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The role of Alveolar macrophages in defense of the lung against Pseudomonas aeruginosa Cheung, Dorothy On Yan 1999

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THE ROLE OF ALVEOLAR MACROPHAGES IN DEFENSE OF THE LUNG AGAINST Pseudomonas aeruginosa by DOROTHY ON YAN CHEUNG B.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1999 © Dorothy On Yan Cheung, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I 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. The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT The objective of this study was to evaluate the role of alveolar macrophages (AM) in clearance of Pseudomonas aeruginosa in mice after intrapulmonary challenge. Alveolar macrophages were depleted by intranasal administration of liposome-encapsulated dichloromethylene diphosphonate (LDMDP). Control mice received an equal volume of phosphate buffered saline encapsulated liposomes (LPBS). 24 hours post-instillation of liposomes, a sublethal dose of P. aeruginosa was inoculated intranasally into mice. Lungs, spleen and liver were obtained from the two groups of mice at different time points; viable bacterial counts and histology were then analyzed. 78 - 88 % of alveolar macrophage depletion did not affect the survival rate of infected mice, or clearance of P. aeruginosa from the lung, spleen or liver, as compared to the control group. Recruitment of neutrophils in the lung was similar in both groups. As well, m vitro experiments have shown that freshly explanted alveolar macrophages were not competent to phagocytose unopsonzied P. aeruginosa, but were able to phagoeytose zymosan particles. Further studies were conducted to assess in situ phagocytic activities of alveolar macrophages. Three hours after the intranasal instillation of P. aeruginosa or other particles, bronchoalveolar lavage (BAL) was performed. Alveolar macrophage phagocytosis of zymosan particles (23%) and latex beads (27%) was much higher than that of P. aeruginosa (0-5%). Neutrophils were recruited to the lung in response to a high bacterial dose. These results suggest that the role of alveolar macrophages in the defense of the lung against P. aeruginosa challenge may not extensively involve their phagocytic activities. iii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables vii List of Figures viii Acknowledgements ix Abbreviations x INTRODUCTION 1 Overview 1 Cystic Fibrosis 1 Pathogenesis of Pseudomonas aeruginosa in Cystic Fibrosis 2 P. aeruginosa 3 a. Secreted virulence factors 3 b. Lipopolysaccharides, the endotoxin 4 Alveolar Macrophages 5 a. Micro-environment in the lower respiratory tract 5 b. Professional phagocytes 5 c. Secretory cells 6 d. Inhibition of AM functions using different particles 9 e. Suppression of AM functions using liposomes 9 f. AM depletion by liposome-encapsulated dichloromethylene 10 diphosphonate in animal models In Vitro Macrophage Phagocytosis 11 RATIONALE AND OBJECTIVE 12 iv MATERIALS AND METHODS 13 P. aeruginosa strains and culture conditions 13 Animals 13 Reagents 13 Preparation of PBS-, DMDP- and FITC-liposomes 14 Intranasal instillation of liposomes 15 Intranasal administration of P. aeruginosa 15 Intranasal administration of latex beads and zymosan particles 16 Bronchoalveolar lavage 16 Cytospin preparations 17 Determination of the numbers of alveolar macrophages and neutrophils in 17 bronchoalveolar lavage Analysis of lung, spleen and liver CFU and histopathology 18 Preparation of murine resident peritoneal macrophages 18 In vitro effects of LPBS, LDMDP, DMDP and LFITC on murine alveolar 19 and peritoneal macrophages In vitro phagocytosis assays 19 In vitro effects of LPBS, LDMDP and DMDP on epithelial cell line A549 20 In situ phagocytosis assays 21 Statistical analysis 21 RESULTS In vitro effects of LPBS, LDMDP and DMDP on murine peritoneal macrophages In vitro phagocytosis of unopsonized P. aeruginosa by murine peritoneal macrophages incubated with LPBS, LDMDP and DMDP for 24 and 48 h In vitro effects of LPBS, LDMDP, DMDP and LFITC on murine alveolar macrophages In vitro phagocytosis of unopsonized P. aeruginosa by murine alveolar macrophages incubated with LPBS, LDMDP and DMDP for 24, 48 and 72 h In vitro effects of LPBS, LDMDP and DMDP on respiratory epithelial cell line A549 The route of administration of LPBS and LDMDP to the airspace in mice In vivo depletion of alveolar macrophages by intranasal instillation of DMDP-liposomes The strain of mice, route of bacterial inoculation and bacterial strains in P. aeruginosa infection model Intranasal inoculation and clearance of P. aeruginosa in untreated mice Effects of AM depletion on P. aeruginosa clearance in vivo In vitro phagocytic activity of freshly explanted alveolar macrophages In situ phagocytic activities of alveolar macrophages DISCUSSION In vitro effects of LPBS, LDMDP and DMDP on macrophages and epithelial cells In vivo depletion of alveolar macrophages by intranasal instillation of DMDP-liposomes Intranasal inoculation and clearance of P. aeruginosa in untreated, nondepleted and AM-depleted mice In vitro phagocytic activities of freshly explanted alveolar macrophages Particle phagocytosis by alveolar macrophages in situ BIBLIOGRAPHY LIST OF TABLES Table 1. Major virulence factors of P. aeruginosa Table 2. Secretory products from macrophages Table 3. In vitro phagocytosis of unopsonized P. aeruginosa by murine peritoneal macropahges incubated with LPBS, LDMDP and DMDP for 24 and 48 h. Table 4. In vitro phagocytosis of unopsonized P. aeruginosa by murine alveolar macropahges incubated with LPBS, LDMDP and DMDP for 24, 48 and 72 h. Table 5. In vitro phagocytic activities of freshly explanted murine alveolar macarophages and resident peritoneal macrophages. viii LIST OF FIGURES Fig. 1. Effects of LPBS, LDMDP and DMDP on murine peritoneal macrophages (PM) 23 in vitro. Fig. 2. Effects of LPBS, LDMDP and DMDP on murine alveolar macrophages (AM) 27 in vitro. Fig. 3. Phagocytosis of liposome-encapsulated FITC by alveolar macrophages. 28 Fig. 4. Depletion of alveolar macrophages in vivo by intranasal (i.n.) instillation of 34 DMDP-liposomes. Fig. 5. Histologic sections of LPBS- and LDMDP-instilled lung in mice. 35 Fig. 6. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. 40 Fig. 7. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. 41 Fig. 8. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. 42 Fig. 9. Effects of alveolar macrophage depletion in B ALB/c mice on P. aeruginosa 45 (FRD1) clearance in vivo. Fig. 10. Effects of alveolar macrophage depletion in BALB/c mice on P. aeruginosa 46 (PAOl) clearance in vivo. Fig. 11. Effects of alveolar macrophage depletion in CD-I mice on P. aeruginosa 47 (PAOl) clearance in vivo. Fig. 12. Histologic sections and BAL cytospin preparations from P. aeruginosa- 48 challenged LPBS- and LDMDP-treated mice. Fig. 13. In vitro phagocytic activities of freshly explanted alveolar macrophages and 51 resident peritoneal macrophages Fig. 14. In situ phagocytic activities of alveolar macrophages. 57 ix ACKNOWLEDGEMENTS I would like to express sincere gratitude to my supervisor Dr. D. P. Speert for his remarkable patience, guidance and vision throughout my project. I also thank my graduate supervisory committee - Drs. J. K. Chantler, W. R. McMaster, and P. Pare, for their help and advice. I appreciate the colleagues in the Speert, Stokes and Mahenthiralingam laboratories, for sharing their knowledge and expertise. Finally, I would like to extend my thanks to my family and friends for their support and encouragement. This project was supported by a studentship from the Canadian Cystic Fibrosis Foundation. ABBREVIATIONS AM = alveolar macrophages BAL = bronchoalveolar lavage CSF = colony stimulating factors DMDP = dichloromethylene diphosphonate i.n. = intranasal i.t. = intratracheal IFN = interferon IL = interleukin LDMDP = liposome-encapsulated dichloromethylene diphosphonate LFITC = liposome-encapsulated fluorescein isothiocyanate LPBS = liposome-encapsulated phosphate buffered saline LPS = lipopolysaccharides LRT = lower respiratory tract MIP-2 = macrophage inflammatory protein-2 NO = nitric oxide PM = peritoneal macrophages PMN = polymorphonuclear leukocytes TNF = tumor necrosis factor TS A = trypticase soy agar 1 INTRODUCTION Overview Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen, that causes chronic infections in patients with cystic fibrosis (CF). Infection 'of the lung is the leading cause of morbidity and mortality in CF (20, 48). Alveolar macrophages (AM), strategically situated at the air-tissue interface in the alveoli and alveolar ducts, provide the first line of defense against inhaled organisms in the lower respiratory tract (LRT). Besides their phagocytic and microbicidal functions, AM also secrete numerous chemical mediators upon stimulation, thereby playing a role in regulating inflammatory reactions in the lung (24). The role of AM in defense of the lung against P. aeruginosa is yet to be elucidated, and is the topic of investigation in this thesis. Cystic Fibrosis The syndrome of cystic fibrosis (CF) of the pancreas was first described by D. H. Anderson in 1938. In her clinical description of the disease, the two fundamental pathophysiologic problems in CF - pancreatic insufficiency with malnutrition and airway infections - are related to the finding that the patients produce extremely viscous secretions. The secretions result in autodigestion of the pancreas through blockage of pancreatic ducts and inability to release digestive enzymes. In the airways, the viscous secretions lead to chronic infection due to numerous types of organisms. In the past few decades, enormous efforts have been made in describing this disease from clinical, pathological and genetic perspectives. CF is now recognized as an autosomal recessive trait found in approximately 1 in 2000 to 2500 Caucasian children and at a much lower rate in Hispanic and black populations. Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for CF, is located on 2 chromosome 7 and its gene product influences the movement of chloride directly or indirectly (17, 48, 59). More than 50 mutations have been found in the putative CFTR gene product. In 68 - 70 % of CF cases, a deletion of three nucleotides causes the protein product of the CFTR to lack an amino acid, phenylalanine at position 508. CF affects various organs, including airways, liver, pancreas, small intestine, reproductive tract and skin (sweat glands). However, pulmonary impairment accounts for 90% of disability and deaths in CF patients and is mainly a result of chronic airway infection (17, 22, 59). Pathogenesis of Pseudomonas aeruginosa in Cystic Fibrosis The CF lung presents a unique environment for potential microbial invasion. A genetic defect in the CFTR results in an ionic imbalance across respiratory epithelium. Consequently, such ionic imbalance leads to altered secretions and eventually thickened mucus in the respiratory airway, providing an environment for bacterial growth. Lung infection is initiated by attachment to and subsequent colonization of mucosal epithelium of the upper respiratory tract by microbes followed by descending infection. Bacterial colonization and infection of the CF lung occurs soon after birth. Staphylococcus aureus is the first detected pathogen, and followed by a number of micro-organisms, with P. aeruginosa the predominant pathogen. P. aeruginosa is recovered from 50 - 70 % of respiratory tract cultures of chronically infected CF patients (9, 17). Adherence of P. aeruginosa to mammalian epithelial cells is promoted by the pili (11). Following the initial lung infection by nonmucoid strains of P. aeruginosa, mucoid strains predominate and are isolated in 50 - 90 % of all CF patients (9). Once acquired, this bacterium is difficult to treat and is never eradicated. Most strains of P. aeruginosa show substantial degrees of resistance to a wide variety of antimicrobial agents, including P-lactams, tetracyclines, chloramphenicol and fluoroquinolones (31). 3 P. aeruginosa a. Secreted virulence products A variety of exoproducts, such as exotoxin A, exoenzyme S, elastase, alkaline protease and phospholipase C, secreted by P. aeruginosa may contribute to its pathogensis (9, 21). Table 1 summarizes major virulence factors of this pathogen. Exotoxin A (ETA) is classified as a member of the adenosine-diphosphate-ribosylating toxins, which include diphtheria toxin, cholera toxin, pertusis toxin and E. coli heat-labile enterotoxin. ETA transfers ADP-ribose to eucaryotic elongation factor 2 (EF2), resulting in the loss of protein synthesis in the target cell. This toxin also impairs macrophage phagocytic function at low concentrations (10 ng/ml). Exoenzyme S (Exo-S) catalyzes the transfer of ADP-ribose to various eucaryotic proteins, but not EF2. It may also act as a functional adhesin for P. aeruginosa adherence to susceptible tissue (9). In both burn and acute lung infection models, Exo-S production correlates with the dissemination of P. aeruginosa from epithelial colonization sites to result in systemic infection (14). P. aeruginosa produces and secretes two proteolytic enzymes - elastase and alkaline protease, with elastase the more active and abundant. Phospholipase C (PLC), a hemolytic toxin, may be associated with the virulence of P. aeruginosa. PLC and alkaline phosphatase act together as a phosphate scavenging system. PLC degrades phospholipids commonly present in eucaryotic cell membranes but not in procaryotic cell membranes. Diacylglycerol, produced in the process of phospholipid degradation, can indirectly cause toxic side effects in animals (9). Table 1 Major virulence (actors of P. aeruginosa. 4 Virulence factor Potential role Pili Specific adherence Flagella Motility Alginate Anti-phagocytosis ?adhesin antibiotic resistance ?immune injury Rhamnolipid Inhibits neutrophils Cilostatic Pigments Fe acquisition Bacteriocins Cilostatic Stimulates neutrophils Proteases (elastase) Invasiveness, cleavage of Ig+C Phospholipase C Invasiveness, colonizatkxi. nutrients stimulates neutrophils Exoenzyme S Invasiveness, local tissue destruction Exotoxin A Invasiveness, tissue destruction anti-phagocytosis Leukocidin Destroys neutrophils K. Grimwood. 1992. J. Pediatr. Child Health 28: 4-11. b. Lipopolysaccharides, the endotoxin The cell wall of P. aeruginosa consists of a lipid bilayer called the outer membrane, which contains several compounds such as lipopolysaccharides (LPS), lipoptroteins (LP), and specialized outer membrane pore-forming proteins. LPS is the endotoxin of Gram-negative bacteria. It provides essential physiological functions for the bacteria (e.g. rigidity of the cell wall, protection against antibiotics and host defenses), and is a potent stimulator of the inflammatory response. P. aeruginosa LPS consists of three regions - lipid A (a lipid moiety), the core region (an oligosaccharide unit up to fifteen monosaccharides) and the O-antigen (polysaccharide of repeating units). LPS, when complexed with the plasma protein, LPS-binding protein (LPB), binds to CD 14 on macrophages. The interaction among LPS-LPB complexes and macrophage receptors leads to the transmission of macrophage-activating signals, and contributes to the inflammatory responses typically associated with endotoxemia. LPS induces the secretion of tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, prostaglandins and cytolytic proteases by macrophages (24). 5 Alveolar Macrophages a. Micro-environment in the lower respiratory tract Alveolar macrophages (AM), located at the interphase between air and lung tissue, provide the first line of cell-mediated defense against microbial invasion in the LRT. AM reside in an environment rich in surfactant, which is produced and secreted by type II epithelial cells. Surfactant forms a film on the luminal surface of the alveoli and thereby reduces surface tension between air and the lung. Surfactant is a unique macromolecular complex of phospholipids and 10% protein. Surfactant consists of at least four surfactant protein (SP)-A, SP-B, SP-C and SP-D, with SP-A the predominant and most abundant. SP-A increases antibacterial and antiviral functions of AM. SP-A enhances serum-dependent phagocytosis by acting as an opsonin, and serum-independent phagocytosis by stimulating macrophages directly without binding to bacteria. In addition, SP-A increases production of superoxide radicals and chemotactic migration of AM (16, 29, 39). b. Professional phagocytes AM are known to be major phagocytes in the LRT. Phagocytosis is categorized into opsonic and nonopsonic mechanisms. Opsonic phagocytosis is mediated by Fc and complement receptors. However, human and mouse AM show minimal expression of receptors for C3b/iC3b (CR1/CR3/CR4). The receptor for IgG (FcyR) is expressed in human AM, but negligibly in mouse AM (3, 53). In addition, the concentrations of complement and immunoglobulin in the LRT are considerably low; therefore, it is likely that AM utilize nonopsonic, instead of Fc or complement receptor-mediated, phagocytosis to eliminate pathogens such as P. aeruginosa from the lung. Ingestion of bacteria in the absence of opsonins depends on the chemical composition of the bacterial surface and the complementary macrophage receptors; pili of P. aeruginosa enhance the bacterium's susceptibility to macrophage ingestion (24). Phagocytosis is an 6 important mechanism to confine the destruction of invading pathogens. The most potentially destructive processes, such as lysosomal enzyme release and production of reactive oxygen and nitrogen intermediates, occur in internal compartments of a few cells (6). The professional phagocytes - macrophages, monocytes, neutrophils and eosinophils - are capable of killing ingested microbes by both oxidative and nonoxidative mechanisms. In the oxygen-dependent microbicidal mechanism, phagocytic cells produce an array of reactive oxygen radicals with the capacity to kill ingested bacteria. AM are effective in producing reactive oxygen intermediates and reactive nitrogen intermediates. Reactive oxygen intermediates are induced during phagocytosis (15). The production of reactive nitrogen intermediates, induced by TMFoc, contributes to the microbicidal function of macrophages (19). AM and neutrophils can also kill bacteria in the absence of an oxidative burst. Proteins with antimicrobial activity are packaged within phagocytic lysosomes and released to the site of ingested bacteria upon phagosome-lysosome fusion. Among these proteins are defensins, cathepsin G, lysozyme, lactoferrin and cationic proteins (9, 24). c. Secretory cells Certain specificity in generation of host inflammation is achieved because phagocytosis is mediated by recognition of ligands on the foreign particle by individual receptors on the phagocytes. AM express various receptors, such as Fc, complement and mannose, at different stages (6). Macrophages not only phagocytose foreign particles but also release various products (Table 2). Cytokines secreted by macrophages include tumor necrosis factor-alpha (TNFa), transforming growth factor, colony-stimulating factors (CSF), interferon (IFN) and the interleukin (IL) families (30, 47). In addition to releasing proinflammatory substances, such as TNFa, nitric oxide (NO) and arachidonic acid metabolites, and recruiting polymorphonuclear leukocytes (PMN) to the lower airway, AM also engulf apoptotic PMN and secrete anti-7 inflammatory mediators (27, 32). TNF-a induces production of cytokines by monocytes and macrophages, and increased protein synthesis in the liver, including complement components and acute-phase proteins. CSF stimulate hematopoietic stem cells or progenitor cells to form colonies, a collection of cells all descending from the same ancestral cell through cell division. Granulocyte-macrophage CSF (GM-CSF) is produced by various cell types, including macrophages, neutrophils, T cells and B cells, in response to stimulation by certain cytokines or other factors during inflammation. GM-CSF is a pleiotropic cytokine, which induces the proliferation, maturation and activation of different hematopoietic cells at various development stages. Monocyte-macrophage CSF (M-CSF or CSF-1) is produced by a number of cell types, such as macrophages, B cells, T cells and endothelial cells upon stimulation, by other cytokines or LPS. M-CSF is particularly important for the proliferation, differentiation and activation of the monocyte-macrophage lineage of hematopoietic cells. Granulocyte CSF (G-CSF) is secreted primarily by T cells, and by monocytes and macrophages which have been stimulated by IL-1, TNF-a, IFN-y or LPS. G-CSF promotes the proliferation, differentiation and activation of the neutrophil lineage of hematopoietic cells. IL-1 induces the proliferation of T cells, synthesis of hepatic acute phase protein (e.g. serum amyloid A, C-reactive proteins, complement components, fibrinogen and antiproteases), and production of metalloproteins, which bind serum iron and zinc essential for bacterial growth. IL-6 promotes T-cell proliferation, enhance phagocytosis and expression of Fey receptors. IL-8 exhibits chemotactic activity for PMN, T cells and basophils. It also stimulates PMN to release lysosomal enzymes and to adhere to endothelium surface. T-and natural killer cells respond to IL-12 with increased cytokine production, primarily IFN-y, a potent enhancer of the microbicidal activity of phagocytic cells. IFN-y activates macrophage secretion of numerous products such as reactive oxygen products, TNF-a and IL-1 (24). However, IFN-y suppresses macrophage nonopsonic and opsonic receptor-mediated 8 phagocytosis, but enhances oxidative radical production (50). IFN-y has diverse effects on macrophage receptor expression: selective Fc receptor expression is enhanced, mannose receptor expression is down-regulated, and complement receptor expression is unaffected. IL-10 is a potent immunosuppressant of macrophage functions by depressing their antigen-presenting capacity. Transforming growth factor-(3 inhibits lymphocyte response and inflammation in general (41, 47). Table 2. Secretory products from macrophages E n z y m e s Lysozyme Acid hydrolases (proteases, nucleases, glycosidases, phosphatases, l ipases, etc) Elastase Col lagenase Plasminogen activator Angiotensin-converting enzyme Media tors Interferons (IFNa, IFNP) Colony-stimulating factors (GM-CSF, M-CSF, G - C 3 F , and others) Interleukins (IL-1, IL-6, IL-8, IL-10, IL-12) Chemokines Tumor necrosis factor a (TNFa) Platelet-derived growth factor Platelet-activating factor (PAF) Transforming growth factor p (TGFp) Angiogenesis factors Nitric oxide Arachidonate derivatives (prostaglandins, leukotrienes) Comp lemen t componen ts C 1 - C 9 Properdin Factors B, D, I, and H Coagu la t i on factors Factors V, VII, IX, and X Prothrombin Thromboplastin React ive oxygen spec ies Hydrogen peroxide Superoxide anion Nitric oxide Singlet oxygen Hydroxyl radicals M isce l l aneous Glutathione Nucleotides (adenosine, thymidine, guanosine, etc) Stites, D. P., A. I. Terr, and T. G. Parslow. 1997. Medical Immunology. 9 ed. Appleton & Lange. Connecticut. 9 d. Inhibition of AM functions using different particles The role of AM in defense of the lung against various pathogens has been investigated by suppression of macrophage functions. Depletion of AM and blocking of phagocytosis in vivo can be achieved by administration of silica particles, carrageenan and dextran sulfate, gadolinium, anti-macrophage antibodies and receptor antagonists, liposomes, and liposome-encapsulated drugs. The interaction of silica with the plasma membrane leads to calcium ion influx, resulting in ATP depletion and cell death. However, sublethal doses of silica induce monocytes or macrophages to produce IL-1, IL-6, TNFa and NO. Both carrageenan and dextran sulfate deplete or suppress phagocytic activity. However, they also have a strong effect on lymphocytes, and enhance the macrophage-mediated effects of LPS-induced septic shock and TNF-a production. Although in vitro exposure of rat AM to gadolinium chloride results in cell death by apoptosis, intravenous injection of the compound in mice does not result in depletion of Kupffer cells in the liver, but enhances the proliferation of non-phagocytic cells. Receptor antagonists and antibodies, directed against macrophage receptors to suppress receptor-mediated phagocytosis, may be internalized and hydrolyzed in a short period of time (56). e. Suppression of AM functions using liposomes In addition to silica particles, carrageenan, dextran sulfate, gadolinium, anti-macrophage antibodies and receptor antagonists, liposomes are used to suppress macrophage functions. Liposomes are artificially prepared spheres, consisting of concentric phospholipid bilayers separated by aqueous compartments. When dispersed in water, phospholipid molecules, (e.g. phosphatidylcholine) find a conformation in which the relatively hydrophilic head groups make up both of the outer parts of each bilayer, while the hydrophobic fatty acid chains are located directly opposed to each other in the inner side of the bilayer (57). In contrast to silica, carrageenan, dextran sulfate and gadolinium chloride, liposomes do not stimulate the basic or 10 LPS-induced production of proinflammatory cytokines and/or NO by macrophages. Aqueous solutions containing hydrophilic molecules, such as dichloromethylene diphosphonate (DMDP), are encapsulated during the preparation of liposomes. The usual fate of liposomes is ingestion and digestion by macrophages. Due to its strong hydrophilic properties, DMDP is not able to escape from the cell by crossing the cell membranes when released intracellularly (56). DMDP (mol. wt. 289) is one of the bisphosphonates used for treating osteolytic bone diseases. Bisphosphonates have a strong infinity for metal ions such as calcium, magnesium and especially iron (13). Liposomally delivered DMDP induces apoptosis in phagocytic cells in vitro and in vivo. Yet, the mechanisms by which DMDP affects cellular metabolism are still unknown. Free and encapsulated DMDP inhibits rather than stimulates cytokine production by macrophages (56). Since liposomes cannot cross capillary walls and other vascular barriers, various macrophage populations can be depleted through the use of different administration routes. AM are depleted by liposome-encapsulated dichloromethylene diphosphonate (LDMDP) delivered to the pulmonary tract in rodent models (2, 5, 23, 27, 32). f. AM depletion by liposome-encapsulated dichloromethylene diphosphonate in animal models The role of macrophages in removing foreign particles and regulating inflammatory reactions has been assessed in studies in which AM are depleted. 70 - 95% of AM can be depleted by administration of LDMDP to the lung in rodent models (2, 5, 23, 32). After inoculation of a normally sublethal dose of Klebsiella pneumoniae, survival in AM-depleted mice decreases dramatically. The increased mortality is accompanied by increased numbers of K. pneumoniae CFU in lung and plasma, compared to untreated mice. 48 h post-infection, PMN recruitment in DMDP-treated lungs is higher than that in control infected lungs, suggesting that decreased K. pneumoniae clearance is not due to impaired PMN influx. As well, neutralization of TNF-a or 11 macrophage inflammatory protein-2 (MIP-2), before infection, reduces the numbers of PMN (5). In addition, depletion of macrophages in rats results in substantial impairment of Pneumocystis carinii clearance compared to liposomal-PBS- or PBS-treated rats (32). 4 h after inoculation of P. aeruginosa to the airspace, PMN counts in lavage fluid from AM-depleted rats are much lower than those from PBS-treated rats. However, after instillation of recombinant MIP-2 into liposomal DMDP-treated rats, the number of PMN recruited into airspaces increased to a level similar to control rats given the same dose of MIP-2 (23). In Vitro Macrophage Phagocytosis In vitro response of macrophages to challenge with P. aeruginosa and other particles has been investigated. Phagocytosis of unopsonized P. aeruginosa by murine peritoneal macrophages and by cultivated mouse and human AM is dependent on the presence of D-glucose; bacterial binding to macrophages occurs in the absence of D-glucose. However, phagocytosis of unopsonized zymosan, opsonized P. aeruginosa, or IgG-coated sheep erythrocytes is not dependent on the presence of glucose in the phagocytic medium (1, 3, 10, 49, 60). The presence of a flagellum is required for phagocytosis, but not binding, of unopsonized P. aeruginosa by murine macrophages. The surface factors flagella, pili and non-pilus adhesins are often absent in P. aeruginosa isolates recovered from chronically infected CF patients. These isolates, lacking a flagellum, are resistant to nonopsonic phagocytosis, while those lacking pili but retaining their motility are susceptible to ingestion (34). 12 OBJECTIVE AND HYPOTHESIS Pseudomonas aeruginosa is the predominant respiratory tract pathogen in patients who have cystic fibrosis. Infection of the lung is the leading cause of death in these patients. Although this disease has been recognized for many years,, and the association between colonization and infection by P. aeruginosa and lung damage has been described, the host-pathogen relationship is not well understood (48). Alveolar macrophages (AM) play a major role in host defense against microbial invasion in the lung. Macrophages not only phagocytose foreign particles but also secrete many cytokines, and thereby assist in modulating immune response (30). In vitro response of alveolar macrophages to challenge with P. aeruginosa has been investigated (1, 3, 10, 60). However, in vivo interactions between P. aeruginosa and alveolar macrophages are not clearly known. Studying the host defense mechanism against P. aeruginosa challenge will contribute to our understanding of the host-parasite relationship. I hypothesize that AM play an important role in defense against P. aeruginosa infection in the lower respiratory tract. The main objective of these studies was to further elucidate the in vivo role of AM in defense of the lung against P. aeruginosa challenge. Firstly, AM were depleted by intranasal (i.n.) instillation of LDMDP in mice. AM-depleted mice received a sublethal dose of P. aeruginosa by i.n. inoculation. The effects of AM depletion on bacterial clearance in lung, spleen and liver, and PMN recruitment to the lung were assessed. Secondly, in vitro phagocytosis of unopsonized P. aeruginosa, zymosan and latex beads by freshly explanted murine AM were compared to that by their peritoneal counterparts. Finally, assessment of in situ phagocytic activities of AM was performed by challenging mice i.n. with unopsonized P. aeruginosa, zymosan particles or latex beads and subsequently obtaining bronchoalveolar lavage (BAL) fluid to evaluate BAL cell numbers and the number of ingested particles per cell in the BAL population. 13 MATERIALS AND METHODS : P. aeruginosa strains and culture conditions. P. aeruginosa strain PI is a nonmucoid derivative of a mucoid isolate from a patient with cystic fibrosis. Strain c2908c was obtained from a CF patient. Strain PAK was obtained from Dr. W. Paranchych (Edmonton, AB), strain PAOl from Dr. B. Holloway (Australia), and FRD1 from Dr. R.E.W. Hancock (Vancouver, BC). Bacteria were kept frozen at -70°C and grown fresh for each experiment. Bacteria were grown overnight in Luria Broth (10 g tryptone [Becton Dickinson and Co., Cockeysville, MD, USA], 5 g yeast extract [Becton Dickinson] and 10 g NaCl [BDH Inc., Toronto, ON] per liter distilled water) at 37°C shaking. Animals. Pathogen-free male CD-I mice weighing 35 to 37 g were obtained from Charles River Breeding Laboratories, St-Constant, Quebec and female 6- to 8-week-old BALB/c mice from the University of British Columbia Animal Care Centre, Vancouver, BC. All mice were housed in pathogen-free conditions in the animal care facility at the Research Institute for Children's and Women's Health, Vancouver, BC. The animal procedures were approved by the University of British Columbia Committee on Animal Care, Vancouver, BC. Reagents Complete medium = RPMI 1640 (Gibco BRL, Grand Island, NY, USA) 10% fetal calf serum (FCS; Gibco BRL) lOmM HEPES (Sigma Chemical Co., St. Louis, MO, USA) penicillin (104 units/ml; StemCell Technologies Inc., Vancouver, BC) streptomycin (10 mg/ml, StemCell Technologies Inc.) 14 Phagocytosis medium (PM) =138 mM NaCl 8.1 mMNa2HP04 1.5mMK2HP04 2.7 mM KC1 0.6 mM CaCl2.2H20 1 mM MgCl2.6H20 (all from Sigma Chemical Co.) Gel-Hank's solution = Hank's Balanced Salt Solution (Gibco BRL) 10% gelatin (Gibco BRL). Preparation of PBS-, DMDP- and FITC-liposomes. Liposomes were prepared as described (57). 86 mg of egg phosphatidylcholine (Sigma Chemical Co.) and 8 mg of cholesterol (Sigma Chemical Co.) were dissolved in 10ml of chloroform (BDH Inc.) in a 500ml round bottom flask. The chloroform was evaporated by low vacuum rotation, and a thin phospholipid film was formed around the flask. At room temperature, the lipid was dispersed in 10 ml of fluorescein isothiocyanate (FITC; Sigma Chemical Co.), phosphate buffered saline (PBS, pH 7.4; Oxoid, Basingstoke, Hampshire, England) or 0.6 M dichloromethylene diphosphonate (2.5g DMDP in 10ml PBS). DMDP was generously provided by Boehringer Mannheim GmbH, Mannheim, Germany. The suspension was kept at room temperature for 2 hr under nitrogen gas, then sonicated for 3 min in a water bath sonicator and kept overnight at 4°C. By centrifugation (Beckman J2-21 Centrifuge, JA20 rotor) at 10,000 X g for 15 min, free/unencapsulated DMDP was removed from DMDP-liposomes (LDMDP), filtered through a 0.2-u.m-syringe filter and stored at -20°C. PBS-, DMDP- and FITC-liposomes, were washed two to three times with 10 ml of PBS and 15 centrifuged at 25,000 X g for 30 min. At the end, the liposomes were resuspended in 4ml of sterile PBS at 4°C under nitrogen gas. Liposomes were used-within 7 days of preparation. Intranasal instillation of liposomes. Mice were anesthetized with ketamine (135mg/kg; MTC Pharmaceuticals, Cambridge, ON) intraperitoneally. 2 u l aliquots of PBS- or DMDP-liposomes were repeatedly placed directly on the external nares for a total volume of 50 u l . The entire procedure required 15 to 20 min per mouse. Mice were allowed to recover from anesthesia before being returned to their cages. To assess the efficiency of LDMDP in depleting alveolar macrophage, bronchoalveolar lavages (BAL) were performed on mice before intranasal challenge with P. aeruginosa. The number of alveolar macrophages in BAL fluid from LDMDP-treated mice was then compared to those from the control mice exposed to PBS-liposomes (LPBS). For histologic examination, sections of the lung were excised and fixed in 10% buffered Formalin (BDH Inc.), and processed at the Anatomic/Surgical Pathology Laboratory, BCs Children's Hospital, Vancouver, BC. The parafin-embedded sections (2-3 um) were stained with hematoxylin and eosin (H & E). Intranasal administration of P. aeruginosa. LPBS and LDMDP were delivered 24 h or 48 h prior to bacterial infections. An aliquot of the overnight shaking culture of P. aeruginosa was centrifuged (Fisher Micro-centrifuge 23 5B) at 13000 X g for 30 sec and resuspended in the same volume of Gel-Hank's solution. Bacteria were then serially diluted to the desired density. Mice were anesthetized with ketamine (135mg/kg) intraperitoneally, after which 2 u l aliquots of bacterial culture (106 - 1010 CFU/ml) were repeatedly placed directly on the external nares for a total volume of 20 u l or 50 ul. Bacteria were kept on ice prior to challenge. The entire procedure required 15 to 20 min per 16 mouse. Mice were allowed to recover from anesthesia before being returned to their cages. Serial dilutions of the infection inoculum were made in Gel-Hank's solution, spread on Trypticase soy agar (TSA; Becton Dickinson and Co.) plates and incubated for 18 h at 37°C. The inoculation dose was then determined by viable bacterial counts. Intranasal administration of latex beads and zymosan particles. Mice were anesthetized with ketamine (135mg/kg) intraperitoneally, after which 2 ul aliquots of latex beads (3 um; Sigma Chemical Co.) or zymosan particles (Sigma Chemical Co.) were placed directly in the nares for a total volume of 20 ul. The entire procedure required 15 to 20 min per mouse. Mice were allowed to recover from anesthesia before being returned to their cages. Zymosan particles were prepared by adding 40 ul of 5% stock (in PBS and 0.02% sodium azide [Sigma Chemical Co.]) to 1 ml of phagocytosis medium. They were then centrifuged (Fisher Micro-centrifuge 235B) at 13,000 X g for 1 min, washed with fresh phagocytosis medium twice and resuspended in 1 ml of the same medium. Bronchoalveolar lavage. At various timed intervals, mice were euthanized with an overdose (0.1ml) of sodium pentobarbital (65mg/ml; MTC Pharmaceuticals) by intraperitoneal injection. The trachea was exposed and intubated with a sterile 22- or 24-gauge catheter (Johnson & Johnson Medical Inc., Arlington, TX, USA). The lungs were lavaged with two 5-ml washes of cold sterile PBS. The bronchoalveolar lavage (BAL) fluid was centrifuged (Jouan CR312) at 395 X g for 15 min at 4°C and resuspended in 1 ml of complete medium. The total BAL cell count, excluding erythrocytes, was determined by Trypan blue exclusion using a hemocytometer. The differential counts of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) were performed on 17 cytocentrifuge preparations stained with Diff-Quick (EM Industries Inc., Gibbstown, NJ, USA). Analysis of variance (ANOVA) was used to assess differences between groups at P<0.05. Cytospin preparations. The cytospin apparatus (assembled in this order) consisted of a clamp, microscope slide (Corning Glass Works, Corning, NY, USA), filter (Shandon Southern Instruments Inc., Sewickley, PA, USA), and funnel. 70 ul of 2 % gelatin was cytocentrifuged (Shandon Cytospin 2) on the microscope slide at 48 X g for 1 min and then 100. ul of BAL fluid for 2 min. The cytospin preparation was then fixed with methanol (Fisher Scientific, NJ, USA) and stained with Diff-Quick. The numbers of AM and PMN in 100 BAL cells were counted and expressed as percentage. Determination of the numbers of alveolar macrophages and neutrophils in bronchoalveolar lavage. An aliquot (10 u.1) of the BAL fluid was taken; an equal volume (lOul) of Trypan blue was added to the aliquot. 10 ul of the 20ul-cell suspension with Trypan blue was transferred to a hemocytometer. The number of cells in the lOul-aliquot, which excluded Trypan blue dye i.e. T(-), was counted in four separate segments of the hemocytometer. The percentage of AM or PMN in a BAL cell population was determined from the number of each type of cell in 100 BAL cells on a cytospin preparation. a) The number of AM or PMN in an aliquot of the BAL fluid was calculated by multiplying the number of T(-) cells in four segments of the hemocytometer by the percentage of AM or PMN in a BAL cell population (100 BAL cells). [# T(-) cells (in 4 segments of the hemocytometer)] [% AM or PMN (from cytospin preparation)] = # T(-) AM or PMN in an aliquot of the BAL suspension b) The total number of AM or PMN in the BAL fluid: the number of T(-) AM or PMN in an aliquot of the BAL suspension (a) was divided by four segments counted, and then multiplied by the dilution factor e.g. 2x if an equal volume of Trypan blue dye was added, then multiplied by the predetermined fixed number of cells per ml (104cells/ml) in a 10ul-aliquot and by the total volume of the BAL suspension. r ~\ # T(-) AM or PMN in an aliquot of the BAL fluid 4 segments counted (hemocytometer) = the total # AM or PMN in the BAL fluid (dilution factor)(104cells/ml)(total vol. BAL fluid) Analysis of lung, spleen and liver CFU and histopathology. Mice were euthanized by cervical exsanguination at various time points. Lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on TSA plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. P. aeruginosa colonies were identified based on the morphology, color and odor of the colonies, and colonies testing positive with the oxidase test. For histologic examination, tissue blocks were excised, fixed, processed and stained with H & E. Preparation of murine resident peritoneal macrophages. Resident macrophages were obtained from the peritonea of 6- to 8-week-old BALB/c mice (49). Explanted macrophages were kept in complete medium. The number of viable cells was determined by Trypan blue exclusion with a hemocytometer. 19 In vitro effects of LPBS, L D M D P , DMDP and L F I T C on murine alveolar and peritoneal macrophages. Alveolar macrophages obtained by BAL and peritoneal macrophages harvested from the peritoneal cavity were kept in complete medium. 105 cells (in approximately 100 u.1) were plated on acid-washed 11-mm diameter round glass coverslips in 24-well plastic tissue culture trays (Becton Dickson and Co.) After 30 min of incubation at 37°C in 5% C0 2, 400 u.1 of complete medium was added to the wells. After the addition of 5, 10 and 15 u.1 of LFITC, LPBS, LDMDP or DMDP, macrophages were incubated at 37°C in 5% C0 2 for 24, 48 and 72 hours. At timed intervals, macrophages were washed twice with PBS, fixed with methanol, stained with Giemsa (BDH Inc., Toronto, ON) and mounted on microscope slides with Entellan (Merck, Darmstadt, Germany). The morphology of untreated and-treated macrophages was assessed microscopically. Macrophages with intact cytoplasm and nuclei were considered healthy, and those with no cytoplasm or shriveled cytoplasm and nuclei unhealthy. At least 60 macrophages per coverslip were scored. Each sample was assayed in duplicate, and the experiments were performed two to three times with different macrophage preparations. Data were expressed as means + SEM. ANOVA was used to evaluate data, and P values of <0.05 were considered significant. In vitro phagocytosis of P. aeruginosa by untreated and treated macrophages was examined as follows. In vitro phagocytosis assays. Alveolar macrophages obtained by BAL and peritoneal macrophages harvested from the peritonea were kept in complete medium. 10s cells (in approximately 100 u,l) were plated on acid-washed 11-mm diameter round glass coverslips in 24-well plastic tissue culture trays. After 30 min of incubation at 37°C in 5% C0 2, 400 u.1 of complete medium was added to the wells. 20 Two hours after adherence to the coverslips, or 24, 48 and 72 hr after incubation with LPBS, LDMDP, and DMDP, macrophages were washed with PBS two times to remove non-adherent cells. 500 ul of glucose-free phagocytosis medium was then added to the wells. 25 ul of latex beads, zymosan suspension, or overnight shaking culture of unopsonized P. aeruginosa (approximately 107 cfu), was added to the wells. Cells were incubated with or without 10 mM D-glucose at 37°C for another hour. Extracellular bacteria were lysed by the addition of 500 ul of cold lysozyme (5mg/ml; Boehringer Mannheim GmbH, Mannheim, Germany) in 0.25 M Tris buffer (pH 8.0; ICN Biomedicals, Aurora, OH) for 6 min 30 sec at room temperature. After a PBS wash, cells were incubated with 500 ul of H2O for 2 min 10 sec, washed with PBS twice and fixed with methanol for at least 15 min. The coverslips were stained with Giemsa stain for 20 min, washed with water, air dried and mounted on glass microscope slides. Phagocytosis was assessed microscopically. At least 60 macrophages per coverslip were scored. The experiments were repeated two to three times, with each sample in duplicate. Data were expressed as means + SEM. Two-sample Student's t-test and ANOVA were used to evaluate data, and P values of <0.05 were deemed significant. In vitro effects of LPBS, LDMDP and DMDP on epithelial cell line A549. Epithelial cell line A549, maintained in F12K (Gibco BRL) / 10 % FCS, was treated with 1 x Trypsin-EDTA (Gibco BRL) for 15 min. The cell suspension was centrifuged (Jouan CR312) at 454 X g for 10 min, and resuspended in F12K/10% FCS. Viable cell counts were determined with a hemocytometer by Trypan blue exclusion. 105 cells (in approximately 100 ul) were plated on acid-washed 11-mm diameter round glass coverslips in 24-well plastic tissue culture trays. After 30 min of incubation at 37°C in 5% C0 2, 400 ul of F12K/10% FCS was added to the wells. After the addition of 15 ul of LPBS, LDMDP or DMDP (0.6 M, 50, 5 and 0.5 mM), A549 21 cells were incubated at 37°C in 5% C0 2 for 24 and 48 h. At timed intervals, cells were washed twice with PBS,.fixed with methanol, stained with Giemsa'and mounted on microscope slides. The morphology of untreated and treated epithelial cells was assessed microscopically. In situ phagocytosis assays. Mice were challenged intranasally with P. aeruginosa, latex beads or zymosan particles as described above. Three hours after instillation, BAL was performed. BAL cells were centrifuged (Jouan CR312) at 395 X g for 15 min at 4°C, resuspended and incubated with 1 ml of lysozyme (5mg/ml) in 0.25 M Tris Buffer, pH 8.0 at room temperature (RT) for 6 min 30 sec. After the addition of 2 ml of PBS, BAL cells were then centrifuged (Jouan CR312) at 395 X g for 15 min at 4°C, resuspended in 1 ml of water and incubated at RT for 2 min 10 sec. After 2 ml of PBS was added, the BAL suspension was centrifuged under the same conditions and resuspended in 1 ml of PBS. The differential counts of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) were performed on Diff-Quick-stained cytocentrifuge preparations of BAL cells. 100 cells were counted, and the results were expressed as percentage. Data were expressed as means + standard errors. Two-sample Student's t-test was used to evaluate data, with P<0.05 deemed significant. Statistical analysis Data were expressed as means + standard errors (SE) or + standard errors of the means (SEM). Analysis of variance (ANOVA) and two-sample Student's t-test were used to analyze data. Data were considered statistically significant if P values were less than or equal to 0.05. 22 RESULTS In vitro effects of LPBS, LDMDP and DMDP on murine peritoneal macrophages. Initial experiments were done to assess the effects of PBS-liposomes, DMDP-liposomes and free DMDP on peritoneal macrophages (PM) in vitro. Experimental conditions were first set up using PM, not AM, due to the abundant number of PM obtained from a preparation compared to that of AM. At timed intervals, the morphology of Giemsa-stained PM was assessed microscopically. PM with intact cytoplasm and nuclei were considered healthy, and those with no cytoplasm or shriveled cytoplasm and nuclei unhealthy. PM, plated on coverslips, were incubated with 5, 10 and 15 ul of LPBS, LDMDP or DMDP (0.6M stock) for 24 and 48 hr. Controls received no treatment. Fig. 1 shows that the majority (94 - 99 %) of untreated (control) and LPBS-treated macrophages looked healthy. After 24 h of incubation with LDMDP (5 and 10 ul), the percentage (33 and 23 %, respectively) of PM with unhealthy characteristics -shriveled cytoplasm and nuclei - was significantly higher than their LPBS-treated counterparts. However, there was no significant difference in morphology between PM incubated with 15 ul of LDMDP and those treated with LPBS. The effects of LDMDP on peritoneal macrophages were evident at 48 h. 48-h incubation with LDMDP (5, 10 and 15 ul) resulted in a substantial percentage (32, 53 and 71%, respectively) of peritoneal macrophages with shriveled cytoplasm and nuclei, compared to the untreated (1%) and LPBS-treated (0-3%) macrophages. As the dose of LDMDP increased, the percentage of unhealthy-looking macrophages increased. At 24 and 48 h, all macrophages incubated with free DMDP became unhealthy, with no visible cytoplasm. 23 Fig. 1. Effects of LPBS, LDMDP and DMDP on murine peritoneal macrophages (PM) /'/; vitro. PM, plated on coverslips, were incubated with 5, 10 and 15 u.1 of LPBS, LDMDP and DMDP (0.6M stock) for 24 h, 48 h. Controls received no .treatment. At timed intervals, cells were washed with PBS, fixed with methanol, stained with Giemsa stain and mounted on microscope slides. The morphology of treated PM was assessed microscopically. % healthy macrophages (solid bars) and unhealthy macrophages (hatched bars) were compared. 100 macrophages per coverslip were scored. The experiments were repeated two times, with each sample in duplicate. Results were expressed as means + SEM. *, significant at P<0.§5 (ANOVA) compared to Value for untreated (control) and LPBS-treated PM. 24 h 120 • Healthy 0 Unhealthy / 7 / />V 48 h 120 ^ ^ 24 In vitro phagocytosis of unopsonized P. aeruginosa by murine peritoneal macrophages incubated with LPBS, LDMDP and DMDP for 24 and 48 h. In addition to their morphology, the nonopsonic phagocytosis of P. aeruginosa (PI) by murine PM incubated with LPBS, LDMDP and DMDP for 24 and 48 h was assessed (Table 3). At 24 and 48 h, untreated and treated macrophages were incubated with unopsonized P. aeruginosa strain PI (approximately 107 cfu) in the presence or absence of 10 mM D-glucose. P. aeruginosa strain PI, a nonmucoid derivative of a mucoid isolate from a patient with cystic fibrosis, has been previously used in the phagocytosis assays performed in our laboratory (1, 10, 49). Extracellular bacteria were lysed by treatment with cold lysozyme. Cells were washed with PBS, fixed and Giemsa-stained. Lysed bacteria were either washed off or remained on coverslips in round shape. Ingested bacteria were rod-shaped and clustered. The number of ingested bacteria per macrophage was scored microscopically. Phagocytosis of unopsonized P. aeruginosa (PI) by macrophages requires the presence of D-glucose (49). At 24 h, the number of P. aeruginosa (PI) ingested by LPBS- and LDMDP-treated PM was similar, but decreased slightly compared to that by untreated PM. DMDP-treated PM had lost their cytoplasm; thus phagocytosis of PI was not observed. Percentage of LDMDP-treated PM with ingested P. aeruginosa (78 - 86.5 %) was lower than that of untreated and LPBS-treated PM with ingested P. aeruginosa (91.5 - 100 %). At 48 h, there was a decrease in the number of ingested PI by PM subjected to LDMDP (7.2 - 8.3 bacteria/macrophage) compared to control and LPBS-treated PM (26.4 and 21 - 27.4 bacteria/macrophage, respectively). The percentage of LDMDP-treated PM (45.5 - 60.5 %) with ingested P. aeruginosa was much lower than that of untreated and LPBS-treated PM (96.5 - 98.5 %). These results were from macrophages capable of phagocytosis i.e. macrophages with intact cytoplasm or nuclei/cytoplasm not shriveled. 25 Table 3. In vitro phagocytosis of unopsonized P. aeruginosa by murine peritoneal macrophages incubated with LPBS, LDMDP and DMDP for 24 and 48 h. Assessment of nonopsonic phagocytosis of P. aeruginosa by peritoneal macrophages (PM) was performed after 24 and 48 h of incubation with LPBS, LDMDP and DMDP. PM were incubated for 60 min with overnight shaking culture of unopsonized P. aeruginosa strain PI (approximately 107 cfu) with or without 10 mM D-glucose. Extracellular bacteria were lysed by the addition of cold lysozyme. After a PBS wash, cells were incubated with H2O, washed with PBS twice and fixed with methanol. The coverslips were stained with Giemsa stain, and mounted on glass microscope slides. At least 60 macrophages per coverslip were scored microscopically. The experiments were repeated two to three times, with each sample in duplicate. Phagocytosis of P. aeruginosa (PI) was negligible in the absence of D-glucose. Data are as means ± SEM. *, significant at P<0.05 (ANOVA) compared to value for untreated (control) and LPBS-treated PM. PI + 10 mM D-glucose (average number of % peritoneal macrophages ingested P. aeruginosa with ingested P. aeruginosa per macrophage) Experimental condition - 24 h 48 h 24 h 48 h Control 19.7 + 0.3 26.4 + 6.5 100.0 ±_0.0 98.5 ±_0.5 LPBS (5 ul) 19.2+1.2 25.2±_6.9 97.5 + 2.5 98.5 ±_1.5 LPBS (10 ul) 14.5 ±_2.4 21.0±_10.8 91.5 +.1.5 97.0 + 3.0 LPBS (15 ul) 18.8±_1.5 27.4 ±.10.6 95.5 ±_0.5 96.5 + 3.5 LDMDP (5 ul) 16.4 ±1.7 8.3 +.3.3* 78.0+10.0 60.5 ±22.5* LDMDP (10 ul) 13.8 ±.1.4 7.2 + 4.2* 80.5 +2.5 45.5 ±22.5* LDMDP (15 ul) 15.4 + 0.4 8.2 + 2.0* 86.5 ±_10.5 55.5 ±.15.5* DMDP (5 ul) 0 0 0 0 DMDP (10 ul) 0 0 0 0 DMDP (15 ul) 0 0 0 26 In vitro effects of LPBS, LDMDP, DMDP and LFITC on murine alveolar macrophages. Further experiments were done to evaluate the effects of PBS-liposomes, DMDP-liposomes and free DMDP on alveolar macrophages (AM) in vitro. The uptake of liposomes by AM was assessed by AM phagocytosis of FITC-encapsulated liposomes (LFITC) in vitro. Alveolar macrophages (AM), plated on coverslips, were incubated with 15 ul of LFITC, LPBS, LDMDP or DMDP (0.6M stock) for 24, 48 and 72 nr. Controls received no treatment. The morphology of Giemsa-stained AM was assessed microscopically. AM with intact cytoplasm and nuclei were considered healthy, and those with no cytoplasm or shriveled cytoplasm and nuclei unhealthy. The results of one representative experiment are shown (Fig. 2) due to high variations among experiments. 100 % of untreated (control) and LPBS-treated macrophages looked healthy, whereas 61, 88 and 61 % of AM showed unhealthy characteristics after incubation with LDMDP for 24, 48 and 72 hours, respectively. All alveolar macrophages, incubated with free DMDP for 24, 48 and 72 hr, became unhealthy i.e. no cytoplasm. The photomicrographs in Fig. 3 show AM phagocytosis of liposomes, as a strong fluorescence was observed in AM incubated with FITC-liposomes for 24 h and 48 h. 27 Fig. 2. Effects of LPBS, LDMDP and DMDP on murine alveolar macrophages (AM) in vitro. AM, plated on coverslips, were incubated with 15 ul of LPBS, LDMDP and DMDP (0.6M stock) for 24 h, 48 h, and 72 h. Controls received no treatment. The morphology of the treated alveolar macrophages was assessed microscopically. 100 macrophages per coverslip were scored. The experiments were repeated two times. % healthy macrophages (solid bars) and unhealthy macrophages (hatched bars) were compared. Results of one representative experiment are shown. 24 h 120 100 A CONTROL L-PBS(15ul) L-DMDP(15ul) Dose (ul) DMDP (15ul) 48 h E CONTROL L-PBS(15ul) L-DMDP (15ul) Dose (ul) DMDP (15ul) 72 h 120 100 E CONTROL L-PBS(15ul) L-DMDP (15ul) Dose (ul) DMDP (15ul) 28 Fig. 3. Phagocytosis of liposome-encapsulated FITC by alveolar macrophages. Alveolar macrophages, plated on coverslips, were incubated with LFITC for 24 and 48 h. At timed intervals, cells were washed with PBS to remove unincorporated liposomes. Cells were then fixed and Giemsa-stained. Phagocytosis was assessed with the aid of a fluorescent microscope. Magnification, X 250. a. 24 h 29 In vitro phagocytosis of unopsonized P. aeruginosa by murine alveolar macrophages incubated with LPBS, LDMDP and DMDP for 24, 48 and 72 h. In addition to their morphology, nonopsonic phagocytosis of P. aeruginosa (PI) by murine alveolar macrophages (AM) incubated with LPBS, LDMDP and DMDP for 24, 48 and 72 h was assessed (Table 4). At timed intervals, macrophages were incubated with unopsonized P. aeruginosa (approximately 107 cfu) in the presence or absence of 10 mM D-glucose. Extracellular bacteria were lysed by treatment with cold lysozyme. Cells were washed with PBS, fixed and Giemsa-stained. The number of ingested bacteria per macrophage was scored microscopically. Phagocytosis of unopsonized P. aeruginosa (PI) by macrophages in the absence of D-glucose is negligible (49). At 24, 48 and 72 h, LPBS-treated AM (2.5 bacteria / macrophage) ingested similar number of P. aeruginosa (PI) as untreated AM (2.0 bacteria / macrophage), whereas LDMDP- and DMDP-treated AM ingested negligible amount of PI (0 -0.07 bacteria / macrophage). Percentages of LDMDP- and DMDP-treated AM with ingested bacteria (0-3 %) were substantially lower than those of control and LPBS-treated PM with phagocytosed PI (38-76 %). The decreased phagocytosis of PI by LDMDP-treated AM might be due to the disruption/suppression of their phagocytic function by LDMDP, although the disruption was not reflected as extensively in their morphology. AM incubated with DMDP had lost their cytoplasm; therefore, ingestion of P. aeruginosa was not observed. 30 Table 4. In vitro phagocytosis of unopsonized P. aeruginosa by murine alveolar macrophages incubated with LPBS, LDMDP and DMDP for 24, 48 and 72 h. Assessment of nonopsonic phagocytosis of P. aeruginosa by alveolar macrophages (AM) was performed after 24, 48 and 72 h of incubation with LPBS, LDMDP and DMDP. Macrophages were incubated for 60 min with overnight shaking culture of unopsonized P. aeruginosa strain PI (approximately 107 cfu) with or without 10 mM D-glucose. Extracellular bacteria were lysed by the addition of cold lysozyme. After a PBS wash, cells were incubated with H2O, washed with PBS twice and fixed with methanol. The coverslips were Giemsa-stained and mounted on glass microscope slides. At least 60 macrophages per coverslip were scored microscopically. Ingestion of P. aeruginosa (PI) was negligible in the absence of D-glucose. Data are as means ± SE. •*, significant at P<0.05 (ANOVA) compared to value for untreated (control) and LPBS-treated AM. PI + 10 mM D-glucose (average number of ingested % alveolar macrophages with P. aeruginosa per macrophage) ingested P. aeruginosa Experimental condition ^4h 48h 72h 24h 48h 72h Control 2.0 ±3.0 4.2 ±4.9 7.8 ±.8.0 38 60 76 LPBS (15 )al) 2.5 ±3.5 2.2 ±2.9 5.4 ±4.9 52 48 54 LDMDP (15 ul) 0.07 ±0.37* 0* 0* 3* 0* 0* DMDP (15 ul) 0 0 0 0 0 0 31 In vitro effects of LPBS, L D M D P and DMDP on respiratory epithelial cell line A549. To determine whether LPBS, LDMDP or DMDP would affect respiratory epithelial cells, an epithelium-derived cell line A549 was used. A549 cells, plated on coverslips, were incubated with 15 ul of LPBS or LDMDP for 24 and 48 h. Changes in the morphology of the respiratory epithelial cell line were not observed after 24 and 48 h of incubation with LPBS and LDMDP. At 4 hr, the percentage of A549 cells with unhealthy characteristics - shriveled nuclei and cytoplasm - increased with increasing concentrations of DMDP i.e. 8 - 15 % of A549 cells incubated with 0.5 and 5 mM of DMDP, 23 - 33 % of A549 cells incubated with 50 mM of DMDP and 80 - 85 % of A549 cells incubated with 0.6 M of DMDP. The route of administration of LPBS and L D M D P to the airspace in mice Alveolar macrophage (AM) depletion in mice is performed by administration of DMDP-liposomes into the airspace of the animals. PBS- and DMDP-liposomes are delivered to the lung by intratracheal (i.t.) instillation (5, 25, 32), intubation (2) and aerosolization (23, 27). Intratracheal instillation involves surgery. Mice are anesthetized with pentobarbital by peritoneal injection. The trachea is incised, and an aliquot of the liposomal preparation is administered via a sterile catheter/needle. The skin incision is then closed with a suture or staple to make the wound accessible for subsequent bacterial inoculation if necessary (5, 25, 32). The delivery of liposomes by i.t. instillation is effective; however, it is difficult and stressful for the mice to recover from multiple surgeries, which may also increase the chance of wound infections. Intubation is the process of administration of agents into the trachea via the mouth. It does not require surgery. However, the efficiency and confidence of delivering the agents into the trachea, not the esophagus, is not guaranteed. In addition, it is difficult to view the tracheal opening via the mouth in mice due to their small size. Intubation is performed more efficiently in larger rodent, such as rats (2). LPBS and LDMDP are also delivered into the lung by aerosol 32 inhalation. Aerosols are generated by a nebulizer, which is driven by compressed air at a flow rate of 10 - 12 litres/min (23, 27). Liposome-encapsulated drug can be uniformly distributed in the lung by aerosolization, but are disrupted during the process, thereby releasing the drug into the airspace (12). Intranasal instillation is an alternative method, which does not involve surgery and thus minimize wound infections. This procedure is used to deliver bacteria into the lung of mice (25, 54), and has been modified to instill liposomes into the airspace of mice in AM-depletion experiments (Fig. 4). In this procedure, small aliquots of the liposomal preparations were placed directly into the nares of the mice, and were breathed into the lung during normal inhalation; therefore, disruption of liposomes due to high flow rate was minimized. Initial experiments of administration of LPBS and LDMDP in mice by i.t. and i.n. instillation showed that the effectiveness of AM depletion by both methods was similar (data not shown). Intranasal instillation was performed in subsequent AM depletion studies because it was easy to carry out, effective in delivery of liposomes and less stressful for the mice. In vivo depletion of alveolar macrophages by intranasal instillation of DMDP-liposomes. In the experiments described in this thesis, mice were administered with LPBS and LDMDP by i.n. instillation. The effect of LDMDP in vivo was evaluated by comparing the number of AM in bronchoalveolar lavage (BAL) fluid from LPBS- and LDMDP-treated mice. 50 ul of LPBS or LDMDP was delivered to mice by i.n. instillation. 24 and 48 h after the instillation of liposomes, BAL was performed and the number of AM in BAL fluid was determined from hemocytometer counts and Diff-Quick-stained cytospin preparations. 24 and 48 h post-instillation, the number of AM recovered from LDMDP-treated mice (4.73 x 104 and 5.40 x 104) was significantly lower than those in LPBS-treated mice (2.63 x 105 and 2.31 x 105), respectively (Fig. 4a). At 24 and 48 h, 78 - 88 % and 74 - 80 % of AM were depleted in LDMDP-treated group, respectively, compared to 0 % in LPBS-treated mice (Fig. 4b). At 72 h, 33 the number of AM in BAL from LDMDP-treated group was slightly increased (data not shown). Lung sections of mice with LPBS and LDMDP instilled were histologically similar, with a slight increase of PMN compared to untreated lungs (Fig 5). 34 Fig. 4. Depletion of alveolar macrophages in vivo by intranasal (i.n.) instillation of DMDP-liposomes. 50 ul of PBS-liposomes (LPBS) or DMDP-liposomes ( L D M D P ) was delivered to mice by i.n. instillation. 24 and 48 h after the instillation of liposomes, B A L was performed and the number of A M in the B A L fluid was determined from hemacytometer counts and Diff-Quick-stained cytospin preparations, (a) Number of alveolar macrophages ( A M ) in the B A L fluid from L P B S - (solid bars) and LDMDP-treated (hatched bars) mice at 24 and 48 h post-instillation, (b) The corresponding number of remaining A M expressed as percentage. Data were expresed as means + S E M . *, significant at ?<0.05 ( A N O V A ) compared to value for LPBS-treated mice. 35 Fig. 5. Histologic sections of LPBS- and LDMDP-instilled lung in mice. 50 u.1 of LPBS or LDMDP was delivered to mice by intranasal instillation. 24 and 48 h after the instillation of liposomes, lung sections from untreated, LPBS- and LDMDP-treated mice were excised and fixed in 10 % buffered Formalin, and processed. The parafin-embedded sections ( 2 - 3 jam) were stained with hematoxylin and eosin. Magnification, X 125. a. untreated b. LPBS-treated 36 37 The strain of mice, route of bacterial inoculation and bacterial strains in P. aeruginosa infection model. Prior to the assessment of AM depletion on P. aeruginosa clearance from the lung, the strain of mice, route of bacterial infection and bacterial strains were determined. Mice of different strains vary in survival rate and bacterial clearance in response to pulmonary P. aeruginosa infection. Mice of the BALB/c strain are more resistant to P. aeruginosa compared to those of the DBA/2, A/J and C57BL/6 strains, in response to intratracheal (i.t.) infection of P. aeruginosa strain 508 (1 x 104 to 3 x 104) enmeshed in agar beads. DBA/2 mice are severely afflicted by the infection, with a high bacterial load and high mortality rate following infection (36). Preliminary experiments of i.t. inoculation of P. aeruginosa strain PI (1.9 x 10 ) in BALB/c and DBA/2 mice showed that the former had cleared the bacteria from the lung by day 1 post-infection, whereas the latter still had a substantial number of P. aeruginosa (3.67 x 10s) in the lung. Due to its high resistance to P. aeruginosa pulmonary challenge, mice of the BALB/c strain were chosen for subsequent infection experiments. The chance of observing differences between AM-depleted and non-depleted infected mice would be higher in a resistant mouse strain (BALB/c) than a susceptible one (DBA/2). This phenomenon is shown in the Mycoplasma pulmonis lung infection model. AM depletion enhances infection in mycoplasma-resistant C57BL/6NCr (C57BL), but not in mycoplasma-susceptible C3H/HeNCr (C3H) (25). The bacterial inoculation into the airspace of the rodents is achieved by various methods: i.t. instillation (5, 23, 32, 36), i.n. instillation (25, 54) and intubation (27). Initial experiments demonstrated that i.t. inoculation of P. aeruginosa was effective in delivering bacteria into the lung of the mice (data not shown). However, the chance of wound infections might increase with multiple surgeries; therefore, it was not the best method to deliver bacteria into the airspace of the animals. Intubation does not require surgery. However, as discussed above, it is difficult to view the tracheal opening via the mouth in mice due to their small size. Intranasal inoculation 38 was determined to be the route of infection in mice because it was easy to perform, effective in bacterial deposition (Figs. 6, 7 & 8), and would minimize wound infections as it would not involve surgery. The P. aeruginosa strains used in the infection model were determined on the basis of several factors i.e. the bacterial strain would colonize in the lung for at least 1 day before being cleared and not cause cytotoxicty in mice. PI, a nonmucoid derivative of a mucoid isolate from a CF patient, has been used in our laboratory for many years. When infected intratracheally at 6.5 x 106 in mice, it was cleared from the lung by day 1 post-infection. PA103 is a cytotoxic strain (46), and hence might complicate the results of lung infection in AM-depleted mice, which were immunocompromised. The two strains of P. aeruginosa, which were used in the infection model, were PA01 and FRD1. PAOl, a laboratory strain, is nonmucoid and non-cytotoxic (46). FRD1, a mucoid strain, was used in infection experiments to examine the clearance of mucoid strains in mice in response to pulmonary challenge with P. aeruginosa. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. The deposition of P. aeruginosa in the lung by intranasal infection, and subsequent bacterial clearance in untreated mice was assessed by inoculating a sublethal dose of P. aeruginosa intranasally in mice which had received no treatment of liposomes. At timed intervals, lungs, spleen and liver were harvested, and serial dilutions were made from the homogenates and plated on TSA plates. Viable bacterial counts were then analyzed. Three hours (Day 0) after intranasal inoculation of P. aeruginosa strains PAOl at 2.55 x 106 (Fig. 6) and FRD1 at 1.9 x 106 (Fig. 7), the numbers of bacteria recovered from the lung of individual mice were consistent and comparable to the initial inoculum doses. The number of P. aeruginosa CFU in the lung had decreased by day 1 post-infection (Fig. 6 & 7) and cleared from the lung by day 4 post-infection (Fig. 7). No viable bacteria were recovered from the spleen or 39 liver in the infected mice (Fig. 6 & 7). A higher dose of P. aeruginosa strain FRD1 (5.5 x 107) was inoculated intranasally in mice (Fig. 8). One day after infection, the number of CFU in lung 8 • 4 did not decrease and increased considerably in spleen (0 to 1.61 x 10 ) and liver (1.57 x 10 to 1.89 x 108). By day 3 post-infection, none of the infected mice survived and viable bacterial count analysis revealed a substantial number of CFU in lung, spleen and liver (data not shown). A lower inoculum dose (106), instead of 107, was chosen in subsequent infection experiments so as to increase the chances of detecting differences between AM-depleted and nondepleted infected mice. 40 Fig. 6. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. B A L B / c mice, which had received no treatment of liposomes, received a sublethal dose (2.55 x 106) of P. aeruginosa strain P A O l intranasally. A t 3 h (day 0) and 24 h (day 1) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on T S A plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. C F U counts from individual mice were represented as B A L B / c 1, B A L B / c 2 and B A L B / c 3 . Data were expressed as means ( B A L B / c ) + SE. lung 3 L l _ o 8 .00 7 .00 6 . 0 0 5 .00 4 . 0 0 3 .00 2 . 0 0 1.00 0 .00 - B A L B / d - B A L B / C 2 - B A L B / C 3 - B A L B / c D a y s Pos t i n fec t i on b spleen O 9 .00 8 .00 7 .00 6 . 0 0 5 .00 4 . 0 0 3 .00 2 . 0 0 1.00 0 .00 • B A L B / c 1 • B A L B / C 2 —A— B A L B / c 3 —H—BALB/c • m— D a y s Pos t i n fec t i on liver _o c ro a> o LL o 9 .00 8 .00 7 .00 6 . 0 0 5 .00 4 . 0 0 3 .00 2 .00 1.00 0 .00 B A L B / d B A L B / C 2 B A L B / C 3 B A L B / c D a y s Pos t i n fec t i on 41 Fig. 7. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. B A L B / c mice, which had received no treatment of liposomes, were inoculated with 1.9 x 10 6 of P. aeruginosa strain F R D 1 intranasally. A t 3 h (day 0), 24 h (day 1) and 96 h (day 4) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on T S A plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. C F U counts from individual mice were represented as B A L B / c 1 and B A L B / c 2 . Data were expressed as means ( B A L B / c ) ± SE . 9.00 0 1 2 3 4 5 Days Postinfection b spleen ! O 9.00 8.00 7.00 -I 6.00 5.00 4.00 3.00 2.00 -I 1.00 0.00 2 3 Days Postinfection B A L B / d B A L B / C 2 B A L B / c liver S .o 3 u. o 9.00 • 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 2 3 Days Postinfection B A L B / d B A L B / C 2 B A L B / c 42 Fig. 8. Intranasal inoculation and clearance of P. aeruginosa in untreated mice. BALB/c mice, which had received no treatment of liposomes, received P. aeruginosa strain FRD1 (5.5 x 107) intranasally. At 3 h (day 0) and 24 h (day 1) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on TSA plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. CFU counts from individual mice were represented as BALB/c land BALB/c2. Data were expressed as means (BALB/c) + SE. a lung O 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 • BALB/d -BALB/c2 -X— BALB/c Days Postinfection b spleen 9.00 8.00 7.00 "° 6.00 O) £ 5.00 c ro E> 4.00 .o [2 3.00 O 2.00 1.00 0.00 - • — B A L B / C 1 • * — B A L B / C 2 -X— BALB/c Days Postinfection C liver .g 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 -BALB/d •BALB /C2 -BALB/c Days Postinfection 43 Effects of AM depletion on P. aeruginosa clearance in vivo. The initial experiments were designed to assess the in vivo role of alveolar macrophages (AM) in clearance of P. aeruginosa from the lung. PBS- and DMDP-liposomes were instilled intranasally (i.n.) 24 h prior to i.n. inoculation of P. aeruginosa in mice. Depletion of AM was confirmed by BAL counts and cytospin preparations on the day of infection. At timed intervals post-infection, lungs, spleen and liver were obtained for viable bacterial counts and histologic examination. Serial dilutions were made from the homogenates and plated on TSA plates. LPBS- and LDMDP-treated BALB/c mice were inoculated intranasally with P. aeruginosa strains FRD1 at 6.0 x 106 (Fig. 9) and PAOl at 2.7 x 107 (Fig. 10). In these two separate experiments, difference in the number of viable bacteria between AM-depleted and nondepleted lungs was not detected. P. aeruginosa was cleared from the lung of both groups in a similar fashion. No viable bacteria were isolated from the spleen or liver in the infected mice at these time points. Infection experiments (Figs. 10 and 11), were performed up to 5 days post-infection to examine clearance of the different bacterial strains. Differences between AM-depleted and nondepleted mice were not expected to be observed because the number of AM would have increased by 5 days after infection i.e. 6 days after instillation of LDMDP (2, 5, 23). After this project had started, the effects of AM depletion on P. aeruginosa pneumonia were published by Kooguchi et al. Four to forty-eight hours after infection of PAOl (5 x 10s) in CD-I mice (35 -37 g in weight), the number of bacteria in the lungs of mice with 95 % of AM depleted is significantly higher than those of nondepleted (saline-treated) mice (27). In light of their results, the infection experiments were repeated under our conditions, except CD-I, instead of BALB/c, mice were used to determine whether the discrepancy between the results of my experiments and those of Kooguchi et al was due to mouse strain, or other factors such as the route of LDMDP administration and/or degree of AM depletion. CD-I are outbred mice i.e. offsprings of parents that are unrelated and randomly mated. BALB/c are inbred mice; their parents are related. CD-I 44 mice are often used in toxicology and drug studies because they represent a population that is not very closely related e.g. humans, and BALB/c mice in controlled experiments in which fewer variables are preferred. LPBS- and LDMDP-treated CD-I mice were infected intranasally with PAOl at 6.9 x 106. However, there was no significant difference in bacterial clearance between AM-depleted and nondepleted CD-I mice (Fig. 11). Recruitment of PMN to the lung in LPBS-and LDMDP-treated CD-I mice with P. aeruginosa (PAOl) inoculated was evaluated from BAL counts and cytospin preparations. The number of PMN isolated from AM-depleted mice was not significantly different from that obtained from nondepleted mice throughout five days post-infection (data not shown). Histologic examination of the lungs from AM-depleted and nondepleted CD-I mice (Fig. 12a,b) revealed increased numbers of PMN compared to AM-depleted and nondepleted uninfected lungs (Fig. 5). Cytospin preparations of BAL cells from LPBS- and LDMDP-treated showed that the recruited PMN were competent to phagocytose P. aeruginosa (Fig. 12c,d). Ingested P. aeruginosa appeared as dark rods (arrows) and usually clustered, with some visible in phagosomes, as those by PMN (Fig. 12c,d), and alveolar and peritoneal macrophages (Fig. 13). The spots in the cytoplasm of AM were not phagocytosed P. aeruginosa because the spots were scattered and not rod in shape; they might be granules or artifacts from being cytocentrifuged onto microscope slides (Fig. 12c,d). Further studies with LPBS- and LDMDP-treated mice challenged intranasally with P. aeruginosa strain PAOl at various doses - 1.32 x 107, 6.5 x 105 and 9 x 103 - did not show any difference in bacterial clearance between the two groups of mice (data not shown). 4 5 Fig. 9. Effects of alveolar macrophage depletion in BALB/c mice on P. aeruginosa (FRD1) clearance in vivo. 24 h after intranasal instillation of PBS- or DMDP-liposomes, BALB/c mice were intranasally challenged with P. aeruginosa strain FRD1 (6.0 x 106). Depletion of AM was confirmed by BAL counts and cytospin preparations on the day of infection. At 3 h (day 0) and 24 h (day 1) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on TSA plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. Data were expressed as means + SE. lung b spleen ! U. o 9.00 8.00 _ 7.00 o I 6.00 -I ra 5.00 OQ | 4.00 " 3.00 2.00 -I 1.00 0.00 Days Postinfection -n— LPBS -X—LDMDP Days Postinfection liver S 3 o Days Postinfection 46 Fig. 10. Effects of alveolar macrophage depletion in BALB/c mice on P. aeruginosa (PAOl) clearance in vivo. 24 h after intranasal instillation of PBS- or DMDP-liposomes, BALB/c mice were intranasally challenged with P. aeruginosa strain PAOl (2.7 x 107). Depletion of AM was confirmed by BAL counts and cytospin preparations on the day of infection. At 3 h (day 0), 24 h (day 1), 48 h (day 2) and 120 h (day 5) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on TSA plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. Data were expressed as means + SE. lung Ol o 3 Days Postinfection b spleen 9.00 8 .00 7 .00 A & 6 .00 o D) k-3 5.00 A 4 . 0 0 3 .00 2 .00 1.00 o.oo m-0 -0— LPBS -X—LDMDP 2 3 Days Postinfection 5 liver 3 O 9 .00 8 .00 -I 7.00 6 .00 5 .00 4 .00 3 .00 2 .00 1.00 o.oo m-0 -B— LPBS -X—LDMDP 3 4 Days Postmleclion 5 47 Fig. 11. Effects of alveolar macrophage depletion in CD-I mice on P. aeruginosa (PAOl) clearance in vivo. 24 h after intranasal instillation of P B S - or DMDP-liposomes, C D - I mice were intranasally challenged with P. aeruginosa strain P A O l (6.9 x 10 6). Depletion of A M was confirmed by B A L counts and cytospin preparations on the day o f infection. At 3 h (day 0), 48 h (day 2) and 120 h (day 5) post-infection, lungs, spleen and liver were removed aseptically and placed in 5ml of Gel-Hank's solution. The tissues were homogenized. Serial 1:10 dilutions of the homogenates were spread on T S A plates and incubated for 18 h at 37°C. Viable bacterial counts were then analyzed. Data were expressed as means + SE. 0 1 2 3 4 5 6 Days Postinfection b spleen D) 3 9.00 8.00 -7.00 -6.00 5.00 -4.00 -3.00 2.00 1.00 A 0.00 k -B— LPBS -X— LDMDP liver S o 3 9.00 8.00 A 7.00 6.00 -I 5.00 4.00 3.00 2.00 1.00 0.00 0 Days Postinfection 2 3 4 Days Postinfection • LPBS -LDMDP 48 Fig. 12. Histologic sections and BAL cytospin preparations from P. aeruginosa-challenged LPBS- and LDMDP-treated mice. 24 h after intranasal (i.n.) instillation of LPBS or LDMDP, CD-I mice were intranasally challenged with P. aeruginosa strain PAOl (6.9 x 106). Depletion of AM was confirmed by BAL counts and cytospin preparations on the day of infection. 3 h after i.n. inoculation of P. aeruginosa, lung sections from (a) LPBS- and (b) LDMDP-treated mice were excised and fixed in 10 % buffered Formalin, processed and stained with hematoxylin and eosin. Magnification, X 125. 3 h post-infection, bronchoalveolar lavage (BAL) fluid were obtained from (c) LPBS- and (d) LDMDP-treated P. aeruginosa-challenged mice. BAL cells were cytocentrifuged onto microscope slides and stained with Diff-Quick. Mag., X 1250. a. lung sections: LPBS-treated 50 In vitro phagocytic activities of freshly explanted alveolar macrophages. In AM-depleted and nondepleted lungs, no significant difference in P. aeruginosa clearance from the lung was observed and PMN recruited to the infected lung phagocytosed P. aeruginosa. Therefore, one of the known functions - phagocytosis - by alveolar macrophages was assessed. In vitro phagocytic abilities of freshly explanted alveolar macrophages and resident peritoneal macrophages (PM) were evaluated. Macrophages, plated on coverslips in glucose-free phagocytosis medium, were incubated with 25 pi of latex beads, zymosan particles, or overnight shaking culture of unopsonized P. aeruginosa strains PI and PAOl (approximately 10 cfu) with or without 10 mM D-glucose. Extracellular bacteria were lysed by the addition of cold lysozyme. The coverslips were then washed, fixed, Giemsa-stained, air dried and mounted on microscope slides. The photomicrographs in Fig. 13 demonstrate the phagocytic activities of freshly explanted AM and resident PM. Extracellular bacteria, which were lysed, unbound and bound, could be distinguished from intracellular bacteria, which appeared as dark and clustered rods, by visual examination of stained macrophages. Results, expressed as number of ingested particles per macrophage, are shown in Table 5. In vitro phagocytosis assays indicated that unopsonized P. aeruginosa were poorly ingested by freshly explanted murine AM from BALB/c and CD-I mice in the presence or absence of D-glucose (1.3 -1.8 and 2.0 - 3.4 bacteria/ macrophage), respectively. While freshly explanted murine resident PM ingested unopsonized P. aeruginosa strain PI (13.9 PI bacteria/macrophage) in the presence of D-glucose, and strain PAOl (5.7 and 8.3 PAOl bacteria/macrophage) with and without glucose, respectively. AM and PM phagocytosed similar numbers of zymosan (3.2 - 3.4 and 1.6 particles/macrophage), respectively. 51 Fig. 13. In vitro phagocytic activities of freshly explanted alveolar macrophages and resident peritoneal macrophages. AM were obtained from BALB/c and CD-I mice and PM from BALB/c mice. Macrophages, in glucose-free phagocytosis medium, were incubated with 25 ul latex beads, zymosan particles, or overnight shaking culture of unopsonized P. aeruginosa strains PI and PAOl (approximately 107 cfu) with or without 10 mM D-glucose. Extracellular bacteria were lysed by the addition of 500 ul of cold lysozyme. After a PBS wash, cells were incubated with 500 u.1 of H2O, washed with PBS twice and fixed with methanol. The coverslips were Giemsa-stained, washed and mounted on glass microscope slides. Ingested bacteria appeared as dark rods (arrows). Magnification, X 1250 . AM - zymosan 53 PM — zymosan PM - latex beads 54 Table 5. In vitro phagocytic activities of freshly explanted murine alveolar macrophages (AM) and resident peritoneal macrophages (PM). AM were obtained from BALB/c and CD-1 mice and PM from BALB/c mice. Macrophages, in glucose-free phagocytosis medium, were incubated with 25 ul latex beads, zymosan particles, or overnight shaking culture of unopsonized P. aeruginosa strains PI and PAOl (approximately 107 cfu) with or without 10 mM D-glucose. Extracellular bacteria were lysed by the addition of cold lysozyme. After a PBS wash, cells were incubated with of H2O, washed with PBS twice and fixed with methanol. The coverslips were Giemsa-stained, washed and mounted on glass microscope slides. Phagocytosis was assessed microscopically. At least 60 macrophages per coverslip were scored. The experiments were repeated two to three times, with each sample in duplicate. Ingestion of particles was scored microscopically. Data were expressed as means + SEM. *, significant at P<0.05 (two sample Student's t-test) versus value for AM. Experimental Average number of ingested condition particles per macrophage BALB/c Alveolar SEM Peritoneal SEM P1 1.3 0.6 0.1 0.0 P1+D-glucose 1.4 0.9 13.9* 2.4 PA01 1.4 0.6 5.7* 1.2 PA01+D-glucose 1.8 0.7 8.3* 0.2 zymosan 3.4 0.2 1.6 0.0 latex beads N/A N/A 2.3 0.4 CD-1 P1 2.1 0.6 P1+D-glucose 3.4 0.6 PA01 2.3 0.5 PA01+D-glucose 2.0 0.6 zymosan 3.2 0.5 55 In situ phagocytic activities of alveolar macrophages. 78 - 88 % of alveolar macrophage depletion did not affect P. aeruginosa clearance from the lung, and PMN recruited to the infected lung phagocytosed P. aeruginosa. As well, in vitro studies demonstrated that freshly explanted alveolar macrophages (AM) were not competent to phagocytose unopsonized P. aeruginosa, but were able to phagocytose zymosan particles. Therefore, further studies were conducted to assess in situ phagocytic ability of AM. P. aeruginosa strains used in in situ experiments included the ones used in the in vivo infection model (FRD1 and PAOl) and in the in vitro phagocytosis assays (PI and PAOl), as well as strains c2908c and PAK. Previous in vitro phagocytosis assays performed in our laboratory have demonstrated that ingestion of c2908c by peritoneal macrophages was glucose-dependent and PAK glucose-independent. Latex beads and zymosan were used as control particles, as in the in vitro assays. Therefore, there were four nonmucoid strains - PI and c2908c (glucose-dependent strains), and PAK and PAOl (glucose-independent strains), one mucoid strain, FRD1, and two control particles, latex beads and zymosan, used in in situ assays (Fig. 14). Three hours after intranasal instillation of P. aeruginosa, zymosan particles or latex beads, BAL was performed. The differential counts of AM and PMN (Fig. 14a) and assessment of particle ingestion by AM and PMN (Fig. 14b) in the BAL population were performed on Diff-Quick-stained cytospin preparations. 100 BAL cells were counted, and the results of the differential counts Of AM and PMN were expressed as percentage (Fig. 14a). In untreated/uninfected control mice, mononuclear cells comprised 99- 100 % of BAL cells and PMN less than 1 %. Recruitment of neutrophils to the lung was substantially increased upon high infection dose of P. aeruginosa -PAK at 108, and PI, c2908c, and FRD1 at 107(16 - 62 %). PMN recruitment to the lung was minimal in mice inoculated with zymosan particles (9 %) and latex beads (4 %). Fig. 14b shows the percentage of AM and PMN with ingested particles in the BAL population. AM phagocytosis 56 of unopsonized P. aeruginosa (0-5 %) was much lower than that of zymosan particles (23 %) and latex beads (27 %), whereas 0.3-13 % PMN phagocytosed P. aeruginosa. 5 7 Fig. 14. In situ phagocytic activities of alveolar macrophages. Three hours after the intranasal instillation of P. aeruginosa, zymosan particles or latex beads, bronchoalveolar lavage (BAL) was performed. The differential counts of alveolar macrophages (AM) and neutrophils (PMN), and assessment of ingested particles by AM and PMN were performed on cytospin preparations stained with Diff-Quick with the aid of a light microscope. Each sample was performed in triplicate. 100 BAL cells were counted. Results were expressed as means + SE. (a) % of AM and PMN in the BAL cell population (b) % of AM and PMN with ingested particles in the BAL cell population. *, significant at P<0.05 (two sample Student's t-test) compared to value for untreated (control) mice. a. percentages of A M and PMN in the BAL cell population 100.0 8. < CQ O 90.0 80.0 70.0 60.0 50.0 -I 40.0 30.0 20.0 10.0 0.0 1 I i i 1 1 p % Macrophages - % PMN $ ^ N<*> ^ <$>°' ^ N# ^ N# 6* ^ 4-' N ' N ' <oa <<P ^ <<P 4P • ^  b. percentages of A M and PMN with ingested particles in the BAL cell population 30.0 Q % Macrophages w/particles B % PMN w /particles 58 DISCUSSION Effective host defense against bacterial infection in the lungs requires the orchestration of inflammatory responses. Alveolar macrophages (AM) provide one of the first lines of defense against microbial invasion in the lower respiratory tract. AM are involved in phagocytosis and regulation of the immune response. The role of AM in host defense of the lung against P. aeruginosa is controversial and is the topic of investigation in this thesis. The role of AM in defense against P. aeruginosa challenge was assessed by three approaches - in vivo AM depletion and subsequent P. aeruginosa infection in mice, in vitro nonopsonic phagocytosis by freshly explanted AM and in situ phagocytic activities of AM. In vitro effects of LPBS, LDMDP and DMDP on macrophages and epithelial cells. Previous in vitro studies have shown that AM actively phagocytose liposomes; AM display the morphology characteristic of ongoing phagocytic activity, such as grossly irregular perimeters and extensive pseudopod formation (43). Uptake of liposomes by AM was demonstrated by the strong fluorescence emitted in AM treated with liposome-encapsulated FITC for 24 and 48 h (Fig. 3). The morphology and phagocytic ability of AM and peritoneal macrophages (PM) were examined after incubation with LPBS, LDMDP and DMDP (all at 5, 10 and 15 ul) in 0.5 ml culture medium (Figs. 1 and 2, Tables 3' and 4). The number of PI ingested by LPBS-treated PM and AM was slightly decreased, with some exceptions, than that by untreated macrophages (Tables 3 and 4). This reduced phagocytosis by LPBS-treated macrophages is likely due to saturation of macrophage phagocytic ability by liposomes. Large liposomes, as prepared in these studies, are selectively phagocytosed by macrophages, whereas small liposomes (<0.2 um) may also be internalized by non-phagocytic cells; thus, the former are more efficient in blocking phagocytosis than the latter. Macrophage phagocytosis is blocked by 59 high doses of blocking agents, such as liposomes and carbon particles, due to saturation of phagocytic capacity. In contrast to carbon particles, which cannot be digested or metabolized by macrophages, liposomes are digested by macrophages; therefore, a phagocytosis blockade by liposomes may be expected to last for a shorter period of time than that induced by carbon particles (56). However, the effects of LDMDP on phagocytic competency by PM, at 48 h (Table 3), and by AM, at all time points (Table 4), were more apparent than those exerted by LPBS, suggesting that a significant decrease in phagocytosis of P. aeruginosa (PI) by macrophages was not only due to liposomes alone. The effects of LDMDP on PM were more evident at 48 h than 24 h (Fig. 1 and Table 3). At 48 h, the changes in the morphology and phagocytic activities of LDMDP-treated PM were significant and dose-dependent, compared to those of untreated and LPBS-treated PM. At 24 h, the effects of LDMDP (5 and 10 ul) on the morphology and phagocytic function of PM were significant, but were not dose-dependent, compared to untreated and LPBS-treated PM. The delayed response of PM to LDMDP in vitro may be due to the fact that macrophages in vitro are not very active; therefore, the time for LDMDP to be ingested, and DMDP to be released into the cytoplasm and to substantially exert its effects on macrophages in vitro requires more than 24 h. The results of one representative in vitro experiment assessing the morphology of AM treated with LPBS, LDMDP and DMDP were shown due to high variations among experiments (Fig. 2). The effects of LDMDP on the morphology of AM were substantial, compared to those of LPBS, but were not enhanced with longer incubation periods. There were no significant changes after 72 h of incubation, compared to those at 24 and 48 h (Fig. 2), which might be due to saturation of liposome uptake by AM, as discussed above. However, a significant inhibition of the phagocytic ability of LDMDP-treated AM, compared to those of untreated and LPBS-treated AM (Table 4), was likely a result of the disruption/suppression of their phagocytic function by LDMDP, although the disruption was not reflected as substantially in their morphology. 60 The results of these in vitro experiments assessing the effects of LPBS, LDMDP and DMDP on AM and PM correlated with those of an in vitro study of rat AM incubated with LPBS, LDMDP and DMDP (2). After 5 days of incubation, the effects of LPBS, LDMDP and DMDP (all at 0.05, 0.5 and 5 ul/ml culture medium) on the number of rat AM, originally seeded at 1.5 x 10s AM/well, in vitro was assessed by obtaining the absorbance at 570 nm. Preliminary experiments have shown that within the range of 0 - 1.5 x 105 AM/well, there is a linear relationship between the number of AM stained with crystal violet and the absorbance at 570 nm, and hence is an accurate way of quantifying the number of AM (2). Although used at different concentrations, the effects of LDMDP on the morphology, phagocytic ability of murine AM and PM (Figs. 1 and 2, and Tables 3 and 4), and adherence to a surface by rat AM (2) have been demonstrated to be more significant and evident than those of LPBS. LDMDP-treated AM and PM had shriveled nuclei and cytoplasm, suggesting that they had undergone apoptosis. Macrophages phagocytose LDMDP, which accumulates in the phagolysosomes, resulting in selective cell death without damaging surrounding cells or tissues (5). LDMDP induces apoptotic cell death in macrophages in vitro and in vivo (37, 56). Apoptosis is characterized by cell shrinkage, condensed nuclei, blebbing of the plasma membrane, compacted cytoplasmic organelles and DNA fragmentation. Apoptotic macrophages are then removed from the circulation by other macrophages, which in turn would be exposed to the effects of LDMDP and undergo apoptosis (37). The changes in cellular metabolism upon accumulation of intracellular DMDP are still unknown. The roles of intracellular calcium and iron (35, 58), and ATP metabolism of the cell (42) have been investigated. AM and PM incubated with 0.6 M DMDP stock showed unhealthy characteristics - no cytoplasm, and hence ingestion of P. aeruginosa was not observed. DMDP-treated AM and PM lost their cytoplasm, indicating that the mechanisms by which DMDP and LDMDP affect macrophages were not the same. A549 respiratory epithelial cells incubated with DMDP (50 mM and 0.6 M), but not 0.5 61 and 5 mM, for 24 and 48 h showed changes in morphology. However, the concentration of DMDP used in in vitro assays was higher than that incorporated into LDMDP. During liposomal preparation, only 1 % of DMDP is encapsulated into LDMDP (57). No change in morphology of epithelial cells incubated to LPBS and LDMDP was observed, suggesting that these liposomes administered in vivo in mice would not likely affect surrounding epithelium in mice; their efforts appear to be limited to macrophages. In vivo depletion of alveolar macrophages by intranasal instillation of DMDP-Iiposomes. At 24 and 48 h, 74 - 88 % of AM were depleted by intranasal (i.n.) instillation of LDMDP (Fig. 4b). At 72 h, the number of AM recovered from LDMDP-treated mice slightly increased (data not shown). These results correlated with the results of AM depletion by administration of LDMDP to the lung using various methods in rodent models (2, 5, 23, 25, 27, 32). Intratracheal (i.t.) insufflation of LDMDP results in reduction of 65 - 80 % of AM in mice 24 h post-instillation and lasts for 5 days (5, 25), and 75 - 85 % of AM in rats 72 h later (25, 32), compared to no significant AM depletion in animals receiving LPBS, PBS, saline, or no treatment (5, 25, 32). Rats intubated with LDMDP are depleted of >70 % of AM within 1 day and lasting for >5 days post-instillation (2). About 95 % of AM in mice and rats are depleted by aerosol inhalation within 3 days of aerosolization (23, 27). By days 3 to 5, the number of AM gradually increases and reaches normal levels in 2 to 4 weeks (2, 5, 23). A high degree (95%) of AM are depleted by LDMDP delivered by aerosol inhalation. However, there are two main concerns of administration of liposome-encapsulated drug by aerosolization, which include disruption of liposomes and uncertainty in the amount of encapsulated drug delivered by different nebulizers (12). The unilamellar liposomes, prepared for nebulization, are obtained after extrusion through a 0.2 pm-pore-size filter. A nebulizer supplied with compressed air at a flow rate of 10 - 12 litres/min is used to generate liposome 62 aerosol (23, 27). Therefore, it is not unexpected that liposomes are disrupted when passed through a nebulizer at such a high flow rate. In addition, various nebulizer types differ in the amount of liposome-encapsulated compounds predicted, by a mathematical lung deposition model, to deposit in the different regions of the lung (12). DMDP, released from disrupted DMDP-liposomes, also affect cells other than AM. Insufflation of DMDP causes AM cell death, deposition of debris in the alveolar space and cytoplasmic edema of alveolar epithelial cells (2). Unilamellar vesicles (<0.2 |j.m), unlike multilamellar liposomes, are internalized by non-phagocytic cells (57). Epithelial cells, which are not professional phagocytic cells, are capable of internalizing particles (44). As discussed earlier, the respiratory epithelial cell line A549 showed unhealthy characteristics after incubation with DMDP for 48 h. Therefore, the striking effect of LDMDP on AM depletion (95%) by aerosolization (23, 27) may be partly due to free DMDP, which is released into the airspace in the aerosol, and this may affect subsequent bacterial clearance in AM-depleted and nondepleted mice as epithelial cells are damaged. Multilamellar liposomes, as prepared in these studies, are predominantly ingested by phagocytic cells, thereby depleting macrophages more efficiently than could their smaller unilamellar counterparts (57). In addition, our in vitro studies showed that there were no significant changes in the morphology of the epithelial cell line A549 incubated with LPBS and LDMDP for 24 and 48 h, suggesting that multilamellar liposomes would not likely affect epithelial cells in vivo. DMDP does not easily cross cell membrane and has a very short half-life in circulation. Therefore, one would assume that liposome-encapsulated DMDP, when phagocytosed by macrophages, does not escape from the cell, and that other tissues are protected (13). 63 Intranasal inoculation and clearance of P. aeruginosa in untreated, nondepleted and A M -depleted mice P. aeruginosa recovered from the lung shortly after i.n. administration was very similar to the initial inoculum, suggesting that this route of infection was sufficient to introduce an intrapulmonary infection in mice (Figs. 6, 7 and 8). However, no significant difference in clearance of P. aeruginosa strain PAOl (Figs. 9, 10 and 11) or neutrophil recruitment (Fig. 12) between AM-depleted and nondepleted BALB/c and CD-I mice was detected. These results did not correlate with previous studies (5, 25, 27). AM depletion results in reduced killing of Mycoplasma pulmonis in resistant strain C57BL, but not in susceptible strain C3H, mice; mouse strains differ significantly in resistance to M. pulmonis (25). 48. h after Klebsiella pneumoniae inoculation, mice with 65 % of AM depleted show increased bacterial counts in lungs compared to nondepleted infected mice (5). However, K. pneumoniae is more virulent than P. aeruginosa; 100 CFU of the former was used to infect mice (5), whereas a much higher dose (106 CFU) of the latter was sufficient to introduce an intrapulmonary infection in mice without being cleared too quickly (Figs. 6 & 7). Therefore, 65 % AM depletion reduces K. pneumoniae clearance in the lung due to high virulence of the bacteria. The discrepancy between my results with P. aeruginosa-infected AM-depleted mice and those by Broug-Holub et al and Hickman-Davis et al may be due to differences in infecting agents. Decreased bacterial clearance has been observed in CD-I mice depleted of 95 % of AM by aerosolization and infected with P. aeruginosa strain PAOl by intubation (27). The discrepancy between my results and those by Kooguchi et al is not likely due to mouse strain, as no significant difference in P. aeruginosa clearance in both AM-depleted BALB/c and CD-I mice was observed, compared to nondepleted mice (Figs. 9, 10 & 11). The extremely high degree of AM depletion (95 %) reported by Kooguchi et al, compared to that reported in this thesis (74 - 88 %), may account for the differences between the results. However, as discussed above, epithelial cells are damaged by free DMDP, which is 64 leaked from disrupted DMDP-liposomes administered by nebulization into the airspace. Bacterial colonization and clearance in mice with AM depleted by aerosolization is different from that in mice with AM depleted by intranasal inoculation. At 48 h post-infection, PMN recruitment in DMDP-treated lungs is higher than that in control infected lungs, suggesting that decreased K. pneumoniae clearance is not due to impaired PMN influx. As well, neutralization of TNF-a or MIP-2, before infection, reduces the numbers of PMN (5). 4 h after inoculation of P. aeruginosa to the airspace, PMN counts in lavage fluid from AM-depleted rats are much lower than those from PBS-treated rats. However, after instillation of recombinant MIP-2 into liposomal DMDP-treated rats, the number of PMN recruited into airspaces increase to a level similar to control rats given the same dose of MIP-2 (23). The discrepancy between the results of PMN recruitment by Broug-Holub et al and Hashimoto et al may be due to the assessment of PMN recruitment at different timed intervals. Recruitment of PMN is impaired at 4 h (23) but later recovered at 48 h post-infection (5). In AM-depleted P. aeruginosa-challenged mice, the number of PMN recovered from BAL fluid at early time points (0 - 6 h) is much lower than that at late phase (24 - 96 h). PMN recruitment in mice with 95 % AM depleted is impaired in early postinfection period, but is recovered in later time points, compared to nondepleted animals. The increase in PMN numbers in AM-depleted lungs at late phase associates with an increase in the number of AM in the BAL fluid (27). These results suggest that AM are important in PMN recruitment to the lung. As shown in histologic sections (Fig. 12), recruitment of PMN to the lung was not impaired in LDMDP-treated mice compared to LPBS-treated mice, suggesting that the remaining 12-26 % AM were sufficient to elicit an inflammatory response, particularly PMN, to infected sites. PMN did not seem to be affected by LDMDP or LPBS, consistent with the results of in vitro and in vivo experiments demonstrating that neutrophils, morphologically and functionally, are not affected by LDMDP (45). In the uninfected or untreated lung, PMN comprise a small 65 proportion of cells in the LRT and macrophages 85 to 98 % (25, 55, 60). However, the number of PMN increases in lungs challenged with P. aeruginosa by i.t. inoculation. AM from normal rats secrete neutrophil chemotactic factors into culture medium upon addition of P. aeruginosa in vitro (40). PMN, but not AM, actively phagocytosed P. aeruginosa, as shown in BAL cytopsin preparations from both groups of mice (Fig. 12c,d), indicating that PMN actively participated in bacterial clearance. PMN, monocytes, and macrophages are described as the "professional phagocytes", determined by the rate and extent to which they are can ingest upon activation (6). PMN are shown to be as phagocytic as AM; the fusion of phagosome and primary lysosome allows the entry of myeloperoxidase, which uses hydrogen peroxide and chloride to make potent antimicrobial substances (38). Killing of P. aeruginosa and K. pneumoniae is reduced in neutropenic mice, demonstrating the importance of PMN in bacterial clearance (33). PMN emigration during an inflammatory response is mediated through the interactions between adhesion molecules on neutrophils and endothelial cells, upon activation by chemotactic factors (24). The major component of the Gram-negative bacterial cell wall, lipopolysaccharide (LPS), activates monocytes and macrophages to secrete proinflammatory mediators, e.g. interleukin-1-beta (IL-1 p) and IL-8 and TNFa (26). The GDI 1/18 integrins on PMN and intercellular adhesion molecule-1 (ICAM-1) on endothelium surface, in the presence of an activator molecule, contribute to the arrest of PMN along the vascular endothelium. Activation of the CD 18 integrins is linked to increased avidity and upregulation of expression on PMN surface. ICAM-1, which is constitutively expressed on endothelial cells, is upregulated by IL-1, TNFa, endotoxin and interferon-y (7). IL-8 displays chemotactic activity for PMN and activates PMN to release lyososmal enzyme and to adhere to endothelial cells. After attachment to endothelium (pavement), PMN insert pseudopodia between the endothelial cells, dissolve the basement membrane (diapedesis), leave the vascular vessel, and migrate along increasing concentrations of chemotactic substances (24). The neutrophilic inflammation elicited in response to an 66 intrapulmonary challenge with P. aeruginosa can be further characterized by measuring levels of PMN by myeloperoxidase activity and of cytokines known to recruit PMN, such as macrophage inflammatory protein-2 (MIP-2), a functional analogue of human IL-8, and TNFa. In vitro phagocytic activities of freshly explanted alveolar macrophages Poor nonopsonic phagocytosis of P. aeruginosa by freshly explanted murine AM (Table 5) correlated well with previously published data on freshly explanted and cultivated murine AM, where the former show negligible phagocytosis of unopsonized P. aeruginosa and the latter increased (10, 60). However, ingestion of zymosan particles and latex beads by freshly explanted AM demonstrated that they were phagocytically competent. The phagocytic activities of freshly explanted AM from BALB/c and CD-I mice were similar, suggesting the poor phagocytic ability of uncultivated AM is not due to mouse strain. Freshly explanted PM ingested unopsonized P. aeruginosa strain PI in the presence of D-glucose and strain PAOl in the presence or absence of glucose. The effect of glucose on nonopsonic phagocytosis of P. aeruginosa was originally demonstrated in murine resident and thioglycolate-elicited peritoneal macrophages (49) and later in human and murine alveolar macrophages (1, 10, 60). The difference in phagocytic competency of freshly explanted alveolar and peritoneal macrophages may be due to their differential glycolytic activities. AM reside in the oxygen-rich environment of lung airways (13% O2) whereas PM dwell in the relatively anaerobic peritoneum (6.5% 02); therefore, their activities of enzymes for oxidative phosphorylation and glycolysis may vary. AM, from rabbit and guinea pig, have greater oxygen utilization, higher activities of cytochrome oxidase and lower glycolytic enzyme activities than PM (28). The association of glucose transport and nonopsonic phagocytosis of P. aeruginosa has been previously reported (1, 10, 60). Phloretin, an inhibitor of faciliated glucose transport, inhibited phagocytosis of unopsonized P. aeruginosa, but not zymosan, by peritoneal macrophages. Phlorizin, an inhibitor of the sodium-67 glucose cotransporters, did not alter glucose transport or ingestion of unopsonized P. aeruginosa and zymosan (1). An increase in uptake of unopsonized P. aeruginosa coincides with increased facilitated glucose transport by murine AM cultivated in vitro (10). However, the association of increased ingestion of unopsonized P. aeruginosa and glucose transport is not seen in cultivated human AM. This inconsistency between human and murine AM may be attributed to their utilization of transported glucose via glycolysis (60). In addition to the implicated role of glycolysis, signal transduction also plays a role in macrophage phagocytic activities (18, 21). The interaction of particles with specific cell surface receptors initiates intracelluar signalling pathways, which subsequently lead to cytoskeleton rearrangement, pseudopod extension, and particle ingestion. Tyrosine kinase and protein kinase C are linked to Fc-mediated phagocytosis, and phosphatidylinositol 3-kinase (PI 3-kinase) to cytoskeleton alteration (21). Previously in vitro studies in our laboratory have shown the inhibitory effects of wortmannin, a potent and irreversible inhibitor of PI 3-kinase, and genistein, an inhibitor of tyrosine kinase, on ingestion of unopsonized P. aeruginosa and zymosan by peritoneal macrophages (data not shown). Binding of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and IL-5 to the corresponding high affinity receptors leads to signal transduction and functional activation of the cells (18). The interaction of glucose transport and signal transduction may be involved in macrophage phagocytosis. GM-CSF promotes facilitated glucose transport by a human melanoma cell line (51). In a myeloid cell line, IL-3 stimulates glucose uptake by actively maintaining the affinity of the plasma memberane glucose transporter for glucose (4). Taken together, the differences in phagocytic competency of AM and PM may be due to their differential abilities to transport and utilize glucose, and initiate the signalling pathways. 68 Particle phagocytosis by alveolar macrophages in situ. In vitro response of AM to P. aeruginosa has been demonstrated in previous studies (1,3, 10, 60), and in vivo response of AM to i.t. challenge of P. aeruginosa in intra-Peyer's patch-immunized and nonimmunized rats (8). A different approach was taken in these studies to assess in situ phagocytic activities of AM in response to challenge by unopsonized P. aeruginosa or other particles, such as zymosan and latex beads. Three hours post-instillation, percentages of AM with ingested unopsonized P. aeruginosa were much lower than those with ingested zymosan particles and latex beads in the BAL population (Fig. 14b). These results indicated that AM were phagocytically competent but were unable to ingest unopsonized P. aeruginosa, consistent with those of in vitro assays showing poor nonopsonic phagocytosis by freshly explanted AM. However, it is worthwhile to note that AM recovered by BAL represent the majority, not all, of AM in the lung. Buret et al have shown that phagocytosis of P. aeruginosa by AM in intra-Peyer's patch-immunized and nonimmunized rats infected with P. aeruginosa (108) decreases substantially at 4 h post-infection, compared to that at 30 min after infection, despite an increase in AM numbers in immune rats. The number of AM is increased by 10-fold in immune rats at 4 h, but not 30 min, post-infection, compared to nonimmune rats. However, the number of PMN increases dramatically in immune (106) and nonimmune (107) rats at 4 h post-infection, compared to that at 30 min post-infection (103 and 104, respectively) (8). The decreased phagocytic activity of AM in rats at 4 h post-infection reported by Buret at al may be due to the recruitment of other immune cells, such as PMN, which migrate to the infected sites and are involved in bacterial clearance. As demonstrated in Fig. 14, PMN, recruited in lungs challenged with high bacterial dose, phagocytosed P. aeruginosa, indicating that PMN actively participated in bacterial clearance. Neutrophil bactericidal activity is higher than that of the unactivated macrophages, due to the robust capacity of the former to produce reactive oxygen radicals (9). These results correlate with in vivo Staphylococcus aureus infection studies 69 demonstrating that a small inoculum (105) can be cleared by. AM alone, whereas an inoculum of 106 elicits a modest influx of PMN to the alveoli, and PMN and AM together have a greater capacity to clear \07 S. aureus (33). The number of PMN elicited in response to latex-bead- and zymosan-challenge was minimal. 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