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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 University of 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 i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1988 ©Janet Louise 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) i i ABSTRACT The i n t e r a c t i o n s of macrophages with Pseudomonas aeruginosa were studied. Five monoclonal antibodies s p e c i f i c f o r porin protein F were tested f o r t h e i r a b i l i t y to opsonize P. aeruginosa f o r complement-independent phagocytosis by u n e l i c i t e d mouse peritoneal macrophages, human peripheral blood monocytes and mouse macrophage c e l l l i n e P388 D 1. A l l f i v e antibodies s i g n i f i c a n t l y increased the l e v e l of b a c t e r i a l uptake over that obtained with the non-opsonic c o n t r o l s . The r e l a t i v e effectiveness of the d i f f e r e n t antibodies was approximately the same i n a l l c e l l types i n d i c a t i n g that the P388 D 1 c e l l s can be used as a model f o r normal macrophages. Of the four monoclonal antibodies d i r e c t e d against s i m i l a r epitopes of protein F, the three IgGl monoclonal antibodies were s u b s t a n t i a l l y more opsonic than the one IgG2a isotype. P. aeruginosa cytotoxin and periplasmic contents caused a s i g n i f i c a n t reduction i n antibody-mediated phagocytosis of P. aeruginosa. Phagocytosis was restored upon pre-incubation with a n t i - c y t o t o x i n serum. Both cytotoxin and periplasmic contents caused d e p o l a r i z a t i o n of the P388 D 1 c e l l membrane, as demonstrated using a p o l a r i z a t i o n - s e n s i t i v e fluorescent probe. These data indicated that P. aeruginosa cytotoxin was l o c a l i z e d i n the periplasm and had the p o t e n t i a l to i n h i b i t macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane. i i i Monoclonal antibodies d i r e c t e d against protein F were also capable of enhancing phagocytosis of i n vivo-grown P. aeruginosa. P. aeruginosa c e l l s taken d i r e c t l y from the i n vivo growth system were s i g n i f i c a n t l y more susceptible to macrophage phagocytosis than were the same c e l l s a f t e r being washed i n buffer. The phagocytosis-promoting f a c t o r could be i s o l a t e d from the supernatant of i n vivo-grown b a c t e r i a and was determined to be f i b r o n e c t i n . Data indicated that promotion by f i b r o n e c t i n of non-opsonic phagocytosis was mediated by d i r e c t a c t i v a t i o n of the macrophages. The tetrapeptide arginine-glycine-aspartate-serine i n the eukaryotic c e l l binding domain of f i b r o n e c t i n was demonstrated to be the macrophage-activating region. Phagocytosis of a mutant P. aeruginosa s t r a i n l a c k i n g surface p i l i could not be enhanced by f i b r o n e c t i n . Furthermore, exogenously added Pseudomonas p i l i was capable of abrogating the enhanced phagocytosis of the wild type s t r a i n observed with f i b r o n e c t i n - a c t i v a t e d macrophages. I t was concluded that Pseudomonas p i l i were the b a c t e r i a l ligands required f o r attachment to f i b r o n e c t i n -activated macrophages i n the i n i t i a l stages of non-opsonic phagocytosis. i v TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i v L i s t of Tables v i i i L i s t of Figures x ACKNOWLEDGMENTS x i i DEDICATION x i i i INTRODUCTION 1 1. Medical importance of Pseudomonas aeruginosa 1 2. Macrophage functions 1 3. Role of macrophages i n P. aeruginosa i n f e c t i o n s 2 4. Mechanism of phagocytosis 4 5. Pseudomonas products which i n h i b i t phagocytosis 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 of t h i s study 9 MATERIALS AND METHODS 1 1 1. B a c t e r i a 1 1 A. B a c t e r i a l s t r a i n s 11 B. Media and i n v i t r o growth conditions 11 C. B a c t e r i a l growth i n vivo 12 D. Preparation of s u b - c e l l u l a r f r a c t i o n s 13 V TABLE OP CONTENTS PAGE E. Sodium dodecyl sulphate (SDS)-polyacrylamide 14 gel electrophoresis ( i ) Protein s t a i n i n g 14 ( i i ) Western immunoblotting 15 F. Protein and 2-keto-3-deoxyoctonate 15 (KDO) assays 2. Macrophages 15 A. Macrophage c e l l l i n e s 15 B. U n e l i c i t e d mouse peritoneal macrophages 16 C. Human peripheral blood monocyte-derived macrophages .. 16 3. Antibody preparation 17 A. A n t i - p r o t e i n F monoclonal antibodies 17 B. Polyclonal sera • 17 ( i ) Anti-cytotoxin serum 17 ( i i ) Anti-exoenzyme S serum 18 ( i i i ) A n t i - f i b r o n e c t i n serum 18 4. Phagocytosis assay I 8 A. Conditions I 8 B. I n h i b i t o r s / a c t i v a t o r s 19 ( i ) Chapter One 19 ( i i ) Chapter Two 19 ( i i i ) Chapter Three 20 5. Fluorescence assay 20 v i TABLE OF CONTENTS PAGE RESULTS 22 Chapter I - Opsonic phagocytosis .of Pseudomonas aeruginosa 22 by macrophages and macrophage c e l l l i n e s 1. Choice of an appropriate model c e l l l i n e 23 2. Establishment of phagocytosis assay conditions 24 3. Opsonic phagcytosis: Influence of antibody subclass 27 4. Summary 30 Chapter II - P. aeruginosa cytotoxin: 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 of macrophages 1. C e l l u l a r 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 of phagocytosis 36 3. Mechanism of macrophage i n a c t i v a t i o n 40 4. Summary 43 Chapter I I I - Fibronectin-mediated a c t i v a t i o n of non-opsonic 46 phagocytosis of P. aeruginosa 1. E f f e c t of i n vivo growth on phagocytosis 46 2. Enhancement of phagocytosis by i n vivo supernatant 49 3. Characterization of the phagocytosis-promoting f a c t o r 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 of macrophages by f i b r o n e c t i n 63 6. Determination of the act i v e domain of f i b r o n e c t i n 67 7. Mechanism of fibronectin-mediated macrophage a c t i v a t i o n .. 69 v i i TABLE OF CONTENTS PAGE 8. E f f e c t of growth conditions on fibronectin-mediated 71 non-opsonic macrophage phagocytosis 9- Determination of the b a c t e r i a l ligand f o r 73 non-opsonic phagocytosis 10. Other b a c t e r i a 75 11. Summary 75 DISCUSSION 82 1. Use of the P388 D^ macrophage c e l l l i n e as a model 82 f o r macrophage studies 2. Role of isotype i n opsonized phagocytosis 82 by macrophages 3. Cytotoxin: l o c a l i z a t i o n and putative r o l e i n i n f e c t i o n ... 84 4. Fibronectin as an a c t i v a t o r of macrophage non-opsonic 88 phagocytosis LITERATURE CITED 96 LIST OF TABLES v i i i PAGE TABLE I Enhancement of as s o c i a t i o n of P. aeruginosa s t r a i n 29 M2 with mouse peritoneal macrophages, P388pi c e l l s , and human peripheral blood monocytes using monoclonal antibodies d i r e c t e d against protein F. II E f f e c t of an t i - c y t o t o x i n and anti-exoenzyme S sera 39 on i n h i b i t i o n of opsonized phagocytosis of P. aeruginosa s t r a i n M2. I l l Depolarization of the P388 D 1 plasma membrane 42 by cytotoxin, valinomycin and osmotic shockate assessed using the fluorescentnt probe diSC3(5). IV Enhancement of the as s o c i a t i o n of i n vivo-grown 51 P. aeruginosa 3 t r a i n M2 with P388 ui c e l l s using supernatant from mouse chambers. V S t a b i l i t y of the phagocytosis-enhancing f a c t o r 52 of i n vivo chamber supernatant. VI Enhancement of the as s o c i a t i o n of P. aeruginosa 53 st r a i n s M2 and H103 with u n e l i c i t e d mouse peritoneal macrophages and the P388j)i c e l l l i n e using i n vivo supernatant from rat chambers. VII In v i t r o growth conditions a f f e c t the a b i l i t y of 55 i n vivo supernatant to enhance as s o c i a t i o n of P. aeruginosa H103 with P388 D 1 c e l l s . VIII Enhancement of the as s o c i a t i o n of P. aeruginosa 58 s t r a i n H103 with P388 Di c e l l s using pooled f r a c t i o n s c o l l e c t e d from an FPLC gel si e v i n g f r a c t i o n a t i o n of i n vivo supernatant from rat chambers. IX 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 phagocytosis- 62 promoting a c t i v i t y of i n vivo supernatant from rat peritoneal chambers and bovine f i b r o n e c t i n . X Time course of emergence of phagocytosis-promoting 64 a c t i v i t y i n rat and mouse H103 and s a l i n e chambers. XI E f f e c t of washing on fibronectin-mediated enhancement 68 of non-opsonic macrophage phagocytosis of P. aeruginosa s t r a i n H103. i x LIST OF TABLES PAGE TABLE XII E f f e c t of a g i t a t i o n during growth on the s u s c e p t i b i l i t y .... 72 of P. aeruginosa s t r a i n H103 to fibronectin-mediated macrophage non-opsonic phagocytosis. XIII Fibronectin-mediated macrophage phagocytosis of 74 P. aeruginosa s t r a i n s H103 (wild type), BLP3 ( p i l i n minus), PAOl-leu ( p i l i n p o s i t i v e ) , and pBPl6l ( p i l i n p o s i t i v e ) . XIV I n h i b i t i o n of fibronectin-mediated macrophage 76 non-opsonic uptake of P. aeruginosa s t r a i n H103 by exogenous PA01 p i l i . X LIST OF FIGURES PAGE FIGURE 1 Opsonized phagocytosis of P. aeruginosa s t r a i n M2 25 by three mouse macrophage c e l l l i n e s , mouse peritoneal macrophages and human peripheral blood monocyte-derived macrophages. 2 Time course of P. aeruginosa a s s o c i a t i o n with P388px 26 i n the presence or absence of ant i - F monoclonal antibody MA2-10. 3 Percent of macrophages associated with s p e c i f i c 28 numbers of b a c t e r i a a f t e r 90 min. 4 SDS-polyacrylamide g e l of cytotoxin and P. aeruginosa 34 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 . 5 Western immunoblot of cytotoxin and P. aeruginosa 35 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 with rabbit a n t i - c y t o t o x i n serum. 6 Western immunoblot of cytotoxin and P. aeruginosa 37 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 with rabbit anti-exoenzyme S serum. 7 Changes i n diSC 3(5) fluorescence a f t e r addition of 41 phosphate-buffered s a l i n e , cytotoxin or valinomycin to diSC 3(5) e q u i l i b r a t e d P388 D 1 c e l l s . 8 E f f e c t of increasing concentrations of cytotoxin or 44 osmotic shockate on the rate of increase of diSC3(5) fluorescence. 9 Description of the i n vivo growth model 47 10 Opsonized phagocytosis of i n vivo- and i n vitro-grown 48 P. aeruginosa s t r a i n M2 by mouse macrophage c e l l l i n e P388 Di. 11 FPLC gel sie v i n g f r a c t i o n a t i o n of i n vivo supernatant 57 from rat chambers - e l u t i o n p r o f i l e at an absorbance of 230 nm. LIST OF FIGURES x i PAGE FIGURE 12 Western immune-blots of f r a c t i o n s c o l l e c t e d from 60 an FPLC g e l si e v i n g f r a c t i o n a t i o n of i n vivo supernatant from rat chambers probed with goat anti-human f i b r o n e c t i n serum. 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 of i n vivo supernatant 61 from mouse chambers - e l u t i o n p r o f i l e at an absorbance of 230 nm. 14 Western immunoblots of the supernatant of b a c t e r i a - 65 and saline-containing chambers that had been incubated i n the peritoneum of mice and rats f o r 4, 24 and 48 hours probed with goat anti-human f i b r o n e c t i n serum. 15 E f f e c t of increasing concentrations of f i b r o n e c t i n 66 and RGDS on the l e v e l of uptake of P. aeruginosa s t r a i n H103 by macrophage c e l l l i n e P388])i-16 E f f e c t of increasing concentrations of f i b r o n e c t i n 70 on the rate of increase of diSC3(5) fluorescence. 17 Enhancement of the a s s o c i a t i o n of E. c o l i with 77 P388ni c e l l s using supernatant from i n vivo-grown E. c o l i . 18 Enhancement of the ass o c i a t i o n of S. aureus with 78 P388uT_ c e l l s using supernatant from i n vivo-grown S. aureus. 19 Western immunoblot of the supernatants of E. c o l i - 79 and S. aureus-containing chambers probed with goat anti-human f i b r o n e c t i n . 20 A conceptual model of f i b r o n e c t i n - a c t i v a t e d 95 macrophage non-opsonic phagocytosis of P. aeruginosa. x i i ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. Bob Hancock, f o r his excellent advice, understanding, and f i n a n c i a l support during the course of my studies. I am s i n c e r e l y g r a t e f u l to Dr. David Speert and Dr. Trevor Trust, who permitted me to work i n t h e i r laboratories f o r extended periods of time. I would l i k e to thank the members of the Biochemistry/ Microbiology Department at the University of V i c t o r i a , the Microbiology Department at the Univ e r s i t y of B r i t i s h Columbia, and the members of my supervisory committee f o r t h e i r help and advice. I am e s p e c i a l l y g r a t e f u l to Dr. Niamh K e l l y who generously donated her time and e f f o r t to the co l l a b o r a t i v e study that led to the data presented i n Chapter Three of t h i s t h e s i s . In p a r t i c u l a r , I wish to thank a l l the members of the Hancock, Tru3t and Speert l a b o r a t o r i e s , who offered me invaluable f r i e n d s h i p , support and guidance during my stay. A s p e c i a l thanks to Rosario Bauzon f o r her excellent typing of t h i s t h e s i s . x i i i DEDICATION This thesis i s dedicated to my parents, Jim and Peggy B a t t e r s h i l l , who have always supported my aspirations and endeavours, and to my husband, Andy, whose constant f r i e n d s h i p , love and support are so very important to me. 1 INTRODUCTION 1. Medical Importance of Pseudomonas aeruginosa Pseudomonas aeruginosa i s an opportunistic pathogen, often causing 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 are e s p e c i a l l y at r i s k include those with severe burns, cancer, diabetes and c y s t i c f i b r o s i s . Indeed, P. aeruginosa remains one of the predominant causes of death from gram negative septicemia i n North America, and the most common bacterium associated with terminal lung disease i n patients with c y s t i c f i b r o s i s (Speert, 1985). The i n t r i n s i c resistance of P. aeruginosa to a n t i b i o t i c treatment contributes s u b s t a n t i a l l y to the high mortality rates observed. P. aeruginosa i s a gram negative b a c t e r i a and thus has two c e l l envelope membranes: the cytoplasmic (or inner) membrane and the outer t membrane. These are separated by a sin g l e sheet of peptidoglycan. The outer membrane i s p a r t i c u l a r l y important i n Pseudomonas i n f e c t i o n s as i t plays a r o l e i n a n t i b i o t i c resistance and contains endotoxic lipopolysaccharide (LPS) and protein antigens. 2. Macrophage Functions Macrophages are often the f i r s t c e l l s to i n f i l t r a t e a s i t e of i n f e c t i o n (Dunn et a l . , 1985). In addition to t h e i r r o l e i n b a c t e r i a l uptake, they play an important part i n amplifying the host immune response. Through t h e i r release of potent 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 procoagulant, pyrogen, in t e r f e r o n , elastase, and plasminogen a c t i v a t o r , inflammation and wound healing occur at an accelerated rate (Schaffner et a l . . 1982). Many of the secreted products of macrophages have functional importance as chemoattractants f o r recruitment of other immune c e l l s into the s i t e of i n f e c t i o n (Schaffner et a l . , 1982). Furthermore, antigen presentation by macrophages i s an important event i n the functioning of T c e l l populations i n both the humoral and c e l l u l a r immune response. In p a r t i c u l a r , macrophages bearing major h i s t o c o m p a t i b i l i t y antigen molecules on t h e i r c e l l surface are highly e f f i c i e n t i n educating an t i g e n - s p e c i f i c T c e l l s . This a c t i v a t i o n of T lymphocytes appears to be dependent on the macrophage secretory product i n t e r l e u k i n 1 (Schaffner et a l . , 1982). Macrophages ingesting immunoglobulin-coated antigens are capable of processing the immunoglobulin Fc fragments into subfragments and secreting these back into the environment. These subfragments are d i r e c t l y mitogenic f o r B c e l l s and have a potent adjuvant e f f e c t on T lymphocytes (Schaffner et a l . , 1982). 3. Role of Macrophages i n P. aeruginosa Infections Macrophages represent a primary l i n e of defense against i n f e c t i o n i n deep tissues such as the lung (Green and Kass, 1964) and the peritoneal c a v i t y (Dunn et a l . , 1985). In the presence of serum opsonins such as antibody and complement, P. aeruginosa can be e f f i c i e n t l y phagocytosed by normal macrophage populations (Reynolds et a l . , 1975). In a recent study, (Mutharia and Hancock, 1983), a series of 3 monoclonal antibodies were rai s e d against protein antigens common to a l l 17 P. aeruginosa serotypes. Monoclonal antibodies d i r e c t e d against one of these outer membrane proteins (protein F) were pro t e c t i v e i n a mouse i n f e c t i o n model and were opsonic f o r human polymorphonuclear c e l l phagocytosis (Hancock et a l . , 1985). Antibodies against protein F thus appear to have p o t e n t i a l as passive vaccines i n animal systems. These data suggested that monoclonal antibodies are acting to opsonize P. aeruginosa f o r phagocytosis i n the mouse i n f e c t i o n model. Both polymorphonuclear leukocytes and c e l l s of the macrophage lineage are capable of ingesting P. aeruginosa (Young and Armstrong, 1972; Reynolds et a l . , 1975). While polymorphonuclear leukocytes are no doubt important i n b a c t e r i a l clearance, 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 functioning immune system. Indeed, the r e s u l t s of two independent studies suggested that the phagocytic action of macrophages i s the major mechanism of eliminating b a c t e r i a i n the i n i t i a l stages of i n f e c t i o n (Green and Kass, 1964; Dunn et a l . , 1985). Although polymorphonuclear leukocytes are capable of s i m i l a r phagocytic clearance, t h e i r absence i n the i n c i p i e n t stages of i n f e c t i o n eliminates them as a p o t e n t i a l f i r s t l i n e of host defense. In the case of some patients with c y s t i c f i b r o s i s , i n i t i a l P. aeruginosa i n f e c t i o n s can be quickly suppressed a f t e r a n t i b i o t i c treatment (Friend, 1986). This may involve not only a n t i b i o t i c action, but also non-immune clearance mechanisms of the host. These l a t t e r mechanisms apparently encompass both the mucociliary system and non-opsonic phagocytosis by pulmonary a l v e o l a r macrophages (Speert, 4 1985). On repeated i n f e c t i o n with P. aeruginosa, lung function of an 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 r a p i d l y deteriorates and b a c t e r i a can no longer be eradicated from the area. Thus, the i n a b i l i t y to c l e a r P. aeruginosa may in d i c a t e , i n part, a defect i n phagocyte uptake and k i l l i n g . 4. Mechanism of Phagocytosis There are three basic steps i n macrophage phagocytosis, (described here with reference to phagocytosis of p a r t i c l e s ) . These are p a r t i c l e attachment, generation and transmission of the phagocytic s i g n a l , and p a r t i c l e ingestion. The f i r s t step involves apposition of the macrophage plasma membrane to the p a r t i c l e surface. This event occurs i n the absence of macrophage c e l l u l a r metabolism and i s l a r g e l y dependent on surface properties of the p a r t i c l e and c e l l . The e f f i c i e n c y of attachment increases several f o l d i f the material i n question i s opsonized with antibody or complement. This i s due to the f a c t that macrophages have at lea s t two classes of surface receptors, the Fc receptors and the C3 receptor. Mouse macrophages and macrophage-like c e l l l i n e s appear to have three d i s t i n c t Fc receptors (Green et a l . , 1985). One bind3 monomeric and aggregated IgG2a, another binds aggregates of IgGl or IgG2b, and the l a s t binds IgG3. The C3 receptor recognizes C3b, the C3 convertase cleavage product of complement component C3- Thus, antigens complexed to IgG and/or C3b bind a v i d l y to macrophages. Non-opsonic phagocytosis involves a s s o c i a t i o n of phagocytes and b a c t e r i a i n the absence of external opsonins (Speert et a l . , 1984). This 5 mode of phagocytosis has been considered to be a r e l a t i v e l y i n e f f i c i e n t process (Aduan and Reynolds, 1979), and thus r e l a t i v e l y unimportant to the clearance of t h i s organism. In conjunction with, or following, antigen recognition and binding, a s i g n a l must be passed to the i n t e r i o r of the c e l l to i n i t i a t e p a r t i c l e ingestion. At present, the existence of a phagocytic s i g n a l i s b a s i c a l l y hypothetical but several theories have been put forward as to p o t e n t i a l components of the s i g n a l (Young, 1985). Receptor-ligand i n t e r a c t i o n has been shown to r e s u l t 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 concentration (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 of the Fc receptor cation channel (Young et a l . , 1983a;1983b; 1984), and a l t e r a t i o n i n e l e c t r i c a l properties of the c e l l (McCann et a l . . 1983; G a l l i n , 1981; Young, 1983c). The l o c a l calcium gradient set up by receptor-ligand binding would determine the p h y s i c a l state of a c t i n v i a g e l s o l i n (Stossel et a l . , 1981), a calcium-activated protein which severs bonds between a c t i n and actin-binding p r o t e i n to e f f e c t a g e l - s o l transformation. This i n turn could regulate the flow of pseudopods around the p a r t i c l e f o r ingestion. This presents a reasonable mechanism by which a c t i n and calcium-dependent a c t i n regulatory proteins could provide the dynamic force f o r phagocytosis. The increase i n i n t e r n a l calcium concentration i s thought to be a r e s u l t of calcium transport into the c e l l and release of calcium from i n t r a c e l l u l a r stores (Young et a l . , 1984). Candidates f o r the i n t e r n a l store include membrane bound calcium (Hoffstein, 1979) and an i n t r a c e l l u l a r p rotein which becomes phosphorylated to release calcium on 6 phagocytic stimulus (Vaux et a l . , 1982). The IgGl/2b Fc receptor of murine macrophages has been shown to be a non-selective ligand-dependent cation channel with high permeability to sodium and potassium and low permeability f o r calcium (Young et a l . , 1983a). This channel could be at le a s t i n part responsible f o r the a l t e r a t i o n i n e l e c t r i c a l properties of the macrophage c e l l ( i . e . membrane hyperpolarization/depolarization). In addition, the Na +K + ATPase pump (Young, 1983c), the Na +Ca + + exchange mechanism (Braquet et a l . , 1985) and C a + + - activated K + conductances (Young, 1983c) have been seen to set up gradients on macrophage stimulation. Macrophages are 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 action p o t e n t i a l s (McCann et a l . , 1983; G a l l i n , 1981). Friedhoff (1983) hypothesized that t h i s movement of ions through s p a t i a l l y separate pumps or channels would produce an e l e c t r i c a l f i e l d thus inducing f l u i d and membrane movement. These membrane order changes have been observed i n response to several phagocytic s t i m u l i (Esfahani et a l . , 1982; Horvath et a l . , 1982; Sandor et a l . , 1981; Larsen et a l . , 1985). Aggregation of p a r t i c l e s on the surface of the macrophage following recognition and binding i s believed to be e s s e n t i a l f o r subsequent i n t e r n a l i z a t i o n ( L e s l i e , 1985; Dower et a l . , 1981; Sandor et a l . , 1981; Petty, 1985). In f a c t , the sulfhydryl-redox model of antibody dependent phagocytosis hypothesizes that d i s u l f i d e l i n k s formed between occupied receptors provides the "zippering" mechanism of phagocytosis (Petty, 1985). 7 5. Pseudomonas Products Which I n h i b i t Phagocytosis One of the ways i n which P. aeruginosa may protect i t s e l f from the host defense system i s through production of phagocytosis-inhibiting toxins (Speert, 1985). It has long been established that s t r a i n s of P. aeruginosa c o l o n i z i n g i n d i v i d u a l s with c y s t i c f i b r o s i s are predominantly mucoid i n phenotype (Doggett, 1969). These organisms produce large amounts of mucoid exopolysaccharide and are more r e s i s t a n t to phagocytosis than t h e i r non-mucoid counterparts (Baltimore and M i t c h e l l , 1980). This resistance has been a t t r i b u t e d to both a d i r e c t t o x i c e f f e c t of mucoid exopolysaccharide on macrophage phagocytosis ( O l i v e r and Weir, 1983) and masking by the mucoid coating of b a c t e r i a l ligands required f o r phagocytosis (Baltimore and M i t c h e l l , 1980). The most t o x i c product of P. aeruginosa appears to be exotoxin A (Woods and Iglewski, 1983). This e x t r a c e l l u l a r l y secreted toxi n i s an e f f i c i e n t i n h i b i t o r of protein synthesis (Iglewski and Kabat, 1975) and has been proven to s i g n i f i c a n t l y reduce macrophage phagocytosis at low concentrations (Pollack and Anderson, 1978). Both leukocidin (cytotoxin; molecular weight 27,000 daltons) and polymorphonuclear leukocyte (PMN) i n h i b i t o r (molecular weight 65,000 daltons) have been observed to compromise phagocytosis by PMN c e l l s (Scharmann et a l . , 1976; Nonoyama et a l . , 1979). I t has not been determined i f e i t h e r of these two proteins are capable of i n h i b i t i n g b a c t e r i a l uptake by macrophages. Previous studies have indicated that 38% of P. aeruginosa s t r a i n s also produce exoenzyme S (Sokol et a l . , 1981), hypothesized to be a protein 8 synthesis i n h i b i t o r (Iglewski et a l . , 1978). Other secreted products such as a l k a l i n e protease, elastase, and phospholipase are believed to be important f a c t o r s causing the l o c a l t i s s u e destruction which precedes invasion of the bloodstream (Sanai et a l . , 1978). 6. Macrophage A c t i v a t i o n Numerous host factors are capable of stimulating macrophages. The r e s u l t s of such stimulation may take the form of increased adherence, receptor-mediated phagocytosis, chemotaxis, b a c t e r i c i d a l a c t i v i t y , or secretion of b i o l o g i c a l l y a c t i v e molecules (Schaffner et a l . , 1982). The mechanism of a c t i v a t i o n appears to involve changes i n e l e c t r o s t a t i c surface charges, a l t e r a t i o n s i n transmembrane p o t e n t i a l ( d e p o l a r i z a t i o n ) , 2+ and s h i f t s i n Ca gradients (Schaffner et a l . , 1982). The macrophage c e l l functions that mediate such a c t i v a t i o n involve dynamic and f l e e t i n g molecular i n t e r a c t i o n s . Thus, the f u n c t i o n a l behavior of the macrophage membrane i s probably dependent on events taking place on timescales ranging from picoseconds to seconds (Lakowicz, 1980). Use of fluorescence spectroscopy of fluorescent molecules inserted into macrophage membranes permits continuous observation of the dynamic i n i t i a l events of a c t i v a t i o n and i n h i b i t i o n of macrophage function. Some of the macrophage a c t i v a t i n g f a ctors studied to date are 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 , prostaglandins, and f i b r o n e c t i n (Schaffner et a l . , 1982; Hogg, 1986; Braquet et a l . , 1985; Wright et a l . , 1983). The a d a p t a b i l i t y of macrophage populations to such 9 s t i m u l i i s a c r u c i a l mechanism of upgrading the host immune response to i n f e c t i o n (Schaffner et a l . , 1982). 7. Fibronectin Fibronectin i s a large dimeric glycoprotein (molecular weight 440,000 daltons) which has s p e c i f i c binding s i t e s f o r mammalian c e l l s , v i r a l glycoproteins, and b a c t e r i a l surfaces (Proctor, 1987). Fibronectin has several b i o l o g i c a l functions including c e l l - t o - c e l l attachment, c l o t s t a b i l i z a t i o n , c e l l d i f f e r e n t i a t i o n and wound healing. The f i b r o n e c t i n network at a s i t e of i n j u r y provides the s c a f f o l d on which the components of connective t i s s u e required f o r wound healing can be deposited (Proctor, 1987). In addition, f i b r o n e c t i n has been shown to a c t i v a t e macrophages f o r increased adherence (Akiyama et a l . , 1981), C3- and Fc-receptor-mediated phagocytosis of coated erythrocytes (Wright et a l . , 1983; Pommier et a l . , 1983), and maintenance of anti-staphylococcal a c t i v i t y (Proctor et a l . , 1985). I t has been hypothesized to act as an opsonin i n the promotion of Staphylococcal uptake (Proctor et a l . , 1982). 8. S p e c i f i c Aims of This Study The objective of t h i s i n v e s t i g a t i o n was to examine the i n t e r a c t i o n of macrophages with P. aeruginosa. To a s s i s t i n t h i s aim, a v i s u a l assay of phagocytosis was established and determination of an appropriate macrophage c e l l l i n e f o r experimentation was made. This assay was used to inv e s t i g a t e the opsonic p o t e n t i a l of Pseudomonas outer membrane protein F - s p e c i f i c monoclonal antibodies with respect to timecourse of b a c t e r i a l 10 uptake and influence of antibody subclass. One of the ways i n which P. aeruginosa may protect i t s e l f from such basic host defenses as opsonized phagocytosis i s v i a production of cytotoxin. This 27,000 dalton a c i d i c p rotein probably acts by forming pores i n the membrane of target c e l l s of the immune system (Scharmann, 1976). This appears to r e s u l t i n increased membrane permeability to small molecules and ions (Baltch et a l . , 1985). Such i n t o x i c a t i o n has been documented i n granulocytes (Baltch et a l . , 1985), endothelial c e l l s (Suttorp et a l . , 1985), and E h r l i c h a s c i t e s tumor c e l l s (Lutz et a l . , 1987). Two of the aims of t h i s study were to determine the c e l l u l a r l o c a l i z a t i o n of cytotoxin and to investigate i t s e f f e c t on macrophage phagocytic function. To further our i n v e s t i g a t i o n of the opsonic p o t e n t i a l of a n t i - p r o t e i n F monoclonal antibodies, the e f f e c t of i n vivo growth of P. aeruginosa on opsonic and non-opsonic phagocytosis was determined. These studies led to the discovery that P. aeruginosa c e l l s taken d i r e c t l y from an i n vivo system were s i g n i f i c a n t l y more susceptible to macrophage phagocytosis than were the same c e l l s a f t e r being washed i n buffer. The phagocytosis-promoting f a c t o r could be i s o l a t e d from the supernatant of centrifuged i n vivo-grown b a c t e r i a ( i n vivo supernatant) and by f r a c t i o n a t i o n of t h i s supernatant was determined to be f i b r o n e c t i n . The objectives of t h i s study were to determine the r o l e of f i b r o n e c t i n i n promotion of phagocytosis and to investigate the b a c t e r i a l ligand required f o r fibronectin-mediated uptake. 11 MATERIALS AND METHODS 1. Bacter i a A. B a c t e r i a l s t r a i n s . Pseudomonas aeruginosa 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 pathogenicity studies ( S t i e r i t z and Holder, 1975) were used as reference s t r a i n s throughout t h i s t h e s i s . Other P. aeruginosa s t r a i n s used were BLP3, a Tn501-induced pilus-minus mutant; pBPl6l, a pilus-expressing s t r a i n containing both the Tn501 mutation and an a d d i t i o n a l plasmid-encoded p i l i n gene; and PAOl-leu, the PA01 parental s t r a i n of these two mutants. A l l three of these s t r a i n s were kind g i f t s from B r i t t a i n Paslowske (University of Alberta, Edmonton, Al b e r t a ) . Cytotoxin was i s o l a t e d from P. aeruginosa s t r a i n 158 by Frieder Lutz (Justus-Liebig University, Giessen, Federal Republic of Germany) and was kindly donated f o r use i n these studies. E s c h e r i c h i a c o l i s t r a i n C600 (Applegard, 1954) was used i n control experiments f o r the cytotoxin studies of Chapter Two. E. c o l i s t r a i n 8239 and Staphylococcus aureus s t r a i n 8529 were c l i n i c a l i s o l a t e s kindly donated by Anthony Chow (University of B r i t i s h Columbia, Vancouver, B.C.). B. Media and i n v i t r o growth conditions. P. aeruginosa s t r a i n s H103, M2 and PAOl-leu were maintained on Trypticase Soy agar (Becton, Dickinson & Co., Cockeysville, MD) or 1% (wt/vol) Proteose Peptone no. 2 agar (Difco Laboratories, De t r o i t , MI). BLP3 and pBPl6l were maintained on Trypticase Soy agar containing 300 ug/ml c a r b e n i c i l l i n . E s c h e r i c h i a c o l i s t r a i n s 12 C600 and 8239, and S. aureus s t r a i n 8529 were maintained on 1% Proteose Peptone no. 2 agar. P r i o r to assay, c e l l s were inoculated from these plates and grown f o r 20 h at 37°C e i t h e r with vigorous shaking (200 rpm) or s t a t i c a l l y (without shaking) i n broth or on plates. Basal medium 2 (BM2)-glucose broth ( G i l l e l a n d et a l . , 1974) was used f o r the experiments of Chapters One and Two, while Trypticase Soy media was used f o r the studies presented i n Chapter Three. Stationary phase c e l l s were washed i n phosphate buffered s a l i n e (PBS), pH 7.2, and resuspended to a a concentration of 1x10 /ml p r i o r to experimentation. C. B a c t e r i a l growth i n vivo. B a c t e r i a l growth i n vivo was kindly performed by Dr. Niamh K e l l y i n our laboratory. B r i e f l y , chambers f o r implantation into mice were constructed from 1 ml polypropylene syringe b a r r e l s as previously described (Day et a l . , 1980). Chambers f o r implantation into rats were s i m i l a r l y constructed from 3 ml polypropylene syringe b a r r e l s ( K e l l y et a l . , 1988). The P. aeruginosa culture used to inoculate the chambers was grown overnight i n Proteose Peptone no. 2 broth 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 to give a chamber inoculum of approximately 10^ b a c t e r i a per ml. Chambers f o r implantation into mice received a volume of 100 v l of the d i l u t e d culture, while chambers f o r rats received 500 u l . The animals were anaesthesized by i n t r a p e r i t o n e a l i n j e c t i o n of sodium pentobarbitol (Somnotol, M.T.C. Pharmaceuticals, Mississauga, Ontario, Canada) at 0.06 mg/g of body weight f o r mice and 0.14 mg/g f o r r a t s . Chambers were implanted, four per animal, through a small l o n g i t u d i n a l i n c i s i o n i n the abdomen of the animal. The chambers 13 were removed a f t e r three days at which stage the b a c t e r i a l cultures had 8 9 reached t h e i r maximal density of 10 - 10 c e l l s / m l ( 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 Petroff-Hausser b a c t e r i a l counting chamber (Hausser S c i e n t i f i c , Blue B e l l , PA) and maintained on i c e u n t i l use. Washed organisms were centrifuged at 12,000 x g and gently resuspended i n PBS twice p r i o r to assay. The f i r s t decanted supernatant from these c e l l s was saved f o r assessment of phagocytosis enhancement ( i n vivo supernatant). To characterize the phagocytosis-promoting f a c t o r , i n vivo supernatant was passed over a Fast Pressure L i q u i d Chromatography (FPLC) Superose 12 ge l s i e v i n g column (Pharmacia, Dorval, Quebec). The flow rate was 30 ml/h and e l u t i o n buffer consisted of 20 mM T r i s (ICN Biomedicals, Cleveland, Ohio), 0.1M NaCl (BDH Chemicals, Toronto, Ontario), pH 7-5. Fractions were c o l l e c t e d , l y o p h i l i z e d , resuspended i n d i s t i l l e d water and dialyzed extensively against d i s t i l l e d water. The f i n a l resuspension volume was the same as the volume of supernatant i n i t i a l l y added to 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. aeruginosa cytotoxin 2+ (Lutz, 1979), osmotic shock f l u i d , prepared by the Mg /freeze-thaw method (Hoshino and Kageyama, 1980; Poole and Hancock, 1984), and inner and outer membranes (Hancock and Nikaido, 1978) were prepared as described previously. These preparations contained, res p e c t i v e l y <0.01, 0.15, 0.24 and 0.43 ug LPS per ug protein. Cytotoxin was obtained from s t r a i n 158, while osmotic shock f l u i d s and membranes were prepared from s t r a i n 14 H103. E. c o l i osmotic shockate was i s o l a t e d from s t r a i n C600 by the method of Neu and Heppel (1965). Growth supernatant samples were prepared from P. aeruginosa s t r a i n H103 c e l l s grown f o r 20 h at 37°C i n shaken BM2-glucose broth (200 rpm). Growth supernatant was decanted a f t e r removal of c e l l s by c e n t r i f u g a t i o n at 13,000 x g f o r 10 min at 4°C. The supernatant was l y o p h i l i z e d and resuspended i n 10 mM T r i s - H C l , pH 7.4 at a 150-fold higher than o r i g i n a l concentration. Extensive d i a l y s i s was performed against 10 mM Tr i s - H C l buffer, pH 7.4 buffer to remove low molecular weight substances. Samples were stored at -70°C u n t i l use. E. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed as described previously (Hancock and Carey, 1979). The acrylamide concentration i n the lower separating gel was 11% (wt/vol) f o r the cytotoxin studies (Chapter Two) and 5% f o r detection of f i b r o n e c t i o n (Chapter Three). Samples were s o l u b i l i z e d i n the presence of 5% (vol/vol) 2-mercaptoethanol (Bio-Rad, Mississauga, Ontario) and were heated at 100°C f o r 10 min i n the absence of SDS. For estimation of molecular weights on SDS-gels, the following molecular weight standards were used, bovine serum albumin (66.2K); ovalbumin (45K); phosphate dehydrogenase (36K); carbonic anhydrase (29K); and trypsinogen (24K). ( i ) Protein s t a i n i n g . Proteins i n SDS-PAGE were stained i n 1% (wt/vol) Coomassie b r i l l i a n t blue dissolved i n g l a c i a l a c e t i c acid:methanol:water at a r a t i o of 1:4.5:4.5. Destaining was performed i n the same s o l u t i o n (without the dye) at a 0.7:2:7-2 r a t i o . 15 ( i i ) Western immunoblotting. Proteins from SDS-polyacrylamide gels were imraunoblotted onto n i t r o c e l l u l o s e paper (0.45 um) as described previously (Mutharia and Hancock, 1983). For development, 20 ul of rabbit a n t i - c y t o t o x i n serum, 30 ul of rabbit anti-exoenzyme S serum, or 100 ug of goat anti-human f i b r o n e c t i n (Sigma, St. Louis, M0) was u t i l i z e d per b l o t . F. Protein and 2-keto-3-deoxyoctonate (KDO) assays. Protein assays were done by the method of Schacterle and Pollack (1973). Determination of lipopolysacharide (LPS) content was made by assuming that the LPS contained 4.3% (wt/wt) of KDO (Darveau and Hancock, 1983). KDO assays were performed by the method of Osborn et a l . (1972) with an a d d i t i o n a l 15 min hydrolysis step i n 50 mM H^SO^. 2. Macrophages A. Macrophage c e l l l i n e s . Three mouse macrophage c e l l l i n e s were assessed f o r t h e i r phagocytic capacity: P388 D 1 > a DBA/2 macrophage tumor c e l l l i n e (American Tissue Culture C o l l e c t i o n #CCL46); PU5-1.8, a Balb/c monocytic, macrophage-like c e l l l i n e (American Tissue Culture C o l l e c t i o n #TIB6l); and DC7, a macrophagetT c e l l f u s ion product. C e l l l i n e s were maintained at 37°C, 10% C0 2 > 80% humidity i n Nunc flat-bottomed f l a s k s (Gibco, Burlington, Ontario) using supplemented RPMI-1640 medium (Gibco). Media was supplemented with 44 mM sodium bicarbonate (Fisher 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 (Gibco), 10 mM Hepes (Terochem Laboratories, Vancouver, B.C.), 0.04% v/v 16 2-mercaptoethanol (Bio-Rad, Mississauga, Ontario), 2 mM L-glutamine (Sigma, St. Louis, MO), 40 units per ml p e n i c i l l i n and 40 mg/ml streptomycin (Gibco), pH 7.2. P r i o r to assay c e l l s were harvested by 5 gentle p i p e t t i n g and resuspended to 5x10 c e l l s / m l i n f r e s h medium. Aliquots (2 ml) of t h i s c e l l suspension were incubated i n 35x10 mm Nunclon ti s s u e culture dishes (Gibco) at 37°C i n 10% C0^. Non-adherent c e l l s were washed o f f j u s t p r i o r to assay. B. O n e l i c i t e d mouse peritoneal macrophages. Peritoneal macrophages were obtained from s i x to eight week old female Balb/c mice. C e l l s were washed from the peritoneal c a v i t y with supplemented RPMI-1640. C e l l s were 67.1% macrophages, 32.5% red blood c e l l s , and 0.4% granulocytes as determined by " D i f f - q u i k M (Canlab, Vancouver, B.C.) s t a i n i n g and v i s u a l inspection. Macrophages were separated from erythrocytes by 5 c e n t r i f u g a t i o n at 1000 RPM f o r 10 min and were resuspended to 5x10 c e l l s / m l i n supplemented RPMI-1640 (above). Aliquots (2 ml) of t h i s c e l l suspension were incubated i n 35x10 mm Nunclon t i s s u e culture dishes (Gibco) at 37°C i n 10% C0 2- A f t e r 20 h non-adherent c e l l s were removed by washing the monolayer gently with RPMI-1640. C. Human peripheral blood monocyte-derived macrophages. Monocytes were prepared from human peripheral blood using the Picoll-paque separation technique as described previously (Boyum, 1968). Monocytes were maintained i n RPMI-1640 medium supplemented with 44 mM sodium bicarbonate, 15% homologous human serum, 40 units per ml p e n i c i l l i n and 40 17 mg/ml streptomycin. C e l l s were kept i n screw cap Teflon j a r s ( S a v i l l e x , Minnetonka, MN) at 37°C, 5% CC>2 f o r approximately 96 h p r i o r to use. 5 These "Day Four" monocytes were washed and resuspended to 5x10 c e l l s / m l i n RPMI-1640 supplemented with 10 mM Hepes and 2 g/1 bovine serum albumin (Sigma). Aliquots of t h i s c e l l suspension (2 ml) were incubated i n Nunclon t i s s u e culture dishes f o r 1 h p r i o r to assay. Non-adherent c e l l s were washed o f f before s t a r t i n g phagocytosis experiments. 3. Antibody Preparation A. A n t i - p r o t e i n F monoclonal antibodies. Monoclonal antibodies were 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 sulphate p r e c i p i t a t i o n as previously described (Mutharia and Hancock, 1983). The following monoclonal antibodies described previously (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 protein F (Mutharia and Hancock, 1985); and MA1-3 s p e c i f i c f o r l i p o p r o t e i n s I/H2 (Hancock et a l . , 1982). The isotype of a l l antibodies was determined as described previously (Mutharia and Hancock, 1985) by double immunodiffusion (Ouchterlony, 1958). B. Polyclonal sera. ( i ) A nti-cytotoxin serum. Rabbit a n t i - c y t o t o x i n sera was generously prepared by F. Lutz by the method of Harboe and I n g i l d (1973) as described by Baltch et a l . (1987) and was used at 13 u l per assay. One u l of t h i s a n t i s e r a was shown to pevent 1 ug of cytotoxin from inducing the swelling of human granulocytes. The cytotoxin preparation 18 used to r a i s e the a n t i s e r a was pure as assessed by sodium dodecyl sulphate electrophoresis, immunodiffusion, and enzymatic methods f o r determining the presence of other known Pseudomonal components (Lutz et a l . , 1987). ( i i ) Anti-exoenzyme S serum. Rabbit anti-exoenzyme S serum was a generous g i f t of T h a l i a Nicas (University of Ottawa, Ottawa, Ontario). ( i i i ) A n t i - f i b r o n e c t i n serum. Goat anti-human f i b r o n e c t i n was purchased from Sigma. 4. Phagocytosis Assay A. Conditions. The v i s u a l inspection phagocytic assay u t i l i z e d was modified from that published by Speert et a l . (1984). B r i e f l y , 1 ml of RPMI-1640 medium without f e t a l c a l f serum, 2-mercaptoethanol, or a n t i b i o t i c (phagocytosis buffer) was added to a washed, cultured macrophage monolayer to give a f i n a l concentration of 1x10^ c e l l s / m l . To assess i n h i b i t i o n or a c t i v a t i o n of opsonic phagocytosis, various preparations were added to the macrophage monolayer 15 min p r i o r to add i t i o n of a n t i - p r o t e i n F monoclonal antibodies and b a c t e r i a l c e l l s . To assess i n h i b i t i o n or a c t i v a t i o n of non-opsonic uptake, preparations were added to the macrophage monolayer 15 min p r i o r to addition of b a c t e r i a and no other opsonins or macrophage ac t i v a t o r s were included i n the system. P. aeruginosa was used i n the assay at a bacteria:macrophage r a t i o of 20:1 and phagocytosis was allowed to occur f o r 90 min (peritoneal macrophages or macrophage c e l l l i n e s ) or 60 min (human peripheral blood monocytes) i n o 10% C0^ at 37 C. Following incubation, c e l l s were scraped from the d i s h with a rubber policeman and resuspended with gentle p i p e t t i n g . 19 Aliquots of t h i s suspension were cytocentrifuged onto a glass s l i d e (Cytospin 2, Shandon Southern Instruments, Inc., Sewickley, PA; 450 rpm, 5 min), and stained with " D i f f - q u i k " f o r viewing under o i l at lOOOx. The number of b a c t e r i a i n each of 60 c e l l s was counted and s t a t i s t i c a l analysis (Student's t test) performed. Speert et a l . (1984) had previously shown that r e s u l t s obtained with t h i s v i s u a l phagocytosis assay gave comparable data to that of chemiluminescence and e l e c t r o n microscopy studies. B. I n h i b i t o r s / a c t i v a t o r s . ( i ) Chapter one. To assess the opsonic capacity of the o a n t i - p r o t e i n F monoclonal antibodies (ELISA t i t e r 10 ), they were u t i l i z e d at 10% of assay volume. Negative controls were MA1-3 (directed at a non-surface exposed protein epitope of P. aeruginosa) and PBS. ( i i ) Chapter two. To assess i n h i b i t i o n of phagocytosis, phosphate buffered s a l i n e (PBS), cytotoxin, or one of the s u b c e l l u l a r f r a c t i o n s was added to the macrophage monolayer 15 min p r i o r to addition of antibody and b a c t e r i a . I n h i b i t o r s were added i n 100 y l volumes [13 ug of cytotoxin, 500 ug of osmotic shockates (from 10*"^  b a c t e r i a l c e l l s ) , 400 ug of growth supernatant (10*"''" c e l l s ) or 500 Ug inner or outer membrane (2x10^ c e l l s ) ] . A n t i - p r o t e i n F monoclonal antibody MA5-8 ( t i t e r 10 ) was used i n a l l phagocytosis v i s u a l assays at a volume of 30 u l per assay. I f used, rabbit a n t i - c y t o t o x i n or rabbit anti-exoenzyme S was preincubated with i n h i b i t o r f o r 5 min p r i o r to ad d i t i o n . Background l e v e l s of b a c t e r i a l a s s o c i a t i o n (obtained i n the 20 presence of PBS alone) were subtracted from the average number of b a c t e r i a associated per macrophage. ( i i i ) Chapter three. To assess enhancement of phagocytosis, PBS, 100 u l of i n vivo supernatant, various concentrations of bovine plasma f i b r o n e c t i n (Sigma) or the arg i n i n e - g l y c i n e - a s p a r t i c acid-serine (RGDS) peptide (Sigma) were added. No a d d i t i o n a l opsonins or macrophage act i v a t o r s were included i n the 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 incubated with supernatant or f i b r o n e c t i n f o r 5 min at room temperature p r i o r to additi o n to the macrophage monolayer. Antibody was used at the recommended r a t i o of 1 ug per ug of f i b r o n e c t i n . When u t i l i z e d , 60 ug/ml of p u r i f i e d PA01 p i l i , prepared by B. Paslowske as described previously (Paranchych et a l . , 1979) was incubated with i n vivo supernatant-, f i b r o n e c t i n - or RGDS-activated macrophages f o r 15 min at 37°C, 10% C0 2 p r i o r to the addit i o n of ba c t e r i a . 5. Fluorescence Assay P388 D 1 c e l l s were resuspended, with a pipette, from the bottom of f l a t bottomed f l a s k s , centrifuged at 1000 rpm f o r 10 min, resuspended at a concentration of 2.5 x 10 6 c e l l s / m l i n fr e s h supplemented RPMI medium, and grown 16 h p r i o r to assay i n screw-cap t e f l o n j a r s . A f t e r gentle resuspension, c e l l s were washed and resuspended i n an experimental s o l u t i o n which approximated the ion composition of RPMI-1640 medium (Rink et a l . , 1980). KC1 was added to a f i n a l concentration of 0.3 mM to create 21 a K + concentration gradient across the plasma membrane. C e l l s were dispensed i n 1 ml assay volumes, and the carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide (diS-C 3(5)) (Molecular Probes, Oregon) was added at a concentration of 2 x l 0 - ^ M. Carbocyanine dyes are l i p o p h i l i c probes which are highly fluorescent i n aqueous environments and minimally fluorescent i n the hydrophobic environment of the membrane (Rink et a l . , 1980). When ion fluxes are generated across the membrane, e i t h e r the probe i s shunted out of the membrane to become more fluorescent (upon dep 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) or more probe i s inserted into the membrane to become less fluorescent (upon hyperpolarization). To test the e f f e c t s of cytotoxin, osmotic shock f l u i d s and f i b r o n e c t i n on the plasma membrane of macrophages, various concentrations were added to c e l l s e q u i l i b r a t e d with diSC 3(5) and the change i n fluorescence ( e x c i t a t i o n at 620 nm; emission at 670 nm) was measured i n a Perkin-Elmer 650-10S Spectrofluorimeter. A l l samples were added i n 100 u l aliquots (volumes equalized with PBS, pH 7.2). The K + ionophore valinomycin (Sigma), at a concentration of 2xl0~^ M was used as a p o s i t i v e c o n t r o l f o r d e p o l a r i z a t i o n . Rates of d e p o l a r i z a t i o n were measured from the maximal slopes obtained from the spectrofluorimeter trace. Maximal rates were always attained within 1 min of stimulus addition. 22 RESULTS CHAPTER I Opsonic Phagocytosis of Pseudomonas aeruginosa by Macrophages and  Macrophage C e l l Lines Pseudomonas aeruginosa i s an opportunistic pathogen capable of producing 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 d e b i l i t a t e d i n d i v i d u a l s . Patients e s p e c i a l l y at r i s k include those with c y s t i c f i b r o s i s , severe burns, cancer or diabetes. Because of i t s high natural resistance to most applicable a n t i b i o t i c s , Pseudomonas i n f e c t i o n s are often f a t a l . As a gram-negative bacterium, P. aeruginosa possesses two c e l l envelope membranes: the cytoplasmic (or inner) membrane and the outer membrane. These layers are separated by a si n g l e layer of peptidoglycan. The outer membrane appears to be important i n Pseudomonas i n f e c t i o n s since i t plays a r o l e i n a n t i b i o t i c resistance and contains the endotoxic lipopolysaccharide (LPS) and protein antigens. Mutharia and Hancock (1983) i s o l a t e d a series of monoclonal antibodies s p e c i f i c f o r epitopes on protein antigens common to a l l 17 P. aeruginosa serotypes. Results indicated that monoclonal antibodies di r e c t e d against one of these outer membrane proteins (protein F) were protective i n mouse i n f e c t i o n models (Hancock et a l . , 1985). Further work demonstrated that f i v e monoclonal antibodies d i r e c t e d against protein F could be separated i n t o two classes, each reacting with a d i f f e r e n t highly conserved surface epitope on the porin (Mutharia and Hancock, 1983). Monoclonal antibodies 23 MA4-4, 2-10, 4-10 and 5-10 were a l l hypothesized to react against one epitope, while MA5-8 was s p e c i f i c f o r a d i s t i n c t epitope. Antibodies against protein F thus appear to have p o t e n t i a l as passive immunotherapeutic agents. Previous data indicated, however, that these monoclonal antibodies f a i l e d to enhance complement-mediated b a c t e r i c i d a l k i l l i n g of P. aeruginosa (Hancock et a l . , 1985). Therefore, since macrophages are one of the primary l i n e s of defense against i n f e c t i o n (Dunn et a l . , 1985; Green and Kass, 1964; Reynolds et a l . , 1975), one might assume that the monoclonal antibodies were act i n g to opsonize the P. aeruginosa f o r phagocytosis i n the mouse i n f e c t i o n model. This Chapter presents data demonstrating that protein F - s p e c i f i c monoclonal antibodies could indeed opsonize P. aeruginosa s t r a i n M2 f o r uptake by macrophages and a macrophage c e l l l i n e . 1. Choice of an appropriate model c e l l l i n e The v i s u a l inspection phagocytic assay u t i l i z e d was modified from that published by Speert et a l . (1984). B r i e f l y , the assay involved a 90 min incubation of macrophages, b a c t e r i a and opsonin ( i f used) together i n a ti s s u e culture dish. Macrophages were then scraped from the dish, resuspended gently, and cytocentrifuged onto a glass s l i d e . Following s t a i n i n g , the number of b a c t e r i a i n each of 60 macrophages were counted and s t a t i s t i c a l comparisons with appropriate controls made using Student's t t e s t . Speert and colleagues (1984) demonstrated that r e s u l t s obtained with t h i s assay gave comparable data to chemiluminescence and ele c t r o n microscopy studies. I n i t i a l l y , three macrophage c e l l l i n e s were assessed 24 f o r t h e i r phagocytic c a p a b i l i t y using the v i s u a l assay and P. aeruginosa s t r a i n M2 opsonized with the protein F - s p e c i f i c monoclonal antibody MA5-10. The three c e l l l i n e s under study were PU5-1.8, a Balb/c macrophage-like monocytic tumor; DC7, a macrophage:T c e l l f u s i o n product; and P388 D 1, a DBA/2 macrophage tumor c e l l l i n e . The l e v e l of phagocytosis obtained i n the presence and absence of antibody was determined f o r a l l three c e l l l i n e s , unelecited mouse peritoneal macrophages, and human peripheral blood monocyte-derived macrophages. The behaviour of c e l l l i n e P388 D 1 most c l o s e l y approximated that of the normal macrophages from both mouse and human o r i g i n (Figure 1). In addition, t h i s c e l l l i n e was co n s i s t e n t l y healthy during prolonged culture and displayed a c e l l u l a r morphology s i m i l a r to that of normal macrophages. P388j^ was thus judged to be an appropriate model f o r u n e l i c i t e d mouse peritoneal macrophages and cultured human peripheral blood monocytes i n assessment of phagocytosis of P. aeruginosa. 2. Establishment of phagocytosis assay conditions To in v e s t i g a t e the time course of b a c t e r i a l uptake i n the presence or absence of monoclonal antibody, the v i s u a l phagocytosis assay was performed as i n Materials and Methods but was halted at four time points f o r assessment. The data obtained from two separate experiments showed that the s a l i n e negative c o n t r o l produced a gradual increase i n phagocytosis from 0 to 30 min. Aft e r 30 min, a plateau region was reached and l i t t l e f u r t h e r uptake was observed f o r the duration of the experiment (Figure 2). Anti-F monoclonal antibody MA2-10 on the other hand produced 25 Bacteria / Macrophage 5 -/ / / / / / / / / / / / / / / / / / / / •A / / / / / / / / / / / / / / / / \A / / / / / / / / / / / / V i i r Sal Ab PUS-1.8 Sal Ab DC-7 SaL Ab P388 DI Sal Ab Mouse Peritoneal Macrophages i i r Sal Ab Human Monocytes Figure 1. Opsonized phagocytosis o f P . aeruginosa s t r a i n M2 by mouse macrophage c e l l l i n e s PU5-1.8, 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 blood monocyte-derived macrophages. Sa l ine (Sal ) or p r o t e i n F - s p e c i f i c monoclonal antibody MA5-10 (Ab) were added to macrophages and b a c t e r i a and incubated at 3 7 ° C i n 10% C 0 2 f o r 90 min (mouse p e r i t o n e a l macrophages or macrophage c e l l l i n e s ) or 60 min (human p e r i p h e r a l blood monocyte-derived macrophages). The averages of three independent experiments are shown. 26 Bacteria per Macrophage MA2-10 0 20 40 60 80 100 Incubation T ime (min) Figure 2. Timecourse of P. aeruginosa a s s o c i a t i o n with P388D D^ i n the presence or absence (saline) of a n t i - F monoclonal antibody MA2-10. The v i s u a l phagocytosis assay was performed as i n Materials and Methods but was halted at 15, 30, 60 and 90 min f o r assessment. The average of 3 independent experiments i s shown. 27 good uptake almost immediately and leveled out between 30 and 60 min. Phagocytosis increased 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 with the monoclonal antibody, producing a biphasic time course of b a c t e r i a l a s s o c i a t i o n . From the r e s u l t s of these experiments, I chose to h a l t these P388 D 1 phagocytic assays at a time at which the average number of bacteria/phagocyte i n the negative controls was v i r t u a l l y s t a t i c (90 min). With the negative c o n t r o l s , s a l i n e and MA1-3 [a monoclonal antibody dire c t e d against an outer membrane epitope that was not surface exposed (Mutharia and Hancock, 1985)], most of the macrophages had phagocytosed few or no b a c t e r i a a f t e r 90 min (Figure 3). This resulted i n a d i s t r i b u t i o n with a large peak at zero and a very low, short shoulder region. When monoclonal antibody MA4-10 was added, t h i s major peak s h i f t e d over to approximately 5, 7 and 11 b a c t e r i a per c e l l f o r human peripheral blood monocytes, P388 D 1 and mouse peritoneal macrophages, re s p e c t i v e l y . The shoulder region also lengthened considerably. 3. Opsonic phagocytosis:influence of antibody subclass The panel of f i v e a n t i - F monoclonal antibodies were assessed f o r t h e i r opsonic capacity using c e l l l i n e P388 D 1, u n e l i c i t e d mouse peritoneal macrophages, or human peripheral blood monocyte-derived macrophages, P. aeruginosa s t r a i n M2 and the v i s u a l assay of phagocytosis. A l l f i v e a n t i - F monoclonal antibodies caused 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 with u n e l i c i t e d mouse peritoneal macrophages i n a l l assays performed (Table I, p<0.01). I n t e r e s t i n g l y , the two most opsonic antibodies, KA4-10 and 5-10 were of the IgGl subclass. 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 . Percent of macrophages assoc ia ted w i th s p e c i f i c numbers of b a c t e r i a a f t e r 90 min. For de ta i l s - of assay see M a t e r i a l s and Methods. Negative c o n t r o l MA1-3 d i s t r i b u t i o n was approximately the same f o r a l l macrophage c e l l types . A d d i t i o n o f monoclonal antibody KA4-10 s h i f t e d the major peak f o r P388^ c e l l s , u n e l i c i t e d mouse p e r i t o n e a l macrophages (mouse per . mo.) and human p e r i p h e r a l blood monocyte-derlved macrophages (human mo.) . Table I. Enhancement of the ass o c i a t i o n of Pseudomonas aeruginosa s t r a i n M2 with mouse peritoneal macrophages, P388^ c e l l s , and human peripheral blood monocytes using monoclonal antibodies dir e c t e d against protein F. Opsonin S p e c i f i c Average number of b a c t e r i a associated/phagocyte (Isotype) Antigen Mouse peritoneal P388j^ Human peripheral macrophages a'° blood monocytes*5 Saline - not done 3-0 + 1.9 2.3 + 1.9 MA1-3 (IgGl) 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 (IgGl) F 5.9 ± 3.6 C 4.7 + 1.7 d 6.1 + 2.7 g MA2-10 (IgGl) F 4.3 ± 3.5 C 5.7 + 2.1 c 9.9 + 6.0 h MA4-10 (IgGl) F 14.6 ± 9.0 C 7.3 + 2.2° 10.0 + 7.5 f ameans ± standard deviations of 3 independent experiments. bmeans ± S.D. of 6 independent experiments. cp<0.01 (by Student's t test) f o r 100% of assays. dp<0.01 i n 2 of 3 assays. ep<0.01 i n 4 of 6 assays, p>0.1 i n 2 of 6 assays, •p p<0.01 i n 5 of 6 assays, p<0.1 i n 1 of 6 assays. gp<0.01 i n 3 of 6 assays. p<0.01 i n 3 of 6 assays, p<0.1 i n 3 of 6 assays. 30 A l l antibodies displayed some a b i l i t y to opsonize P. aeruginosa s t r a i n M2 f o r phagocytosis by mouse macrophage c e l l l i n e , P388 D 1 (Table I ) . Monoclonal antibodies MA4-10, 2-10 and 5-8 produced a phagocytic index which was s i g n i f i c a n t l y higher than the negative c o n t r o l values i n a l l assays performed (p<0.01). The two most e f f i c i e n t monoclonal antibodies, MA4-10 and 2-10, were again of the IgGl subclass. MA5-10 and 4-4 were each found to be s i g n i f i c a n t l y opsonic i n two of three experiments. For cultured human peripheral blood monocytes, the average number of associated b a c t e r i a was s i g n i f i c a n t l y increased i n at le a s t four of s i x assays i n the presence of monoclonal antibodies MA4-10, 2-10, 5-8 and 4-4 (Table I ) . Again, the most opsonic antibodies, MA4-10 and 2-10 were of the IgGl subclass. 4. Summary Three macrophage c e l l l i n e s were assessed f o r t h e i r phagocytic c a p a b i l i t y using P. aeruginosa s t r a i n M2 opsonized with the protein F - s p e c i f i c monoclonal antibody MA 5-10. Of the three, the behaviour of mouse macrophage c e l l l i n e P388 D 1 most c l o s e l y approximated that of normal u n e l i c i t e d mouse peritoneal macrophages and cultured human peripheral blood monocyte-derived macrophages. P388j^ was thus judged to be an appropriate model f o r normal macrophages i n assessment of phagocytosis of P. aeruginosa. A timecourse of b a c t e r i a l uptake i n the presence of monoclonal antibody indicated that opsonic phagocytosis followed a biphasic timecourse. Good uptake was observed immediately and lev e l e d out between 30 and 60 min. Phagocytosis was seen to increase 31 s i g n i f i c a n t l y a f t e r 60 min. A panel of f i v e a n t i - F monoclonal antibodies were assessed f o r t h e i r opsonic capacity. A l l f i v e antibodies caused 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 with mouse peritoneal macrophages and P388j^ c e l l s , while four of the f i v e were opsonic f o r phagocytosis by human peripheral blood monocyte-derived macrophages. The most e f f e c t i v e antibodies were of the IgGl subclass. 32 CHAPTER II P. aeruginosa Cytotoxin: L o c a l i z a t i o n and In a c t i v a t i o n of Macrophages In the case of patients with c y s t i c f i b r o s i s , P. aeruginosa frequently causes p e r s i s t e n t lung i n f e c t i o n , i n d i c a t i n g that the host immune system i s incapable of c l e a r i n g the b a c t e r i a . As macrophages are important i n defense against lung i n f e c t i o n , i t has been suggested that these phagocytes may not be functioning c o r r e c t l y i n the lungs of patients with c y s t i c f i b r o s i s (Speert, 1985). One of the ways i n which P. aeruginosa may protect i t s e l f from such basic host defenses i s through production of a cytotoxin. P. aeruginosa cytotoxin, previously named leukocidin (Scharmann, 1976), has been i s o l a t e d from autolysates of P. aeruginosa c e l l s and appears to be associated with a l l i s o l a t e s of P. aeruginosa (Baltch et a l . , 1987). I t inact i v a t e s eukaryotic c e l l s by forming lesions or pores i n the membrane of target c e l l s of the immune system (Lutz et a l . , 1987). This r e s u l t s i n increased plasma membrane permeability to small molecules and ions (Scharmann, 1976). Such i n t o x i c a t i o n has been documented i n granulocytes (Baltch et a l . , 1985), endothelial c e l l s (Suttorp et a l . , 1985), E h r l i c h a s c i t e s tumor c e l l s (Lutz et a l . , 1987) and human leukemic c e l l s (Sasak and Lutz, 1985). In the case of granulocytes, treatment with the cytotoxin causes an i n h i b i t i o n of the a b i l i t y of the granulocytes to k i l l P. aeruginosa c e l l s (Baltch et a l . , 1985). The experiments of t h i s chapter were designed to determine 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 of cytotoxin and to examine i t s e f f e c t on macrophages. Towards t h i s end, 33 various b a c t e r i a l c e l l compartments were tested f o r the presence of cytotoxin, and osmotic shock f l u i d (periplasmic contents) and a p u r i f i e d preparation of cytotoxin were observed f o r t h e i r i n t e r a c t i o n with mouse macrophage c e l l l i n e P388 D 1. 1. C e l l u l a r l o c a l i z a t i o n of cytotoxin Cytotoxin was previously i s o l a t e d from c e l l u l a r autolysates (e.g. F i g . 4, lane 1), which were the supernatant f r a c t i o n s of stationary phase P. aeruginosa c e l l s , resuspended i n phosphate-buffered s a l i n e , and incubated f o r 56 h at 37°C (Lutz, 1979; Scharmann, 1976). A v a r i e t y of experiments were performed to obtain release of cytotoxin 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 with which cytotoxin was associated, although they d i d e s t a b l i s h that cytotoxin was c e l l - a s s o c i a t e d and could be released by various l y s i s techniques (Scharmann, 1976). To determine the c e l l u l a r l o c a l i z a t i o n of cytotoxin i n P. aeruginosa, several c e l l compartments were tested f o r i t s presence using SDS-polyacrylamide g e l electrophoresis and Western b l o t t i n g techniques (Figures 4,5). While rabbit a n t i - c y t o t o x i n serum reacted i n Western immunoblots with heavily overloaded inner and outer membrane protein preparations, no discernable bands were seen at the molecular weight of cytotoxin ( F i g . 5, lanes 4 and 5). S i m i l a r l y , i n 150-fold concentrated growth supernatants, the only immunolabelled band i n ant i - c y t o t o x i n b l o t s was at approximately 56 kD ( F i g . 5, lane 3). These membrane and growth supernatant proteins cross-reacted strongly with anti-exoenzyme S serum (Figure 6, lanes 3, 4, 5). 34 S 1 2 3 4 5 Figure 4. Coomassie-stained SDS polyacrylamide g e l of cytotoxin and P. aeruginosa s t r a i n H103 su 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+ cytotoxin; lane 2, Mg /freeze-thaw osmotic shockate; lane 3, growth supernatant; lane 4, inner membrane; lane 5, outer membrane. Running positions of molecular weight standards (lane S) are marked to the l e f t of the g e l . The running p o s i t i o n of the 28 kD cytotoxin band i s indicated on the r i g h t . The amounts loaded per lane were 2.8 ug of cytotoxin and 40 Ug of each of the other samples. Figure 5. Western immunoblot of cytotoxin and P. aeruginosa s t r a i n H103 su b c e l l u l a r f r a c t i o n s probed with rabbit a n t i - c y t o t o x i n serum. Lane 1, 2+ p u r i f i e d cytotoxin; lane 2, Mg /freeze-thaw osmotic shockate; lane 3, growth supernatant; lane 4, inner membrane; lane 5, outer membrane. The amounts loaded per lane were 2.8 ug of cytotoxin and 40 ug of each of the other samples. Compared to the osmotic shockate, 12 times as many c e l l s were required to produce 40 ug of growth supernatant and 20 times as many c e l l s were required to produce 40 ug of inner and outer membrane. 36 In contrast osmotic shockates, prepared by the Mg /freeze-thaw method of Hoshino and Kageyama (1980) [which i s the preferred method f o r i s o l a t i o n of the periplasmic contents of P. aeruginosa (Poole and Hancock, 1984) ], contained a polypeptide band of approximately 28 kD which reacted r e l a t i v e l y strongly with a n t i - c y t o t o x i n sera on Western immunoblots ( F i g . 5, lane 2; a minor species of 27 kD was also evident on some blots and may have been a p r o t e o l y t i c breakdown product of cytotoxin). The presence of a 28 kD crossreactive band i n the periplasmic f r a c t i o n was reproducibly obtained with 8 separate preparations of osmotic shockate. No cross-reative bands were seen on the anti-exoenzyme S b l o t (Figure 6, lane 2). 2. Cytotoxin i n h i b i t o n of phagocytosis In previous i n v e s t i g a t i o n s , cytotoxin had been shown to cause swelling and l y s i s of polymorphonuclear leukocytes (Baltch et a l . , 1985; Scharmann, 1976), pulmonary artery endothelial c e l l s (Suttorp et a l . , 1985) and E h r i i c h a s c i t e s tumor c e l l s (Lutz et a l . , 1987). In the case of polymorphonuclear leukocytes, cytotoxin was demonstrated to i n h i b i t the b a c t e r i c i d a l a c t i v i t y of these c e l l s against P. aeruginosa (Baltch et a l . , 1985) . To confirm that osmotic shockates contained a cytotoxin, I determined the e f f e c t s of sub-lethal concentrations of cytotoxin and osmotic shockates on the a b i l i t y of the macrophage c e l l l i n e P388 D 1 to phagocytose P. aeruginosa c e l l s . P u r i f i e d cytotoxin was used i n the assay at 13 yg/ml. This concentration d i d not appear to 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 of the 37 1 2 3 4 5 Figure 6. Western immunoblot of cytotoxin and P. aeruginosa s t r a i n H103 su b c e l l u l a r f r a c t i o n s probed with rabbit anti-exoenzyme S serum. Lane 1, 2+ p u r i f i e d cytotoxin; lane 2, Mg /freeze-thaw osmotic shockate; lane 3, growth supernatant; lane 4, inner membrane; lane 5, outer membrane. The amounts loaded per lane were 2.8 ug of cytotoxin and 40 ug of each of the other samples. 38 macrophages during the assay, as assessed by the a b i l i t y of macrophages to adhere to glass surfaces, although minor swelling was occasionally observed. At higher concentrations, cytotoxin resulted i n increasing c e l l damage and l y s i s . Lower concentrations caused reduced phagocytosis, although maximal e f f e c t s were seen at 13 ug/ml. At a b a c t e r i a to macrophage r a t i o of 20 to 1, 8.0 opsonized P. aeruginosa c e l l s became associated per untreated macrophage (Table I I ) . Addition of p u r i f i e d cytotoxin r e s u l t e d i n a 95% decrease i n opsonized phagocytosis. Osmotic shockate was also i n h i b i t o r y , decreasing phagocytosis by 71%. Addition of rabbit a n t i - c y t o t o x i n restored phagocytosis to approximately 90 per cent i n each of these cases (Table I I ) . This a n t i s e r a was incapable of promoting P. aeruginosa uptake on i t s own. In contrast, anti-exoenzyme S was unable to influence the i n h i b i t i o n of 2+ phagocytosis by cytotoxin or Mg /freeze-thaw shockate. Although P. aeruginosa growth supernatant displayed s i g n i f i c a n t phagocytosis i n h i b i t i o n (Table I I ) , the e f f e c t could be completely negated using e i t h e r a n t i - c y t o t o x i n or anti-exoenzyme S. This confirmed that the a n t i - c y t o t o x i n sera contained antibodies that cross-reacted with exoenzyme S (see F i g . 5, lane 3 and d i s c u s s i o n ) . Inner and outer membrane preparations increased uptake l e v e l s over and above those obtained i n the antibody c o n t r o l . As a negative c o n t r o l , osmotic shock f l u i d from Esch e r i c h i a c o l i s t r a i n C600 was also examined i n t h i s assay. While 97% i n h i b i t i o n of phagocytosis was observed, addition of a n t i - c y t o t o x i n serum di d not restore phagocytosis i n t h i s case (Table I I ) . 39 Table I I . E f f e c t of an t i - c y t o t o x i n and anti-exoenzyme S sera on i n h i b i t i o n of opsonized phagocytosis of P. aeruginosa s t r a i n M2 Bacteri a associated per macrophage (%) In h i b i t o r No an t i - c y t o t o x i n Anti-cytotoxin Anti-exoenzyme S treated treated None Cytotoxin P. aeruginosa osmotic shockate P. aeruginosa growth supernatant P. aeruginosa inner membrane P. aeruginosa outer membrane E. c o l i osmotic shockate 8.0 (100) b 0.4 ( 5 ) d 2.3 ( 2 9 ) d 2.3 ( 2 9 ) d 16.4 (205) 16.0 (200) 0.2 ( 3 ) d 7.0 (88) 7.3 (91) 7.0 (88) ND ND 1.3 ( I 6 ) d ND 0.4 ( 5 ) d 0.3 ( 4 ) d 7.4 (93) ND ND ND ^ h e input r a t i o of b a c t e r i a : macrophage c e l l s i n the assay was 20:1. Sixty macrophages per assay were assessed f o r numbers of associated b a c t e r i a . Data represents the mean of 4 to 9 independent assays. ND - not determined. ^Number i n brackets i s the percent 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 to that i n the absence of i n h i b i t o r . CA si n g l e experiment with monoclonal antibody MA5-8 as opsonin and three experiments without opsonin demonstrated that the an t i - c y t o t o x i n sera was unable to increase phagocytosis by non-cytotoxin-treated macrophages. dp<0.01 (Student's t tes t ) that uptake was s i g n i f i c a n t l y lower than that obtained i n the absence of i n h i b i t o r f o r a l l assays performed. 40 3. Mechanism of macrophage i n a c t i v a t i o n Previous studies have suggested that the mode of act i o n of cytotoxin was to produce d i s c r e t e membrane lesions (Lutz et a l . , 1987) which allowed passage of small molecules and ions into and out of the target c e l l (Scharmann, 1976). It i s l i k e l y that such a l t e r a t i o n s i n membrane permeability caused d i s s i p a t i o n ( i . e . depolarization) of ion gradients required to s i g n a l phagocytosis [e.g. a C a 2 + gradient (Young et a l . , 1984)]. Carbocyanine dyes [ i . e . diSC 3(5)] are l i p o p h i l i c probes which are highly fluorescent i n aqueous environments and minimally fluorescent i n the hydrophobic environment of the membrane. When ion fluxes are generated across the membrane, the probe i s e i t h e r shunted out of the membrane to become more fluorescent (on de p o l a r i z a t i o n or current with the concentration gradient) or more probe i s inserted i n t o the membrane to become less fluorescent (on hyperpolarization or current against the concentration gradient). This type of dye was thus used to investigate ion fluxes across the phagocytic membranes of the macrophage c e l l . Using one of these p o l a r i z a t i o n - s e n s i t i v e fluorescent probes [ d i S C ^ S ) ] , i t was determined that cytotoxin and osmotic shock f l u i d d i d indeed produce strong d e p o l a r i z a t i o n of the P388 D 1 plasma membrane (Figure 7, Table I I I ) . This d e p o l a r i z a t i o n was s i m i l a r to that caused by the p o s i t i v e c o n t r o l ionophore, valinomycin, i n d i s s i p a t i o n of an imposed high K + concentration gradient (Figure 7, Table I I I ) . The rate of de p o l a r i z a t i o n increased as a function of the concentration of cytotoxin or osmotic shockate added and reached a plateau at higher concentrations Figure 7. Changes i n diSC.j(5) fluorescence a f t e r a d d i t i o n of phosphate-buffered s a l i n e , cytotoxin (35 yg/ml) or valinomycin (2xlO _ 6M) to c e l l s of the macrophage c e l l l i n e P388 D 1 that had been e q u i l i b r a t e d i n the presence of 2xlO~ 6M d i S C ^ S ) . Depolarization i s indicated by an increase i n fluorescence. 42 Table I I I . Depolarization of the P388 D 1 plasma membrane by cytotoxin, valinomycin and osmotic shockate assessed using the fluorescent probe diSC.(5) A d d i t i o n 1 Net fluorescence increase I n i t i a l rate of fluorescence >b ( a r b i t r a r y units) increase (units/min) None Valinomycin Cytotoxin P. aeruginosa osmotic shockate P. aeruginosa growth supernatant P. aeruginosa LPS E. c o l i osmotic shockate 0 7.4 7.2 6.6 0 0 0 6.1 5.2 7.2 0 0 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 across the P388j)i plasma membrane, to the following f i n a l concentrations: valinomycin, 2xlO~^M; cytotoxin, 35 ug/ml; osmotic shockates, 500 ug/ml; growth supernatant, 400 ug/ml; LPS, 385 ug/ml. These concentrations were chosen to demonstrate the maximal possible e f f e c t s ; lower concentrations caused sub-maximal e f f e c t s as seen i n F i g . 8. *>Data represent the means of three independent experiments performed on separate days. 43 (Figure 8). As controls, the osmotic shockate from E. c o l i (Table III) and the concentrated growth supernatant from P. aeruginosa were tested and f a i l e d to produce any d e p o l a r i z a t i o n of the macrophage plasma membrae. LPS u t i l i z e d at a concentration f i v e times that found i n Mg 2 + freeze/thaw osmotic shockate (385 ug LPS per assay) caused no measurable dep o l a r i z a t i o n . 4. Summary P. aeruginosa cytotoxin and periplasmic contents caused a s i g n i f i c a n t i n h i b i t i o n of opsonic phagocytosis of P. aeruginosa s t r a i n M2 by mouse macrophage c e l l l i n e P388 D 1. Phagocytosis was restored i n each case upon pre-incubation of e i t h e r preparation with a n t i - c y t o t o x i n serum. Although growth supernatant also caused a s i g n i f i c a n t reduction i n opsonic phagocytosis, i t s e f f e c t s were negated by pre-incubation with an anti-exoenzyme S preparation. Other c e l l u l a r f r a c t i o n s tested were not i n h i b i t o r y f o r macrophage phagocytosis. Both cytotoxin and periplasmic contents caused d e p o l a r i z a t i o n of the P388 D 1 plasma membrane, as demonstrated using a p o l a r i z a t i o n - s e n s i t i v e probe. S i m i l a r c o r r e l a t i o n s were not observed f o r other P. aeruginosa f r a c t i o n s . The rate of de p o l a r i z a t i o n increased as a function of the concentration of cytotoxin or osmotic shockate added and reached a plateau l e v e l at higher concentrations. Using Western immunoblotting techniques i t was determined that the only c e l l u l a r f r a c t i o n containing an i d e n t i f i a b l e cytotoxin p r o t e i n was osmotic shockate. These data indicated that P. aeruginosa Depolarization Rate (units/min) 8 -i — O s m o t i c S h o c k a t e 0 20 40 60 80 100 120 140 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 like those shown in Fig. 7 and represent the means of 3 different experiments. 45 cytotoxin i s l o c a l i z e d i n the periplasm and has the p o t e n t i a l to i n h i b i t macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane. 46 CHAPTER H I Fibronectin-Mediated A c t i v a t i o n of Non-Opsonic Phagocytosis of  P. aeruginosa To extend the studies of Chapter One, the a b i l i t y of a n t i - p r o t e i n F monoclonal antibodies to mediate phagocytosis of i n vivo-grown P. aeruginosa was determined. Using the method of Day et a l . (1980), (Figure 9), P. aeruginosa c e l l s were grown f o r 3 days i n the peritoneal c a v i t y of laboratory mice or r a t s . B a c t e r i a were contained i n 1-cm long p l a s t i c chambers sealed at both ends with 0.22 ym membrane f i l t e r s allowing free exchange of peritoneal f l u i d s and b a c t e r i a l products while p r o h i b i t i n g immune c e l l access or b a c t e r i a l escape. Upon removal of chambers, b a c t e r i a could be separated from the f l u i d i n the chamber by c e n t r i f u g a t i o n . This decanted f l u i d was c a l l e d the i n vivo supernatant. 1. E f f e c t of i n vivo growth on phagocytosis To study the e f f e c t of i n vivo growth on phagocytosis, P. aeruginosa s t r a i n M2 was grown i n v i t r o i n r a p i d l y agitated broth or i n vivo using, the chamber implant model ( F i g . 9). S u s c e p t i b i l i t y to both non-opsonic and opsonic phagocytosis was determined. I t was found that unwashed i n vivo-grown b a c t e r i a showed s i g n i f i c a n t l y higher phagocytosis than unwashed i n vltro-grown organisms ( F i g . 10). P. aeruginosa c e l l s taken d i r e c t l y from the i n vivo growth system were s i g n i f i c a n t l y more susceptible to non-opsonic phagocytosis than were the same P. aeruginosa c e l l s a f t e r being washed twice i n buffer ( F i g . 10). Washing the i n vitro-grown 47 10 bacteria in saline I Chamber sealed on both ends with 0.22 um filters Insertion into mouse or rat peritoneal cavity 1 72 hours D Contents | Centrifugation Bacteria in vivo supernatant Figure 9. D e s c r i p t i o n of the i n v ivo growth model. 10 b a c t e r i a l c e l l s i n p h y s i o l o g i c a l s a l i n e were sealed i n t o polypropylene chambers u s i n g 0.22 um f i l t e r s . 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 o f laboratory mice or r a t s and incubated i n v ivo f o r 72 hours . B a c t e r i a l cu l tures were harvested and could be separated from the i n v ivo supernatant by c e n t r i f u g a t i o n at 12,000 x g f o r 10 min. 48 Figure 10. Opsonized phagocytosis of i n vivo- and i n vitro-grown P. aeruginosa 3 t r a i n M2 by mouse macrophage c e l l l i n e P388 D 1 > M2 c e l l s were washed (w) or unwashed (uw), with (+Ab) and without the ad d i t i o n of a n t i - p r o t e i n F monoclonal antibody MA5-8. Each column represents the average uptake observed i n 3 independent 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 protein P - s p e c i f i c monoclonal antibody MA5-8 resulted i n a s i g n i f i c a n t increase (p<0.01) i n phagocytosis of both i n v i t r o and i n vivo-grown b a c t e r i a compared with unopsonized organisms ( F i g . 10). This further suggested that a n t i - F monoclonal antibodies have p o t e n t i a l as passive immunotherapeutic agents. An i n t e r e s t i n g f i n d i n g of these studies was that the average increase i n b a c t e r i a per macrophage r e s u l t i n g from opsonization appeared to 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 regardless of whether they were unwashed or washed. This suggested that the i n vivo-grown organisms may have had a reduced surface exposure of protein F r e l a t i v e to i n vitro-grown c e l l s . 2. Enhancement of phagocytosis by i n vivo supernatant I n i t i a l l y , I wished to determine i f the decreased phagocytosis of washed i n vivo-grown b a c t e r i a was r e l a t e d to e f f e c t s of the washing procedure on the organisms, or due to removal of some phagocytosis-promoting f a c t o r i n the i n vivo supernatant. To determine i f i n vivo supernatant could be added back to the washed b a c t e r i a and s t i l l f a c i l i t a t e uptake of b a c t e r i a by macrophages, the v i s u a l assay of phagocytosis 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 studies to provide co n t i n u i t y with the above study. 50 It was observed that there was indeed a phagocytosis-promoting f a c t o r obtained from i n vivo chambers which could be separated 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 at 12,000 x g f o r 10 min and added back to again 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 with the macrophage (Table IV). At a b a c t e r i a to macrophage r a t i o of 20 to 1, an average of 2.9 washed b a c t e r i a became associated per macrophage as compared to 7.7 b a c t e r i a taken up i n the presence of i n vivo supernatant. This d i f f e r e n c e was found to be s t a t i s t i c a l l y s i g n i f i c a n t i n 7 out of 8 assays performed (p<0.005, Student's t test) and marginally s i g n i f i c a n t (p<0.1) i n the other assay. This phagocytosis enhancing f a c t o r was t i t e r a b l e i n t h i s system as decreasing amounts of added supernatant resulted i n progressively decreasing l e v e l s of b a c t e r i a l uptake (Table IV). In a ser i e s of experiments designed to investigate some of the phys i c a l properties of the phagocytosis-promoting f a c t o r , i t was determined that i n vivo supernatant retained i t s a c t i v i t y best when stored at -70°C (Table V). In addition, the a c t i v e component of i n vivo supernatant was extremely heat stable and could withstand a temperature of 60°C f o r 30 min or even 100°C f o r 10 min (Table V). To determine i f the observed phenomena were r e s t r i c t e d to the mouse i n vivo system or t h i s p a r t i c u l a r s t r a i n of P. aeruginosa, the same studies were c a r r i e d out using rat-grown b a c t e r i a . Almost i d e n t i c a l r e s u l t s were obtained i n the rat model u t i l i z i n g both s t r a i n M2 and laboratory wild-type s t r a i n H103 (Table VI). A d d i t i o n a l l y , rat supernatant from i n vivo chambers could e f f e c t i v e l y promote uptake of s t r a i n H103 grown i n v i t r o on Trypticase Soy agar (Table VI). S i m i l a r r e s u l t s were obtained 51 Table IV. Enhancement of the as s o c i a t i o n of i n vivo-grown P. aeruginosa M2 with P388 n i c e l l s using supernatant from mouse chambers Treatment of i n vivo Addition to b a c t e r i a B a c t e r i a associated grown M2 per macrophage unwashed PBS 9.4 ± 3.8 a washed PBS 2.9 ± 1.4 washed 100% i n vivo supernatant 7.7 ± 3.4 b washed 75% i s vivo supernatant 6.1 c washed 50% i n vivo supernatant 4.3 d washed 25% i n vivo supernatant 3.8 d ap<0.005 (by Student's t test) i n 8/8 assays when compared to the "washed + PBS" c o n t r o l . Dp<0.005 i n 7/8 assay, p<0.1 i n 1/8 assays when compared to the "washed + PBS" c o n t r o l . cp<0.005 i n the one assay performed when compared to the "washed + PBS" co n t r o l . d n o t s i g n i f i c a n t l y greater than the "washed + PBS" c o n t r o l . 52 Table V. S t a b i l i t y of the phagocytosis-enhancing f a c t o r of i n vivo chamber supernatant. Test condition Average number of b a c t e r i a associated per macrophage +M2 + i n vivo UW 7.6 +M2 + i n vivo W + PBS 2.5 +M2 + i n vivo W + i n vivo supernatant 6.4 +M2 + i n vivo W + stored supenatant (-70°C) 7.1 +M2 + i n vivo W + stored supernatant (-20°C) 4.5 +M2 + i n vivo W + supernatant treated @ 60°C, 30 min 5.9 +M2 + i n vivo W + supernatant treated @ 100°C, 10 min 9.1 53 Table VI. Enhancement of the as s o c i a t i o n of P. aeruginosa s t r a i n s M2 and H103 with u n e l i c i t e d mouse peritoneal macrophages and the P388p^ macrophage c e l l l i n e using i n vivo supernatant from rat chambers Bacteria associated per phagocyte Macrophage c e l l type B a c t e r i a l c e l l type a Unwashed Washed b a c t e r i a Washed b a c t e r i a b a c t e r i a + PBS + rat i n vivo supernatant P388 D 1 i n vivo grown M2 7 . 3 ± 3 . 3 b 3.012.3 1 0 . 6 ± 3 . 7 b P388 D 1 i n vivo grown H103 9.0 ± 3.5 b 2.2 ± 1.8 6.6 ± 1.8° P388 Di i n v i t r o grown H103 - 5.8 ± 2.3 13.9 ± 5 . l b Mouse i n v i t r o grown H103 - 2.1 ± 1.0 7.0 ± 3.1 b peritoneal macrophages a i n v i t r o b a c t e r i a were grown on Trypticase Soy agar pla t e s , i n vivo b a c t e r i a were grown i n peritoneal chambers i n r a t s . bp<0.005 (by Student's t test) i n 3/3 assays performed when compared to the "washed + PBS" c o n t r o l . 54 using i n yjLtro-grown s t r a i n H103 and u n e l i c i t e d mouse peritoneal macrophages. In t h i s case, addition of rat i n vivo supernatant increased b a c t e r i a l a s s o c i a t i o n from 2.1 to 7.0 b a c t e r i a per phagocyte. These differences were found to be s t a t i s t i c a l l y s i g n i f i c a n t i n a l l assays performed (p<0.005, Student's t t e s t ) . I t was thus evident that the mouse macrophage c e l l l i n e P388 D 1 could be used as a model f o r u n e l i c i t e d mouse peritoneal macrophages i n t h i s system. Thus, the remainder of these studies centered on measuring promotion of a s s o c i a t i o n of i n vitro-grown s t r a i n H103 with P388 D 1 c e l l s . This p a r t i c u l a r system was chosen due to the r e l a t i v e ease of working with i n vitro-grown organisms. However, due to the d i f f e r e n t responses of i n vivo-grown organisms observed i n F i g . 10 and Table VI, i t was necessary to determine how growth conditions a f f e c t e d i n vivo 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 agitated (200 rpm) Trypticase Soy broth or on a plate of Trypticase Soy agar. While uptake of agitated b a c t e r i a could not be promoted using i n vivo supernatant from rat peritoneal chambers, phagocytosis of plate-grown organisms was s i g n i f i c a n t l y enhanced (Table VII). Thus, s t r a i n H103 c e l l s grown on Trypticase Soy agar plates were used f o r most of the subsequent studies. 55 Table VII. In v i t r o growth conditions a f f e c t the a b i l i t y of i n vivo supernatant to enhance a s s o c i a t i o n of P. aeruginosa H103 with P388„, c e l l s H103 growth condition Addition to b a c t e r i a B a c t e r i a associated per macrophage Agitated broth (TSB) PBS 8.2 + 0.3 Agitated broth (TSB) Rat chamber supernatant 7.2 + 0.4 a Plate (TSA) PBS 5.9 + 2.2 Plate (TSA) Rat chamber supernatant 13.9 + 4.3 b aNot 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 con t r o l i n 3/3 assays performed (Student's t t e s t ) . Dp<0.005 i n 3/3 assays when compared to the PBS c o n t r o l . 56 3. Characterization of the phagocytosis-promoting f a c t o r To further characterize the phagocytosis-promoting f a c t o r , rat i n vivo supernatant was f r a c t i o n a t e d on a Fast Pressure L i q u i d 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 four pools ( F i g . 11). A f t e r extensive d i a l y s i s and concentration, the phagocytosis-promoting a c t i v i t y of these pools was assessed. I t was observed that Pool A strongly enhanced phagocytosis, Pool B was less e f f e c t i v e , while Pools C and D were i n e f f e c t i v e (Table V I I I ) . These data established that the f a c t o r was of high molecular weight. Thus, three possible candidates were considered f o r the phagocytosis promoting f a c t o r , antibody, complement and f i b r o n e c t i n , each of which had been previously shown to promote phagocytosis (Proctor, 1987). The ease of d i s s o c i a t i o n of the f a c t o r from c e l l s (by c e n t r i f u g a t i o n and washing) and the use of naive mice i n the experiments seemed to r u l e out antibody. In addition, the a c t i v i t y was stable to heating to 100°C f o r 10 min (Table 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. Therefore, we tested f o r the presence of f i b r o n e c t i n using a commercially-available anti-human f i b r o n e c t i n antibody preparation. Use of t h i s a n t i s e r a was possible due to the strong evolutionary and antigenic conservation of f i b r o n e c t i n (Peterson and Skorstengaard, 1985). By Western immunoblotting with goat anti-human f i b r o n e c t i n a n t i s e r a , i n vivo supernatant and Pool A reacted strongly, Pool B, reacted less strongly and Pools C and D f a i l e d to react. Thus, the order of r e a c t i v i t y matched the a b i l i t y to enhance phagocytosis (Table V I I I ) . Pool A was p a r t i a l l y resolved by FPLC into two peaks, 1 and 2 ( F i g . 11). Fibronectin was 57 Figure 11. FPLC gel sie v i n g f r a t i o n a t i o n of i n vivo supernatant from rat chambers. The e l u t i o n p r o f i l e at an absorbance of 230 nm i s shown. Peaks were c o l l e c t e d i n 4 pools (A-D). Peaks 1 and 2 of pool A are l a b e l l e d . 58 Table VIII. Enhancement of the as s o c i a t i o n with P388 D 1 c e l l s using pooled FPLC gel sie v i n g f r a c t i o n a t i o n rat chambers of P. aeruginosa s t r a i n H103 fr a c t i o n s c o l l e c t e d from an of i n vivo supernatant from Addition Ba c t e r i a associated per macrophage PBS 5.0 ± 0.4 i n vivo supernatant 13.5 ± 1.4 a Pool A supernatant 10.5 ± 1.9 b Pool B supernatant 8.5 ± 5.7 C Pool C supernatant 4.2 ± 0.9 d Pool D supernatant 5.6 ± 0.1 d ap<0.005 (by Student's t test) i n 11/11 assays performed when compared to the PBS c o n t r o l . bp<0.005 i n 2/2 assays when compared to the PBS c o n t r o l . cp<0.005 i n 1/2 assays when compared to the PBS con t r o l , p>0.5 i n the other assay. d 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 the PBS control i n 2/2 assays. 59 present i n peak 2 ( F i g . 12, lane 2) but not peak 1 ( F i g . 12, lane 1). Small amounts of f i b r o n e c t i n were detectable i n Pool B ( F i g . 12, lane 3) but none i n pools C or D. This suggested that the f i b r o n e c t i n content and thus the phagocytosis-promoting a c t i v i t y of pool B was probably due to incomplete separation of peak 2 and Pool B. The FPLC f r a c t i o n a t i o n p r o f i l e of mouse i n vivo supernatant was b a s i c a l l y i d e n t i c a l to that of the rat i n vivo supernatant ( F i g . 13). To confirm f i b r o n e c t i n as the phagocytosis-promoting f a c t o r , a n t i - f i b r o n e c t i n sera was incubated with i n vivo 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, to the l e v e l of the PBS c o n t r o l , the phagocytosis-promoting a b i l i t y of the i n vivo supernatant (Table IX). A commercially a v a i l a b l e preparation of bovine plasma f i b r o n e c t i n was tested f o r phagocytosis promoting a c t i v i t y at 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 to 14.1 b a c t e r i a per macrophage (Table IX). I f f i b r o n e c t i n was incubated with a n t i - f i b r o n e c t i n p r i o r to assay, phagocytosis was not s i g n i f i c a n t l y enhanced (Table IX). 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 present to allow production or possibly a c t i v a t i o n of t h i s f a c t o r , saline-containing chambers were incubated i n vivo and t h e i r f l u i d contents assessed f o r phagocytosis-promotion. I t was observed that s a l i n e chamber supernatants from both mice and rats were indeed capable of enhancing phagocytosis of 60 1 2 3 Figure 12. Western immunoblots of f r a c t i o n s c o l l e c t e d from an FPLC g e l siev i n g f r a c t i o n a t i o n of i n vivo supernatant from rat chambers probed with goat-anti-human f i b r o n e c t i n serum. Lane 1, FPLC peak 1; lane 2, FPLC peak 2; lane 3, FPLC pool B. 61 Figure 13. FPLC g e l sie v i n g f r a c t i o n a t i o n of i n vivo supernatant from mouse chambers. The e l u t i o n p r o f i l e at an absorbance of 230 nm i s shown. 62 Table IX. 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 phagocytosis-promoting a c t i v i t y of i n vivo supernatant from rat peritoneal chambers and bovine f i b r o n e c t i n Addition to i n v i t r o -grown H103 Bacteri a associated/macrophage No With a n t i - 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 PBS i n vivo supernatant 100 ug bovine f i b r o n e c t i n s a l i n e chamber supernatant 5.2 ± 0.7 12.2 ± 1.5 a 14.1 ± 4.6 a 15.3 ± 5.0 a not done 6.5 ± 1.8 b 5.5 ± 2.4 b 10.3 ± 3.0° ^ ^ . 0 0 5 (Student's t test) i n 4/4 assays performed when compared to the PBS c o n t r o l . 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 the PBS control i n 2 to 4 assays; p<0.005 when compared to the assay i n the absence of a n t i - f i b r o n e c t i n . cp<0.005 when compared to the assay i n the absence of a n t i - f i b r o n e c t i n . 63 i n vitro-grown P. aeruginosa s t r a i n H103 from 4-5 to 24.1 and 11.5 b a c t e r i a per macrophage, re s p e c t i v e l y (Table X). The phagocytosis promoting f a c t o r i n saline-containing chamber supernatants was apparently f i b r o n e c t i n since a n t i - f i b r o n e c t i n antibodies s i g n i f i c a n t l y reduced the a b i l i t y of these supernatants to promote phagocytosis (Table IX; p<0.005). To e s t a b l i s h the time at which the phagocytosis-promoting f a c t o r appeared i n vivo, b a c t e r i a - and saline-containing chambers were incubated i n the peritoneum of mice and rats f o r 4, 8, 16, 24, 44 and 68 h. Supernatants from these chambers were tested f o r phagocytosis-enhancement. In both s a l i n e and b a c t e r i a l chambers, phagocytosis-promoting a c t i v i t y appeared within 4 h i n mice and within 16 h i n rats (Table X). The degree of phagocytosis 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 bacteria-containing and saline-containing chambers i n e i t h e r animal system, although 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 of f i b r o n e c t i n i n these chambers correlated well with the a b i l i t y to enhance 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 . F ibronectin was not detectable i n Western blots of rat 4 h supernatants but was e a s i l y seen at 24 h ( F i g . 14). In mice, however, t h i s p rotein was found at a sub s t a n t i a l concentration at a l l time points ( F i g . 14). 5. A c t i v a t i o n of macrophages by f i b r o n e c t i n To determine the threshold l e v e l of f i b r o n e c t i n required f o r a c t i v a t i o n of macrophage-mediated non-opsonic uptake, various concentrations were incubated with the P3881.1 c e l l s p r i o r to add i t i o n of 64 Table X. Time course of emergence of phagocytosis-promoting a c t i v i t y i n rat and mouse H103 and s a l i n e chambers Time of implantation of chambers i n animals p r i o r to harvesting of supernatant Bacte r i a associated per macrophage using supernatant of chambers from:  Mice Rats Bact e r i a - S a l i n e - B a c t e r i a - S a l i n e -containing containing containing containing chambers chambers chambers chambers 0 (PBS control) 4.5 4 18.4 a 8 21.6 a 16 24.7 a 24 22.3 a 44 25.3 a 68 ' 25.5 a 20.2 a 3.3 2.9 24. l a 4.6 2.6 26.3 a 7.4 a 6.8 a 23.4 a 11.8 a 7.5 a 21.8 a 12.0 a 10.8 a 24. l a 13.4 a 11.! ap<0.005 (by Student's t tes t ) compared to the PBS c o n t r o l ; a l l other data not s i g n i f i c a n t l y d i f f e r e n t . 65 1 2 3 4 5 0 7 8 9 10 11 12 13 Figure 14. 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 i 11 inn—i i 111fin—i i 111mi—i i i nun—i i 1111in—i i 11mi 1 10 100 1000 10000 100000 1000000 Concentration Fibronectin or RGDS (nm) Figure 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 of uptake of P. aeruginosa s t r a i n H103 by macrophage c e l l l i n e P388^. Each data point represents the average uptake observed i n 3 independent experiments. 67 Pseudomonas ( F i g . 15). I t was found that while concentrations of 13 nM and lower caused no response, a s i g n i f i c a n t l e v e l of phagocytosis enhancement was observed at concentration at and above 27 nM. Maximal a c t i v a t i o n of macrophages was obtained at concentrations around 50 nM f i b r o n e c t i n . It had already been established that f i b r o n e c t i n was not acting as a t y p i c a l opsonin since i t could 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 resuspension i n buffer ( F i g . 10, Table XI). However, experiments i n which f i b r o n e c t i n was added to macrophages f o r 15 min at 37°C, and then the macrophages washed twice p r i o r to ad d i t i o n of b a c t e r i a and assessment of phagocytosis y i e l d e d a very d i f f e r e n t r e s u l t . In t h i s case, washing f a i l e d to prevent a c t i v a t i o n of macrophages f o r increased uptake of P. aeruginosa (Table XI). 6. Determination of the a c t i v e domain of f i b r o n e c t i n Fibronectin i s a large, dimeric glycoprotein with numerous s p e c i f i c binding s i t e s f o r mammalian c e l l s and b a c t e r i a l surfaces (Proctor, 1987). One of these regions of the molecule, the eukaryotic c e l l binding domain, has been shown to i n t e r a c t with various mammalian c e l l types including macrophages (Brown and Goodwin, 1988). A four amino acid sequence, arginine-glycine-aspartate-serine (RGDS), has been proven to be the smallest portion of the eukaryotic c e l l binding domain capable of i n t e r a c t i o n with mammalian c e l l s (Brown and Goodwin, 1988). As such, t h i s sequence was considered a l i k e l y candidate f o r the macrophage-activating region of the f i b r o n e c t i n molecule. 68 Table XI. E f f e c t of washing on fibronectin-mediated enhancement of non-opsonic macrophage phagocytosis of P. aeruginosa s t r a i n H103 Addition to b a c t e r i a Addition to macrophage Treatment p r i o r to mixing of b a c t e r i a with macrophages  Average number of b a c t e r i a associated per macrophage PBS F i b r o n e c t i n a F i b r o n e c t i n a PBS PBS PBS PBS PBS F i b r o n e c t i n 0 Fibronectin 8-none none ba c t e r i a washed none macrophage washed 3.7 ± 0.8 10.1 ± 0.1 b 4.8 + 1.8 C 8.9 ± 3.3 b 10.0 + 0.4 b aSource of f i b r o n e c t i n was i n vivo peritoneal chamber supernatant from rats i n which the only phagocytosis-promoting f a c t o r was f i b r o n e c t i n . bp<0.005 (Student's t test) i n a l l assays performed (2-4 i n d i v i d u a l experiments) when compared to the PBS c o n t r o l . c 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 the PBS con t r o l i n 2/2 assays. 69 To test t h i s hypothesis, a commercially a v a i l a b l e preparation of RGDS was incubated with the macrophages f o r 15 min p r i o r to addi t i o n of bac t e r i a . This four amino acid sequence s i g n i f i c a n t l y 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 i n a concentration-dependent manner (Fi g . 15). The maximal l e v e l of enhancement was s i m i l a r to that obtained with a p u r i f i e d bovine f i b r o n e c t i n preparation ( F i g . 15) and when used at a concentration of 100 uM was found to be s t a t i s t i c a l l y greater than the PBS cont r o l i n a l l four assays performed (p<0.005 by Student's t test) 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 the value obtained i n the presence of f i b r o n e c t i n . Concentrations as small as 2 uM RGDS were capable of s i g n i f i c a n t l y enhancing phagocytosis. 7. Mechanism of fibronectin-mediated macrophage a c t i v a t i o n The i n i t i a l step i n macrophage phagocytosis involves receptor-mediated i n t e r a c t i o n s of the macrophage plasma membrane with the b a c t e r i a l surface. For f i b r o n e c t i n to ac t i v a t e macrophages f o r enhanced phagocytosis, subsequent to f i b r o n e c t i n binding a s i g n a l must be passed to the i n t e r i o r of the c e l l to a c t i v a t e the macrophage and presumably upregulate non-opsonic receptors. Past studies have suggested that phagocytic a c t i v a t i o n signals involve the generation of ion fluxes across the macrophage membrane (Young et 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 fluorescent probe diSCg(5), i t was determined that f i b r o n e c t i n d i d indeed produce d e p o l a r i z a t i o n of the P388 D 1 plasma membrane ( F i g . 16). The rate of ion f l u x generation increased as a function of the concentration of f i b r o n e c t i n added. The rates of de p o l a r i z a t i o n attained 70 Depolarization Rate (units/min) 8 -r 7 -6 -5 -4 -3 -2 -1 -0 50 100 150 200 250 Concentration of Fibronectin (nM) Figure 16. Ef f ec t o f increas ing concentrat ions of f i b r o n e c t i n on the rate of increase ( i . e . d e p o l a r i z a t i o n ) of diSCg(5) f luorescence . The rates were c a l c u l a t e d from traces l i k e those shown i n F i g . 7 and represent the means of 2 independent experiments. 71 at higher f i b r o n e c t i n concentrations were s i m i l a r to that caused by the p o s i t i v e c o n t r o l ionophore, valinomycin, i n d i s s i p a t i o n of an imposed high K + concentration gradient. I t should be noted that fibronectin-mediated ion fluxes were generated v i r t u a l l y immediately across the macrophage membrane, and maximal rates were attained within 1 min of stimulus addition. 8. E f f e c t of growth conditions on fibronectin-mediated non-opsonic  macrophage phagocytosis. It had already been determined that fibronectin-mediated a c t i v a t i o n of macrophage phagocytosis was observed with b a c t e r i a grown on agar plates, but not with b a c t e r i a grown i n broth with rapid shaking (Table VII). It was necessary to determine i f the decreased phagocytosis of shaken b a c t e r i a was r e l a t e d to e f f e c t s of growth i n broth, or due to the rapid a g i t a t i o n of the organisms i n the broth c u l t u r e . P. aeruginosa s t r a i n H103 was grown e i t h e r i n r a p i d l y agitated (200 rpm) Trypticase Soy broth, s t a t i c Trypticase Soy broth or on a plate of Trypticase Soy agar and a v i s u a l assay of fibronectin-enhanced phagocytosis was performed. I t was observed that while a commercially a v a i l a b l e f i b r o n e c t i n preparation and fibr o n e c t i n - c o n t a i n i n g i n vivo supernatant were able to s i g n i f i c a n t l y increase phagocytosis of s t a t i c broth- and plate-grown organisms, uptake of agitated b a c t e r i a could not be promoted using e i t h e r preparation (Table XII). Pseudomonas grown on agar was marginally more susceptible to phagocytosis than the s t a t i c broth c u l t u r e . At a b a c t e r i a to macrophage r a t i o of 20 to 1, an average of 17.3 and 14.5 b a c t e r i a became associated 72 Table XII. The e f f e c t of a g i t a t i o n during growth on the s u s c e p t i b i l i t y of P. aeruginosa s t r a i n H103 to fibronectin-mediated macrophage non-opsonic phagocytosis Growth Average number of bacteria/macrophage Condition + PBS + i n vivo supernatant + f i b r o n e c t i n Shaken broth 7.2 6.4 b 7.6° S t a t i c broth 7.2 14.5 a 11.0 a Agar plate 7.2 17.3 a 13.6 a ap<0.005 (student's t test) when compared to the PBS co n t r o l i n 2/2 ( i n vivo supernatant) and 1/1 (f i b r o n e c t i n ) assays. 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 the PBS control i n 2/2 ( i n vivo supernatant) and 1/1 (f i b r o n e c t i n ) assays. 73 per P388 D 1 mouse macrophage c e l l (respectively) i n the presence of i n vivo supernatant. This small d i f f e r e n c e i n uptake was echoed i n the presence of commercially a v a i l a b l e f i b r o n e c t i n (Table XII). These data established that vigorous shaking during growth was capable of removing or suppressing expression of the b a c t e r i a l ligand required 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 uptake. 9• Determination of the b a c t e r i a l ligand f o r non-opsonic phagocytosis In previous studies, i t was suggested that Pseudomonas adherence to human buccal e p i t h e l i a l c e l l s (Woods et a l . , 1980) and polymorphonuclear leukocytes (Paranchych et a l . , 1986) was p i l u s mediated. Therefore the p o s s i b i l i t y was considered that p i l i were the b a c t e r i a l ligand involved i n fibronectin-mediated macrophage a c t i v a t i o n . P. aeruginosa s t r a i n s H103 (wild type) and BLP3 (a pilus-minus mutant constructed by transposon Tn501 i n s e r t i o n into the cloned chromosomal gene followed by gene replacement) were grown i n vivo i n chambers or on Trypticase Soy agar and assessed 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. While uptake of the pilus-minus mutant could not be promoted using i n vivo supernatant, f i b r o n e c t i n , or RGDS, phagocytosis of the wild type s t r a i n was s i g n i f i c a n t l y enhanced by a l l of these three preparations (Table XIII). In c o n t r o l experiments, the PA01 parent s t r a i n of t h i s Tn501 mutation and a pilus-expressing s t r a i n containing both the Tn501 mutation and an a d d i t i o n a l plasmid-encoded p i l i n gene (pBPl6l) were each susceptible to f i b r o n e c t i n - a c t i v a t e d non-opsonic uptake (Table XIII). 74 Table XIII. Fibronectin-mediated macrophage phagocytosis of P. aeruginosa s t r a i n s H103 (wild type), BLP3 ( p i l i n minus), PAOl-leu ( p i l i n p o s i t i v e ) , and pBPl6l ( p i l i n p o s i t i v e ) P. aeruginosa Growth Average number of bacteria/macrophage s t r a i n condition Unwashed Washed Washed Washed Washed +PBS +in vivo +fi b r o n e c t i n +RGDSa supernatant H103 rat peritoneum 9.0 b (wild type) TSA 2.2 6.0 6.6 b 11.2 b 6.9 b 13.5 b ND 10.6 b BLP3 rat peritoneum 4-5° ( p i l i n minus) TSA 6.4 5.2 6.3 C 4.8 C 7.1 c 4.7° ND 5.2 C PAOl-leu TSA ( p i l i n positve) 5.0 10. l b 14.2 b 10.7 b pBP161 TSA ( p i l i n p o s i t i v e ) 4.8 7.0 b 8.9 b 9.3 b ^D - not done. bp<0.005 (Student's t test) when compared assays performed. to the " washed +PBS" co n t r o l i n 2/2 c 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 the PBS co n t r o l i n 2/2 assays performed. 75 To confirm p i l i as the non-opsonic phagocytosis b a c t e r i a l ligand, 60 ug/ml of p u r i f i e d p i l i was added to i n vivo supernatant; f i b r o n e c t i n - or RGDS-activated macrophages and incubated f o r 15 min p r i o r to assay. This exogenously added p i l i s i g n i f i c a n t l y reduced, to the l e v e l of the PBS c o n t r o l , the phagocytosis-promoting a b i l i t y of a l l 3 preparations (Table XIV). 10. Other b a c t e r i a 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 uptake was r e s t r i c t e d to P. aeruginosa, c l i n i c a l i s o l a t e s of Es c h e r i c h i a c o l i and Staphylococcus aureus were grown i n the mouse chamber system and assessed using the v i s u a l phagocytosis assay ( F i g . 17,18). I t was determined that the i n vivo supernatant i s o l a t e d with each organism was capable of s i g n i f i c a n t l y enhancing uptake of e i t h e r i n vivo- or agitated i n vitro-grown b a c t e r i a . Both preparations of i n vivo supernatant were proven to contain s u b s t a n t i a l quantities of f i b r o n e c t i n as assessed using Western immunoblotting techniques ( F i g . 19). 11. Summary The monoclonal antibody MA5-8, di r e c t e d against porin protein F was capable of s i g n i f i c a n t l y enhancing phagocytosis of i n vivo-grown P. aeruginosa. P. aeruginosa c e l l s taken d i r e c t l y from the i n vivo growth system were s i g n i f i c a n t l y more susceptible to macrophage phagocytosis than were the same c e l l s a f t e r being washed i n buffer. It was demonstrated that a phagocytosis-promoting f a c t o r was found i n the supernatant obtained 76 Table XIV. I n h i b i t i o n of fibronectin-mediated macrophage non-opsonic uptake of P. aeruginosa s t r a i n H103 by exogenous PA01 p i l i Average number of bacteria/macrophage Macrophage a c t i v a t o r without p i l i exogenous p i l i None (PBS control) 7.1 6.71 In vivo supernatant 16.0 6.7 a Fibronectin RGDS 19.1 14.2 5.5s 5.0E ap<0.005 (Student's t test) s i g n i f i c a n t l y lower than the "without p i l i " c o n t r o l i n 2 independent assays. Dnot s i g n i f i c a n t l y lower than the "without p i l i " c o ntrol i n 2 independent assays. 77 Bacteria per Macrophage 25 -i in vivo in vitro Figure 17. Enhancement of the a s s o c i a t i o n of E. c o l i with P388 D 1 c e l l s using the supernatant from rat chambers of i n vivo-grown E. c o l l . 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 a d d i t i o n of i n vivo supernatant. Each column represents the average uptake observed i n 2 independent experiments. 78 Figure 18. Enhancement of the association of S. aureus with P388 D 1 cells using the supernatant from rat chambers of in vivo-grown S. aureus. Bacterial cells were washed (w) or unwashed (uw), with (+SN) and without the addition of in vivo supernatant. Each column represents the average uptake observed in 2 Independent experiments. 79 MIL 1 2 3 Figure 19. Western immune-blots of the supernatants of E. c o l i - and S. aureus-containing rat chambers probed with goat-anti-human f i b r o n e c t i n serum. Lane 1, E. c o l i i n vivo supernatant; lane 2, S. aureus i n vivo supernatant; lane 3, p u r i f i e d bovine f i b r o n e c t i n . 80 from chambers incubated i n the peritoneal c a v i t y of laboratory mice or r a t s . The phagocytosis-promoting f a c t o r was e f f e c t i v e with both s t r a i n s of P. aeruginosa tested, using both u n e l i c i t e d mouse peritoneal macrophages and the P388 D^ mouse macrophage c e l l l i n e as the phagocytic c e l l . Phagocytosis enhancement was observed with i n vivo-grown b a c t e r i a and with b a c t e r i a grown i n v i t r o on agar plates but not with b a c t e r i a grown i n v i t r o with rapid a g i t a t i o n . Supernatants from mice and rats were fra c t i o n a t e d using a Fast Pressure L i q u i d Chromatography g e l exclusion column. The phagocytosis-promoting 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 . Furthermore, a n t i - f i b r o n e c t i n sera negated the phagocytosis-promoting a c t i v i t i e s of i n vivo chamber supernatant, while commercial bovine f i b r o n e c t i n was i t s e l f capable of promoting phagocytosis. The concentration of f i b r o n e c t i n increased i n both rat and mouse per i t o n e a l chambers with time, coincident with the a b i l i t y of chamber supernatants to promote phagocytosis. I t was concluded that f i b r o n e c t i n was the phagocytosis-promoting f a c t o r of chamber supernatants. B a c t e r i a l presence i n the peritoneal chambers was not required to e l i c i t f i b r o n e c t i n uptake in t o the chambers. It was demonstrated that concentrations as low as 27 nM f i b r o n e c t i n produced s i g n i f i c a n t enhancement of macrophage phagocytosis. Washing of f i b r o n e c t i n - t r e a t e d macrophages d i d not prevent phagocytosis enhancement, but washing of 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 . The tetrapeptide a r g i n i n e - g l y c i n e - a s p a r t i c acid-serine, which comprises the eukaryotic c e l l binding domain of f i b r o n e c t i n , was also capable of promoting b a c t e r i a l uptake. Fibronectin caused d e p o l a r i z a t i o n of the P388 plasma 81 membrane, as demonstrated using a p o l a r i z a t i o n - s e n s i t i v e fluorescent probe. These data i n d i c a t e that promotion by f i b r o n e c t i n of non-opsonic phagocytosis i s mediated by a c t i v a t i o n of the macrophages. While f i b r o n e c t i n was able to s i g n i f i c a n t l y increase phagocytosis of P. aeruginosa grown i n s t a t i c broth, uptake of agitated b a c t e r i a could not be promoted. Phagocytosis of a mutant s t r a i n lacking surface p i l i could not be enhanced by f i b r o n e c t i n regardless of growth conditions. Furthermore, 60 yg/ml of exogenously added Pseudomonas p i l i was capable of abrogating the enhanced phagocytosis of the wild type s t r a i n observed with f i b r o n e c t i n - a c t i v a t e d macrophages. It was concluded that Pseudomonas p i l i were the b a c t e r i a l ligands required f o r attachment to fi 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 stages of non—opsonic phagocytosis. Fibronectin-mediated macrophage non-opsonic uptake was not r e s t r i c t e d to P. aeruginosa, but could be reproduced using i n vivo-grown E. c o l i or S. aureus. In these cases, however, uptake of agitated i n vitro-grown b a c t e r i a could be e f f i c i e n t l y enhanced using i n vivo supernatant. Both preparations of i n vivo supernatant were proven to contain substantial quantities of f i b r o n e c t i n as assessed using Western immunoblotting techniques. 82 DISCUSSION In t h i s thesis three types of i n t e r a c t i o n between macrophages and P. aeruginosa were studied. The f i r s t dealt with opsonic phagocytosis and le d to the establishment of an appropriate model c e l l l i n e f o r subsequent studies. The second set of experiments investigated one of the ways i n which Pseudomonas may protect i t s e l f from phagocytosis: v i a l i b e r a t i o n of a cytotoxin. These studies were designed to determine the c e l l u l a r l o c a l i z a t i o n of t h i s p rotein and to elucidate i t s e f f e c t on macrophage function. The f i n a l section of my thesis addressed fibronectin-mediated a c t i v a t i o n of non-opsonic phagocytosis of P. aeruginosa. Bach of these three i n t e r a c t i o n s w i l l be discussed separately below. 1. Use of the P388 p i macrophage c e l l l i n e as a model f o r macrophage  studies It was c l e a r from the data presented i n Table I and Figures 1-3 that monoclonal antibodies d i r e c t e d against porin protein F could act to opsonize P. aeruginosa f o r phagocytosis by mouse peritoneal macrophages, P388 D 1 c e l l s and human peripheral blood monocytes. There appeared to be some advantage i f antibody and phagocytes were of the same species. This was indicated when MA5-10 f a i l e d 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 three of s i x experiments performed on human c e l l s . This was not a phenomenon a t t r i b u t a b l e to isotype as other IgGl antibodies functioned quite well i n these t e s t s . 83 Certain trends could be seen i n the a b i l i t y of s p e c i f i c monoclonal antibodies to mediate phagocytosis by a l l three macrophage c e l l types. For example, MA4-10 always resulted i n the highest phagocytic index, MA5-8 always scored i n the middle to high region and MA4-4 was cons i s t e n t l y one of the two weakest opsonins. I t i s thus apparent that the mouse macrophage c e l l l i n e P388 D 1 can be used as a model f o r u n e l i c i t e d mouse peritoneal macrophages and cultured human peripheral blood monocytes. The appropriateness of t h i s model was confirmed i n subsequent studies of fi b r o n e c t i n - a c t i v a t e d macrophage non-opsonic phagocytosis. In these experiments, s i m i l a r l e v e l s of fibronectin-stimulated b a c t e r i a l uptake were observed f o r both P388 D 1 c e l l s or u n e l i c i t e d mouse peritoneal macrophages (Table VI). 2. Role of isotype i n opsonized phagocytosis by macrophages Previous data has suggested that there are at le a s t two d i s t i n c t highly conserved epitopes on porin protein F (Mutharia and Hancock, 1985). Monoclonal antibodies MA4-4, 2-10, 4-10 and 5-10 were a l l hypothesized to react against one epitope, while MA5-8 was s p e c i f i c f o r the other. Of the four monoclonal antibodies d i r e c t e d against a s i m i l a r epitope, the three IgGl monoclonal antibodies were s u b s t a n t i a l l y more opsonic than the one IgG2a isotype (Table I ) . In the past, there has been some dispute as to the opsonic p o t e n t i a l of IgGl. This isotype has been reported to 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 (Ralph et a l . , 1980; Schneider et a l . , 1981; Unkeless and Eisen, 1975) and a high a f f i n i t y f o r i s o l a t e d Fc receptors i n column systems (Schneider et a l . , 84 1981). It i s important to note that while macrophages bind monomeric IgGl with a r e l a t i v e l y low a f f i n i t y , high molecular weight aggregates of IgGl bind at le a s t as well as IgG2a and IgG2b (Heusser et a l . , 1977). This suggests that the IgGl-bacteria complex i s seen as an "agggregate" of immunoglobulin by the macrophage i n my system. Of the three IgGl antibodies hypothesized to be di r e c t e d against a common epitope of protein F, one of these, MA4-10, produced s u b s t a n t i a l l y better opsonophagocytosis i n a l l c e l l types tested. This may have been due to a better geometric complementarity between the antigen-binding pocket of MA4-10 and i t s corresponding antigenic determinant. A d d i t i o n a l l y , microheterogeneity i n the protein structure or g l y c o s y l a t i o n patterns of the Fc portion of MA4-10 may have produced a better binding a f f i n i t y f o r the macrophage Fc receptor. In e i t h e r case, increased a s s o c i a t i o n of b a c t e r i a with the macrophage would be the expected r e s u l t . These studies demonstrated that monoclonal antibodies d i r e c t e d against p r o t e i n F are capable of opsonizing P. aeruginosa f o r phagocytosis by a l l three macrophage c e l l types tested. This supports the proposal that the mechanism of protection a f f o r t e d by monoclonal antibodies i n vivo (Hancock et a l . , 1985) i s through opsonization f o r phagocytosis. The s p e c i f i c i t y and effectiveness of these a n t i - F monoclonal antibodies provides them with s u b s t a n t i a l p o t e n t i a l as immunotherapeutic agents. 3. Cytotoxin: l o c a l i z a t i o n and putative r o l e i n i n f e c t i o n The data presented i n Chapter Two of t h i s thesis i n d i c a t e that P. aeruginosa cytotoxin i s l o c a l i z e d i n the periplasmic space of t h i s 85 organism. Thus, osmotic shockates reproducibly demonstrated a 28 kD protein 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 cytotoxin (Pig. 5). In addition, osmotic shockates and cytotoxin both i n h i b i t e d opsonized phagocytosis of P. aeruginosa by macrophages, a phenomenon that could be abrogated by a n t i - c y t o t o x i n sera (Table I I ) . Furthermore, both preparations caused the formation of channels i n the macrophage membrane as assessed 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 fluorescent probe diSC^tS) (Table I I I , Figs. 7,8). None of the other c e l l f r a c t i o n s demonstrated s i m i l a r c o r r e l a t i o n s i n a c t i v i t y with p u r i f i e d cytotoxin. For example, 150-fold concentrated growth supernatants contained a 56 kD band that reacted with a n t i - c y t o t o x i n sera ( F i g . 5) and t h i s preparation could i n h i b i t phagocytosis (Table II) but was unable to depolarize the macrophage membrane (Table I I I ) . By use of anti-exoenzyme S antiserum ( F i g . 6), i t was possible to completely abrogate t h i s phagocytosis i n h i b i t i o n (Table II) and I ascribe t h i s crossreactive band to another Pseudomonas  aeruginosa toxin, exoenzyme S. These r e s u l t s suggest that exoenzyme S was the major macrophage i n h i b i t i n g t o x i n i n growth supernatant. Presumably other secreted Pseudomonal toxins such as exoproteases, lipases and exotoxin A were not i n f l u e n c i n g macrophages i n t h i s system. The f a c t that the a n t i - c y t o t o x i n sera recognizes the 56 kD band ( F i g . 5) may be due to c r o s s r e a c t i v i t y of cytotoxin with exoenzyme S or due to the presence of minor contaminating antibodies to exoenzyme S i n a n t i - c y t o t o x i n serum. Nevertheless, the a b i l i t y of cytotoxin and the 2+ Mg /freeze-thaw osmotic shockate to i n h i b i t macrophage phagocytosis was 86 not r e l a t e d to the presence of exoenzyme S i n these preparations, since anti-exoenzyme S sera d i d not react with these preparations on Western immunoblots ( F i g . 6) and d i d not reverse the i n h i b i t o r y e f f e c t s of these preparations i n phagocytosis of opsonized P. aeruginosa c e l l s (Table I I ) . It should be noted that i n s p i t e of the f a c t that the an t i - c y t o t o x i n sera recognized other bands i n complex protein preparations (growth supernatant, inner and outer membranes) only the 28 kD protein was reproducibly detected i n osmotic shockate. The i n a b i l i t y of LPS to cause d e p o l a r i z a t i o n of the macrophage membrane i n the fluorescence assay (Table III) eliminated the p o s s i b i l i t y that contaminating LPS i n the osmotic shock preparation was responsible f o r the observed r e s u l t s . Furthermore, inner and outer membrane f r a c t i o n s , containing large amounts of LPS, had the a b i l i t y to increase phagocytosis (Table I I ) . This suggested that LPS could a c t i v a t e macrophages, as previously described by others (Nowakowski et a l . , 1980). There i s sub s t a n t i a l precedent f o r the importance of periplasmic toxins i n pathogenesis. For example, Shiga toxin of S h i g e l l a dysenteriae 1 (Donahue-Rolfe and Keutsch, 1983), verotoxin ( s h i g a - l i k e toxin) of E. c o l i (Karmali et a l . , 1985) and h e a t - l a b i l e enterotoxin of E. c o l i (Evans et a l . , 1974) are a l l periplasmic, as indicated by polymyxin release experiments. The periplasmic l o c a t i o n of cytotoxin does not necessari l y argue against a ro l e f o r t h i s toxin i n pathogenesis of P. aeruginosa i n f e c t i o n s . Indeed, i t s l o c a t i o n i n the periplasm may be part of a population strategy of P. aeruginosa to allow the organism to maintain i t s presence at an i n f e c t i o n s i t e . In response to e i t h e r host 87 b a c t e r i c i d a l defense mechanisms (incl u d i n g complement-mediated l y s i s and phagocytosis) or a n t i b i o t i c therapy, cytotoxin and other periplasmic constituents may be released by l y s i s or damage of the outer membrane. Such l i b e r a t i o n of cytotoxin would have a s i g n i f i c a n t e f f e c t on host phagocytic c e l l s as suggested by the previously-observed k i l l i n g and l y s i s of neutrophils (Baltch et a l . , 1985; Scharmann et a l . , 1976), the strong d e p o l a r i z a t i o n of the macrophage membrane ( F i g . 8) and the reduced a b i l i t y of treated macrophages to phagocytose P. aeruginosa (Table I I ) . These l a s t two e f f e c t s may indeed be analogous since d e p o l a r i z a t i o n would d i s s i p a t e ion gradients required to s i g n a l i n i t i a t i o n of phagocytosis (Young et a l . , 1984) and thus r e s u l t i n the diminished uptake observed i n Table I I . It should be noted that cytotoxin appears to be marginally more ac t i v e against neutrophils as compared to macrophages. While concentrations as low as 6 ug/ml have been reported to cause complete l y s i s of granulocytes (Baltch et a l . , 1985), 13 ug/ml produced complete i n h i b i t i o n of phagocytosis but l i t t l e l y s i 3 i n macrophages. I t i s possible that macrophages di s p l a y an increased resistance to t h i s toxin. L i b e r a t i o n of cytotoxin by dead and dying P. aeruginosa c e l l s may be an important f a c t o r i n the persistent lung i n f e c t i o n of i n d i v i d u a l s with c y s t i c f i b r o s i s . Through i n h i b i t i o n of phagocytosis, these organisms may be capable of protecting the b a c t e r i a l population from a primary defense mechanism of the lung. 88 4. Fibronectin as an a c t i v a t o r of macrophage non-opsonic phagocytosis I n i t i a l studies demonstrated that the protein F - s p e c i f i c monoclonal antibody MA5-8 was capable of opsonizing i n vivo-grown P. aeruginosa f o r phagocytosis by mouse macrophage c e l l l i n e P388 D 1 ( F i g . 10). This f u r t h e r suggested that a n t i - F monoclonal antibodies have su b s t a n t i a l p o t e n t i a l as passive immunotherapeutic agents. An i n t e r e s t i n g f i n d i n g of these studies was that the average increase i n b a c t e r i a per phagocyte r e s u l t i n g from opsonization appeared to 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 . This suggested that the i n vivo-grown organisms may have had a reduced surface exposure of protein F r e l a t i v e to i n vitro-grown c e l l s . These studies also demonstrated that a phagocytosis promoting f a c t o r was found i n the supernatant obtained from P. aeruginosa chambers incubated i n the peritoneal c a v i t y of laboratory mice and r a t s . This f a c t o r could be separated 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 to again f a c i l i t a t e a s s o c i a t i o n of P. aeruginosa with mouse u n e l i c i t e d p e r i t o n e a l macrophages or macrophage c e l l l i n e P388p^ (Tables IV and VI). The ease of removal of t h i s f a c t o r suggested that i t was not an opsonin such as antibody or complement, which t y p i c a l l y have very high a f f i n i t i e s and thus remain attached to c e l l s during c e n t r i f u g a t i o n . Furthermore, treatment of i n vivo supernatant at 100°C f o r 10 minutes f a i l e d to e f f e c t phagocytosis enhancement (Table V). This treatment would have in a c t i v a t e d any complement or antibody proteins present. 89 This phenomenon was reproduced with two s t r a i n s of P. aeruginosa of d i f f e r e n t serotypes, using e i t h e r mice or rats as the i n vivo chamber host. A d d i t i o n a l l y , rat i n vivo supernatant could e f f e c t i v e l y promote uptake of plate-grown b a c t e r i a by P388^ c e l l s or u n e l i c i t e d mouse peri t o n e a l macrophages. The s i m i l a r r e s u l t s obtained with these two macrophage c e l l types confirmed that mouse macrophage c e l l l i n e P388 D 1 was an appropriate model f o r u n e l i c i t e d mouse peritoneal macrophages, as previously demonstrated i n the studies of opsonized phagocytosis of P. aeruginosa (see Chapter One). The f a c t that f i b r o n e c t i n c o p u r i f i e d with the phagocytosis-promoting a c t i v i t y using a gel s i e v i n g column suggested to us that f i b r o n e c t i n might be the f a c t o r i n question. Fibronectin has been shown previously to a c t i v a t e macrophages f o r increased adherence (Akiyama et a l . , 1981), C3 and Fc-receptor mediated phagocytosis of coated erythrocytes (Wright et a l . . 1983; Pommier et a l . , 1983), and maintenance of anti-staphylococcal a c t i v i t y (Proctor et a l . , 1985). However, no reports of i t s e f f e c t s on non-opsonic phagocytosis or on phagocytosis of a gram negative bacterium have appeared to date. In my studies, the phagocytosis-promoting a c t i v i t y of a commercially-available f i b r o n e c t i n preparation and the negating e f f e c t s of a n t i - f i b r o n e c t i n antibodies (Table IX) strongly supported the conclusion that f i b r o n e c t i n was the a c t i v e component of i n vivo supernatant. The s i m i l a r phagocytosis enhancement produced by i n vivo supernatants from chambers containing b a c t e r i a or s a l i n e (Table X) suggested that b a c t e r i a l presence was not required to e l i c i t the f i b r o n e c t i n response. 90 Confirmation of t h i s was obtained using Western b l o t t i n g techniques ( F i g . 14). These data suggested that f i b r o n e c t i n increased i n concentration over time i n the p e r i t o n e a l chambers, possibly due to s u r g i c a l i n j u r y or implantation of a f o r e i g n body (the chamber), but not n e c e s s a r i l y i n response to the b a c t e r i a . There i s a 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 (Grinnel, 1984). I t should be noted that P. aeruginosa commonly i n i t i a t e s i n f e c t i o n s at s i t e s of i n j u r y , including wounds and burns. The presence of f i b r o n e c t i n e a r l y i n the mouse time course experiments may have indicated a higher l e v e l of f i b r o n e c t i n i n the normal peritoneum of 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 have had more e f f i c i e n t d e l i v e r y of f i b r o n e c t i n to the s i t e of i n j u r y . The lower maximal l e v e l s of b a c t e r i a l a s s o c i a t i o n with macrophages found using rat i n vivo supernatant may however, r e f l e c t a macrophage preference f o r homologous f i b r o n e c t i n since the macrophages used were derived from mice. The data presented i n Table XI and F i g . 15 demonstrated that f i b r o n e c t i n acts as a macrophage a c t i v a t o r to stimulate increased phagocytosis of P. aeruginosa. The f a c t that macrophages retained enhanced a b i l i t y to phagocytose P. aeruginosa following incubation with and subsequent removal of f i b r o n e c t i n - c o n t a i n i n g i n vivo supernatant strongly favored t h i s i n t e r p r e t a t i o n . In contrast, treatment of b a c t e r i a with t h i s supernatant and subsequent washing p r i o r to mixing with macrophages resulted i n only background l e v e l s of phagocytosis. Furthermore, the tetrapeptide eukaryotic c e l l binding domain was s u f f i c i e n t to a c t i v a t e macrophages, and 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 reminiscent of other macrophage a c t i v a t o r s . It i s thus apparent that f i b r o n e c t i n acts not as an opsonin, l e c t i n or ligand i n t h i s system, but acts d i r e c t l y on the macrophages to stimulate uptake. Fibronectin i s a large dimeric glycoprotein whose structure can be divided into several f u n c t i o n a l domains. These areas have been named according to the substances which bind i n that region of the molecle (Proctor, 1987). The eukaryotic cell-adhesion region had been previously shown to i n t e r a c t with various mammalian c e l l types including macrophages (Brown and Goodwin, 1988). A four amino a c i d sequence, RGDS, i n t h i s c e l l binding domain has been shown to i n t e r a c t with the mammalian c e l l - s u r f a c e glycoprotein H b / I I I a (Brown and Goodwin, 1988). In our studies, a commercially a v a i l a b l e RGDS preparation was able to s i g n i f i c a n t l y increase non-opsonic uptake of Pseudomonas by mouse macrophage c e l l l i n e P388j^ (F i g . 15). Although 80 f o l d higher molar concentrations of RGDS than f i b r o n e c t i n were required to observe t h i s e f f e c t , t h i s i s consistent with previous studies (Pierschbacher and Ruoslahti, 1984) and the notion that the structure of the tetrapeptide would be conformationally constrained i n the i n t a c t f i b r o n e c t i n molecule. Using the p o l a r i z a t i o n - s e n s i t i v e fluorescent probe d i S C 3 ( 5 ) , i t was determined that f i b r o n e c t i n could generate a strong ion f l u x across the macrophage membrane at concentrations as low as 27 nM ( F i g . 16). This r e s u l t correlated well with studies of the concentration requirements of f i b r o n e c t i n i n promoting phagocytosis ( F i g . 15). While maximal phagocytosis was observed at concentrations around 50 nM, the rate of ion f l u x generation increased as a function of the l e v e l of f i b r o n e c t i n to at 92 le a s t 230 nM. This suggested that while higher concentrations of f i b r o n e c t i n can produce greater ion f l u x over the macrophage membrane, lower concentrations and t h e i r correspondingly lower rates of ion f l u x were s u f f i c i e n t to maximally enhance phagocytosis at a b a c t e r i a to macrophage r a t i o of 20:1. While these studies do not prove i t , t h i s perturbation of ion gradients across the membrane may be the phagocytic 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 uptake of P. aeruginosa. The data presented i n Table XIII demonstrated that Pseudomonas p i l i were the b a c t e r i a l ligands 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 phagocytosis. The f a c t that exogenously added p i l i could abrogate the a b i l i t y of f i b r o n e c t i n to enhance phagocytosis by mouse macrophage c e l l l i n e P388 n i strongly favoured t h i s i n t e r p r e t a t i o n (Table XIV). Fibronectin promotion of macrophage phagocytosis was observed with b a c t e r i a grown on agar plates, to a l e s s e r extent with organisms grown i n s t a t i c broth, and not a l l with r a p i d l y agitated cultures (Table XII). As a g i t a t i o n i s an accepted method of removing p i l i from Pseudomonas, one might expect progressively fewer p i l i per c e l l on increasing a g i t a t i o n . In our studies, f i b r o n e c t i n was unable to enhance uptake of a s t r a i n of P. aeruginosa lacking surface p i l i (Table XIII). This s t r a i n was constructed by transposon Tn501 i n s e r t i o n into the cloned chromosomal p i l i n gene followed by gene replacement. In control experiments, the PA01 parent s t r a i n of t h i s Tn501-induced mutant and a pilus-expressing s t r a i n containing both the Tn501 mutation and an a d d i t i o n a l plasmid-encoded p i l i n gene (pBPl6l) were each susceptible to 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 (Table XIII). This confirmed that the absence of enhanced phagocytosis was not due to properties of t h i s p a r t i c u l a r parent s t r a i n or to transposon Tn501 sequences. This data i s consistent with previous studies showing that heavily p i l i a t e d s t r a i n s of P. aeruginosa were more susceptible to polymorphonuclear leukocyte (PMN) phagocytosis than less p i l i a t e d s t r a i n s (Paranchych et a l . , 1986). It was also determined that exogenous p i l i were capable of i n h i b i t i n g PMN non-opsonic uptake of t h i s organism (Paranchych et a l . , 1986). This suggests that P. aeruginosa p i l i are the b a c t e r i a l ligand to which both PMN and macrophage populations attach to i n i t i a t e non-opsonic phagocytosis. McEachran and I r v i n (1985) had determined that buccal e p i t h e l i a l c e l l s d i s p l a y at least two classes of receptors f o r binding of P. aeruginosa. The f i r s t i s a low copy number-high a f f i n i t y receptor f o r p i l i , while the second i s a high copy number-low a f f i n i t y receptor which may recognize alginate, the primary component of mucoid exopolysaccharide. I t seemed u n l i k e l y that increased mucoid exopolysaccharide was responsible f o r the increased uptake observed with statically-grown organisms, however, as organisms which produce large amounts of alginate are more r e s i s t a n t to phagocytosis than non-mucoid b a c t e r i a (Baltimore and M i t c h e l l , 1980; Simpson et a l . , 1988). Furthermore, alginate from some st r a i n s of P. aeruginosa i s incapable of binding even to buccal e p i t h e l i a l c e l l s (Doig et a l . , 1987). Baltimore and M i t c h e l l (1980) hypothesized that mucoid coating may mask b a c t e r i a l ligands required f o r e f f i c i e n t phagocytosis. Perhaps surface-expressed p i l i are these c r u c i a l b a c t e r i a l ligands. 94 Studies performed using E. c o l i and S. aureus indicated that f i b r o n e c t i n - c o n t a i n i n g i n vivo supernatant was capable of s i g n i f i c a n t l y enhancing macrophage phagocytosis of both i n vivo- and agitated i n vitro-grown b a c t e r i a ( F i g . 17,18). It i s possible that a g i t a t i o n i s incapable of removing the E. c o l i or S. aureus b a c t e r i a l ligands to which activated macrophages bind. 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 be acting as a ligand or opsonin i n promoting a s s o c i a t i o n of these organisms with the phagocyte. Indeed, previous investigators have suggested that f i b r o n e c t i n can indeed opsonize Staphylococcus f o r phagocytosis (Proctor et a l . , 1982). This seems u n l i k e l y i n the case of E. c o l i . as f i b r o n e c t i n i s unable to mediate attachment of t h i s organism to polymorphonuclear leukocytes (Proctor et a l . , 1985). Non-opsonic uptake may be a important clearance mechanism at s i t e s of P. aeruginosa i n f e c t i o n . These areas often display t i s s u e i n j u r y and would therefore possess a high l o c a l concentration of f i b r o n e c t i n (Grinnel, 1984). As such, i n f i l t r a t i n g macrophages could be activated by f i b r o n e c t i n to enhance t h e i r a s s o c i a t i o n with b a c t e r i a v i a surface-expressed p i l i . A conceptual model of f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic phagocytosis i s presented i n Figure 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. aeruginosa from the s i t e of i n f e c t i o n . In addition, t h i s would provide a rapid source of macrophage-processed P. aeruginosa antigens f o r presentation to the appropriate subsets of T-lymphocytes, allowing i n i t i a t i o n of the c e l l u l a r and humoral immune responses. 95 Fibronectin activates macrophages Non-activated macrophage (receptors are inactive (-») and cannot bind to bacterial surface pili) Activated macrophage (active receptors (A) can now bind to surface-expressed pili and promote uptake) Figure 20. A conceptual model of f i b r o n e c t i n - a c t i v a t e d macrophage non-opsonic phagocytosis of P. aeruginosa. Non-activated macrophages have in a c c e s s i b l e , i n a c t i v e or absent Pseudomonas p i l i receptors and thus cannot bind to the b a c t e r i a l surface. 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