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Macrophage interaction with Pseudomonas aeruginosa Kluftinger, Janet Louise 1988

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MACROPHAGE INTERACTION WITH PSEUDOMONAS AERUGINOSA  By  JANET LOUISE KLUFTINGER B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1984  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY  We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1988 ©Janet L o u i s e K l u f t i n g e r , 1988  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ii ABSTRACT  The studied.  i n t e r a c t i o n s o f macrophages w i t h Pseudomonas a e r u g i n o s a were F i v e monoclonal a n t i b o d i e s s p e c i f i c f o r p o r i n p r o t e i n F were  t e s t e d f o r t h e i r a b i l i t y t o o p s o n i z e P. a e r u g i n o s a f o r complement-independent p h a g o c y t o s i s by u n e l i c i t e d mouse p e r i t o n e a l macrophages, human p e r i p h e r a l b l o o d monocytes and mouse macrophage line P388 . D 1  cell  A l l f i v e a n t i b o d i e s s i g n i f i c a n t l y i n c r e a s e d the l e v e l  b a c t e r i a l uptake over t h a t o b t a i n e d w i t h the non-opsonic r e l a t i v e e f f e c t i v e n e s s o f the d i f f e r e n t a n t i b o d i e s was same i n a l l c e l l  types i n d i c a t i n g t h a t the P 3 8 8  model f o r normal macrophages.  D 1  controls.  approximately  of The the  c e l l s can be used as  a  Of the f o u r monoclonal a n t i b o d i e s d i r e c t e d  a g a i n s t s i m i l a r e p i t o p e s o f p r o t e i n F, the t h r e e I g G l monoclonal a n t i b o d i e s were s u b s t a n t i a l l y more o p s o n i c than the one  IgG2a i s o t y p e .  P. a e r u g i n o s a c y t o t o x i n and p e r i p l a s m i c c o n t e n t s caused r e d u c t i o n i n antibody-mediated P h a g o c y t o s i s was  p h a g o c y t o s i s o f P.  a  significant  aeruginosa.  r e s t o r e d upon p r e - i n c u b a t i o n w i t h a n t i - c y t o t o x i n serum.  Both c y t o t o x i n and p e r i p l a s m i c c o n t e n t s caused d e p o l a r i z a t i o n o f the P388  D 1  c e l l membrane, as demonstrated u s i n g a p o l a r i z a t i o n - s e n s i t i v e  f l u o r e s c e n t probe.  These d a t a i n d i c a t e d t h a t P. a e r u g i n o s a c y t o t o x i n was  l o c a l i z e d i n the p e r i p l a s m and had the p o t e n t i a l t o  inhibit  macrophage-mediated p h a g o c y t o s i s , p o s s i b l y by p e r t u r b i n g i o n g r a d i e n t s a c r o s s the macrophage plasma membrane.  iii  Monoclonal enhancing  a n t i b o d i e s d i r e c t e d a g a i n s t p r o t e i n F were a l s o c a p a b l e o f  p h a g o c y t o s i s o f i n vivo-grown  P. a e r u g i n o s a .  P.  aeruginosa  c e l l s taken d i r e c t l y from the i n v i v o growth system were s i g n i f i c a n t l y more s u s c e p t i b l e t o macrophage p h a g o c y t o s i s than were the same c e l l s b e i n g washed i n b u f f e r .  The p h a g o c y t o s i s - p r o m o t i n g  i s o l a t e d from the supernatant o f i n vivo-grown to  be f i b r o n e c t i n .  non-opsonic macrophages.  p h a g o c y t o s i s was The  mediated  determined  by f i b r o n e c t i n o f  by d i r e c t a c t i v a t i o n o f the  t e t r a p e p t i d e a r g i n i n e - g l y c i n e - a s p a r t a t e - s e r i n e i n the  e u k a r y o t i c c e l l b i n d i n g domain o f f i b r o n e c t i n was macrophage-activating  region.  s t r a i n lacking surface p i l i Furthermore,  f a c t o r c o u l d be  b a c t e r i a and was  Data i n d i c a t e d t h a t promotion  after  exogenously  demonstrated  P h a g o c y t o s i s o f a mutant P.  c o u l d not be enhanced by  added Pseudomonas p i l i was  I t was  a c t i v a t e d macrophages i n the i n i t i a l  aeruginosa  capable of abrogating with  concluded t h a t Pseudomonas  were the b a c t e r i a l l i g a n d s r e q u i r e d f o r attachment  the  fibronectin.  the enhanced p h a g o c y t o s i s o f the w i l d type s t r a i n observed f i b r o n e c t i n - a c t i v a t e d macrophages.  t o be  pili  to f i b r o n e c t i n -  stages o f non-opsonic  phagocytosis.  iv TABLE OF CONTENTS PAGE ABSTRACT  i i  TABLE OF CONTENTS  iv  L i s t o f Tables List  viii  of Figures  x  ACKNOWLEDGMENTS  x i i  DEDICATION  xiii  INTRODUCTION  1  1.  M e d i c a l importance o f Pseudomonas  aeruginosa  2.  Macrophage f u n c t i o n s  1  3.  Role o f macrophages i n P. a e r u g i n o s a i n f e c t i o n s  2  4.  Mechanism o f p h a g o c y t o s i s  4  5.  Pseudomonas  7  6.  Macrophage a c t i v a t i o n  8  7.  Fibronectin  9  8.  S p e c i f i c aims o f t h i s study  9  products which i n h i b i t p h a g o c y t o s i s  MATERIALS AND METHODS 1.  1  1  1  Bacteria  1  1  A.  Bacterial strains  11  B.  Media and i n v i t r o growth c o n d i t i o n s  11  C.  B a c t e r i a l growth i n v i v o  12  D.  Preparation of sub-cellular fractions  13  V  TABLE OP CONTENTS PAGE E.  Sodium d o d e c y l s u l p h a t e ( S D S ) - p o l y a c r y l a m i d e gel  electrophoresis  (i)  Protein staining  14  Western immunoblotting  15  P r o t e i n and 2-keto-3-deoxyoctonate  15  (ii) F.  14  (KDO) assays 2.  3.  Macrophages A.  Macrophage c e l l  B.  U n e l i c i t e d mouse p e r i t o n e a l macrophages  16  C.  Human p e r i p h e r a l b l o o d monocyte-derived macrophages ..  16  A.  A n t i - p r o t e i n F monoclonal  B.  Polyclonal sera  15  17 antibodies  17 •  17  A n t i - c y t o t o x i n serum  17  (ii)  Anti-exoenzyme S serum  18  (iii)  A n t i - f i b r o n e c t i n serum  18  P h a g o c y t o s i s assay  I  8  A.  Conditions  I  8  B.  Inhibitors/activators  19  (i)  Chapter One  19  (ii)  Chapter Two  19  Chapter Three  20  (iii) 5.  lines  Antibody p r e p a r a t i o n  (i)  4.  15  F l u o r e s c e n c e assay  20  vi TABLE OF CONTENTS PAGE RESULTS Chapter  22 I - Opsonic p h a g o c y t o s i s .of Pseudomonas a e r u g i n o s a by macrophages and macrophage c e l l  22  lines  1.  Choice o f an a p p r o p r i a t e model c e l l  2.  E s t a b l i s h m e n t o f p h a g o c y t o s i s assay c o n d i t i o n s  24  3.  Opsonic p h a g c y t o s i s : I n f l u e n c e o f a n t i b o d y s u b c l a s s  27  4.  Summary  30  Chapter  line  23  I I - P. a e r u g i n o s a c y t o t o x i n : l o c a l i z a t i o n and  32  i n a c t i v a t i o n o f macrophages 1.  Cellular l o c a l i z a t i o n of cytotoxin  33  2.  Cytotoxin i n h i b i t i o n o f phagocytosis  36  3.  Mechanism o f macrophage i n a c t i v a t i o n  40  4.  Summary  43  Chapter  I I I - F i b r o n e c t i n - m e d i a t e d a c t i v a t i o n o f non-opsonic  46  p h a g o c y t o s i s o f P. a e r u g i n o s a 1.  E f f e c t o f i n v i v o growth on p h a g o c y t o s i s  46  2.  Enhancement o f p h a g o c y t o s i s by i n v i v o supernatant  49  3.  C h a r a c t e r i z a t i o n o f the phagocytosis-promoting  56  4.  Requirement f o r b a c t e r i a  59  5.  A c t i v a t i o n o f macrophages by f i b r o n e c t i n  63  6.  D e t e r m i n a t i o n o f t h e a c t i v e domain o f f i b r o n e c t i n  67  7.  Mechanism o f f i b r o n e c t i n - m e d i a t e d macrophage a c t i v a t i o n  factor  ..  69  vii TABLE OF CONTENTS PAGE 8.  E f f e c t o f growth c o n d i t i o n s on f i b r o n e c t i n - m e d i a t e d non-opsonic  9-  macrophage  71  phagocytosis  D e t e r m i n a t i o n o f the b a c t e r i a l l i g a n d f o r non-opsonic  73  phagocytosis  10.  Other b a c t e r i a  75  11.  Summary  75  DISCUSSION 1.  82  Use o f t h e P388 ^ macrophage c e l l D  l i n e as a model  82  f o r macrophage s t u d i e s 2.  Role o f i s o t y p e i n o p s o n i z e d p h a g o c y t o s i s  82  by macrophages 3.  Cytotoxin:  l o c a l i z a t i o n and p u t a t i v e r o l e i n i n f e c t i o n  4.  F i b r o n e c t i n as an a c t i v a t o r o f macrophage non-opsonic  ...  84 88  phagocytosis LITERATURE  CITED  96  viii LIST OF TABLES PAGE TABLE I  II  Ill  IV  Enhancement o f a s s o c i a t i o n o f P. a e r u g i n o s a s t r a i n M2 w i t h mouse p e r i t o n e a l macrophages, P388pi c e l l s , and human p e r i p h e r a l b l o o d monocytes u s i n g monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t p r o t e i n F.  29  E f f e c t o f a n t i - c y t o t o x i n and anti-exoenzyme S s e r a on i n h i b i t i o n o f o p s o n i z e d p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n M2.  39  D e p o l a r i z a t i o n o f the P 3 8 8 plasma membrane by c y t o t o x i n , v a l i n o m y c i n and osmotic shockate a s s e s s e d u s i n g the f l u o r e s c e n t n t probe d i S C 3 ( 5 ) .  42  Enhancement o f t h e a s s o c i a t i o n o f i n vivo-grown P. a e r u g i n o s a 3 t r a i n M2 w i t h P 3 8 8 i c e l l s u s i n g supernatant from mouse chambers.  51  S t a b i l i t y o f the phagocytosis-enhancing o f i n v i v o chamber s u p e r n a t a n t .  52  D 1  u  V  VI  VII  factor  Enhancement o f t h e a s s o c i a t i o n o f P. a e r u g i n o s a s t r a i n s M2 and H103 w i t h u n e l i c i t e d mouse p e r i t o n e a l macrophages and t h e P388j)i c e l l l i n e u s i n g i n v i v o supernatant from r a t chambers.  53  I n v i t r o growth c o n d i t i o n s a f f e c t t h e a b i l i t y o f i n v i v o supernatant t o enhance a s s o c i a t i o n o f P. a e r u g i n o s a H103 w i t h P 3 8 8 cells.  55  Enhancement o f the a s s o c i a t i o n o f P. a e r u g i n o s a s t r a i n H103 w i t h P 3 8 8 i c e l l s u s i n g p o o l e d f r a c t i o n s c o l l e c t e d from an FPLC g e l s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant from r a t chambers.  58  A n t i - f i b r o n e c t i n i n h i b i t i o n of phagocytosispromoting a c t i v i t y o f i n v i v o supernatant from r a t p e r i t o n e a l chambers and b o v i n e f i b r o n e c t i n .  62  Time c o u r s e o f emergence o f p h a g o c y t o s i s - p r o m o t i n g a c t i v i t y i n r a t and mouse H103 and s a l i n e chambers.  64  E f f e c t o f washing on f i b r o n e c t i n - m e d i a t e d enhancement o f non-opsonic macrophage p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n H103.  68  D 1  VIII  D  IX  X  XI  ix LIST OF TABLES PAGE TABLE XII  XIII  XIV  E f f e c t o f a g i t a t i o n d u r i n g growth on the s u s c e p t i b i l i t y o f P. a e r u g i n o s a s t r a i n H103 t o f i b r o n e c t i n - m e d i a t e d macrophage non-opsonic p h a g o c y t o s i s .  ....  72  F i b r o n e c t i n - m e d i a t e d macrophage p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n s H103 ( w i l d t y p e ) , BLP3 ( p i l i n minus), P A O l - l e u ( p i l i n p o s i t i v e ) , and p B P l 6 l ( p i l i n p o s i t i v e ) .  74  I n h i b i t i o n o f f i b r o n e c t i n - m e d i a t e d macrophage non-opsonic uptake o f P. a e r u g i n o s a s t r a i n H103 by exogenous PA01 p i l i .  76  X  LIST OF FIGURES PAGE FIGURE 1  Opsonized p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n M2 by t h r e e mouse macrophage c e l l l i n e s , mouse p e r i t o n e a l macrophages and human p e r i p h e r a l b l o o d monocyted e r i v e d macrophages.  25  2  Time c o u r s e o f P. a e r u g i n o s a a s s o c i a t i o n w i t h P388px i n t h e presence o r absence o f a n t i - F monoclonal a n t i b o d y MA2-10.  26  3  P e r c e n t o f macrophages a s s o c i a t e d w i t h s p e c i f i c numbers o f b a c t e r i a a f t e r 90 min.  28  4  SDS-polyacrylamide g e l o f c y t o t o x i n s t r a i n H103 s u b c e l l u l a r f r a c t i o n s .  34  5  Western immunoblot o f c y t o t o x i n and P. a e r u g i n o s a s t r a i n H103 s u b c e l l u l a r f r a c t i o n s probed w i t h r a b b i t a n t i - c y t o t o x i n serum.  35  6  Western immunoblot o f c y t o t o x i n and P. a e r u g i n o s a s t r a i n H103 s u b c e l l u l a r f r a c t i o n s probed w i t h r a b b i t anti-exoenzyme S serum.  37  7  Changes i n d i S C ( 5 ) f l u o r e s c e n c e a f t e r a d d i t i o n o f phosphate-buffered s a l i n e , cytotoxin or valinomycin to d i S C ( 5 ) e q u i l i b r a t e d P 3 8 8 cells.  41  8  E f f e c t of increasing concentrations of cytotoxin or osmotic shockate on the r a t e o f i n c r e a s e o f diSC3(5) fluorescence.  44  9  Description  47  3  3  10  and P. a e r u g i n o s a  D 1  o f t h e i n v i v o growth model  Opsonized p h a g o c y t o s i s o f i n v i v o - and i n v i t r o - g r o w n P. a e r u g i n o s a s t r a i n M2 by mouse macrophage c e l l line P388 i.  48  FPLC g e l s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant from r a t chambers - e l u t i o n p r o f i l e a t an absorbance o f 230 nm.  57  D  11  xi LIST OF  FIGURES PAGE  FIGURE 12  Western immune-blots o f f r a c t i o n s c o l l e c t e d from an FPLC g e l s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant from r a t chambers probed w i t h goat anti-human f i b r o n e c t i n serum.  60  13  FPLC g e l s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant from mouse chambers - e l u t i o n p r o f i l e a t an absorbance o f 230 nm.  61  14  Western immunoblots o f the supernatant o f b a c t e r i a and s a l i n e - c o n t a i n i n g chambers t h a t had been i n c u b a t e d i n the peritoneum o f mice and r a t s f o r 4, 24 and 48 hours probed w i t h goat anti-human f i b r o n e c t i n serum.  65  15  E f f e c t of increasing concentrations of f i b r o n e c t i n and RGDS on the l e v e l o f uptake o f P. a e r u g i n o s a s t r a i n H103 by macrophage c e l l l i n e P388])i-  66  16  E f f e c t of increasing concentrations of f i b r o n e c t i n on the r a t e o f i n c r e a s e o f diSC3(5) f l u o r e s c e n c e .  70  17  Enhancement o f the a s s o c i a t i o n o f E. c o l i w i t h P388ni c e l l s u s i n g supernatant from i n v i v o grown E. c o l i .  77  18  Enhancement o f the a s s o c i a t i o n o f S. aureus w i t h P388 T_ c e l l s u s i n g supernatant from i n v i v o grown S. aureus.  78  19  Western immunoblot o f the s u p e r n a t a n t s o f E. c o l i and S. a u r e u s - c o n t a i n i n g chambers probed w i t h goat anti-human f i b r o n e c t i n .  79  20  A c o n c e p t u a l model o f f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic p h a g o c y t o s i s o f P. a e r u g i n o s a .  95  u  xii  ACKNOWLEDGEMENTS  I wish t o thank my s u p e r v i s o r , Dr. Bob Hancock, f o r h i s e x c e l l e n t a d v i c e , u n d e r s t a n d i n g , and f i n a n c i a l support d u r i n g t h e c o u r s e o f my studies. I am s i n c e r e l y g r a t e f u l t o Dr. David Speert and Dr. T r e v o r T r u s t , who p e r m i t t e d me t o work i n t h e i r l a b o r a t o r i e s f o r extended p e r i o d s o f time. I would l i k e t o thank t h e members o f t h e B i o c h e m i s t r y / M i c r o b i o l o g y Department a t t h e U n i v e r s i t y o f V i c t o r i a , t h e M i c r o b i o l o g y Department a t t h e U n i v e r s i t y o f B r i t i s h Columbia, and t h e members o f my s u p e r v i s o r y committee f o r t h e i r h e l p and a d v i c e . I am e s p e c i a l l y g r a t e f u l t o Dr. Niamh K e l l y who g e n e r o u s l y donated h e r time and e f f o r t t o t h e c o l l a b o r a t i v e study t h a t l e d t o t h e d a t a p r e s e n t e d i n Chapter Three o f this thesis. In p a r t i c u l a r , I wish t o thank a l l t h e members o f t h e Hancock, T r u 3 t and Speert l a b o r a t o r i e s , who o f f e r e d me i n v a l u a b l e f r i e n d s h i p , support and guidance d u r i n g my s t a y . A s p e c i a l thanks t o R o s a r i o Bauzon f o r h e r excellent typing of t h i s thesis.  xiii  DEDICATION  T h i s t h e s i s i s d e d i c a t e d to my who  have always supported my  p a r e n t s , Jim and Peggy  a s p i r a t i o n s and endeavours, and  husband, Andy, whose c o n s t a n t f r i e n d s h i p , l o v e and important  to  me.  Battershill,  support  to  my  a r e so very  1  INTRODUCTION  1.  M e d i c a l Importance o f Pseudomonas Pseudomonas  aeruginosa  a e r u g i n o s a i s an o p p o r t u n i s t i c pathogen, o f t e n c a u s i n g  l i f e - t h r e a t e n i n g i n f e c t i o n s i n immunocompromised  patients.  Individuals  who a r e e s p e c i a l l y a t r i s k i n c l u d e those w i t h severe burns, d i a b e t e s and c y s t i c f i b r o s i s . predominant causes America,  cancer,  Indeed, P. a e r u g i n o s a remains one o f the  o f death from gram n e g a t i v e s e p t i c e m i a i n North  and the most common b a c t e r i u m a s s o c i a t e d w i t h t e r m i n a l l u n g  disease i n patients with c y s t i c f i b r o s i s  ( S p e e r t , 1985).  The  intrinsic  r e s i s t a n c e o f P. a e r u g i n o s a t o a n t i b i o t i c treatment c o n t r i b u t e s s u b s t a n t i a l l y t o the h i g h m o r t a l i t y r a t e s  observed.  P. a e r u g i n o s a i s a gram n e g a t i v e b a c t e r i a and thus has two envelope membranes:  cell  the c y t o p l a s m i c ( o r i n n e r ) membrane and the o u t e r  t membrane.  These a r e s e p a r a t e d by a s i n g l e sheet o f p e p t i d o g l y c a n .  o u t e r membrane i s p a r t i c u l a r l y  important  i n Pseudomonas  The  i n f e c t i o n s as i t  p l a y s a r o l e i n a n t i b i o t i c r e s i s t a n c e and c o n t a i n s e n d o t o x i c l i p o p o l y s a c c h a r i d e (LPS) and p r o t e i n a n t i g e n s .  2.  Macrophage F u n c t i o n s Macrophages a r e o f t e n the f i r s t  infection uptake,  (Dunn e t a l . , 1985).  they p l a y an important  response.  c e l l s to i n f i l t r a t e a s i t e of  In a d d i t i o n to t h e i r r o l e i n b a c t e r i a l p a r t i n a m p l i f y i n g the host  Through t h e i r r e l e a s e o f potent  immune  immunomodulators and enzymes,  such as i n t e r l e u k i n 1, lysozyme, f i b r o n e c t i n , complement components,  2  p r o c o a g u l a n t , pyrogen, i n t e r f e r o n , e l a s t a s e , and plasminogen  activator,  i n f l a m m a t i o n and wound h e a l i n g o c c u r a t an a c c e l e r a t e d r a t e ( S c h a f f n e r e t al..  1982).  Many o f the s e c r e t e d p r o d u c t s o f macrophages have f u n c t i o n a l  importance as c h e m o a t t r a c t a n t s f o r r e c r u i t m e n t o f o t h e r immune c e l l s the  site of infection  ( S c h a f f n e r e t a l . , 1982).  into  Furthermore, a n t i g e n  p r e s e n t a t i o n by macrophages i s an important event i n t h e f u n c t i o n i n g o f T c e l l p o p u l a t i o n s i n b o t h t h e humoral and c e l l u l a r  immune response.  p a r t i c u l a r , macrophages b e a r i n g major h i s t o c o m p a t i b i l i t y molecules on t h e i r c e l l  surface are highly e f f i c i e n t  antigen-specific T c e l l s . dependent al.,  In  antigen  i n educating  T h i s a c t i v a t i o n o f T lymphocytes appears t o be  on t h e macrophage s e c r e t o r y product i n t e r l e u k i n 1 ( S c h a f f n e r e t  1982). Macrophages i n g e s t i n g immunoglobulin-coated a n t i g e n s a r e c a p a b l e o f  p r o c e s s i n g t h e immunoglobulin Fc fragments i n t o subfragments and s e c r e t i n g these back i n t o t h e environment.  These subfragments a r e d i r e c t l y  m i t o g e n i c f o r B c e l l s and have a p o t e n t adjuvant e f f e c t on T lymphocytes (Schaffner et a l . ,  3.  1982).  Role o f Macrophages i n P. a e r u g i n o s a I n f e c t i o n s Macrophages r e p r e s e n t a p r i m a r y l i n e o f defense a g a i n s t i n f e c t i o n i n  deep t i s s u e s such as t h e l u n g (Green and Kass, 1964) and t h e p e r i t o n e a l cavity  (Dunn e t a l . , 1985).  I n t h e presence o f serum opsonins such as  a n t i b o d y and complement, P. a e r u g i n o s a can be e f f i c i e n t l y phagocytosed by normal macrophage p o p u l a t i o n s (Reynolds e t a l . ,  1975).  In a r e c e n t study, ( M u t h a r i a and Hancock, 1983), a s e r i e s o f  3  monoclonal a n t i b o d i e s were r a i s e d a g a i n s t p r o t e i n a n t i g e n s common t o a l l 17 of  P. a e r u g i n o s a s e r o t y p e s .  Monoclonal  a n t i b o d i e s d i r e c t e d a g a i n s t one  these o u t e r membrane p r o t e i n s ( p r o t e i n F) were p r o t e c t i v e i n a mouse  i n f e c t i o n model and were o p s o n i c f o r human polymorphonuclear phagocytosis  (Hancock e t a l . , 1985).  cell  A n t i b o d i e s a g a i n s t p r o t e i n F thus  appear t o have p o t e n t i a l as p a s s i v e v a c c i n e s i n animal systems. d a t a suggested  t h a t monoclonal a n t i b o d i e s a r e a c t i n g t o o p s o n i z e  P. a e r u g i n o s a f o r p h a g o c y t o s i s i n t h e mouse i n f e c t i o n model. polymorphonuclear  1975).  Both  l e u k o c y t e s and c e l l s o f the macrophage l i n e a g e a r e  c a p a b l e o f i n g e s t i n g P. a e r u g i n o s a al.,  These  (Young and Armstrong, 1972; Reynolds e t  While polymorphonuclear  l e u k o c y t e s a r e no doubt important i n  b a c t e r i a l c l e a r a n c e , macrophages must be regarded as a c r u c i a l component of  an e f f i c i e n t l y f u n c t i o n i n g immune system.  independent  s t u d i e s suggested  Indeed, t h e r e s u l t s o f two  t h a t the p h a g o c y t i c a c t i o n o f macrophages i s  the major mechanism o f e l i m i n a t i n g b a c t e r i a i n the i n i t i a l i n f e c t i o n (Green and Kass, polymorphonuclear  1964; Dunn e t a l . , 1985).  In  l i n e o f host  stages o f i n f e c t i o n e l i m i n a t e s them as a  defense.  t h e case o f some p a t i e n t s w i t h c y s t i c f i b r o s i s ,  P. a e r u g i n o s a i n f e c t i o n s can be q u i c k l y suppressed treatment  Although  leukocytes are capable o f s i m i l a r phagocytic clearance,  t h e i r absence i n t h e i n c i p i e n t potential f i r s t  stages o f  ( F r i e n d , 1986).  initial  after  antibiotic  T h i s may i n v o l v e not o n l y a n t i b i o t i c  but a l s o non-immune c l e a r a n c e mechanisms o f the h o s t .  These  action,  latter  mechanisms a p p a r e n t l y encompass b o t h t h e m u c o c i l i a r y system and non-opsonic  p h a g o c y t o s i s by pulmonary a l v e o l a r macrophages ( S p e e r t ,  4  1985).  On r e p e a t e d  i n f e c t i o n w i t h P. a e r u g i n o s a ,  i n d i v i d u a l with c y s t i c f i b r o s i s  lung f u n c t i o n o f an  r a p i d l y d e t e r i o r a t e s and b a c t e r i a can no  l o n g e r be e r a d i c a t e d from the a r e a .  Thus, t h e i n a b i l i t y  to clear  P. a e r u g i n o s a may i n d i c a t e , i n p a r t , a d e f e c t i n phagocyte uptake and killing.  4.  Mechanism o f P h a g o c y t o s i s There a r e t h r e e b a s i c steps i n macrophage p h a g o c y t o s i s ,  here w i t h r e f e r e n c e t o p h a g o c y t o s i s  of p a r t i c l e s ) .  These a r e p a r t i c l e  attachment, g e n e r a t i o n and t r a n s m i s s i o n o f the p h a g o c y t i c particle ingestion.  The f i r s t  s i g n a l , and  s t e p i n v o l v e s a p p o s i t i o n o f t h e macrophage  plasma membrane t o t h e p a r t i c l e s u r f a c e . of  (described  T h i s event  occurs  i n t h e absence  macrophage c e l l u l a r metabolism and i s l a r g e l y dependent on s u r f a c e  p r o p e r t i e s o f t h e p a r t i c l e and c e l l .  The e f f i c i e n c y o f attachment  i n c r e a s e s s e v e r a l f o l d i f the m a t e r i a l i n q u e s t i o n i s opsonized antibody  o r complement.  with  T h i s i s due t o the f a c t t h a t macrophages have a t  l e a s t two c l a s s e s o f s u r f a c e r e c e p t o r s , the Fc r e c e p t o r s and t h e C3 receptor.  Mouse macrophages and macrophage-like c e l l  three d i s t i n c t  Fc r e c e p t o r s (Green e t a l . , 1985).  aggregated IgG2a, another binds  IgG3.  product  l i n e s appear t o have  One bind3 monomeric and  b i n d s aggregates o f I g G l o r IgG2b, and t h e l a s t  The C3 r e c e p t o r r e c o g n i z e s  o f complement component C3-  C3b, the C3 c o n v e r t a s e  Thus, a n t i g e n s  cleavage  complexed t o IgG  and/or C3b b i n d a v i d l y t o macrophages. Non-opsonic p h a g o c y t o s i s  i n v o l v e s a s s o c i a t i o n o f phagocytes and  b a c t e r i a i n t h e absence o f e x t e r n a l opsonins  (Speert e t a l . , 1984).  This  5  mode o f p h a g o c y t o s i s process  (Aduan and  clearance  has  been c o n s i d e r e d  Reynolds, 1979), and  to be a r e l a t i v e l y  thus r e l a t i v e l y unimportant to  the  o f t h i s organism.  In c o n j u n c t i o n w i t h ,  or f o l l o w i n g , a n t i g e n  r e c o g n i t i o n and  s i g n a l must be passed t o the i n t e r i o r o f the c e l l ingestion.  inefficient  At p r e s e n t ,  h y p o t h e t i c a l but  to i n i t i a t e  the e x i s t e n c e o f a p h a g o c y t i c  a  particle  signal i s basically  s e v e r a l t h e o r i e s have been put forward as t o p o t e n t i a l  components o f the s i g n a l (Young, 1985). been shown to r e s u l t concentration  binding,  Receptor-ligand  i n t e r a c t i o n has  i n regional v a r i a t i o n s i n s u b c e l l u l a r calcium  (Smith et a l . , 1985;  Young et a l . , 1984;  Vaux ejb a l . , 1982),  a c t i v a t i o n o f the Fc r e c e p t o r c a t i o n channel (Young et a l . , 1983a;1983b; 1984), and 1983;  a l t e r a t i o n i n e l e c t r i c a l p r o p e r t i e s o f the c e l l  Gallin,  The  1981;  Young, 1983c).  l o c a l calcium gradient  s e t up by r e c e p t o r - l i g a n d b i n d i n g would  determine the p h y s i c a l s t a t e o f a c t i n v i a g e l s o l i n a calcium-activated  (McCann et a l . .  ( S t o s s e l et a l . , 1981),  p r o t e i n which severs bonds between a c t i n  a c t i n - b i n d i n g p r o t e i n to e f f e c t a g e l - s o l transformation.  and  This i n turn  c o u l d r e g u l a t e the f l o w o f pseudopods around the p a r t i c l e f o r i n g e s t i o n . This presents  a reasonable  mechanism by which a c t i n and  a c t i n regulatory proteins could provide phagocytosis.  The  the dynamic f o r c e f o r  increase i n i n t e r n a l calcium  concentration  t o be a r e s u l t o f c a l c i u m t r a n s p o r t i n t o the c e l l and from i n t r a c e l l u l a r s t o r e s  calcium-dependent  (Young et a l . , 1984).  i n t e r n a l s t o r e i n c l u d e membrane bound c a l c i u m  i s thought  release of  Candidates f o r the  ( H o f f s t e i n , 1979)  i n t r a c e l l u l a r p r o t e i n which becomes p h o s p h o r y l a t e d  calcium  and  an  to release calcium  on  6  phagocytic stimulus The  (Vaux e t a l . , 1982).  IgGl/2b Fc r e c e p t o r  non-selective  o f murine macrophages has been shown t o be a  ligand-dependent c a t i o n channel w i t h h i g h p e r m e a b i l i t y t o  sodium and p o t a s s i u m and low p e r m e a b i l i t y 1983a).  This  alteration  channel c o u l d be a t l e a s t  i n e l e c t r i c a l properties  hyperpolarization/depolarization). (Young, 1983c), the N a C a +  and  Ca  + +  - activated K  s e t up g r a d i e n t s  +  f o r calcium  (Young e t a l . ,  i n part responsible  f o r the  o f t h e macrophage c e l l  ( i . e . membrane  I n a d d i t i o n , the N a K  ATPase pump  +  +  exchange mechanism (Braquet e t a l . , 1985)  + +  conductances (Young, 1983c) have been seen t o  on macrophage s t i m u l a t i o n .  Macrophages a r e e l e c t r i c a l l y  e x c i t a b l e showing r e c t i f y i n g conductances and a c t i o n p o t e n t i a l s al.,  1983; G a l l i n , 1981).  Friedhoff  (1983) h y p o t h e s i z e d t h a t  (McCann e t  this  movement o f i o n s through s p a t i a l l y s e p a r a t e pumps o r channels would produce an e l e c t r i c a l f i e l d  thus i n d u c i n g  f l u i d and membrane movement.  These membrane o r d e r changes have been observed i n response t o s e v e r a l phagocytic s t i m u l i al.,  (Esfahani  e t a l . , 1982; Horvath e t a l . , 1982; Sandor e t  1981; L a r s e n e t a l . , 1985). A g g r e g a t i o n o f p a r t i c l e s on the s u r f a c e  r e c o g n i t i o n and b i n d i n g internalization Petty,  1985).  i s believed  o f the macrophage f o l l o w i n g  t o be e s s e n t i a l f o r subsequent  ( L e s l i e , 1985; Dower e t a l . , 1981; Sandor e t a l . , 1981; In f a c t , the sulfhydryl-redox  model o f a n t i b o d y dependent  p h a g o c y t o s i s h y p o t h e s i z e s t h a t d i s u l f i d e l i n k s formed between o c c u p i e d receptors 1985).  p r o v i d e s t h e " z i p p e r i n g " mechanism o f p h a g o c y t o s i s  (Petty,  7  5.  Pseudomonas Products Which I n h i b i t One  Phagocytosis  o f the ways i n which P. a e r u g i n o s a may  host defense  p r o t e c t i t s e l f from  system i s through p r o d u c t i o n o f p h a g o c y t o s i s - i n h i b i t i n g  t o x i n s ( S p e e r t , 1985).  I t has  l o n g been e s t a b l i s h e d t h a t s t r a i n s o f  P. a e r u g i n o s a c o l o n i z i n g i n d i v i d u a l s w i t h c y s t i c f i b r o s i s predominantly  the  mucoid i n phenotype (Doggett,  1969).  are  These organisms  produce l a r g e amounts o f mucoid e x o p o l y s a c c h a r i d e and a r e more r e s i s t a n t t o p h a g o c y t o s i s than t h e i r non-mucoid c o u n t e r p a r t s ( B a l t i m o r e and M i t c h e l l , 1980).  T h i s r e s i s t a n c e has been a t t r i b u t e d t o b o t h a d i r e c t  t o x i c e f f e c t o f mucoid e x o p o l y s a c c h a r i d e on macrophage p h a g o c y t o s i s ( O l i v e r and Weir, 1983)  and masking by the mucoid c o a t i n g o f b a c t e r i a l  ligands required f o r phagocytosis  ( B a l t i m o r e and M i t c h e l l ,  The most t o x i c product o f P. a e r u g i n o s a appears (Woods and I g l e w s k i , 1983). efficient  1980).  t o be e x o t o x i n A  T h i s e x t r a c e l l u l a r l y s e c r e t e d t o x i n i s an  i n h i b i t o r o f p r o t e i n s y n t h e s i s ( I g l e w s k i and Kabat, 1975)  has been proven t o s i g n i f i c a n t l y reduce macrophage p h a g o c y t o s i s a t  and low  c o n c e n t r a t i o n s ( P o l l a c k and Anderson, 1978). Both l e u k o c i d i n ( c y t o t o x i n ; m o l e c u l a r weight 27,000 d a l t o n s ) and polymorphonuclear  l e u k o c y t e (PMN)  d a l t o n s ) have been observed (Scharmann e t a l . , 1976; determined  inhibitor  ( m o l e c u l a r weight 65,000  t o compromise p h a g o c y t o s i s by PMN  Nonoyama e t a l . , 1979).  i f e i t h e r o f these two  cells  I t has not been  p r o t e i n s are capable of  inhibiting  b a c t e r i a l uptake by macrophages. P r e v i o u s s t u d i e s have i n d i c a t e d t h a t 38% o f P. a e r u g i n o s a s t r a i n s produce exoenzyme S ( S o k o l e t a l . ,  1981), h y p o t h e s i z e d t o be a p r o t e i n  also  8  synthesis i n h i b i t o r  (Iglewski et a l . ,  1978).  Other s e c r e t e d p r o d u c t s  such  as a l k a l i n e p r o t e a s e , e l a s t a s e , and p h o s p h o l i p a s e are b e l i e v e d t o be important f a c t o r s c a u s i n g the l o c a l t i s s u e d e s t r u c t i o n which precedes i n v a s i o n o f the b l o o d s t r e a m  6.  (Sanai et a l . ,  1978).  Macrophage A c t i v a t i o n Numerous host f a c t o r s a r e c a p a b l e o f s t i m u l a t i n g macrophages.  r e s u l t s o f such s t i m u l a t i o n may  take the form o f i n c r e a s e d adherence,  r e c e p t o r - m e d i a t e d p h a g o c y t o s i s , chemotaxis, s e c r e t i o n o f b i o l o g i c a l l y a c t i v e molecules mechanism o f a c t i v a t i o n appears  The  bactericidal activity, (Schaffner et a l . ,  or  1982).  The  t o i n v o l v e changes i n e l e c t r o s t a t i c  s u r f a c e charges, a l t e r a t i o n s i n transmembrane p o t e n t i a l  (depolarization),  2+ and s h i f t s  i n Ca  gradients (Schaffner et a l . ,  c e l l f u n c t i o n s t h a t mediate molecular i n t e r a c t i o n s .  1982).  The macrophage  such a c t i v a t i o n i n v o l v e dynamic and  fleeting  Thus, the f u n c t i o n a l b e h a v i o r o f the macrophage  membrane i s p r o b a b l y dependent on events t a k i n g p l a c e on t i m e s c a l e s r a n g i n g from p i c o s e c o n d s t o seconds  (Lakowicz, 1980).  Use o f f l u o r e s c e n c e  s p e c t r o s c o p y o f f l u o r e s c e n t m o l e c u l e s i n s e r t e d i n t o macrophage membranes p e r m i t s continuous o b s e r v a t i o n o f the dynamic i n i t i a l  events o f a c t i v a t i o n  and i n h i b i t i o n o f macrophage f u n c t i o n . Some o f the macrophage a c t i v a t i n g f a c t o r s s t u d i e d t o date a r e i n t e r f e r o n y. macrophage i n h i b i t i n g f a c t o r , p r o s t a g l a n d i n s , and fibronectin  (Schaffner et a l . ,  Wright e t a l . ,  1983).  1982;  Hogg, 1986;  Braquet e t a l . ,  1985;  The a d a p t a b i l i t y o f macrophage p o p u l a t i o n s t o such  9  s t i m u l i i s a c r u c i a l mechanism o f upgrading infection  7.  the host immune response  to  ( S c h a f f n e r e t a l . , 1982).  Fibronectin F i b r o n e c t i n i s a l a r g e d i m e r i c g l y c o p r o t e i n ( m o l e c u l a r weight 440,000  d a l t o n s ) which has  s p e c i f i c b i n d i n g s i t e s f o r mammalian c e l l s ,  g l y c o p r o t e i n s , and b a c t e r i a l s u r f a c e s ( P r o c t o r , 1987).  viral  Fibronectin  s e v e r a l b i o l o g i c a l f u n c t i o n s i n c l u d i n g c e l l - t o - c e l l attachment, stabilization,  c e l l d i f f e r e n t i a t i o n and wound h e a l i n g .  The  has  clot  fibronectin  network a t a s i t e o f i n j u r y p r o v i d e s the s c a f f o l d on which the components o f c o n n e c t i v e t i s s u e r e q u i r e d f o r wound h e a l i n g can be d e p o s i t e d ( P r o c t o r , 1987).  In a d d i t i o n , f i b r o n e c t i n has been shown t o a c t i v a t e macrophages  f o r i n c r e a s e d adherence (Akiyama e t a l . , 1981), C3Fc-receptor-mediated 1983;  p h a g o c y t o s i s of coated e r y t h r o c y t e s (Wright et a l . ,  Pommier et a l . , 1983), and maintenance o f a n t i - s t a p h y l o c o c c a l  activity  ( P r o c t o r e t a l . , 1985).  opsonin i n the promotion  8.  and  I t has been h y p o t h e s i z e d t o a c t as  an  o f S t a p h y l o c o c c a l uptake ( P r o c t o r e t a l . , 1982).  S p e c i f i c Aims o f T h i s Study The  o b j e c t i v e o f t h i s i n v e s t i g a t i o n was  macrophages w i t h P. a e r u g i n o s a . p h a g o c y t o s i s was macrophage c e l l  To a s s i s t  t o examine the i n t e r a c t i o n o f i n t h i s aim,  a v i s u a l assay  of  e s t a b l i s h e d and d e t e r m i n a t i o n o f an a p p r o p r i a t e l i n e f o r e x p e r i m e n t a t i o n was  made.  T h i s assay was  used  i n v e s t i g a t e the o p s o n i c p o t e n t i a l o f Pseudomonas o u t e r membrane p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s w i t h r e s p e c t t o timecourse o f b a c t e r i a l  to  10  uptake and P.  i n f l u e n c e of antibody subclass.  a e r u g i n o s a may  protect  One  o f the ways i n which  i t s e l f from such b a s i c host  opsonized phagocytosis i s v i a production  of c y t o t o x i n .  defenses This  as  27,000 d a l t o n  a c i d i c p r o t e i n p r o b a b l y a c t s by forming pores i n the membrane o f c e l l s o f the immune system (Scharmann, 1976). i n c r e a s e d membrane p e r m e a b i l i t y al.,  1985).  et a l . , 1985), e n d o t h e l i a l c e l l s a s c i t e s tumor c e l l s  T h i s appears t o r e s u l t i n  to s m a l l molecules and  Such i n t o x i c a t i o n has  ions  (Lutz et a l . , 1987).  Two  o f the aims o f t h i s  study  to  function.  i n v e s t i g a t i o n o f the o p s o n i c p o t e n t i a l o f a n t i - p r o t e i n  F monoclonal a n t i b o d i e s ,  the e f f e c t o f i n v i v o growth o f P. a e r u g i n o s a  non-opsonic p h a g o c y t o s i s was  the d i s c o v e r y  (Baltch  ( S u t t o r p et a l . , 1985), and E h r l i c h  i n v e s t i g a t e i t s e f f e c t on macrophage p h a g o c y t i c  o p s o n i c and  ( B a l t c h et  been documented i n g r a n u l o c y t e s  were to determine the c e l l u l a r l o c a l i z a t i o n o f c y t o t o x i n and  To f u r t h e r our  target  t h a t P.  determined.  on  These s t u d i e s l e d t o  a e r u g i n o s a c e l l s taken d i r e c t l y from an i n v i v o  system were s i g n i f i c a n t l y more s u s c e p t i b l e to macrophage p h a g o c y t o s i s than were the same c e l l s a f t e r b e i n g washed i n b u f f e r . p h a g o c y t o s i s - p r o m o t i n g f a c t o r c o u l d be  The  i s o l a t e d from the supernatant  c e n t r i f u g e d i n vivo-grown b a c t e r i a ( i n v i v o s u p e r n a t a n t ) and f r a c t i o n a t i o n o f t h i s supernatant was  determined to be  of  by  fibronectin.  The  o b j e c t i v e s o f t h i s study were t o determine the r o l e o f f i b r o n e c t i n i n promotion of p h a g o c y t o s i s and f o r fibronectin-mediated  to i n v e s t i g a t e the b a c t e r i a l l i g a n d  uptake.  required  11  MATERIALS AND METHODS  1.  Bacteria A.  Bacterial strains.  Pseudomonas a e r u g i n o s a PA01 s t r a i n H103, a  laboratory serotype 5 i s o l a t e  (Nicas and Hancock, 1980) and M2, a s t r a i n  t r a d i t i o n a l l y used f o r mouse p a t h o g e n i c i t y s t u d i e s ( S t i e r i t z and Holder, 1975) were used as r e f e r e n c e s t r a i n s throughout  this thesis.  Other  P. a e r u g i n o s a s t r a i n s used were BLP3, a Tn501-induced p i l u s - m i n u s mutant; p B P l 6 l , a p i l u s - e x p r e s s i n g s t r a i n c o n t a i n i n g b o t h t h e Tn501 mutation and an a d d i t i o n a l plasmid-encoded  p i l i n gene; and P A O l - l e u , t h e PA01 p a r e n t a l  s t r a i n o f these two mutants.  A l l t h r e e o f these s t r a i n s were k i n d g i f t s  from B r i t t a i n Paslowske ( U n i v e r s i t y o f A l b e r t a , Edmonton, A l b e r t a ) . C y t o t o x i n was i s o l a t e d from P. a e r u g i n o s a s t r a i n 158 by F r i e d e r L u t z ( J u s t u s - L i e b i g U n i v e r s i t y , G i e s s e n , F e d e r a l R e p u b l i c o f Germany) and was k i n d l y donated  f o r use i n t h e s e  Escherichia c o l i experiments  studies.  s t r a i n C600 (Applegard, 1954) was used i n c o n t r o l  f o r t h e c y t o t o x i n s t u d i e s o f Chapter Two.  and S t a p h y l o c o c c u s aureus  s t r a i n 8529 were c l i n i c a l  E. c o l i  isolates  donated by Anthony Chow ( U n i v e r s i t y o f B r i t i s h Columbia,  B.  Media and i n v i t r o growth c o n d i t i o n s .  s t r a i n 8239  kindly  Vancouver, B.C.).  P. a e r u g i n o s a s t r a i n s H103,  M2 and P A O l - l e u were m a i n t a i n e d on T r y p t i c a s e Soy agar  (Becton, D i c k i n s o n  & Co., C o c k e y s v i l l e , MD) o r 1% (wt/vol) P r o t e o s e Peptone no. 2 agar  (Difco  L a b o r a t o r i e s , D e t r o i t , MI). BLP3 and p B P l 6 l were m a i n t a i n e d on T r y p t i c a s e Soy agar c o n t a i n i n g 300 ug/ml c a r b e n i c i l l i n .  Escherichia c o l i  strains  12  C600 and 8239, and S. aureus Peptone no. 2 agar.  s t r a i n 8529 were m a i n t a i n e d on 1%  Proteose  P r i o r t o assay, c e l l s were i n o c u l a t e d from  these  p l a t e s and grown f o r 20 h a t 37°C e i t h e r w i t h v i g o r o u s s h a k i n g (200 or s t a t i c a l l y  (without shaking) i n b r o t h o r on p l a t e s .  (BM2)-glucose  broth ( G i l l e l a n d et a l . ,  o f Chapters One  and Two,  phosphate b u f f e r e d s a l i n e c o n c e n t r a t i o n o f 1x10  C.  B a s a l medium 2  used f o r the  experiments  used f o r the  S t a t i o n a r y phase c e l l s were washed i n  (PBS), pH 7.2,  and resuspended  to a  /ml p r i o r t o e x p e r i m e n t a t i o n .  B a c t e r i a l growth i n v i v o .  performed  was  w h i l e T r y p t i c a s e Soy media was  s t u d i e s p r e s e n t e d i n Chapter Three.  a  1974)  rpm)  B a c t e r i a l growth i n v i v o was  by Dr. Niamh K e l l y i n our l a b o r a t o r y .  Briefly,  kindly  chambers f o r  i m p l a n t a t i o n i n t o mice were c o n s t r u c t e d from 1 ml p o l y p r o p y l e n e s y r i n g e b a r r e l s as p r e v i o u s l y d e s c r i b e d (Day e t a l . ,  1980).  Chambers f o r  i m p l a n t a t i o n i n t o r a t s were s i m i l a r l y c o n s t r u c t e d from 3 ml syringe barrels  ( K e l l y e t a l . , 1988).  i n o c u l a t e the chambers was  polypropylene  The P. a e r u g i n o s a c u l t u r e used  to  grown o v e r n i g h t i n P r o t e o s e Peptone no. 2 b r o t h  and d i l u t e d i n p h y s i o l o g i c a l s a l i n e t o g i v e a chamber inoculum o f a p p r o x i m a t e l y 10^ b a c t e r i a per ml.  Chambers f o r i m p l a n t a t i o n i n t o mice  r e c e i v e d a volume o f 100 v l o f the d i l u t e d c u l t u r e , w h i l e chambers f o r r a t s r e c e i v e d 500  ul.  The animals were a n a e s t h e s i z e d by  i n j e c t i o n o f sodium p e n t o b a r b i t o l (Somnotol, M i s s i s s a u g a , O n t a r i o , Canada) a t 0.06 0.14  mg/g  for rats.  mg/g  intraperitoneal  M.T.C. P h a r m a c e u t i c a l s ,  o f body weight  f o r mice and  Chambers were implanted, f o u r per animal, through  s m a l l l o n g i t u d i n a l i n c i s i o n i n the abdomen o f the a n i m a l .  The  chambers  a  13  were removed a f t e r t h r e e days a t which stage the b a c t e r i a l c u l t u r e s 8 reached t h e i r maximal d e n s i t y o f 10  had  9 - 10  cells/ml  ( K e l l y et a l . ,  1988). Unwashed i n vivo-grown  Pseudomonas c e l l s were counted i n a  P e t r o f f - H a u s s e r b a c t e r i a l c o u n t i n g chamber (Hausser S c i e n t i f i c , Blue PA) and m a i n t a i n e d on i c e u n t i l use. 12,000 x g and g e n t l y resuspended  Washed organisms  i n PBS  were c e n t r i f u g e d a t  t w i c e p r i o r to a s s a y .  decanted s u p e r n a t a n t from t h e s e c e l l s was  Bell,  The  saved f o r assessment  first  of  p h a g o c y t o s i s enhancement ( i n v i v o s u p e r n a t a n t ) . To c h a r a c t e r i z e the p h a g o c y t o s i s - p r o m o t i n g f a c t o r , i n v i v o supernatant was  passed over a F a s t P r e s s u r e L i q u i d Chromatography (FPLC) Superose  g e l s i e v i n g column (Pharmacia, D o r v a l , Quebec). and e l u t i o n b u f f e r c o n s i s t e d o f 20 mM O h i o ) , 0.1M  NaCl  (BDH  Tris  The f l o w r a t e was  e x t e n s i v e l y a g a i n s t d i s t i l l e d water.  30  ml/h  (ICN B i o m e d i c a l s , C l e v e l a n d ,  Chemicals, T o r o n t o , O n t a r i o ) , pH 7-5.  were c o l l e c t e d , l y o p h i l i z e d , resuspended  12  Fractions  i n d i s t i l l e d water and  dialyzed  The f i n a l r e s u s p e n s i o n volume  was  the same as the volume o f supernatant i n i t i a l l y added t o the column.  D.  Preparation of s u b - c e l l u l a r f r a c t i o n s .  P. a e r u g i n o s a c y t o t o x i n 2+  ( L u t z , 1979),  osmotic shock f l u i d ,  method (Hoshino and Kageyama, 1980;  p r e p a r e d by the Mg  Poole and Hancock, 1984),  and o u t e r membranes (Hancock and N i k a i d o , 1978) previously. and 0.43 158,  /freeze-thaw and i n n e r  were p r e p a r e d as d e s c r i b e d  These p r e p a r a t i o n s c o n t a i n e d , r e s p e c t i v e l y <0.01, 0.15,  ug LPS per ug p r o t e i n .  C y t o t o x i n was  o b t a i n e d from  0.24  strain  w h i l e osmotic shock f l u i d s and membranes were p r e p a r e d from  strain  14  H103.  E. c o l i osmotic shockate was  method o f Neu  and Heppel  (1965).  from P. a e r u g i n o s a s t r a i n H103 BM2-glucose b r o t h (200 rpm).  i s o l a t e d from s t r a i n C600 by  the  Growth supernatant samples were p r e p a r e d  c e l l s grown f o r 20 h a t 37°C i n shaken Growth supernatant was  decanted  after  removal o f c e l l s by c e n t r i f u g a t i o n a t 13,000 x g f o r 10 min a t 4°C. supernatant was  l y o p h i l i z e d and resuspended  i n 10 mM  1 5 0 - f o l d h i g h e r than o r i g i n a l c o n c e n t r a t i o n . performed  a g a i n s t 10 mM  m o l e c u l a r weight  E.  substances.  at a  was  b u f f e r t o remove low  Samples were s t o r e d a t -70°C u n t i l  use.  Sodium d o d e c y l s u l p h a t e ( S D S ) - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s .  SDS-polyacrylamide previously  g e l e l e c t r o p h o r e s i s was  (Hancock and Carey, 1979).  lower s e p a r a t i n g g e l was Two)  T r i s - H C l , pH 7.4  Extensive d i a l y s i s  T r i s - H C l b u f f e r , pH 7.4  The  performed  as d e s c r i b e d  The a c r y l a m i d e c o n c e n t r a t i o n i n the  11% ( w t / v o l ) f o r the c y t o t o x i n s t u d i e s  and 5% f o r d e t e c t i o n o f f i b r o n e c t i o n (Chapter T h r e e ) .  (Chapter  Samples were  s o l u b i l i z e d i n the presence o f 5% ( v o l / v o l ) 2-mercaptoethanol  (Bio-Rad,  M i s s i s s a u g a , O n t a r i o ) and were heated a t 100°C f o r 10 min  i n the  o f SDS.  the f o l l o w i n g  For e s t i m a t i o n o f m o l e c u l a r weights on SDS-gels,  m o l e c u l a r weight ovalbumin  standards were used, bovine serum albumin  (45K); phosphate  absence  (66.2K);  dehydrogenase (36K); c a r b o n i c anhydrase  (29K);  and t r y p s i n o g e n (24K). (i)  Protein staining.  P r o t e i n s i n SDS-PAGE were s t a i n e d i n 1%  ( w t / v o l ) Coomassie b r i l l i a n t b l u e d i s s o l v e d i n g l a c i a l acid:methanol:water  at a r a t i o o f 1:4.5:4.5.  acetic  D e s t a i n i n g was  the same s o l u t i o n (without the dye) a t a 0.7:2:7-2 r a t i o .  performed i n  15  (ii)  Western immunoblotting.  P r o t e i n s from S D S - p o l y a c r y l a m i d e  g e l s were imraunoblotted onto n i t r o c e l l u l o s e paper (0.45 um) as d e s c r i b e d previously  ( M u t h a r i a and Hancock,  1983).  F o r development,  20 u l o f  r a b b i t a n t i - c y t o t o x i n serum, 30 u l o f r a b b i t anti-exoenzyme S serum, o r 100 ug o f goat anti-human f i b r o n e c t i n  (Sigma, S t . L o u i s , M0) was  u t i l i z e d per b l o t .  F.  P r o t e i n and 2-keto-3-deoxyoctonate (KDO) a s s a y s .  P r o t e i n assays  were done by t h e method o f S c h a c t e r l e and P o l l a c k (1973). of  Determination  l i p o p o l y s a c h a r i d e (LPS) c o n t e n t was made by assuming t h a t t h e LPS  c o n t a i n e d 4.3% (wt/wt) o f KDO (Darveau and Hancock,  1983).  KDO assays  were performed by t h e method o f Osborn e t a l . (1972) w i t h an a d d i t i o n a l 15 min h y d r o l y s i s s t e p i n 50 mM H^SO^.  2.  Macrophages A.  Macrophage c e l l  lines.  Three mouse macrophage c e l l  assessed f o r t h e i r phagocytic capacity: tumor c e l l  line  P388  D 1 >  l i n e s were  a DBA/2 macrophage  (American T i s s u e C u l t u r e C o l l e c t i o n #CCL46); PU5-1.8, a  B a l b / c monocytic, macrophage-like c e l l  line  C o l l e c t i o n # T I B 6 l ) ; and DC7, a macrophagetT l i n e s were m a i n t a i n e d a t 37°C, 10% C 0 flat-bottomed f l a s k s  2 >  (American T i s s u e cell  f u s i o n product.  Cell  80% h u m i d i t y i n Nunc  (Gibco, B u r l i n g t o n , O n t a r i o ) u s i n g  RPMI-1640 medium ( G i b c o ) .  Culture  supplemented  Media was supplemented w i t h 44 mM  sodium  b i c a r b o n a t e ( F i s h e r S c i e n t i f i c , Vancouver, B.C.), 10% v/v f e t a l c a l f  serum  ( G i b c o ) , 10 mM Hepes (Terochem L a b o r a t o r i e s , Vancouver, B.C.), 0.04% v/v  16  2-mercaptoethanol (Bio-Rad, (Sigma, S t . L o u i s , MO), streptomycin  M i s s i s s a u g a , O n t a r i o ) , 2 mM  L-glutamine  40 u n i t s per ml p e n i c i l l i n and 40 mg/ml  ( G i b c o ) , pH 7.2.  P r i o r t o assay c e l l s were h a r v e s t e d  by  5 g e n t l e p i p e t t i n g and resuspended t o 5x10 Aliquots  (2 ml)  c e l l s / m l i n f r e s h medium.  o f t h i s c e l l s u s p e n s i o n were i n c u b a t e d i n 35x10 mm  t i s s u e c u l t u r e d i s h e s (Gibco) a t 37°C i n 10% C0^. were washed o f f j u s t p r i o r t o  B.  Non-adherent  Nunclon  cells  assay.  O n e l i c i t e d mouse p e r i t o n e a l macrophages.  P e r i t o n e a l macrophages  were o b t a i n e d from s i x t o e i g h t week o l d female B a l b / c mice.  C e l l s were  washed from the p e r i t o n e a l c a v i t y w i t h supplemented RPMI-1640.  C e l l s were  67.1%  as  macrophages, 32.5%  determined  by " D i f f - q u i k  inspection.  r e d b l o o d c e l l s , and 0.4% (Canlab, Vancouver, B.C.)  M  granulocytes s t a i n i n g and  visual  Macrophages were s e p a r a t e d from e r y t h r o c y t e s by 5  c e n t r i f u g a t i o n a t 1000  RPM  f o r 10 min  and were resuspended t o  c e l l s / m l i n supplemented RPMI-1640 (above). s u s p e n s i o n were i n c u b a t e d i n 35x10 mm (Gibco) a t 37°C i n 10% C0 2  5x10  A l i q u o t s (2 ml) o f t h i s  cell  Nunclon t i s s u e c u l t u r e d i s h e s  A f t e r 20 h non-adherent c e l l s were  removed by washing the monolayer g e n t l y w i t h RPMI-1640.  C.  Human p e r i p h e r a l b l o o d monocyte-derived macrophages.  were prepared  Monocytes  from human p e r i p h e r a l b l o o d u s i n g the P i c o l l - p a q u e  s e p a r a t i o n t e c h n i q u e as d e s c r i b e d p r e v i o u s l y (Boyum, 1968). were m a i n t a i n e d  i n RPMI-1640 medium supplemented w i t h 44 mM  Monocytes sodium  b i c a r b o n a t e , 15% homologous human serum, 40 u n i t s per ml p e n i c i l l i n and  40  17  mg/ml s t r e p t o m y c i n .  C e l l s were kept i n screw cap T e f l o n j a r s ( S a v i l l e x ,  Minnetonka, MN) a t 37°C, 5% CC> f o r a p p r o x i m a t e l y 96 h p r i o r t o u s e . 2  5  These "Day Four" monocytes were washed and resuspended t o 5x10  cells/ml  i n RPMI-1640 supplemented w i t h 10 mM Hepes and 2 g/1 b o v i n e serum albumin (Sigma).  Aliquots of this c e l l  Nunclon t i s s u e c u l t u r e d i s h e s were washed o f f b e f o r e  3.  Antibody A.  s u s p e n s i o n (2 ml) were i n c u b a t e d i n  f o r 1 h p r i o r t o assay.  Non-adherent  cells  s t a r t i n g p h a g o c y t o s i s experiments.  Preparation  A n t i - p r o t e i n F monoclonal a n t i b o d i e s .  Monoclonal a n t i b o d i e s  c o l l e c t e d from murine a s c i t e s and p u r i f i e d by ammonium p r e c i p i t a t i o n as p r e v i o u s l y d e s c r i b e d f o l l o w i n g monoclonal a n t i b o d i e s  (Mutharia  described  sulphate  and Hancock, 1983).  previously  were  The  (Mutharia and  Hancock, 1985) were used: MA4-4, MA5-10, MA2-10, MA5-8 and MA4-10, s p e c i f i c f o r p r o t e i n F (Mutharia  and Hancock, 1985); and MA1-3  f o r l i p o p r o t e i n s I/H2 (Hancock e t a l . , 1982). antibodies 1985)  was determined as d e s c r i b e d  specific  The i s o t y p e o f a l l  previously  (Mutharia  and Hancock,  by double immunodiffusion ( O u c h t e r l o n y , 1958).  B.  Polyclonal (i)  sera.  A n t i - c y t o t o x i n serum.  R a b b i t a n t i - c y t o t o x i n s e r a was  g e n e r o u s l y p r e p a r e d by F. L u t z by t h e method o f Harboe and I n g i l d (1973) as d e s c r i b e d One  by B a l t c h e t a l . (1987) and was used a t 13 u l p e r a s s a y .  u l o f t h i s a n t i s e r a was shown t o pevent 1 ug o f c y t o t o x i n from  inducing  t h e s w e l l i n g o f human g r a n u l o c y t e s .  The c y t o t o x i n  preparation  18  used  t o r a i s e the a n t i s e r a was  pure as a s s e s s e d by sodium d o d e c y l  e l e c t r o p h o r e s i s , immunodiffusion, the presence  Anti-exoenzyme S serum.  generous g i f t  o f T h a l i a Nicas  (iii)  4.  from  Rabbit anti-exoenzyme S serum was  Goat anti-human f i b r o n e c t i n  was  Sigma.  Assay  Conditions.  The  v i s u a l i n s p e c t i o n p h a g o c y t i c assay u t i l i z e d  m o d i f i e d from t h a t p u b l i s h e d by Speert e t a l . (1984). RPMI-1640 medium without antibiotic  a  ( U n i v e r s i t y o f Ottawa, Ottawa, O n t a r i o ) .  A n t i - f i b r o n e c t i n serum.  Phagocytosis A.  methods f o r d e t e r m i n i n g  o f o t h e r known Pseudomonal components ( L u t z e t a l . , 1987).  (ii)  purchased  and enzymatic  sulphate  Briefly,  f e t a l c a l f serum, 2-mercaptoethanol,  ( p h a g o c y t o s i s b u f f e r ) was  1 ml  was of  or  added t o a washed, c u l t u r e d  macrophage monolayer t o g i v e a f i n a l c o n c e n t r a t i o n o f 1x10^  cells/ml.  To assess i n h i b i t i o n o r a c t i v a t i o n o f o p s o n i c p h a g o c y t o s i s , v a r i o u s p r e p a r a t i o n s were added t o the macrophage monolayer 15 min p r i o r t o a d d i t i o n o f a n t i - p r o t e i n F monoclonal a n t i b o d i e s and b a c t e r i a l c e l l s . a s s e s s i n h i b i t i o n o r a c t i v a t i o n o f non-opsonic  uptake,  To  p r e p a r a t i o n s were  added t o the macrophage monolayer 15 min p r i o r t o a d d i t i o n o f b a c t e r i a  and  no o t h e r opsonins  or macrophage a c t i v a t o r s were i n c l u d e d i n the system.  P. a e r u g i n o s a was  used  and p h a g o c y t o s i s was o r macrophage c e l l  i n the assay a t a bacteria:macrophage  a l l o w e d t o o c c u r f o r 90 min  l i n e s ) o r 60 min  r a t i o of  ( p e r i t o n e a l macrophages  (human p e r i p h e r a l b l o o d monocytes) i n  o 10% C0^  a t 37 C.  F o l l o w i n g i n c u b a t i o n , c e l l s were scraped from  d i s h w i t h a rubber policeman  20:1  and resuspended  the  with gentle p i p e t t i n g .  19  Aliquots  o f t h i s s u s p e n s i o n were c y t o c e n t r i f u g e d  (Cytospin  onto a g l a s s  slide  2, Shandon Southern Instruments, I n c . , S e w i c k l e y , PA;  450  rpm,  5 min), and s t a i n e d w i t h " D i f f - q u i k " f o r v i e w i n g under o i l a t lOOOx. number o f b a c t e r i a i n each o f 60 c e l l s was analysis  (Student's t t e s t ) performed.  previously  shown t h a t  counted and  The  statistical  S p e e r t e t a l . (1984) had  r e s u l t s o b t a i n e d w i t h t h i s v i s u a l p h a g o c y t o s i s assay  gave comparable d a t a t o t h a t o f chemiluminescence  and e l e c t r o n  microscopy  studies.  B.  Inhibitors/activators. (i)  Chapter one.  To a s s e s s the o p s o n i c c a p a c i t y  o f the  o a n t i - p r o t e i n F monoclonal  antibodies  u t i l i z e d a t 10% o f assay volume. at a n o n - s u r f a c e exposed (ii) phosphate  f r a c t i o n s was  N e g a t i v e c o n t r o l s were MA1-3  p r o t e i n e p i t o p e o f P. a e r u g i n o s a ) and  Chapter two.  buffered  (ELISA t i t e r 10 ), they were (directed PBS.  To a s s e s s i n h i b i t i o n o f p h a g o c y t o s i s ,  s a l i n e (PBS), c y t o t o x i n ,  o r one o f the s u b c e l l u l a r  added t o the macrophage monolayer 15 min p r i o r t o a d d i t i o n  o f a n t i b o d y and b a c t e r i a . [13 ug o f c y t o t o x i n , bacterial cells),  I n h i b i t o r s were added i n 100 y l volumes  500 ug o f osmotic shockates  400 ug o f growth supernatant  Ug i n n e r o r o u t e r membrane (2x10^  cells)].  (from 10*"^  (10*"''" c e l l s ) o r  Anti-protein  F  500  monoclonal  a n t i b o d y MA5-8 ( t i t e r 10 ) was used i n a l l p h a g o c y t o s i s v i s u a l assays at a volume o f 30 u l per a s s a y . I f used, r a b b i t a n t i - c y t o t o x i n o r r a b b i t anti-exoenzyme addition.  S was  p r e i n c u b a t e d w i t h i n h i b i t o r f o r 5 min p r i o r t o  Background l e v e l s o f b a c t e r i a l a s s o c i a t i o n  ( o b t a i n e d i n the  20  presence o f PBS a l o n e ) were s u b t r a c t e d from t h e average number o f b a c t e r i a a s s o c i a t e d p e r macrophage. (iii)  Chapter t h r e e .  To a s s e s s enhancement o f p h a g o c y t o s i s , PBS,  100 u l o f i n v i v o s u p e r n a t a n t , v a r i o u s c o n c e n t r a t i o n s o f b o v i n e plasma fibronectin  (Sigma) o r t h e a r g i n i n e - g l y c i n e - a s p a r t i c a c i d - s e r i n e (RGDS)  p e p t i d e (Sigma) were added.  No a d d i t i o n a l opsonins o r macrophage  a c t i v a t o r s were i n c l u d e d i n t h e system. When u t i l i z e d , goat anti-human f i b r o n e c t i n (Sigma) was i n c u b a t e d w i t h supernatant o r f i b r o n e c t i n f o r 5 min a t room temperature a d d i t i o n t o t h e macrophage monolayer.  prior to  Antibody was used a t t h e  recommended r a t i o o f 1 ug p e r ug o f f i b r o n e c t i n . When u t i l i z e d , 60 ug/ml o f p u r i f i e d PA01 p i l i , B. Paslowske  p r e p a r e d by  as d e s c r i b e d p r e v i o u s l y (Paranchych e t a l . ,  1979) was  i n c u b a t e d w i t h i n v i v o s u p e r n a t a n t - , f i b r o n e c t i n - o r RGDS-activated macrophages f o r 15 min a t 37°C, 10% C 0  2  p r i o r to the a d d i t i o n o f  bacteria.  5.  F l u o r e s c e n c e Assay P388  D 1  c e l l s were resuspended,  w i t h a p i p e t t e , from t h e bottom o f  f l a t bottomed f l a s k s , c e n t r i f u g e d a t 1000 rpm f o r 10 min, resuspended c o n c e n t r a t i o n o f 2.5 x 1 0  6  c e l l s / m l i n f r e s h supplemented  and grown 16 h p r i o r t o assay i n screw-cap  teflon jars.  r e s u s p e n s i o n , c e l l s were washed and resuspended s o l u t i o n which approximated et a l . ,  1980).  at a  RPMI medium, After gentle  i n an e x p e r i m e n t a l  t h e i o n c o m p o s i t i o n o f RPMI-1640 medium (Rink  KC1 was added t o a f i n a l c o n c e n t r a t i o n o f 0.3 mM t o c r e a t e  21  a K  +  c o n c e n t r a t i o n g r a d i e n t a c r o s s the plasma membrane.  dispensed  i n 1 ml assay volumes, and  3,3'-dipropylthiodicarbocyanine Oregon) was  the carbocyanine  iodide (diS-C (5)) 3  added at a c o n c e n t r a t i o n o f 2 x l 0 ^ M. -  C e l l s were  dye  ( M o l e c u l a r Probes, Carbocyanine dyes a r e  l i p o p h i l i c probes which a r e h i g h l y f l u o r e s c e n t i n aqueous environments minimally  f l u o r e s c e n t i n the hydrophobic environment o f the membrane  et a l . , 1980).  When i o n f l u x e s a r e generated  and  (Rink  a c r o s s the membrane, e i t h e r  the probe i s shunted out o f the membrane to become more f l u o r e s c e n t (upon d e p o l a r i z a t i o n , decreasing e l e c t r i c a l p o t e n t i a l gradients across  the  plasma membrane) o r more probe i s i n s e r t e d i n t o the membrane to become l e s s f l u o r e s c e n t (upon h y p e r p o l a r i z a t i o n ) . To t e s t the e f f e c t s o f c y t o t o x i n , osmotic  shock f l u i d s and  fibronectin  on the plasma membrane o f macrophages, v a r i o u s c o n c e n t r a t i o n s were added to c e l l s e q u i l i b r a t e d with d i S C ( 5 ) 3  ( e x c i t a t i o n at 620  nm;  and  e m i s s i o n at 670  the change i n f l u o r e s c e n c e nm)  was  measured i n a  650-10S S p e c t r o f l u o r i m e t e r .  A l l samples were added i n 100  (volumes e q u a l i z e d w i t h PBS,  pH 7.2).  The  (Sigma), at a c o n c e n t r a t i o n o f 2 x l 0 ~ ^ M was for depolarization.  K  +  ionophore  Perkin-Elmer  ul aliquots  valinomycin  used as a p o s i t i v e c o n t r o l  Rates of d e p o l a r i z a t i o n were measured from the  maximal s l o p e s o b t a i n e d from the s p e c t r o f l u o r i m e t e r t r a c e . were always a t t a i n e d w i t h i n 1 min  of stimulus a d d i t i o n .  Maximal r a t e s  22  RESULTS  CHAPTER I  Opsonic P h a g o c y t o s i s Macrophage C e l l  o f Pseudomonas a e r u g i n o s a by Macrophages  Lines  Pseudomonas a e r u g i n o s a producing  and  i s an o p p o r t u n i s t i c pathogen c a p a b l e  life-threatening infections i n debilitated  of  individuals.  P a t i e n t s e s p e c i a l l y a t r i s k i n c l u d e those w i t h c y s t i c f i b r o s i s , burns,  cancer o r d i a b e t e s .  severe  Because o f i t s h i g h n a t u r a l r e s i s t a n c e t o most  a p p l i c a b l e a n t i b i o t i c s , Pseudomonas i n f e c t i o n s a r e o f t e n f a t a l . As a gram-negative b a c t e r i u m , envelope  membranes:  membrane. The it  P. a e r u g i n o s a possesses  the c y t o p l a s m i c  two  ( o r i n n e r ) membrane and  cell the o u t e r  These l a y e r s a r e s e p a r a t e d by a s i n g l e l a y e r o f p e p t i d o g l y c a n .  o u t e r membrane appears t o be important  i n Pseudomonas i n f e c t i o n s s i n c e  p l a y s a r o l e i n a n t i b i o t i c r e s i s t a n c e and  lipopolysaccharide Mutharia  c o n t a i n s the  endotoxic  (LPS) and p r o t e i n a n t i g e n s .  and Hancock (1983) i s o l a t e d a s e r i e s o f monoclonal a n t i b o d i e s  s p e c i f i c f o r e p i t o p e s on p r o t e i n a n t i g e n s common t o a l l 17 P. serotypes. one  aeruginosa  R e s u l t s i n d i c a t e d t h a t monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t  o f these o u t e r membrane p r o t e i n s ( p r o t e i n F) were p r o t e c t i v e i n mouse  i n f e c t i o n models (Hancock e t a l . , 1985).  F u r t h e r work demonstrated t h a t  f i v e monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t p r o t e i n F c o u l d be i n t o two  separated  c l a s s e s , each r e a c t i n g w i t h a d i f f e r e n t h i g h l y conserved  e p i t o p e on the p o r i n ( M u t h a r i a and Hancock, 1983).  surface  Monoclonal a n t i b o d i e s  23  MA4-4, 2-10, 4-10 and 5-10 were a l l h y p o t h e s i z e d t o r e a c t a g a i n s t one e p i t o p e , w h i l e MA5-8 was s p e c i f i c f o r a d i s t i n c t  epitope.  A n t i b o d i e s a g a i n s t p r o t e i n F thus appear t o have p o t e n t i a l as p a s s i v e immunotherapeutic agents.  P r e v i o u s d a t a i n d i c a t e d , however, t h a t these  monoclonal a n t i b o d i e s f a i l e d t o enhance complement-mediated k i l l i n g o f P. a e r u g i n o s a (Hancock e t a l . , 1985). macrophages a r e one o f t h e primary (Dunn e t a l . , 1985; Green and Kass,  bactericidal  Therefore, since  l i n e s o f defense a g a i n s t i n f e c t i o n 1964; Reynolds e t a l . , 1975), one  might assume t h a t t h e monoclonal a n t i b o d i e s were a c t i n g t o o p s o n i z e t h e P. a e r u g i n o s a f o r p h a g o c y t o s i s i n t h e mouse i n f e c t i o n model. presents data demonstrating  This  Chapter  t h a t p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s  c o u l d indeed o p s o n i z e P. a e r u g i n o s a s t r a i n M2 f o r uptake by macrophages and a macrophage c e l l  1.  line.  Choice o f an a p p r o p r i a t e model c e l l The  line  v i s u a l i n s p e c t i o n p h a g o c y t i c assay u t i l i z e d was m o d i f i e d from t h a t  p u b l i s h e d by Speert e t a l . (1984).  Briefly,  t h e assay i n v o l v e d a 90 min  i n c u b a t i o n o f macrophages, b a c t e r i a and opsonin tissue culture dish. resuspended  ( i f used) t o g e t h e r i n a  Macrophages were then scraped from t h e d i s h ,  g e n t l y , and c y t o c e n t r i f u g e d onto a g l a s s s l i d e .  Following  s t a i n i n g , t h e number o f b a c t e r i a i n each o f 60 macrophages were and  s t a t i s t i c a l comparisons w i t h a p p r o p r i a t e c o n t r o l s made u s i n g  t test.  Speert and c o l l e a g u e s (1984) demonstrated t h a t r e s u l t s  counted Student's obtained  w i t h t h i s assay gave comparable d a t a t o chemiluminescence and e l e c t r o n microscopy  studies.  Initially,  t h r e e macrophage c e l l l i n e s were a s s e s s e d  24  for  t h e i r p h a g o c y t i c c a p a b i l i t y u s i n g t h e v i s u a l assay and P. a e r u g i n o s a  s t r a i n M2 o p s o n i z e d w i t h t h e p r o t e i n F - s p e c i f i c monoclonal a n t i b o d y MA5-10.  The t h r e e c e l l  macrophage-like  l i n e s under study were PU5-1.8, a B a l b / c  monocytic  tumor; DC7, a macrophage:T c e l l  and P 3 8 8 , a DBA/2 macrophage tumor c e l l D 1  p h a g o c y t o s i s o b t a i n e d i n t h e presence determined  f o r a l l three c e l l  line.  line P388  D 1  product;  The l e v e l o f  and absence o f a n t i b o d y was  l i n e s , u n e l e c i t e d mouse p e r i t o n e a l  macrophages, and human p e r i p h e r a l b l o o d monocyte-derived behaviour o f c e l l  fusion  most c l o s e l y approximated  macrophages.  The  that o f the  normal macrophages from both mouse and human o r i g i n ( F i g u r e 1 ) . I n addition, this c e l l  l i n e was c o n s i s t e n t l y h e a l t h y d u r i n g p r o l o n g e d  culture  and d i s p l a y e d a c e l l u l a r morphology s i m i l a r t o t h a t o f normal macrophages.  P388j^ was thus judged  t o be an a p p r o p r i a t e model f o r  u n e l i c i t e d mouse p e r i t o n e a l macrophages and c u l t u r e d human p e r i p h e r a l b l o o d monocytes i n assessment o f p h a g o c y t o s i s o f P. a e r u g i n o s a .  2.  E s t a b l i s h m e n t o f p h a g o c y t o s i s assay c o n d i t i o n s To i n v e s t i g a t e the time c o u r s e o f b a c t e r i a l uptake i n t h e presence o r  absence o f monoclonal a n t i b o d y , the v i s u a l p h a g o c y t o s i s assay was performed for  as i n M a t e r i a l s and Methods but was h a l t e d a t f o u r time p o i n t s  assessment.  The d a t a o b t a i n e d from two s e p a r a t e experiments  t h a t the s a l i n e n e g a t i v e c o n t r o l produced p h a g o c y t o s i s from 0 t o 30 min. and  little  (Figure 2).  showed  a gradual increase i n  A f t e r 30 min, a p l a t e a u r e g i o n was reached  f u r t h e r uptake was observed  f o r the d u r a t i o n o f the experiment  A n t i - F monoclonal a n t i b o d y MA2-10 on t h e o t h e r hand  produced  25 Bacteria / Macrophage  5-  / / / / / /  /  i i Sal Ab PUS-1.8  F i g u r e 1.  / / / / / / / / / r  Sal Ab DC-7  Opsonized p h a g o c y t o s i s  macrophage c e l l l i n e s  / / / /  •A  / / / / / / / / / / / / / / / /  SaL Ab P388 DI  /  / / / / /  \A Sal Ab Mouse Peritoneal Macrophages  / / / /  / /  V i  i r Sal Ab Human Monocytes  o f P . a e r u g i n o s a s t r a i n M2 by mouse  P U 5 - 1 . 8 , DC-7 and P388p^, u n e l e c i t e d  mouse  p e r i t o n e a l macrophages, and human p e r i p h e r a l b l o o d m o n o c y t e - d e r i v e d macrophages.  Saline (Sal)  o r p r o t e i n F - s p e c i f i c monoclonal a n t i b o d y  MA5-10 (Ab) were added t o macrophages and b a c t e r i a and i n c u b a t e d a t i n 10% C 0 lines)  2  37°C  f o r 90 min (mouse p e r i t o n e a l macrophages o r macrophage c e l l  o r 60 min (human p e r i p h e r a l b l o o d m o n o c y t e - d e r i v e d macrophages).  The averages  o f t h r e e independent experiments a r e shown.  26  B a c t e r i a p e r Macrophage  MA2-10  20  0  40  60  80  100  Incubation Time (min)  Figure  2.  Timecourse o f P. a e r u g i n o s a a s s o c i a t i o n w i t h P388D ^ i n t h e  p r e s e n c e o r absence  D  ( s a l i n e ) o f a n t i - F monoclonal a n t i b o d y MA2-10.  v i s u a l p h a g o c y t o s i s assay was performed as i n M a t e r i a l s was h a l t e d a t 15, 30, 60 and 90 min f o r assessment. independent experiments i s shown.  The  and Methods b u t  The average o f 3  27  good uptake almost Phagocytosis  immediately  and l e v e l e d out between 30 and 60 min.  i n c r e a s e d s i g n i f i c a n t l y a f t e r 60 min when c e l l s were  opsonized w i t h t h e monoclonal a n t i b o d y , p r o d u c i n g a b i p h a s i c time  course  of  bacterial association.  I chose  to  h a l t these P 3 8 8  D 1  From t h e r e s u l t s o f these experiments,  p h a g o c y t i c assays a t a time a t which the average  number o f b a c t e r i a / p h a g o c y t e i n the n e g a t i v e c o n t r o l s was v i r t u a l l y  static  (90 m i n ) . With t h e n e g a t i v e c o n t r o l s , s a l i n e and MA1-3  [ a monoclonal a n t i b o d y  d i r e c t e d a g a i n s t an o u t e r membrane e p i t o p e t h a t was not s u r f a c e exposed ( M u t h a r i a and Hancock, 1985)], most o f t h e macrophages had few o r no b a c t e r i a a f t e r 90 min ( F i g u r e 3 ) .  phagocytosed  This resulted i n a  d i s t r i b u t i o n w i t h a l a r g e peak a t z e r o and a very low, s h o r t s h o u l d e r region.  When monoclonal a n t i b o d y MA4-10 was added, t h i s major peak  s h i f t e d over t o approximately  5, 7 and 11 b a c t e r i a p e r c e l l f o r human  p e r i p h e r a l b l o o d monocytes, P 3 8 8 respectively.  3.  Opsonic  D 1  and mouse p e r i t o n e a l macrophages,  The s h o u l d e r r e g i o n a l s o lengthened c o n s i d e r a b l y .  p h a g o c y t o s i s : i n f l u e n c e o f antibody subclass  The p a n e l o f f i v e a n t i - F monoclonal a n t i b o d i e s were a s s e s s e d f o r t h e i r o p s o n i c c a p a c i t y u s i n g c e l l l i n e P 3 8 8 , u n e l i c i t e d mouse p e r i t o n e a l D 1  macrophages, o r human p e r i p h e r a l b l o o d monocyte-derived  macrophages,  P. a e r u g i n o s a s t r a i n M2 and the v i s u a l assay o f p h a g o c y t o s i s . a n t i - F monoclonal a n t i b o d i e s caused  A l l five  s i g n i f i c a n t l y higher b a c t e r i a l  a s s o c i a t i o n w i t h u n e l i c i t e d mouse p e r i t o n e a l macrophages i n a l l assays performed  ( T a b l e I , p<0.01).  I n t e r e s t i n g l y , t h e two most o p s o n i c  a n t i b o d i e s , KA4-10 and 5-10 were o f t h e IgGl s u b c l a s s .  28  % of Macrophages 80 ->  0-1  2-3  4-5  6-7  8-9  10-1112-13 14-15 16-17  +18  Number of Bacteria / Macrophage  Figure 3.  P e r c e n t o f macrophages a s s o c i a t e d w i t h s p e c i f i c  b a c t e r i a a f t e r 90 m i n .  numbers o f  For d e t a i l s - o f a s s a y see M a t e r i a l s and Methods.  N e g a t i v e c o n t r o l MA1-3 d i s t r i b u t i o n was a p p r o x i m a t e l y t h e same f o r a l l macrophage c e l l t y p e s .  A d d i t i o n o f monoclonal a n t i b o d y KA4-10 s h i f t e d  major peak f o r P 3 8 8 ^ c e l l s ,  the  u n e l i c i t e d mouse p e r i t o n e a l macrophages  (mouse p e r . mo.) and human p e r i p h e r a l b l o o d m o n o c y t e - d e r l v e d macrophages (human m o . ) .  Table I.  Enhancement o f t h e a s s o c i a t i o n o f Pseudomonas a e r u g i n o s a M2 w i t h mouse p e r i t o n e a l macrophages, P 3 8 8 ^ c e l l s ,  strain  and human  p e r i p h e r a l b l o o d monocytes u s i n g monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t p r o t e i n F.  Opsonin (Isotype)  Specific Antigen  Average number o f b a c t e r i a Mouse p e r i t o n e a l  associated/phagocyte  P388j^  Human p e r i p h e r a l  macrophages '°  b l o o d monocytes*  a  not done  3-0 + 1.9  2.3 + 1.9  H2/I  1.6 ± 1.2  3.1 + 2.9  3.8 + 2.0  MA4-4 (IgG2a)  F  4.6 ± 3.4  C  4.3 + 2.2  d  5.2 + 3.0  e  MA5-8 (IgG2b)  F  5.3 ± 2.7  C  5.8 + 3.4  C  6.4 + 5.2  f  MA5-10 ( I g G l )  F  5.9 ± 3.6  C  4.7 + 1.7  d  6.1 + 2.7  g  MA2-10 ( I g G l )  F  4.3 ± 3.5  C  5.7 + 2.1  MA4-10 ( I g G l )  F  C  7.3 + 2.2°  -  Saline MA1-3  (IgGl)  14.6  ± 9.0  a  means ± s t a n d a r d d e v i a t i o n s o f 3 independent  experiments.  b  means ± S.D. o f 6 independent  c  p<0.01 (by Student's  d  p<0.01 i n 2 o f 3 a s s a y s .  e  p<0.01 i n 4 o f 6 a s s a y s , p>0.1 i n 2 o f 6 a s s a y s ,  experiments.  t t e s t ) f o r 100% o f a s s a y s .  •p p<0.01 i n 5 o f 6 a s s a y s , p<0.1 i n 1 o f 6 a s s a y s . g  p<0.01 i n 3 o f 6 a s s a y s . p<0.01 i n 3 o f 6 a s s a y s , p<0.1 i n 3 o f 6 a s s a y s .  c  9.9 + 6.0 10.0  + 7.5  f  h  5  30  A l l a n t i b o d i e s d i s p l a y e d some a b i l i t y  t o o p s o n i z e P. a e r u g i n o s a  M2 f o r p h a g o c y t o s i s by mouse macrophage c e l l Monoclonal  line, P388  a n t i b o d i e s MA4-10, 2-10 and 5-8 produced  strain  (Table I ) .  D 1  a phagocytic  index  which was s i g n i f i c a n t l y h i g h e r than t h e n e g a t i v e c o n t r o l v a l u e s i n a l l assays performed  (p<0.01).  The two most e f f i c i e n t monoclonal a n t i b o d i e s ,  MA4-10 and 2-10, were a g a i n o f the I g G l s u b c l a s s .  MA5-10 and 4-4 were  each found t o be s i g n i f i c a n t l y o p s o n i c i n two o f t h r e e  experiments.  For c u l t u r e d human p e r i p h e r a l b l o o d monocytes, the average  number o f  a s s o c i a t e d b a c t e r i a was s i g n i f i c a n t l y i n c r e a s e d i n a t l e a s t f o u r o f s i x assays i n t h e presence (Table I ) .  o f monoclonal a n t i b o d i e s MA4-10, 2-10, 5-8 and 4-4  Again, t h e most o p s o n i c a n t i b o d i e s , MA4-10 and 2-10 were o f  the I g G l s u b c l a s s .  4.  Summary Three macrophage c e l l  capability  l i n e s were a s s e s s e d f o r t h e i r  phagocytic  u s i n g P. a e r u g i n o s a s t r a i n M2 o p s o n i z e d w i t h t h e p r o t e i n  F - s p e c i f i c monoclonal a n t i b o d y MA 5-10. mouse macrophage c e l l  line P388  D 1  Of t h e t h r e e , t h e b e h a v i o u r o f  most c l o s e l y  approximated  that of  normal u n e l i c i t e d mouse p e r i t o n e a l macrophages and c u l t u r e d human p e r i p h e r a l b l o o d monocyte-derived  macrophages.  P388j^ was thus  judged  t o be an a p p r o p r i a t e model f o r normal macrophages i n assessment o f p h a g o c y t o s i s o f P. a e r u g i n o s a . presence  A timecourse o f b a c t e r i a l uptake i n t h e  o f monoclonal a n t i b o d y i n d i c a t e d t h a t o p s o n i c  followed a b i p h a s i c timecourse.  phagocytosis  Good uptake was observed  l e v e l e d out between 30 and 60 min.  immediately and  P h a g o c y t o s i s was seen t o i n c r e a s e  31  significantly  after  60 min.  A p a n e l o f f i v e a n t i - F monoclonal a n t i b o d i e s were a s s e s s e d f o r t h e i r opsonic c a p a c i t y . bacterial  A l l f i v e a n t i b o d i e s caused  significantly  higher  a s s o c i a t i o n w i t h mouse p e r i t o n e a l macrophages and P388j^  c e l l s , w h i l e f o u r o f t h e f i v e were o p s o n i c f o r p h a g o c y t o s i s by human p e r i p h e r a l b l o o d monocyte-derived  macrophages.  a n t i b o d i e s were o f t h e I g G l s u b c l a s s .  The most  effective  32  CHAPTER I I  P. a e r u g i n o s a C y t o t o x i n : L o c a l i z a t i o n and I n a c t i v a t i o n o f Macrophages In the case o f p a t i e n t s w i t h c y s t i c f i b r o s i s , P. a e r u g i n o s a f r e q u e n t l y causes p e r s i s t e n t is  lung i n f e c t i o n ,  i n d i c a t i n g t h a t the host immune system  i n c a p a b l e o f c l e a r i n g the b a c t e r i a .  defense a g a i n s t l u n g i n f e c t i o n ,  As macrophages a r e important i n  i t has been suggested  that these  phagocytes may not be f u n c t i o n i n g c o r r e c t l y i n t h e lungs o f p a t i e n t s w i t h cystic fibrosis  ( S p e e r t , 1985).  One o f t h e ways i n which P. a e r u g i n o s a may p r o t e c t i t s e l f from b a s i c host defenses  i s through p r o d u c t i o n o f a c y t o t o x i n .  such  P. a e r u g i n o s a  c y t o t o x i n , p r e v i o u s l y named l e u k o c i d i n (Scharmann, 1976), has been i s o l a t e d from a u t o l y s a t e s o f P. a e r u g i n o s a c e l l s and appears a s s o c i a t e d w i t h a l l i s o l a t e s o f P. a e r u g i n o s a  ( B a l t c h e t a l . , 1987).  i n a c t i v a t e s e u k a r y o t i c c e l l s by forming l e s i o n s o r pores  This results i n  i n c r e a s e d plasma membrane p e r m e a b i l i t y t o s m a l l molecules  a s c i t e s tumor c e l l s  c y t o t o x i n causes  and i o n s  Such i n t o x i c a t i o n has been documented i n g r a n u l o c y t e s  ( B a l t c h e t a l . , 1985), e n d o t h e l i a l c e l l s  and L u t z , 1985).  It  i n t h e membrane  o f t a r g e t c e l l s o f t h e immune system ( L u t z e t a l . , 1987).  (Scharmann, 1976).  t o be  ( S u t t o r p e t a l . , 1985), E h r l i c h  ( L u t z e t a l . , 1987) and human leukemic  I n t h e case o f g r a n u l o c y t e s , treatment  cells  (Sasak  with the  an i n h i b i t i o n o f t h e a b i l i t y o f t h e g r a n u l o c y t e s t o k i l l  P. a e r u g i n o s a c e l l s  ( B a l t c h e t a l . , 1985).  c h a p t e r were d e s i g n e d  t o determine  The experiments  of t h i s  the b a c t e r i a l c e l l u l a r l o c a l i z a t i o n o f  c y t o t o x i n and t o examine i t s e f f e c t on macrophages.  Towards t h i s end,  33  v a r i o u s b a c t e r i a l c e l l compartments were t e s t e d f o r the presence o f c y t o t o x i n , and osmotic shock f l u i d preparation  o f c y t o t o x i n were observed f o r t h e i r i n t e r a c t i o n w i t h mouse  macrophage c e l l  1.  ( p e r i p l a s m i c c o n t e n t s ) and a p u r i f i e d  line  P388 . D 1  Cellular l o c a l i z a t i o n of cytotoxin C y t o t o x i n was p r e v i o u s l y i s o l a t e d from c e l l u l a r a u t o l y s a t e s  (e.g. F i g .  4, l a n e 1 ) , which were the supernatant f r a c t i o n s o f s t a t i o n a r y phase P. a e r u g i n o s a c e l l s , incubated  resuspended i n p h o s p h a t e - b u f f e r e d s a l i n e , and  f o r 56 h a t 37°C ( L u t z , 1979; Scharmann, 1976).  A variety of  experiments were performed t o o b t a i n r e l e a s e o f c y t o t o x i n but these methods d i d not d e f i n i t i v e l y e s t a b l i s h the c e l l u l a r compartment w i t h which c y t o t o x i n was a s s o c i a t e d ,  a l t h o u g h they d i d e s t a b l i s h t h a t c y t o t o x i n was  c e l l - a s s o c i a t e d and c o u l d be r e l e a s e d by v a r i o u s (Scharmann, 1976).  lysis  techniques  To determine t h e c e l l u l a r l o c a l i z a t i o n o f c y t o t o x i n i n  P. a e r u g i n o s a , s e v e r a l c e l l compartments were t e s t e d f o r i t s presence u s i n g SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and Western b l o t t i n g t e c h n i q u e s ( F i g u r e s 4,5). While r a b b i t a n t i - c y t o t o x i n serum r e a c t e d i n Western immunoblots w i t h h e a v i l y o v e r l o a d e d i n n e r and o u t e r protein preparations, weight o f c y t o t o x i n concentrated  membrane  no d i s c e r n a b l e bands were seen a t the m o l e c u l a r  ( F i g . 5, lanes 4 and 5 ) . S i m i l a r l y , i n 1 5 0 - f o l d  growth s u p e r n a t a n t s , t h e o n l y  immunolabelled band i n  a n t i - c y t o t o x i n b l o t s was a t a p p r o x i m a t e l y 56 kD ( F i g . 5, l a n e 3 ) . membrane and growth supernatant p r o t e i n s anti-exoenzyme S serum ( F i g u r e 6, lanes  cross-reacted 3, 4, 5 ) .  These  strongly with  34  S  F i g u r e 4.  1  Coomassie-stained  2  3  4  5  SDS p o l y a c r y l a m i d e g e l o f c y t o t o x i n and  P. a e r u g i n o s a s t r a i n H103 s u b c e l l u l a r f r a c t i o n s .  Lane 1, p u r i f i e d  2+ c y t o t o x i n ; l a n e 2, Mg  /freeze-thaw  osmotic shockate; l a n e 3, growth  supernatant; l a n e 4, i n n e r membrane; lane 5, o u t e r membrane. p o s i t i o n s o f m o l e c u l a r weight the g e l . the r i g h t .  standards  Running  ( l a n e S) a r e marked t o t h e l e f t o f  The r u n n i n g p o s i t i o n o f the 28 kD c y t o t o x i n band i s i n d i c a t e d on The amounts loaded p e r l a n e were 2.8 ug o f c y t o t o x i n and 40  Ug o f each o f the o t h e r  samples.  F i g u r e 5.  Western immunoblot o f c y t o t o x i n and P. a e r u g i n o s a s t r a i n H103  s u b c e l l u l a r f r a c t i o n s probed w i t h r a b b i t a n t i - c y t o t o x i n serum.  Lane 1,  2+ purified  c y t o t o x i n ; l a n e 2, Mg  /freeze-thaw osmotic shockate; l a n e 3,  growth s u p e r n a t a n t ; l a n e 4, i n n e r membrane; l a n e 5, o u t e r membrane.  The  amounts l o a d e d p e r l a n e were 2.8 ug o f c y t o t o x i n and 40 ug o f each o f the o t h e r samples.  Compared t o t h e osmotic shockate, 12 times as many  c e l l s were r e q u i r e d t o produce 40 ug o f growth s u p e r n a t a n t and 20 times as many c e l l s were r e q u i r e d t o produce 40 ug o f i n n e r and o u t e r membrane.  36  In c o n t r a s t osmotic shockates, prepared by method of Hoshino and  /freeze-thaw  Kageyama (1980) [which i s the p r e f e r r e d method f o r  i s o l a t i o n o f the p e r i p l a s m i c contents 1984) ], c o n t a i n e d  the Mg  a polypeptide  o f P.  aeruginosa  (Poole and  Hancock,  band o f a p p r o x i m a t e l y 28 kD which r e a c t e d  r e l a t i v e l y s t r o n g l y w i t h a n t i - c y t o t o x i n s e r a on Western immunoblots ( F i g . 5, l a n e 2; a minor s p e c i e s o f 27 kD was and may  with 8 separate  preparations  was  o f osmotic shockate.  c r o s s - r e a t i v e bands were seen on the anti-exoenzyme S b l o t ( F i g u r e  lane  6,  2).  Cytotoxin  i n h i b i t o n of  In p r e v i o u s and  The  c r o s s r e a c t i v e band i n the p e r i p l a s m i c f r a c t i o n  reproducibly obtained  2.  on some b l o t s  have been a p r o t e o l y t i c breakdown product o f c y t o t o x i n ) .  presence o f a 28 kD  No  also evident  phagocytosis  i n v e s t i g a t i o n s , c y t o t o x i n had been shown t o cause s w e l l i n g  l y s i s o f polymorphonuclear l e u k o c y t e s  1976), pulmonary a r t e r y e n d o t h e l i a l c e l l s E h r i i c h a s c i t e s tumor c e l l s  ( B a l t c h et a l . , 1985; ( S u t t o r p et a l . , 1985)  (Lutz et a l . , 1987).  polymorphonuclear l e u k o c y t e s ,  c y t o t o x i n was  To c o n f i r m  and  In the case o f  demonstrated t o i n h i b i t  b a c t e r i c i d a l a c t i v i t y o f these c e l l s a g a i n s t P. a e r u g i n o s a 1985) .  Scharmann,  t h a t osmotic shockates c o n t a i n e d  determined the e f f e c t s o f s u b - l e t h a l c o n c e n t r a t i o n s  (Baltch et a l . ,  a cytotoxin, I of c y t o t o x i n  and  osmotic shockates on the a b i l i t y o f the macrophage c e l l l i n e P 3 8 8 phagocytose P.  aeruginosa  P u r i f i e d c y t o t o x i n was concentration  d i d not  the  D 1  to  cells. used i n the assay at 13 yg/ml.  This  appear t o s i g n i f i c a n t l y a l t e r the v i a b i l i t y o f  the  37  1  F i g u r e 6.  2  3  4  5  Western immunoblot o f c y t o t o x i n and P. a e r u g i n o s a  s t r a i n H103  s u b c e l l u l a r f r a c t i o n s probed w i t h r a b b i t anti-exoenzyme S serum.  Lane 1,  2+ purified  c y t o t o x i n ; l a n e 2, Mg  growth s u p e r n a t a n t ;  /freeze-thaw  osmotic  shockate;  l a n e 3,  l a n e 4, i n n e r membrane; l a n e 5, o u t e r membrane.  The  amounts l o a d e d p e r l a n e were 2.8 ug o f c y t o t o x i n and 40 ug o f each o f the o t h e r  samples.  38  macrophages d u r i n g the assay, as a s s e s s e d by the a b i l i t y adhere t o g l a s s s u r f a c e s , a l t h o u g h minor s w e l l i n g was observed.  o f macrophages t o  occasionally  At h i g h e r c o n c e n t r a t i o n s , c y t o t o x i n r e s u l t e d i n i n c r e a s i n g  damage and l y s i s .  Lower c o n c e n t r a t i o n s caused  reduced  a l t h o u g h maximal e f f e c t s were seen a t 13 ug/ml. macrophage r a t i o o f 20 t o 1, 8.0  c y t o t o x i n r e s u l t e d i n a 95% shockate was  phagocytosis,  At a b a c t e r i a t o  o p s o n i z e d P. a e r u g i n o s a c e l l s  a s s o c i a t e d per u n t r e a t e d macrophage ( T a b l e I I ) . decrease  a n t i s e r a was  i n opsonized phagocytosis.  a l s o i n h i b i t o r y , d e c r e a s i n g p h a g o c y t o s i s by  90 per cent i n each o f t h e s e cases i n c a p a b l e o f promoting  c o n t r a s t , anti-exoenzyme S was  became  A d d i t i o n of p u r i f i e d Osmotic  71%.  A d d i t i o n of r a b b i t a n t i - c y t o t o x i n r e s t o r e d phagocytosis approximately  cell  to  (Table I I ) .  This  P. a e r u g i n o s a uptake on i t s own.  In  unable t o i n f l u e n c e the i n h i b i t i o n o f 2+  p h a g o c y t o s i s by c y t o t o x i n o r Mg  /freeze-thaw  P. a e r u g i n o s a growth supernatant  displayed significant  inhibition  shockate.  Although phagocytosis  ( T a b l e I I ) , the e f f e c t c o u l d be c o m p l e t e l y negated  a n t i - c y t o t o x i n o r anti-exoenzyme S.  using either  T h i s confirmed t h a t the  a n t i - c y t o t o x i n s e r a c o n t a i n e d a n t i b o d i e s t h a t c r o s s - r e a c t e d w i t h exoenzyme S (see F i g . 5, l a n e 3 and d i s c u s s i o n ) .  Inner and o u t e r membrane  p r e p a r a t i o n s i n c r e a s e d uptake l e v e l s over and above those o b t a i n e d i n the antibody c o n t r o l . Escherichia c o l i  As a n e g a t i v e c o n t r o l , osmotic s t r a i n C600 was  i n h i b i t i o n o f p h a g o c y t o s i s was did  not r e s t o r e p h a g o c y t o s i s  shock f l u i d  a l s o examined i n t h i s assay.  observed,  i n t h i s case  from While  97%  a d d i t i o n o f a n t i - c y t o t o x i n serum (Table I I ) .  39  Table I I .  E f f e c t o f a n t i - c y t o t o x i n and anti-exoenzyme S s e r a on i n h i b i t i o n o f o p s o n i z e d p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n M2  B a c t e r i a a s s o c i a t e d p e r macrophage (%)  Inhibitor  No a n t i - c y t o t o x i n  Anti-cytotoxin treated  None  8.0  Cytotoxin  0.4 ( 5 )  P. a e r u g i n o s a shockate  osmotic  P. a e r u g i n o s a growth supernatant  (100) d  b  Anti-exoenzyme S treated  ND 7.0 (88)  0.4 ( 5 )  d  d  2.3 ( 2 9 )  d  7.3 (91)  0.3 ( 4 )  2.3 ( 2 9 )  d  7.0 (88)  7.4 (93)  P. a e r u g i n o s a i n n e r membrane  16.4 (205)  ND  ND  P. a e r u g i n o s a o u t e r membrane  16.0 (200)  ND  ND  E. c o l i osmotic shockate  0.2 ( 3 )  d  1.3 ( I 6 )  d  ND  ^ h e i n p u t r a t i o o f b a c t e r i a : macrophage c e l l s i n t h e assay was 20:1. S i x t y macrophages p e r assay were a s s e s s e d f o r numbers o f a s s o c i a t e d bacteria. Data r e p r e s e n t s the mean o f 4 t o 9 independent a s s a y s . ND - not determined. ^Number i n b r a c k e t s i s t h e p e r c e n t b a c t e r i a l a s s o c i a t i o n r e l a t i v e t o t h a t i n t h e absence o f i n h i b i t o r . C  A s i n g l e experiment w i t h monoclonal a n t i b o d y MA5-8 as o p s o n i n and t h r e e experiments without o p s o n i n demonstrated t h a t the a n t i - c y t o t o x i n s e r a was unable t o i n c r e a s e p h a g o c y t o s i s by n o n - c y t o t o x i n - t r e a t e d macrophages.  d  p<0.01 (Student's t t e s t ) t h a t uptake was s i g n i f i c a n t l y lower than t h a t o b t a i n e d i n t h e absence o f i n h i b i t o r f o r a l l assays  performed.  40  3.  Mechanism o f macrophage i n a c t i v a t i o n Previous  s t u d i e s have suggested t h a t the mode o f a c t i o n o f c y t o t o x i n  was t o produce d i s c r e t e membrane l e s i o n s (Lutz e t a l . , 1987) which a l l o w e d passage o f s m a l l molecules and ions i n t o and out o f t h e t a r g e t (Scharmann, 1976). permeability  It i s likely  cell  t h a t such a l t e r a t i o n s i n membrane  caused d i s s i p a t i o n ( i . e . d e p o l a r i z a t i o n ) o f i o n g r a d i e n t s  r e q u i r e d t o s i g n a l p h a g o c y t o s i s [e.g. a C a  2 +  gradient  (Young e t a l . ,  1984)]. Carbocyanine dyes [ i . e . d i S C ( 5 ) ] 3  highly fluorescent  a r e l i p o p h i l i c probes which a r e  i n aqueous environments and m i n i m a l l y  the hydrophobic environment o f t h e membrane. generated across  gradient)  become l e s s f l u o r e s c e n t concentration ion  gradient).  fluxes across  When i o n f l u x e s a r e  the membrane, t h e probe i s e i t h e r shunted out o f t h e  membrane t o become more f l u o r e s c e n t concentration  fluorescent i n  (on d e p o l a r i z a t i o n o r c u r r e n t w i t h t h e  o r more probe i s i n s e r t e d i n t o t h e membrane t o (on h y p e r p o l a r i z a t i o n o r c u r r e n t  against the  T h i s type o f dye was thus used t o i n v e s t i g a t e  the phagocytic  membranes o f t h e macrophage c e l l .  Using one o f these p o l a r i z a t i o n - s e n s i t i v e f l u o r e s c e n t probes [diSC^S)],  i t was determined t h a t c y t o t o x i n and osmotic shock f l u i d d i d  indeed produce s t r o n g d e p o l a r i z a t i o n o f the P 3 8 8 ( F i g u r e 7, T a b l e I I I ) .  +  concentration  gradient  d e p o l a r i z a t i o n increased  plasma membrane  T h i s d e p o l a r i z a t i o n was s i m i l a r t o t h a t caused by  the p o s i t i v e c o n t r o l ionophore, v a l i n o m y c i n , high K  D 1  i n d i s s i p a t i o n o f an imposed  ( F i g u r e 7, T a b l e I I I ) .  The r a t e o f  as a f u n c t i o n o f t h e c o n c e n t r a t i o n  or osmotic shockate added and reached a p l a t e a u  at higher  of cytotoxin concentrations  F i g u r e 7.  Changes i n diSC.j(5) f l u o r e s c e n c e a f t e r a d d i t i o n o f  phosphate-buffered  s a l i n e , c y t o t o x i n (35 yg/ml) o r v a l i n o m y c i n  ( 2 x l O M ) t o c e l l s o f the macrophage c e l l l i n e P 3 8 8 _6  e q u i l i b r a t e d i n the presence o f 2xlO~ M d i S C ^ S ) . 6  i n d i c a t e d by an i n c r e a s e i n f l u o r e s c e n c e .  D 1  t h a t had been  Depolarization i s  42  Table I I I .  D e p o l a r i z a t i o n o f the P 3 8 8 v a l i n o m y c i n and  osmotic  f l u o r e s c e n t probe  Net Addition  D 1  plasma membrane by c y t o t o x i n ,  shockate  the  diSC.(5)  fluorescence increase >b (arbitrary units)  1  assessed using  Initial  rate of fluorescence  increase  (units/min)  None  0  0  Valinomycin  7.4  6.1  Cytotoxin  7.2  5.2  6.6  7.2  0  0  0  0  P. a e r u g i n o s a shockate  osmotic  P. a e r u g i n o s a growth supernatant P.  aeruginosa  E. c o l i  osmotic  LPS shockate  P r e p a r a t i o n s were added, a f t e r diSC3(5) had e q u i l i b r a t e d a c r o s s the P388j)i plasma membrane, to the f o l l o w i n g f i n a l c o n c e n t r a t i o n s : v a l i n o m y c i n , 2xlO~^M; c y t o t o x i n , 35 ug/ml; osmotic shockates, 500 ug/ml; growth supernatant, 400 ug/ml; LPS, 385 ug/ml. These c o n c e n t r a t i o n s were chosen to demonstrate the maximal p o s s i b l e e f f e c t s ; lower c o n c e n t r a t i o n s caused sub-maximal e f f e c t s as seen i n F i g . 8. *>Data r e p r e s e n t the means o f t h r e e independent experiments performed s e p a r a t e days.  on  43  (Figure 8).  As c o n t r o l s , the osmotic shockate from E. c o l i  (Table I I I )  and the c o n c e n t r a t e d growth supernatant from P. a e r u g i n o s a were t e s t e d  and  f a i l e d t o produce any d e p o l a r i z a t i o n o f the macrophage plasma membrae. LPS u t i l i z e d a t a c o n c e n t r a t i o n f i v e times t h a t found i n M g  2 +  freeze/thaw osmotic shockate (385 ug LPS per assay) caused no  measurable  depolarization.  4.  Summary P. a e r u g i n o s a c y t o t o x i n and p e r i p l a s m i c c o n t e n t s caused a  significant  i n h i b i t i o n o f o p s o n i c p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n M2 by mouse macrophage c e l l  line P388 . D 1  P h a g o c y t o s i s was  r e s t o r e d i n each case  upon p r e - i n c u b a t i o n o f e i t h e r p r e p a r a t i o n w i t h a n t i - c y t o t o x i n serum. A l t h o u g h growth supernatant a l s o caused a s i g n i f i c a n t  reduction i n opsonic  p h a g o c y t o s i s , i t s e f f e c t s were negated by p r e - i n c u b a t i o n w i t h an anti-exoenzyme  S preparation.  Other c e l l u l a r f r a c t i o n s t e s t e d were not  i n h i b i t o r y f o r macrophage p h a g o c y t o s i s .  Both c y t o t o x i n and  c o n t e n t s caused d e p o l a r i z a t i o n o f the P 3 8 8 demonstrated  D 1  periplasmic  plasma membrane, as  u s i n g a p o l a r i z a t i o n - s e n s i t i v e probe.  were not observed f o r o t h e r P. a e r u g i n o s a f r a c t i o n s .  Similar  correlations  The r a t e o f  d e p o l a r i z a t i o n i n c r e a s e d as a f u n c t i o n o f the c o n c e n t r a t i o n o f c y t o t o x i n or  osmotic shockate added and reached a p l a t e a u l e v e l a t h i g h e r  concentrations.  U s i n g Western immunoblotting  t e c h n i q u e s i t was  t h a t the o n l y c e l l u l a r f r a c t i o n c o n t a i n i n g an i d e n t i f i a b l e p r o t e i n was  osmotic shockate.  determined  cytotoxin  These d a t a i n d i c a t e d t h a t P. a e r u g i n o s a  Depolarization Rate (units/min)  8 -i  —  Osmotic  0  20  40  60  80  100  Shockate  120  140  Protein Concentration (ug/ml)  Figure 8.  Effect of Increasing concentrations  of cytotoxin or  2+ Mg  freeze-thaw osmotic shockate on the rate of increase (I.e.  depolarization) of diSC^CS) fluorescence.  The rates were calculated  from traces l i k e those shown i n F i g . 7 and represent the means of 3 d i f f e r e n t experiments.  45  c y t o t o x i n i s l o c a l i z e d i n t h e p e r i p l a s m and has the p o t e n t i a l t o i n h i b i t macrophage-mediated p h a g o c y t o s i s , across  p o s s i b l y by p e r t u r b i n g i o n g r a d i e n t s  the macrophage plasma membrane.  46  CHAPTER  HI  F i b r o n e c t i n - M e d i a t e d A c t i v a t i o n o f Non-Opsonic P h a g o c y t o s i s o f P.  aeruginosa To extend the s t u d i e s o f Chapter One,  monoclonal  the a b i l i t y o f a n t i - p r o t e i n F  a n t i b o d i e s t o mediate p h a g o c y t o s i s o f i n  P. a e r u g i n o s a was  determined.  vivo-grown  U s i n g the method o f Day  e t a l . (1980),  ( F i g u r e 9 ) , P. a e r u g i n o s a c e l l s were grown f o r 3 days i n the p e r i t o n e a l c a v i t y o f l a b o r a t o r y mice o r r a t s .  B a c t e r i a were c o n t a i n e d i n 1-cm  p l a s t i c chambers s e a l e d a t both ends w i t h 0.22  long  ym membrane f i l t e r s  a l l o w i n g f r e e exchange o f p e r i t o n e a l f l u i d s and b a c t e r i a l p r o d u c t s w h i l e p r o h i b i t i n g immune c e l l a c c e s s o r b a c t e r i a l escape.  Upon removal  chambers, b a c t e r i a c o u l d be s e p a r a t e d from the f l u i d  i n the chamber by  centrifugation.  1.  T h i s decanted  E f f e c t o f i n v i v o growth on  f l u i d was  c a l l e d the i n v i v o s u p e r n a t a n t .  phagocytosis  To study the e f f e c t o f i n v i v o growth on p h a g o c y t o s i s , P. s t r a i n M2  was  aeruginosa  grown i n v i t r o i n r a p i d l y a g i t a t e d b r o t h o r i n v i v o u s i n g ,  the chamber implant model ( F i g . 9 ) . and o p s o n i c p h a g o c y t o s i s was vivo-grown  of  S u s c e p t i b i l i t y to both  determined.  I t was  non-opsonic  found t h a t unwashed i n  b a c t e r i a showed s i g n i f i c a n t l y h i g h e r p h a g o c y t o s i s than unwashed  i n v l t r o - g r o w n organisms  ( F i g . 10).  P. a e r u g i n o s a c e l l s taken  directly  from the i n v i v o growth system were s i g n i f i c a n t l y more s u s c e p t i b l e t o non-opsonic  p h a g o c y t o s i s than were the same P. a e r u g i n o s a c e l l s  b e i n g washed t w i c e i n b u f f e r ( F i g . 10).  after  Washing the i n v i t r o - g r o w n  47  10  bacteria i n saline  I Chamber sealed on both ends with 0.22 um filters  Insertion into mouse or rat peritoneal cavity 1  72 hours  D Contents |  Bacteria  Figure 9.  i n vivo  supernatant  D e s c r i p t i o n o f the i n v i v o growth model.  i n p h y s i o l o g i c a l s a l i n e were s e a l e d 0.22  Centrifugation  um f i l t e r s .  10  bacterial  cells  i n t o p o l y p r o p y l e n e chambers u s i n g  Chambers were i n s e r t e d i n t o the p e r i t o n e a l c a v i t y  l a b o r a t o r y mice o r r a t s and i n c u b a t e d i n v i v o f o r 72 h o u r s .  Bacterial  c u l t u r e s were h a r v e s t e d and c o u l d be s e p a r a t e d from t h e i n v i v o s u p e r n a t a n t by c e n t r i f u g a t i o n a t 12,000 x g f o r 10 m i n .  of  48  F i g u r e 10.  Opsonized  p h a g o c y t o s i s o f i n v i v o - and i n v i t r o - g r o w n  P. a e r u g i n o s a 3 t r a i n M2 by mouse macrophage c e l l  line P388  D 1 >  M2  cells  were washed (w) o r unwashed (uw), w i t h (+Ab) and without the a d d i t i o n o f anti-protein  F monoclonal  average uptake observed  a n t i b o d y MA5-8.  i n 3 independent  Each column r e p r e s e n t s t h e experiments.  49  b a c t e r i a resulted i n a small, i n s i g n i f i c a n t a l t e r a t i o n  (p>0.5 by Student's  t test) i n phagocytosis. Experiments u t i l i z i n g the p r o t e i n P - s p e c i f i c monoclonal resulted i n a significant  a n t i b o d y MA5-8  i n c r e a s e (p<0.01) i n p h a g o c y t o s i s o f both i n  v i t r o and i n vivo-grown b a c t e r i a compared w i t h unopsonized ( F i g . 10).  organisms  T h i s f u r t h e r suggested t h a t a n t i - F monoclonal a n t i b o d i e s have  p o t e n t i a l as p a s s i v e immunotherapeutic t h e s e s t u d i e s was r e s u l t i n g from  agents.  An i n t e r e s t i n g f i n d i n g o f  t h a t the average i n c r e a s e i n b a c t e r i a per macrophage  o p s o n i z a t i o n appeared  t o be lower f o r i n vivo-grown  than  f o r i n v i t r o - g r o w n b a c t e r i a r e g a r d l e s s o f whether they were unwashed o r washed.  T h i s suggested t h a t the i n vivo-grown  organisms may  have had  a  reduced s u r f a c e exposure o f p r o t e i n F r e l a t i v e t o i n v i t r o - g r o w n c e l l s .  2.  Enhancement o f p h a g o c y t o s i s by i n v i v o supernatant Initially,  I wished t o determine i f the d e c r e a s e d p h a g o c y t o s i s o f  washed i n vivo-grown b a c t e r i a was  r e l a t e d t o e f f e c t s o f the  washing  procedure on the organisms, o r due t o removal o f some p h a g o c y t o s i s promoting f a c t o r i n the i n v i v o s u p e r n a t a n t .  To determine i f i n v i v o  supernatant c o u l d be added back t o the washed b a c t e r i a and  still  f a c i l i t a t e uptake o f b a c t e r i a by macrophages, the v i s u a l assay o f p h a g o c y t o s i s was  performed.  Mouse i n vivo-grown M2  c e l l s and mouse  macrophage c e l l l i n e P388p^ were used i n these i n i t i a l p r o v i d e c o n t i n u i t y w i t h the above study.  studies to  50  I t was  observed t h a t t h e r e was  indeed a p h a g o c y t o s i s - p r o m o t i n g  factor  o b t a i n e d from i n v i v o chambers which c o u l d be s e p a r a t e d from the b a c t e r i a e a s i l y by c e n t r i f u g a t i o n a t 12,000 x g f o r 10 min and added back t o a g a i n f a c i l i t a t e b a c t e r i a l a s s o c i a t i o n w i t h the macrophage ( T a b l e I V ) . b a c t e r i a t o macrophage r a t i o o f 20 t o 1, an average o f 2.9 became a s s o c i a t e d per macrophage as compared t o 7.7 the presence o f i n v i v o s u p e r n a t a n t . statistically  significant  i n 7 out o f 8 assays performed  T h i s p h a g o c y t o s i s enhancing f a c t o r was  washed b a c t e r i a  b a c t e r i a taken up i n  T h i s d i f f e r e n c e was  Student's t t e s t ) and m a r g i n a l l y s i g n i f i c a n t  At a  found t o be (p<0.005,  (p<0.1) i n the o t h e r a s s a y .  t i t e r a b l e i n t h i s system  as  d e c r e a s i n g amounts o f added supernatant r e s u l t e d i n p r o g r e s s i v e l y d e c r e a s i n g l e v e l s o f b a c t e r i a l uptake In  a s e r i e s o f experiments  (Table IV).  d e s i g n e d t o i n v e s t i g a t e some o f the  p h y s i c a l p r o p e r t i e s o f the p h a g o c y t o s i s - p r o m o t i n g f a c t o r , determined at  i t was  t h a t i n v i v o supernatant r e t a i n e d i t s a c t i v i t y b e s t when s t o r e d  -70°C ( T a b l e V ) .  supernatant was  In a d d i t i o n , the a c t i v e component o f i n v i v o  extremely heat s t a b l e and c o u l d w i t h s t a n d a temperature  60°C f o r 30 min o r even 100°C f o r 10 min To determine  of  (Table V).  i f the observed phenomena were r e s t r i c t e d t o the mouse i n  v i v o system o r t h i s p a r t i c u l a r s t r a i n o f P. a e r u g i n o s a , the same s t u d i e s were c a r r i e d out u s i n g rat-grown b a c t e r i a .  Almost  o b t a i n e d i n the r a t model u t i l i z i n g both s t r a i n M2 w i l d - t y p e s t r a i n H103  (Table V I ) .  i d e n t i c a l r e s u l t s were and  laboratory  A d d i t i o n a l l y , r a t supernatant from i n  v i v o chambers c o u l d e f f e c t i v e l y promote uptake o f s t r a i n H103 v i t r o on T r y p t i c a s e Soy agar ( T a b l e V I ) .  grown i n  S i m i l a r r e s u l t s were o b t a i n e d  51  T a b l e IV.  Enhancement o f t h e a s s o c i a t i o n o f i n vivo-grown P. a e r u g i n o s a M2 w i t h P 3 8 8  Treatment o f i n v i v o  c e l l s u s i n g supernatant from mouse chambers  ni  Addition to bacteria  grown M2  a  Bacteria  associated  p e r macrophage  unwashed  PBS  9.4 ± 3 . 8  washed  PBS  2.9 ± 1.4  washed  100%  i n v i v o supernatant  7.7 ± 3.4  washed  75%  i s v i v o supernatant  6.1  washed  50% i n v i v o supernatant  4.3  d  washed  25%  i n v i v o supernatant  3.8  d  a  b  c  p<0.005 (by Student's t t e s t ) i n 8/8 assays when compared t o t h e "washed + PBS" c o n t r o l .  D  p<0.005 i n 7/8 assay, p<0.1 i n 1/8 assays when compared t o the "washed + PBS" c o n t r o l .  c  p<0.005 i n t h e one assay performed control.  d  not  significantly  when compared t o t h e "washed + PBS"  g r e a t e r than t h e "washed + PBS" c o n t r o l .  52  T a b l e V.  S t a b i l i t y o f the phagocytosis-enhancing f a c t o r o f i n v i v o chamber  Test  supernatant.  condition  Average number o f b a c t e r i a a s s o c i a t e d p e r macrophage  +M2 + i n v i v o UW  7.6  +M2 + i n v i v o W + PBS  2.5  +M2 + i n v i v o W + i n v i v o supernatant  6.4  +M2 + i n v i v o W + s t o r e d supenatant  7.1  +M2 + i n v i v o W + s t o r e d supernatant  (-70°C) (-20°C)  4.5  +M2 + i n v i v o W + supernatant  t r e a t e d @ 60°C, 30 min  5.9  +M2 + i n v i v o W + supernatant  t r e a t e d @ 100°C, 10 min  9.1  53  Table VI.  Enhancement o f the a s s o c i a t i o n o f P. a e r u g i n o s a s t r a i n s M2 and H103 w i t h u n e l i c i t e d mouse p e r i t o n e a l macrophages and the P388p^ macrophage c e l l rat  l i n e u s i n g i n v i v o supernatant  chambers  Bacteria associated Macrophage  Bacterial  cell  cell  type  from  type a  Unwashed bacteria  per phagocyte  Washed b a c t e r i a + PBS  Washed  bacteria  + r a t i n vivo supernatant  P388  D 1  i n v i v o grown M2  7.3±3.3  P388  D 1  i n v i v o grown H103  9.0 ± 3 . 5  b  b  3.012.3  10.6±3.7  2.2 ± 1.8  6.6 ± 1.8°  P388 i  i n v i t r o grown H103  -  5.8 ± 2.3  13.9 ± 5 . l  Mouse peritoneal macrophages  i n v i t r o grown H103  -  2.1 ± 1.0  7.0 ± 3 . 1  D  a  b  i n v i t r o b a c t e r i a were grown on T r y p t i c a s e Soy agar p l a t e s , i n v i v o b a c t e r i a were grown i n p e r i t o n e a l chambers i n r a t s . p<0.005 (by Student's t t e s t ) i n 3/3 assays performed the "washed + PBS" c o n t r o l .  when compared t o  b  b  b  54  u s i n g i n yjLtro-grown s t r a i n H103 and u n e l i c i t e d mouse p e r i t o n e a l macrophages.  I n t h i s case, a d d i t i o n o f r a t i n v i v o s u p e r n a t a n t i n c r e a s e d  b a c t e r i a l a s s o c i a t i o n from 2.1 t o 7.0 b a c t e r i a p e r phagocyte. d i f f e r e n c e s were found t o be s t a t i s t i c a l l y performed (p<0.005, Student's t t e s t ) . mouse macrophage c e l l  line P388  D 1  significant  These  i n a l l assays  I t was thus e v i d e n t t h a t t h e  c o u l d be used as a model f o r  u n e l i c i t e d mouse p e r i t o n e a l macrophages i n t h i s  system.  Thus, t h e remainder o f t h e s e s t u d i e s c e n t e r e d on measuring promotion o f a s s o c i a t i o n o f i n v i t r o - g r o w n s t r a i n H103 w i t h P 3 8 8  D 1  cells.  This  p a r t i c u l a r system was chosen due t o t h e r e l a t i v e ease o f working w i t h i n v i t r o - g r o w n organisms.  However, due t o the d i f f e r e n t responses o f i n  vivo-grown organisms observed i n F i g . 10 and T a b l e V I , i t was n e c e s s a r y to determine how growth c o n d i t i o n s a f f e c t e d i n v i v o supernatant-promoted phagocytosis.  S t r a i n H103 was grown e i t h e r i n r a p i d l y a g i t a t e d  T r y p t i c a s e Soy b r o t h o r on a p l a t e o f T r y p t i c a s e Soy agar.  (200 rpm)  While uptake  o f a g i t a t e d b a c t e r i a c o u l d not be promoted u s i n g i n v i v o supernatant from r a t p e r i t o n e a l chambers, p h a g o c y t o s i s o f plate-grown organisms was s i g n i f i c a n t l y enhanced ( T a b l e V I I ) .  Thus, s t r a i n H103 c e l l s grown on  T r y p t i c a s e Soy agar p l a t e s were used f o r most o f the subsequent  studies.  55  Table VII.  I n v i t r o growth c o n d i t i o n s  affect  the a b i l i t y o f i n vivo  supernatant t o enhance a s s o c i a t i o n o f P. a e r u g i n o s a H103 w i t h P388„,  cells  H103 growth c o n d i t i o n  Addition to bacteria  Bacteria per  associated  macrophage  Agitated  b r o t h (TSB)  PBS  8.2 + 0.3  Agitated  b r o t h (TSB)  Rat chamber supernatant  7.2 + 0 . 4  P l a t e (TSA)  PBS  5.9 + 2.2  P l a t e (TSA)  Rat chamber supernatant  13.9 + 4.3  a  N o t s i g n i f i c a n t l y d i f f e r e n t from t h e PBS c o n t r o l i n 3/3 assays performed (Student's t t e s t ) .  D  p<0.005 i n 3/3 assays when compared t o the PBS c o n t r o l .  b  a  56  3.  C h a r a c t e r i z a t i o n o f the p h a g o c y t o s i s - p r o m o t i n g  factor  To f u r t h e r c h a r a c t e r i z e the p h a g o c y t o s i s - p r o m o t i n g supernatant was  f r a c t i o n a t e d on a F a s t P r e s s u r e L i q u i d  factor, rat i n vivo Chromatography  (FPLC) Superose 12 g e l s i e v i n g column and peaks were c o l l e c t e d i n f o u r p o o l s ( F i g . 11).  A f t e r e x t e n s i v e d i a l y s i s and c o n c e n t r a t i o n , the  phagocytosis-promoting observed  a c t i v i t y o f these p o o l s was  assessed.  It  t h a t P o o l A s t r o n g l y enhanced p h a g o c y t o s i s , P o o l B was  e f f e c t i v e , w h i l e P o o l s C and D were i n e f f e c t i v e These d a t a e s t a b l i s h e d t h a t the f a c t o r was  was less  (Table V I I I ) . of high molecular  weight.  Thus, t h r e e p o s s i b l e c a n d i d a t e s were c o n s i d e r e d f o r the p h a g o c y t o s i s promoting  f a c t o r , a n t i b o d y , complement and f i b r o n e c t i n , each o f which had  been p r e v i o u s l y shown t o promote p h a g o c y t o s i s ( P r o c t o r , 1987). o f d i s s o c i a t i o n o f the f a c t o r from c e l l s  ease  (by c e n t r i f u g a t i o n and washing)  and the use o f n a i v e mice i n the experiments In a d d i t i o n , the a c t i v i t y was  The  seemed t o r u l e out  s t a b l e t o h e a t i n g t o 100°C f o r 10  antibody. min  ( T a b l e V ) , thus r u l i n g out complement which i s i n a c t i v a t e d at t h i s temperature.  T h e r e f o r e , we  t e s t e d f o r the presence o f f i b r o n e c t i n u s i n g a  c o m m e r c i a l l y - a v a i l a b l e anti-human f i b r o n e c t i n a n t i b o d y p r e p a r a t i o n . o f t h i s a n t i s e r a was  p o s s i b l e due  conservation of f i b r o n e c t i n Western immunoblotting  t o the s t r o n g e v o l u t i o n a r y and  ( P e t e r s o n and Skorstengaard,  1985).  Use  antigenic By  w i t h goat anti-human f i b r o n e c t i n a n t i s e r a , i n v i v o  supernatant and P o o l A r e a c t e d s t r o n g l y , P o o l B, r e a c t e d l e s s s t r o n g l y P o o l s C and D f a i l e d ability  to react.  Thus, the o r d e r o f r e a c t i v i t y matched the  t o enhance p h a g o c y t o s i s ( T a b l e V I I I ) .  r e s o l v e d by FPLC i n t o two  peaks,  and  P o o l A was  1 and 2 ( F i g . 11).  partially  Fibronectin  was  57  F i g u r e 11. chambers.  FPLC g e l s i e v i n g  frationation  o f i n v i v o supernatant from r a t  The e l u t i o n p r o f i l e a t an absorbance o f 230 nm i s shown.  were c o l l e c t e d  i n 4 pools  (A-D).  Peaks  Peaks 1 and 2 o f p o o l A a r e l a b e l l e d .  58  Table VIII.  Enhancement o f t h e a s s o c i a t i o n of P. a e r u g i n o s a s t r a i n H103 with P 3 8 8  D 1  c e l l s u s i n g p o o l e d f r a c t i o n s c o l l e c t e d from an  FPLC g e l s i e v i n g rat  f r a c t i o n a t i o n o f i n v i v o supernatant from  chambers  Addition  B a c t e r i a associated per macrophage  PBS  a  b  5.0 ± 0.4  i n v i v o supernatant  13.5 ± 1.4  a  P o o l A supernatant  10.5 ± 1.9  b  P o o l B supernatant  8.5 ± 5.7  C  P o o l C supernatant  4.2 ± 0.9  d  P o o l D supernatant  5.6 ± 0 . 1  d  p<0.005 (by Student's t t e s t ) i n 11/11 assays performed when compared t o t h e PBS c o n t r o l .  p<0.005 i n 2/2 assays when compared t o t h e PBS c o n t r o l .  c  p<0.005 i n 1/2 assays when compared t o the PBS c o n t r o l , p>0.5 i n t h e o t h e r a s s a y .  d  not  significantly different  than t h e PBS c o n t r o l i n 2/2 a s s a y s .  59  p r e s e n t i n peak 2 ( F i g . 12, l a n e 2) but not peak 1 ( F i g . 12, l a n e 1 ) . S m a l l amounts o f f i b r o n e c t i n were d e t e c t a b l e i n P o o l B ( F i g . 12, l a n e 3) but none i n p o o l s C o r D.  T h i s suggested  thus the p h a g o c y t o s i s - p r o m o t i n g  t h a t the f i b r o n e c t i n content and  a c t i v i t y o f p o o l B was p r o b a b l y due t o  incomplete s e p a r a t i o n o f peak 2 and P o o l B.  The FPLC f r a c t i o n a t i o n  p r o f i l e o f mouse i n v i v o supernatant was b a s i c a l l y the r a t i n v i v o supernatant  i d e n t i c a l to that of  ( F i g . 13).  To c o n f i r m f i b r o n e c t i n as the p h a g o c y t o s i s - p r o m o t i n g  factor,  a n t i - f i b r o n e c t i n s e r a was i n c u b a t e d w i t h i n v i v o supernatant f o r 5 min p r i o r to phagocytosis assays.  The a n t i s e r a s i g n f i c a n t l y reduced,  l e v e l o f the PBS c o n t r o l , the p h a g o c y t o s i s - p r o m o t i n g v i v o supernatant  t o the  a b i l i t y o f the i n  (Table I X ) .  A commercially a v a i l a b l e p r e p a r a t i o n o f b o v i n e plasma f i b r o n e c t i n was t e s t e d f o r p h a g o c y t o s i s promoting a c t i v i t y a t 230 nM. This concentration of f i b r o n e c t i n increased b a c t e r i a l a s s o c i a t i o n with P388  D 1  c e l l s from 5.2 t o 14.1 b a c t e r i a per macrophage ( T a b l e I X ) . I f  f i b r o n e c t i n was i n c u b a t e d w i t h a n t i - f i b r o n e c t i n p r i o r t o assay, p h a g o c y t o s i s was not s i g n i f i c a n t l y enhanced ( T a b l e I X ) .  4.  Requirement f o r b a c t e r i a To determine i f b a c t e r i a must be p r e s e n t t o a l l o w p r o d u c t i o n  o r p o s s i b l y a c t i v a t i o n o f t h i s f a c t o r , s a l i n e - c o n t a i n i n g chambers were i n c u b a t e d i n v i v o and t h e i r f l u i d c o n t e n t s a s s e s s e d f o r phagocytosis-promotion.  I t was observed t h a t s a l i n e chamber  from both mice and r a t s were indeed capable o f enhancing  supernatants  phagocytosis o f  60  1 2 3  F i g u r e 12.  Western immunoblots o f f r a c t i o n s c o l l e c t e d from an FPLC g e l  s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant from r a t chambers probed w i t h goat-anti-human f i b r o n e c t i n serum. 2; lane 3, FPLC p o o l B.  Lane 1, FPLC peak 1; l a n e 2, FPLC peak  61  F i g u r e 13.  FPLC g e l s i e v i n g f r a c t i o n a t i o n o f i n v i v o supernatant  mouse chambers.  The e l u t i o n p r o f i l e a t an absorbance  from  o f 230 nm i s shown.  62  T a b l e IX.  Anti-fibronectin of  i n h i b i t i o n o f phagocytosis-promoting a c t i v i t y  i n v i v o supernatant from r a t p e r i t o n e a l  chambers and b o v i n e  fibronectin  Addition to i n vitro-  Bacteria  grown H103  associated/macrophage  No  PBS  With  anti-fibronectin  anti-fibronectin  5.2 ± 0.7  not done  i n v i v o supernatant  12.2  ± 1.5  a  6.5 ± 1.8  b  100 ug b o v i n e  14.1  ± 4.6  a  5.5 ± 2.4  b  15.3  ± 5.0  a  fibronectin  s a l i n e chamber supernatant  ^ ^ . 0 0 5 (Student's t t e s t ) to t h e PBS c o n t r o l .  10.3  ± 3.0°  i n 4/4 assays performed when compared  b  n o t s i g n i f i c a n t l y d i f f e r e n t from t h e PBS c o n t r o l i n 2 t o 4 a s s a y s ; p<0.005 when compared t o t h e assay i n the absence o f a n t i - f i b r o n e c t i n .  c  p<0.005 when compared t o t h e assay i n the absence  of anti-fibronectin.  63  i n v i t r o - g r o w n P. a e r u g i n o s a s t r a i n H103 from 4-5 t o 24.1 and 11.5 b a c t e r i a p e r macrophage, r e s p e c t i v e l y promoting  (Table X).  The p h a g o c y t o s i s  f a c t o r i n s a l i n e - c o n t a i n i n g chamber s u p e r n a t a n t s was a p p a r e n t l y  f i b r o n e c t i n s i n c e a n t i - f i b r o n e c t i n a n t i b o d i e s s i g n i f i c a n t l y reduced the a b i l i t y o f these supernatants t o promote p h a g o c y t o s i s  ( T a b l e IX; p<0.005).  To e s t a b l i s h the time a t which the p h a g o c y t o s i s - p r o m o t i n g appeared  factor  i n v i v o , b a c t e r i a - and s a l i n e - c o n t a i n i n g chambers were i n c u b a t e d  i n the peritoneum Supernatants  o f mice and r a t s f o r 4, 8, 16, 24, 44 and 68 h.  from these chambers were t e s t e d f o r  phagocytosis-enhancement.  In b o t h s a l i n e and b a c t e r i a l chambers, p h a g o c y t o s i s - p r o m o t i n g appeared  w i t h i n 4 h i n mice and w i t h i n 16 h i n r a t s  activity  (Table X).  The  degree  o f p h a g o c y t o s i s enhancement d i d not d i f f e r s i g n i f i c a n t l y between b a c t e r i a - c o n t a i n i n g and s a l i n e - c o n t a i n i n g chambers i n e i t h e r system,  animal  a l t h o u g h mouse supernatants were s u b s t a n t i a l l y more a c t i v e  than  t h e i r rat counterparts. The emergence o f f i b r o n e c t i n i n these chambers c o r r e l a t e d w e l l w i t h the a b i l i t y t o enhance b a c t e r i a l a s s o c i a t i o n w i t h P 3 8 8  D 1  cells.  F i b r o n e c t i n was not d e t e c t a b l e i n Western b l o t s o f r a t 4 h s u p e r n a t a n t s but was e a s i l y  seen a t 24 h ( F i g . 14).  In mice, however, t h i s p r o t e i n was  found a t a s u b s t a n t i a l c o n c e n t r a t i o n a t a l l time p o i n t s ( F i g . 14).  5.  A c t i v a t i o n o f macrophages by f i b r o n e c t i n To determine  the t h r e s h o l d l e v e l o f f i b r o n e c t i n r e q u i r e d f o r  a c t i v a t i o n o f macrophage-mediated non-opsonic  uptake, v a r i o u s  c o n c e n t r a t i o n s were i n c u b a t e d w i t h the P388 .  c e l l s prior to addition of  1  1  64  T a b l e X.  Time c o u r s e o f emergence o f p h a g o c y t o s i s - p r o m o t i n g a c t i v i t y i n r a t and mouse H103 and s a l i n e chambers  Time o f i m p l a n t a t i o n  B a c t e r i a a s s o c i a t e d p e r macrophage  o f chambers i n  using  supernatant o f chambers from:  animals p r i o r t o  Mice  Rats  harvesting of  Bacteria-  Saline-  Bacteria-  Saline-  supernatant  containing  containing  containing  containing  chambers  chambers  chambers  chambers  0 (PBS c o n t r o l )  4.5  4  18.4  a  20.2  8  21.6  a  24. l  16  24.7  a  26.3  a  24  22.3  a  23.4  44  25.3  a  21.8  25.5  a  68  a  '  24. l  a  a  3.3  2.9  4.6  2.6 6.8  a  a  7.5  a  12.0  a  10.8  a  13.4  a  7.4  a  a  11.8  a  a  11.!  p<0.005 (by Student's t t e s t ) compared t o t h e PBS c o n t r o l ; a l l o t h e r d a t a not s i g n i f i c a n t l y d i f f e r e n t .  65  12  Figure 14.  3 4  5  0  7 8  9 10 11 12 13  Western immune-blots after SDS-polyacrylamide gel  electrophoresis of the supernatant of bacteria- and saline-containing chambers that had been incubated in the peritoneum of mice and rats for 4, 24 and 48 hours probed with goat-anti-human fibronectin serum.  Lanes 1-3,  supernatant from bacteria-containing chambers incubated in rat peritoneal cavities for 4, 24 and 68 hours, respectively; lanes 4-6, supernatant from saline-containing chambers incubated in rat peritoneal cavities for 4, 24 and 68 hours, respectively; lanes 7-9, supernatant from bacteria-containing chambers incubated in mouse peritoneal cavities for 4, 24 and 68 hours, respectively; lanes 10-12, supernatant from saline-containing chambers incubated in mouse peritoneal cavities for 4, 24 and 68 hours, respectively; lane 13, purified bovine fibronectin.  66  Bacteria per Macrophage  RGDS  2-  0 i—i  1  i 11 inn—i  10  i 111fin—i  100  i 111mi—i i i nun—i i 1111in—i i 11mi  1000  10000  100000 1000000  Concentration Fibronectin or RGDS (nm)  F i g u r e 15.  E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s o f f i b r o n e c t i n and RGDS on  the l e v e l o f uptake o f P. a e r u g i n o s a s t r a i n H103 by macrophage c e l l P388^.  line  Each d a t a p o i n t r e p r e s e n t s t h e average uptake o b s e r v e d i n 3  independent  experiments.  67  Pseudomonas ( F i g . 15).  I t was  found t h a t w h i l e c o n c e n t r a t i o n s o f 13  and lower caused no response, a s i g n i f i c a n t enhancement was  l e v e l of phagocytosis  observed a t c o n c e n t r a t i o n a t and above 27 nM.  a c t i v a t i o n o f macrophages was  nM  Maximal  o b t a i n e d a t c o n c e n t r a t i o n s around  50  nM  fibronectin. I t had a l r e a d y been e s t a b l i s h e d t h a t f i b r o n e c t i n was  not a c t i n g as a  t y p i c a l o p s o n i n s i n c e i t c o u l d be removed from b a c t e r i a by  simple  c e n t r i f u g a t i o n and r e s u s p e n s i o n i n b u f f e r ( F i g . 10, T a b l e X I ) . experiments  i n which f i b r o n e c t i n was  However,  added t o macrophages f o r 15 min a t  37°C, and then the macrophages washed t w i c e p r i o r t o a d d i t i o n o f b a c t e r i a and assessment o f p h a g o c y t o s i s y i e l d e d a v e r y d i f f e r e n t In  t h i s case, washing f a i l e d t o prevent a c t i v a t i o n o f macrophages f o r  i n c r e a s e d uptake  6.  result.  o f P. a e r u g i n o s a ( T a b l e X I ) .  D e t e r m i n a t i o n o f the a c t i v e domain o f f i b r o n e c t i n F i b r o n e c t i n i s a l a r g e , d i m e r i c g l y c o p r o t e i n w i t h numerous  specific  b i n d i n g s i t e s f o r mammalian c e l l s and b a c t e r i a l s u r f a c e s ( P r o c t o r , 1987). One  o f these r e g i o n s o f the m o l e c u l e ,  the e u k a r y o t i c c e l l b i n d i n g domain,  has been shown t o i n t e r a c t w i t h v a r i o u s mammalian c e l l macrophages (Brown and Goodwin, 1988). arginine-glycine-aspartate-serine  types  A f o u r amino a c i d  including  sequence,  (RGDS), has been proven t o be  the  s m a l l e s t p o r t i o n o f the e u k a r y o t i c c e l l b i n d i n g domain capable o f i n t e r a c t i o n w i t h mammalian c e l l s sequence was  (Brown and Goodwin, 1988).  c o n s i d e r e d a l i k e l y c a n d i d a t e f o r the  r e g i o n o f the f i b r o n e c t i n  molecule.  As such,  this  macrophage-activating  68  T a b l e XI.  E f f e c t o f washing on f i b r o n e c t i n - m e d i a t e d enhancement o f non-opsonic  Addition to bacteria  macrophage p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n  Addition to  Treatment p r i o r  Average number o f  macrophage  to mixing of  bacteria  b a c t e r i a with  per macrophage  associated  macrophages  PBS  PBS  none  3.7 ± 0.8 10.1 ± 0 . 1  Fibronectin  a  PBS  none  Fibronectin  a  PBS  b a c t e r i a washed  4.8 +  1.8  C  none  8.9 ± 3.3  b  PBS  Fibronectin  PBS  Fibronectin 8  0  macrophage washed  10.0 +  0.4  b  b  a  S o u r c e o f f i b r o n e c t i n was i n v i v o p e r i t o n e a l chamber supernatant from r a t s i n which the o n l y p h a g o c y t o s i s - p r o m o t i n g f a c t o r was f i b r o n e c t i n .  b  p<0.005 (Student's t t e s t ) i n a l l assays performed experiments) when compared t o the PBS c o n t r o l .  c  not  H103  (2-4 i n d i v i d u a l  s i g n i f i c a n t l y d i f f e r e n t from the PBS c o n t r o l i n 2/2 a s s a y s .  69  To t e s t t h i s h y p o t h e s i s , a commercially  a v a i l a b l e p r e p a r a t i o n o f RGDS  was i n c u b a t e d w i t h t h e macrophages f o r 15 min p r i o r t o a d d i t i o n o f bacteria.  T h i s f o u r amino a c i d sequence s i g n i f i c a n t l y i n c r e a s e d b a c t e r i a l  a s s o c i a t i o n with P 3 8 8  D 1  c e l l s i n a concentration-dependent  manner  ( F i g . 15). The maximal l e v e l o f enhancement was s i m i l a r t o t h a t o b t a i n e d w i t h a p u r i f i e d bovine f i b r o n e c t i n p r e p a r a t i o n ( F i g . 15) and when used a t a c o n c e n t r a t i o n o f 100 uM was found PBS c o n t r o l i n a l l f o u r assays  t o be s t a t i s t i c a l l y g r e a t e r than t h e  performed (p<0.005 by Student's  t t e s t ) and  not s i g n i f i c a n t l y d i f f e r e n t from t h e v a l u e o b t a i n e d i n t h e presence o f fibronectin.  C o n c e n t r a t i o n s as s m a l l as 2 uM RGDS were c a p a b l e o f  s i g n i f i c a n t l y enhancing  7.  phagocytosis.  Mechanism o f f i b r o n e c t i n - m e d i a t e d macrophage a c t i v a t i o n The  initial  s t e p i n macrophage p h a g o c y t o s i s  involves  receptor-mediated  i n t e r a c t i o n s o f t h e macrophage plasma membrane w i t h t h e b a c t e r i a l surface.  F o r f i b r o n e c t i n t o a c t i v a t e macrophages f o r enhanced  phagocytosis,  subsequent t o f i b r o n e c t i n b i n d i n g a s i g n a l must be passed t o  the i n t e r i o r o f t h e c e l l  t o a c t i v a t e t h e macrophage and presumably  u p r e g u l a t e non-opsonic r e c e p t o r s .  Past s t u d i e s have suggested  that  phagocytic a c t i v a t i o n signals involve the generation o f i o n fluxes across the macrophage membrane (Young e t a l . , 1984).  Using the p o l a r i z a t i o n -  s e n s i t i v e f l u o r e s c e n t probe d i S C g ( 5 ) , i t was determined d i d indeed produce d e p o l a r i z a t i o n o f t h e P 3 8 8 16).  D 1  that f i b r o n e c t i n  plasma membrane ( F i g .  The r a t e o f i o n f l u x g e n e r a t i o n i n c r e a s e d as a f u n c t i o n o f t h e  c o n c e n t r a t i o n o f f i b r o n e c t i n added.  The r a t e s o f d e p o l a r i z a t i o n a t t a i n e d  70  Depolarization Rate (units/min)  8 -r  7-  6 -  5 -  4 -  3-  2 -  1 -  0  50  100  150  200  250  Concentration of Fibronectin (nM)  F i g u r e 16.  E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s o f f i b r o n e c t i n on the  of increase  (i.e.  d e p o l a r i z a t i o n ) of diSCg(5) fluorescence.  were c a l c u l a t e d from t r a c e s means o f 2 independent  The r a t e s  l i k e those shown i n F i g . 7 and r e p r e s e n t  experiments.  rate  the  71  at h i g h e r f i b r o n e c t i n c o n c e n t r a t i o n s were s i m i l a r t o t h a t caused by the p o s i t i v e c o n t r o l ionophore,  valinomycin,  K  I t s h o u l d be noted  +  concentration gradient.  i o n f l u x e s were generated  i n d i s s i p a t i o n o f an imposed h i g h that fibronectin-mediated  v i r t u a l l y immediately  a c r o s s t h e macrophage  membrane, and maximal r a t e s were a t t a i n e d w i t h i n 1 min o f s t i m u l u s addition.  8.  E f f e c t o f growth c o n d i t i o n s on f i b r o n e c t i n - m e d i a t e d non-opsonic macrophage  phagocytosis.  I t had a l r e a d y been determined t h a t f i b r o n e c t i n - m e d i a t e d a c t i v a t i o n o f macrophage p h a g o c y t o s i s  was observed  w i t h b a c t e r i a grown on agar p l a t e s ,  but not w i t h b a c t e r i a grown i n b r o t h w i t h r a p i d shaking I t was n e c e s s a r y  t o determine i f t h e decreased  (Table V I I ) .  phagocytosis  o f shaken  b a c t e r i a was r e l a t e d t o e f f e c t s o f growth i n b r o t h , o r due t o t h e r a p i d a g i t a t i o n o f the organisms i n t h e b r o t h c u l t u r e .  P. a e r u g i n o s a  strain  H103 was grown e i t h e r i n r a p i d l y a g i t a t e d (200 rpm) T r y p t i c a s e Soy b r o t h , s t a t i c T r y p t i c a s e Soy b r o t h o r on a p l a t e o f T r y p t i c a s e Soy agar and a v i s u a l assay observed  of fibronectin-enhanced  t h a t w h i l e a commercially  phagocytosis  I t was  a v a i l a b l e f i b r o n e c t i n p r e p a r a t i o n and  f i b r o n e c t i n - c o n t a i n i n g i n v i v o supernatant increase phagocytosis  was performed.  were a b l e t o s i g n i f i c a n t l y  o f s t a t i c b r o t h - and plate-grown organisms, uptake  o f a g i t a t e d b a c t e r i a c o u l d not be promoted u s i n g e i t h e r p r e p a r a t i o n (Table X I I ) . phagocytosis  Pseudomonas grown on agar was m a r g i n a l l y more s u s c e p t i b l e t o than the s t a t i c b r o t h c u l t u r e .  At a b a c t e r i a t o macrophage  r a t i o o f 20 t o 1, an average o f 17.3 and 14.5 b a c t e r i a became a s s o c i a t e d  72  Table XII.  The e f f e c t o f a g i t a t i o n d u r i n g growth on t h e s u s c e p t i b i l i t y o f P. a e r u g i n o s a s t r a i n H103 t o f i b r o n e c t i n - m e d i a t e d macrophage non-opsonic  phagocytosis  Growth Condition  Average number o f + PBS  bacteria/macrophage  + i n v i v o supernatant  + fibronectin  Shaken b r o t h  7.2  6.4  7.6°  S t a t i c broth  7.2  14.5  a  11.0  a  Agar p l a t e  7.2  17.3  a  13.6  a  b  a  p<0.005 ( s t u d e n t ' s t t e s t ) when compared t o t h e PBS c o n t r o l i n 2/2 ( i n v i v o s u p e r n a t a n t ) and 1/1 ( f i b r o n e c t i n ) a s s a y s .  b  n o t s i g n i f i c a n t l y d i f f e r e n t than t h e PBS c o n t r o l i n 2/2 ( i n v i v o s u p e r n a t a n t ) and 1/1 ( f i b r o n e c t i n ) a s s a y s .  73  per P 3 8 8  D 1  mouse macrophage c e l l  vivo supernatant. presence  ( r e s p e c t i v e l y ) i n the presence  T h i s s m a l l d i f f e r e n c e i n uptake was  o f commercially  of i n  echoed i n the  a v a i l a b l e f i b r o n e c t i n (Table X I I ) .  These d a t a e s t a b l i s h e d t h a t v i g o r o u s shaking d u r i n g growth was of  removing o r s u p p r e s s i n g e x p r e s s i o n o f the b a c t e r i a l l i g a n d r e q u i r e d f o r  f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic  9•  capable  uptake.  D e t e r m i n a t i o n o f the b a c t e r i a l l i g a n d f o r non-opsonic In  p r e v i o u s s t u d i e s , i t was  human b u c c a l e p i t h e l i a l c e l l s l e u k o c y t e s (Paranchych p o s s i b i l i t y was  suggested  t h a t Pseudomonas adherence t o  (Woods e t a l . , 1980)  e t a l . , 1986)  was  phagocytosis  and  polymorphonuclear  p i l u s mediated.  T h e r e f o r e the  c o n s i d e r e d t h a t p i l i were the b a c t e r i a l l i g a n d i n v o l v e d i n  f i b r o n e c t i n - m e d i a t e d macrophage a c t i v a t i o n .  P. a e r u g i n o s a s t r a i n s  H103  ( w i l d type) and BLP3 (a p i l u s - m i n u s mutant c o n s t r u c t e d by t r a n s p o s o n Tn501 i n s e r t i o n i n t o the c l o n e d chromosomal gene f o l l o w e d by gene  replacement)  were grown i n v i v o i n chambers o r on T r y p t i c a s e Soy agar and a s s e s s e d f o r t h e i r s u s c e p t i b i l i t y to fibronectin-enhanced phagocytosis. of  While  the p i l u s - m i n u s mutant c o u l d not be promoted u s i n g i n v i v o  f i b r o n e c t i n , o r RGDS, p h a g o c y t o s i s o f the w i l d type s t r a i n  uptake  supernatant,  was  s i g n i f i c a n t l y enhanced by a l l o f these t h r e e p r e p a r a t i o n s ( T a b l e X I I I ) . In  c o n t r o l experiments,  the PA01  parent s t r a i n o f t h i s Tn501 mutation  a p i l u s - e x p r e s s i n g s t r a i n c o n t a i n i n g both the Tn501 mutation a d d i t i o n a l plasmid-encoded  and  and  an  p i l i n gene ( p B P l 6 l ) were each s u s c e p t i b l e t o  f i b r o n e c t i n - a c t i v a t e d non-opsonic  uptake ( T a b l e X I I I ) .  74  Table XIII.  F i b r o n e c t i n - m e d i a t e d macrophage p h a g o c y t o s i s o f P. a e r u g i n o s a s t r a i n s H103 ( w i l d t y p e ) , BLP3 ( p i l i n minus), p o s i t i v e ) , and p B P l 6 l ( p i l i n  P. a e r u g i n o s a  Growth  strain  PAOl-leu  (pilin  positive)  Average number o f  bacteria/macrophage  condition Unwashed  Washed  Washed  +PBS  +in v i v o  Washed  Washed  +fibronectin  +RGDS  supernatant  H103 ( w i l d type)  r a t peritoneum TSA  BLP3 r a t peritoneum ( p i l i n minus) TSA  9.0  b  4-5°  2.2 6.0  6.6 11.2  6.4 5.2  6.3 4.8  PAOl-leu TSA ( p i l i n positve)  5.0  pBP161 TSA ( p i l i n positive)  4.8  ^D  6.9 13.5  b  b  7.1 4.7°  c  C  C  10. l  7.0  b  b  b  b  14.2  b  - not done.  b  p<0.005 ( S t u d e n t ' s t t e s t ) when compared to a s s a y s performed.  c  not  significantly different  t h e "washed +PBS" c o n t r o l i n 2/2  than t h e PBS c o n t r o l i n 2/2 assays performed.  b  ND 5.2  10.7  b  8.9  ND 10.6  C  b  9.3  b  a  75  To c o n f i r m p i l i  as the non-opsonic  ug/ml o f p u r i f i e d p i l i RGDS-activated exogenously  p h a g o c y t o s i s b a c t e r i a l l i g a n d , 60  was added t o i n v i v o s u p e r n a t a n t ; f i b r o n e c t i n - o r  macrophages and i n c u b a t e d f o r 15 min p r i o r t o assay.  added p i l i  s i g n i f i c a n t l y reduced,  c o n t r o l , the phagocytosis-promoting  This  t o the l e v e l o f the PBS  a b i l i t y of a l l 3 preparations  (Table XIV).  10.  Other  bacteria  To determine  i f f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic  was r e s t r i c t e d t o P. a e r u g i n o s a , c l i n i c a l  uptake  i s o l a t e s o f E s c h e r i c h i a c o l i and  S t a p h y l o c o c c u s aureus were grown i n the mouse chamber system and a s s e s s e d u s i n g the v i s u a l p h a g o c y t o s i s assay ( F i g . 17,18).  I t was determined  the i n v i v o supernatant  i s o l a t e d w i t h each organism was c a p a b l e o f  s i g n i f i c a n t l y enhancing  uptake  vitro-grown b a c t e r i a . proven  of e i t h e r i n vivo- or agitated i n  Both p r e p a r a t i o n s o f i n v i v o supernatant were  t o c o n t a i n s u b s t a n t i a l q u a n t i t i e s o f f i b r o n e c t i n as a s s e s s e d u s i n g  Western immunoblotting  11.  that  techniques  ( F i g . 19).  Summary The monoclonal  a n t i b o d y MA5-8, d i r e c t e d a g a i n s t p o r i n p r o t e i n F was  c a p a b l e o f s i g n i f i c a n t l y enhancing P. a e r u g i n o s a .  p h a g o c y t o s i s o f i n vivo-grown  P. a e r u g i n o s a c e l l s taken d i r e c t l y from the i n v i v o growth  system were s i g n i f i c a n t l y more s u s c e p t i b l e t o macrophage p h a g o c y t o s i s were the same c e l l s a f t e r b e i n g washed i n b u f f e r . that a phagocytosis-promoting  I t was  than  demonstrated  f a c t o r was found i n the supernatant  obtained  76  T a b l e XIV.  I n h i b i t i o n o f f i b r o n e c t i n - m e d i a t e d macrophage  non-opsonic  uptake o f P. a e r u g i n o s a s t r a i n H103 by exogenous PA01  pili  Average number o f bacteria/macrophage  Macrophage  activator  without  pili  exogenous  7.1  6.7  16.0  6.7  a  Fibronectin  19.1  5.5  s  RGDS  14.2  5.0  None (PBS c o n t r o l )  In  a  D  vivo supernatant  1  E  p<0.005 (Student's t t e s t ) s i g n i f i c a n t l y lower than t h e "without c o n t r o l i n 2 independent a s s a y s . n o t s i g n i f i c a n t l y lower than t h e "without p i l i " independent a s s a y s .  control i n 2  pili  pili"  77  Bacteria per Macrophage 25  -i  in vivo  F i g u r e 17.  in vitro  Enhancement o f t h e a s s o c i a t i o n o f E . c o l i w i t h P 3 8 8  u s i n g t h e s u p e r n a t a n t from r a t chambers o f i n vivo-grown  D 1  cells  E. c o l l .  B a c t e r i a l c e l l s were washed (w) o r unwashed (uw), w i t h (+SN) and w i t h o u t the a d d i t i o n o f i n v i v o s u p e r n a t a n t . uptake observed i n 2 independent  Each column r e p r e s e n t s the a v e r a g e  experiments.  78  Figure 18.  Enhancement of the association of S. aureus with P388  D1  c e l l s using the supernatant from r a t chambers of i n vivo-grown S. aureus. B a c t e r i a l c e l l s were washed (w) or unwashed (uw), with (+SN) and without the addition of i n vivo supernatant. uptake observed i n 2 Independent  Each column represents the average  experiments.  79  MIL 1  F i g u r e 19.  2  3  Western immune-blots o f the supernatants o f E. c o l i -  S. a u r e u s - c o n t a i n i n g r a t chambers probed w i t h goat-anti-human serum.  Lane 1, E. c o l i  i n v i v o supernatant; l a n e 2, S. aureus  supernatant; l a n e 3, p u r i f i e d bovine  fibronectin.  and  fibronectin i n vivo  80  from chambers i n c u b a t e d i n the p e r i t o n e a l c a v i t y o f l a b o r a t o r y rats.  The p h a g o c y t o s i s - p r o m o t i n g  o f P. a e r u g i n o s a t e s t e d ,  f a c t o r was  e f f e c t i v e w i t h both s t r a i n s  u s i n g both u n e l i c i t e d mouse p e r i t o n e a l  macrophages and the P 3 8 8 ^ mouse macrophage c e l l D  cell.  mice o r  P h a g o c y t o s i s enhancement was  l i n e as the p h a g o c y t i c  observed w i t h i n vivo-grown b a c t e r i a  and w i t h b a c t e r i a grown i n v i t r o on agar p l a t e s but not w i t h b a c t e r i a grown i n v i t r o w i t h r a p i d a g i t a t i o n . f r a c t i o n a t e d u s i n g a Fast column.  from mice and r a t s were  P r e s s u r e L i q u i d Chromatography g e l  The p h a g o c y t o s i s - p r o m o t i n g  Furthermore,  Supernatants  f a c t o r c o - p u r i f i e d with f i b r o n e c t i n .  a n t i - f i b r o n e c t i n s e r a negated  the  phagocytosis-promoting  a c t i v i t i e s o f i n v i v o chamber s u p e r n a t a n t , w h i l e commercial f i b r o n e c t i n was  i t s e l f c a p a b l e o f promoting  concentration of f i b r o n e c t i n increased chambers w i t h time, c o i n c i d e n t  phagocytosis.  The  w i t h the a b i l i t y o f chamber s u p e r n a t a n t s  I t was  phagocytosis-promoting  f a c t o r o f chamber s u p e r n a t a n t s .  chambers was  bovine  i n both r a t and mouse p e r i t o n e a l  promote p h a g o c y t o s i s .  i n the p e r i t o n e a l  exclusion  concluded t h a t f i b r o n e c t i n was  not r e q u i r e d  to  the  Bacterial  presence  t o e l i c i t f i b r o n e c t i n uptake  i n t o the chambers. I t was produced  demonstrated t h a t c o n c e n t r a t i o n s as low as 27 nM  s i g n i f i c a n t enhancement o f macrophage p h a g o c y t o s i s .  fibronectin Washing o f  f i b r o n e c t i n - t r e a t e d macrophages d i d not prevent p h a g o c y t o s i s enhancement, but washing o f f i b r o n e c t i n - t r e a t e d b a c t e r i a d i d . arginine-glycine-aspartic binding uptake.  a c i d - s e r i n e , which comprises  domain o f f i b r o n e c t i n , was Fibronectin  The  tetrapeptide the e u k a r y o t i c c e l l  a l s o c a p a b l e o f promoting  caused d e p o l a r i z a t i o n o f the P388  bacterial  plasma  81  membrane, as demonstrated probe.  using a p o l a r i z a t i o n - s e n s i t i v e  These d a t a i n d i c a t e t h a t promotion  p h a g o c y t o s i s i s mediated While f i b r o n e c t i n was  by f i b r o n e c t i n o f  non-opsonic  by a c t i v a t i o n o f the macrophages. able to s i g n i f i c a n t l y increase phagocytosis of  P. a e r u g i n o s a grown i n s t a t i c b r o t h , uptake be promoted.  fluorescent  o f a g i t a t e d b a c t e r i a c o u l d not  P h a g o c y t o s i s o f a mutant s t r a i n l a c k i n g s u r f a c e p i l i  could  not be enhanced by f i b r o n e c t i n r e g a r d l e s s o f growth c o n d i t i o n s . Furthermore, of  60 yg/ml o f exogenously  added Pseudomonas p i l i was  a b r o g a t i n g the enhanced p h a g o c y t o s i s o f the w i l d type s t r a i n  w i t h f i b r o n e c t i n - a c t i v a t e d macrophages. pili  I t was  observed  concluded t h a t Pseudomonas  were the b a c t e r i a l l i g a n d s r e q u i r e d f o r attachment  f i b r o n e c t i n — a c t i v a t e d macrophages i n the i n i t i a l  capable  to  stages o f  non—opsonic  phagocytosis. F i b r o n e c t i n - m e d i a t e d macrophage non-opsonic to  P. a e r u g i n o s a , but c o u l d be reproduced  S. aureus.  uptake was  not  u s i n g i n vivo-grown  In these c a s e s , however, uptake  restricted E. c o l i  of agitated i n vitro-grown  b a c t e r i a c o u l d be e f f i c i e n t l y enhanced u s i n g i n v i v o s u p e r n a t a n t .  Both  p r e p a r a t i o n s o f i n v i v o supernatant were proven t o c o n t a i n s u b s t a n t i a l q u a n t i t i e s o f f i b r o n e c t i n as a s s e s s e d u s i n g Western techniques.  or  immunoblotting  82  DISCUSSION  In t h i s t h e s i s t h r e e types o f i n t e r a c t i o n between macrophages and P. a e r u g i n o s a were s t u d i e d .  The f i r s t  d e a l t w i t h o p s o n i c p h a g o c y t o s i s and  l e d t o the e s t a b l i s h m e n t o f an a p p r o p r i a t e model c e l l studies.  The second  s e t o f experiments  l i n e f o r subsequent  i n v e s t i g a t e d one o f the ways i n  which Pseudomonas may p r o t e c t i t s e l f from p h a g o c y t o s i s : v i a l i b e r a t i o n o f a cytotoxin.  These s t u d i e s were d e s i g n e d t o determine  the c e l l u l a r  l o c a l i z a t i o n o f t h i s p r o t e i n and t o e l u c i d a t e i t s e f f e c t on macrophage function.  The f i n a l s e c t i o n o f my t h e s i s addressed f i b r o n e c t i n - m e d i a t e d  a c t i v a t i o n o f non-opsonic  p h a g o c y t o s i s o f P. a e r u g i n o s a .  Bach o f t h e s e  t h r e e i n t e r a c t i o n s w i l l be d i s c u s s e d s e p a r a t e l y below.  1.  Use o f the P 3 8 8  p i  macrophage c e l l l i n e as a model f o r macrophage  studies I t was c l e a r from the d a t a p r e s e n t e d i n T a b l e I and F i g u r e s 1-3 t h a t monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t p o r i n p r o t e i n F c o u l d a c t t o o p s o n i z e P. a e r u g i n o s a f o r p h a g o c y t o s i s by mouse p e r i t o n e a l macrophages, P388  D 1  c e l l s and human p e r i p h e r a l b l o o d monocytes.  There appeared  t o be  some advantage i f a n t i b o d y and phagocytes were o f t h e same s p e c i e s . was i n d i c a t e d when MA5-10 f a i l e d  This  to s i g n i f i c a n t l y increase b a c t e r i a l  uptake i n t h r e e o f s i x experiments  performed  on human c e l l s .  T h i s was not  a phenomenon a t t r i b u t a b l e t o i s o t y p e as o t h e r I g G l a n t i b o d i e s f u n c t i o n e d q u i t e w e l l i n these  tests.  83  C e r t a i n t r e n d s c o u l d be seen i n t h e a b i l i t y o f s p e c i f i c monoclonal a n t i b o d i e s t o mediate p h a g o c y t o s i s by a l l t h r e e macrophage c e l l For example, MA4-10 always r e s u l t e d i n t h e h i g h e s t p h a g o c y t i c always s c o r e d i n t h e middle  line P388  D 1  index, MA5-8  t o h i g h r e g i o n and MA4-4 was c o n s i s t e n t l y one  o f t h e two weakest o p s o n i n s . macrophage c e l l  types.  I t i s thus apparent  t h a t t h e mouse  c a n be used as a model f o r u n e l i c i t e d mouse  p e r i t o n e a l macrophages and c u l t u r e d human p e r i p h e r a l b l o o d monocytes. a p p r o p r i a t e n e s s o f t h i s model was c o n f i r m e d  i n subsequent s t u d i e s o f  f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic p h a g o c y t o s i s . experiments,  The  I n these  s i m i l a r l e v e l s o f f i b r o n e c t i n - s t i m u l a t e d b a c t e r i a l uptake  were observed  f o r both P 3 8 8  D 1  c e l l s o r u n e l i c i t e d mouse p e r i t o n e a l  macrophages ( T a b l e V I ) .  2.  Role o f i s o t y p e i n o p s o n i z e d P r e v i o u s d a t a has suggested  h i g h l y conserved 1985).  phagocytosis  by macrophages  t h a t t h e r e a r e a t l e a s t two d i s t i n c t  e p i t o p e s on p o r i n p r o t e i n F ( M u t h a r i a and Hancock,  Monoclonal a n t i b o d i e s MA4-4, 2-10, 4-10 and 5-10 were a l l  hypothesized the o t h e r .  t o r e a c t a g a i n s t one e p i t o p e , w h i l e MA5-8 was s p e c i f i c f o r Of t h e f o u r monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t a s i m i l a r  e p i t o p e , t h e t h r e e I g G l monoclonal a n t i b o d i e s were s u b s t a n t i a l l y more o p s o n i c than t h e one IgG2a i s o t y p e ( T a b l e I ) .  I n t h e p a s t , t h e r e has been  some d i s p u t e as t o t h e o p s o n i c p o t e n t i a l o f I g G l .  T h i s i s o t y p e has been  r e p o r t e d t o have a low a f f i n i t y f o r i n t a c t macrophage c e l l s 1980;  Schneider  e t a l . , 1981; Unkeless  (Ralph e t a l . ,  and E i s e n , 1975) and a h i g h  a f f i n i t y f o r i s o l a t e d Fc r e c e p t o r s i n column systems ( S c h n e i d e r e t a l . ,  84  1981).  I t i s important t o note t h a t w h i l e macrophages b i n d monomeric  IgGl  w i t h a r e l a t i v e l y low a f f i n i t y , h i g h m o l e c u l a r weight aggregates o f IgGl b i n d a t l e a s t as w e l l as IgG2a and IgG2b (Heusser e t a l . , 1977).  This  suggests t h a t t h e I g G l - b a c t e r i a complex i s seen as an "agggregate" o f immunoglobulin by t h e macrophage i n my system. antibodies  h y p o t h e s i z e d t o be d i r e c t e d a g a i n s t  F, one o f these, in a l l c e l l  Of t h e t h r e e  IgGl  a common e p i t o p e  of protein  MA4-10, produced s u b s t a n t i a l l y b e t t e r opsonophagocytosis  types t e s t e d .  T h i s may have been due t o a b e t t e r  complementarity between t h e a n t i g e n - b i n d i n g corresponding a n t i g e n i c determinant.  geometric  pocket o f MA4-10 and i t s  A d d i t i o n a l l y , microheterogeneity i n  the p r o t e i n s t r u c t u r e o r g l y c o s y l a t i o n p a t t e r n s  o f t h e Fc p o r t i o n o f  MA4-10 may have produced a b e t t e r b i n d i n g a f f i n i t y f o r t h e macrophage Fc receptor.  I n e i t h e r case, i n c r e a s e d  macrophage would be t h e expected  a s s o c i a t i o n o f b a c t e r i a with the  result.  These s t u d i e s demonstrated t h a t monoclonal a n t i b o d i e s p r o t e i n F are capable o f opsonizing t h r e e macrophage c e l l  and  types t e s t e d .  T h i s supports t h e p r o p o s a l  a l . , 1985) i s through o p s o n i z a t i o n  f o r phagocytosis.  e f f e c t i v e n e s s o f these a n t i - F monoclonal a n t i b o d i e s  s u b s t a n t i a l p o t e n t i a l as immunotherapeutic  3.  Cytotoxin: The  against  P. a e r u g i n o s a f o r p h a g o c y t o s i s by a l l  mechanism o f p r o t e c t i o n a f f o r t e d by monoclonal a n t i b o d i e s et  directed  that the  i n vivo  (Hancock  The s p e c i f i c i t y provides  them w i t h  agents.  l o c a l i z a t i o n and p u t a t i v e r o l e i n i n f e c t i o n  d a t a p r e s e n t e d i n Chapter Two o f t h i s t h e s i s i n d i c a t e t h a t  P. a e r u g i n o s a c y t o t o x i n i s l o c a l i z e d i n t h e p e r i p l a s m i c  space o f t h i s  85  organism.  Thus, osmotic  shockates  r e p r o d u c i b l y demonstrated  a 28  kD  p r o t e i n with antigenic c r o s s r e a c t i v i t y with p u r i f i e d c y t o t o x i n ( P i g . 5). In a d d i t i o n , osmotic  shockates and c y t o t o x i n both i n h i b i t e d  opsonized  p h a g o c y t o s i s o f P. a e r u g i n o s a by macrophages, a phenomenon t h a t c o u l d be abrogated by a n t i - c y t o t o x i n s e r a ( T a b l e I I ) .  Furthermore,  p r e p a r a t i o n s caused  i n the macrophage membrane  the f o r m a t i o n o f channels  both  as a s s e s s e d by the d e p o l a r i z a t i o n - s e n s i t i v e f l u o r e s c e n t probe d i S C ^ t S ) (Table I I I , F i g s .  7,8).  None o f the o t h e r c e l l f r a c t i o n s demonstrated a c t i v i t y with p u r i f i e d cytotoxin.  similar correlations i n  For example, 1 5 0 - f o l d c o n c e n t r a t e d  growth s u p e r n a t a n t s c o n t a i n e d a 56 kD band t h a t r e a c t e d w i t h a n t i - c y t o t o x i n s e r a ( F i g . 5) and t h i s p r e p a r a t i o n c o u l d i n h i b i t phagocytosis  ( T a b l e I I ) but was  membrane ( T a b l e I I I ) . was  unable t o d e p o l a r i z e the macrophage  By use o f anti-exoenzyme S a n t i s e r u m ( F i g . 6 ) , i t  p o s s i b l e t o c o m p l e t e l y abrogate t h i s p h a g o c y t o s i s  inhibition  ( T a b l e I I ) and I a s c r i b e t h i s c r o s s r e a c t i v e band to another Pseudomonas a e r u g i n o s a t o x i n , exoenzyme S.  These r e s u l t s suggest t h a t exoenzyme S  the major macrophage i n h i b i t i n g t o x i n i n growth s u p e r n a t a n t . o t h e r s e c r e t e d Pseudomonal t o x i n s such as e x o p r o t e a s e s , e x o t o x i n A were not i n f l u e n c i n g macrophages i n t h i s  was  Presumably  lipases  and  system.  The f a c t t h a t the a n t i - c y t o t o x i n s e r a r e c o g n i z e s the 56 kD band ( F i g . 5) may due  be due  t o c r o s s r e a c t i v i t y o f c y t o t o x i n w i t h exoenzyme S o r  t o the presence o f minor c o n t a m i n a t i n g a n t i b o d i e s t o exoenzyme S i n  a n t i - c y t o t o x i n serum.  N e v e r t h e l e s s , the a b i l i t y o f c y t o t o x i n and  the  2+ Mg  /freeze-thaw  osmotic shockate t o i n h i b i t macrophage p h a g o c y t o s i s  was  86  not  r e l a t e d t o t h e presence o f exoenzyme S i n these p r e p a r a t i o n s , s i n c e  anti-exoenzyme S s e r a d i d not r e a c t w i t h these p r e p a r a t i o n s on Western immunoblots  ( F i g . 6) and d i d not r e v e r s e t h e i n h i b i t o r y e f f e c t s o f these  preparations  i n phagocytosis  I t s h o u l d be noted  o f opsonized  P. a e r u g i n o s a  inability  that contaminating t h e observed  shockate.  o f LPS t o cause d e p o l a r i z a t i o n o f t h e macrophage  membrane i n t h e f l u o r e s c e n c e assay  for  (growth  i n n e r and o u t e r membranes) o n l y the 28 kD p r o t e i n was  r e p r o d u c i b l y d e t e c t e d i n osmotic The  (Table I I ) .  t h a t i n s p i t e o f t h e f a c t t h a t the a n t i - c y t o t o x i n s e r a  r e c o g n i z e d o t h e r bands i n complex p r o t e i n p r e p a r a t i o n s supernatant,  cells  ( T a b l e I I I ) e l i m i n a t e d the p o s s i b i l i t y  LPS i n t h e osmotic  results.  shock p r e p a r a t i o n was r e s p o n s i b l e  Furthermore, i n n e r and o u t e r membrane  f r a c t i o n s , c o n t a i n i n g l a r g e amounts o f LPS, had t h e a b i l i t y t o i n c r e a s e phagocytosis  (Table I I ) .  T h i s suggested  t h a t LPS c o u l d a c t i v a t e  macrophages, as p r e v i o u s l y d e s c r i b e d by o t h e r s There i s s u b s t a n t i a l precedent toxins i n pathogenesis.  (Nowakowski e t a l . , 1980).  f o r t h e importance o f p e r i p l a s m i c  For example, S h i g a t o x i n o f S h i g e l l a  1 (Donahue-Rolfe and Keutsch,  dysenteriae  1983), v e r o t o x i n ( s h i g a - l i k e t o x i n ) o f  E. c o l i ( K a r m a l i e t a l . , 1985) and h e a t - l a b i l e e n t e r o t o x i n o f E. c o l i (Evans e t a l . , 1974) a r e a l l p e r i p l a s m i c , as i n d i c a t e d by polymyxin r e l e a s e experiments.  The p e r i p l a s m i c l o c a t i o n o f c y t o t o x i n does not  n e c e s s a r i l y argue a g a i n s t a r o l e f o r t h i s t o x i n i n pathogenesis P. a e r u g i n o s a  infections.  Indeed, i t s l o c a t i o n i n t h e p e r i p l a s m may be  p a r t o f a p o p u l a t i o n s t r a t e g y o f P. a e r u g i n o s a maintain  of  i t s presence a t an i n f e c t i o n s i t e .  t o a l l o w t h e organism t o  I n response t o e i t h e r  host  87  b a c t e r i c i d a l defense mechanisms ( i n c l u d i n g complement-mediated l y s i s and p h a g o c y t o s i s ) o r a n t i b i o t i c therapy, c y t o t o x i n and o t h e r p e r i p l a s m i c c o n s t i t u e n t s may be r e l e a s e d by l y s i s o r damage o f t h e o u t e r membrane. Such l i b e r a t i o n o f c y t o t o x i n would have a s i g n i f i c a n t e f f e c t on host p h a g o c y t i c c e l l s as suggested  by t h e p r e v i o u s l y - o b s e r v e d k i l l i n g and l y s i s  o f n e u t r o p h i l s ( B a l t c h e t a l . , 1985; Scharmann e t a l . , 1976), t h e s t r o n g d e p o l a r i z a t i o n o f t h e macrophage membrane ( F i g . 8) and t h e reduced o f t r e a t e d macrophages t o phagocytose P. a e r u g i n o s a last  two e f f e c t s may indeed be analogous  (Table I I ) .  ability  These  s i n c e d e p o l a r i z a t i o n would  d i s s i p a t e i o n gradients required t o s i g n a l i n i t i a t i o n o f phagocytosis (Young e t a l . , 1984) and thus r e s u l t Table I I .  i n t h e d i m i n i s h e d uptake observed i n  I t s h o u l d be noted t h a t c y t o t o x i n appears  t o be m a r g i n a l l y more  a c t i v e a g a i n s t n e u t r o p h i l s as compared t o macrophages.  While  c o n c e n t r a t i o n s as low as 6 ug/ml have been r e p o r t e d t o cause complete l y s i s o f g r a n u l o c y t e s ( B a l t c h e t a l . , 1985), 13 ug/ml produced i n h i b i t i o n of phagocytosis but l i t t l e  lysi3  i n macrophages.  complete  It i s  p o s s i b l e t h a t macrophages d i s p l a y an i n c r e a s e d r e s i s t a n c e t o t h i s  toxin.  L i b e r a t i o n o f c y t o t o x i n by dead and d y i n g P. a e r u g i n o s a c e l l s may be an important  f a c t o r i n the p e r s i s t e n t lung i n f e c t i o n o f i n d i v i d u a l s  cystic fibrosis.  with  Through i n h i b i t i o n o f p h a g o c y t o s i s , these organisms may  be capable o f p r o t e c t i n g t h e b a c t e r i a l p o p u l a t i o n from a primary mechanism o f t h e l u n g .  defense  88  4.  F i b r o n e c t i n as an a c t i v a t o r o f macrophage non-opsonic p h a g o c y t o s i s Initial  s t u d i e s demonstrated t h a t t h e p r o t e i n F - s p e c i f i c  monoclonal  a n t i b o d y MA5-8 was c a p a b l e o f o p s o n i z i n g i n vivo-grown P. a e r u g i n o s a f o r p h a g o c y t o s i s by mouse macrophage c e l l  line P388  D 1  ( F i g . 10).  This  f u r t h e r suggested t h a t a n t i - F monoclonal a n t i b o d i e s have s u b s t a n t i a l p o t e n t i a l as p a s s i v e immunotherapeutic  agents.  An i n t e r e s t i n g f i n d i n g o f  these s t u d i e s was t h a t the average i n c r e a s e i n b a c t e r i a p e r phagocyte r e s u l t i n g from o p s o n i z a t i o n appeared t o be lower f o r i n vivo-grown than f o r i n vitro-grown b a c t e r i a .  T h i s suggested t h a t t h e i n vivo-grown  organisms may have had a reduced s u r f a c e exposure o f p r o t e i n F r e l a t i v e t o i n vitro-grown c e l l s . These s t u d i e s a l s o demonstrated t h a t a p h a g o c y t o s i s promoting  factor  was found i n t h e supernatant o b t a i n e d from P. a e r u g i n o s a chambers i n c u b a t e d i n t h e p e r i t o n e a l c a v i t y o f l a b o r a t o r y mice and r a t s .  This  f a c t o r c o u l d be s e p a r a t e d e a s i l y from b a c t e r i a by c e n t r i f u g a t i o n and added back t o a g a i n f a c i l i t a t e a s s o c i a t i o n o f P. a e r u g i n o s a w i t h mouse u n e l i c i t e d p e r i t o n e a l macrophages o r macrophage c e l l ( T a b l e s IV and V I ) .  l i n e P388p^  The ease o f removal o f t h i s f a c t o r suggested t h a t i t  was not an o p s o n i n such as a n t i b o d y o r complement, which t y p i c a l l y have v e r y h i g h a f f i n i t i e s and thus remain a t t a c h e d t o c e l l s centrifugation.  during  Furthermore, treatment o f i n v i v o supernatant a t 100°C  f o r 10 minutes f a i l e d t o e f f e c t p h a g o c y t o s i s enhancement ( T a b l e V ) . treatment would have i n a c t i v a t e d any complement o r a n t i b o d y p r o t e i n s present.  This  89  T h i s phenomenon was different  reproduced w i t h two  s t r a i n s o f P. a e r u g i n o s a o f  s e r o t y p e s , u s i n g e i t h e r mice o r r a t s as the i n v i v o chamber  host.  A d d i t i o n a l l y , r a t i n v i v o supernatant c o u l d e f f e c t i v e l y promote  uptake  o f plate-grown  b a c t e r i a by P 3 8 8 ^ c e l l s o r u n e l i c i t e d mouse  p e r i t o n e a l macrophages. macrophage c e l l was  The  s i m i l a r r e s u l t s o b t a i n e d w i t h these  types confirmed t h a t mouse macrophage c e l l  line  P388  D 1  an a p p r o p r i a t e model f o r u n e l i c i t e d mouse p e r i t o n e a l macrophages, as  p r e v i o u s l y demonstrated  i n the s t u d i e s o f o p s o n i z e d p h a g o c y t o s i s o f  P. a e r u g i n o s a (see Chapter  One).  The f a c t t h a t f i b r o n e c t i n c o p u r i f i e d w i t h the a c t i v i t y u s i n g a g e l s i e v i n g column suggested be the f a c t o r i n q u e s t i o n .  phagocytosis-promoting  t o us t h a t f i b r o n e c t i n might  F i b r o n e c t i n has been shown p r e v i o u s l y t o  a c t i v a t e macrophages f o r i n c r e a s e d adherence (Akiyama e t a l . , and F c - r e c e p t o r mediated al..  1983;  activity  1981),  C3  p h a g o c y t o s i s o f c o a t e d e r y t h r o c y t e s (Wright e t  Pommier et a l . , 1983), and maintenance o f a n t i - s t a p h y l o c o c c a l  (Proctor et a l . ,  non-opsonic  1985).  However, no r e p o r t s o f i t s e f f e c t s  on  p h a g o c y t o s i s o r on p h a g o c y t o s i s o f a gram n e g a t i v e b a c t e r i u m  have appeared of  two  to date.  In my  s t u d i e s , the p h a g o c y t o s i s - p r o m o t i n g  activity  a c o m m e r c i a l l y - a v a i l a b l e f i b r o n e c t i n p r e p a r a t i o n and the n e g a t i n g  e f f e c t s o f a n t i - f i b r o n e c t i n a n t i b o d i e s ( T a b l e IX) s t r o n g l y supported c o n c l u s i o n t h a t f i b r o n e c t i n was  the  the a c t i v e component o f i n v i v o  supernatant. The  s i m i l a r p h a g o c y t o s i s enhancement produced  by i n v i v o s u p e r n a t a n t s  from chambers c o n t a i n i n g b a c t e r i a o r s a l i n e ( T a b l e X) suggested b a c t e r i a l presence was  not r e q u i r e d t o e l i c i t the f i b r o n e c t i n  that  response.  90  C o n f i r m a t i o n o f t h i s was ( F i g . 14).  o b t a i n e d u s i n g Western b l o t t i n g t e c h n i q u e s  These d a t a suggested t h a t f i b r o n e c t i n i n c r e a s e d  concentration  over time i n the p e r i t o n e a l chambers, p o s s i b l y due  s u r g i c a l i n j u r y or i m p l a n t a t i o n necessarily  in to  o f a f o r e i g n body (the chamber), but  i n response t o the b a c t e r i a .  There i s a  not  considerable  precedent f o r t h i s , as f i b r o n e c t i n i s commonly found at wound s i t e s ( G r i n n e l , 1984).  I t s h o u l d be  noted t h a t P.  a e r u g i n o s a commonly  i n f e c t i o n s at s i t e s o f i n j u r y , i n c l u d i n g wounds and The may  initiates  burns.  presence o f f i b r o n e c t i n e a r l y i n the mouse time c o u r s e experiments  have i n d i c a t e d a h i g h e r l e v e l o f f i b r o n e c t i n i n the normal peritoneum  o f mice as compared to r a t s .  A l t e r n a t i v e l y , the mouse system may  more e f f i c i e n t d e l i v e r y o f f i b r o n e c t i n t o the  s i t e of i n j u r y .  have  The  lower  maximal l e v e l s o f b a c t e r i a l a s s o c i a t i o n w i t h macrophages found u s i n g i n v i v o supernatant may  d a t a p r e s e n t e d i n T a b l e XI and  F i g . 15 demonstrated  f i b r o n e c t i n a c t s as a macrophage a c t i v a t o r t o s t i m u l a t e p h a g o c y t o s i s o f P.  aeruginosa.  The  enhanced a b i l i t y t o phagocytose P. and  aeruginosa following  favored  this interpretation.  w i t h t h i s supernatant and  i n vivo  In c o n t r a s t ,  from mice.  that  increased  f a c t t h a t macrophages  subsequent removal o f f i b r o n e c t i n - c o n t a i n i n g  strongly  retained  incubation  with  supernatant  treatment o f  bacteria  subsequent washing p r i o r to m i x i n g w i t h  macrophages r e s u l t e d i n o n l y background l e v e l s o f p h a g o c y t o s i s . Furthermore, the sufficient  rat  however, r e f l e c t a macrophage p r e f e r e n c e f o r  homologous f i b r o n e c t i n s i n c e the macrophages used were d e r i v e d The  had  tetrapeptide  eukaryotic  t o a c t i v a t e macrophages, and  c e l l binding  domain  was  f i b r o n e c t i n caused macrophage  91  d e p o l a r i z a t i o n i n a manner r e m i n i s c e n t  o f o t h e r macrophage a c t i v a t o r s . I t  i s thus apparent t h a t f i b r o n e c t i n a c t s not as an opsonin, l e c t i n o r l i g a n d in  t h i s system, b u t a c t s d i r e c t l y on t h e macrophages t o s t i m u l a t e Fibronectin i s a large dimeric  g l y c o p r o t e i n whose s t r u c t u r e c a n be  d i v i d e d i n t o s e v e r a l f u n c t i o n a l domains. according  t o t h e substances which b i n d  ( P r o c t o r , 1987).  The e u k a r y o t i c  These areas have been named  i n that region o f the molecle  c e l l - a d h e s i o n r e g i o n had been p r e v i o u s l y  shown t o i n t e r a c t w i t h v a r i o u s mammalian c e l l (Brown and Goodwin, 1988).  uptake.  types i n c l u d i n g macrophages  A f o u r amino a c i d sequence, RGDS, i n t h i s  cell  b i n d i n g domain has been shown t o i n t e r a c t w i t h t h e mammalian c e l l - s u r f a c e g l y c o p r o t e i n H b / I I I a (Brown and Goodwin, 1988).  I n our s t u d i e s , a  c o m m e r c i a l l y a v a i l a b l e RGDS p r e p a r a t i o n was a b l e t o s i g n i f i c a n t l y non-opsonic uptake o f Pseudomonas by mouse macrophage c e l l (Fig.  15).  A l t h o u g h 80 f o l d h i g h e r molar c o n c e n t r a t i o n s  increase  l i n e P388j^  o f RGDS than  f i b r o n e c t i n were r e q u i r e d t o observe t h i s e f f e c t , t h i s i s c o n s i s t e n t previous  studies  (Pierschbacher  and R u o s l a h t i ,  1984) and t h e n o t i o n  the s t r u c t u r e o f t h e t e t r a p e p t i d e would be c o n f o r m a t i o n a l l y  with that  constrained i n  the i n t a c t f i b r o n e c t i n m o l e c u l e . U s i n g t h e p o l a r i z a t i o n - s e n s i t i v e f l u o r e s c e n t probe d i S C ( 5 ) , 3  i t was  determined t h a t f i b r o n e c t i n c o u l d g e n e r a t e a s t r o n g i o n f l u x a c r o s s t h e macrophage membrane a t c o n c e n t r a t i o n s  as low as 27 nM ( F i g . 16).  r e s u l t c o r r e l a t e d well with studies o f the concentration f i b r o n e c t i n i n promoting p h a g o c y t o s i s ( F i g . 15). p h a g o c y t o s i s was observed a t c o n c e n t r a t i o n s f l u x generation  increased  This  requirements o f  While maximal  around 50 nM, t h e r a t e o f i o n  as a f u n c t i o n o f t h e l e v e l o f f i b r o n e c t i n t o a t  92  l e a s t 230 nM.  T h i s suggested  that while higher concentrations o f  f i b r o n e c t i n can produce g r e a t e r i o n f l u x over the macrophage membrane, lower c o n c e n t r a t i o n s and t h e i r c o r r e s p o n d i n g l y lower r a t e s o f i o n f l u x were s u f f i c i e n t t o maximally macrophage r a t i o o f 20:1.  enhance p h a g o c y t o s i s a t a b a c t e r i a t o  While  these s t u d i e s do not prove  i t , this  p e r t u r b a t i o n o f i o n g r a d i e n t s a c r o s s t h e membrane may be t h e p h a g o c y t i c a c t i v a t i o n s i g n a l which t r i g g e r s enhanced non-opsonic P. a e r u g i n o s a .  The d a t a p r e s e n t e d i n T a b l e X I I I demonstrated t h a t  Pseudomonas p i l i  were t h e b a c t e r i a l l i g a n d s f o r f i b r o n e c t i n - a c t i v a t e d  macrophage non-opsonic c o u l d abrogate  phagocytosis.  The f a c t t h a t exogenously  added  pili  t h e a b i l i t y o f f i b r o n e c t i n t o enhance p h a g o c y t o s i s by mouse  macrophage c e l l ( T a b l e XIV).  uptake o f  line P388  n i  s t r o n g l y favoured t h i s  F i b r o n e c t i n promotion  interpretation  o f macrophage p h a g o c y t o s i s was  observed w i t h b a c t e r i a grown on agar p l a t e s , t o a l e s s e r e x t e n t  with  organisms grown i n s t a t i c b r o t h , and not a l l w i t h r a p i d l y a g i t a t e d cultures  (Table X I I ) .  As a g i t a t i o n i s an a c c e p t e d method o f removing  from Pseudomonas, one might expect p r o g r e s s i v e l y fewer p i l i increasing In of  pili  p e r c e l l on  agitation.  our s t u d i e s , f i b r o n e c t i n was unable  P. a e r u g i n o s a l a c k i n g s u r f a c e p i l i  t o enhance uptake o f a s t r a i n  (Table X I I I ) .  T h i s s t r a i n was  c o n s t r u c t e d by t r a n s p o s o n Tn501 i n s e r t i o n i n t o t h e c l o n e d chromosomal p i l i n gene f o l l o w e d by gene replacement.  I n c o n t r o l experiments,  the PA01  parent s t r a i n o f t h i s Tn501-induced mutant and a p i l u s - e x p r e s s i n g s t r a i n c o n t a i n i n g both t h e Tn501 mutation  and an a d d i t i o n a l plasmid-encoded  pilin  gene ( p B P l 6 l ) were each s u s c e p t i b l e t o f i b r o n e c t i n - a c t i v a t e d macrophage  93  non-opsonic  uptake ( T a b l e X I I I ) .  enhanced p h a g o c y t o s i s was  T h i s confirmed  not due  t h a t the absence o f  to properties of t h i s p a r t i c u l a r  parent  s t r a i n o r t o t r a n s p o s o n Tn501 sequences. T h i s d a t a i s c o n s i s t e n t w i t h p r e v i o u s s t u d i e s showing t h a t h e a v i l y piliated  s t r a i n s o f P. a e r u g i n o s a were more s u s c e p t i b l e t o  polymorphonuclear (Paranchych  l e u k o c y t e (PMN)  e t a l . , 1986).  I t was  were c a p a b l e o f i n h i b i t i n g PMN (Paranchych  e t a l . , 1986).  p h a g o c y t o s i s than l e s s p i l i a t e d  non-opsonic  T h i s suggests  b a c t e r i a l l i g a n d t o which both PMN i n i t i a t e non-opsonic determined  a l s o determined  phagocytosis.  t h a t exogenous  uptake of t h i s  t h a t P. a e r u g i n o s a p i l i  a r e the  and macrophage p o p u l a t i o n s a t t a c h t o McEachran and I r v i n  r e c e p t o r s f o r b i n d i n g o f P. a e r u g i n o s a . number-high a f f i n i t y r e c e p t o r f o r p i l i , number-low a f f i n i t y r e c e p t o r which may component of mucoid e x o p o l y s a c c h a r i d e .  (1985) had  The f i r s t  classes of  i s a low copy  w h i l e the second  i s a h i g h copy  r e c o g n i z e a l g i n a t e , the  primary  I t seemed u n l i k e l y t h a t i n c r e a s e d  r e s p o n s i b l e f o r the i n c r e a s e d uptake  w i t h s t a t i c a l l y - g r o w n organisms,  pili  organism  t h a t b u c c a l e p i t h e l i a l c e l l s d i s p l a y a t l e a s t two  mucoid e x o p o l y s a c c h a r i d e was  strains  observed  however, as organisms which produce l a r g e  amounts o f a l g i n a t e a r e more r e s i s t a n t t o p h a g o c y t o s i s than non-mucoid b a c t e r i a ( B a l t i m o r e and M i t c h e l l , Furthermore,  1980;  a l g i n a t e from some s t r a i n s o f P. a e r u g i n o s a i s i n c a p a b l e o f  b i n d i n g even t o b u c c a l e p i t h e l i a l c e l l s and M i t c h e l l  Simpson e t a l . , 1988).  (Doig e t a l . , 1987).  (1980) h y p o t h e s i z e d t h a t mucoid c o a t i n g may  ligands required f o r e f f i c i e n t phagocytosis. pili  a r e these c r u c i a l b a c t e r i a l l i g a n d s .  Baltimore  mask b a c t e r i a l  Perhaps s u r f a c e - e x p r e s s e d  94  S t u d i e s performed  u s i n g E. c o l i and S. aureus  f i b r o n e c t i n - c o n t a i n i n g i n v i v o supernatant was  indicated  capable o f  that  significantly  enhancing macrophage p h a g o c y t o s i s o f b o t h i n v i v o - and a g i t a t e d i n v i t r o - g r o w n b a c t e r i a ( F i g . 17,18).  I t i s possible that a g i t a t i o n i s  i n c a p a b l e o f removing the E. c o l i o r S. aureus b a c t e r i a l l i g a n d s t o which a c t i v a t e d macrophages b i n d .  A l t e r n a t i v e l y , f i b r o n e c t i n may  l i g a n d o r o p s o n i n i n promoting phagocyte.  Indeed,  a s s o c i a t i o n o f these organisms  p r e v i o u s i n v e s t i g a t o r s have suggested  can indeed o p s o n i z e S t a p h y l o c o c c u s f o r p h a g o c y t o s i s 1982).  be a c t i n g as a  that  w i t h the fibronectin  (Proctor et a l . ,  T h i s seems u n l i k e l y i n the case o f E. c o l i . as f i b r o n e c t i n i s  unable t o mediate attachment leukocytes  (Proctor et a l . ,  Non-opsonic uptake may P. a e r u g i n o s a i n f e c t i o n .  o f t h i s organism  to  polymorphonuclear  1985). be a important c l e a r a n c e mechanism a t s i t e s o f  These areas o f t e n d i s p l a y t i s s u e i n j u r y  and  would t h e r e f o r e possess a h i g h l o c a l c o n c e n t r a t i o n o f f i b r o n e c t i n ( G r i n n e l , 1984).  As such, i n f i l t r a t i n g macrophages c o u l d be a c t i v a t e d  by  f i b r o n e c t i n t o enhance t h e i r a s s o c i a t i o n w i t h b a c t e r i a v i a surface-expressed p i l i . macrophage non-opsonic  A c o n c e p t u a l model o f f i b r o n e c t i n - a c t i v a t e d p h a g o c y t o s i s i s p r e s e n t e d i n F i g u r e 20.  This  system would promote e f f i c i e n t non-opsonic  clearance of p i l i a t e d  P. a e r u g i n o s a from the s i t e o f i n f e c t i o n .  In a d d i t i o n , t h i s would p r o v i d e  a r a p i d source o f macrophage-processed P. a e r u g i n o s a a n t i g e n s f o r p r e s e n t a t i o n t o the a p p r o p r i a t e subsets o f T-lymphocytes, i n i t i a t i o n o f the c e l l u l a r and humoral immune responses.  allowing  95  Fibronectin activates macrophages  Activated macrophage (active receptors (A) can now bind to surface-expressed pili and promote uptake)  Non-activated macrophage (receptors are inactive (-») and cannot bind to bacterial surface pili)  F i g u r e 20.  A c o n c e p t u a l model o f f i b r o n e c t i n - a c t i v a t e d macrophage  non-opsonic  p h a g o c y t o s i s o f P. a e r u g i n o s a .  i n a c c e s s i b l e , i n a c t i v e o r absent cannot  Pseudomonas p i l i  bind t o the b a c t e r i a l surface.  and t h e a c t i v e Pseudomonas p i l i  N o n - a c t i v a t e d macrophages have r e c e p t o r s and thus  F i b r o n e c t i n a c t i v a t e s macrophages  r e c e p t o r s can now mediate attachment o f  the phagocyte and P. a e r u g i n o s a v i a s u r f a c e - e x p r e s s e d p i l i .  Agitated  P. a e r u g i n o s a would have reduced  association  of  quantities of p i l i  b a c t e r i a and macrophage would be reduced.  and thus  96  LITERATURE CITED  1.  Aduan, R.P., and H.Y. Reynolds. 1979. I n "Pseudomonas a e r u g i n o s a : c l i n i c a l m a n i f e s t a t i o n s o f i n f e c t i o n and c u r r e n t t h e r a p y . " (Doggett, R.G., ed.) pp. 135-156. 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