y s i s o f f o r c e i n c r e a s e p e r m i n . i n d u c e d b y h i s t a m i n e a n d OA was a n a l y z e d u s i n g a 3 - f a c t o r a n a l y s i s o f v a r i a n c e . The f a c t o r s were 1) d o s e l e v e l ( i . e . h i s t a m i n e , OA) 2) g r o u p ( C , S, T) a n d 3) s u b j e c t s . T h i s d e s i g n , a l t h o u g h c o m p l i c a t e d , a l l o w s t h e d o s e a n d g r o u p v a r i a t i o n s t o be v i e w e d a f t e r c o n t r o l l i n g f o r s u b j e c t v a r i a t i o n . I t s h o u l d be n o t e d t h a t t h e s u b j e c t s a r e n e s t e d w i t h i n t h e g r o u p s w h i l e g r o u p i n g and d o s e l e v e l a r e f u l l y c r o s s e d . T h i s a n a l y s i s was c a r r i e d o u t f o r e a c h o f t h e f o u r t i s s u e t y p e s (UT, LT, B r , a n d P a r ) . O r t h o g o n a l c o n t r a s t s were u s e d t o a s s e s s d i f f e r e n c e s b e t w e e n g r o u p s when s i g n i f i c a n t r e s u l t s were o b t a i n e d f r o m t h e ANOVA. The a s s e s s m e n t o f t o t a l h i s t a m i n e c o n t e n t was done u s i n g a two-way a n a l y s i s o f v a r i a n c e w i t h s u b j e c t s n e s t e d i n g r o u p s ( C , S, T ) . A f t e r a s i g n i f i c a n t r e s u l t D u n c a n ' s m u l t i p l e r a n g e t e s t was u s e d . F o r t h e PCA e x p e r i m e n t t h e b l u e - a r e a was e x a m i n e d a t e a c h s e r u m d i l u t i o n b y an u n p a i r e d T - t e s t b e t w e e n S and T g r o u p s a n d a l l t h e b l u e a r e a s i n t h e S a n d T g r o u p s were c o m p a r e d u s i n g a t h r e e - w a y ANOVA. The l a r g e s t a n d a r d e r r o r o f t h e mean i s an i n d i c a t i o n o f t h e l a r g e i n t e r - a n i m a l v a r i a t i o n . The O A - i n d u c e d h i s t a m i n e r e l e a s e was c o m p a r e d f o r S a n d T g r o u p s - 40 -a t e a c h c o n c e n t r a t i o n o f OA u s i n g an u n p a i r e d o n e - t a i l e d T - t e s t . The t e s t was done o n e - t a i l e d s i n c e i t c o u l d o n l y be e x p e c t e d t h a t t h e T v a l u e w o u l d be l e s s t h a n t h e S v a l u e . A l t h o u g h t h e s t a n d a r d e r r o r o f t h e mean v a l u e s v a r i e d f r o m S t o T g r o u p s t h e d i f f e r e n c e was n o t deemed s u f f i c i e n t t o q u e s t i o n t h e v a l i d i t y o f t h e T - t e s t . A t h r e e - w a y ANOVA was u s e d t o c o m p a r e t h e S g r o u p t o t h e T g r o u p . - 41 -CHAPTER 4. RESULTS P a r t 4.1: D e v e l o p m e n t o f T o l e r a n c e 4.1.1) A s s e s s m e n t by V i s u a l G r a d i n g S y s t e m F i g . 4.1 shows a p l o t o f t h e d a i l y mean v i s u a l g r a d e o f t h e g r o u p o f 28 g u i n e a p i g s f o r t h e f i r s t 43 d a y s o f a e r o s o l c h a l l e n g e . The f i r s t two weeks e x h i b i t t h e h i g h e s t r e s p o n s e s w i t h r e a c t i v i t y d r o p p i n g o f f s t e a d i l y t h e r e a f t e r . By 34 d a y s t o l e r a n c e t o t h a t r e g i m e o f a n t i g e n c h a l l e n g e was e s t a b l i s h e d a n d no v i s i b l e r e a c t i o n t o t h e a e r o s o l i z e d a n t i g e n c o u l d be d e t e c t e d . On t h e 3 5 t h day o f c h a l l e n g e OA f r o m a new b o t t l e was a d m i n i s t e r e d . T h i s p r o v o k e d a m a r k e d l y i n c r e a s e d r e s p o n s e f o r t h e n e x t t h r e e c h a l l e n g e s . T h i s i n c r e a s e d r e s p o n s e was s h o r t - l i v e d . 4.1.2) A s s e s s m e n t o f R e a c t i v i t y by L u n g M e c h a n i c s F i g u r e s 4.2 and 4.3 show t h e d o s e - r e s p o n s e c u r v e s f o r d y n a m i c c o m p l i a n c e ( C d y n ) a n d p u l m o n a r y r e s i s t a n c e (RL) a t 1, 22, a n d 43 d a y s a f t e r t h e i n i t i a l c h a l l e n g e . W i t h i n c r e a s i n g t i m e o f a e r o s o l e x p o s u r e h i g h e r d o s e s o f OA w e r e r e q u i r e d t o e l i c i t d e c r e a s e s i n C d yn a n d i n c r e a s e s i n RL. C o m p a r i s o n o f C d y n and RL on d a y s 1, 22, a n d 43 a t t h e OA c o n c e n t r a t i o n o f 2.2 x 1 0 ~ 4 M shows a s i g n i f i c a n t l y (ANOVA, D u n c a n ' s T - t e s t p < 0 . 0 1 ) d e c r e a s i n g r e s p o n s e ( T a b l e 4 . 1 ) . - 42 -i i i i i i i r — i I I I I 1 — I 1 I I I I I I I 0 2 U 6 8 10 12 U 16 18 20 22 2U 26 28 30 32 34 3 6 38 <V0 42 <U T i m e ( d a y s ) F i g u r e 4.1. The d a i l y mean v i s u a l g r a d e +_ SEM o f 2 8 - g u i n e a p i g s c h r o n i c a l l y c h a l l e n g e d w i t h OA a e r o s o l f o r 43 d a y s . S e e t e x t f o r e x p l a n a t i o n o f g r a d i n g s y s t e m . 110 J I x 1 0 - 7 x 1 0 - 6 x 1 0 " 5 x 1 0 _ i x 1 0 ~ 3 x 1 0 - 3 ' i Ovalbumin (M) i i F i g u r e 4.2. O A - i n d u c e d c h a n g e s i n l u n g m e c h a n i c s d u r i n g 43 d a y s o f c h r o n i c a n t i g e n a e r o s o l . c h a l l e n g e . Mean +_ SEM % o f C o n t r o l C d y n i s shown f o r 1 day (N=6) (A), 22 d a y s (N=8) ( • ) a n d 43 d a y s (N=7) ( • ) o f t h e c h r o n i c a e r o s o l c h a l l e n g e p e r i o d . $ Cdyn s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 1 ) a t 2.2 x 1 0 " 4 M OA c o m p a r i n g e a c h o f d a y s 1, 22 a n d 43 t o t h e o t h e r s . - 44 -c o O 600 500 H 400 300 A 200 100 I I I I ' 1 2.2 2.2 2.2 2.2 2.2 8.8 x10 - 7 x10"6 x10 - 5 x10 _ i x10"3 x10 - 3 Ovalbumin (M) F i g u r e 4.3. O A - i n d u c e d c h a n g e s i n l u n g m e c h a n i c s d u r i n g 43 d a y s o f c h r o n i c a n t i g e n a e r o s o l c h a l l e n g e . Mean + SEW % o f C o n t r o l RL i s shown f o r 1 day (N=6) ( A ) , 22 d a y s (N=8) ( + ) a n d 43 d a y s (N=7) ( • ) o f t h e c h r o n i c a e r o s o l c h a l l e n g e p e r i o d , & RL s i g n i f i c a n t l y d i f f e r e n t (p<0.01) a t 2.2 x 10"4 M OA c o m p a r i n g e a c h o f d a y s 1, 22 a n d 43 t o t h e o t h e r s . - 45 -T a b l e 4.1. Lung M e c h a n i c s A s s e s s m e n t o f T o l e r a n c e D e v e l o p m e n t Time D u r i n g Mean % D e c r e a s e Cdyn Mean % I n c r e a s e RL _+ SEM C h r o n i c + SEM a t 2.2 x 1 0 " * M OA a t 2.2 x 1 0 ~ 4 M OA E x p o s u r e Day 1 79 + 10 * 450 + 200 * N=8 Day 22 40 + 8 * 87 + 50 * N=6 Day 43 11 + 6 * 40 + 14 * N=8 * S i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o f t h e o t h e r d a y s ( p < 0 . 0 1 ) - 46 -4.1.3) E f f e c t o f C h a l l e n g e L a y - O f f s on T o l e r a n c e The e f f e c t s o f c h a l l e n g e l a y - o f f s on t o l e r a n c e were a s s e s s e d v i s u a l l y a n d by l u n g m e c h a n i c s . F i g . 4.4 c o m p a r e s t h e e f f e c t s o f 4 t h r o u g h 24 d a y s l a y - o f f w i t h t h e mean v i s u a l g r a d e f o r t h o s e g u i n e a p i g s r e g u l a r l y c h a l l e n g e d . I n c r e a s e d r e a c t i v i t y was o b s e r v e d f o r t h e 10 day l a y - o f f p e r i o d ( u n p a i r e d T - t e s t p < 0 . 0 5 ) . T a b l e 4.2 c o m p a r e s t h e mean % o f c o n t r o l _+ SEM Cdyn and RL a t 2.2 x 1 0 ~ 4 M OA f o r 4, 6, 8, 10 a n d 24 d a y s l a y - o f f . Some g r o u p s ( 4 a n d 8 d a y s ) r e c o v e r s i g n i f i c a n t r e a c t i v i t y ( D u n n e t t ' s T - t e s t p < 0 . 0 5 ) , b u t n e v e r r e g a i n t h e i r i n i t i a l l e v e l s . I n c r e a s i n g l a y - o f f t i m e d o e s n o t r e s u l t i n a p r o g r e s s i v e l o s s o f t o l e r a n c e . P a r t 4.2: I n v e s t i g a t i o n s o f t h e M e c h a n i s m o f T o l e r a n c e 4.2.1) I n V i t r o A i r w a y Smooth M u s c l e S t u d i e s F i g . 4.5 i l l u s t r a t e s s a m p l e UT t o l e r a n t a n d s e n s i t i z e d r e c o r d e r t r a c i n g s o f a c o m p l e t e e x p e r i m e n t . T i s s u e c o n t r a c t i o n s b e g a n w i t h i n one m i n . o f t h e a d d i t i o n o f a g o n i s t t o t h e o r g a n b a t h . R e g a r d l e s s o f t h e g r o u p o r d r u g u s e d t o p r o v o k e c o n t r a c t i o n , t h e m a g n i t u d e o f grams r e s p o n s e i n d u c e d was l a r g e s t f o r UT, s l i g h t l y l e s s f o r L T , s l i g h t l y l e s s a g a i n f o r B r , a n d much l e s s f o r P a r ( F i g . 4.6, 4 . 7 ) . However, when r e s p o n s e s were e x p r e s s e d a s a p e r c e n t a g e o f t h e maximum t i s s u e c o n t r a c t i o n i n d u c e d by c a r b a c h o l o r h i s t a m i n e , t h e UT, i _ 47 -1 . 0 0 -1 i 0.90 • x J 0 . 8 0 -0 ' l I I I I I I I I i 0 2 4 6 8 10 12 K 16 18 20 Time (weeks) F i g u r e 4.4. T h e w e e k l y mean v i s u a l g r a d e + SEM o f 26 g u i n e a p i g s c h a l l e n g e d a n d w i t h h e l d f r o m c h a l l e n g e o v e r a p e r i o d o f 20 weeks. F o u r a n i m a l s s e r v e d a s c o n t r o l s a n d were m a i n t a i n e d on t h e 5 d a y s / w e e k c h a l l e n g e s c h e d u l e , w h i l e o t h e r g r o u p s o f 4, 5, 4, 5 a n d 4 a n i m a l s w e r e l a i d o f f c h a l l e n g e f o r 4 ( A ) , 6 ( O ) , 8 ( 0 ) , 10 ( A ) , a n d 24 ( # ) d a y s r e s p e c t i v e l y . (X) r e p r e s e n t s t h e mean r e s p o n s e o f t h e g r o u p o f g u i n e a p i g s a e r o s o l c h a l l e n g e d f o r one week. S i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l a f t e r 10 d a y s l a y - o f f ( p < 0 . 0 5 ) . - 48 -T a b l e 4.2. Lung M e c h a n i c s A s s e s s m e n t o f E f f e c t o f L a y - O f f s on T o l e r a n c e Mean % o f C o n t r o l C d yn Mean % o f C o n t r o l RL + SEM Time + SEM a t 2.2 x 10-4 M OA a t 2.2 X 10"4 M OA Day 1 C h r o n i c 22 + 10 550 +_ 200 E x p o s u r e Day 43 C h r o n i c 89 _+ 6 140 + 14 E x p o s u r e 4 d a y s 49 + 13 * 190 + 34 * l a y - o f f 6 d a y s 80 + 16 110 + 30 l a y - o f f 8 d a y s 43 + 17 * 210 + 49 * l a y - o f f 10 d a y s 65 + 14 160 + 30 l a y - o f f 24 d a y s 61 + 15 160 + 23 l a y - o f f * S i g n i f i c a n t l y d i f f e r e n t f r o m Day 43 ( p < 0 . 0 5 ) . - 50 -CO E CC k. O) 0 CO c o a CO a> QC CD c E CO +-» CO I 2.50 -t 2.00 1.50 H 1 00 0 .50 H 0 J 5 . 0 x 1 0 - 7 5 . 5 x 1 0 - 6 Histamine (M) 5 . 5 x 1 0 " 5 2 . 2 x 1 0 ' ' Carbachol (M) F i g u r e 4.6. Mean grams r e s p o n s e + SEM t o h i s t a m i n e a n d c a r b a c h o l by c o n t r o l t i s s u e s UT (N=10) ( # ) , LT (N=10) ( • ) , B r (N=10) ( X ) , a n d P a r (N=10) ( • ) . - 51 CO E ca *—' CO c O Q. CO CD QC < O 2 . 0 0 . 1 . 5 0 J 1 . 0 0 J 0 . 5 0 0 J 1.1 x 1 0 " ' 2 1 . 2 x 1 0 " " 1 . 2 x 1 0 " ' ° 1.2 x 10 ~ 9 1 . 2 x 1 0 ~ 8 1 . 2 x 1 0 " 7 Ovalbumin (M) F i g u r e 4.7. Mean grams r e s p o n s e + SEM t o o v a l b u m i n by s e n s i t i z e d t i s s u e s UT (N=10) ( • ) , LT (N=10) ( • ) , B r (N=10) ( X ) , a n d P a r (N=10) - 52 -£ Histamine (M) i F i g u r e 4.8. Mean % o f maximum h i s t a m i n e r e s p o n s e t o h i s t a m i n e by c o n t r o l t i s s u e s UT (N=10) (•), LT (N=10) ( • ) , B r (N=10) (X) an d P a r (N=10) ( • ) . - 53 -F i g u r e 4.9. Mean % o f maximum c a r b a c h o l r e s p o n s e + SEM t o o v a l b u m i n by s e n s i t i z e d t i s s u e s UT (N=10) ( # ) , LT (N=10) ( • ) , B r (N=10) (X) and' P a r (N=10) ( • ) . - 54 -LT and B r r e s p o n s e s were s i m i l a r , b u t P a r r e m a i n e d d i s t i n c t ( F i g . 4.8, 4 . 9 ) . F o r t h i s r e a s o n , UT, LT a n d B r d a t a a r e c o m b i n e d f o r c o m p a r i n g c o n t r o l , t o l e r a n t and s e n s i t i z e d g r o u p s . F i g . 4.10 c o m p a r e s t h e r e s p o n s e s o f c o n t r o l , s e n s i t i z e d a n d t o l e r a n t t i s s u e s t o i n c r e a s i n g d o s e s o f h i s t a m i n e . No d i f f e r e n c e i n a i r w a y s m o o t h m u s c l e c o n t r a c t i l i t y b e t w e e n t h e g r o u p s c a n be d e t e c t e d . C o m p a r i s o n o f r a t e s o f O A - i n d u c e d t i s s u e c o n t r a c t i o n show t h a t e x c e p t f o r t o l e r a n t b r o n c h i , w h i c h h a d a s i g n i f i c a n t l y g r e a t e r (ANOVA p < 0 . 0 1 ) maximum r a t e o f c o n t r a c t i o n ( 0 . 3 6 +_ 0.04 g / m i n . ) t h a n s e n s i t i z e d b r o n c h i ( 0 . 1 3 +_ 0.04 g/min.) r a t e s o f f o r c e g e n e r a t i o n were s i m i l a r f o r s e n s i t i z e d and t o l e r a n t t i s s u e s . Maximum r a t e s o f c o n t r a c t i o n o f s e n s i t i z e d t i s s u e s w ere e l i c i t e d a t m i d d o s e - r e s p o n s e c u r v e c o n c e n t r a t i o n s and f o r t o l e r a n t t i s s u e s a t u p p e r d o s e - r e s p o n s e c u r v e c o n c e n t r a t i o n s . A l l c o n t r a c t i o n s w ere s u s t a i n e d a n d r e q u i r e d s e v e r a l h o u r s o f w a s h i n g b e f o r e t h e t i s s u e s r e l a x e d t o t h e b a s e l i n e s t a t e . R e p e t i t i o n o f t h e OA d o s e - r e s p o n s e c u r v e p r o d u c e d c o n t r a c t i o n s w h i c h were g r e a t l y r e d u c e d o r n o n - e x i s t e n t . F i g . 4.11 c o m p a r e s c o n t r o l , s e n s i t i z e d a n d t o l e r a n t a i r w a y s m o o t h m u s c l e r e s p o n s e s t o OA. L a c k o f c o n t r o l s m o o t h m u s c l e r e s p o n s e t o OA shows t h a t OA d o e s n o t c a u s e a n y n o n - s p e c i f i c c o n t r a c t i o n . C o m p a r i s o n o f t i s s u e f r o m s e n s i t i z e d a n i m a l s w i t h t i s s u e f r o m t o l e r a n t a n i m a l s shows a s i g n i f i c a n t l y d e c r e a s e d r e s p o n s e by t h e t o l e r a n t t i s s u e o v e r t h e OA d o s e r a n g e s t u d i e d (ANOVA, D u n c a n ' s T - t e s t p < 0 . 0 0 1 a t 1.2 x 10-10 M OA t o 1.2 x 10-7 M). - 55 -cc co co CD CO c o a CO CD OC CD c co CO I CD CO c o a CO CD rr CD CO CO E E " x CO 100. 90-1 8 0 J J 70 J 60J 50 J i.0 30 20 J 1 0 J I I I 5.0x10 7 5.5 x10"6 5.5x10"5 Histamine (M) F i g u r e 4.10. Mean % o f maximum t i s s u e r e s p o n s e _+ SEM t o h i s t a m i n e by C o n t r o l , T o l e r a n t a n d S e n s i t i z e d g r o u p s . UT, LT a n d B r ( ) d a t a h a v e b e e n c o m b i n e d s o t h a t N=30 f o r e a c h o f C o n t r o l ( • ) , S e n s i t i z e d (A), a n d T o l e r a n t ( • ) g r o u p s . N=10 f o r P a r t i s s u e ( — — ) i n e a c h o f C o n t r o l ( • ) , S e n s i t i z e d (A), and T o l e r a n t ( # ) g r o u p s . - 56 -Ovalbumin (M) F i g u r e 4.11. Mean % o f maximum t i s s u e r e s p o n s e _+ SEM t o o v a l b u m i n by C o n t r o l ( • ) , T o l e r a n t ( • ) , a n d S e n s i t i z e d (A) g r o u p s . UT, LT a n d B r d a t a h a v e b e e n c o m b i n e d s o t h a t N=30 f o r e a c h o f t h e g r o u p s . •O S i g n i f i c a n t d e c r e a s e (p < 0 . 0 0 1 ) i n r e s p o n s e b y t o l e r a n t t i s s u e o v e r t h e OA d o s e r a n g e 1.2 x 1 0 ~ 1 0 M t o 1.2 x 1 0 " 7 M. - 57 -4.2.2) T o t a l T i s s u e H i s t a m i n e C o n t e n t T a b l e 4.3 shows t h e r e s u l t s f o r t h e l u n g t i s s u e h i s t a m i n e c o n t e n t i n t o l e r a n t , a c u t e l y s e n s i t i z e d and c o n t r o l g u i n e a p i g s . The t o l e r a n t g r o u p o f a n i m a l s h a d s i g n i f i c a n t l y (AMOVA, D u n c a n ' s T - t e s t p < 0 . 0 5 ) more h i s t a m i n e ( 5 . 7 4 +_ 0 . 7 6 / j g h i s t a m i n e / g wet t i s s u e ) t h a n t h e c o n t r o l ( 3 . 0 5 +_ 0.33) o r s e n s i t i z e d ( 3 . 3 0 +_ 0.38) g r o u p s . 4.2.3) P a s s i v e C u t a n e o u s A n a p h y l a x i s F i g . 4.12 shows t h e PCA r e a c t i o n f r o m a g u i n e a p i g w h i c h h a d a s i x h o u r s e n s i t i z a t i o n w i t h s e r u m a n t i b o d y . No r e s p o n s e was e l i c i t e d by s a l i n e o r u n d i l u t e d c o n t r o l s e r u m . C o m p a r i s o n o f t h e a r e a s o f b l u e i n g f r o m t o l e r a n t a nd s e n s i t i z e d s e r a shows no g r o s s l y a p p a r e n t s i g n i f i c a n t d i f f e r e n c e i n a n t i b o d y l e v e l s i n s e r a f r o m t o l e r a n t a n d s e n s i t i z e d a n i m a l s . T a b l e 4.4 p r e s e n t s t h e mean a r e a s o f r e a c t i o n (mm ) o f e a c h s e r u m d i l u t i o n f r o m t o l e r a n t a n d s e n s i t i z e d s e r a . C o m p a r i s o n o f t h e s e n s i t i z e d w i t h t h e t o l e r a n t g r o u p o v e r a l l t h e c o n c e n t r a t i o n s shows t h e t o l e r a n t g r o u p t o h a v e s i g n i f i c a n t l y l e s s a r e a o f b l u e i n g t h a n t h e s e n s i t i z e d g r o u p (ANOVA p < 0 . 0 5 ) . By an u n p a i r e d T - t e s t , t h e g r o u p s w ere t h e same e x c e p t a t t h e 1/100 d i l u t i o n ( p < 0 . 0 5 ) . The r e s u l t s f r o m t h e f o u r 48 h o u r and s e v e n day s e n s i t i z a t i o n s were i m p o s s i b l e t o q u a n t i f y , s i n c e s y s t e m i c s e n s i t i z a t i o n o f t h e a n i m a l s o c c u r r e d , a n d o n l y two a n i m a l s s u r v i v e d t h e a n t i g e n c h a l l e n g e T a b l e 4.3. T o t a l Lung P a r e n c h y m a l H i s t a m i n e T i s s u e jmg h i s t a m i n e / g wet l u n g _+ SEW C o n t r o l 3.05 + 0.33 S e n s i t i z e d 3.30 + 0.38 T o l e r a n t 5.74 + 0.76 * * S i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l and S e n s i t i z e d ( p < 0 . 0 5 ) . - 59 -SENSITIZED SERUM 1/50 SALINE CONTROL SENSITIZED SERUM 1/100 SENSITIZED SERUM 1/400 SENSITIZED SERUM 1/200 TOLERANT SERUM 1/50 CONTROL SERUM UNDILUTED TOLERANT SERUM 1/100 TOLERANT SERUM 1/400 TOLERANT SERUM 1/200 F i g u r e 4.12. C o m p a r i s o n o f s e r a f r o m s e n s i t i z e d a nd t o l e r a n t g u i n e a p i g s by a s i x h o u r p a s s i v e c u t a n e o u s a n a p h y l a x i s s e n s i t i z a t i o n . T a b l e 4.4. S i x H o u r PCA C o m p a r i s o n o f S e n s i t i z e d a n d T o l e r a n t S e r a . A r e a o f B l u e i n g Serum mm2 +_ SEM D i l u t i o n S e n s i t i z e d * T o l e r a n t 1/50 364 + 35 321 + 23 1/100 222 + 20 188 + 21 1/200 164 + 20 144 + 18 1/400 119 + 27 107 22 * G r o u p s s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r ( P < 0 . 0 5 ) - 61 -t o p r o v i d e a r e a s o f b l u e i n g . The a r e a s o f b l u e i n g were more d i f f u s e t h a n t h e a r e a s f r o m t h e 6 h o u r s e n s i t i z a t i o n , b u t a p p r o x i m a t e c o m p a r i s o n o f t h e s e a r e a s d i d n o t show a r e d u c e d b l u e i n g r e a c t i o n f r o m t o l e r a n t s e r a . U n f o r t u n a t e l y , more s e r u m was n o t a v a i l a b l e , s o t h e I g G ^ a n d t h e I g E s t u d i e s c o u l d n o t be r e p e a t e d w i t h PCA o r an a l t e r n a t e a s s a y m e t hod. 4.2.4) L y m p h o c y t e T r a n s f e r s T a b l e 4.5 shows t h e v i s u a l g r a d e a s s i g n e d t o e a c h o f t h e l y m p h o c y t e r e c i p i e n t s . A l l f o u r g u i n e a p i g s w h i c h r e c e i v e d l y m p h o c y t e s f r o m t o l e r a n t a n i m a l s r e a c t e d w i t h s t r o n g b r o n c h o c o n s t r i c t i o n t o t h e OA a e r o s o l c h a l l e n g e . T h r e e o f t h e f o u r a n i m a l s w h i c h r e c e i v e d l y m p h o c y t e s f r o m n o n - s e n s i t i z e d c o n t r o l a n i m a l s r e a c t e d s t r o n g l y . 4.2.5) I n V i t r o A n t i g e n - I n d u c e d H i s t a m i n e R e l e a s e T a b l e 4.6 shows t h e r e s u l t s f o r t h e i n v i t r o h i s t a m i n e r e l e a s e f r o m t i s s u e f r o m a c u t e l y s e n s i t i z e d n o n - e x p o s e d a n i m a l s and c h r o n i c a l l y e x p o s e d t o l e r a n t a n i m a l s . The g r o u p s h a v e a s i m i l a r maximum r e l e a s e o f h i s t a m i n e , w i t h a p p r o x i m a t e l y 20 % a n t i g e n - i n d u c e d r e l e a s e o f h i s t a m i n e o b s e r v e d w i t h t h e t h r e e h i g h e s t OA c o n c e n t r a t i o n s . The r e l e a s e o f h i s t a m i n e i n r e s p o n s e t o 1.1 x 1 0 ~ 9 M OA i s s i g n i f i c a n t l y l e s s ( 0 . 3 4 +_ 0 . 2 7 y u g h i s t a m i n e r e l e a s e d / g t i s s u e , 1.0 _+ 0.7 % h i s t a m i n e r e l e a s e d ) f o r t h e t o l e r a n t g r o u p t h a n f o r t h e s e n s i t i z e d g r o u p ( 1 . 2 +_ 0 . 3 > i g - 62 -h i s t a m i n e r e l e a s e d / g t i s s u e , 5.1 +_ 1.4 % h i s t a m i n e r e l e a s e d ) ( u n p a i r e d T - t e s t p < 0 . 0 5 ) . C o m p a r i s o n o f t h e two g r o u p s o v e r a l l t h e OA c o n c e n t r a t i o n s u s i n g a t h r e e - w a y ANOVA shows no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e g r o u p s . T h e s e r e s u l t s a r e shown g r a p h i c a l l y i n F i g . 4.13. - 63 Table 4 . 5 . Visual Grade Assessment of Lymphocyte Transfer Effect on Response to OA Challenge Lymphocytes Donated By: Tolerant guinea pigs Control guinea pigs Visual 2 2 2 2 2 2 2 0 Grade T a b l e 4.6. O v a l b u m i n - I n d u c e d H i s t a m i n e R e l e a s e f r o m C h opped G u i n e a P i g Lung O v a l b u m i n (M) G r o u p 2.2 x 1 0 " 8 1.1 x I O - 9 2.2 x 10-7 2.2 x 1 0 " 6 2.2 x 10-5 2.2 x i o - 4 S e n s i t i z e d 1.2 + 0.3 * 3.3 + 0.7 4.2 + 1.0 5.0 + 2.3 6.1 + 2.8 6.9 + 3.3 N=4 ( 5 . 1 + 1.4) ( 1 1 . 6 + 1.3) (14.8 + 1.6) ( 1 6 . 6 + 2.5) (19.6 + 3.1) ( 2 2 . 4 + 3.4) T o l e r a n t 0.34 + 0.27a 2.6 + 0.8 4.7 + 1.2 4.9 + 1.1 5.4 + 1.0 5.5 + 1.2 N=4 ( 1 . 0 + 0 . 7 ) b ( 7 . 7 + 2.5) ( 1 2 . 4 + 3.4) (20.4 + 5.8) (16.9 + 2.6) ( 1 9 . 7 + 4.6) a V a l u e s a r e mean_/jg h i s t a m i n e r e l e a s e d / g wet l u n g +_ SEM b V a l u e s a r e mean % r e l e a s e o f h i s t a m i n e + SEM * S i g n i f i c a n t l y d i f f e r e n t f r o m T o l e r a n t t i s s u e a t t h i s OA c o n c e n t r a t i o n ( p < 0 . 0 5 ) - 65 2 8 J 24-1 Ovalbumin (M) F i g u r e 4.13. C o m p a r i s o n o f % h i s t a m i n e r e l e a s e i n d u c e d by o v a l b u m i n f o r S e n s i t i z e d (N=4) ( A ) a n d T o l e r a n t (N=4) ( § ) g r o u p s . O S i g n i f i c a n t d i f f e r e n c e (p <0.05) b e t w e e n t h e g r o u p s a t 1.1 x 1 0 ~ 9 M OA. - 66 -CHAPTER 5. DISCUSSION The d e v e l o p m e n t o f a s t a t e o f n o n - r e a c t i v i t y ( t o l e r a n c e ) t o a n t i g e n c h a l l e n g e i n s e n s i t i z e d , c h r o n i c a l l y a e r o s o l c h a l l e n g e d g u i n e a p i g s h a s b e e n n o t e d p r e v i o u s l y ( 3 9 , 125-127, 130, 145) a n d h a s i n t h i s s e r i e s o f e x p e r i m e n t s b e e n f u r t h e r e x p l o r e d . F o r many, t h e g o a l o f t h e u s e o f g u i n e a p i g a n a p h y l a x i s i s t h e d e v e l o p m e n t o f a m o d e l o f c h r o n i c human a s t h m a . W h i l e t h e l o s s o f a n a n a p h y l a c t i c r e s p o n s e t o a n t i g e n c h a l l e n g e p r e c l u d e s t h e u s e o f g u i n e a p i g a n a p h y l a x i s f o r t h i s p u r p o s e , t h e u n d e r s t a n d i n g o f t h e m e c h a n i s m o f t h i s t o l e r a n c e h o l d s p o s s i b i l i t i e s f o r i n t e r f e r e n c e a n d s u b s e q u e n t d e v e l o p m e n t o f a c h r o n i c m o d e l . I n a d d i t i o n , i t may s u g g e s t o t h e r a r e a s f o r i n v e s t i g a t i n g t h e e t i o l o g y a n d t r e a t m e n t o f human a s t h m a . O v e r t h e weeks o f d a i l y a e r o s o l c h a l l e n g e s e v e r a l f e a t u r e s o f t h e g u i n e a p i g m o d e l became a p p a r e n t . One o f t h e m o s t s t r i k i n g i s t h e l a r g e v a r i a t i o n i n i n h e r e n t r e a c t i v i t y among t h e a n i m a l s . T h i s l a r g e v a r i a t i o n i s p r e s e n t i n b o t h o u t b r e d H a r t l e y g u i n e a p i g s a n d i n b r e d S t r a i n 13 g u i n e a p i g s . The a n i m a l s c o u l d be c l a s s i f i e d a s r a n g i n g f r o m " h i g h r e s p o n d e r " t o "low r e s p o n d e r " g u i n e a p i g s ( F i g . 5 . 1 ) . Once t h e y h a d d e v e l o p e d a t o l e r a n t s t a t e , some a n i m a l s o c c a s i o n a l l y h a d s p o r a d i c e p i s o d e s o f ' b r e a k t h r o u g h ' r e a c t i v i t y . T h e s e w o u l d o n l y l a s t one d a y , w e r e n o t l i n k e d t o t h e ' b r e a k t h r o u g h s ' by o t h e r g u i n e a p i g s a n d h a d no a p p a r e n t t e m p o r a l c y c l e . T h e r e was a l s o v a r i a t i o n i n t h e p a t t e r n o f r e s p o n s e i n t h e f i r s t two weeks o f a n t i g e n e x p o s u r e . R o u g h l y 50 % o f t h e g u i n e a p i g s o - 67 -1 oH Mean Visual Grade/Exposure j i F i g u r e 5.1. T h i r t y - t w o g u i n e a p i g s were a s s i g n e d a v i s u a l g r a d e f o r r e a c t i v i t y t o c h r o n i c o v a l b u m i n c h a l l e n g e o v e r a p e r i o d o f f i v e t o s e v e n weeks. The t o t a l o f a l l g r a d e s was d i v i d e d by t h e number o f e x p o s u r e s t o p r o v i d e a d i s t r i b u t i o n o f r e a c t i v i t y among t h e g r o u p o f a n i m a l s . See t e x t f o r e x p l a n a t i o n o f g r a d i n g s y s t e m . - 68 -e x h i b i t e d t h e i r s t r o n g e s t r e s p o n s e i n t h e f i r s t week o f c h a l l e n g e , w h i l e t h e o t h e r 50 % r e s p o n d e d m o s t s t r o n g l y d u r i n g t h e s e c o n d week. T h i s v a r i a t i o n s u g g e s t s t h a t n o t a l l a n i m a l s a r e r a p i d l y t h o r o u g h l y s e n s i t i z e d by t h e i n t r a p e r i t o n e a l OA/HKP i n j e c t i o n . T h o s e a n i m a l s w h i c h do n o t r e a c t u n t i l t h e s e c o n d week may be f u r t h e r s e n s i t i z e d by i n h a l a t i o n o r j u s t r e q u i r e a l o n g e r s e n s i t i z a t i o n t i m e t o p r o d u c e a v i s i b l e r e s p o n s e t o c h a l l e n g e . O t h e r i n v e s t i g a t o r s h a v e s u c c e s s f u l l y u s e d a e r o s o l d e l i v e r y o f a n t i g e n a s a s e n s i t i z a t i o n m e t h o d ( 1 1 1 , 1 2 6 ) . A l t h o u g h i n t r a p e r i t o n e a l i n j e c t i o n o f a n t i g e n i s m o s t common ( 1 1 1 , 127, 1 2 8 ) , f e e d i n g o f a n t i g e n ( 1 3 0 ) a n d p a s s i v e s e n s i t i z a t i o n (146) h a v e a l s o b e e n u s e d . A b d o m i n a l p r o c e s s i n g o f a n t i g e n seems t o o c c u r i n e a c h o f t h e a c t i v e s e n s i t i z a t i o n m e t h o d s ( 4 5 ) . A e r o s o l c h a l l e n g e t o m i m i c t h e c o n d i t i o n s o f p r o v o c a t i o n o f human a l l e r g i c a s t h m a i s t h e m o s t common method o f a s s e s s i n g t h e l e v e l o f b r o n c h o c o n s t r i c t i v e r e a c t i v i t y . D e p e n d i n g upon t h e s c h e d u l e o f a n t i g e n e x p o s u r e , t h e d i m i n i s h e d r e a c t i v i t y i n r e s p o n s e t o c h r o n i c a e r o s o l c h a l l e n g e o c c u r s a s e a r l y a s t h e s e c o n d week o f a n t i g e n e x p o s u r e ( 1 1 1 , 146) o r a s l a t e a s t h e f o u r t h week ( 1 4 7 ) . D u r i n g t h e c o u r s e o f r e p e a t e d a e r o s o l e x p o s u r e s i t was o b s e r v e d t h a t t h e l e n g t h o f t i m e b e f o r e o n s e t o f c y a n o s i s a n d b r o n c h o c o n s t r i c t i o n i n c r e a s e d . I n t h e f i r s t few c h a l l e n g e s , r e s p o n s e s w ere e l i c i t e d w i t h i n t h e f i r s t s e v e r a l m i n u t e s . A f t e r two weeks o f c h a l l e n g e , numerous a n i m a l s w e r e t a k i n g a s l o n g a s 40-50 m i n . b e f o r e e x h i b i t i n g t h e i r r e a c t i o n . T h i s phenomenon i n d i c a t e s t h e g r a d u a l o n s e t o f t o l e r a n c e , a n d seems t o be r e l a t e d t o t h e d o s e o r c o n c e n t r a t i o n o f - 69 -t h e a n t i g e n b e i n g p r o c e s s e d by t h e a n i m a l , s i n c e P a r e e t a l ( 1 1 1 ) f o u n d t h a t by i n c r e a s i n g t h e c o n c e n t r a t i o n o f OA i n t h e a e r o s o l t h e i n i t i a l l e v e l o f b r o n c h o c o n s t r i c t i o n c o u l d be m a i n t a i n e d . T h e l o n g e r t h e a n i m a l s i n h a l e a n t i g e n , t h e l a r g e r t h e c o n c e n t r a t i o n w i l l be w i t h i n t h e l u n g s . T h i s a p p a r e n t d o s e - d e p e n d e n c y may i n v o l v e a c o n c e n t r a t i o n g r a d i e n t o f a n t i g e n i n t h e mucus, a n d / o r c o m p e t i t i o n f o r a n t i g e n - s p e c i f i c IgG-^ o r I g E a t t h e l e v e l o f t h e m a s t c e l l . As t o l e r a n c e d e v e l o p s , t h e minimum amount o f a n t i g e n r e q u i r e d t o e l i c i t a r e s p o n s e i n c r e a s e s , t h u s d e m a n d i n g more t i m e a n d an i n c r e a s e d OA c o n c e n t r a t i o n a t t h e t a r g e t s i t e . T h e r e may a l s o be an i n c r e a s e d e f f i c i e n c y o f r e m o v a l o f t h e a n t i g e n f r o m t h e a i r w a y , p e r h a p s b y i m p r o v e d t r a n s p o r t o f t h e a n t i g e n - l a d e n mucus. As an o b j e c t i v e means o f a s s e s s i n g b r o n c h o c o n s t r i c t i v e r e a c t i v i t y l u n g m e c h a n i c s w e r e s t u d i e d on t h e c h r o n i c a l l y c h a l l e n g e d g u i n e a p i g s . T h i s p r o v i d e d a c o r r o b o r a t i o n f o r t h e d e v e l o p m e n t o f t o l e r a n c e a s a s s e s s e d by t h e v i s u a l g r a d i n g . The s t a b i l i t y o r l a b i l i t y o f t o l e r a n c e when a n t i g e n e x p o s u r e s a r e t e r m i n a t e d i s o f i n t e r e s t . H i c k s and S k e l d o n ( 1 2 8 ) h a v e r e p o r t e d t h a t when g u i n e a p i g s a r e d e s e n s i t i z e d by r e p e a t e d c h a l l e n g e a f t e r a 30-60 da y i n i t i a l s e n s i t i z a t i o n t i m e , r e s e n s i t i z a t i o n i s i m p a i r e d a f t e r a 7 d a y l a y - o f f . H e r x h e i m e r ( 1 2 7 ) h a s r e p o r t e d t h a t a f t e r d e s e n s i t i z a t i o n , r e s e n s i t i z a t i o n o c c u r s t o a v a r i a b l e d e g r e e d e p e n d i n g u p o n t h e number o f d a y s w i t h o u t c h a l l e n g e a n d t h e i n d i v i d u a l g u i n e a p i g . I n t h e a e r o s o l l a y - o f f e x p e r i m e n t s d e s c r i b e d h e r e , w i t h h o l d i n g t h e a e r o s o l f o r s e v e r a l d a y s d o e s n o t r e s u l t i n a u n i f o r m r e t u r n t o t h e s e n s i t i v e - 70 -s t a t e . R e s p o n s e s f r o m some o f t h e 22 o r i g i n a l l y u n r e s p o n s i v e a n i m a l s w h i c h were w i t h h e l d f r o m OA e x p o s u r e f o r 4-24 d a y s do show an i n c r e a s e d b r o n c h o c o n s t r i c t i o n i n r e s p o n s e t o t h e a e r o s o l when a s s e s s e d by e i t h e r a v i s u a l g r a d e o r l u n g m e c h a n i c s . Some p o s s i b l e m e c h a n i s m s f o r t o l e r a n c e i n c l u d e d e c r e a s e d s m o o t h m u s c l e s e n s i t i v i t y t o h i s t a m i n e , d e p l e t i o n o f m e d i a t o r s i n m a s t c e l l s , a l t e r e d m a s t c e l l f u n c t i o n , o r an i m m u n o l o g i c a l l y m e d i a t e d m e c h a n i s m . The i n v i t r o a i r w a y s m o o t h m u s c l e s t u d y was u n d e r t a k e n t o i n v e s t i g a t e t h e p o s s i b i l i t y o f a d e c r e a s e d s m o o t h m u s c l e r e s p o n s e t o h i s t a m i n e i n t o l e r a n t a n i m a l s . C o m p a r i s o n o f t h e f o r c e s e l i c i t e d b y c o n t r a c t i o n o f t h e t i s s u e s e g m e n t s a t t h e m a x i m a l h i s t a m i n e d o s e showed t h e s e p a t t e r n s ( F i g . 4.6) i n a l l o f t h e g r o u p s ( C , T, and S ) : UT > L T ; UT > B r , P a r ; LT = B r ; LT > P a r ; B r > P a r . T h e s e d i f f e r e n c e s may be a t t r i b u t e d t o v a r i o u s t i s s u e c h a r a c t e r i s t i c s s u c h a s : d e c r e a s i n g s m o o t h m u s c l e i n l o w e r a i r w a y s , more c o n n e c t i v e t i s s u e i n l o w e r a i r w a y s , c h a n g e s i n a i r w a y h i s t a m i n e r e c e p t o r p o p u l a t i o n , c h a n g e s i n r e c e p t o r r e s p o n s i v e n e s s , v a r i a t i o n s i n s i z e o f t i s s u e a n d v a r i a t i o n s i n c u t t i n g o f t i s s u e . C o n v e r s i o n o f t h e d a t a t o p e r c e n t a g e o f m a x i m a l t i s s u e r e s p o n s e ( u s u a l l y e l i c i t e d by c a r b a c h o l b u t s o m e t i m e s by maximum h i s t a m i n e d o s e ) n o r m a l i z e s f o r t h e s e d i f f e r e n c e s s o t h a t UT, L T a n d B r r e s p o n s e s i n a l l t h e g r o u p s become t h e same. The p a r e n c h y m a , h o w e v e r , r e m a i n s s i g n i f i c a n t l y d i f f e r e n t (ANOVA, O r t h o g o n a l c o n t r a s t s p < 0 . 0 5 ) f r o m UT, L T and B r i n a l l g r o u p s . A t l o w h i s t a m i n e d o s e s t h e p a r e n c h y m a a c h i e v e s a l a r g e r p r o p o r t i o n o f i t s maximum t h a n do UT, LT a n d B r . - 71 -P o s s i b l e r e a s o n s f o r t h i s c h a r a c t e r i s t i c o f a p p a r e n t l y g r e a t e r h i s t a m i n e s e n s i t i v i t y by P a r may be an i n c r e a s e d number o f h i s t a m i n e r e c e p t o r s a n d / o r i n c r e a s e d r e c e p t o r r e s p o n s i v e n e s s , o r l a r g e r n umbers o f a l p h a - ^ r e c e p t o r s ( 1 6 ) . A n o t h e r p o s s i b i l i t y i s t h a t t h e r e i s l e s s r e s i s t a n c e t o c o n t r a c t i o n f r o m t h e l a c k o f c a r t i l a g e i n p a r e n c h y m a , s o s m a l l s m o o t h m u s c l e c o n t r a c t i o n s a r e more e a s i l y o b s e r v e d as a d e c r e a s e d t i s s u e l e n g t h . The c r i t i c a l c o n c l u s i o n f r o m t h i s s t u d y o f i n v i t r o s m o o t h m u s c l e r e s p o n s e t o h i s t a m i n e , h o w e v e r , i s t h a t t h e T o l e r a n t , S e n s i t i z e d , a n d C o n t r o l g r o u p s d i s p l a y e q u a l r e a c t i v i t y t o h i s t a m i n e . The s e c o n d p a r t o f t h i s e x p e r i m e n t i n v e s t i g a t e d t h e r e s p o n s e s o f t h e t i s s u e s t o OA. The c o n t r a c t i l e r e s p o n s e s o b t a i n e d f r o m e a c h t i s s u e o f e a c h g r o u p showed t h e f e a t u r e s o f t h e t y p i c a l S c h u l t z - D a l e r e a c t i o n ( 1 4 8 ) . T h e s e a r e a r a p i d o n s e t o f r e s p o n s e , s e n s i t i v i t y t o low OA c o n c e n t r a t i o n s , s u s t a i n e d c o n t r a c t i o n s r e q u i r i n g l o n g w a s h i n g t o rem o v e , a n d d e s e n s i t i z a t i o n o f t h e t i s s u e t o a s e c o n d e x p o s u r e t o OA. The r i g h t w a r d s h i f t i n t h e " t o l e r a n t " OA d o s e - r e s p o n s e c u r v e s u g g e s t s a d e c r e a s e d m e d i a t o r r e l e a s e f r o m mast c e l l s i n r e s p o n s e t o t h e OA s t i m u l u s . The c h a r a c t e r o f r e s p o n s e t o OA o f t h e a i r w a y s m o o t h m u s c l e a p p e a r s u n c h a n g e d b e t w e e n t o l e r a n t a n d s e n s i t i z e d a n i m a l s . T h e r a t e s o f c o n t r a c t i o n a r e t h e same f o r t o l e r a n t a n d s e n s i t i z e d g r o u p s e x c e p t f o r t h e t o l e r a n t b r o n c h i a l r a t e o f c o n t r a c t i o n w h i c h i s s i g n i f i c a n t l y g r e a t e r (ANOVA, O r t h o g o n a l c o n t r a s t s p < 0 . 0 1 ) t h a n t h e s e n s i t i z e d b r o n c h i a l r a t e o f c o n t r a c t i o n . T h e r e l e v a n c e o f t h i s o b s e r v a t i o n i s u n c e r t a i n s i n c e o n l y t h e b r o n c h i a l t i s s u e s e g m e n t s - 72 -d i s p l a y e d t h i s p r o p e r t y . I t may i n d i c a t e t h a t an a n a p h y l a c t i c r e a c t i o n i n g u i n e a p i g s i s s t r o n g e s t i n t h e b r o n c h i due t o a l t e r e d r e c e p t o r s . A common f i n d i n g i n human a s t h m a i s h y p e r p l a s i a a n d h y p e r t r o p h y o f a i r w a y s m o o t h m u s c l e . T h e s e o r g a n b a t h s t u d i e s g i v e i n d i r e c t e v i d e n c e t h a t s u c h a i r w a y s m o o t h m u s c l e c h a n g e s h a v e n o t o c c u r r e d i n t h e c h r o n i c a l l y O A - e x p o s e d , t o l e r a n t g u i n e a p i g s . T h i s c o n c l u s i o n c a n be drawn f r o m t h e f a c t t h a t t h e maximum f o r c e g e n e r a t e d b y t o l e r a n t , s e n s i t i z e d a n d c o n t r o l a n i m a l s i s t h e same. S e c o n d a r i l y , t h e r a t e s o f c o n t r a c t i o n a r e s i m i l a r e x c e p t f o r t h e p r e v i o u s l y m e n t i o n e d b r o n c h i a l s e g m e n t s . The p o s s i b i l i t y t h a t t o l e r a n c e may be due t o d e c r e a s e d m e d i a t o r s i n t h e l u n g was a p p r o a c h e d by c o m p a r i n g l u n g h i s t a m i n e c o n t e n t s o f c o n t r o l , s e n s i t i z e d a n d t o l e r a n t a n i m a l s . The e m p h a s i s p u t on h i s t a m i n e i n t h e s e e x p e r i m e n t s d o e s n o t i n t e n d t o i g n o r e t h e c o n t r i b u t i o n o f o t h e r m e d i a t o r s o f a n a p h y l a x i s , b u t t h e a c t i o n s o f h i s t a m i n e a r e r e l a t i v e l y w e l l u n d e r s t o o d , i t c a n be r e a d i l y a s s a y e d , a n d i t h a s b e e n shown t o be an i m p o r t a n t i n d u c e r o f a i r w a y c o n s t r i c t i o n i n g u i n e a p i g s ( 1 4 9 - 1 5 1 ) . The f i n d i n g t h a t h i s t a m i n e was n o t d e p l e t e d i n c h r o n i c a l l y c h a l l e n g e d a n i m a l s was a l s o r e p o r t e d b y B r o d e r ( 1 4 6 ) . H owever, i n o u r g r o u p o f t o l e r a n t H a r t l e y g u i n e a p i g s , t h e h i s t a m i n e c o n t e n t was s i g n i f i c a n t l y g r e a t e r t h a n t h a t o f c o n t r o l a n d s e n s i t i z e d a n i m a l s . T h i s s u g g e s t s t h a t t h e r e may be a me c h a n i s m i n h i b i t i n g t h e r e l e a s e o f h i s t a m i n e . A n o t h e r p o s s i b l e n o n - i m m u n o l o g i c a l l y m e d i a t e d m e c h a n i s m h a s b e e n - 73 -i n v e s t i g a t e d by M c C a i g and S o u h r a d a ( 3 5 ) . T h e r e s t i n g membrane p o t e n t i a l o f g u i n e a p i g a i r w a y s m o o t h m u s c l e was o b s e r v e d t o d e c r e a s e w i t h c h r o n i c i n h a l a t i o n o f a e r o s o l i z e d a l b u m i n . I n a d d i t i o n , a i r w a y s m o o t h m u s c l e d e p o l a r i z e d t o a l e s s e r e x t e n t i n r e s p o n s e t o h i s t a m i n e and OA a s t h e p e r i o d o f c h r o n i c c h a l l e n g e i n c r e a s e d . P r e s e n t d a t a i s n o t c o m p a t i b l e w i t h s u c h c h a n g e s s i n c e t h e a i r w a y s m o o t h m u s c l e r e s p o n s i v e n e s s t o h i s t a m i n e was u n c h a n g e d . W i t h no a p p a r e n t p h y s i o l o g i c m e c h a n i s m h a v i n g b e e n shown, i m m u n o l o g i c m e c h a n i s m s were c o n s i d e r e d . Some p o s s i b l e e x p l a n a t i o n s f o r t h e d e v e l o p m e n t o f t o l e r a n c e i n c l u d e a d e c r e a s e i n h o m o c y t o t r o p i c a n t i b o d y l e v e l s , t h e p r e s e n c e o f a b l o c k i n g a n t i b o d y , d e c r e a s e d a n t i b o d y r e c e p t o r s on mast c e l l s , o r s u p p r e s s o r c e l l s o r f a c t o r s a c t i n g t o p r e v e n t mast c e l l m e d i a t o r r e l e a s e . Many o t h e r i n v e s t i g a t o r s h a v e a t t e m p t e d t o c o r r e l a t e t h e PCA t i t r e w i t h t h e l e v e l o f a n a p h y l a c t i c r e s p o n s e ( 5 2 , 53, 126, 128, 1 3 0 ) . T h e s e s t u d i e s h a v e shown t h a t t h e r e i s no c o r r e l a t i o n b e t w e e n t h e r a p i d i t y o f t h e r e s p o n s e a nd t h e PCA t i t r e ( 1 2 6 ) . F u r t h e r , t h e a b s e n c e o r d i m i n i s h m e n t o f a r e s p o n s e d o e s n o t c o r r e l a t e w i t h a d e c r e a s e d PCA t i t r e ( 1 2 6 , 128, 1 3 0 ) . The o n l y p o s i t i v e c o r r e l a t i o n b e t w e e n PCA r e a c t i o n s a n d a n a p h y l a x i s i s t h a t when a n a p h y l a c t i c r e s p o n s e s a r e p r e s e n t , s o a r e PCA r e a c t i o n s . T h e r e a r e t h r e e h o m o c y t o t r o p i c a n t i b o d i e s known i n t h e g u i n e a p i g . T h e s e a r e I g G ^ _ ( 1 5 2 ) , I g G - ^ ( 1 5 3 ) a nd r e a g i n i c ( 5 6 , 154-156) a n t i b o d i e s . The p r e s e n c e o f t h e s e a n t i b o d i e s c a n be d e t e c t e d b y a l l o w i n g d i f f e r e n t t i m e s f o r s e n s i t i z a t i o n , a n d h e a t i n g some s e r u m s a m p l e s . I g G j _ a n t i b o d i e s a r e - 74 -s t a b l e t o h e a t i n g a t 5 6 ° C f o r 1 h o u r a n d a r e p r e s e n t i n mg/ml l e v e l s i n t h e s e r u m o f g u i n e a p i g s ( 1 5 7 ) . T h e I g G l g c a n be d e t e c t e d a f t e r a 4 h o u r s e n s i t i z a t i o n p e r i o d , b u t p e r s i s t s i n t h e s k i n f o r o n l y 1-4 d a y s ( 5 4 , 157) w h i l e I g G - ^ c a n be d e t e c t e d a f t e r 18 h o u r s o f s e n s i t i z a t i o n ( 5 3 ) and p e r s i s t s f o r up t o 7 d a y s ( 5 3 , 55) a l t h o u g h b e c o m i n g i n c r e a s i n g l y weak. I g E e x i s t s i n o n l y ng/ml q u a n t i t i e s i n s e r u m , i s l a b i l e t o h e a t i n g a t 5 6 ° C f o r 1 h o u r , a n d p e r s i s t s i n s k i n f o r s e v e r a l weeks ( 1 5 7 ) . More t h a n 18 h o u r s i s r e q u i r e d f o r s e n s i t i z a t i o n ( 5 3 ) . The s i x h o u r s e n s i t i z a t i o n t h e r e f o r e shows o n l y I g G - ^ a n t i b o d y , and t h e 4 day s e n s i t i z a t i o n w i t h h e a t e d s e r u m shows o n l y I g G - ^ a n t i b o d y . The 7 day s e n s i t i z a t i o n shows p r i m a r i l y t h e p r e s e n c e o f I g E , a l t h o u g h some c o n t r i b u t i o n f r o m r e s i d u a l I g G l h may be p r e s e n t . ^ G ^ g was shown t o be s i g n i f i c a n t l y d e c r e a s e d i n t o l e r a n t g u i n e a p i g s e r u m , t h u s p r o v i d i n g a p o s s i b l e m e c h a n i s m f o r d e c r e a s e d r e a c t i v i t y . A l t h o u g h t h e r e d o e s n o t seem t o be a l a r g e d i f f e r e n c e b e t w e e n t h e two g r o u p s , t h e r e i s a c o n s i s t e n t t r e n d t o s l i g h t l y s m a l l e r a r e a s o f b l u e i n t h e t o l e r a n t g r o u p . What t h i s means i n t e r m s o f a c t u a l d i f f e r e n c e s i n Ab t i t r e i s n o t known, and e v e n i f i t w e r e , t h e e x a c t r e l a t i o n s h i p b e t w e e n Ab q u a n t i t y a n d m a s t c e l l r e l e a s e o f m e d i a t o r s i s unknown. I f , f o r i n s t a n c e , a l l t h e F c r e c e p t o r s a r e s a t u r a t e d a t t h e l e v e l o f Ab p r e s e n t i n t h e t o l e r a n t g r o u p o r a t an e v e n l o w e r Ab l e v e l , m e d i a t o r r e l e a s e w o u l d n o t be i n h i b i t e d i n t h e t o l e r a n t g r o u p , e v e n t h o u g h l e s s Ab was c i r c u l a t i n g . No d i r e c t a s s a y s f o r b l o c k i n g a n t i b o d i e s were c a r r i e d o u t , s i n c e e x p e r i m e n t s by B l a n c h a r d e t a l ( 1 2 6 ) i n d i c a t e d t h a t a l t h o u g h - 75 -p r e c i p i t a t i n g a n t i b o d i e s were p r e s e n t i n t h e s e r u m o f n o n - r e s p o n s i v e a n i m a l s , t h e s e d i d n o t b l o c k t h e PCA r e a c t i o n . A n o t h e r p o s s i b i l i t y f o r an a n t i b o d y - m e d i a t e d m e c h a n i s m o f t o l e r a n c e h a s b e e n s u g g e s t e d by A n d r e e t a l ( 1 4 5 ) i n t h e i r s y s t e m o f m i c e i n t r a g a s t r i c a l l y i m m u n i z e d w i t h SRBC. T h e i r r e s u l t s i n d i c a t e t h e f o r m a t i o n o f IgA immune c o m p l e x e s w i t h e n t e r i c i m m u n i z a t i o n . T h i s p r o d u c e s a t o l e r o g e n i c e f f e c t w h i c h c a n be t r a n s f e r r e d t o v i r g i n r e c i p i e n t s by way o f s e r u m . A t t e m p t s a t t r a n s f e r r i n g s e r u m f r o m n o n - r e s p o n s i v e g u i n e a p i g s t o a c u t e l y s e n s i t i z e d g u i n e a p i g s h a s s o f a r f a i l e d t o i n h i b i t t h e a n a p h y l a c t i c r e s p o n s e ( 1 2 6 ) . The p o s s i b i l i t y t h a t IgA i s f o r m e d and a c t s l o c a l l y i n t h e l u n g mucosa as a " b l o c k i n g a n t i b o d y " c a n n o t be r u l e d o u t , h o w e v e r . The p r e s e n c e o f s u p p r e s s o r c e l l s h a s b e e n w e l l d o c u m e n t e d i n v a r i o u s s y s t e m s ( 1 5 8 - 1 6 2 ) a n d i t s r o l e i n t h e d e v e l o p m e n t o f i m m u n o l o g i c a l n o n - r e s p o n s i v e n e s s h a s s u p p o r t i n n u m e r o u s s y s t e m s . A s u b - p o p u l a t i o n o f T - c e l l s h a v i n g a number o f f u n c t i o n s h a s b e e n a c t i v a t e d w i t h ConA i n v i t r o . The a c t i o n s o f t h i s p o p u l a t i o n o f c e l l s i n c l u d e s u p p r e s s i o n o f a n t i b o d y p r o d u c t i o n ( 1 6 3 , 1 6 4 ) , s u p p r e s s i o n o f c y t o t o x i c l y m p h o c y t e g e n e r a t i o n ( 1 6 5 , 1 6 6 ) , s u p p r e s s i o n o f T - c e l l m i t o g e n i c r e s p o n s e ( 1 6 7 ) a n d s u p p r e s s i o n o f m i x e d l y m p h o c y t e c u l t u r e (MLC) ( 1 6 8 ) . M i l l e r e t a l ( 1 4 7 ) f o u n d t h a t a n t i g e n - s p e c i f i c s u p p r e s s o r T - c e l l s f r o m O A - f e d m i c e c o u l d i n d u c e s u p p r e s s i o n o f h u m o r a l a n d CMI r e s p o n s e s when t r a n s f e r r e d t o n o r m a l r e c i p i e n t s . Ngan and K i n d ( 1 6 9 ) d e t e c t e d s u p p r e s s o r T - c e l l s f o r I g E a n d I g G i n P e y e r ' s p a t c h e s o f m i c e made t o l e r a n t by t h e o r a l a d m i n i s t r a t i o n o f o v a l b u m i n . A s i m i l a r - 76 -f i n d i n g h a s b e e n r e p o r t e d by C h i o r a z z i , F o x and K a t z ( 1 7 0 ) where I g E " n o n - r e s p o n d e r " mouse s t r a i n s w ere c o n v e r t e d t o I g E " r e s p o n d e r s " b y t h e e l i m i n a t i o n o f s u p p r e s s o r T - c e l l a c t i v i t y . The m e c h a n i s m o f a c t i o n o f s u p p r e s s o r c e l l s i s n o t c o m p l e t e l y u n d e r s t o o d , b u t t h e p r o d u c t i o n o f s u p p r e s s o r f a c t o r s i s a s t r o n g p o s s i b i l i t y . K a t z and c o - w o r k e r s ( 1 7 1 , 172) h a v e d e t e c t e d a n o n - i m m u n o g l o b u l i n " s u p p r e s s i v e f a c t o r o f a l l e r g y " ( S F A ) p r e s e n t i n t h e s e r u m and a s c i t e s f l u i d s o f C F A - i n o c u l a t e d l o w I g E r e s p o n d e r m i c e . X - i r r a d i a t i o n o f l o w - r e s p o n d e r m i c e n e a r t h e t i m e o f p r i m a r y i m m u n i z a t i o n c a u s e s an i n c r e a s e i n I g E p r o d u c t i o n , a n d r e s u l t s i n e n h a n c e d p r i m a r y o r a d o p t i v e s e c o n d a r y I g E a n t i b o d y r e s p o n s e s . G i v i n g s e r u m w i t h S F A t o i r r a d i a t e d m i c e a b o l i s h e d t h e i r r a d i a t i o n - e n h a n c e d r e s p o n s e s . T r a n s f e r o f SFA s e r a t o h i g h r e s p o n d e r m i c e , h o w e v e r , showed t h e s e m i c e t o be r e l a t i v e l y u n a f f e c t e d by t h e s u p p r e s s i v e a c t i v i t y . K o n t i a i n e n e t a l ( 1 7 3 ) h a v e a l s o r e p o r t e d a s u p p r e s s o r f a c t o r p r o d u c e d i n v i t r o w h i c h when i n j e c t e d a b o u t t h e t i m e o f K L H - a n t i g e n a d m i n i s t r a t i o n i n h i b i t s t h e i n v i t r o IgM a n d I g G r e s p o n s e t o t r i n i t r o p h e n y l KLH. O f most i n t e r e s t , h o w e v e r , i s t h e p r e s e n c e o f a human s u p p r e s s i v e f a c t o r o f a l l e r g y w h i c h h a s b e e n d e m o n s t r a t e d by K a t z ( 1 7 4 ) i n an i n v i t r o s y s t e m i n w h i c h a d d i t i o n o f human SFA i n h i b i t e d I g E a n t i b o d y s y n t h e s i s b y human p e r i p h e r a l m o n o n u c l e a r c e l l s . A r e g u l a t o r y l i n k b e t w e e n t h e m e d i a t o r s o f a n a p h y l a x i s a n d t h e c e l l s o f t h e immune s y s t e m h a s b e e n d e t e c t e d by t h e d i s c o v e r y o f l y m p h o c y t e s w h i c h b i n d h i s t a m i n e ( 1 7 5 - 1 7 9 ) . S u p p r e s s o r c e l l s g e n e r a t e d by h i s t a m i n e h a v e b e e n shown t o s u p p r e s s t h e i n v i t r o p r o l i f e r a t i v e - 77 -r e s p o n s e t o a n t i g e n (MLC) and m i t o g e n ( 1 8 0 ) . R e p o r t s o f l y m p h o c y t e s b e a r i n g h i s t a m i n e r e c e p t o r s h a v e shown t h a t t h e y a l s o m o d u l a t e i n v i t r o r e l e a s e o f h i s t a m i n e f r o m b a s o p h i l s ( 1 8 1 ) , l y m p h o c y t e - m e d i a t e d c y t o t o x i c i t y ( 1 8 2 ) , a n t i g e n - i n d u c e d MIF ( 1 8 3 ) a n d l y m p h o c y t e p r o l i f e r a t i o n ( 1 8 4 ) . A n o t h e r m e d i a t o r w h i c h a p p e a r s t o be i n v o l v e d i n t h e s t i m u l a t i o n o f n o n - s p e c i f i c T s u p p r e s s o r c e l l s i s p r o s t a g l a n d i n E ( 1 8 5 - 1 8 7 ) . PGE i s known t o be p r e s e n t d u r i n g a n a p h y l a x i s a s a b r o n c h i a l s m o o t h m u s c l e r e l a x e r , s o i t a p p e a r s t h a t i t may f u n c t i o n i n two ways t o i n h i b i t t h e a n a p h y l a c t i c r e s p o n s e . The p r e s e n c e o f s u p p r e s s o r c e l l s was n o t d e m o n s t r a t e d i n t h e p r o t o c o l f o l l o w e d i n t h i s e x p e r i m e n t . However, t h i s i s n o t p r o o f t h a t s u p p r e s s o r c e l l s a r e n o t p r e s e n t . I t i s p o s s i b l e t h a t a d i f f e r e n t p r o t o c o l , p e r h a p s u s i n g a d i f f e r e n t s e n s i t i z a t i o n t i m e , m ethod o f l y m p h o c y t e i s o l a t i o n , r o u t e o f l y m p h o c y t e a d m i n i s t r a t i o n , m e t h o d o f a c t i v i t y a s s e s s m e n t o r i r r a d i a t e d r e c i p i e n t s w o u l d a l l o w d e t e c t i o n o f s u p p r e s s o r a c t i v i t y . The most o b v i o u s i n d i c a t o r o f s u p p r e s s o r a c t i v i t y , h o w e v e r , w o u l d be d e c r e a s e d a n t i b o d y p r o d u c t i o n , and t h e r e s u l t s f r o m PCA s t u d i e s s u g g e s t t h a t t h i s i s s o . P e r h a p s d a t a f r o m I g G j ^ and I g E a s s a y s w o u l d s u p p o r t t h i s . T h e r e may be a s w e l l , h o w e v e r , an a l t e r n a t i v e m e c h a n i s m o f s u p p r e s s i v e a c t i v i t y . As p r e v i o u s l y d e s c r i b e d ( 1 8 1 ) , l y m p h o c y t e s b e a r i n g h i s t a m i n e r e c e p t o r s h a v e b e e n shown t o i n h i b i t h i s t a m i n e r e l e a s e f r o m b a s o p h i l s i n v i t r o . I t i s p o s s i b l e , t h e r e f o r e , t h a t o t h e r c e l l t y p e s may be d i r e c t l y o r i n d i r e c t l y i n f l u e n c e d by a s u p p r e s s i v e m e c h a n i s m . The s t u d y o f i n v i t r o a n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e f r o m - 78 -c h o p p e d g u i n e a p i g l u n g s u g g e s t s t h a t t h e r e i s a m e c h a n i s m w h e r e b y t h e n o r m a l r e l e a s e o f h i s t a m i n e f r o m m a s t c e l l s i s i n h i b i t e d i n a n i m a l s u n r e s p o n s i v e i n v i v o t o a g i v e n l e v e l o f OA a n t i g e n a e r o s o l . M a s t c e l l s f r o m t o l e r a n t g u i n e a p i g s r e l e a s e s i g n i f i c a n t l y l e s s h i s t a m i n e t h a n a c u t e l y s e n s i t i z e d g u i n e a p i g s when e x p o s e d t o a low c o n c e n t r a t i o n o f OA. L e s s h i s t a m i n e i s r e l e a s e d b o t h q u a n t i t a t i v e l y a n d a s a p e r c e n t a g e r e l e a s e . A t t h e t i m e t h e s e e x p e r i m e n t s were c a r r i e d o u t , t h e h i s t a m i n e r e l e a s e d a t t h e l o w e s t OA c o n c e n t r a t i o n u s e d was n e a r t h e l o w e r l i m i t o f t h e s e n s i t i v i t y o f t h e h i s t a m i n e a s s a y . F o r t h i s r e a s o n , l o w e r c o n c e n t r a t i o n s o f OA c o u l d n o t be u s e d . However, a new h i s t a m i n e a s s a y w i t h g r e a t e r s e n s i t i v i t y i s now b e i n g d e v e l o p e d w h i c h w o u l d a l l o w f u r t h e r s t u d i e s a t l o w e r OA c o n c e n t r a t i o n s . A l t h o u g h o n l y f o u r g u i n e a p i g s were s t u d i e d , t h e a n i m a l a t t h e ' h i g h r e s p o n d e r s ' e n d o f t h e d i s t r i b u t i o n ( > 0.71, F i g . 5.1) r e l e a s e d a p p r e c i a b l y more h i s t a m i n e ( 1 2 8 ng) a t t h e l o w e s t OA d o s e t h a n t h o s e i n t h e ' l o w e r r e s p o n d e r s ' p o r t i o n o f t h e d i s t r i b u t i o n ( 0 - 0 . 4 0 ) , ( i . e . 2, 9, 14 ng h i s t a m i n e ) . T h i s s u g g e s t s t h a t t h e a n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e f r o m c h o p p e d l u n g s i s a f a i r l y s e n s i t i v e a s s a y o f t h e d e g r e e o f t o l e r a n c e . The c a l c u l a t i o n o f t o t a l h i s t a m i n e c o n t e n t / g wet l u n g f r o m t h e t i s s u e u s e d f o r a n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e p r o d u c e d v a l u e s 6-10 f o l d l a r g e r t h a n t h e p r e v i o u s t o t a l h i s t a m i n e a s s a y . T h i s p l a c e d t h e t o t a l h i s t a m i n e c o n t e n t f r o m t h e S t r a i n 13 g r o u p i n t o t h e r a n g e r e p o r t e d by o t h e r s ( 5 3 ) . The r e a s o n f o r t h i s d i f f e r e n c e i s n o t c l e a r , s i n c e t h e o n l y d i f f e r e n c e b e t w e e n t h e two g r o u p s i s t h a t t h e f i r s t - 79 -e x p e r i m e n t i n v o l v e d H a r t l e y g u i n e a p i g s w h i c h h a d b e e n a e r o s o l c h a l l e n g e d f o r 8-12 months a n d t h e s e c o n d e x p e r i m e n t i n v o l v e d S t r a i n 13 g u i n e a p i g s w h i c h h a d b e e n a e r o s o l c h a l l e n g e d f o r 9 m o n t h s . R a t h e r t h a n an i n a b i l i t y t o r e s p o n d , t o l e r a n c e a p p e a r s t o be an a c t i v e i n h i b i t i o n o f a s y s t e m w h i c h i s f u l l y c a p a b l e o f a c t i o n . E v i d e n c e f r o m i n v i t r o a n d i n v i v o s t u d i e s s u p p o r t t h i s . I n t h e h i g h e r c o n c e n t r a t i o n r a n g e o f O A - i n d u c e d h i s t a m i n e r e l e a s e f r o m c h o p p e d l u n g , t h e t o l e r a n t t i s s u e i s a b l e t o r e l e a s e a s much h i s t a m i n e a s t h e s e n s i t i v e t i s s u e . I n v i v o , P a r e e t a l ( 1 1 1 ) showed t h a t a n i m a l s r e c o v e r e d t h e i r r e s p o n s i v e n e s s i f t h e c o n c e n t r a t i o n o f i n h a l e d a n t i g e n was i n c r e a s e d p r o g r e s s i v e l y . T h u s , t h e m e c h a n i s m o f t o l e r a n c e i s c o n n e c t e d t o t h e q u a n t i t y o f a n t i g e n n o r m a l l y p r e s e n t i n t h e s y s t e m . I t seems, h o w e v e r , t o r e q u i r e more t h a n a s i m p l e i n c r e a s e o f a n t i g e n i n t h e s y s t e m t o o v e r c o m e t h e m e c h a n i s m o f t o l e r a n c e . T h i s i s s u g g e s t e d by an o b s e r v a t i o n made d u r i n g t h e p e r i o d o f c h r o n i c a e r o s o l c h a l l e n g e . A g r o u p o f t o l e r a n t g u i n e a p i g s was e x p o s e d t o OA f r o m a new b o t t l e . The f o l l o w i n g day t h e m a j o r i t y o f g u i n e a p i g s e x h i b i t e d s e v e r e a n a p h y l a c t i c r e s p o n s e s ( F i g . 4 . 1 ) . I t i s p o s s i b l e t h a t t h e new b o t t l e o f OA ( o f a d i f f e r e n t L o t #) was d e l i v e r i n g more a n t i g e n p e r w e i g h t t h a n t h e o l d b o t t l e , due t o t h e h a n d l i n g o f t h e o l d b o t t l e , w h i c h p r e d i s p o s e d t o much a b s o r p t i o n o f w a t e r by t h e l y o p h i l i z e d OA. T h i s s u g g e s t s t h a t some " p r o c e s s i n g " o f t h e a n t i g e n must o c c u r t o e q u i p t h e s y s t e m t o r e s p o n d t o t h e new l e v e l o f a n t i g e n ( s ) . T h e r e may a l s o h a v e b e e n new o r c o n t a m i n a t i n g a n t i g e n s i n h i g h e r c o n c e n t r a t i o n i n t h e new b o t t l e w h i c h a c t e d as a b o o s t e r and s t i m u l a t e d a r a p i d r e s p o n s e on t h e - 80 -f o l l o w i n g d a y . The f a c t t h a t t h e r e w a s n ' t a s t r o n g r e s p o n s e on t h e day o f i n i t i a l e x p o s u r e c o r r e l a t e s w i t h t h e h y p o t h e s i s o f an a c t i v e m e c h a n i s m o f i n h i b i t i o n , r a t h e r t h a n i n s u f f i c i e n t q u a n t i t i e s o f a n t i g e n f o r a g i v e n q u a n t i t y o f a v a i l a b l e a n t i b o d y . The e x p e r i m e n t s u n d e r t a k e n t o e x p l o r e t h e d e v e l o p m e n t o f t o l e r a n c e h a v e c o n f i r m e d o b s e r v a t i o n s made by o t h e r s ( 3 9 , 1 1 1 , 12 5 - 1 2 7 , 130, 1 45) t h a t d u r i n g c h r o n i c a e r o s o l c h a l l e n g e , t h e b r o n c h o c o n s t r i c t i v e r e s p o n s e o f s e n s i t i z e d g u i n e a p i g s becomes p r o g r e s s i v e l y l e s s r a p i d a n d l e s s s e v e r e u n t i l e v e n t u a l l y t h e a n i m a l s a r e u n r e a c t i v e t o t h e u s u a l c h a l l e n g e p r o c e d u r e , i . e . a s t a t e o f ' t o l e r a n c e ' a r i s e s . However, w h i l e many o b s e r v a t i o n s o f t h i s h a v e b e e n made i n t e r m s o f l e n g t h e n i n g ' m i c r o s h o c k ' o r ' p r e c o n v u l s i o n ' t i m e ( 5 2 , 127, 128, 146) o r i n t e r m s o f p u l m o n a r y m e c h a n i c s b e f o r e a n d a f t e r a n t i g e n c h a l l e n g e ( 1 1 1 , 115, 1 1 6 ) , e x p e r i m e n t s h a v e n o t p r e v i o u s l y b e e n c a r r i e d o u t w h i c h f o l l o w e d t h e g r a d u a l d e c r e a s e i n v i s i b l e r e a c t i v i t y w i t h t h e l u n g m e c h a n i c s p a r a m e t e r s o f Cdyn a n d RL. T h i s work h a s shown t h a t C d yn a nd RL a r e a g o o d o b j e c t i v e a s s e s s m e n t o f g u i n e a p i g c h a n g i n g a n a p h y l a c t i c r e s p o n s e s t o a e r o s o l c h a l l e n g e . The c o n c l u s i o n drawn f r o m t h e s e e x p e r i m e n t s t h a t w i t h h o l d i n g a e r o s o l c h a l l e n g e p r o d u c e s o n l y a v a r i a b l e r e s e n s i t i z a t i o n c o n f i r m s o b s e r v a t i o n s made by H i c k s and S k e l d o n ( 1 2 8 ) and H e r x h e i m e r ( 1 2 7 ) . F i n d i n g s f r o m t h i s work t h a t a i r w a y s m o o t h m u s c l e f r o m t o l e r a n t a n i m a l s i s a s r e s p o n s i v e t o h i s t a m i n e - i n d u c e d c o n t r a c t i o n b u t l e s s r e s p o n s i v e t o o v a l b u m i n - i n d u c e d c o n t r a c t i o n t h a n s e n s i t i z e d a n i m a l s was f i r s t r e p o r t e d i n an a b s t r a c t ( 1 8 8 ) . T h i s d e f i n i t i v e l y e l i m i n a t e s t h e - 81 -p o s s i b i l i t y t h a t ' t o l e r a n c e ' i s due t o d e c r e a s e d a i r w a y s m o o t h m u s c l e r e s p o n s e t o h i s t a m i n e . T h i s h a s s i n c e b e e n c o n f i r m e d b y Hedman a n d A n d e r s s o n ( 1 8 9 ) . The s t u d i e s o f h i s t a m i n e c o n t e n t i n t h e l u n g p a r e n c h y m a o f t o l e r a n t , s e n s i t i z e d and c o n t r o l g u i n e a p i g s h a s shown a t e n d e n c y f o r t o l e r a n t a n i m a l s t o h a v e h i g h e r l e v e l s o f h i s t a m i n e , t h u s d e c r e a s i n g t h e l i k e l i h o o d o f m e d i a t o r d e p l e t i o n b e i n g t h e m e c h a n i s m o f t o l e r a n c e . No o t h e r r e p o r t s o f t h i s phenomenon h a v e b e e n made. Some i n v e s t i g a t o r s h a v e r e p o r t e d a p o o r c o r r e l a t i o n b e t w e e n l e v e l o f r e a c t i v i t y a n d PCA ( 1 2 6 ) o r h a e m a g g l u t i n a t i n g ( 1 2 8 ) r e s u l t s . O t h e r s h a v e r e p o r t e d t h a t u n r e a c t i v i t y i s a s s o c i a t e d w i t h a d e c r e a s e i n IgG-^ as a s s e s s e d b y PCA ( 1 3 0 , 1 9 0 ) . T h i s d a t a i s s u p p o r t i v e o f t h e l a t t e r c o n c l u s i o n . A n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e was o b s e r v e d i n t h e s e e x p e r i m e n t s t o be d e c r e a s e d i n T o l e r a n t c h o p p e d l u n g t i s s u e when a l o w l e v e l o f a n t i g e n was u s e d . An e x p e r i m e n t by Hedman a n d A n d e r s s o n ( 1 8 9 ) c o m p a r i n g a n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e b e t w e e n t o l e r a n t a nd s e n s i t i z e d g u i n e a p i g s showed no d i f f e r e n c e s i n r e l e a s e a t an OA c o n c e n t r a t i o n o f 2 x 1 0 ~ 7 M. T h i s i s n o t u n e x p e c t e d , a s d a t a shown h e r e i n d i c a t e s t h a t h i s t a m i n e r e l e a s e s a r e c o m p a r a b l e a t h i g h l e v e l s o f OA. T h e s e e x p e r i m e n t s s u g g e s t t h a t q u a n t i t a t i v e l y l e s s m e d i a t o r s a r e r e l e a s e d i n t h e t i s s u e f r o m t o l e r a n t a n i m a l s o r t h a t t h e r e i s a d e c r e a s e d t i s s u e r e s p o n s e t o r e l e a s e d m e d i a t o r s o t h e r t h a n h i s t a m i n e . I n s u p p o r t o f t h e l a t t e r h y p o t h e s i s , Hedman and A n d e r s s o n ( 1 8 9 ) h a v e - 82 -r e p o r t e d t h a t i n v i t r o r e s p o n s e s o f t r a c h e a f r o m a n i m a l s d e s e n s i t i z e d by a 7 day r e g i m e o f OA i n j e c t i o n s e x h i b i t e d a c o n t r a c t i l e r e s p o n s e t o SRS-A w h i c h was 38 % o f t h a t shown by t r a c h e a s f r o m s e n s i t i z e d a n i m a l s . However, t h i s d o e s s t i l l n o t e x c l u d e t h e p o s s i b i l i t y t h a t u pon a n t i g e n c h a l l e n g e l e s s h i s t a m i n e and SRS-A c o u l d be r e l e a s e d f r o m t h e t i s s u e . A m e c h a n i s m a c t i n g a t t h e l e v e l o f t h e mast c e l l p r o v i d e s a p o s s i b l e e x p l a n a t i o n f o r t h e d a t a . One s u c h m e c h a n i s m w o u l d be a d e c r e a s e i n t h e number o f F c r e c e p t o r s . T h i s i s a n o n - s p e c i f i c m e c h a n i s m , and w o u l d i n t e r f e r e w i t h t h e r e a c t i v i t y o f mast c e l l s i n r e s p o n s e t o an a n t i g e n t o w h i c h t h e g u i n e a p i g h a d j u s t b e e n s e n s i t i z e d . A n o t h e r m e c h a n i s m w o u l d be a d i r e c t s u p p r e s s i o n o f mast c e l l m e d i a t o r r e l e a s e by a s u p p r e s s o r c e l l - m a s t c e l l c o n t a c t , o r v i a a s p e c i f i c o r n o n - s p e c i f i c s o l u b l e s u p p r e s s o r f a c t o r a c t i n g on t h e m a s t c e l l membrane. G i v e n t h a t a h i s t a m i n e - i n d u c e d s u p p r e s s o r c e l l i s known t o i n h i b i t h i s t a m i n e r e l e a s e f r o m b a s o p h i l s ( 1 8 1 ) t h i s i s a d i s t i n c t l y p o s s i b l e m e c h a n i s m . I n c o n c l u s i o n , t h e t o l e r a n c e o b s e r v e d i n g u i n e a p i g s c h r o n i c a l l y e x p o s e d t o an a e r o s o l o f OA a n t i g e n a p p e a r s t o m a n i f e s t a t l e a s t i n p a r t by an i n h i b i t i o n o f a n t i g e n - i n d u c e d h i s t a m i n e r e l e a s e f r o m mast c e l l s . The m e c h a n i s m by w h i c h t h i s o c c u r s i s n o t y e t d e f i n e d , b u t may i n c l u d e a d e c r e a s e d l e v e l o f h o m o c y t o t r o p i c Ab. However, a s w i t h a n y s y s t e m i n v o l v i n g p r o t e i n s y n t h e s i s t h e r e a p p e a r s t o be a b a l a n c e o f r e p r e s s i o n a n d a c t i v a t i o n f a c t o r s t o c o n t r o l t h e a n a p h y l a c t i c - 83 -r e s p o n s e . T h i s may p r o v i d e f o r g r a d a t i o n s i n r e s p o n s e , a s w e l l a s t h e phenomenon o f ' b r e a k t h r o u g h ' r e a c t i v i t y , a n d t h e w i d e v a r i a t i o n i n r e a c t i v i t y among a n i m a l s . REFERENCES - 84 -1. M c Fadden ER J r , R o l a n d RH J r . A s t h m a : p e r s p e c t i v e s , d e f i n i t i o n , a n d c l a s s i f i c a t i o n . I n : F i s h m a n AP, e d , P u l m o n a r y d i s e a s e s a n d d i s o r d e r s . New Y o r k : McGraw H i l l , 1 980:564. 2. S c a d d i n g J F . The d e f i n i t i o n o f a s t h m a : g e n e r a l i n t r o d u c t i o n . I n : P o r t e r R, B i r c h J , e d s , I d e n t i f i c a t i o n o f a s t h m a . E d i n b u r g h -L o n d o n : C h u r c h i l l L i v i n g s t o n e , 1971:13. 3. Hogg J C , P a r e PD, B o u c h e r R, M i c h o u d M-C, G u e r z o n G, M o r o z L . P a t h o l o g i c a b n o r m a l i t i e s i n a s t h m a . I n : L i c h t e n s t e i n LM, A u s t e n KF, e d s , A s t h m a . New Y o r k : A c a d e m i c P r e s s , I n c , 1 9 7 7 : 1 . 4. Hogg J C . B r o n c h i a l a s t h m a . I n : T h u r l b e c k WM, A b e l l MR e d s , T h e l u n g : I A P M o n o g r a p h No. 1 9 . B a l t i m o r e : W i l l i a m s a n d W i l k i n s , 1 9 7 8 : 1 8 0 . 5. G o l d WM. A s t h m a . I n : P i e r c e AK e d , B a s i c s o f RD. New Y o r k , A m e r i c a n T h o r a c i c S o c i e t y , V o l . 4, No. 3. 6. M i l l s J E , W i d d i c o m b e J G . 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