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Role of complement receptor type 3 in mycobacterium tuberculosis pathogenesis Melo, Maico DaPonte 1999

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Role of Complement Receptor Type 3 in M y c o b a c t e r i u m tuberculosis Pathogenesis by Maico DaPonte Melo B.Sc , The University of Victoria, 1996 A thesis submitted in partial fulfillment 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 June 1999 © Maico DaPonte Melo, 1999 in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Tofko /o<U/ a l ^ A / g ^ ^ K < _ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) A B S T R A C T Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, is a facultative intracellular pathogen capable of survival in macrophages. Several macrophage receptors including complement receptor type 3 (CR3, CDI lb/CD 18) are reportedly capable of binding M T B . It has been suggested that M T B survival in macrophages is determined by the receptor to which M T B binds before being phagocytosed. A CDI lb knockout mouse model was used as a source of macrophages to test whether CR3 is essential for the association of M T B with macrophages and to examine whether the absence of CR3 on macrophages alters the intracellular fate of M T B . Together, these research objectives were aimed at improving our understanding of the role CR3 plays in the pathogenesis of tuberculosis. Studies using both alveolar macrophages (AM) and peritoneal macrophages (PM) showed CR3 was not essential for M T B binding by macrophages, either in the presence or absence of serum. However, CR3 was involved in the association of M T B with macrophages in the absence of serum and was important for the efficient binding of M T B under conditions where serum was present. Compared to macrophages expressing CDI lb (CDI lb+/+), CDI lb-knockout (CDI lb-/-) macrophages were less able to associate with M T B , either opsonically or non-opsonically. Under opsonic conditions, the enhanced binding of M T B to macrophages was mediated by a heat-labile serum component, as heat-inactivation of serum abrogated the increased binding. Using immunoglobulin-deficient serum to opsonize M T B demonstrated that the increased binding of M T B was not mediated by immunoglobulins. The role of either the classical i i or alternative complement pathways in mediating the increased binding of M T B by P M was also investigated. The observations made here indicate that, in the presence of low serum concentrations, increased binding of M T B by CDI lb+/+ P M is mediated predominantly via activation of the classical pathway but is independent of immunoglobulins. The intracellular survival and replication of M T B following phagocytosis by either CDI lb-/- P M or CDI lb+/+ P M was investigated and no significant difference in the replication of M T B was detected where similar numbers of M T B were ingested. Therefore, the observations made here do not support the hypothesis that M T B exploits CR3 as a means to evade being killed by macrophages. As an alternative approach to the CDI lb knockout mouse model, an alveolar macrophage cell line, MH-S, was tested for its suitability as a model to investigate the role of CR3 in mediating the interaction of M T B with macrophages. Initially, the characterization of MH-S binding properties was performed. However, the principle objective with the MH-S cell line was to isolate CR3-negative populations of MH-S as a means to investigate the role of CR3 in the association of M T B . M T B association assays showed that the association of M T B with MH-S cells was comparable with mouse A M . In the presence of serum, increased M T B binding by MH-S cells was also mediated by a heat-labile serum component and competitive inhibition assays using anti-CR3 monoclonal antibodies showed that CR3 on MH-S cells is a quantitatively important receptor for M T B . However, attempts to isolate stable CR3-negative populations of MH-S cells using immunoselection were not successful and therefore, I was unable to use the MH-S cell line as a model to further characterize the role of CR3 in M T B pathogenesis. iii TABLE OF CONTENTS Abstract i i List of Figures vii List of Tables viii List of Abbreviations ix Acknowledgments x C H A P T E R 1 Introduction 1 1.1 Mycobacterium tuberculosis (MTB) .1 1.2 Macrophage receptors for M T B 2 1.2.1 Complement receptors 2 1.2.1.1 Complement receptor type 1 (CR1, CD35) 4 1.2.1.2 Complement receptor type 3 (CR3, CDllb/CD18) 4 1.2.1.3 Complement receptor type 4 (CR4, p 150/95) 7 1.2.2 Other quantitatively important macrophage receptors for M T B 8 1.3 Survival and intracellular replication of M T B 9 1.4 Specific Aims 10 1.5 Experimental Approach 11 C H A P T E R 2 Materials and Methods 13 2.1 Mycobacteria 13 2.2 Mice 13 2.2.1 C D I lb Knockout mice 13 2.2.2 CDI lb Expressing mice 14 2.2.3 BALB/c mice 14 2.3 Isolation of primary macrophages from mice 14 2.3.1 Isolation of peritoneal macrophages 14 2.3.2 Isolation of alveolar macrophages 15 iv 2.4 Genotyping of CR3-positive and CR3-negative mice 16 2.4.1 D N A preparation 16 2.4.2 PCR amplification of D N A sequences specific for the genomes of either C D 1 lb-/- or CD 1 lb+/+ mice 17 2.4.3 Agarose gel electrophoresis of PCR genotyping products 18 2.5 Flow Cytometric Analysis 18 2.5.1 Flow cytometric analysis of CDI lb+/+ and CDI lb-/- macrophages 18 2.5.2 Antibodies used to probe for surface antigen expression .19 2.6 Alveolar macrophage cell line, MH-S 20 2.6.1 Isolation of CR3-negative populations of MH-S 21 2.7 Particles for probing macrophage receptors 21 2.7.1 Fc receptors 21 2.7.2 Complement receptors 21 2.7.3 Lectin-like receptors 22 2.8 In vitro assays for association of particles with macrophages 22 2.9 In vitro survival and replication of M T B following phagocytosis by either CDI lb+/+ or CDI lb-/- macrophages 25 2.10 Statistical analysis 27 C H A P T E R 3 Results: Role of CR3 in M T B pathogenesis investigated using CDI lb-knockout and CDI lb-expressing mouse macrophages 28 3.1 Genotyping of C D 11 b-/- and C D 11 b+/+ mice 28 3.2 Flow cytometric analysis of CDI lb-/- and CDI lb+/+ macrophages 29 3.3 Association of zymosan, EIgG, and E l g M C with P M from CDI lb-/- and CDI lb+/+mice 31 3.4 Association of M T B with P M from CD 1 lb-/- and CD 1 lb+/+ mice 33 3.4.1 Non-opsonic association of M T B with P M 33 3.4.2 Opsonic association of M T B with P M 34 3.4.3 Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with P M 36 3.4.4 Association of pre-opsonized M T B versus association of M T B in the presence of serum with P M 39 3.4.5 Role of complement in the association of M T B with P M 40 3.5 Association of zymosan, EIgG, and ElgMC' with A M from CDI lb-/- and CDI lb+/+ mice 45 3.6 Association of M T B with A M from CDI lb-/- and CDI lb+/+ mice 47 3.6.1 Non-opsonic association of M T B with A M 47 3.6.2 Opsonic association of M T B with A M 48 3.6.3 Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with A M before and after in vitro differentiation 50 3.6.4 Comparison of M T B binding to Day 0 A M from C57BL/6 and B A L B / c mice 54 3.7 Comparison of M T B survival and replication following phagocytosis by either C D 1 lb-/- P M or C D 1 lb+/+ P M 57 C H A P T E R 4 Discussion: Role of CR3 in M T B pathogenesis investigated using CDI lb-/- and CDI lb+/+ mouse macrophages 60 C H A P T E R 5 Results: Interaction of M T B with MH-S Cells: a comparison with primary macrophages 71 5.1 Comparison of M T B binding to MH-S cells, A M , and P M 71 5.2 MH-S cells, A M , and P M binding of EIgG, E l g M C and zymosan particles 73 5.3 MH-S cells, A M , and P M binding of M T B in the presence of serum 75 5.4 Effect of serum heat-inactivation on the association of M T B with MH-S cells . . . 78 5.5 Role of CR3 in the association of M T B with MH-S cells 80 5.6 Isolation of CR3-negative population of MH-S cells 82 C H A P T E R 6 Discussion: Interaction of M T B with MH-S Cells: a comparison with primary macrophages 84 C H A P T E R 7 Conclusion 88 R E F E R E N C E S 90 APPENDIX I Analysis of R A G serum 97 APPENDIX II Sample calculation for M T B growth rate constant 98 vi LIST OF FIGURES Page Figure 1. PCR genotyping of CD 1 lb+/+ and C D 1 lb-/- mice. 28 Figure 2. Flow cytometric analysis of macrophages obtained from CDI lb+/+ and CDI lb-/-mice. 30 Figure 3. Binding of zymosan, EIgG, and E l g M C to P M . 32 Figure 4. Non-opsonic association of M T B with CDI lb+/+ P M and CDI l b - / - P M . 33 Figure 5. Opsonic association of M T B with CDI lb+/+ P M and CDI l b - / - P M . 35 Figure 6. Effects of heat-inactivated and immunoglobulin-negative serum on the binding of M T B to CD 1 lb+/+ P M and CD 1 lb-/- P M . 38 Figure 7. Binding of zymosan, EIgG, and E l g M C to A M . 46 Figure 8. Non-opsonic association of M T B with CDI lb+/+ A M and CDI l b - / - A M . 47 Figure 9. Opsonic association of M T B with CDI lb+/+ A M and CDI l b - / - A M . 49 Figure 10. Effects of heat-inactivated and immunoglobulin-negative serum on the binding of M T B to CDI lb+/+ A M and CDI lb-/- A M before and after in vitro differentiation 53 Figure 11. Comparison of M T B binding to day 0 CDI lb+/+ A M and day 0 B A L B / c A M in the presence or absence of serum. 56 Figure 12. Comparison of M T B survival and replication following phagocytosis by either CD 1 lb+/+ or CD 1 lb-/- P M in vitro. 58 Figure 13. Binding of M T B and latex beads to P M , A M , and MH-S cells. 72 Figure 14. Binding of zymosan, EIgG, and E l g M C to P M , A M , and MH-S cells. 74 Figure 15. Binding of M T B by P M , A M , and MH-S cells in the presence of normal B A L B / c mouse serum. 77 vii Figure 16. Effect of serum heat inactivation on the binding of M T B by MH-S cells. 79 Figure 17. Role of complement receptor type 3 (CR3) in binding of M T B by MH-S cells in the presence of normal B A L B / c mouse serum. 81 Figure 18. Isolation of CR3-negative subpopulations of MH-S cells. 83 LIST OF TABLES Page Table 1. Binding of M T B to P M either in the presence of serum or following pre-opsonization of M T B with serum. 40 Table 2. Role of complement on binding of M T B to CD 1 lb+/+ P M and CDI l b - / - P M . 44 Table 3. C F U and calculated population doubling time for M T B following phagocytosis by either C D 1 lb+/+ P M or CD 11 b-/- P M in vitro. 59 viii List of Abbreviations A M Alveolar macrophage B C G Bacille Calmette-Guerin BCRICWH British Columbia Research Institute for Children's and Women's Health C F U Colony forming units CRs Complement receptors CR1 Complement receptor type 1 CR3 Complement receptor type 3 CR4 Complement receptor type 4 D G V B Dextrose gelatin veronal buffer EDTA Ethylenediamine-tetraacetic acid E G T A Ethylene glycol bis (2-aminoethyl ether)-N,N,N',N',-tetraacetic acid EIgG Sheep erythrocyte coated with IgG ElgMC' Sheep erythrocyte coated with IgM and complement FCS Fetal calf serum HI Heat-inactivated mouse serum from C57BL/6 mice (CDI lb+/+ strain) HEBMS Heat-inactivated mouse serum from B A L B / c mice IgG Immunoglobulin G LFA-1 Leukocyte function antigen-1 L A M Lipoarabinomannan M B P Mannose binding protein MH-S Murine alveolar macrophage cell line MOI Multiplicity of infection M R Mannose receptor M T B Mycobacterium tuberculosis NMS Normal mouse serum obtained from C57BL/6 mice (CDI lb+/+ strain) NBMS Normal mouse serum obtained from B A L B / c mice PBS Phosphate buffered saline PCR Polymerase chain reaction PHS Pooled human serum from healthy PPD-negative volunteers P M Peritoneal macrophages R A G RAG-1 (-/-) mouse serum ScR Scavenger receptor S E M Standard error of the mean I X Acknowledgments I would like to thank my wife, Naomi, for being a constant source of support and comfort, my parents, Jose and Gloria, for instilling in me the desire and diligence to achieve my goals, my supervisory committee, Drs. Janet Chantler, Dana Devine, and Robert McMaster, for their help and guidance and my supervisor, Dr. Richard Stokes, for the opportunity to be a part of his research team, allowing me to explore and expand my abilities, and for many positive experiences I enjoyed as a graduate student. CHAPTER 1 I N T R O D U C T I O N 1.1 Mycobacterium tuberculosis ( M T B ) M T B , the causative agent of tuberculosis, is a Gram-positive bacterium containing GC-rich D N A that, like other mycobacteria, produces a cell wall of exceptional thickness and unusually low permeability [1]. Mycobacterial cell walls contain large amounts of C60-C90 fatty acids and mycolic acids that are covalently linked to arabinogalactan. These features of the mycobacterial cell wall are believed to contribute to the intrinsic resistance of mycobacteria to therapeutic agents. Indeed, mycobacteria are problem pathogens primarily because they are resistant to most common antibiotics and chemotherapeutic agents. Among antibiotics, M T B is susceptible only to aminoglycosides and rifamycins, and among general chemotherapeutic agents, M T B is susceptible to fluoroquinolones. However, resistance to these agents occurs at a relatively high frequency when these drugs are used singly. Therefore, combinations of drugs are used to treat tuberculosis and the combination of isonazid-rifampin has been one of the most effective and widely used treatments of tuberculosis. Although therapies for tuberculosis have existed for several decades, the emergence of drug resistant strains of M T B along with the ongoing ADDS epidemic continues to make tuberculosis a serious public health threat and a leading cause of death due to an infectious agent [2]. In general terms, M T B is described as a facultative intracellular pathogen which preferentially infects host macrophages. Most researchers and clinicians would agree that three critical steps are required for M T B to establish an infection; 1) binding of M T B to host cells, 2) internalization of M T B by host cells and 3) survival and replication of M T B 1 within host cells [3]. Despite recognizing that these three steps are critical for the establishment of an infection, much remains to be learned about the host-pathogen relationship. For example, little is known about the capacity of the human immune system to eliminate M T B . The majority of M T B infected individuals do not develop disease, and before chemotherapies were available, approximately half of tuberculosis patients recovered with bed rest alone [4]. The latter observation suggests that host factors play a significant role in determining the outcome of an infection. Since binding and subsequent internalization of M T B are prerequisites for establishing disease, characterizing the interaction of M T B with host macrophages is arguably the most logical starting point to further our understanding of tuberculosis pathogenesis. 1.2 Macrophage Receptors for M T B The initial interaction between host macrophages and M T B is an important first step in the pathogenesis of tuberculosis and is mediated by specific macrophage membrane receptors and ligands present on the surface of M T B . Much attention has been given to characterizing the macrophage receptors involved in mediating the binding and phagocytosis of M T B . Macrophage receptors reported to mediate binding of M T B include the complement receptors C R I , CR3, CR4, mannose receptor (MR), lipopolysacharide receptor (CD14), and scavenger receptors [5-14]. However, it remains to be shown definitively whether any one of these receptors is essential for the binding of M T B by macrophages. 2 1.2.1 Complement Receptors The role of complement receptors (CRs) in mediating the association of M T B with macrophages has been the focus of numerous reports [5,6,12,13]. Binding of M T B by CRs involves both opsonic and non-opsonic interactions. In the presence of serum, M T B binding occurs predominantly via CR1, CR3, and CR4 [5,6]. M T B is also reported to bind CR3 and CR4 in the absence of serum through direct interaction of bacterial ligands with these receptors [12,14,15]. The serum-independent interaction of M T B with macrophages is considered to be important in the early stages of pulmonary infection because the lung is an environment in which serum opsonins are relatively scarce or absent. In the presence of serum, M T B , like many bacteria, is reported to activate the alternative pathway of the complement system, resulting in opsonization of M T B with C3b and iC3b [6]. Bacteria that are sufficiently coated with these serum-derived ligands bind to CR1, CR3, and CR4 and are subsequently phagocytosed in membrane-bound phagosomes [6,10]. However, unlike other bacteria, M T B has developed an additional mechanism for acquiring opsonic C3 peptides. Using equine serum, Schorey et al. identified a heat-resistant serum component that enhances the uptake of M T B by macrophages [16]. In their study, Schorey et al. report that pathogenic mycobacteria, including M T B , uniquely recruit the complement fragment C2a to form a C3 convertase and generate opsonically active C3 cleavage products in the absence of early activation components of either the alternative or classical complement pathways [16]. The predominant opsonin generated by scavenging C2a is believed to be C3b rather than iC3b, as mycobacteria that were 3 opsonized by this mechanism bound predominantly to CR1 rather than to CR3 or CR4 [16]. The observation that M T B is capable of acquiring opsonic C3 proteins by at least two different mechanisms suggests that M T B has evolved to seek out the intra-macrophage environment. It is also interesting to note that the novel mechanism for acquiring C3 peptides described by Schorey et al. leads to deposition of predominantly C3b on the M T B surface [16]. Deposition of C3b and not iC3b would favor binding via CR1 and not CR3 or CR4. It has been speculated that routing of M T B to different receptors plays a role in dictating the intracellular fate of M T B following phagocytosis [12,17,18]. 1.2.1.1 Complement receptor type 1 (CR1, CD35) Complement receptor type 1 (CR1) is a monomeric transmembrane protein that binds preferentially to C3b and C4b but also reportedly binds iC3b, albeit less efficiently [19]. CR1 possesses complement regulatory activity and can mediate phagocytosis of bound particles, but its capacity for signal transduction or cell activation has not been thoroughly characterized. It is believed that CR1 -mediated entry provides intracellular pathogens a distinct advantage for intracellular survival following phagocytosis because CR1-mediated phagocytosis is not accompanied by an oxidative burst [20]. Leishmania major, an intracellular protozoan, is reportedly an example of a pathogen that exploits this feature of CRl-mediated phagocytosis. Previous studies have demonstrated that when L. major promastigotes enter macrophages through CR1 they survive and replicate whereas they are killed when entry is mediated through alternate receptors [21]. Thus, entry through CR1 4 and evasion of the respiratory burst may provide one means of successfully infecting macrophages. 1.2.1.2 Complement receptor type 3 (CR3, C D l l b / C D 1 8 , Mac-1) CR3 is a heterodimer belonging to the leukocyte 62-integrin protein family that share the same CD 18 beta chain linked to one of three alpha chain types. The other 62-integrins are LFA-1 (CDI la/CD18) and CR4 (CDI lc/CD18). CR3 is reported to possess at least two different binding sites; namely, the iC3b binding site and a glycan binding site near the C-terminus of the CDI lb chain [22,23]. CR3 is involved in numerous physiological functions including cellular adhesion, cell signaling, cooperation with other receptors, and cytotoxic reactions [24]. Recently, the availability of a CR3 knockout mouse model has provided researchers with a means to investigate the numerous functions of CR3 [25-28]. To date, the work published from experiments using CR3 knockout models has been largely restricted to the role of CR3 in mediating cellular adhesion events. However, recently the role of CR3 in mediating cytotoxic activation of leukocytes in response to opsonized target cells was examined [28]. The glucan binding properties and capacity for cytotoxic activation in normal and CDI lb-deficient mice were characterized and the authors report that mouse CR3 behaves similarly to human CR3 with respect to these functions [22,23,28]. Namely, CDI lb-/- leukocytes did not bind 6-glucans or respond to 6-glucan priming for cytotoxicity as well as CDI lb+/+ leukocytes [28]. In addition to mediating cellular adhesion and numerous other functions, CR3 is also believed to be an important receptor for several intracellular pathogens, including M T B . 5 Previous studies have strongly suggested that there is more than one mode of interaction between M T B and CR3 on murine macrophages [12]. Using two different anti-CR3 monoclonal antibodies, Stokes et al. provided evidence that non-opsonized M T B binds CR3 at a site distinct from the iC3b-binding site [12]. Subsequent studies suggested that the non-opsonic binding of M T B to CR3 occurs via interactions of M T B capsular polysaccharide with the B-glucan binding site near the C terminus of CDI lb [13]. The opsonic and non-opsonic interaction between M T B and CR3 was also seen in experiments with monocyte derived macrophages [6]. Evidence for the non-opsonic binding of M T B with macrophages has been criticized because investigators could not rule out the contribution of endogenous complement proteins. Therefore, unambiguous evidence that non-opsonic interactions occur between M T B and CR3 was obtained in studies in which CR3 was expressed in a non-macrophage background, so that endogenous synthesis of C3 by macrophages could not interfere [13]. Chinese hamster ovary (CHO) cells transfected with C D 18 and C D I lb were shown to bind M T B in the absence of serum [13]. The CR3-expressing CHO model has also been used to investigate strain dependent differences in the interaction of M T B with CR3 and recently differences in the thickness and composition of capsular polysaccharides have been reported to determine the mode of binding of M T B to mammalian cells [15]. Although the experiments using CR3-expressing CHO cells were carefully performed, the major drawback of transfected cell line models is that they cannot be used to address events following binding and therefore are of limited utility for studying the pathogenesis of M T B . 6 1.2.1.3 Complement receptor type 4 (CR4, pl50/95) CR4, also a member of the 62 integrin family, shares many physiologic properties with CR3. Initial evidence for the iC3b-receptor activity of CR4 was the successful isolation of CR4, along with CR3, by iC3b-Sepharose affinity chromatography of solubilized U937 cells [29]. Subsequently, a variety of CR4-mediated functions, common with CR3, have been reported [24]. One feature that distinguishes CR4 from CR3 is that during maturation of tissue macrophages, including alveolar macrophages, the expression of CR3 decreases while that of CR4 increases [10,30]. Although the reason for this apparent switch from CR3 expression to CR4 remains unknown, it may have implications in the pathogenesis of tuberculosis and numerous other diseases. Recently, it has been reported that CR4 is a major receptor for M T B under non-opsonic conditions [14]. Similar to the CR3-expressing CHO model used by Cywes et al. [13], Zaffran et al. assessed the binding of M T B to CR4-transfected CHO and found that CR4 could mediate the binding of M T B [14]. Additional evidence for the role of CR4 in binding M T B was inferred from observations which showed that M T B binding and CR4 expression were higher for monocyte derived macrophages (MDMs) compared to fresh monocytes [14]. Zaffran et al. also examined the capacity of CR4 to initiate signal transduction pathways following M T B binding, reporting that an anti-CD 1 lc antibody specifically blocked the phosphorylation of a 60-kDa protein in M D M s exposed to M T B [14]. Additional experiments may provide insight into the role of CR4 in the pathogenesis of tuberculosis but at the present time the CR4 story is quite premature. 7 1.2.2 Other Quantitatively Important Macrophage Receptors for M T B In addition to the CRs, the mannose receptor (MR), lipopolysaccharide receptor (CD14), and scavenger receptors have also been reported to mediate the binding of M T B to macrophages. The macrophage M R is a monomeric transmembrane protein, with an extracellular domain containing eight carbohydrate-recognition domains characteristic of calcium-dependent lectins [31]. A quantitatively important role for M R in binding and phagocytosis of M T B was demonstrated by competitive inhibition, downregulation of apical mannose receptors by adherence of macrophages to mannan-coated coverslips, and blocking with a polyclonal anti-MR antibody [8]. C D 14, a phosphatidylinositol glycan-linked membrane protein, is best known and characterized as the high affinity receptor for lipopolysaccharides of gram-negative bacteria. However, CD 14 can also bind lipoarabinomannan (LAM) of M T B [11,32]. Although internalization of M T B by microglial cells is reportedly mediated by CD 14 [32], because CD 14 is a lipid-anchored membrane protein without transmembrane and cytoplasmic domains, it is unclear whether CD 14 is capable of mediating phagocytosis without the cooperation of another membrane protein. It has been suggested that CR3 cooperates with C D 14 to facilitate the internalization of particles bound to C D 14 [33]. Macrophage scavenger receptors bind poly anionic macromolecules and particles, including lipopolysaccharides of gram-negative bacteria and lipoteichoic acid of gram-positive bacteria [9,34,35]. Experiments using competitive inhibitors have implicated class A 8 scavenger receptors as quantitatively important receptors for M T B [17]. However, similar to CD 14, it is not known whether scavenger receptors can activate the cytoskeleton to internalize bacteria or whether they bind bacteria with phagocytosis being mediated by other receptors. 1.3 Survival and Intracellular Replication of M T B The survival and intracellular replication of M T B within host macrophages are documented features of the pathogenesis of tuberculosis; however, the means by which M T B evades being killed by macrophages remains unclear. There are several general models that could explain how M T B avoids being killed by macrophages. One model proposes that M T B , like other intracellular pathogens, exploits specific receptors to gain entry into macrophages without eliciting the activation of microbicidal mechanisms. Candidate receptors for such a strategy include both CR1 and CR3. These receptors can bind a diverse group of intracellular pathogens and phagocytosis mediated by either CR1 or CR3 has been reported to occur without eliciting an oxidative burst [20,36,37]. If M T B exploits such a strategy it would not need to evolve other mechanisms for intracellular survival beyond those needed to acquire essential nutrients. An alternative model suggests that M T B has evolved mechanisms to block effective microbicidal mechanisms. An example of this type of strategy is demonstrated by the observation that M T B interferes with the maturation of the macrophage phago-lysosome complex by preventing phagosome acidification [38]. It is also possible that M T B has evolved intrinsic resistance to macrophage microbicidal mechanisms. Evidence for this is reported in a study that 9 found M T B to be resistant to reactive nitrogen intermediates under physiological conditions [39]. There is also increasing evidence that apoptosis can affect the outcome of certain host cell-pathogen interactions [40]. Recently, it was reported that infection of human A M with M T B promotes apoptosis [41]. However, the role of apoptosis in M T B infections is poorly understood and based on previous studies it is not clear whether promoting apoptosis is advantageous to the host or the pathogen. Therefore, determining which of each of these models applies to M T B will have an important impact on improving our understanding of M T B pathogenesis. 1.4 Specific Aims The principal aim of this research project was to determine whether CDI lb is essential for the association of M T B with murine macrophages in vitro. There was also an interest to investigate what effect the absence of CDI lb would have on the survival and replication of M T B following infection of murine macrophages in vitro. Collectively, these research objectives were designed to further our understanding of the role CR3 plays in the pathogenesis of tuberculosis. 1.5 Experimental Approach Several experimental models exist for investigating the role of receptors in mycobacteria-macrophage interactions; however, there is no consensus on which model is best. Much of the previous work has relied on the use of antibodies recognizing macrophage receptors, as 10 competitive inhibitors, to characterize the role receptors play in binding M T B [6,12,17]. These studies assume that the antibodies do not affect macrophage function beyond blocking of receptor binding sites and in cases where antibodies are used to direct binding there is no means to ensure all target receptors are blocked. Thus, studies using antibodies cannot be considered definitive. To overcome the limitations associated with using antibodies, knockout animal models can be used to accurately test the role of individual receptors in binding M T B . Receptor knockout animal models could also provide a means to further investigate the role of specific macrophage receptors in the pathogenesis of tuberculosis. To investigate the role of CR3 in M T B pathogenesis, a CDI lb knockout mouse strain (CDI lb-/-) was used as a source of macrophages for M T B association assays and M T B survival and replication experiments in vitro. To accurately assess the role of CR3 in M T B pathogenesis, a genetically similar mouse strain expressing CR3 (CDI lb+/+) was used as a control in all experiments and a comparison between CDI lb-/- and CDI lb+/+ macrophages was performed. In addition, the expression of surface receptors on macrophages obtained from either CDI lb-/- or CDI lb+/+ mice was analyzed by flow cytometry and binding of defined control particles was used to characterize CDI lb-/- and CD 11 b+/+ macrophage receptor function. As an alternative approach to the CDI lb knockout mouse model, a murine alveolar macrophage cell line (MH-S) was tested as a candidate cell line for investigating macrophage-mycobacteria interactions. It was previously reported that CR3-positive and 11 CR3-negative populations of MH-S could be isolated and that MH-S exhibited several biochemical and physiological features in common with its in vivo counterpart the alveolar macrophage [42,43]. Therefore, the initial aim was to determine whether MH-S was a suitable model for investigating mycobacteria-macrophage interactions and if so, could MH-S be used to investigate the contribution of CR3 in binding M T B . In addition, the MH-S line presents the possibility of isolating several different receptor-negative subpopulations to investigate the involvement of other receptors in mediating the association of M T B with macrophages. 12 C H A P T E R 2 M A T E R I A L S A N D M E T H O D S 2.1 Mycobacteria M T B , strain Erdman (Trudeau Mycobacterial Collection (TMC), #107; American Type Culture Collection (ATCC) #35801, Rockville, MD); was grown to late log phase in Proskauer and Beck medium supplemented with 0.05% Tween 80. Batch cultures were aliquoted into 1.5 ml Sarstedt tubes and stored at -70°C. The number of viable mycobacteria in a batch was estimated by assessing the colony forming units (CFU) from representative tubes. For each batch, two representative tubes were thawed, contents passed through a 25-gauge needle 10 times to break up clumps of bacteria, serially diluted in normal saline plus 0.1% Tween 80, and plated on Middlebrook 7H10 agar (Difco, Detroit, MI). Plates were incubated at 37°C in sealed bags and monitored for 2-3 weeks at which time colonies were enumerated and CFU/ml calculated for the respective batch. 2.2 Mice A l l mice strains were maintained in a specific pathogen-free animal facility at the British Columbia Research Institute for Children's and Women's Health (BCRICWH). For all experiments described herein 6- to 8-week old mice were used. 2.2.1 C D l l b Knockout Mice CR3-negative macrophages were obtained from CDI lb knockout mice (CDI lb-/-) that were made available as part of a collaboration with Glaxo-Wellcome Ltd., U K . Details regarding the creation of the CDI lb-/- mice are confidential and under the proprietorship 13 of Glaxo-Wellcome Ltd. Initially, six breeding pairs of CDI lb-/- mice were received from Glaxo-Wellcome Ltd. and bred at the BCRICWH animal facility. 2.2.2 C D l l b Expressing Mice The CR3-positive mouse strain (CDI lb+/+) is of the same genetic background as the CDI lb knockout strain (both are derived from C57BL/6 mouse strain). With the exception of being treated with a null vector the CDI lb+/+ mice are identical to C57BL/6 mice and were also provided by Glaxo-Wellcome Ltd. Age matched CDI lb+/+ mice were used as controls in all experiments designed to assess the role of CR3 in M T B pathogenesis. Initially, six breeding pairs of CDI lb+/+ mice were received from Glaxo-Wellcome Ltd. and bred at the BCRICWH animal facility. 2.2.3 B A L B / c Mice For experiments aimed at characterizing the association properties of MH-S with M T B , B A L B / c mice were used as a source of A M and P M for comparison with MH-S because MH-S are B A L B / c in origin [43]. B A L B / c mice were purchased from the University of British Columbia Animal Facility and maintained at the BCRICWH animal facility. 2.3 Isolation of Primary Macrophages from Mice 2.3.1 Isolation of Peritoneal Macrophages Peritoneal macrophages (PM) were obtained from mice as follows; mice were killed by cervical dislocation and positioned on an operating stage. Surgical scissors and tweezers were then used to reflect the skin and expose the peritoneal cavity without perforating the 14 peritoneal lining. Five milliliters of supplemented RPMI (RPMI1640 medium [Gibco, Grand Island, NY] plus 10% vol/vol fetal calf serum, 10 m M L-glutamine, and 10 m M sodium pyruvate [Gibco]) that had been loaded into a 5-cc syringe fitted with a 25 gauge needle was then slowly injected into the left side of the peritoneum. The needle was removed and the body cavity massaged for 30-45 seconds. The peritoneal wash fluid was removed from the right side of the peritoneum using a 23-gauge needle and placed in a 50-ml conical tube on ice. Where more than one mouse was used per experiment, the contents of the peritoneal washes were pooled and their leukocytes counted and adjusted to the desired concentration in supplemented RPMI. Typical yield of P M from 6- to 8-week old mice was in the 2-4 x 106 range. 2.3.2 Isolation of Alveolar Macrophages Alveolar macrophages (AM) were isolated from mice as follows; 6- to 8-week old mice were injected intra-peritoneally with a lethal dose of pentabarbitol, and the lower respiratory tree and heart removed and placed in ice cold PBS + 1 m M EDTA. A 22-gauge catheter (Critikon, Tampa, FL) was inserted into the trachea and tied off with silk thread. The catheter-lungs were supported in a jig and lavaged with 10 ml of PBS + 1 m M EDTA, using 1 ml aliquots at a time. The washings were pooled and then pelleted by centrifuging at 800 x g for 10 minutes at 4°C. The cell pellet was then resuspended in supplemented RPMI at 1 x 106 cells/ml, and 100 ul aliquots were placed onto 13 mm coverslips lying in wells of 24-well plates. For Day 0 A M , the cells were allowed to adhere for 2 hours at 37°C in 95%air/5% C 0 2 , washed and 1 ml of supplemented RPMI added to each well. Cells were then used immediately in experiments. For Day 4 A M , the 15 cells were returned to the incubator (37°C, 95%air/5%C02) following the addition of 1 ml of supplemented RPMI containing streptomycin and penicillin and maintained for 4 days before being washed and used in experiments. Typical yield of A M from 6- to 8-week old mice was in the 0.5-1 x 106 range. 2.4 Genotyping of CR3-positive and CR3-negative Mice 2.4.1 D N A preparation Initially, representative mice from our CDI lb-/- and CDI lb+/+ colonies were randomly selected for genotyping and after the initial screening, tissue samples from mice used in experiments were stored in liquid nitrogen for periodic retrospective analysis. Genomic D N A from mouse tissue was prepared using conventional D N A isolation techniques. Tissues (lungs and heart) were minced and frozen in liquid nitrogen following excision and either stored or used immediately for D N A preparation. Between 0.5 and 1.0 g of tissue was then ground using a prechilled mortar and pestle and suspended in 1.2 ml digestion buffer (100 m M NaCl, 10 m M Tris-HCl (pH 8), 25 m M E D T A (pH 8), 0.5% SDS, 0.1 mg/ml proteinase K) per 0.1 g of tissue. Samples were then incubated with shaking at 50°C for 18 hours in tightly capped tubes and thoroughly extracted with an equal volume of phenol/chloroform/isoamyl alcohol. Samples were then centrifuged at 1700 x g for 10 minutes and the aqueous (top) layer was transferred to a new tube and xh vol of 7.5 M ammonium acetate and 2 vol (of original amount of top layer) of 100% ethanol added. Following the addition of ethanol the D N A immediately precipitates to form a stringy white precipitate which was recovered by centrifugation at 1700 x g for 2 minutes. The pelleted D N A was then rinsed with 70% ethanol, dried, and resuspended in TE buffer 16 (solubilization enhanced by heating at 65°C for 4 hours). The yield and purity of the D N A was then assessed by spectrophotometric analysis (A280/260 readings). 2.4.2 Polymerase chain reaction (PCR) amplification of D N A sequences specific for the genomes of either CDllb+/+ or C D l l b - / - mice. D N A samples obtained from our CDI lb+/+ and CDI lb-/- mouse colonies were screened to confirm their genetic status as follows. In sterile, DNase-free 0.5 ml Eppendorf tubes, 2 pi of mouse D N A at a concentration of 0.1 ug/ul was added followed by the addition of 5 ul of primer mix (primer mix contained equal molar quantities of primers 856, 2924, and 2506) to each tube sitting on ice. Each tube then received 30 pi of PCR master mix and was overlaid with mineral oil. Controls for the PCR genotyping included running single primer reactions for each of the three primers prior to making aliquots of the primer mix. The PCR master mix composition was as follows: 25 pi dNTPs (100 uM), 13 pi MgCh; (50 mM), 52 pi Gibco PCR buffer, 215 pi sterile distilled water, and 5 pi Taq polymerase (Gibco). Oligonucleotide primers were a generous gift from Mr. Graham Hagger (Glaxo-Wellcome), see below for primer sequence. Oligonucleotide primer sequences Primer 856: 5'- TAT CTT C T A G T G ATT TCC C C A G T A -3' Primer 2924: 5'- TGT A G A C A G CGC CCT G A T TCT CCT -3' Primer 2506: 5'- C C C A C C CCT TCC C A G CCT C G A GC -3' 17 Thermocycle conditions: PCR was performed on a Perkin-Elmer Thermal Cycler with the following thermal cycle parameters: 94°C for 5 minutes, {94°C for 1 minute, 64°C 1 for minute, 72°C 1 for minute} x 35 cycles, 72°C for 5 minutes, and hold at 8°C. 2.4.3 Agarose gel electrophoresis of P C R genotyping products Following the thermal cycling procedure, PCR products were loaded onto 1.5% agarose gels (0.75 grams of agarose/50 ml TBE) and electrophoresed for 90 minutes at 80V. Electrophoresed bands were visualized by ethidium bromide staining and U V illumination on a gel documentation system (BioRad). The size of the PCR products was determined by comparing the electrophoretic mobility of the PCR products with that of known standards (100-bp ladder, Catalogue* 170-8353, BioRad, Mississauga, ON). 2.5 Flow Cytometric Analysis 2.5.1 Flow cytometric analysis of CDllb+/+ and C D l l b - / - macrophages The expression of surface antigens on CDI lb+/+ and CDI lb-/- P M was investigated using flow cytometric analysis. Peritoneal lavage technique was used to isolate peritoneal exudate cells from mice. Macrophages were prepared as follows; peritoneal exudate cells in supplemented RPMI were adhered to 15 cm bacteriologic polystyrene plates at 2-3 x 107 cells/plate for 2 hours at 37°C, 5% CO2. After 2 hours, nonadherent cells were removed by washing in supplemented RPMI. Fresh supplemented RPMI was added to the plates which were then incubated overnight at 37°C, 5% CO2. Adherent cells were prepared for flow cytometry by washing the plates twice with 0.5 mM E D T A in PBS plus 0.02% glucose; plates containing 20-ml of 0.5 mM EDTA in PBS plus 0.02% D-glucose were then chilled 18 for 60 minutes on ice and cells were removed by scraping plates with a cell scraper (Falcon). Cells were washed and resuspended in binding medium (138 m M NaCl, 8.1 m M Na 2 HP0 4 , 1.5 m M K H 2 P 0 4 , 2.7 m M KC1, 0.6 m M CaCl 2 , ImM M g C l 2 , and 5.5 m M D-glucose) at 106 viable cells/ml. Cells were pelleted and resuspended in wash buffer (RPMI with 2% vol/vol FCS, 20 mM HEPES, and 20 m M azide) at 106 cells per ml. Aliquots of 5 x 105 cells in a total volume of 1.25 ml wash buffer were pelleted and resuspended in 100 pi of wash buffer plus the appropriate primary antibodies being tested. Cells were kept on ice for 45 minutes with occasional mixing, washed twice in 1.5 ml wash buffer, and incubated in 100 pi of an appropriate FITC-conjugated secondary antibody at 10 pg/ml for 45 minutes on ice. Cells were then washed once in 1.5 ml wash buffer and once in 1.5 ml PBS, fixed in 0.5 ml 1.5% para-formaldehyde in PBS, and read on a Becton-Dickson FACScalibur Cytometer with the following settings: FL1 (600 V), SS (350 V), with 0% compensation. The mean fluorescence intensity values, based on arbitrary units from a scale of 0-1000, are reported and discussed in terms of ratios compared to the relevant controls. 2.5.2 Antibodies used to probe for surface antigen expression The monoclonal antibodies used in this study were, unless stated otherwise, prepared from hybridomas obtained from A T C C and were as follows: M1/9.3.4HL.2 {rat IgG2b, anti-mouse FcRII [44]}; Ml/70.15.11.5.HL {rat IgG2b, anti-mouse CDI lb, recognizing an epitope that binds iC3b [44]}; 5C6 {rat IgG2b, anti-mouse CDI lb, recognizing epitope that is involved in macrophage attachment and spreading, but is distinct from that which 19 binds iC3b [45,46]}; M17/4.4.11.9 [rat IgG2a, recognizing C D l l a [47]}, was obtained from Developmental Studies Hybridoma Bank; 8C12 {rat IgG from nude mouse ascites, anti-mouse CRI [48]}, a generous gift from Dr. T. Kinoshita (Osaka University, Japan); 2F8 {rat IgG2b, anti-murine scavenger receptor [49]}, a generous gift from Dr. S. Gordon (Oxford University, UK) ; and N418 {hamster IgG, anti-mouse CDI lc [50]} was purchased from Cedarlane Laboratories Ltd. (Hornby, ON). Controls for the mAb were as follows: IR863 for the rat IgG {nonspecific rat IgG, purchased from Cedarlane Laboratories Ltd.} and HM00 {non-specific hamster IgG, purchased from Cedarlane Laboratories Ltd.} for the hamster IgG. Following incubation with the primary antibody, cells were incubated with either donkey anti-rat IgG FITC or goat anti-hamster IgG FITC secondary antibodies {Jackson ImmunoResearch, West Grove, PA}, as appropriate. 2.6 Alveolar Macrophage Cell Line, M H - S The morphological, cytochemical, and functional characteristics of MH-S, an alveolar macrophage cell line, have been documented [43]. MH-S cell line was obtained from NIH (Bethesda, MD) and cultured in supplemented RPMI. The cells were cultured to confluence in Falcon tissue culture flasks prior to being used in binding assays and were maintained in culture up to a maximum of 4 weeks at 37°C in 95% air/5% C 0 2 . For binding assays, MH-S cells were removed from culture flasks by scraping with a Falcon cell scraper, enumerated using trypan blue viability staining, and the cell concentration adjusted to 1 x 106 cells/ml with fresh media. MH-S were then seeded at 1 x 105 cells per coverslip, and incubated for 16-20 h at 37°C, 95%air/5% C 0 2 . 20 2.6.1 Isolation of CR3-negative populations of M H - S Sub-populations of MH-S cells were isolated using magnetic selection of antibody coated beads according to the protocol recommended by the manufacturers of Dynabeads (Dynal Inc., Lake Success, NY) . Briefly, 5x l0 6 cells were suspended with a cocktail of rat anti-mouse CR3 monoclonal antibodies (Ml/70 and 5C6; see section 2.5.2 for details) and incubated at 4°C for 30 minutes with mixing. Pretreated cells were centrifuged at 800 x g for 10 minutes and resuspended in wash buffer (PBSG +1% FCS). Cells were then washed two times in wash buffer to remove all unbound antibody. The washed cells were then resuspended with anti-rat IgG-coated magnetic beads and incubated for 30 minutes at 4°C with mixing. Following this second incubation step, CR3-positive cells were pelleted by applying a magnet to the cell suspension. The magnet was repeatedly applied for 1-2 minutes to remove all the magnetic beads from the suspension. CR3-negative cells remaining in the supernatant were washed and seeded in tissue culture flasks with fresh media. These CR3-negative cells were then subjected to the above procedure repeatedly in an attempt to isolate a stable homogenous CR3-negative population of cells. 2.7 Particles for Probing Macrophage Receptors 2.7.1 Fc Receptors The function of Fc receptors was investigated using EIgG. Two millilitres of sheep erythrocytes (SRBC) at 109/ml in D G V B (2.5% D-glucose, 0.15 m M CaCl 2 , 0.5 m M MgCL., 0.05% gelatin in veronal buffer, pH 7.4 to 7.6 was incubated with 1 pi of anti-SRBC IgG fraction (Cordis, Miami, FL) for 30 minutes at 37°C and 30 minutes at 0°C, 21 washed three times in D G V B , resuspended at 10 /ml in D G V B , and stored at 4°C for no more than 2 days. 2.7.2 Complement Receptors Complement receptors were identified using E l g M C . Two millilitres of SRBC at 109/ml in E D T A - D G V B (2.5% D-glucose, 10 m M EDTA, and 0.05% gelatin in veronal buffer, pH 7.4 to 7.6) was incubated with 50 pi anti-SRBC IgM fraction (Cordis) for 30 minutes at 37°C and 30 minutes 0°C, washed three times with D G V B , and suspended at 109/ml in D G V B (EIgM). Fifty microlilitres of C5-deficient human serum (Sigma, St. Louis, MO) was added to 500 ul EIgM and incubated for 60 minutes at 37°C This was then diluted to 20 ml with E D T A - D G V B and kept on ice for 10 minutes, washed four times in D G V B , resuspended to 108/ml in D G V B , and stored at 4°C for up to 2 weeks. 2.7.3 Lectin-like Receptors Zymosan particles (prepared from bakers yeast, Sigma, St. Louis, MO) were used to probe for lectin-like phagocytic receptors. Zymosan was suspended at 5% wt/vol in PBS plus 0.02% sodium azide and stored at 4°C until use. Before use, an aliquot of stock was washed three times to remove azide and suspended in binding medium at a concentration of 5 x 107 particles/ml. 2.8 In vitro Assay for Association of Particles with macrophages For all cell types, adherent cells were washed twice using binding medium. Following the washes, a 250 pi aliquot of binding medium was added to each well and the cells were acclimatized for 10 minutes at 37°C, 95% air/5% C 0 2 . After acclimatizing, a further 250 22 u.1 of binding medium containing particles to be tested was added to the monolayers. The particles (zymosan, EIgG, E lgMC' ) had been previously pelleted and resuspended in binding medium at 2 x 107 /ml (zymosan), and 4 x 107 /ml (EIgG and E l g M C ) . For a preliminary series of experiments with MH-S and primary macrophages from B A L B / c mice, latex beads were also used in binding assays to assess non-specific phagocytic function. In these experiments, latex beads were pelleted and resuspended in binding medium at 2 x 10 /ml. In both opsonic and non-opsonic binding assays, M T B particles were also pelleted and resuspended in binding medium prior to being added to monolayers. M T B intended for P M monolayers was resuspended in binding medium at 2 x 107 /ml whereas M T B intended for A M or MH-S monolayers was resuspended at 2 x 108 /ml. The final multiplicity of infection (MOI) for the control particles was as follows: 50 (zymosan and latex) and 100 (EIgG and E l g M C ) . In either non-opsonic or opsonic binding assays, the MOI of M T B for A M and MH-S monolayers was 500 whereas the MOI of M T B for P M monolayers was 50. The reason for using lOx more M T B with MH-S and A M compared to P M is based on previous observation that P M bind and ingest M T B much more readily than A M [18]. Following the addition of the particles to be tested, the 24-well plates containing macrophage monolayers were gently rocked for 1 hour at 37°C, 95% air/5% C O 2 (Nutator, Becton Dickinson, Mountain View, CA), followed by a further 2 hours stationary at 37°C, 95% air/5% C O 2 . The monolayers were then washed twice using binding medium, fixed for 10 minutes in 10% formaldehyde in ethanol, mounted cell side up on glass slides, and stained with auromine-o and acridine orange for M T B or Giemsa for latex, zymosan, EIgG, and E l g M C . 23 In the majority of experiments where the effects of serum on M T B binding to macrophages was assessed, opsonization of M T B was achieved by incubating the various sera with M T B and macrophages directly. In one clearly identified series of experiments M T B was pre-opsonized in the presence of 10% serum for 30 minutes at 37°C, then pelleted and resuspended in binding medium before being added to the macrophage monolayers. The latter series of experiments was designed to test whether co-incubation of sera affected M T B association beyond M T B opsonization. In experiments using either C D I lb-/- or CDI lb+/+ macrophages, normal mouse serum (NMS) refers to serum obtained from CDI lb+/+ mice and R A G serum was obtained from RAG-1 (-/-) mice. RAG-1 (-/-) mice, kindly provided by Dr. Lome Clarke (University of British Columbia, Canada), are deficient in mature B and T lymphocytes [51] and therefore do not produce immuno-globulins. Previously, serum IgM levels have been shown to be undetectable in R A G serum [51] and here, the IgG-negative status of R A G serum was confirmed using standard ELISA techniques (Appendix I). For experiments with MH-S and primary macrophages from B A L B / c mice, normal mouse serum (NBMS) refers to serum obtained from B A L B / c mice. A l l mouse sera were isolated and prepared as described previously [52]. Heat-inactivated NMS (HI) and NMBS (HQ3MS) were treated at 56°C for 30 minutes to render the sera deficient for complement activation [53]. In experiments testing the effects of anti-CR3 monoclonal antibodies on the binding of M T B to MH-S, antibodies were used at a concentration of 10 pg/ml and added immediately before M T B aliquots (see section 2.5.2 for description of antibodies). 24 For experiments aimed at characterizing the role of complement fixation pathways in mediating M T B binding, various sera depleted of complement components were added directly to the macrophage monolayers. Factor B-depleted and C2-depleted human sera were purchased (Quidel Corp., San Diego, CA). E D T A and E G T A were added to give a final concentration of 10 m M in the binding medium [6,54]. The control serum in these experiments was normal pooled human serum (PHS), obtained from healthy PPD-negative volunteers at BCRICWH. The binding of EIgG, E l g M C , and zymosan was assessed by counting the total number of particles associated with 100 macrophages (Association Index). Whereas the association of mycobacteria with macrophages was determined by counting the percentage of macrophages with 0, 1 to 5, 6 to 10, 11-30 or more than 30 associated bacteria, as previously described [12]. The purpose of these studies was to characterize the association between M T B and mouse macrophages, so no attempt was made to differentiate attachment from ingestion. However, previous experiments have shown that the majority (greater than 90%) of mycobacteria associated with macrophages after the 3 h incubation period used in these experiments are ingested (unpublished observations, Stokes laboratory). 2.9 In vitro Survival and Replication of M T B following Phagocytosis by either CDllb+/+ or C D l l b - / - Macrophages P M were seeded at 5x l0 5 per coverslip in 1.0 ml of supplemented RPMI, adhered for 2 hours, washed, and incubated in 1.0 ml of media for 16-18 hours at 37°C in 95% air/ 5% 25 CC<2 prior to being used in experiments. M T B intracellular growth assays were conducted in parallel with M T B binding assays so that a comparison between M T B growth and M T B uptake could be made. Additionally, because CDI lb-/- P M were observed to be less efficient at phagocytosing M T B , I tested MOIs of 10 and 20 for CDI lb-/- P M whereas for CDI lb+/+ P M only an MOI of 10 was used. The MOI of 20 for CDI lb-/- P M was chosen because it would lead to approximately the same number of ingested bacteria as the MOI of 10 for CDI lb+/+ P M . As described for the binding assays (section 2.8), P M were exposed to M T B for 3 hours at 37°C in binding medium. To eliminate extracellular bacteria from the system, coverslips were washed twice in binding medium and then transferred to new 24-well plates containing 1.0 ml supplemented RPMI per well. Day 0 coverslips were processed immediately whereas the remaining coverslips (Day 1, 4 and 7) were incubated for the appropriate length of time at 37°C in 95% air/5% C O 2 before being processed. Coverslips were processed as follows. Coverslips were removed from wells and placed individually in separate 14-ml snap-top tubes containing 1.0 ml of PBS + 0.1% Tween 80. A sterile glass rod was then used to fragment the coverslips. Fragmented coverslips were then sonicated for 10 seconds to lyse macrophages and release M T B . This brief sonication has been shown not to affect the viability of M T B (unpublished observation, Stokes laboratory). The sonicates were then serially diluted in PBS + 0.1% Tween 80 and 50 pi aliquots of the diluents were then plated onto 7H11 plates. For Day 1, 4, and 7 the supernatants from each well, which contained macrophages that had lifted off from the coverslips, were sonicated briefly, serially diluted in PBS + 0.1% Tween 80 and 50 pi 26 aliquots of the diluents were then plated onto 7H11 plates. Plates were incubated at 37°C in sealed bags and monitored for 2-3 weeks and then colonies were enumerated and CFU/ml calculated. The C F U for Days 1, 4, and 7 post-infection were determined by combining the C F U values obtained for coverslips and supernatants for each respective condition. It has been observed previously that approximately 90% of viable M T B in the supernatants are associated with macrophages and that M T B does not replicate in supplemented RPMI over seven days (unpublished observations, Stokes laboratory). 2.10 Statistical Analysis Data are expressed as mean + S E M . When applicable, the Student's t test for independent means was used to evaluate binding data. For experiments where several different treatments were being compared, analysis of variance of the means was performed using the Bonferroni correction. Differences were considered significant at P < 0.05. 27 CHAPTER 3 R E S U L T S : Role of CR3 in M T B Pathogenesis Investigated using C D l l b - / - and CDllb+/+ Mouse Macrophages 3.1 Genotyping of C D l l b - / - and C D l l b +/+ Mice Genotyping of CDI lb-/- and CDI lb+/+ mouse strains used in these experiments was performed using a protocol developed by Mr. Graham Haggar at Glaxo-Wellcome Ltd. Representative mice were randomly selected from either the CDI lb+/+ or CDI 1 bu-rnouse colonies for genotyping. CDI lb+/+ mice yielded an expected 613-bp band upon PCR screening whereas CDI lb-/- mice produced an expected 418-bp band (Figure 1). The exact nature of the target vector and creation of the CDI lb-/- mouse strain is confidential and under the proprietorship of Glaxo-Wellcome Ltd. L 1 2 3 4 5 6 7 8 L 9 10 -613 bp (CDllb+/+) -418 bp (CDllb-/-) Figure 1. An example of PCR Genotyping of CDI lb+/+ and CDI lb-/- mice. PCR products were loaded in a 1.5% agarose gel, electrophoresed for 90 minutes at 80V, and then visualized under U V illumination following ethidium-bromide staining. Lanes 1-4 (CDI lb-/- D N A from our colony), and 9 (CDI lb-/- D N A from Glaxo-Wellcome Ltd.) with expected 418-bp band, lanes 5-8 (CDI lb+/+ D N A from our colony), and 10 (CDI lb+/+ D N A from Glaxo-Wellcome Ltd.) with expected 613-bp band. 3 pi of 100-bp D N A standard ladder (BioRad, Catalogue # 170-8353) loaded in the two lanes denoted L . 28 3.2 Flow cytometric analysis of C D l l b - / - and CDllb+/+ macrophages Flow cytometry was used to investigate the expression of surface antigens on macrophages from CDI lb-/- and CDI lb+/+ mice. Using two different anti-CR3 monoclonal antibodies (Ml/70 and 5C6), recognizing different epitopes on CR3,1 found that the expression of either CR3 epitope on P M from the CDI lb-/- mice was negligible compared to P M from CDI lb+/+ mice (Figure 2). Similarly, the expression of CD18, the common chain of the 62 integrins, on CDI lb-/- P M was also negligible compared to CDI lb+/+ P M (Figure 2). The expression of the leukocyte common antigen (CD45) was comparable between macrophages obtained from either CDI lb-/- or CDI lb+/+ mice with mean fluorescence values approximately 5x that of their respective controls (Figure 2). The expression of the macrophage marker F4/80, CD35, CDI la, CDI lc, and scavenger receptor were also comparable between macrophages from either CDI lb-/- or CDI lb+/+ mice (Figure 2). With the exception of CDI lc , the observations reported here are in close agreement with those made by Glaxo-Wellcome Ltd. (personal communication with Mr. G. Haggar). Scientists at Glaxo-Wellcome observed that peripheral blood leukocytes from CDI lb-/-mice express approximately 5x less CDI lc than those obtained from CDI lb+/+ mice. A possible explanation for this difference is that CDI lc expression varies between peripheral blood leukocytes and P M . Based on the flow cytometry data, CDI lc expression is low on CDI lb+/+ P M (Figure 2) and therefore it is possible that the low expression of CDI lc does not allow for the detection of an appreciable difference in CDI l c expression between CDI lb+/+ P M and CDI lb-/- P M . 29 Figure 2. Flow Cytometric Analysis of Macrophages Obtained from CDI lb+/+ and CDI lb-/- Mice. P M were analyzed by flow cytometry for the expression of CD45 (LCA), CDI lb (CR3), C D l l c (CR4), CD18 (p2-integrin common chain), CD35 (CR1), CDI l a (LFA-1), F4/80 (macrophage marker), ScR (scavenger receptor) and compared with either non-specific rat antibody (IgG control) or non-specific hamster antibody (HMOO) controls as appropriate." Mean fluorescence intensity values are reported for samples where 2500-3000 events were read on a Becton-Dickson FACScalibur cytometer. 30 3.3 Association of Zymosan, EIgG, and ElgMC' with PM from CDllb-/- and CDllb+/+ mice Although flow cytometric analysis provides a means to probe for the expression of surface receptors, it does not provide information on the functional status of receptors. Therefore, association of control particles was used to probe for the expression of functional receptors for glycans (Zymosan), IgG (EIgG), and complement component iC3b ( E l g M C ) on the surface of P M obtained from either CDI lb-/- or CDI lb+/+ mice. Under the conditions used for these experiments, CDI lb-/- P M associated with EIgG and E l g M C at levels comparable to CDI lb+/+ P M , but the binding of zymosan was significantly less for CDI lb-/- P M compared to CDI lb+/+ P M (P = 0.003). CDI lb-/- P M bound zymosan less efficiently than CDI lb+/+ P M with association indices of 1028 ± 32 and 1474 + 34, respectively (Figure 3). The association indices of EIgG for CDI lb-/- and CDI lb+/+ P M were 1160 + 138 and 1260 + 56, respectively (Figure 3). The association indices of E l g M C for CDI lb-/- and CDI lb+/+ P M were 762 + 112 and 997 ± 78, respectively (Figure 3). Based on the observation that CDI lb+/+ P M express high levels of CR3 whereas the CDI lb-/- do not express CR3 and because E l g M C is reported to be a means to probe for CR3 function, I expected to see a significant difference in the binding of E l g M C between CDI lb-/- and CDI lb+/+ P M . However, the observed difference in E l g M C binding by P M of either phenotype was not statistically significant (P > 0.05). 31 1600 Zymosan EIgG ElgMC Figure 3. Binding of Zymosan, EIgG, and E l g M C ' to P M . P M obtained from either C D I lb+/+ or C D I lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was then tested. Binding was assessed as the number 6f particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n = 6 (3 separate experiments with duplicate coverslips in each experiment). 32 3.4 Association of MTB with PM from CDllb-/- and CDllb+/+ mice. 3.4.1 Non-opsonic Association of MTB with PM Under non-opsonic conditions, the association of M T B with C D l l b - / - P M was significantly less than that observed for CDI lb+/+ P M (P < 0.008 for all comparisons). The percentage of CDI lb-/- P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 60 + 4.4%, 13.7 + 2.5%, 0.3 + 0.2%, respectively (Figure 4). Whereas, the percentage of CDI lb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 73 + 2.5%, 28 + 2.7%, and 2+1%, respectively (Figure 4). 1 0 0 1 or more > 10 bacteria > 30 bacteria Figure 4. Non-opsonic association of M T B with CDI lb+/+ P M and CDI lb-/- P M . P M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind M T B in a serum-free environment was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, and greater than 30 bacteria is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). 33 3.4.2 Opsonic Association of MTB with PM Similar to previously reported observations, the binding of M T B by P M was enhanced by the addition of serum [18]. In the presence of serum, there was a more dramatic difference between the association of M T B with CDI lb-/- P M and with CDI lb+/+ P M than that seen under non-opsonic conditions (compare Figures 4 and 5). In the presence of 1% NMS, 88 + 2.3% of CDI lb-/- P M associated with one or more bacterium whereas 100 + 0.2% of CDI lb+/+ P M were observed to associate with one or more bacterium (Figure 5; P < 0.001 at the level of binding at least one bacterium). The percentage of P M associated with greater than 10 bacteria were 51 + 4.6% and 98 ± 3.3% for CDI lb-/- P M and CDI lb+/+ P M , respectively (Figure 5; P < 0.001 at the greater than 10 bacteria level). CDI lb-/- P M were approximately 9x less efficient at binding M T B than CDI lb+/+ P M with 6.2 + 2.3% and 45 + 3.3% of macrophages binding greater than 30 bacteria, respectively (Figure 5; P < 0.001 at the greater than 30 bacteria per macrophage level). 34 1 or more >10 bacteria >30 bacteria Figure 5 . Opsonic association of M T B with CDI lb+/+ P M and CDI lb-/- P M . P M obtained from either C D I lb+/+ or C D I lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 h. The ability of these macrophages to bind M T B in the presence of 1% normal mouse serum (NMS) was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). Note: the S E M for CDI lb+/+ P M binding M T B at the level of 1 or more M T B is 0.2%. 35 3.4.3 Effects of heat-inactivated and immunoglobulin-negative serum on association of MTB with PM. Heat-inactivated normal mouse serum and RAG-1 (-/-) mouse serum were used to investigate further the enhanced binding of M T B by P M in the presence of serum opsonins. Heat-inactivation (56°C for 30 minutes) of serum abrogates activation of complement and other serum components which are sensitive to the heat treatment [53]. R A G serum, obtained from mice which do not possess differentiated T and B cells, is immunoglobulin-deficient (IgG-negative status confirmed by ELISA, Appendix I) and therefore provides a means to investigate the contribution of natural antibody in mediating the association of M T B with macrophages. R A G serum also provides a means to evaluate the role antibody-mediated complement pathway activation plays in enhancing the association of M T B with macrophages. In the presence of heat-inactivated serum, the binding of M T B by P M of either phenotype was significantly less than that seen when normal mouse serum was used (P < 0.00005) and was similar to levels of M T B binding seen under non-opsonic conditions (Figure 6). When NMS was used to opsonize M T B , approximately half of the CDI lb-/- P M and nearly all CDI lb+/+ P M were associated with greater than 10 bacteria (Figure 6). However, when heat-inactivated serum is used, less than 20% of CDI lb-/- P M and only 33 + 3.4 % of CDI lb+/+ P M associate with greater than 10 bacteria (Figure 6). The observed abrogation of enhanced binding following heat treatment of serum is in agreement with previous reports [6,18] and is additional evidence supporting the role of complement proteins in mediating M T B binding under conditions where serum is present. 36 For P M of either phenotype, no appreciable difference in the binding of M T B was observed when R A G serum was compared to NMS (Figure 6; P > 0.05 for M T B binding at all levels). In the presence of 1% R A G serum, the percentage of CDllb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 98 + 5.1, 92 + 5.1, and 35.3 ± 3.1, respectively (Figure 6). For M T B binding to CDI lb-/- P M in the presence of 1% R A G serum, the percentage of cells with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 88 + 3.2, 58 + 4.0, and 3.2 + 1.0, respectively (Figure 6). Under the conditions used here, the observations indicate that enhanced binding of M T B seen in the presence of serum is independent of immunoglobulins. 37 1 or more >10 bacteria >30 bacteria 1 or more >10 bacteria >30 bacteria Figure 6. Effects of heat-inactivated and immunoglobulin-negative serum on binding of M T B to CDI lb+/+ and CDI lb-/- P M . P M obtained from either CDI lb+/+ or CDI lb-A-mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind M T B either in the absence (-) or presence of mouse sera was tested. The sera used in these binding assays were normal mouse serum (NMS), heat-inactivated (HI) normal mouse serum, and serum deficient for immunoglobulins (RAG), all at a concentration of 1 % in binding medium. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, and greater than 30 bacteria is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 38 3.4.4 Association of pre-opsonized MTB versus association of MTB in the presence of serum with PM. It has been reported previously that, compared to serum-free binding, the binding of M T B by human monocytes is enhanced when M T B is either pre-opsonized with serum or when serum is added to the binding medium [6]. However, to determine whether the presence of serum in the binding medium had an effect on the enhanced binding of M T B that could not be explained solely by M T B opsonization, a comparison of M T B binding between pre-opsonized M T B and M T B in the presence of serum was performed. In this series of experiments, M T B binding to either CDI lb+/+ P M or CDI lb-/- P M was assessed in assays testing either NMS or R A G serum (Table 1). The observations indicate that serum mediated binding of M T B by P M was comparable whether M T B was pre-opsonized with serum or binding occurred in the presence of serum. No significant difference in the percentage of CDI lb+/+ P M binding M T B was observed between assays in which M T B was either pre-opsonized with serum or incubated with serum (P > 0.05 for all conditions and at all levels of M T B binding). In the presence of NMS or R A G serum, approximately half of the CDI lb+/+ P M in the monolayer were associated with greater than 30 M T B (Table 1). Similarly, when M T B was pre-opsonized with either NMS or R A G seriim, approximately half of the CDI lb+/+ P M were associated with greater than 30 M T B (Table 1). Consistent with observations reported in previous sections, the binding of M T B by CDI lb-/- P M was significantly less than that observed for CDI lb+/+ P M (P < 0.0013 for all conditions tested). Similar to CDI lb+/+ P M , there was also no significant difference in M T B binding to CDI lb-/- P M as a result of pre-opsonization or incubation of M T B with either NMS or R A G serum (P > 0.05 at all levels 39 of M T B binding). In the presence of serum or following pre-opsonization with serum approximately 40% of CDI lb-/- P M were observed to associate with greater than 10 M T B whereas, less than 5% of CDI lb-/- P M were observed to associate with M T B at the greater than 30 level (Table 1). Table 1. Association of MTB with PM: A comparison between pre-opsonized MTB and MTB in the presence of serum . Percent Macrophages Binding MTB CDllb+/+ CDllb-/-Bacteria/ macrophage 0 1- 10 11 -30 >30 0 1-10 11-30 >30 NMS 1.8 ± 0.8 1.6 ± 1.2 35 ± 4.3 62 ± 3.6 14 ± 1 . 9 46 ± 5 . 1 37 ± 4 . 5 3.8 ± 2 . 8 Pre-NMS 1.0 ± 0.7 2.1 ± 1.0 38 ± 2.6 60 ± 3 . 2 19 ± 2 . 6 40 ± 3 . 4 39 ± 3 . 2 3.3 ± 1.4 R A G 1.5 ± 0.5 1.6 ± 0 . 8 37 ± 2.8 60 ± 3 . 2 15 ± 1.7 46 ± 4 . 1 39 ± 4 . 0 1.0 ± 0 . 6 Pre-RAG 2.3 ± 1.1 4.3 ± 2 . 2 44 ± 2.5 50 ± 3 . 4 16 ± 1.8 46 ± 4 . 6 37 ± 3 . 1 l.i 3 ± 0 . 6 Association of MTB in the presence of 1% normal mouse serum (NMS) or 1% RAG mouse serum (RAG) and association of MTB pre-opsonized with 10% normal mouse serum (Pre-NMS) or pre-opsonized with 10% RAG mouse serum (Pre-RAG) was assessed. The mean + SEM for n = 4 (2 separate experiments with duplicate coverslips in each experiment is reported). Binding of MTB was measured as the percentage of PM in the monolayer that bound 0, 1-10, 11-30, or greater than 30 bacteria. 40 3.4.5 Role of Complement in the Association of M T B with P M . Thus far, the results obtained indicated that the enhanced binding of M T B to P M in the presence of serum was due to a heat-labile serum component present in immunoglobulin-negative and immunoglobulin-positive serum, suggesting complement activation and fixation that was independent of serum immunoglobulins. The role of complement in mediating enhanced M T B binding by macrophages has been studied extensively [6,16,54]; however, the precise mechanism by which M T B activates complement is still not fully understood. Hetland et al. reported that anti-LAM immunoglobulins play a significant role in mediating the activation of the classical complement pathway and suggested that this was a means for M T B to increase its uptake by macrophages [54]. On the other hand, Schlesinger et al. reported that M T B activates the alternative complement pathway through interactions of M T B surface molecules with complement proteins [6]. Recently, using human monocytes and equine serum, Schorey et al. reported that M T B is capable of scavenging C2a to create a novel C3 convertase that leads to deposition of C3b on the M T B surface and enhanced uptake of M T B by human monocytes [16]. Due to the recent developments regarding the role of complement in mediating enhanced M T B binding by macrophages and because the role of complement in these interactions is not fully understood, I was interested in investigating whether a specific activation pathway of complement, either the classical or alternative pathway, was responsible for the serum-mediated enhanced binding of M T B that I had observed in my studies. Enhanced M T B binding resulting from the activation of either complement pathway is believed to be due to the deposition of opsonic C3 proteins on M T B . Both the classical and alternative 41 pathways are dependent upon M g 2 + for the formation of their respective C3 convertases, C4b2a and C3bBb [55-57]. However, the classical pathway differs from the alternative pathway in that it is dependent upon C a 2 + for the formation of the Clqrs complex [57,59]. E D T A which chelates both C a 2 + and M g 2 + is therefore reported to be a means to inactivate both the classical and alternative pathways of complement whereas E G T A which chelates C a 2 + but not M g 2 + is a means to inactivate the classical pathway while leaving the alternative pathway intact [6,55]. In addition to using chelating agents to investigate the role of complement in mediating M T B binding in the presence of serum, I used factor B-depleted and C2-depleted human serum to assess the roles of either the alternative or classical pathways, respectively. For these experiments, pooled human serum (PHS) from healthy PPD-negative volunteers was used as the control serum because neither factor B-depleted nor C2-depleted mouse sera were commercially available at the time of these studies. It has been shown previously that for mouse P M , the serum-mediated enhanced binding of M T B occurs when either normal human serum or normal mouse serum are employed (unpublished observations Stokes laboratory). In the current series of experiments, the enhanced binding of M T B by P M seen with NMS was also observed with PHS, confirming that opsonins derived from human serum are compatible with the mouse model employed in these experiments. In the presence of either E D T A or EGTA, the association of M T B with P M dropped to levels below those seen with PHS and were also considerably lower than levels of M T B binding seen under serum-free conditions (Table 2). Less than 6% of CDI lb+/+ P M were observed to associate with M T B at the greater than 10 bacteria per 42 macrophage level when either chelating agent was present in the binding medium (Table 2). In contrast, nearly all CDI lb+/+ P M associated with greater than 10 bacteria in the presence of 1% PHS and under serum-free conditions 61 + 5.4 % of the CDI lb+/+ P M monolayer associated with greater than 10 bacteria (Table 2). The effects of E D T A and E G T A were similar for CDI lb-/- P M (Table 2). Based on these observations, it appears that EDTA and E G T A have much broader effects on M T B binding than complement pathway inactivation. To further assess the contribution of either the alternative or classical pathways in mediating the enhanced binding of M T B to macrophages, factor B-depleted and C2-depleted serum were used as a source of opsonins. Compared to PHS, the association of M T B with CDI lb+/+ P M was less when either factor B-depleted or C2-depleted serum were used as a source of opsonins (Table 2). However, the difference seen with C2-depleted serum was more dramatic. In the presence of 1% C2-depleted serum, the percentage of CDI lb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria was 90 + 8.6%, 63 ± 4.1%, and 6.0 ± 1.7%, respectively (Table 2). There was approximately 8x less macrophages associated with M T B at the greater than 30 level when C2-depleted serum was used instead of PHS as a source of opsonins. On the other hand, substituting factor B-depleted serum for PHS lead to a moderate decrease in M T B binding with approximately a two-fold reduction in the percentage of macrophages associated with greater than 30 bacteria (Table 2). Under the conditions tested, it appears that the enhanced binding of M T B to CDI lb+/+ P M is mediated predominantly through activation of the classical complement pathway. 43 Unlike the CDI lb+/+ P M , the role of either the alternative or classical pathways in mediating M T B binding to CDI lb-/- P M appears equivalent. In the presence of 1% C2-depleted serum, the percentage of CDI lb-/- P M associated with 1 or more, greater than 10, or greater than 30 bacteria was 65 ± 7.0%, 24 + 4.3%, and 2.0 + 0.9%, respectively (Table 2). When factor B-depleted serum was substituted for PHS, the percentage of cells binding 1 or more, greater than 10, or greater than 30 bacteria was 71 + 9.0%, 28 + 5.7%, and 1.5 + 0.6%, respectively (Table 2). Compared to PHS, there was approximately a two-fold reduction in the percentage of macrophages binding M T B at the greater than 10 bacteria per macrophage level when either factor B-depleted or C2-depleted serum was used as a source of opsonins. However, based on the observation that CDI lb is required for efficient serum mediated binding of M T B by macrophages, the use of CDI lb-/- P M to investigate the contribution of either the classical or alternative pathways introduces a degree of complexity that makes the data somewhat equivocal. 44 Table 2. Role of Complement in the Association of M T B with P M Percent Macrophages Binding M T B CDllb+/+ C D l l b - / -Bacteria /macrophage 0 1-10 11-30 >30 0 1 -10 11-30 >30 No serum 12 + 2.2 27 ± 2.2 49 ± 4.2 1 2 ± 1.2 41 ± 5.8 38 ± 4 . 9 19 ± 2 . 0 1.7 ± 1.0 PHS 2.0 ± 0.4 2.8 ± 1.2 48 ± 3.7 47 ± 3.5 17 ± 5.2 22 ± 2 . 7 51 ± 3 . 9 10 ± 3 . 4 PHS+EDTA 58 + 4.1 36 + 4.0 5.0 ± 3 . 2 0 ± 0 59 ± 5.5 38 ± 4 . 7 2.8 ± 1.4 0 ± 0 PHS+EGTA 58 + 3.5 40 ± 4.1 2.5 ± 1 0 ± 0 53 ± 6.9 41 ± 5 . 2 6.2 ± 2 . 6 0 ± 0 Factor B (-) 3.3 + 1.2 8.0 ± 2.8 63 ± 1.7 26 ± 3 . 2 29 ± 3.9 43 ± 3 . 3 27 ± 5 . 1 1.5 ± 1 C2 (-) 9.7 + 1.0 28 ± 4.5 57 ± 2.4 6.0 ± 1.7 36 ± 4.7 40 ± 2 . 7 22 ± 3.4 2.0 ± 0 . 9 Association of M T B in the presence of binding medium alone [No serum], 1% pooled human serum [PHS], 1% PHS with lOmM E D T A [PHS+EDTA] or 1% PHS with lOmM E G T A [PHS+EGTA] and in the presence of either 1% Factor B depleted human serum [Factor B(-)] or C2 depleted human serum [C2(-)] was assessed. The mean + S E M for n = 6 (3 separate experiments each with duplicate coverslips is reported). Binding of M T B was measured as the percentage of P M in the monolayer that bound 0, 1-10, 11-30, or greater than 30 bacteria. 3.5 Association of Zymosan, EIgG, and E l g M C with A M from C D l l b - / - and CDllb+/+ mice. Control particles were also used to probe for functional receptors on A M . Under the conditions used here, no significant difference in the association of zymosan, EIgG, or E l g M C was observed between CDI lb-/- A M and CDI lb+/+ A M (Figure 7; P = 0.25, 0.18, and 0.95 for zymosan, EIgG, and E l g M C , respectively). Both CDI lb-/- A M and CDI lb+/+ A M associated with zymosan very efficiently, with association indices of 2062 45 + 76 and 2203 + 82, respectively (Figure 7). The levels of zymosan binding are similar to those reported previously for Day 4 mouse A M [18]. As reported previously, A M did not bind EIgG as avidly as zymosan [18]. Here the association indices of EIgG for CDI lb-/-A M and CDI lb+/+ A M were 1702 ± 154 and 1322 ± 210, respectively (Figure 7). Similar to the observations with P M , no significant difference in E l g M C binding was observed for CDI lb-/- A M and CDI lb+/+ A M (P > 0.05), with association indices of 425 ± 77 and 433 + 102, respectively (Figure 7). 2500 Zymosan EIgG E l g M C Figure 7. Binding of Zymosan, EIgG, and E l g M C to A M . A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was tested. Binding was assessed as the number of particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n 6 (3 separate experiments with duplicate coverslips in each experiment). 46 3.6.1 Non-opsonic Association of M T B with A M Under non-opsonic conditions, the binding of M T B by CDI lb-/- A M was significantly less than that observed for CDI lb+/+ A M (P = 0.007 at the level of binding at least one bacterium). For CDI lb+/+ A M , 46 + 3.8% bound at least one bacterium whereas only 33.2 + 1.9% of CDI lb-/- A M were observed to bind at least one bacterium (Figure 8). At the level of 10 or more bacteria per macrophage, approximately 5% of CDI lb+/+ A M bound greater than 10 bacteria whereas only 1% of CDI lb-/- A M monolayers were observed to bind greater than 10 bacteria (Figure 8). 100 CQ CT C CQ CD11b+/+ AM CD11b- / - AM 1 or more > 10 bacteria> 30 bacteria Figure 8. Non-opsonic association of M T B with CDI lb+/+ A M and CDI lb-/- A M . A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B in a serum-free environment was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 10 (5 separate experiments with duplicate coverslips from each experiment). 47 3.6.2 Opsonic Association of M T B with A M In the presence of normal mouse serum (NMS), M T B binding for either CDI lb-/- A M or CDI lb+/+ A M increased compared to binding under non-opsonic conditions (P < 0.008 for all comparisons between non-opsonic and opsonic binding for A M of either phenotype). Similar to non-opsonic conditions, M T B binding by CDI lb+/+ A M was significantly greater than that seen with CDI lb-/- A M (P = 0.003 and 0.004 at the level of binding at least 1 bacterium and greater than 10 bacteria, respectively). With 1% NMS present in the binding medium, 51.1 + 5.9% of CDI lb-/- A M bound at least 1 bacterium and 13 + 2.7% bound greater than 10 bacteria (Figure 9). The increased association of M T B with A M was more dramatic for CDI lb+/+ A M . In the presence of 1% NMS, 71 + 2.3% of CDI lb+/+ A M bound at least 1 bacterium and 25.2 ± 2.5% bound greater than 10 bacteria (Figure 9). 48 Figure 9. Opsonic association of M T B with CDI lb+/+ A M and CDI lb-/- A M . A M obtained from either C D I lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B in the presence of 1% normal mouse serum (NMS) was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 10 (5 separate experiments with duplicate experiments from each experiment). 49 3.6.3 Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with A M before and after in vitro differentiation. Heat-inactivated serum and R A G serum were again used to test the effects of serum on the enhanced binding of M T B by macrophages. Compared to NMS, the binding of M T B by Day 4 CDI lb+/+ A M was significantly less when HI was used a source of opsonins (P < 0.005 at all levels of M T B binding). For Day 4 CDI lb+/+ A M , the percentage of macrophages binding 1 or more, greater than 10, or greater than 30 bacteria per macrophage in the presence of 1% HI was 42 + 1.7%, 2.5 + 1.0%, and 0%, respectively (Figure 10). Likewise, M T B binding by Day 4 CDI lb-/- A M was significantly less when HI was used as a source of opsonins instead of NMS (P < 0.007 at all levels of M T B binding). The percentage of Day 4 CDI lb-/- A M binding 1 or more, greater than 10, or greater than 30 bacteria in the presence of 1% HI was 35 ± 5.9%, 0.3 + 0.3%, and 0%, respectively (Figure 10). For A M of either phenotype the binding of M T B in the presence of HI was comparable to levels seen under non-opsonic conditions (Figure 10). The observations made here are in agreement with a previous report [18] and provide additional evidence supporting the role of complement proteins in mediating the enhanced binding of M T B under conditions where serum is present. Similar to the results obtained with P M , no significant difference in the binding of M T B was observed for A M of either phenotype when R A G serum was used instead of NMS as a source of opsonins in binding assays (P > 0.05 at all levels of M T B binding). In the presence of 1% R A G serum, the percentage of Day 4 CDI lb+/+ A M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 85 + 1.7%, 48 + 50 5.4%, and 12 ± 3.9%, respectively (Figure 10). For M T B binding to Day 4 CDI lb-/- A M in the presence of 1% R A G serum, the percentage of macrophages associated with 1 or more, greater than 10, or greater than 30 bacteria was 75 + 1.9%, 31 + 6.1%, and 3.8 + 1.8%, respectively (Figure 10). Therefore, similar to the observations with P M , the enhanced binding of M T B by A M of either phenotype appears to be mediated by a heat-labile serum component but is independent of immunoglobulins. In addition to testing the effects of serum on M T B binding, I also examined the effects of in vitro differentiation of A M on M T B binding. Previous studies have shown that CR3 expression, along with the expression of several other receptors is low for Day 0 A M but culturing of A M in vitro leads to an increase in CR3, and other receptor expression, by Day 4 A M [18]. Based on this observation, I was interested in testing the hypothesis that increased M T B binding by Day 4 A M was due to an increase in CR3 expression. The association of M T B with Day 0 and Day 4 A M from either CDI lb-/- or CDI lb+/+ mice was assessed. Presumably, if increased expression of CR3 was exclusively responsible for the enhanced binding of M T B by Day 4 A M , an increase in M T B binding would be observed with CDI lb+/+ A M differentiation but not with CDI lb-/- A M . However, in this series of experiments the association of M T B with A M of either phenotype increased with in vitro differentiation. Both CDI lb+/+ and CDI lb-/- Day 4 A M were capable of binding M T B more efficiently than their Day 0 counterparts (Figure 10). In the presence of NMS, the binding of M T B by Day 0 CDI lb+/+ A M was significantly less that that observed for Day 4 CDI lb+/+ A M (P = 0.001 at the level of binding at least one bacterium). The percentage of Day 0 CDI lb+/+ A M binding 1 or more, greater than 10, or greater than 30 51 bacteria in the presence of NMS was 58 + 4.0%, 7.8 ± 3.5%, and 0.3 ± 0.3%, respectively (Figure 10). Likewise, M T B binding in the presence of NMS by Day 0 CDI lb-/- A M was significantly less than that observed with Day 4 CDI lb-/- A M (P < 0.001). The percentage of Day 0 CDI lb-/- A M binding 1 or more, greater than 10, or greater than 30 M T B was 41 + 1.6%, 1.8 ± 0.7%, and 0%, respectively (Figure 10). The increased binding of M T B by Day 4 A M was also observed under non-opsonic conditions (Figure 10). Therefore, based on these observations the serum-mediated increased binding of M T B that accompanies differentiation cannot be attributed exclusively to CR3. In fact, the data suggest that the majority of M T B binding by A M , either in the presence or absence of serum, is mediated by receptors other than CR3. 52 1 or more > 10 > 30 1 or more > 10 Bacteria Per Macrophage > 30 Figure 10. Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with A M before and after in vitro differentiation. A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered and cultured for 2 hours (Day O) or 4 days (Day 4). The ability of these macrophages to bind M T B either in the absence (-) or presence of mouse sera was assessed. The sera used in these binding assays were normal mouse serum (NMS), heat-inactivated normal mouse serum (HI), or serum deficient in immuno-globulins (RAG). A l l sera used at 1% concentrations in binding medium. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 4 (2 separate experiments with duplicate coverslips from each experiment). 53 3.6.4 Comparison of M T B binding between C 5 7 B L / 6 and B A L B / c Day 0 A M In addition to demonstrating that receptors other than CR3 play a quantitatively important role in the binding of M T B by A M , the in vitro differentiation experiments also identified that Day 0 A M from either CDI lb+/+ or CDI lb-/- mice respond to the addition of serum with increased M T B binding (Figure 10). The latter observation is in contrast to previous reports which found that Day 0 A M from B A L B / c do not respond to the addition of serum with enhanced binding of M T B [18]. In the present studies, the binding of M T B in the presence of 1% NMS by either CDI lb+/+ or CDI lb-/- Day 0 A M was significantly greater than M T B binding under non-opsonic conditions (P = 0.007 and 0.046 for Day 0 CDI lb+/+ A M and Day 0 CDI lb-/- A M , respectively). Because Day 0 A M from either CDI lb+/+ or CDI lb-/- mice appeared to behave differently with respect to M T B binding in the presence of serum than Day 0 A M from B A L B / c mice, I was interested in investigating this difference. The association of M T B with Day 0 A M obtained from CDI lb+/+ mice of C57BL/6 background was compared with Day 0 A M from B A L B / c mice. In the absence of serum, Day 0 A M from either C57BL/6 mice or B A L B / c mice behaved similarly with respect to M T B binding (P = 0.63 for comparison of binding at least one bacterium under non-opsonic conditions). The percentage of Day 0 A M from C57BL/6 or B A L B / c mice binding at least one bacterium under non-opsonic conditions were 15.3 + 3.2% and 13.0 + 3.0%, respectively (Figure 11). In the presence of serum, Day 0 C57BL/6 A M responded to the addition of serum with increased M T B binding whereas, similar to previous reports [18] Day 0 B A L B / c A M did not (Figure 11). The observed difference in M T B binding 54 between Day 0 C57BL/6 A M and Day 0 B A L B / c A M in the presence of serum was statistically significant and not dependent upon the mouse serum source (P = 0.002 and 0.001 for 1% N B M S and NMS, respectively). The serum-mediated enhanced binding of M T B by Day 0 C57BL/6 A M was seen using serum from either C57BL/6 or B A L B / c mice (Figure 11). The percentage of Day 0 C57BL/6 A M associated with at least one bacterium was 40.2 ± 3.3% and 42.0 ± 4.0% for 1% NBMS and 1% NMS, respectively (Figure 11). In contrast, neither C57BL/6 mouse serum nor B A L B / c mouse serum caused Day 0 B A L B / c A M to respond with increased M T B binding (Figure 11). The percentage of Day 0 B A L B / c A M associated with at least one bacterium was 15.5 + 4.6% and 15.2 + 2.3% for 1% NBMS and 1% NMS, respectively (Figure 11). The observed difference in M T B binding between Day 0 C57BL/6 A M and Day 0 B A L B / c A M emphasizes the role host factors may play in determining the outcome of an infection with M T B . 55 100 (-) 1%NBMS 1%NMS Figure 11. Comparison of M T B binding to Day 0 C57BL/6 A M and Day 0 B A L B / c A M in the presence and absence of serum. A M obtained from either CDI lb+/+ or B A L B / c mice were adhered to coverslips for 2 hours. The ability of these macrophages to bind M T B either in the absence of serum (-), presence of 1% B A L B / c mouse serum (1%NBMS) or 1% CDI lb+/+ mouse serum (1% NMS) was tested. The mean percentage + S E M of the macrophages binding at least one bacterium is reported for n = 4 (2 separate experiments with duplicate coverslips from each experiment). 56 3.7 Comparison of M T B Survival and Replication following Phagocytosis by either C D l l b - / - or CD11+/+ P M . To assess whether CR3-mediated entry provides M T B an advantage for intracellular survival and replication in macrophages, colony forming units (CFU) of M T B at 0, 1, 4, and 7 days following in vitro phagocytosis by either CDI lb-/- P M or CDI lb+/+ P M were determined. When P M of either phenotype were exposed to M T B at the same multiplicity of infection (MOI), the C F U of M T B at 7 days post-infection was significantly greater for CDI lb+/+ P M compared to CDI lb-/- P M (P = 0.031). However, based on the M T B binding data presented above, the higher C F U of M T B seen with CDI lb+/+ P M was possibly due to the increased efficiency with which CDI lb+/+ P M bind and internalize M T B . This claim is supported by comparing the C F U at Day 0 for the two macrophage populations infected with M T B at an MOI of 10. The C F U of M T B from CDI lb+/+ P M and CDI lb-/- P M at this point were 9.2 x 104 ± 1.0 x 103 and 6.3 x 104 ± 2.5 x 103, respectively (Figure 12 and Table 3), a significantly different uptake (P = 0.008). An estimated 4-5 population doublings occurred over the seven days for M T B that had been ingested by P M of either phenotype and the calculated doubling times for M T B from CDI lb+/+ P M and CDI lb-/- P M were 34.3 hours and 37.8 hours, respectively (Table 3). To control for the contribution of the initial number of M T B ingested, M T B survival and replication was also assessed for CDI lb-/- P M exposed to twice the MOI of M T B . This was designed to seed the same number of M T B in macrophages of either phenotype. Using an MOI of 20 for CDI lb-/- P M and an MOI of 10 for CDI lb+/+ P M resulted in the internalization of similar numbers of M T B , as measured by C F U on Day 0 (P = 0.143). 57 The corresponding C F U of M T B for CDI lb+/+ P M and CDI lb-/- P M on Day 0 were determined to be 9.2 x 104 + 1.0 x 103 and 1.0 x 105 + 5.0 x 103, respectively (Table 3). A comparison of the C F U at Days 1,4, and 7 following M T B phagocytosis demonstrated that, when similar numbers of M T B were internalized by either CDI lb-/- P M or CDI lb+/+ "i P M , no significant difference in the C F U of M T B was observed (Figure 12 and Table 3; P 5e+6 o 4e+6 • 3e+6 -2e+6 -1e+6 -0e+0 -10CD11b+/+ 10CD11b-/-20CD11b-/ -0 1 4 7 D a y s P o s t I n f e c t i o n Figure 12. Comparison of M T B survival and replication following phagocytosis by either CDI lb-/- P M or C D I lb+/+ P M in vitro. P M obtained from either C D I lb-/- or C D I lb+/+ mice were adhered to coverslips overnight and exposed to M T B at the indicated MOIs for 3 hours in a serum-free environment. The CFU/ml of M T B for P M monolayers was then determined immediately (Day 0) or at 1,4, and 7 days post infection. The mean CFU/ml + S E M from two separate experiments with duplicate coverslips in each is shown. CFU/ml of M T B for CDI lb+/+ P M monolayers infected at a dose of 10 M T B per macrophage [10CD1 lb+/+] and CDI lb-/- P M monolayers infected at either 10 [10CD1 lb-/-] or 20 [20CDllb-/-] M T B per macrophage are shown. 58 Table 3. C F U and Calculated Population Doubling Time for M T B following Phagocytosis by either CDllb+/+ P M or C D l l b - / - P M in vitro CFU/ml at Number of Days Post Infection Mean (+ SEM) Doubling P M 0 1 4 7 Time (hours) 10CDllb+/+ 9.2 x 104 (1.0 x 103) 1.6 x 105 (6.5 x 103) 5.6 x 105 (2.1 x 104) 2.7 x 106 (2.1 x 105) 34.3 lOCDllb-/- 6.3 x 104 (2.5 x 103) 8.8 x 104 (6.5 x 103) 6.8 x 105 (1.4 x 104) 1.4 x 106 (1.4 x10 s) 37.8 20CDllb-/- 1.0 x 105 (5.0 xlO 3) 2.0 x 105 (1.7 x 104) 8.8 x 105 (1.6 x 105) 3.1 x 106 (7.5 x 105) 34.2 The CFU/ml and calculated population doubling times for MTB following phagocytosis by either CDI lb+/+ PM or CDI lb-/- PM in vitro are reported. PM from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips overnight and exposed to MTB at the indicated MOIs for 3 hours in a serum-free environment. The CFU/ml of MTB for PM monolayers was then determined immediately (Day 0) or at 1,4, and 7 days post infection. The mean CFU/ml + SEM from two separate experiments with duplicate coverslips in each is shown. CFU/ml of MTB for CDI lb+/+ PM monolayers infected at a dose of 10 MTB per macrophage [10CDllb+/+] and CDllb-/- PM monolayers infected at either 10 [lOCDllb-/-] or 20 [20CD1 lb-/-] MTB per macrophage are shown. The population doubling times for MTB were calculated using CFU values at Day 7 and Day 0 for each condition and the equation N t = N 0e k t which describes logarithmic growth rates where N t = number of bacteria at time = t, N 0 = number of bacteria at time = 0, k = growth rate constant, t = time and e = natural logarithm. 59 C H A P T E R 4 DISCUSSION: Role of CR3 in M T B Pathogenesis Investigated using C D l l b - / - and CDllb+/+ Mouse Macrophages The binding of M T B to host macrophages is the initial step in the establishment of infection. This important first step is the focus of much research based on the observation that other intracellular pathogens are able to survive inside macrophages following phagocytosis via some, but not all, receptor mediated pathways [60-63]. It has been speculated that M T B exploits CR3 to gain entry into macrophages without activating macrophage microbicidal functions because CR3-promoted phagocytosis can occur without triggering an oxidative burst [64,65]. In this study the contribution of CR3 to the binding of M T B by murine macrophages was examined using a CDI lb knockout mouse model and the role of complement in mediating the binding of M T B under conditions where serum is present was investigated. The hypothesis that M T B exploits CR3 as a means to favor its intracellular survival was also tested by comparing the growth of intracellular M T B up to 7 days following phagocytosis by either CDI lb-/- P M or CDl lb+/+PM. Particle binding experiments were used to characterize macrophage receptor function. These assays demonstrated that CDI lb-/- P M were less able to bind zymosan compared to their CDI lb+/+ counterparts (Figure 3). The difference in zymosan binding between CDI lb-/- P M and CDI lb+/+ P M is in agreement with recently reported observations and may be attributed to the lectin-like properties of CR3 [28]. No difference in the association of EIgG or E l g M C was detected between CDI lb-/- P M and CDI lb+/+ P M (Figure 3). 60 The conditions used to prepare E l g M C particles are designed to coat the erythrocytes with iC3b, the principal ligand for CR3, therefore it is surprising to find that CDI lb-/- P M associated with E l g M C at levels comparable to CDI lb+/+ P M . It is possible that P M from the CDI lb knockout mouse have compensated for the absence of CDI lb by expressing higher than normal amounts of other receptors with common binding specificity. Presumably, if this were the case the CDI lb knockout macrophages would express greater amounts of CR4 or CR1 because these receptors have binding functions in common with CR3. However, the flow cytometry data do not support the idea that the CDI lb knockout mouse compensated for the loss of CR3 by expressing greater amounts of CR4, CR1, or any of several other receptors that were measured (Figure 2). It is also possible that normal levels of CR1 and/or CR4 present on CDI lb-/- P M take over the task of binding E l g M C . The latter could be tested using competitive ligands, such as anti-CRl and anti-CR4 antibodies, for the complement binding sites on CR1 and CR4. Although the protocol used to generate E l g M C was designed to favor the deposition of iC3b, E l g M C may also have C3b on their surface and therefore comparable levels of E l g M C binding between CDI lb+/+ P M and CDI lb-/- P M may also be due to CR1-mediated binding to C3b deposited on E l g M C . The association of M T B with CDI lb-/- P M in the absence of serum was significantly less than that observed for CDI lb+/+ P M (P = 0.008). Approximately 20% fewer macrophages bound M T B in CDI lb-/- monolayers compared to CDI lb+/+ monolayers (Figure 4). This observation indicates that CR3 is a quantitatively important receptor for M T B under serum-free conditions but that CR3 does not account for the majority of binding. These observations also indirectly support the hypothesis that other macrophage 61 receptors are important for the non-opsonic binding of M T B . Alternate candidate receptors for the non-opsonic binding of M T B include MR, CD14, and CR4 [8,14,32]. Dissecting the relative contribution of each of these receptors in mediating the interaction of M T B with macrophages and the outcomes of these interactions will be critically important in increasing our understanding of tuberculosis. Although CR3 was shown to play a limited role in the non-opsonic binding of M T B , a dramatic difference in M T B binding was observed between CDI lb-/- P M and CDI lb+/+ P M in the presence of serum. At the greater than 30 bacteria per macrophage level, CDI lb-/- P M associated with M T B approximately 9x less efficiently than did CDI lb+/+ P M (Figure 5). This observation confirms the importance of CR3 for the efficient uptake of opsonised M T B and suggests that CR3 is the predominant receptor for M T B in the presence of serum. Presumably, the enhanced binding of M T B to CR3 in the presence of serum is mediated by the interaction of opsonic iC3b that has been deposited on M T B and the iC3b-binding site of CR3. Whether CR3 functions as the primary receptor or cooperates with other receptors to mediate the uptake of M T B was not investigated. However, recently, it has been proposed that CR3 cooperates with other macrophage receptors including Fc receptors and C D 14 to mediate the internalization of bound particles [33]. One plausible model suggests that alternate receptors, such as CD 14, serve as scouts for CR3, thus expanding the ligand binding repertoire of CR3 [33]. Because CD 14 has been reported to bind L A M of M T B [11,32] this model has interesting implications for the role of CR3 in the pathogenesis of 62 tuberculosis. Future experiments using knockout mice with deficiencies in multiple receptors could be used to investigate receptor cooperation in M T B phagocytosis. The binding of M T B in the presence of serum was further investigated using heat-inactivated serum (HI) and R A G serum. The latter is obtained from RAG-1 (-/-) mice and does not contain immunoglobulins [51] whereas HI does not have the capacity for complement activation [53]. When HI was used as a source of opsonins, the association of M T B with P M of either phenotype returned to levels similar to non-opsonic association (Figure 6), suggesting that, for both CDI lb+/+ and CDI lb-/- P M , complement activation was responsible for the enhanced binding of M T B . Binding studies with R A G serum demonstrated that immunoglobulins were not important for the enhanced binding of M T B seen in the presence of NMS. Contrary to claims made by Hetland et al. [55], the observations made here indicate that immunoglobulin-mediated classical complement pathway activation is not important for the enhanced uptake of M T B by macrophages. Due to recent developments and controversy concerning the role of complement in mediating the enhanced binding of M T B to macrophages I was interested in characterizing the role of either the classical or alternative complement pathways in mediating M T B binding. Using either factor B-depleted or C2-depleted human serum to evaluate, respectively, the contribution of the alternative or classical complement pathways, I found that with CDI lb+/+ P M the majority of M T B binding was enhanced via activation of the classical and not the alternative complement pathway. C2-depletion greatly reduced the enhanced binding of M T B and brought binding to levels comparable to those seen when 63 serum was absent. There was approximately a nine-fold decrease in the percentage of macrophages associated with greater than 30 bacteria when C2-depleted serum was used as a source of opsonins instead of PHS (Table 2). On the other hand, the decrease in M T B binding seen with factor B-depleted serum was approximately two-fold at the greater than 30 bacteria per macrophage level (Table 2). The experiments described here do not support the claim that enhanced binding of M T B by macrophages is mediated primarily through activation of the alternative pathway [6] or by the immunoglobulin-mediated classical pathway [54]. A possible explanation for the disagreement with these earlier reports is that Schlesinger et al. examined the role of complement in M T B binding using factor B-depleted serum but did not assess the effects of C2-depleted serum on M T B binding [6]. Whereas, Hetland et al. draw their conclusions from experiments using chelating agents and serum from tuberculoid and non-tuberculoid individuals to determine whether B C G activates either the classical or alternative pathways of complement [54]. I studied the effects of E D T A and E G T A on the binding of M T B because these chelating agents have been widely used as a means to investigate the contribution of either the classical or alternative pathways [6, 54]. At the concentration used to inactivate complement, EDTA and E G T A have an effect on the binding of M T B to macrophages beyond complement inactivation. Indeed, the cationic-dependent function of several macrophage receptors, including CR3, has been documented [66,67]. Therefore, in light of the observations made here, previous work using E D T A or E G T A as a means to investigate the opsonic binding of pathogens by macrophages should be revisited and tested with the appropriate factor depleted sera. 64 Taken together, the observations with R A G serum and C2-depleted serum indicate that, in the presence of normal serum, enhanced binding of M T B by macrophages is mediated predominantly via activation of the classical complement pathway and occurs independently of immunoglobulins. The immunoglobulin-independent enhanced binding of M T B by macrophages in the presence of serum may be attributed to the complement activating potential of the mannose-binding protein (MBP) [68]. M B P , a lectin specific for mannose and iV-acetylglucosamine, has structural homology with C l q and reportedly activates the classical complement pathway [68]. Therefore, it is conceivable that binding of M B P to mannose residues on M T B triggers the deposition of opsonic C3 peptides on the M T B surface, in a C2-dependent manner, which leads to enhanced binding of M T B by macrophages. In addition, the observation that M T B has developed an invasion strategy which is based on the acquisition of opsonic C3 peptides by scavenging C2a to create a novel C3 convertase [16] could also explain some of the results presented here. Contrary to CDI lb+/+ P M , the role of either the classical or alternative pathways in mediating enhanced binding of M T B appears to be equivalent with CDI lb-/- P M . When either factor B-depleted or C2 depleted serum were used there was a significant decrease in the binding of M T B compared to PHS (Table 2). At the present time, no definitive explanation for the observed difference in complement activation between CDI lb-/- P M and CDI lb+/+ P M can be presented. However, the observation that CDI lb-/- P M behave differently than CDI lb+/+ P M with respect to complement-mediated M T B binding supports the hypothesis that CR3 is an important receptor in mediating the opsonic association of M T B with macrophages. 65 Previously, Stokes et al. reported that the phenotype of a macrophage greatly affects its interaction with mycobacteria and demonstrated that P M , although widely cited in the literature as a model to investigate mycobacteria-macrophage interactions, do not behave like A M with respect to M T B binding [18]. In the current studies, the role of CR3 in M T B binding was initially assessed with experiments using P M . There are several reasons why P M were used initially, including the feasibility of their isolation and the fact that P M are widely cited in the literature as models for investigating macrophage properties. However, I was also interested in characterizing the role of CR3 in M T B binding using A M because murine A M closely resemble the resident human alveolar macrophage phenotype [18] and A M are the initial host cell in clinical M T B infections. Particle binding assays failed to demonstrate a difference in the binding of any of the control particles between A M of either phenotype. Unlike P M , no difference in zymosan binding was observed between CDI lb-/- A M and CDI lb+/+ A M (Figure 7). Compared to P M , A M do not express high levels of CR3 [12, 18], therefore the low expression of CR3 on A M may not allow for the detection of the contribution of CR3 to binding zymosan. However, A M associated with zymosan very efficiently and presumably the binding of zymosan by A M is mediated by other receptors such as the MR. Like P M , no significant difference in EIgG or E l g M C binding was detected between CDI lb-/- A M and CDI lb+/+ A M . Again the absence of a detectable difference between macrophages of either phenotype to associate with E l g M C was unexpected. However, it has been shown previously that A M do not associate with E l g M C very efficiently [18] and therefore, it is 66 also possible that the low expression of CR3 on A M does not allow one to detect differences in the association of E l g M C between CDI lb-/- and CDI lb+/+ A M . The pathophysiologic relevance of M T B infection in the lung makes characterizing the non-opsonic interaction of M T B with host macrophages an important area of research. Comparable with the observations that were made with P M , CDI lb-/- A M were less able to associate with M T B compared to their CDI lb+/+ counterparts. Of particular interest was the observation that the absence of CDI lb on A M depressed M T B binding modestly under non-opsonic conditions. This observation is in disagreement with a previous report by Cywes et al. which suggested that CR3 is the major receptor for M T B under non-opsonic conditions [13]. In fact, the observations made here suggest that the majority of M T B binding to A M occurs independently of CR3, thereby providing support for the claim that alternate receptors such as CR4 or M R are responsible for mediating the binding of M T B under non-opsonic conditions [14,69]. The role of CR3 in mediating the efficient uptake of M T B in the presence of serum was also demonstrated for A M . CDI lb-/- A M associated with M T B less efficiently than CDI lb+/+ A M in the presence of serum. Examining the effects of serum on the association of M T B with A M demonstrated that, similar to our observations with P M , the enhanced binding of M T B under opsonic conditions is mediated by a heat-labile serum component(s) but is independent of serum immunoglobulins. This observation indirectly supports the hypothesis that enhanced binding of M T B by macrophages is mediated by immunoglobulin-independent complement activation. However, the role of the classical 67 versus the alternative pathway for complement activation and C3b fixation in mediating the binding of M T B to A M was not examined because previous work has shown that mouse A M do not respond to human serum with increased binding of M T B (unpublished observations Stokes laboratory). This factor, in conjunction with the unavailability of C2-depleted or factor B- depleted mouse sera made the relevant experiments difficult to perform. The apparent serum incompatibility of murine A M but not murine P M with human serum cannot yet be readily explained. It is possible that differences in the expression and/or specificity of CRs on murine A M compared to P M do not permit efficient complement activation when human serum is used as a source of opsonins. For example, mouse CRI does not recognize human complement proteins [70-72]. Therefore, if CRI on mouse A M is important for the serum-mediated binding of M T B , the serum incompatibility may explain the requirement for mouse serum as a source of opsonins for M T B binding by mouse A M . An alternative explanation is that M T B binding to A M in the presence of serum is not predominantly complement mediated and that the heat sensitive serum factor(s) responsible for the enhanced binding of M T B by A M is present in mouse serum but not present in human serum. Previous reports have suggested that the increased expression of surface receptors, including CR3, which accompanies differentiation of A M are responsible for increased binding of M T B [18]. Stokes et al. previously reported that Day 0 A M bind M T B poorly and that M T B binding is not enhanced by the addition of serum to the binding medium [18]. However, after four days of differentiation in vitro, A M respond to the addition of serum with enhanced binding of M T B [18]. In the present study, I was interested in testing 68 whether the enhanced binding seen by Day 4 A M in the presence of serum was due to the increased expression of CR3. Based on the observations, it appears that a portion of the enhanced binding could be attributed to CR3 but that the majority of M T B binding by either Day 0 or Day 4 A M was not mediated by CR3. In addition, I also found that murine Day 0 A M from different strains of mice do not behave uniformly with respect to M T B binding. Day 0 A M obtained from either CDI lb-/- or CDI lb+/+ mice, both of which are of similar genetic background as C57BL/6 mice, responded to the addition of serum with enhanced M T B binding. Whereas, similar to previous reports, Day 0 A M from B A L B / c mice did not respond to the addition of serum with enhanced M T B binding [18]. The difference in M T B binding seen between Day 0 A M from B A L B / c mice and C57BL/6 mice was due to functional differences in the A M themselves as these differences were observed when serum from either mouse strain was used as a source of opsonins. Further experiments would be required to provide a clear explanation for the observed difference in M T B binding between Day 0 A M from C57BL/6 and B A L B / c mice. However, it is possible that the increased binding of M T B , in the presence of serum, by Day 0 A M from C57BL/6 compared to Day 0 A M B A L B / c was due to differences in receptor expression. The latter could be examined using flow cytometric analysis and presumably Day 0 A M from C57BL/6 mice would express greater amounts of receptors which mediate the binding of M T B in the presence of serum. The M T B survival and replication experiments performed here assessed the role of CR3 in determining the intracellular viability of M T B following phagocytosis under non-opsonic conditions. Non-opsonic conditions were used for infecting macrophages in an attempt to 69 mimic the initial interaction of M T B with human A M . Admittedly, the use of P M is less desirable than A M . However, based on the previous observations that A M do not express high levels of CR3 whereas P M do express high levels of CR3,1 felt that for these experiments P M would provide a better means to assess the role of CR3. In testing whether the absence of CR3 affects the intracellular fate of M T B , no significant difference was observed between CDI lb-/- P M and CDI lb+/+ P M in the survival and replication of M T B following phagocytosis of similar numbers of M T B (P > 0.05 for all comparisons). Therefore the observations made here do not support the hypothesis that CR3-mediated entry provides M T B with an advantage for intracellular survival and replication. It would be interesting to see if the same conclusion would be made following phagocytosis under opsonic conditions. It has been reported previously that binding to the iC3b-binding site on CR3 leads to phagocytosis without an accompanying oxidative burst [36] whereas, engagement of both the 0-glucan binding site and the iC3b binding site on CR3 is accompanied by an oxidative burst [22,23,28]. Therefore, it is possible that CR3 alters the intracellular fate of M T B but only does so under conditions where serum is present. The latter could be tested by modifying the survival and replication experiments described here to compare the viability of M T B following phagocytosis in the presence of serum. 70 C H A P T E R 5 R E S U L T S : Interaction of M T B with M H - S Cells Compared to Primary Macrophages 5.1 Comparison of M T B binding to M H - S cells, A M and P M . Similar to previously reported findings [12], the observations made here demonstrated that the binding of M T B to different macrophage phenotypes varied. Under non-opsonic conditions, 79.0 + 12% of resident B A L B / c P M were associated with at least one bacterium compared to only 36.0 ± 8 . 1 % of B A L B / c A M (Figure 13; P < 0.001). Of significant interest to the overall aim of testing whether the MH-S cell line was a suitable model for investigating macrophage-MTB interactions was the observation that MH-S cells behaved similarly to their in vivo counterpart, the mouse A M , with respect to M T B binding under non-opsonic conditions. Under the conditions tested, 35.7 + 12.7% of M H -S monolayer population bound at least one bacterium (Figure 13). No significant difference was observed among the three macrophage populations in their ability to bind latex particles (Figure 13; P > 0.3 for comparisons of all three macrophage cell types). This demonstrated that the decreased binding of M T B by MH-S cells and A M was not due to a global inability of these two cell populations to bind particles. 71 MTB Latex Figure 13. Binding of M T B and Latex Beads to P M , A M , and MH-S cells. P M obtained from BALB/c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M obtained from BALB/c mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B or 0.8 um polyvinyl latex beads (Latex) in a serum-free environment was then assessed. The percentage of the macrophage population binding > 1 M T B or > 1 latex bead is shown. The mean + S E M is shown for n = 12 (6 experiments with duplicate coverslips from each experiment). 7 2 5.2 M H - S , A M , and P M binding of EIgG, E l g M C and zymosan particles. Binding of control particles was used to probe for the expression of functional receptors for IgG (EIgG), complement component iC3b (E lgMC) , and glycans (zymosan) on the surface of the three macrophage populations. Under the conditions used for these experiments, P M did not bind E l g M C as efficiently as zymosan or EIgG (Figure 14) with approximately 40% of population binding E l g M C compared to nearly 100% of the population binding EIgG or zymosan. A M bound zymosan particles very efficiently with approximately 100% of A M ingesting an average of 20-25 zymosan particles (Figure 14). The association of EIgG with A M was moderate whereas the association of E l g M C with A M was negligible (Figure 14). MH-S cells were observed to bind EIgG and zymosan moderately but the binding of E l g M C by MH-S cells was also negligible (Figure 14). The data demonstrate that, compared with A M , MH-S cells exhibit similar binding properties with respect to EIgG and E l g M C but bind zymosan less efficiently (P for EIgG and E l g M C association = 0.13 and 0.47, respectively and P < 0.001 for zymosan association). Compared to P M , MH-S cells exhibit an impaired ability to bind all three particles tested, however, the difference in EIgG binding was not significant (P > 0.05 for EIgG and P = 0.007 and 0.008 for zymosan and E l g M C , respectively). 73 2500 Zymosan EIgG ElgMC Figure 14. Binding of Zymosan, EIgG, and E lgMC' to P M , A M , and MH-S cells. P M obtained from B A L B / c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M from B A L B / c mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was tested. Binding was assessed as the number of particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n = 6 (3 separate experiments with duplicate coverslips in each experiment). Note: the binding of E l g M C by MH-S was negligible with an association index of4.7 + 5.0. 74 5.3 M H - S cells, A M and P M binding of M T B in the presence of serum. Because MH-S cells behaved similarly to A M with respect to M T B binding in a serum-free environment I was interested in characterizing M T B binding by MH-S cells in the presence of serum. With increasing serum concentrations of 0%, 1%, and 5%, the percentage of A M binding M T B were 36 ± 2.6%, 66.7 ± 8.3%, and 83 ± 4.3%, respectively (Figure 15). In the presence of serum, an increase in the average number of M T B per A M was also observed. Approximately 40% of the A M contained greater than 10 bacteria in the presence of 5% serum compared to less than 1% when no serum was present (Figure 15). The percentage of MH-S cells binding M T B also increased significantly in the presence of serum (P = 0.041), but the increased binding of M T B was less than that seen with A M . With increasing serum concentrations of 0%, 1%, and 5% the percentage of MH-S binding M T B were 40.3 ±3.0%, 49.2 ± 3.9%, and 52.5 ± 3.4%, respectively (Figure 15). Unlike A M , the increase in the percentage of MH-S cells binding M T B in the presence of serum did not include the majority of cells. Instead, a subpopulation of MH-S cells responded to the addition of serum with increased binding of M T B . In the presence of 5% N B M S , approximately 20% of MH-S cells associated with greater than 10 bacteria compared to less than 2% when no serum was present (Figure 15). Neither MH-S nor A M were able to associate with M T B to levels seen with P M . As previously reported, the binding of M T B by P M was dramatically enhanced by the addition of serum [12]. With increasing concentrations of serum of 0%, 1%, and 5% the percentage of P M binding M T B were 66.7 ± 1.6%, 85.5 ± 3.0%, and 96.5 ± 0.7%, respectively (Figure 15). The enhancement of M T B binding in the presence of serum also corresponded with a marked increase in the average number of bacterium per macrophage. 75 In the presence of 5% normal mouse serum, approximately 90% of the P M contained greater than 10 bacterium compared to less than 30% of cells binding greater than 10 bacterium when no serum was present (Figure 15). 76 100 • 80 • 60 • 40 20 CQ • 0 1— 100 T 80 c 60 Bindi 40 20 0 100 80 60 40 20 MH-S > 1 bacter ium w — > 10 bacter ia (-) 1 % N B M S 5 % N B M S Figure 15. Binding of M T B by P M , A M , and MH-S cells in the presence of normal B A L B / c mouse serum (NBMS). P M obtained from B A L B / c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M obtained from B A L B / c mice were adhered to coverslips and cultured for 4 days. The binding of M T B was assessed in the absence of serum (-) or presence of 1% or 5% N B M S . The mean percentage of the macrophages binding either at least 1 or greater than 10 M T B is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). 77 5.4 Effect of serum heat-inactivation on the association of M T B with M H - S cells The enhanced binding of M T B by macrophages seen in these experiments could be attributed to several different macrophage receptors. However, it is generally accepted that complement receptors mediate this enhanced binding because heat treatment of the serum abrogates the enhancement [6,18]. Therefore to test whether complement activation was responsible for the enhanced binding of M T B by MH-S cells I compared the binding of M T B by MH-S cells in the presence of N B M S and HIBMS. In this series of experiments the percentage of MH-S cells associated with at least one bacterium was 56.8 + 5.6% and 55.4 + 4.0% for 1% and 5% N B M S , respectively (Figure 16). When HIBMS was used as a source of opsonins, the percentage of MH-S associated with at least one bacterium dropped to 25.2 ± 4.3% and 33.2 + 3.0% for 1% HIBMS and 5% HIBMS, respectively (Figure 16). The association of M T B with MH-S in the presence of HIBMS was comparable to that seen under non-opsonic conditions, with 34.2 + 8.7% of MH-S associated with at least one bacterium in the absence of serum (Figure 16). In addition, when HIBMS was used as a source of opsonins, less than 0.5% of MH-S cells associated with greater than 10 bacteria (Figure 16). Therefore the observations made here indicate that the serum-mediated enhanced binding of M T B by MH-S is mediated by a heat-labile serum component and suggests that, similar to primary macrophages, M T B binding was enhanced by opsonization with complement proteins. 78 100 Figure 16. Effect of serum heat inactivation on the binding of M T B by MH-S cells. MH-S cells were adhered to coverslips in serum-supplemented medium for 18 h. The binding of M T B in the absence of serum or presence of 1% N B M S , 1% heat-inactivated B A L B / c mouse serum (HIBMS), 5% N B M S , or 5% HIBMS was assessed. The mean percentage of MH-S cells binding either at least 1 or greater than 10 M T B is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 79 5.5 Role of CR3 in the association of M T B with MH-S cells Based on the observation that approximately 20% of MH-S cells responded to the addition of serum with increased binding of M T B , I hypothesized that this subpopulation of cells represented a CR3-positive population of MH-S. To test this hypothesis, I assessed the effects of two different anti-CR3 monoclonal antibodies on the enhanced binding of M T B by MH-S. Compared with a non-specific rat IgG control (IR863) antibody, both Ml /70 and 5C6, two different anti-CR3 monoclonal antibodies recognizing different epitopes on CR3, inhibited the binding of M T B by MH-S significantly (P = 0.016 and 0.023 for M l / 7 0 and 5C6, respectively). The percentage of MH-S cells associated with one or more M T B was reduced from approximately 40% to 17.2 + 5.3% and 16.0 + 6.8% in the presence of Ml /70 and 5C6, respectively (Figure 17). 80 100 Figure 17. Role of complement receptor type 3 (CR3) in binding of M T B by MH-S cells in the presence of N B M S . MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. The effects of anti-CR3 monoclonal antibodies on the binding of M T B in presence of 1% N B M S with non-specific rat IgG (Control) or with anti-CR3 monoclonal antibodies (Ml/70 and 5C6) was tested. The mean percentage of MH-S cells binding at least 1 or greater than 10 M T B is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 81 5.6 Isolation of CR3-negative population of M H - S cells Binding studies with anti-CR3 antibodies indicated that CR3 played a major role in mediating the binding of M T B by MH-S cells under opsonic conditions. However, because binding assays using antibodies as competitive inhibitors cannot be considered definitive, I wanted to dissect the role of CR3 in mediating M T B binding using CR3-negative and CR3-positve populations of MH-S cells. In the original publication describing MH-S cells the authors report that approximately 10% of MH-S cells are Mac-1 positive and that CR3-negative and CR3-positive subpopulations of MH-S cells were isolated using a cell sorter [42]. Based on this information, I attempted to isolate CR3-negative and CR3-positive subpopulations of MH-S cells using immunoselection. Using this method as a means to isolate receptor negative cell populations, one would expect to see an increase in the proportion of CR3-negative cells with each successive round of immunoselection. However, the ratio of CR3-negative to CR3-positive cells for rounds one, two, and three of immunoselection were 4.9:1, 6.1:1, and 2.6:1, respectively (Figure 18). Thus, contrary to the previous report [42], I was unable to isolate stable CR3-positive and CR3-negative subpopulations of MH-S cells and based on the observations made over several separate trials it appears that the expression of CR3 on MH-S cells is transient or at the very least fluctuates within a given MH-S population. 82 Round 1 Round 2 Round 3 Figure 18. Isolation of CR3-negative subpopulations of MH-S cells. MH-S cells were exposed to a cocktail of rat anti-mouse CR3 monoclonal antibodies and MH-S cells which bound anti-CR3 monoclonal antibodies were magnetized by exposing the cells to iron beads coated with sheep anti-rat immunoglobulins. CR3-expressing cells were then removed from the cell suspension using a magnetic apparatus. The resulting CR3-negative MH-S cells were then cultured for 24-48 hours and representatives of this population were again subjected to the immunoselectiOn procedure. The percentage of CR3-negative [CR3(-)] and CR3-positive [CR3(+)] cells is reported for three successive rounds of immunoselection. 83 C H A P T E R 6 DISCUSSION: Interaction of M T B with M H - S Cells Compared to Primary Macrophages The binding properties of MH-S cells, an immortalized alveolar macrophage cell line, were compared with primary murine macrophages to determine whether the MH-S cell was a suitable model for investigating macrophage-MTB interactions. The ultimate objective of these experiments was to use CR3-negative and CR3-positive populations of MH-S cells as a means to dissect the role of CR3 in mediating the binding of M T B with macrophages. Using an immortalized cell line as a means to investigate the contribution of CR3 in mediating the binding of M T B by macrophages was also seen to be advantageous from a cost perspective as well as reducing the use of animals for experimental purposes. Characterizing the non-opsonic binding of M T B by MH-S cells demonstrated that the association of M T B with MH-S was similar to that seen with A M . In the absence of serum, the distribution in the binding of M T B by MH-S was also similar to A M , in that less than 1% of the cells in the monolayer bound greater than 10 bacteria (Figure 14). Neither MH-S nor A M bound M T B as well as P M (Figure 12). It has been shown previously that the binding of M T B either opsonically or non-opsonically to P M is significantly greater than that seen with A M [18]. P M and A M were used in these experiments as a means to compare the binding properties of MH-S cells to other macrophage models and as examples of how M T B binding differs among different macrophage phenotypes. 84 Particle binding experiments demonstrated that MH-S cells behave similarly to A M with respect to EIgG and E l g M C ' binding (Figure 13). However, one striking difference in the binding properties of MH-S cells compared to A M was the reduced ability to bind zymosan by MH-S. Compared to A M the binding of zymosan by MH-S was reduced approximately 6-fold, with association indices of 2334 + 174 and 392 + 11, respectively (Figure 13). Recently, CR3 has been reported to be an important receptor for unopsonized zymosan and the binding of zymosan by CR3 is believed to occur at the CR3 glycan binding site [28]. However, several observations suggest that the majority of zymosan binding by macrophages occurs independently of CR3. First, flow cytometry data indicate that A M and MH-S have similar CR3 expression [42] but A M associated with zymosan much more efficiently than MH-S (Figure 14). Second, E l g M C association assays demonstrated that CR3 function on A M was poor but this did not correlate with poor zymosan binding (Figure 14). Third, P M associated with zymosan less efficiently than A M but CR3 function, measured here by E l g M C binding (Figure 14), and CR3 expression, measured by flow cytometric analysis and reported previously [12,18], indicates that CR3 expression and function is greater for P M than A M . The observations made here support the claim that the binding of zymosan by A M reflects the role of lectin-like receptors, other than CR3, on macrophages in the normal immuno-surveillance of lungs in vivo [18]. The binding of fungal pathogens by A M has been documented [73,74] and presumably the mechanism for binding fungal pathogens is common to zymosan binding. A possible explanation for the inferior binding of zymosan 85 by MH-S cells is that due to continuous culturing in vitro the MH-S cell line has lost the physiological response to stimuli that drive the expression of receptors for zymosan. Investigating the association of M T B with MH-S cells under opsonic conditions demonstrated that, similar to P M and A M , the enhanced binding of M T B by MH-S cells was due to complement activation since mild heat treatment of serum abrogated the enhanced binding (Figure 16). However, contrary to observations with P M and A M , MH-S cells did not respond uniformly to the addition of serum. Over several separate experiments the percentage of MH-S cells that responded to the addition of serum with increased M T B binding ranged from 10-25% (Figure 15 and 16). It is possible that these serum-responsive cells represent a CR3-positive subpopulation of MH-S. This claim would be consistent with the original report on the phenotypic characteristics of MH-S, where the authors report that approximately 10% of the MH-S parental strain are Mac-1 positive [42]. Indeed, previous studies have identified the quantitatively important role of CR3 in mediating MTB-macrophage interactions [12,13]. Therefore, I considered the possibility that a subpopulation of CR3-expressing MH-S cells was responsible for the increased binding of M T B . In binding assays using two different anti-CR3 monoclonal antibodies, Ifound that M T B binding by MH-S cells was blocked significantly (P < 0.02 for either antibody). An approximate 2-fold reduction in the percentage of MH-S cells associated with M T B was observed in the presence of either Ml /70 or 5C6 (Figure 17). Presumably the inhibition caused by 5C6, the non-iC3b site binding monoclonal antibody, was due to steric effects on CR3 whereas Ml /70 inhibition was due to competitive inhibition with the iC3b-binding site on CR3. 86 Having demonstrated that anti-CR3 monoclonal antibodies could dramatically reduce the binding of M T B by MH-S cells, I was interested in characterizing further the role of CR3 in mediating the binding of M T B . Specifically, I wanted to isolate CR3-negative and CR3-positive populations in the MH-S cell line and compare the binding properties of these two different MH-S subpopulations. However, after several rounds of immunoselection and numerous attempts I was unable to isolate stable CR3-negative populations of MH-S cells. In fact, contrary to previous reports [42] the observations made here indicate that CR3 was transiently expressed on MH-S cells as successive rounds of immunoselection failed to enrich the cell population for CR3 negative cells (Figure 17). Based on the latter observation, I did not pursue using the MH-S cell line as a model to assess the role of CR3 in mediating the interaction of M T B with macrophages. 87 C H A P T E R I C O N C L U S I O N Collectively, the evidence presented here indicates that CR3 is not essential for the binding of M T B either opsonically or non-opsonically by murine macrophages in vitro. Under opsonic conditions, CR3 was required for the efficient uptake of M T B by macrophages. However, the absence of CR3 did not alter the intracellular viability of M T B that had been internalized under serum-free conditions. Although the experiments performed here did not examine the role of CR3 in vivo, given the numerous functions of CR3, including the recruitment of monocytes and neutrophils to sites of inflammation and the observation that CR3 is required for the efficient uptake of M T B in the presence of serum, it is possible that CR3 plays an important role in pathogenesis of tuberculosis. Evidence for the latter would require performing in vivo experiments comparing the pathological hallmarks of M T B in CDI lb-/- and CDI lb+/+ animal models. Future experiments using the CDI lb-/- mouse model should facilitate a fuller understanding of the role of CR3 in the pathogenesis of tuberculosis. In addition, experiments using animal models with deficiencies in other receptors should clarify whether alternate receptors are required for the association of M T B with macrophages and whether or not other receptors are exploited by M T B as a means to avoid macrophage microbicidal mechanisms. The work with the MH-S cell line did not rule out the potential of MH-S cells to be used as a model for investigating M T B pathogenesis. However, based on the observations presented here, caution should be exercised when using MH-S cells to characterize the receptor-ligand interactions between M T B and macrophages. 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Unbound serum antigens were washed three times with wash buffer (PBS + 0.05% Tween 20) and then blocked with 1% milk powder in wash buffer for 30 minutes at room temperature. Alkaline-phosphatase conjugated goat anti-mouse IgG secondary was then added (1/1000 with wash buffer) to each well and incubated for 2 hours at room temperature. After this incubation step, wells were washed 3 times with wash buffer and alkaline-phosphatase substrate para-Nitrophenyl phosphate (Sigma Product No. 9389) was added. Absorbance (A405nm) readings were then made each 30 minutes up to 1.5 hours. The A405nm readings reported above are representative of the averages of duplicate wells from one ELISA. 97 APPENDIX II: Sample Calculation of MTB Population Doubling Time where N t = number of bacteria at time = t N 0 = number of bacteria at time = 0 k = growth rate constant t = time To find k, use Eq. l and values for N(7) and No Sample calculation for 10CD1 lb+/+ M T B data: 2.74e6 = 9.2e4ek(7) k = 0.4848 day 1 To calculate for t; one population doubling implies Nt = 2No and therefore substituting 2No for Nt gives equation 2. Eq.2 2No = Noekt Also have 2 = e k t and therefore, t = ln2 / k The doubling time for M T B from 10CD1 lb+/+ is calculated as follows: t = ln2/0.4848 day-1 = 1.4296 days or 34.3 hours Similarly, it is possible to calculate the population doubling times for M T B from the 10CD1 lb-/- and 20CD1 lb-/- monolayers. The calculated population doubling times for M T B from lOCDl lb - / - and 20CDllb- / - monolayers were 37.8 hours and 34.2 hours, respectively. Eq. 1 N t = N0e' 98 

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