UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Absence of complement receptor 3 results in reduced binding and ingestion of Mycobacterium tuberculosis… Rooyakkers, Amanda Wilhelmina Johanna 2004

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_2004-0613.pdf [ 4.1MB ]
JSON: 831-1.0091684.json
JSON-LD: 831-1.0091684-ld.json
RDF/XML (Pretty): 831-1.0091684-rdf.xml
RDF/JSON: 831-1.0091684-rdf.json
Turtle: 831-1.0091684-turtle.txt
N-Triples: 831-1.0091684-rdf-ntriples.txt
Original Record: 831-1.0091684-source.json
Full Text

Full Text

ABSENCE OF COMPLEMENT RECEPTOR 3 RESULTS IN REDUCED BINDING AND INGESTION OF MYCOBACTERIUM  TUBERCULOSIS  BUT HAS NO  SIGNIFICANT EFFECT ON THE INDUCTION OF REACTIVE OXYGEN AND NITROGEN INTERMEDIATES OR ON THE SURVIVAL OF THE BACTERIA IN RESIDENT AND INTERFERON-GAMMA ACTIVATED MACROPHAGES by AMANDA WILHELMINA JOHANNA ROOYAKKERS B.Sc, The University of British Columbia, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine Program; Department of Medicine) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 2004 © Amanda Wilhelmina Johanna Rooyakkers, 2004  JUBCL FACULTY OF GRADUATE STUDIES  THE UNIVERSITY OF BRITISH COLUMBIA  Library Authorization  3  1  In p r e s e n t i n g this t h e s i s in partial fulfillment of the r e q u i r e m e n t s for a n a d v a n c e d d e g r e e at the University o f British C o l u m b i a , I a g r e e that t h e Library shall m a k e it freely a v a i l a b l e for r e f e r e n c e a n d study. I further a g r e e that p e r m i s s i o n for e x t e n s i v e c o p y i n g of this t h e s i s for s c h o l a r l y p u r p o s e s m a y b e g r a n t e d by the h e a d of my d e p a r t m e n t or b y his o r h e r r e p r e s e n t a t i v e s . It is u n d e r s t o o d that c o p y i n g or publication of this t h e s i s for f i n a n c i a l g a i n s h a l l not b e a l l o w e d without m y written p e r m i s s i o n .  N a m e of A u t h o r (please print)  D a t e (dd/mm/yyyy)  Title of T h e s i s :  I n y r K a n of />i/ce>ba.cter^urn tub-erculesfs But H A S no £\^y\\-^'ca.nt &$ect on -Hie )r\(LucMcri of Qeac^e. Oxu^eri ar\A vl'tWo^en Inter medifl-fou or on jhc ^urvivTcl -f B>Adrcv^ )V| Resident a.v\6 \n+erjcfon - y m » y i < i ^cri'vAt-ed YV\a.cr6pha.^ej c  Degree:  Department of The  mCksA&f V V l M l C i V\  o\  £dey\C^  Year:  SIO0H-  £  University of British C o l u m b i a  Vancouver, B C  Canada  grad.ubc.ca/forms/?formlD=THS  p a g e 1 of 1  last updated: 20-Jul-04  -the  ABSTRACT  The interaction of host macrophage (M<> | ) and Mycobacterium tuberculosis (Mtb) is mediated by cell surface receptors and is important in establishing intracellular infection. M§s are part of the mammalian defenses against microbial infection and can kill invading organisms via reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Using a Complement Receptor 3 (CR3) knockout mouse model I have examined whether the presence of CR3 affected the binding and uptake of Mtb by resident and activated M(|), the survival of the ingested bacteria and the induction of ROI and RNI during this interaction. I have shown that, although CR3 plays a definitive role in the uptake of Mtb, the receptor played no role in the subsequent survival of the bacteria. Thefindingheld true for resident M<|)s in the absence and presence of serum opsonins and also in Interferon-gamma (IFN-y) activated M<|>s. Activation of M<|) populations with IFN-y significantly inhibited the growth of Mtb in host M<|)s and enhanced the respiratory burst and production of nitric oxide (NO). However, the presence of CR3 was not found to be critical in any of these mechanisms. Furthermore, I demonstrated that the control of intracellular growth of Mtb in IFN-y activated M(|)s was not mediated by a direct effect of NO.  ii  T A B L E OF CONTENTS  Abstract  ii  List of Figures  •  List of Tables  •  Abbreviations  •  Acknowledgments  vi vi vii viii  c  CHAPTER 1.  INTRODUCTION  1  1.1 Mycobacterium tuberculosis  1  1.2 Mycobacteria-Host Cell Interaction  1  1.3 Complement Receptor 3  2  1.4 Macrophage Defense Mechanisms  :  1.5 Project Objective  4 :  5  CHAPTER 2.  MATERIALS & METHODS  7  2.1 Mycobacteria  7  2.2 Mice  •  2.3 Murine Genotyping 2.3.1 DNA Extraction 2.3.2 PCR Genotyping 2.3.3 Agarose Gel Electrophoresis 2.4 Bone Marrow-Derived Macrophages 2.4.1 L-conditionedMedia  iii  8 9 10 11 ..12 12 ....13  2.5 In Vitro Growth of Mycobacterium tuberculosis in  Bone Marrow-Derived Macrophages  13  2.6 Nitric Oxide Production from Bone Marrow-Derived Macrophages  15  2.7 Superoxide Production from Bone Marrow-Derived Macrophages  16  2.8 Intracellular Mycobacterium tuberculosis Growth in the Presence of Reactive Nitrogen and Oxygen Intermediate Inhibitors ;  18  2.9 Statistics  18  CHAPTER 3. RESULTS 3.1 Growth oi Mycobacterium tuberculosis in Resident Macrophages  19  3.2 The Role Of Serum Opsonins in the Growth Of Mycobacterium tuberculosis in Resident Macrophages  23  3.3 The Role Of Complement Receptor 3 in ImmuneActivated Macrophages  25  3.4 Complement Receptor 3 Does Not Contribute To The Respiratory Burst Of Macrophages  29  3.5 Nitric Oxide ProductionfromResident and ImmuneActivated Macrophages  32  3.6 Nitric Oxide Does Not Play a Role in the Growth Inhibition of Mycobacterium tuberculosis by  Interferon-y Activated Macrophages 3.7 The Role Of The Respiratory Burst in Macrophage Control of Mycobacterial Growth  35 36  CHAPTER 4. DISCUSSION  39  iv  C H A P T E R  5.  CONCLUSION  57  R E F E R E N C E S  5  v  9  LIST OF FIGURES  Figure 1. Non-Opsonic Growth of Mtb in Resident CR3 WT and KO Mfys  20  Figure 2. The Effect of Serum Opsonization on the Growth of Mtb in Resident M(j)s  24  Figure 3. The Role of CR3 in the M(|) Respiratory Burst  30  LIST OF TABLES  Table 1. Effect of IFN-y Immune Activation and the Addition of LPS on the Growth of Mtb in BMM4> in the Absence and Presence of CR3 Table 2. NO Production From WT and KO M<|) Monolayers During Intracellular Replication of Mtb Table 3. Effect of the NO Inhibitor L-NMMA on the Growth Of Mtb in Resident and IFN-y Activated M<|)s Relative to the Presence of CR3  vi  26 33 37  LIST OF ABBREVIATIONS AG AIDS ATCC BMM<t> BSA CAT CFU CR3 DMSO DPI DT ES FCS HIV IFN-y IL iNOS katG KO L-NMMA LPS M-CSF M(|) MOI NAC NADPH NBT NMS NO PBS-Tween PCR PMA RNI ROI SEM SOD TAE TB WHO WT ZYM  Aminoguanidine Acquired immune deficiency syndrome American type culture collection Bone marrow-derived macrophages Bovine serum albumin Catalase Colony-forming units Complement receptor 3 Dimethylsulphoxide Diphenyleneiodonium chloride Doubling time Embryo-derived stem cells Fetal calf serum Human immunodeficiency virus Interferon gamma Interleukin Inducible nitric oxide synthase Mycobacterium tuberculosis catalase-peroxidase gene Knockout (complement receptor 3 deficient) N -monomethyl-L-arginine Lipopolysaccharide Macrophage colony stimulating factor Macrophage Multiplicity of infection N-acetyl L-cysteine Nicotinamide adenine dinucleotide phosphate Nitro-blue tetrazolium Normal mouse serum Nitric oxide Phosphate-buffered saline plus 0.1% Tween-80 Polymerase chain reaction Phorbol 12-myristate 13-acetate Reactive nitrogen intermediates Reactive oxygen intermediates Standard error of the mean Superoxide dismutase Tris-acetate-EDTA Tuberculosis World Health Organization Wild-type (complement receptor 3 sufficient) Zymosan G  vii  ACKNOWLEGEMENTS I would like to thank my family for being a source of support and encouragement, examination committee members Drs. Yossef Av-Gay, Dana Devine and Alice Mui, and my supervisor, Dr. Richard W. Stokes, for welcoming me to his laboratory and allowing me to have a wonderful learning experience as a graduate student. This work was supported by a British Columbia Lung Association grant, an equipment grantfromthe TB Veterans Association of British Columbia and the Network Centres of Excellence (Canadian Bacterial Diseases Network).  viii  C H A P T E R 1. INTRODUCTION  1.1 Mycobacterium tuberculosis  Tuberculosis (TB), once considered a disease of the past, is growing at such a rate that in 1993 the World Health Organization (WHO) declared the epidemic a global emergency (1). TB is responsible for annual deaths amounting to more than 2 million people and is the number one cause of death due to an infectious bacterium. About 2 billion people, one-third of the world's total population, is currently infected and a person becomes newly infected with TB every second of every day. The resurgence of TB is due to a multitude of factors including HIV/AIDS, international travel and immigration via countries endemic with TB, increases in poverty and homelessness, the emergence of drug and multi drug-resistant TB and poor adherence to TB drug treatment therapies. It has been estimated that if the spread of TB is not brought under control, between the years of 2002 and 2020, "approximately 100 million people will be newly infected, over 150 million people will get sick, and 36 million will die of TB" (1). Mycobacterium tuberculosis (Mtb), the causative agent of disease, is an airborne disease spread through the air, like the common cold, via droplets (i.e. coughing, sneezing) from a person with active disease. It is estimated that a person sufferingfromactive TB will infect between 10 and 15 people per year (2).  1.2 Mycobacteria-Host Cell Interaction  Once inside the human host, Mtb has evolved the ability to survive and grow inside the host macrophage (M<(>s). M(j>s are phagocytic cells of the immune system and are one of the most important subsets of immune cells involved in the elimination of pathogenic microbes.  1  Therefore, the interaction between host M<j)s and bacteria is important for the successful establishment of infection. The initial interaction of host M§ and tuberculosis bacilli is mediated by cell surface receptors on the outer membrane of the M«j), including the mannose receptor, the glucan receptor, toll-like receptors, surfactant protein receptors, scavenger receptors, and the complement receptors 1, 3 and 4 (3-9). Complement Receptor 3 (CR3) is of particular interest because it has been proposed as a major route of entry for Mtb into host cells both in the absence and presence of serum (10, 11).  1.3 Complement Receptor 3  CR3 (CDllb/CD18, Mac-1, a p2, Mo-1) is a heterodimeric member of the (32- integrin m  family and is composed of a CDllb alpha chain non-covalently linked to a CD 18 beta chain. The a chain of CR3 has a molecular mass of 165kDa while the P-chain is 95kDa (12). It is expressed on monocytes, neutrophils, M<|>s, natural killer cells, eosinophils and basophils (13, 14) and to a lesser extent in some populations of T- and B-cells (15). Functions attributed to CR3 include leukocyte adhesion, hematopoietic cell migration, chemotaxis, phagocytosis and induction of the oxidative burst (13, 16, 17).  CR3 possesses the key integrin family  characteristic in that it is capable of undergoing an activation event in response to intracellular signalling events ("inside-out" signalling) that can change its specificity for different ligands as well as its function in mediating adhesion, phagocytosis and extracellular toxicity (18-20). The multitude of functions attributed to CR3 are controlled by these "inside-out" and "outside-in" signaling pathways that regulate the exposure of CR3 binding sites and the receptor's association with the actin cytoskeleton of the cell (21).  CR3 binds a variety of ligands including the  complement component iC3b, ICAM-2, ICAM-1/CD54, factor X, haemagglutinin and  2  fibrinogen as well as polysaccharides such as zymosan (ZYM) and lipopolysaccharide (LPS) (22-28). The multi ligand-binding capability of CR3 allows the receptor to bind a diverse repertoire of pathogens including Bordetella pertussis, Ancylostoma caninum, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Salmonella spp. (25, 29-31).  Two distinct functional domains mediate the binding characteristics of CR3: the I- or A-domain, located in the a subunit, containing the site that mediates the binding and phagocytosis of iC3bcoated particles (32, 33) and the cation-independent P-glucan binding lectin site located Cterminal to the I-domain (34, 35) which is responsible for binding ZYM and polysaccharide moieties on the surface of bacteria under nonopsonic conditions. CR3 plays a role in phagocytosis of opsonized bacteria in addition to phagocytosis via recognition of carbohydrate moieties on the surface of bacteria via two distinct mechanisms: type I phagocytosis under nonopsonic conditions and type II under opsonic conditions (36). In addition to being able to change its ligand binding capabilities CR3 can also be down- or upregulated on cell surfaces by stimulation with certain activating agents, such as interferongamma (IFN-y) or phorbol 12-myristate 13-acetate (PMA), respectively (8,11,15, 37). Previous work has shown that, in both the absence and presence of serum opsonins, CR3 is a major route of entry for mycobacteria (8, 38, 39). Using a CD1 lb knockout mouse model it was shown that approximately 40-50% of bacilli binding to host M<|)s can be attributed to this receptor (10). Mtb can bind to CR3 in a serum and complement independent fashion via surface polysaccharides that bind directly to the CR3 p-glucan lectin site (40, 41). Differences in the polysaccharide composition between different Mtb strains play a role in the binding of Mtb to CR3 (42) and these differences may play a role in the pathogenicity of certain strains.  3  1.4 Macrophage Defense Mechanisms M<j)s are considered to be part of the mammalian defense system against microorganisms and, normally, resident Mfys are microbicidal when infected with any given microorganism. They act as effector cells capable of killing invading organisms via a number of different microbicidal mechanisms, of which reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) are considered to be among the most effective. Normally, internalization of a pathogen in a plasma membrane derived phagosome initiates intracellular signaling pathways that result in the activation of NADPH oxidase which, in turn, generates the superoxide anion (43) and other ROIs (hydrogen peroxide, hydroxyl radical and singlet oxygen). Nitric oxide (NO) is producedfromL-arginine by inducible nitric oxide synthase (iNOS), an enzyme that is also activated upon infection. In addition to their individual toxicities, Superoxide can also combine with NO to produce peroxynitrite, a very strong oxidant, which can inhibit the growth of pathogenic bacteria.  Under certain conditions, mycobacteria are  susceptible to both ROI (44) and RNI (45), but possess mechanisms that enable them to withstand M<> | defenses and survive and replicate within the M<|) (46). The control of Mtb growth in vivo is mediated by Thl-immunity that consists of CD4 and CD8 T cells. These cells secrete Thl-type cytokines, including IFN-y and IL-12, that upregulate M<j> function and enhance the ability of these cells to control mycobacterial growth (47, 48). It is thought that M<> | s activated by these cytokines are able to control the intracellular replication of mycobacteria through the production of effector molecules such as ROI and RNI. The involvement of NO in the control of Mtb is supported by the finding that iNOS knockout mice are unable to control the growth of Mtb and succumb to infection much earlier than WT mice (49, 50). Mice treated with iNOS inhibitors such as N-monomethyl-L-arginine  4  (L-NMMA) or aminoguanidine (AG) are also unable to control mycobacterial growth when infected with Mtb and die much sooner than untreated mice (51, 52). Furthermore, NO plays an important role in the killing of mycobacteria by human alveolar Mtys and inhibition of the NO response to infection results in a reduced ability of the M<)| to control bacterial replication (53). NO has been shown to be both bactericidal and bacteriostatic to mycobacteria and other intracellular pathogens (54-56). CR3 has been indicated to play a direct role in the production of NOfromM<)>s (57, 58) and microglia (59), as well as in response to infection with pathogenic bacteria (60). ROI are also believed to play a role in the survival of mycobacteria in vivo, even though Mtb are relatively resistant to killing by ROIs in vitro (61). When infected with Mtb, phox knockout mice, which lack the phagocyte oxidase gene essential for superoxide production from NADPH oxidase, display enhanced bacterial growth in the lungs (62). However, this increased susceptibility is only transient and diminishes with the onset of acquired immunity (63). Ng et al determined that it is the presence of a catalase-peroxidase (katG) in Mtb that protects the bacteria from the deleterious effects of the respiratory burst (46) suggesting that although ROI may contribute to the control of mycobacterial growth, it is the other M<|) defense mechanisms, such as RNI, that determine the outcome of mycobacterial infection. The observation that phagocytosis mediated by CR3 alone does not generate an oxidative burst (64-66) has resulted in the suggestion that CR3 allows pathogenic bacteria to enter the M<j> without an accompanying respiratory burst and can act as a "stealth receptor", allowing for the successful establishment of infection.  1.5 Project Objective  5  To further elucidate the role that CR3 may have in the pathogenesis of Mtb this project was designed to examine the role that CR3 plays in the intracellular survival of Mtb and whether or not this receptor is critically involved in the production of ROI and RNI, two of the microbicidal mechanisms that M<j>s use to control intracellular growth.  6  CHAPTER 2. MATERIALS & METHODS 2.1 Mycobacteria Mycobacterium tuberculosis  strain Erdman (Trudeau Mycobacterial Collection (TMC)  #107; American Type Culture Collection (ATCC) # 35811) was grown to late-log phase in modified Proskaur and Beck medium (per litre - KH2PO4 5.0g, asparagine 5.0g, MgS04-7H_0 0.6g, magnesium citrate l.Og, ferric ammonium citrate 0.2g, glucose 2.0g, lOOmM sodium pyruvate 5.0ml, glycerol 20ml and 10ml of 10% Tween 80 - adjusted to pH 7.4 with NaOH or KOH) in a static culture with intermittent shaking every two-three days over a two-week period. Batch cultures were divided into 1ml aliquots and stored at -70°C for the remainder of the experiments.  Viability for each batch was determined from the frozen stock; twelve  representative vials were thawed, pelleted for 5 minutes at 14900xg and 0.5ml of supernatant were removed. The vials were re-spun and the remaining 0.5ml of media was removed. The pellet was resuspended in 1.0ml of Phagocytosis Medium (138mM NaCl, 8.1mM Na2HP04, 1.5mM  KH2PO4,  2.7mM KC1, 0.6mM CaCl , LOmM MgC 1 and 5.5mM D-glucose) and probe2  2  sonicated for a ten-second burst using a VC50T 50 watt ultrasonic processor and 6mm probe (Sonics & Materials; Danbury, CT) to disperse any mycobacterial clumps.  For each  corresponding vial, the suspension was serially diluted (10" through 10") into phosphate1  7  buffered saline solution plus 0.1% Tween 80 (PBS-Tween) and plated onto Middlebrook 7H10 (DIFCO; Oakville, ON) agar plates. Plates were incubated at 37°C for 18-21 days after which colony-forming units (CFU) were enumerated. The ratio of bacteria to M<|) present during infection (bacteria:M<j)), termed the multiplicity of infection (MOI), was calculated for all experiments based on the average CFU/ml count for the twelve representative vials.  7  2.2 Mice Both complement receptor 3 expressing (WT) and complement receptor 3 deficient (KO) mice were used for this project. Two distinct sources were used to provide the mice needed for experiments. Early experiments were performed using mice sourced through a collaboration with Glaxo-Wellcome UK. Mice derivedfromthis source are herein referred to as Glaxo mice. Glaxo knockout mice are of C57BL/6 background and are deficient in the CD1 lb protein portion of the CR3 heterodimer, and therefore do not express functional CR3 on cell surfaces. The CD1 lb gene was disrupted using homologous recombination in embryo-derived stem (ES) cells (10). In brief, a portion of CD1 lb exons 13 and 14 were eliminated by introducing a neomycin resistance gene that included a translational stop signal. This insertion truncates CD1 lb at the Cterminal/transmembrane end of the protein and therefore prevents insertion and expression of the molecule at the cell surface. HM-1 ES cells were transfected with a targeting vector and resistant clones were screened using Southern Hybridization. Clones with the CD lib gene knocked out were injected into C57BL/6J blastocysts and were subsequently injected into foster mothers, creating CDllb null offspring. Male null offspring were mated with C57BL/6J wildtype females, producing heterozygote progeny later used to produce offspring of +/+, +/- and -/genotypes. Six Glaxo CD1 lb heterozygous (CD1 lb +/-) breeding pairs were obtained and bred to obtain both CD1 lb+/+ and CD1 lb-/- mice. Midway through the project, due to difficulties maintaining a breeding colony and the inability to directly purchase the mice needed, the mouse source was changed to Jackson Laboratories (Bar Harbor, ME). Homozygous breeding pairs (+/+ and -/-) were purchased but we were unable to continue breeding these pairs in house and both WT and KO mice were  8  subsequently purchased directly from Jackson Laboratories for the remainder of the project. KO mice (strain B6.l29S4-Itgam'  , stock number 003991, deposited by donating investigator  mIMyd  Tanya N. Mayadas) have a targeted mutation in the CDllb gene, also called integrin alpha M (itgam), located on chromosome 7, cytoband F4 and are herein referred to as Itgam mice (16). Briefly, a targeting vector was used to replace a portion of the Itgam gene exon that encodes the translational initiation codon and 15 amino acids of the signal peptide. A neomycin resistant construct was electroporated into 129S4/SvJae-derived ES cells, which were subsequently injected into C57BL/6 blastocysts producing chimeric animals. These B6:129S4 chimeras were backcrossed to C57BL/6 for a rninimum of ten generations resulting in an Itgam KO on a C57BL/6 background. Itgam KO mice do not express the CDllb protein portion of the CR3 heterodimer on cell surfaces and are therefore functionally CR3 deficient. WT mice (strain C57BL/6J, stock number 000664) were purchased at the same time as the KO mice and were age- and sex-matched for each set of experiments. All mice used for experiments were maintained in a specific pathogen-free animal housing facility following Canadian Committee on Animal Care Guidelines.  2.3 Murine Genotyping  Glaxo mice were genotyped at the age of 4-6 weeks to determine the CDllb gene profiles of the offspring of the heterozygous breeding partners. Itgam mice ordered from Jackson Laboratories are genetically identified at source.  9  2.3.1 DNA Extraction DNA samples were obtained from skin samples of the ear punch used to visually identify the offspring. To extract the DNA, the ear punch sample was placed in 150ul of digestion buffer (lOOmM NaCl, lOmM Tris-HCl (pH8), 25mM EDTA (pH8), 0.5% SDS, O.lmg.ml proteinase K) in a 1.5ml sterile Eppendorf tube and incubated overnight (approx 18 hours) at 55°C with constant tube rotation.  The resulting digest was centrifuged at 14900xg to remove any  undigested pieces and/or hair and the clear supernatant was transferred to a new sterile tube. Fifteen jul (1/10 the volume of the cleared supernatant) of 3.0M potassium acetate was added to the tube and mixed. This mix was then thoroughly extracted using an equal total volume (165 pi) of phenol/chloroform/isoamyl alcohol and spun at 14900xg for 10 min in a microfuge. The top (aqueous) layer was transferred to a new sterile tube and Vz volume (approximately 70ul) of isopropanol was added. The aqueous/isopropanol solution was left to stand for 0.5-1.0 hours at room temperature (or 15 min at -20°C) at which time the tube was centrifuged at 14900xg for 5 minutes to pellet out the DNA. The supernatant was removed, the pellet washed with 70% ethanol, centrifuged again under the same conditions followed by careful removal of the ethanol using a pipette. The tube was covered with parafilm punched with small holes and the clean DNA pellet was allowed to air dry at room temperature. This DNA pellet was then resuspended in 20uL of lOmM Tris-HCl pH 8.0 and the yield and purity of the DNA was assessed using a spectrophotometer  (A260/280  comparison).  10  2.3.2 PCR Genotyping DNA samples from the Glaxo mice were analyzed using Polymerase Chain Reaction (PCR) to amplify the regions of the genome that correspond to both the CDllb WT and KO genotypes. The PCR primers used were as follows: Primer 856  5' - TAT CTT CTA GTG ATT TCC CCA GTA - 3'  Primer 2506 5' - CCC ACC CCT TCC CAG CCT CGA GC - 3' Primer 2924 5' - TGT AGA CAG CGC CCT GAT TCT CCT - 3' Sterile, Dnase/RNase-free 0.5ml ultra-thin walled PCR tubes were used for all PCR reactions. Two ul of ear punch-extracted DNA was aliquotted into the tube followed by 5ul of a primer mix (primer mix was an equimolar combination of all three CD1 lb-specific primers; 856, 2924 and 2506 which, as aliquotted, provided a final amount of 25 pmol of each primer per PCR reaction). Thirty ul of a PCR master mix was then added to each tube. The master mix contained 25ul lOOuM dNTPs, 13ul 50mM MgCl , 52ul lOx GIBCO PCR buffer (Invitrogen; 2  Burlington, ON), 215ul sterile distilled/deionized (ddH_0) water and 5ul of GIBCO Taq Polymerase (master mix prepared for batches of tubes in multiples of ten).  PCR controls were  performed by repeating the same conditions, replacing the DNA with sterile ddH_0. PCR was performed using a Bio-Rad Gene Cycler (Bio-Rad; Mississiauga, ON) under the following conditions: a denaturation step of 5 minutes at 94°C, followed by 35 cycles of [1 minute at 94°C, 1 minute at 60°C, 1 minute at 72°C], finished with 5 minutes at 72°C. Reaction tubes were removedfromthe thermal cycler immediately upon reaction completion and were stored at 4°C until examination.  11  2.3.3 Agarose Gel Electrophoresis  PCR products were analyzed using 1.5% agarose gels (0.75g agarose in 50ml TAE). Fifteen ul of the finished PCR reaction was loaded onto the gel and electrophoresed for 60-90 minutes at 80V. Bands were visualized by staining the gel with ethidium bromide followed by UV illumination and photographic record using a Bio-Rad gel documentation (GelDoc) system. Known standards (100 base-pair ladder; Invitrogen) were loaded onto the gel along with the PCR samples so that the size of the PCR products could be determined. Expected size products are 600 bp for the WT genotype and 400 bp for the CD1 lb KO genotype.  2.4 Bone Marrow-Derived Macrophages  Bone marrow-derived macrophages (BMM(|)) were used for all experiments and were isolated from mice using the following procedure. Mice were sacrificed by anaesthetization followed by cervical dislocation. Using aseptic technique, the femur, tibia and humerus were removed, cleaned and surface sterilized with 70% ethanol, both ends cutfromthe bones and using a 25-gauge needle, the bone marrow was flushed into a petri-dish using RPMI 1640 medium plus 10% L-conditioned media (see below), 10% heat-inactivated Fetal Bovine Serum (GIBCO Burlington, ON), lOmM L-glutamine (GIBCO) and lOmM sodium pyruvate (GIBCO) (Supplemented RPMI). Bone marrow washes were pooled and dispersed by repeated passage through an 18-gauge needle. The resulting suspension was pelleted at lOOOxg for 5 minutes and resuspended in 5 ml 0.17M ammonium chloride (pH 7.2) for 5 minutes to lyse red blood cells. The cells were pelleted again, resuspended in 25 ml of supplemented RPMI, transferred to a 150 cm tissue culture flask and incubated for three hours at 37°C, 5% CO2 to deplete non-stem cells. 2  12  After 3 hours the non-adherent stem cells were removed, counted and plated on glass coverslips in 24-well tissue culture plates at a concentration of 2.5 x 10 cells in 1ml of supplemented 5  RPMI. Cells were incubated for 7 days' at 37°C in 5% C0 adding an additional 0.5ml of 2  supplemented media to each well on Day 5. Cells were used after 7 days of differentiation in vitro.  2.4.1 L-conditioned Media  As a source of M<(>-colony stimulating factor (M-CSF), L929 cells (ATCC #CCL-1) were grown in cRPMI (RPMI 1640 medium containing 10% heat-treated FCS, lOmM L-glutamine and lOmM sodium pyruvate). Cells at 5xl0 per ml in a total volume of 125ml were seeded into 3  a 150 cm flaskand incubated at 37°C, 5% CC_. On Day 7, or when the monolayer becomes 2  confluent, whichever was later, the supernatant was collected and filtered through a 0.22 uM filter and stored at -20°C until use.  2.5 In Vitro Growth of Mycobacterium tuberculosis in Bone Marrow-Derived Macrophages  The growth of Mtb was followed over 7 days in BMM<(> from WT and KO mice with or without prior activation with IFN-y. Following the maturation of BMM<|> (above) the overlay of supplemented RPMI was replaced with 0.5 ml cRPMI. Immune-activated M<j>s were prepared by the addition of 250 units/ml of murine recombinant IFN-y (Research Diagnostics Inc; Flanders, NJ). IFN-y was stored at -70°C as a stock solution at 2500U/uL in 0.5x PBS pH 8.0 plus lmg/ml bovine serum albumin (BSA) (Roche Diagnostics; Laval, QC), a carrier protein used to stabilize IFN-y and prevent absorption to vial walls. Resident BMMcj) were therefore maintained by changing the supplemented media to cRPMI and adding BSA at the same concentration as  13  that added with IFN-y. BMM<|) were left for 16 hours at 37°C, 5% G0 before infection with 2  Mtb. Prior to adding to BMM(|>, Mtb was pelleted and resuspended in 1.0ml of Phagocytosis Medium and clumps were dispersed by syringing the suspension up and down 10 times using a 25-gauge needle and 1 ml syringe.  The Mtb suspension was diluted to the appropriate  concentration using Phagocytosis Medium. As needed, LPS (E.coli serotype 026:B6. SIGMA; St Louis, MO), at a final concentration of 4 pg/ml, was added to the monolayer immediately prior to the addition of Mtb. Mtb was then added to the BMM<j) monolayers in 100 uL aliquots at the concentration needed to obtain each reported multiplicity of infection (MOI). Following addition of the mycobacteria, the monolayers were incubated at 37°C, 5% CO2 on a Clay Adams Brand nutating platform (Becton Dickinson; Sparks, MD) for 1 hour followed by stationary incubation for 2 hours. After the 3-hour infection, the supernatant was aspirated off and unbound mycobacteria were removed by washing the monolayers twice with 0.5 ml Phagocytosis Medium. Three replicate coverslips were transferred to 14 ml polystyrene tubes containing 2 ml of PBS-Tween for processing. Remaining coverslips were transferred to new tissue-culture plates containing 1 ml of fresh cRPMI, with or without supplemental IFN-y and LPS as appropriate, and re-incubated prior to processing on subsequent days. For processing infected BMM<j> monolayers, the coverslips were crushed in the tubes containing PBS-Tween, followed by probe sonication for 10 seconds to disrupt M<j>s and break up any clumps of mycobacteria. The mycobacterial suspension was then serially diluted in PBS-Tween and plated in duplicate on 7H10 quad plates. Plates were incubated at 37°C for 18-21 days after which CFU were enumerated.  14  Processing BMM(|) monolayers on Days 4 and 7 was essentially the same except that the overlay and the 2 washesfromthe monolayer were transferred to a 14ml polystyrene tube along with the corresponding coverslip. This ensured that the total contents of the well were assessed and not just the bacteria present in the remaining adherent cells of the monolayer. Preliminary studies have shown that, as infections progress, M<j>s detachfromthe monolayer but still contain viable intracellular bacteria (38). The coverslip was again broken, the contents of the tube probe sonicated for 10 seconds, serially diluted and plated in duplicate on 7H10 plates. CFU were again enumerated after 18-21 days. To calculate the rate at which mycobacteria grow in the monolayers, the doubling times (DT) for 7-day growth of Mtb in BMM(|> were calculated using the equation N = Noe where N kt  t  t  = number of bacteria at time t (7 days), No = number of bacteria at time 0 (after 3 hour infection), k = growth rate constant, t time in hours and e = natural logarithm.  2.6 Nitric Oxide Production from Bone Marrow-Derived Macrophages  The Griess Assay was used to measure nitrite (N02), a stable end product of NO -  production (67). Samples were takenfromthe same wells used to monitor Mtb growth in M(|>s. Control groups were comprised of either Phagocytosis Medium alone (negative control) or LPS alone (positive control). Following 1, 4 or 7 days after infection of BMM(|>, lOOul aliquots of supernatant were removedfromeach of the culture wells and transferred to a 96-well plate to which 50 ul of Griess Reagent 1 (1% (w/v) sulphanilamide dissolved in 2.5% phosphoric acid (H3PO4)) and 50 ul of Griess Reagent 2 (0.1% (w/v) naphthylethylenediamine dihydrochloride dissolved in 2.5% phosphoric acid (H3PO4)) were added sequentially and mixed. It is recommended that cell-free  15  supernatants be used for the Griess Assay, however preliminary experiments showed that removing cellsfromthe supernatant prior to addition of the Griess reagents had no effect on assay outcome, so the step was eliminated. Replicate wells containing fresh cRPMI were included to calculate background values for each plate. Standards were preparedfroma 2 mM stock of NaNC_ in cRPMI (0.014 g dissolved in 100 mlfreshlymade media) to give a range of 1 uM to 128 uM. After a 10-minute incubation at room temperature, the absorbance values of all samples were read using a microplate reader (Bio-Rad; model: Benchmark) with a measurement wavelength of 550 nm and a reference filter of 655 nm. The data was analyzed against a standard curve plotted in Excel using the trendline function and setting the y-intercept to the background value.  2.7 Superoxide Production from Bone Marrow-Derived Macrophages  The respiratory burstfromBMM<|) was measured using Nitro-Blue Tetrazolium (NBT), a formazan salt that is reduced in the presence of superoxide to form an insoluble blue deposit inside the M<)> (68). NBT (SIGMA) wasfreshlyprepared at a concentration of 1.25 mg/ml and pre-warmed to 37°C prior to use. BMM(|> monolayers were used following 16h incubation with or without IFNy (see above). The media was aspirated off, monolayers were washed 2 times with 1 ml of Phagocytosis Medium and 300 pi of NBT solution was added to the well followed by 100 ul of the trigger being tested; Mtb, PMA at 250 ng/ml, ZYM at an MOI of 50:1 or Phagocytosis Medium as a negative control. PMA is a member of the phorbol ester family and elicits a generalized respiratory burst when added to Mfys. ZYM is a yeast cell wall preparation known to bind to M<|) and induce a respiratory burst. LPS was also added to some experimental groups at a  16  concentration of 4 ug/ml. BMM<|) were incubated with the different agents for 1 hour nutating, 2 hours stationary. The supernatant solution was then aspirated off, the monolayers washed 2 times with 1 ml of Phagocytosis Medium, followed by 2 washes with 1 ml of methanol (to ensure removal of all unreduced NBT) and air drying.  The reduced NBT deposits were  solubilized by the addition of 120 ul/well of 2M KOH followed by 140 ul/well dimethyl sulphoxide (DMSO). After mixing, the resultant blue solution was quantitated at OD570nm using a Bio-Rad microplate reader. NBT background values were determined by incubating M<> | monolayers with the NBT solution but without a trigger. A standard curve was obtained by solubilizing 1 mg of powdered NBT in 1 ml of 1.2:1.4 KOH:DMSO, which reduced the powder to the blue color, and serially diluting the solution through to 0.98 ug/ml. Replicate wells of each standard solution were added to each 96-well plate at the time of reading. The data were again analyzed against a standard curve plotted in Excel using the trendline function and setting the y-intercept to the background value. Diphenyleneiodonium chloride (DPI) (SIGMA), an NADPH oxidase inhibitor, was used to determine the actual amount of NBT reduction due strictly to the metabolic products of the respiratory burst of the M(|>s (i.e. used to determine background). DPI was stored as a stock solution (10 mg/ml in DMSO) and prior to infection the DPI stock was diluted 1:100 in cRPMI and added to the wells. DPI-control wells were incubated with 10 uM DPI during the 3-hour infection and the NBT readings from these wells were subtracted from the corresponding experimental well to reflect only the DPI-inhibitable value of superoxide production for each condition.  17  2.8 Intracellular Mycobacterium tuberculosis Growth in the Presence of Reactive Nitrogen and Oxygen Intermediate Inhibitors  The in vitro growth of Mtb in M<> | monolayers was assessed in the presence of RNI inhibitors to determine the role that RNI play in the inhibition of Mtb growth. N -monomethylL-argiriine (L-NMMA) is an arginine analogue that inhibits the production of NO from arginine by binding to the active site of inducible nitric oxide synthase (iNOS) (69-71). Stock L-NMMA (SIGMA) was prepared at a concentration of 25-50 mg/ml in PBS and stored at -20°C until use. Following preparation of BMMij) monolayers (see above), L-NMMA was added to the wells, to a final concentration of 1 uM or 5 uM, immediately prior to addition of the mycobacteria as necessary. The presence of L-NMMA was maintained in tissue culture after initial infection and throughout complete experiments. To inhibit ROI, DPI (10 uM), N-acetyl L-cysteine (NAC) (SIGMA) (5 and 10 uM) or superoxide dismutase (SOD) (SIGMA) (150 U/ml) plus catalase (CAT) (Roche) (6000 U/ml) was added to the BMMcj) monolayer immediately prior to the addition of Mtb. The antioxidants remained in the wells for the entire 7 days of growth, blocking ROI-production accordingly. Coverslips plus supernatant were processed as described above.  2.9 Statistics  One or two-way ANOVA followed by Tukey's multiple comparison test was performed, as appropriate, using GraphPad PRISM statistical analysis software. considered significant with a P value < 0.05.  18  Differences were  CHAPTER 3. RESULTS 3.1 Growth of Mycobacterium tuberculosis in Resident Macrophages To determine whether CR3 plays a role in the in vitro growth of Mtb, resident BMM<|> from both Glaxo (Fig.lA) and Itgam (Fig.lB&C) background mice, were infected with Mtb at two different MOIs, which were arbitrarily designated High (50:1 bacteria:M<j)) (Fig.lA and B) and Low (20:1) (Fig 1C). Because previous work in our lab had indicated that 40-50% of binding of Mtb to resident M<|)s is due to CR3 (Melo 2000), I also infected KO M<|)s at double the MOI for both the High and Low MOIs to compensate for the deficiency in binding in the KO M<t>s. CFU/ml were enumerated immediately after a 3-hour infection (DAY 0) and subsequently at 4 and 7 days post-infection (In preliminary experiments CFU/ml were also enumerated 24 hours after infection (Day 1) for the High MOI groups, but results did not contribute any additional information so were not carried out for Low MOI and are not shown).  For the Glaxo Mice: Uptake of viable Mtb at Day 0, measured by CFUs, in the KO M<> | s infected at 50:1 was lower (3.82e ± 6.3e ) than the WT 5  3  MIIJS  (4.46e ± 1.2e) when infected at the same MOI, 5  4  however, this observation was not statistically significant (P>0.05) (Fig 1 A). Uptake of Mtb in KO M(|>s infected at 100:1 (6.07e + 2.6e ) was significantly higher (PO.001) than WT M<> |s 5  4  infected at 50:1 (4.46e ± 1.2e), indicating that doubling the MOI to try correct for the expected 5  4  40-50% binding deficiency overcompensates for the difference in binding due to the absence or presence of CR3. Uptake in KO M<> | s infected at 100:1 was significantly higher than uptake in KO 50:1 M<> | s (PO.001). At Day 4, KO 50:1 (4.30e ± 2.9e ) was significantly lower than WT 6  19  5  CFU/ml b  cn CD  +  9-  ro  Ol  O  b CD + Ol  b CD +  b CD + CO  b CD +  co b CD +  —>•  —1-  b CD + -vl  K o CD + >sj  - I  —>•  CD  CD  •sj  S~J  +  +  _ "n  § S3 ri- CT  _  i  - t o  a » ? Q o » cn  _- 3- 2.  co 3-  S. _  -»• -»•  CO CO CO CO O l Ji.  o  ->•->• o b) -si co  "sj CO 5 P 3 —* H- 1+ 1 +  >  CQ  >< o  HXXXXXX3  J—  tS3555533335]  >  X  O a>  o  s<  =? O  ro  0)  D o  o £ u> S o P P =f  O  -si  O  7s 7s >  OOq  3  Ji.  o  CD  cn D CO ssj  555335553333^X135555353-I  ro = S  i 58 s ° °_i o *  Ts ^  OOH O 5' fl) _ 01 CD  a>  o S5 S5 o PP  9-o a c 3  CD  <"> CD =5 X CD 3  3  *  Y//M  tz CT  O  5' o 0}  3  s<  J*.  CD  / / / / / / / / / / / / .  =r  O _  II  ca  «  D 0)  V)  ,  2 ^  > o  ////////////).  \/////M  Z Z Z Z Z Z Z Z Z Z Z 2 J H Z Z Z Z Z Z 3  00  5 _: \  V  © _  a) <  X CD O Q.  3  <  a. < C Q CD  D  £  oo:  j*. ro ro  o o o  n> a» ft3 w "  D o c cx  5'  (Q  ro ro ro js.Ji.cn co co i+ i+ i +  => s^ o wII  0)  7s 7s  Q) ^ 3  a) ? O a. O _  <D  "  cn  -'bin  >> S ^ cu 3 u  H  E Z Z Z Z Z Z Z Z Z Z Z Z  Q . -eCrt _ . w m 3 a.  S  Ts <?  O O q ^ w cn O O o  o  CQ  co ro co co C D p b co ro 1+ i+ 1+  »  a  S  o p  00 00 O l  3  o  > D  IH  CD s<  Ji.  CD CO  a  0) s< ssi  ^ ^ ^ ^ ^ ^ ^ ^ ^ ^  3  1  ? 3  »° 8  o  ft S" CD C L  20  50:1 (6.44e ± 2.5e ) (PO.001) and KO 100:1 (6.33e ± 1.4e) was no longer significantly higher 6  5  6  5  than WT 50:1, suggesting that the bacteria in the KO lvtys may not be growing as quickly as those in the WT M<|>s. By Day 7 the pattern of Mtb uptake remained, however there was no longer any statistical difference between CFUs from WT 50:1 and KO 50:1 or 100:1 (1.26e ± 7  6.8e , 1.03e ± 8.5e and 1.30e ± 5.5e , respectively). Overall, KO 50:1 was lower and KO 5  7  5  7  5  100:1 was the same or slightly higher than WT 50:1 over the week infection, with varying statistical significance. KO 50:1 was statistically lower (PO.001) than KO 100:1 at each time point sampled during infection.  For the Itgam Mice: Uptake of Mtb at DAY 0 in the KO 50:1fttyswas lower (1.81e ± 2.3e ) than the WT 5  4  50:1 M(j)s (1.94e ± 2.0e ), however, like that seen in Glaxo BMM(|), this observation was not 5  4  statistically significant (P>0.05) (Fig IB). Similarly, uptake of Mtb in KO Mcfjs infected at 20:1 (1.96e ± 1.7e) was lower than WT M<> | s infected at 20:1 (2.33e ± 1.6e), but again not 4  3  4  3  significantly (P>0.05) (Fig 1C). Uptake of Mtb in KOftfysinfected at 100:1 (3.00e ± 3.4e ) 5  4  was significantly higher (PO.01) than WT M<j)s infected at 50:1, mirroring the effect seen in the Glaxo mice which indicated that doubling the MOI overcompensates for the difference in binding between WT and KO M<|>s. Uptake of Mtb in KO Mfo infected at 40:1 (3.33e ± 3.2e ) 4  3  was also higher (PO.05) than WT M<> | s infected at 20:1. Uptake of Mtb at the High MOI was approximately 10 times higher than uptake at the Low MOI for both WT and KO M(j)s at DAY 0. At Days 4 and 7 the pattern seen at Day 0 was still observed: Mtb CFU counts in the KO wells were lower (but not significantly, p>0.05) than those in the WT wells infected at the same MOI,  21  while doubling the MOI resulted in higher counts than in the WT Mfy for both High and Low MOIs. Uptake in the Glaxo-derived M<j>s was significantly higher (PO.0001) than Itgam in the populations infected at the same MOI, demonstrating that the different B6 backgrounds and/or differences in the CR3 gene knockout have an effect on mycobacterial uptake. Because it was not possible to infect WT and KO M<> | with exactly the same numbers of bacteria, making it hard to make direct comparisons between groups, I also assessed the growth of Mtb in vitro using the bacterial doubling time (DT) as a measure of growth (Fig.l). The DT examines the relationship between the number of bacteria at Day 0 and the number of bacteria at Day 7. There was no significant difference (P>0.05) in the DT between Glaxo WT 50:1, KO 50:1, and KO 100:1 (34.9 ±0.9, 35.7 ± 1.7 and 38.1 ± 1.6 hours, respectively) or Itgam WT 50:1, KO 50:1, and KO 100:1 (30.2 ± 1.5, 29.9 ± 1.0 and 33.0 ± 1.1 hours, respectively) nor between the DT in Itgam WT 20:1, KO 20:1 and KO 40:1 with DT of 25.1 ± 0.5, 24.8 ± 0.8 and 24.8 ± 0.8 hours, respectively. Overall, DT in the Glaxo groups were longer than DT in the Itgam groups infected at the same MOI and DT at the High MOI were longer than in the Low MOI group, indicating that the bacteria replicated more slowly, perhaps as a result of the initial higher load of bacteria in the M<|). In both Glaxo and Itgam M<|) populations, the DT in the KO M<j)s infected at a MOI of 100:1 were slightly, although not significantly, longer than in Mfys infected at 50:1. This further supports the conclusion that the difference in DT was a result of the high bacterial load in the M(j> and not due to CR3 deficiency. Although there was a quantitative difference between the numbers of Mtb in infected resident monolayers, the patterns of growth observed between Glaxo and Itgam, High and Low MOI groups and WT and KO Mfys were the same and indicated that although CR3 plays a role in  22  the initial binding of the tuberculosis bacilli to the M(j), it had no significant effect on the growth of Mtb once inside resident Mfys. Higher bacterial load in the resident monolayer affected the DT of the bacteria, but did not change the finding that CR3 played no role in the growth of Mtb in resident M(|>s.  3.2 The Role O f Serum Opsonins in the Growth O f Mycobacterium tuberculosis in Resident Macrophages  Data in Fig. 1 showed that CR3 played no role in the growth of Mtb following nonopsonic uptake by resident Mfys. Because CR3 has two different binding sites (12), and uptake via the lectin-like binding site of CR3 had no effect on the growth of Mtb in resident M<(>s, I was interested in determining if binding and uptake via the second, iC3b, binding site on CR3 would result in a different outcome for the bacteria. As seen in Fig.2, resident BMM<j> from the Itgam background were infected with Mtb in the presence of 1% normal mouse serum (NMS) to coat the mycobacteria with complement proteins, again at both High and Low sets of MOIs. In the High MOI group, KO 50:1 took up 25% less bacteria than WT 50:1 and KO 100:1 took up 10% less than WT 50:1, although neither reduction was determined to be statistically significant (P>0.05). At a Low MOI, KO 20:1 took up 50% less than WT 20:1 (PO.001) and KO 40:1 took up 15% less than WT 20:1 (P>0.05). Unlike non-opsonic conditions, doubling the MOI was not able to completely restore the deficiency of Mtb uptake via CR3 KO Mfys under opsonic conditions. By Day 7 WT 50:1 CFU counts were not significantly differentfromeither KO 50:1 or KO 100:1 in the High MOI group (1.41e ± 1.5e, 1.38e ± 8.7e and 1.37e ± Lie , 7  6  7  6  7  6  respectively). At Low MOI the CFU values for WT 20:1, KO 20:1 and KO 40:1 (1.17e ± Lie , 7  7  7  6  ft  1.07e' ± 2.3e° and 1.53e' ± 2.1e°, respectively) were also not statistically different at Day 7. 23  CFU/ml o  ro  b  CD  +  cn b CO  cn b CD  cn  CT)  +  +  1  —»•  b CD  cn CD  +  +  I 3  r+ QJ. OT #0) d CD (£3 = CD CD C <5 3 fl) T 3 CD M CD CD S O • w -e- CQD. 3—-I o a c o t o 5 ? a : rn e m u a ; a . OT 5" OJ CD ^S <D O "3 CD _  n  5-3  RT  ffi  3  cr «-* mS.  3  CD 9) CD a . OT 3 Q_ O 1+  0)  3 ?  9 —  m<3"  ii  ^ 0) 0) =» _ CD 3 1 a. o o. 3= c CD 3 CD CO 3 Q .  3 ©  A, 1  5  E'S CD  cif  ai  f  3 ~ CL "I ID W O Cg OT Q . G CD ~ c r Q) CD  m  O  =• CD «-•• CD O s fl) T J OT CD O — fl) 35' 5  5  " 3 O 5TOT (DI  2.3-3 ^ • o  cn  °CD  P  OT  L Z Z Z Z Z Z Z Z z H  O  3>  ~ 3"  si  CO  as 3  a  1  =  S °  °  §  =  R"  3  3"  o  3 ?  3  ^ 3.  as  ^ CD  4^. ro N J 0 0 0  to to co  OT n-  *  oo  si 3  7\  0  W  a- 5  as  f-*- 3 °1— O Q^ 3" O CQ O £ 3 § rO CB S . CO T 3 CD OT =»  O) o ^ o H- HM -1 C D to b b 1+ O  a <z  o m r; Z  o  D > -< o 3  D > -<  m  D > -<  O  OTP  24  Again, I looked at the bacterial growth rates of Mtb in WT and KO Mtys to further determine whether CR3 plays a role in growth of serum in resident M<j)s under opsonic conditions. The DT for WT 50:1, KO 50:1 and KO 100:1 (31.5 ± 3.1, 29.4 ± 1.4 and 30.8 ± 0.5, respectively) were not statistically different. The calculated DT for WT 20:1, KO 20:1 and KO 40:1 (30.0 ± 1.0, 26.7 ± 2.2 and 27.0 ± 0.9, respectively) were also not statistically different from each other. The collective data represented in Figure 2 indicates that in the presence of serum opsonins, the initial bacterial burden in M(j)s affected the growth rate of intracellular Mtb and, as for non-opsonic binding, CR3 played a role in uptake but not the growth of Mtb in resident M<> |s for either of two different MOIs.  3.3 The Role O f Complement Receptor 3 in Immune-Activated Macrophages  In Mtb resistant WT mice, such as C57BL/6, the growth of Mtb is controlled starting approximately 20 days into infection (72). The inhibition of Mtb growth in vivo is controlled by acquired T-cell mediated immunity which is responsible for the subsequent production of IFN-y and other Thl-type cytokines (47) which activate M<j) functions that control the growth of Mtb. To determine if CR3 possibly plays a role in infection.after the onset of acquired immunity I looked at the growth of Mtb in BMM<j) that had been activated by exposure to IFN-y and IFN-y plus LPS (Table 1). I examined growth of Mtb in immune-activated Mtys in Glaxo BMM<j> infected at High MOI and Itgam BMM<|> at Low MOI. Firstly it was observed, in both Glaxo and Itgam backgrounds, that treatment of BMM<|> with IFN-y had a slight inhibitory effect on the uptake of Mtb at Day 0. IFN-y treatment resulted in 0-5% fewer bacteria being taken up in Glaxo BMM<(> and 15-40% fewer bacteria in Itgam BMM<(> during the initial infection in both WT and KO M<|)s, however neither reduction in uptake 25  x X  Oi  1+ cn —^  *  O) _^  1  x X °  o  O  l+ ?°  -vl C O  ->• C O IO  l+  i+ 9* to ^ 5?x^  ->. x O  o X  co co  —»•  _A  O  o  ^  i+ r*  i+  CO  oo O § g  i+ fn cn  1+ cn  j?  X  O  § 1  x X O o  1+  1+ C O _>. 00 CD 1 A  CO  o  <» o>  CO  *  co  O  *  *  O  to  O  o  1+  *  CO  §s  1+  Oi  to  X o  1+ * to C O to co x X  o  — _ l l  Oi  i+  x x X* — O o  cn  § 1  l+ ?>  *k  s  CD I O co Oi x X  ±5.1  55.1  CO -«J k> bo b  to  to  o  o  k  x X  IFN-y Activated M(|)s plus LPS  KO40:1  o  co ~-J  i+ cn cn 00 to ^  1+ 4*.  o  js. C O  o  o  S  to  IO  5  l+  o  CO  cn  l+ CO  IO  CO  1+ «> cn t O  x X  *  °  O  O  *  o  i+ F>  o  § 1 Ow  cn  1+  ^  oo tO  1+ * -  1+ to  x>£  00  -U  co 00 S> X  "  ->• —v  i+ ^ 00 I O x X O O  cn  31.7  ±2.1  CO  -vl  1+  CO _>. C O  1+  CO  cn Ik  cn  ^  5  1+ <©  1+ w  CO  CO  CO  1+ . co JO 0 0  bo  ^ x  x  1+ w to 00  1+ *»  x X O O  ->•  cn  1+  CO  CO ^1 _>. cn  26  CO —* ° o  X  1+ " co Oi co O* x X o o  cn  1+  CO  cn  ^  —1 D  5' °  3d'  m  ->• C O  x X O o 1+ ! ° to t O x>X>  i+ r ° ->• C O  x^S?  o  S D r >  vl  0  ^  o  l <  cn  1+ h>  i+ ! °  CO C O to cn^ L J . cn x X x X o  w  1+ to o  l+ h> to ^ g x °  cn  cn  1+ C O _>. o  ^ O) 00 x X  *  S?x* ° o  1+  o  CO  I  -e-  Q.  CO  -—^  3  CD  i+  V  CD CO Q.' CD  " *. i+  Q o  CD CT  to  p  o <  CO  o  o  o  >  p  x X O o  o  ^  io oo  o  CO  x>£  1+ *k  7\ O  i+ h» 00  i+ P° cn - x l  o  o  <" o>  i+ cn oo cn  °  cn  ?  o  1+ N co 00  -vl  i+  x X —i _li O o  5? X —^ _ A °  ^  i+ w CD 00 co M x X O o  O  cn  l+ P°  i+ ?> to O  GLAXO  <D  i+ 9  ? S  Resident Mrjjs  l+ 7* to C O  w  WT50:1  cn  i+ * to to co C O x X  KO50:1  cn  l+ «*> cn Oi  ->• C O  KO 100:1  O  i+ P  IFN-y Activated M<t)S  o  o  WT50:1  °  KO50:1  WT50:1 l+ <*» to O )  KO 100:1  KO50:1 l+ «*> ->• o co 00 x X  IFN-y Activated M(|>s plus LPS  KO 100:1 H- f" oo C O !^ to x X  0  o  5  Oi  1+ to  P  O  I  bo oo oo 00  1+ to o cn cn 1 A  —i a CD CT  3d  H  O >  s  was statistically significant (P>0.05). Addition of LPS to the IFN-y activated M<j)s at time of infection resulted in a statistically significant reduction (P<0.001) of bacterial uptake in both Glaxo and Itgam-derived BMM()), with approximately 20% and 50% less bacteria being taken up, respectively, in both resident WT and KO IVtys. The pattern of Mtb uptake I observed in resident Mfys was also seen in IFN-y activated M<j>s; in the Glaxo-derived M(|>s, uptake of Mtb at Day 0 in IFN-y activated KO M<))s infected at an MOI of 50:1 was lower than IFN-y activated WT Mtys infected at an MOI of 50:1, however the difference was not statistically significant. Uptake in IFN-y activated KO Mfys infected at an MOI of 100:1 was significantly higher (PO.001) than IFN-y activated WT M(j)s infected at an MOI of 50:1, indicating that CR3 was indeed responsible for partial uptake of Mtb into the M((), but Mtb can also invade the M§ via other cell surface receptors. The pattern of uptake in IFN-y activated Itgam-derived M<(>s was qualitatively the same as that seen in Glaxo-derived Mfys. At Day 0 uptake of Mtb in KO 20:1 Mfys was lower, but not statistically different, than WT 20:1 Mr>s. Uptake in KO 40:1 M<|)s was significantly higher (PO.001) than WT 20:1 Mtys, again showing that CR3 was not essential for Mtb invasion. The same pattern of Mtb uptake at Day 0 was also mirrored in IFN-y activated Mfys infected in the presence of LPS; in Glaxo-derived M<f>s KO 50:1 was lower than WT 50:1, whereas KO 100:1 was higher, with only the latter being significant (P<0.001). In Itgam background M<])s KO 20:1 was lower than WT 20:1, whereas KO 40:1 was higher however, in this instance, neither difference was statistically significant. Four days after infection, in both IFN-y activated and IFN-y activated plus LPS M(j) populations, the CFU patterns were maintained in both the Glaxo and Itgam, WT and KO monolayers; KO 50:1 and KO 20:1 lower than WT 50:1 and WT 40:1, respectively, and KO 100:1 and KO 40:1  27  higher, respectively, but the differences at this timepoint were not statistically different. At Day 7, the number of bacteria in the IFN-y activated or IFN-y activated plus LPS M(|) monolayers with respect to the presence of CR3 follows the pattern revealed by Day 4; there was no statistical difference in the numbers of Mtb in WT or KO monolayers in M<> | s derived from either Glaxo or Itgam backgrounds. Seven days post infection the effect of immune-activation on the growth of Mtb in M<j) monolayers was quite apparent.  IFN-y activated Mtys were much more efficient at  controlling Mtb growth, reflected in bacterial loads from IFN-y activated WT and KO M(|) monolayers that were 2-4 fold lower in the Glaxo-background M<(>s and 5-6 fold lower in the Itgam background, than that of the matched resident monolayer. The same decrease in bacterial load applied to monolayers infected in the presence of IFN-y and LPS, when compared to resident M(()s. The DT of Mtb in IFN-y activated WT and KO M<> | monolayers were significantly longer (P<0.0001) than in resident M<J>s for both Glaxo and Itgam Mfys, reflecting the decreased bacterial numbers in the monolayers by Day 7. LPS was also added to enhance the IFN-y activation of the M<|) monolayers to help control the growth of Mtb, and although the numbers of bacteria at Day 7 were similar to those of Mtys activated with IFN-y alone, when the decreased uptake of Mtb at Day 0 was taken into consideration LPS did not enhance the control of bacterial growth over IFN-y treatment alone. In the Itgam Mfys in particular, the addition of LPS was actually detrimental to the control of bacterial growth by IFN-y activated Mfys, decreasing the DT so that, although they still replicate significantly more slowly (P<0.001) than bacteria in resident M(j)s, the DT in the presence of IFN-y and LPS was shorter than in the presence of IFN-y  28  alone (PO.05).  In the Glaxo IFN-y activated M<|) monolayers, there was no significant  difference in the doubling times between WT 50:1, KO 50:1 and KO 100:1 in the absence of LPS (47.1 ± 4.4, 49.8 + 4.2 and 58.4 ± 5.2, respectively) or in the presence of LPS (47.0 ± 3.1, 55.8 ± 8.2 and 55.1 + 5.1, respectively), however in both groups of activated Glaxo Ivtys the DT in the KO Mfys were somewhat longer than the DT in WT Mtys. Within the Itgam IFN-y activated M<> | monolayers, there was no significant difference in the doubling times between WT 20:1, KO 20:1 and K0 40:1 (37.4 ± 1.5, 35.6 + 1.6 and 37.4 ± 3.5 hours for IFN-y activated M(|>s, and 33.1 ± 1.6, 30.7 ± 1.5 and 31.7 ± 2.1 for IFN-y activated Mtys plus LPS, respectively). These results indicated that CR3 does not play a role in the growth of Mtb in IFN-y activated M<|>s.  3.4  Complement Receptor 3 Does Not Contribute To The Respiratory Burst O f  Macrophages  The respiratory burst consists of the production of superoxide radicals and other toxic oxygen metabolites and it is widely reported that the respiratory burst is an effective antimicrobial mechanism used by Mfys to control intracellular bacterial replication. Also of interest is the possibility that CR3 allows pathogenic bacteria to enter the M(j) without an accompanying respiratory burst (65, 73). The ability of resident and IFN-y activated M<> | s to reduce NBT was examined using three distinct triggers: PMA, ZYM and Mtb in comparison to Mfys incubated with no trigger at all (Fig.3). Both Itgam and Glaxo M<j)s demonstrated basal NADPH-dependent NBT reduction in the absence of any trigger, which increased when the cells were incubated with IFN-y prior to stimulation. In Glaxo-derived Mfys, superoxide production was slightly lower but not significantly different between resident WT and KO (3.0 + 0.5 vs 2.3 ± 0.3) or IFN-y activated WT and KO 29  DPI-inhibitable NBT reduction (ng/ml/2.5e5 cells)  30  DPI-inhibitable NBT reduction (ng/ml/2.5e5 cells) a  (9.2 ± 0.7 vs 8.6 ±1.1) M(|>s triggered with PMA. NBT reduction elicited by ZYM by WT and KO resident (5.8 + 0.4 vs 4.6 ± 0.6, respectively) and IFN-y activated M<|)s (12.7 ± 0.3 vs 11.5 ± 0.3, respectively) were also not significantly different within each group. The superoxide response to Mtb was not significantly different between WT or KO Mfys in either resident or IFN-y activated M<> | populations. There was a small increase in the reduction of NBT in KO M(|>s incubated with Mtb at 100:1 MOI over WT and KO 50:1, suggesting that higher Mtb levels may be able to elicit a superoxide response however, this result was not statistically significant so I am unable to make definitive conclusions about this observation.  Also of note was the  observation that Mtb did hot elicit a superoxide response above basal levels (no trigger) in either the resident or IFN-y activated M<> | populations. In resident Mfys, the respiratory burstfromKO M<> | s infected at an MOI of 50:1 (1.9 ± 0.8) did not differ significantlyfromWT Mfys infected at an MOI of 50:1 (1.8 + 0.6). KO 100:1 (2.5 + 0.4) was slightly higher but, also not significantly differentfromWT 50:1. The pattern was repeated in IFN-y activated WT and KO Mfys. Neither KO 50:1 nor KO 100:1 were significantly differentfromWT 50:1 (4.7 ± 0.4, 5.7 ± 0.6 and 4.4 ± 0.9, respectively).  In the resident population in particular, the NBT reduction elicited by  incubation with Mtb was lower, but not significantly, than that of untriggered M<|)S indicating that Mtb may actually reduce or inhibit the superoxide responsefromhost Mfys. In Itgam-derived M<j>s the superoxide production elicited in response to PMA treatment was also not significantly different between resident WT and KO M<j)s (6.9 ± 0.4 vs 5.1 ± 0.6) or between IFN-y activated WT and KO Mfys (11.5 ± 0.8 vs 10.5 ± 1.1, respectively) although again, in both cases, the KO M(j>s had slightly lower respiratory bursts. Levels of NBT reduction elicited by ZYM between resident WT and KO M<j)s (4.6 ± 0.4 vs 3.9 ± 0.7) or between IFN-y  31  activated WT and KO Jvtys (7.5 ± 0.7 vs 7.0 ± 0.7, respectively) were also slightly lower in the KO M<j)s but still not significantly different. The ability of Mtys to produce superoxide in response to infection with Mtb did not differ significantly between resident WT and KO M^s or between IFN-y activated WT and KO M(|>s. In resident Mtys, the respiratory burst from KO 50:1 (2.0 ± 0.4) or KO 100:1 (1.7 ± 0.3) did not differ significantly from WT 50:1 (1.7 ± 0.3). In IFN-y activated WT and KO M<|>s, neither KO 50:1 nor KO 100:1 were significantly different from WT 50:1 (4.4 ± 0.7, 5.2 ± 0.6 and 5.0 ± 0.5, respectively). Also, as seen in the Glaxoderived M<)>s, Mtb did not elicit a respiratory burst significantly above background levels in either the resident or IFN-y activated M(|> populations. IFN-y activation significantly increased (PO.001) the overall ability of the M§ to produce a respiratory burst in both the Glaxo and Itgam WT and KO populations for the other triggers tested (PMA and ZYM). Addition of LPS prior to Mtb infection was tested but did not affect the production of the respiratory burst and therefore data was not shown. Except for a higher response to PMA in resident Itgam BMM<|) and significantly stronger ROI production in response to ZYM in IFN-y activated Glaxo BMM<|), there were no major differences in ROI productionfromGlaxo or Itgam-derived BMM<|), in the absence or presence ofCR3.  3.5 Nitric Oxide Production from Resident and Immune-Activated Macrophages  To explore whether CR3 plays a role in another M§ defense mechanism, I measured the production of NO from WT and KO resident and immune-activated Mfys (Table 2). As a function of NO production, nitrite levels were testedfromculture supernatant at 1, 4 and 7 days  32  W  H  to  •8 g d  co o  ft  3-  T3 2  ^  CD  CC »  CO  0) CD  O  o  co X  o Ts  o  Q co X  I?  1  CO X  o  o  CO  CD (0 (D  7s  3 3  O  9 ^  o o  CD 0  O  7s  4 > o < a  5  c •a  X  o  7s  o  O  o co  co X o  CO X  o  o  2 <; r>  Nil  o  CO X  CO X  o  3- ° ^  Q  O  O  3  cn o  o  o o  cn o  Ol  o  o o  oi o  Ol O  g'1? 2. g. c/i a "WIS  •5 S? & «  S  "i! 11  3  i  ro  i+  ro p>  ro  i+  o -*  ro  i+  bo co 00  ro  i+  ro O ji bo  ro  i+  co io  ro  1+  Ji co  cn  i+  ro  1+  in-  fo  a B << c —  _J,  CO  r-V  CD CO  9^-§ OQ H CD g-§ s e e3 CD  co  <•*  oI U)  ro  i+  o  _x  CD ~sl  1+ IO 0 7*  O  01  ro  i+  p  o co ©  1+  co _>. w  CO  1+  p  i+  a) ©  bo on  co ©  ro  1+  ro bo  1+  —. ?» a  ro  T=  CO  -«J  CD  (fl  Ct  CO  CD •>>  * 2L  —  13 o CJ  o  CD •O ft  OQ K)  a-  S CD  CQ CO  3  CC  o  CQ  co 3 7s O  CQ CO  CQ  co 3 7s O  3  CQ CO  CQ CO  3  3  o  CQ CO  Ji.  o  ji  ro o  ro o  ji  ro o  ro o  o  ro o  o  CO  if  o  §  -r.  a. g  CD  3  3  3  7;  (Q CO  CQ CO  1  CD  ro o  os o 1-1  3  i+ co ro O co ro  1+  CO  i+  co  po ro ro ro co ro 1. CO  i+  _>. cn ^ b>  i+  CD  ro  i+  o T* co ji.  i+  o Ko  1+ _k  o •  ^ ro  —.  r  o Ko  c  CD CO  CD  o 3 ON  1+ CO cn  £t  1+  ro co  CO  j>  1+ -Ft  i+  ro  ji  co  D  ID t ,  ro J>  1+ -k  i+  ro  P P ^ co CO Ko ro  33  i+  o Ko  i+  _j,  co ^*  1+  ro  o io  —» 7+  •cos  D  s << C CD CO  post-infection.  Nitrite is stable in solution and levels accumulate over the 7 day period  representing the total NO response of the Mcj)s due to Mtb infection. NO production from resident BMM(j) remained at the lower end of assay detection over 7 days and was not significantly different between KO and WT M(j)s at either 4 or 7 days post-infection. IFN-y activation significantly increased the NO response from M<f) monolayers (PO.0001) when compared to resident cells.  The addition of LPS to IFN-y activated cells decreased the  production of NO from Glaxo-derived McJ>s (PO.0001) but increased the NO response above that of IFN-y activation alone (PO.0001) in the Itgam populations. At both Day 4 and Day 7, NO productionfromthe IFN-y activated Glaxd-derived M<> |s were not significantly different between WT and KO, regardless of the MOI. The same pattern appeared in IFN-y activated M(j>s in the presence of LPS. There was not a significant difference in the production of NOfromWT and KO Mrj>s. NO productionfromthese monolayers was lower with the addition of LPS in both the WT and KO M<|)s, suggesting that, in the Glaxo monolayers, LPS may have played a role in reducing the NO response to Mtb. The production of NO from Itgam-derived M(|)s was slightly different from Glaxoderived M<|)S. At Day 4 nitrite detectedfromIFN-y activated KO 20:1 M<j>s was slightly lower but not significantly different from the nitrite levels in WT 20:1 IFN-y activated Mfys. NO productionfromKO 40:1 was significantly higher than NO productionfromWT 20:1 (P<0.05) and KO 40:1 was also significantly higher than KO 20:1 (PO.001), indicating that Mtb infection of MfJ)s may have had a dose effect on NO productionfromthese M<j>s. By day 7, in these immune activated populations, NO productionfromKO 20:1 M<> | monolayers was significantly lower (PO.01) than that of WT 20:1 M<> | monolayers and NO from KO 40:1 infected  34  M§ monolayers remained significantly higher than production from WT 20:1 (PO.01) and KO 20:1 infected M<j>s (PO.001), suggesting that CR3 did play a role in NO production from Itgamderived BMMfJ). In the Itgam M<j)s, LPS treatment in addition to IFN-y activation during Mtb infection universally increased the NO response in both WT and KO. At both days 4 and 7 postinfection NO production from IFN-y activated Mrj>s treated with LPS in conjunction with Mtb was 2-3 times higher than IFN-y activated Mfys infected with Mtb alone.  There was no  significant difference in NO production between WT and KO M(|>s treated with IFN-y and LPS at either Day 4 or Day 7, indicating that, in the Itgam monolayers, the addition of LPS elicited maximal CR3-independent NO production. In preliminary experiments NO production measured at Day 0 (3 hours after infection) was not detectable and at Day 1 (24 hours after infection) was very low. There were also no significant differences between NO produced from resident and IFN-y activated M<j>s at either of these times points (data not shown).  3.6  Nitric Oxide Does Not Play a Role in the Growth Inhibition of Mycobacterium  tuberculosis by Interferon-y Activated Macrophages  As there appeared to be a correlation between the induction of NO and the control of Mtb growth in IFN-y activated MfJ>s, I examined the growth of Mtb in both resident and IFN-y activated Itgam M<j>s in the presence of the iNOS inhibitor, L-NMMA. To determine the direct correlation between NO production and growth, nitrite levels were sampled from the same wells that CFUs were quantitated. The presence of 5mM L-NMMA during infection had no effect on the uptake of Mtb at Day 0 in either the resident or IFN-y activated M(j) populations. The absence or presence of L-NMMA did not significantly affect the growth of Mtb in resident M<j) 35  as measured by CFU at Day 7 or by DT (Table 3). As expected, NO levels were at the lower end of assay detection and were not significantly different between resident Mfys with or without LNMMA treatment. As reported above, IFN-y activation significantly (PO.0001) decreased the ability of Mtb to grow (lengthened the DT) and significantly increased the production of NO from M(|> monolayers (PO.0001).  The addition of 5mM L-NMMA to IFN-y activated  monolayers reduced the production of NO to baseline/resident Mfy level but did not influence the ability of IFN-y activated M<J>s to control mycobacterial growth (Table 3). DT of Mtb in these monolayers remained unaffected when L-NMMA was added and the CFU counts were not significantly different between monolayers treated with IFN-y or with IFN-y plus L-NMMA (Table 3). Of particular interest, is the observation that the presence of L-NMMA greatly reduced the loss of M<>| from the monolayers left untreated or treated with IFN-y alone (data not shown). In the latter groups, the MfJ) were found to detach from the coverslips and remain suspended in the supernatant. These detached cells contain viable Mtb and account for the vast majority of the CFU present in the supernatant (38). Preliminary experiments had been initially carried out using ImM L-NMMA. However, inhibition of NO production in IFN-y activated M<> | was incomplete at this concentration presenting the possibility that the residual RNI production could explain the continued antimycobacterial activity of IFN-y activated M<j>s (data not shown).  3.7 The Role O f The Respiratory Burst in Macrophage Control of Mycobacterial Growth  In an attempt to determine if ROI play a role in the control of mycobacterial growth I analyzed intracellular Mtb growth in the presence of various antioxidants. Unfortunately, all the antioxidants tested were either toxic to the Mfys or were unable to inhibit the intracellular  36  Table 3 - Effect of the NO Inhibitor L-NMMA on the Growth Of Mtb in Resident and IFN-y Activated Mrj)S Relative to the Presence of CR3. Strain  MOI  Treatment  DAY0 (CFU/ml)  DAY 7 (CFU/ml)  DOUBLING TIME (h)  Nitric Oxide Levels (uM)  ITGAM WT  20:1  Resident  2.41x10 ± 7.2x10  9.89 x10 ±1.0x10  31.4 ±1.3  2.6 ±0.2  ITGAM KO  20:1  Resident  1.88x10 + 1.2x10  1.03x10 ± 1.6x10  29.8 ±2.6  2.7 ±0.3  ITGAM KO  40:1  Resident  3.84 x10 ± 2.4x10  2.00x10 ±2.1x10  29.9 ±2.2  2.0 ±0.2  ITGAM WT  20:1  Resident + 5 mM NMMA  2.34x10 ± 8.6x10  1.24x10 ± 1.7x10  29.5 ±1.9  2.4 ±0.3  ITGAM KO  20:1  Resident + 5 mM NMMA  1.99 x10 ± 1.4x10  1.02x10 ±1.5x10  30.4 ±2.8  1.9 ±0.2  ITGAM KO  40:1  Resident + 5 mM NMMA  3.97x10 ± 2.8x10  2.21 x10 ± 3.0x10  29.4 ±2.0  2.4 ±0.3  ITGAM WT  20:1  IFN-y Activated  2.39 x10 ± 1.3x10  43.6 ±3.0  19.1 ±1.6  ITGAM KO  20:1  IFN-y Activated  1.67x10 + 9.9X10  3.13x10 ± 4.0x10  40.1 ±3.5  16.0 ±1.8  ITGAM KO  40:1  IFN-y Activated  3.37x10 ±1.9x10  7.35x10 ±1.0x10  38.2 ±2.3  26.2 ±2.3  ITGAM WT  20:1  2.64x10 ±1.0x10  4.29x10 ± 6.5x10  42.6 ±5.0  2.7 ±0.3  ITGAM KO  20:1  2.03x10 ±1.0x10  3.81 x10 ± 4.6x10  40.0 ±3.1  2.4 ±0.2  ITGAM KO  40:1  7.12x10 ± 9.7x10  40.3 ±3.3  3.6 ±0.5  4  5  2  5  4  3  4  3  4 2  4 3  4 3  4 3  4  2  4  IFN-y Activated  + 5 mM NMMA IFN-y Activated  + 5 mM NMMA IFN-y Activated  + 5 mM NMMA  3  4 3  4  3  3.84x10 ± 1.7x10  4 3  6 5  6  5  6  5  6 5  6 5  3.42x10 ± 3.2x10  5 4  5 4  5  5  s 4  5 4  s 4  N O T E : n i t r i t e v a l u e s s a m p l e d from s a m e w e l l s as C F U w e r e e n u m e r a t e d D a t a s h o w n f o r b o t h C F U / m l a n d N i t r i t e v a l u e s a r e m e a n ± S E M f o r n = 9 r e p l i c a t e s from 3 s e p a r a t e e x p e r i m e n t s performed in triplicate.  37  respiratory burst. DPI, used in the NBT assays to successfully inhibit ROI over 3 hours, exhibited toxicity starting 24 hours after infection, and by Day 4 of growth the M(j) monolayer had deteriorated completely. Another antioxidant, NAC, also exhibited toxicity starting 24 hours after infection. NAC was also tested as an alternative to DPI in the NBT assays, however, it was found to be a strong reducing agent and therefore unusable in experiments designed to measure the respiratory burst. Finally, the combination of SOD + CAT was tested, however this was not able to inhibit the deposition of the formazan salt in the NBT assay nor did it have any effect on Mtb replication when added to the McJ> monolayers during Mtb growth assessment (all data not shown) so it was determined to be inadequate for my experimental procedures.  38  CHAPTER 4. DISCUSSION The purpose of the work reported here was to elucidate the role of CR3 in the pathogenesis of Mtb. CR3 has been suggested as a major player in the infection of M<|)s due to its binding capabilities and attributed roles in the formation of the respiratory burst and the production of RNI. Using two genetically different, but phenotypically similar CR3 KO mice I have examined the role that CR3 plays in the pathogenesis of Mtb. I observed quantitative, but few major qualitative differences between the two mouse sources, which indicated that although the different genetic KO background affected some of the observations made, it did not make a difference in the overall conclusion I was able to make. Interaction of CR3 with its ligand can promote rapid phagocytosis and subsequent degradation of the particle once internalization has occurred (74). Previous work in our lab has shown a 40% decrease in non-opsonic binding and 50% decrease in opsonic binding of Mtb binding to M<j>s in the absence of CR3 (10). In the current study I observed a similar decrease in the number of bacteria taken up by resident KO M<> | s but could not replicate the 40% decrease reported previously for non-opsonic binding, in either the Glaxo or Itgam backgrounds. There are a number of possible explanations for the difference: 1.) it is known that M<j) phenotype determines the non opsonic binding of Mtb to murine M<j>s (8). BMM<|> were used in the current study whereas the previous study had utilized alveolar and peritoneal M<|>s. Thus the difference in binding and uptake may be a function of the different M<|) types used in each set of experiments. 2.) An additional explanation may lie in the different techniques used to assess binding of Mtb. In the current study bacterial numbers were assessed by measuring CFU associated with the M<> | population. In the earlier study (10), bacterial association with M(|> was  39  assessed microscopically, a method that cannot distinguish live and dead bacteria. As all mycobacterial cultures contain a percentage of dead bacteria there is a possibility that CR3 binds dead bacteria more efficiently than other receptors, resulting in a relatively higher percentage of dead bacteria binding to WT Mtys than KO Mtys. This would result in a large difference in Mtb binding between WT and KO M<> | s when assessed microscopically but when CFU are enumerated, which can only count live bacteria, the difference would no longer be apparent. Regardless of the quantitative differences between this body of work and previous data I was still able to conclude that CR3 plays a role in binding of Mtb to Mfys in the absence of serum. There was a clear difference in non-opsonic binding and uptake between the Glaxo and Itgam BMM<|), indicating that the genetic background did play a role in the uptake of Mtb in the absence or presence of CR3. Glaxo BMM<f> took up approximately 2 times more bacteria than Itgam BMM(j> at the same MOI. There may be a difference in the surface receptor activities between Glaxo and Itgam-derived M<|)s that could account for the difference in binding and uptake between the two Mfj> sources. Itgam BMM<|> infected at the High MOI took up almost 10 times more bacteria than Itgam BMMfj) infected at the Low MOI. This shows that MOI played an important role in determining the amount of mycobacterial uptake by BMM(|>. The difference in MOI between the High and Low MOI groups, however, was only 2.5-fold so I did not expect to see a 10-fold difference in binding between the two groups. The observation that the increased binding to BMM(j) was not directly proportional with respect to the increase in bacterial number present in the well suggests that the efficiency of Mtb uptake was enhanced with increasing bacterial numbers, probably due to increased Mtb-MrJ) contact. Another explanation is that different  40  batches of Mtb may exhibit different binding properties with BMMcj), as the Mtb used for the High and Low MOIs were grown as separate cultures. Whereas the DT between WT and KO were not statistically different within experiments, there was a noticeable difference in the DT between Glaxo BMM(|> infected at the High MOI and Itgam BMM<|) infected at High and Low MOI. The difference in DT appeared to be related to the observed pattern of Mtb uptake between the groups. As the uptake of Mtb decreased between the BMM<j) populations the DT also decreased; Glaxo High MOI DT > Itgam High MOI DT > Itgam Low MOI DT, indicating that high bacterial numbers were detrimental to intracellular Mtb replication. In both the Glaxo and Itgam Mcj) populations, the KO 100:1 DT was slightly longer than KO 50:1 DT which further supports the suggestion that the difference in DT was a result of higher bacterial load in the M<j). The role of CR3 in mycobacterial uptake became clearer in the presence of serum opsonins. I observed a 50% reduction of Mtb binding in the absence of CR3 at a LOW MOI and a 30% reduction in Mtb binding in the absence of CR3 at High MOI. With the presence of serum in the M(|) monolayers I could not completely restore KO M(j> binding of Mtb to WT levels at either MOI tested, matching results from Cywes et al who observed that Mtb bound to CR3 in, at least partly, a serum independent manner (41). I was able to restore Mtb binding to near WT levels by doubling the MOI in the CR3 KO M<() but because I was unable to fully restore binding, or observe overcompensation as was seen for non-opsonic uptake, the additional uptake in KO Mcj>s was most likely due to complement-mediated binding to other complement receptors, such as CRI and CR4, and I therefore conclude that CR3 was a dominant receptor for Mtb binding in the presence of serum opsonins. These results indicated that CR3 played a major (although not exclusive) role in Mtb uptake under opsonic conditions but, under non-opsonic conditions,  41  alternative cell-surface receptors were better able to compensate for the reduced uptake of Mtb in the absence of CR3. The difference in uptake seen following infection with High and Low MOIs, both in the absence and presence of serum, also indicated that increasing the number of Mtb available for infection could at least partially overcome the loss of binding due to CR3 deficiency and that other cell surface receptors were sufficient in allowing Mtb invasion of host M(j)s. Because the initial interaction between Mtb and host cells in the lung is in a relatively serum-free environment (75), non-opsonic uptake of Mtb probably plays a more important role in the initial interaction of CR3 and Mtb and so I chose to focus the rest of my studies on the role of CR3 in the absence of serum. Whereas I have confirmed that CR3 has a significant role in the binding and uptake of Mtb by resident M<j>s, I also showed that CR3 does not play a significant role in the subsequent survival and replication of the bacteria. Mtb was able to replicate in CR3-deficient Mtys at rates comparable to those seen in WT M<j>s, irrespective of which of the two CR3 knockouts was utilized, the MOI used or whether serum was present during uptake. I have shown that Mtb does not gain any selective advantage, nor a significant disadvantage, by entering resident M<|)s via CR3. Because I showed that the lack of CR3 did not significantly affect the growth of Mtb within resident Mfys I wanted to examine the possibility that entry via CR3 may play a role after the onset of acquired immunity in the host. The onset of T-cell mediated immunity in the host is characterized by the activation of host M<j)S by T-cells via cytokines such as IFN-y and TNF-oc. To mimic the host events that take place during acquired immunity I treated M(() monolayers with IFN-y. As expected, treatment of BMM(() with IFN-y successfully activated the Mtys so that they  42  were better able to control Mtb growth (45, 76, 77). IFN-y treatment also resulted in fewer bacteria being taken up during the initial infection in both Glaxo and Itgam WT and KO M<j>s, confirming earlier reports that IFN-y plays a key role in host immunity against intracellular pathogens and has been shown to alter the expression of cell surface receptors (37, 78-80). IFNy treatment has been specifically shown to reversibly inactivate CR3 and reduce adherence and ingestion of mycobacteria (11, 37) and other particles (81), however this explanation for the reduction of Mtb binding in the presence of IFN-y is only applicable in WT BMMcj). IFN-y treatment also down-regulates the function of other complement receptors in addition to CR3 (11) and it was therefore more likely that the reduction of Mtb binding in the presence of IFN-y was due to an overall down-regulation of the receptors used for Mtb entry. The effect of IFN-y on Mtb binding was less obvious in the Glaxo BMM(|) populations than in the Itgam BMM<|) populations, perhaps because of the higher MOI, and therefore increased binding, of Mtb to M<> |s which in turn, diminished the effects of IFN-y activation receptor binding. An alternative explanation is that M<(>s derived from the Glaxo background were less susceptible to the effects of IFN-y on surface receptor expression. LPS was also used in conjunction with IFN-y to enhance the activation of Mtys. This treatment significantly reduced the binding of Mtb to both Glaxo and Itgam M<|>s. LPS has been shown to reduce the capability of CR3 on murine M(j)s to bind and phagocytose complement opsonized particles (82) and CR3 has been shown to act as a receptor for LPS (28) through a site distinct from the iC3b site. It was possible, therefore, that addition of LPS to the monolayers inhibited binding of Mtb by competing for the same lectin binding site or that LPS causes a conformational change in CR3 that changes the binding capabilities of CR3, rendering it less  43  efficient in binding Mtb. However, because a decrease in binding in the presence of LPS was also seen in KO M<j>s lacking CR3 it is more likely that LPS enhances M(j> activation by IFN-y thus increasing the inhibition of binding seen by IFN-y through increased down regulation of receptors. LPS has also been shown to inhibit the expression of M<j) surface receptors such as the scavenger receptor and it may be via LPS-mediated down-regulation of this and other receptors that the decrease in Mtb binding is seen (83). Although uptake of Mtb was affected by the absence of CR3 in activated Mcf> populations, I did not observe any concomitant differences in intracellular replication. In both IFN-y and IFN-y plus LPS treated Itgam M<))s there was a slight, but statistically insignificant, reduction in the DT of Mtb in KO M<> | s infected at the same MOI as WT, suggesting that entry via CR3 may inhibit replication of the bacteria in the host cell. When the MOI was increased in the KO M(()s, however, this difference was diminished or eliminated altogether, indicating that the slower replication in the WT M(() was more likely due to the presence of additional Mtb in the cell and was not related to the absence or presence of CR3. However, in Glaxo BMMf the opposite phenomenon was observed. In IFN-y plus LPS treated GlaxO M<> | s there was a slight, but statistically insignificant, increase in the doubling time of Mtb in KO M<> | s infected at the same MOI as WT, suggesting that entry via CR3 may enhance the replication of the bacteria in the host cell. The DT in KO M(|>s were somewhat longer than the DT in WT M<> | s indicating that, in the Glaxo M<|) populations, entry via CR3 improved bacterial replication and was advantageous to Mtb. In the IFN-y activated Glaxo KO BMM<|) increasing the MOI in the KO M<j>sfrom50:1 to 100:1 further increased the DT, again suggesting that an increase in the initial Mtb load can have a negative impact on the subsequent growth of intracellular mycobacterial growth.  44  However, as there was no statistical difference between Mtb growth in resident or activated WT or KO M(|>s at any MOI, or in either the Glaxo or Itgam populations, I therefore conclude that entry via CR3 did not play a significant role in the intracellular survival of Mtb and it was not the absence, or presence of CR3 alone that effected bacterial replication. I examined whether CR3 played a role in the respiratory burst of M<j>s and if this receptor does indeed act as a "stealth receptor" in either resident or immune-activated M<j>s. CD 1 lb-Amice have been shown to be deficient in oxygen radical formation and neutrophils have been used to show that a striking increase in oxygen radical generation accompanies CDllb/CD18 mediated phagocytosis (16). More specifically, CR3 has been shown to play a dominant role in the stimulation of a neutrophil superoxide burst mediated by ZYM and P-glucan particles (84). Further evidence in the role of CR3 in the respiratory burst is the observation that patients with a genetic deficiency in CR3 have neutrophils that do not have a respiratory response to ZYM (85). I examined whether CR3 played a role in the induction of the respiratory burst during Mtb invasion of M<|>s. Using PMA, a potent receptor-independent stimulator of the respiratory burst, I found that KO M<> | s were not inherently deficient in the production of an internal respiratory burst and as expected, treatment of the cells with IFN-y significantly increased the respiratory burstfromboth WT and KO M(j)s. ZYM was used as a receptor-mediated particle control and contrary to earlier reports (10, 16) I found no significant difference in the ability of KO M<> | s to produce a respiratory burst in either resident or IFN-y activated M()>s. I think that the differences found in my current study were due, in part, to differences in the M<> | types examined (BMM<|> in this study versus peritoneal M<|) and neutrophils) and the assay utilized (intracellular production of ROI in this study versus extracellular production of ROI). Of note, in both  45  resident and activated PMA and ZYM stimulated M<)>s, the KO cells exhibited a slightly lowered respiratory burst, however, the differences were not statistically significant. I found no significant difference in the production of superoxide measured between resident WT and KO M<))s or between IFN-y activated WT and KO Mfys when triggered with Mtb. My results indicated that Mtb alone did not elicit a respiratory burst in either resident or activated M«j)s. Mtb possess a multitude of virulence factors that may allow them to evade the respiratory burst, such as superoxide dismutase (sodB) and catalase (katG) that can detoxify oxygen radicals (86, 87), and lipoarabinomannan that can scavenge ROI (88, 89), therefore, it is likely that Mtb are resistant to the effects of any ROI producedfromIFN-y activated Mfys (90). Consequently, it has been found that oxygen radical scavengers have no effect on the antimycobacterial activity of Mfys and mice deficient in the NADPH-oxidase complex are still able to control mycobacterial infection indicating that ROI probably do not play a significant role in the killing of Mtb (47, 61, 91). Other reports have also suggested that mycobacteria may invade Mtys without eliciting a respiratory burst (92, 93). I believe that this is because one or more of the above listed virulence factors either breaks down or inhibits the production of intracellular ROIs during phagocytosis. More recently, it has been shown that the production of ROI plays no role in controlling the growth of Mtb unless the bacteria is specifically deficient in the mechanism used (katG) to counteract this host defense mechanism (46). Although CR3 has been shown previously to play a role in the respiratory burst I have shown that, under certain conditions, alternative phagocytic receptors allowed particle uptake into the M(j) and an accompanying internal respiratory burst in the absence of CR3. However, in the case of Mtb, this was most likely redundant because the bacteria were not shown to specifically elicit a respiratory burst and would be resistant to it if there was one. It is also  46  probable that different cell types or different M(() populations may have different respiratory burst responses, as it has been shown that mycobacteria are phagocytosed by different receptors depending on the M(|) type encountered (7). Also, of interest is the observation that superoxide production in response to nonopsonic uptake of ZYM and mycobacteria depends on which of the binding epitopes of the receptor is engaged (35, 94). There is also evidence from neutrophil populations that phagocytosis is triggered by CR3 via simultaneous recognition of the iC3b site in conjunction with polysaccharide binding via the lectin site which induces a primed receptor state (95), indicating that more than one trigger is needed to produce a respiratory burst. Uptake of ZYM also occurs via the mannose (96) and P-glucan receptors (97) including dectin-1 (98) and it may be via these receptors or in cooperation with these receptors that the accompanying internal respiratory burst occurs. The differences in contribution of CR3 to the respiratory burst may also be due to the activation state of the receptor. CR3 exists in two distinct conformational states, active and non-active, that regulate the binding capabilities of the receptor (99). .In response to certain agonists CR3 undergoes a rapid activation that exposes new binding epitopes on the cells surface (8, 100) and it may be the activation state of the receptor that determines the M<)> response/production of ROI. It has also been shown that engagement of different epitopes of CR3 determines whether a respiratory burst will accompany phagocytosis (19, 35, 101). CR3 has also been shown to interact with Fey receptors to facilitate neutrophil functions (102) and it has been shown that cooperation between CR3 and the Fey receptors receptor is required for the generation of a synergistic respiratory burst (66). Binding of specific peptide motifs have been shown to indirectly influence the binding activity of CR3 most likely through interactions with other surface receptors (103). Finally, it has also been shown that mycobacteria can enter the M(j> via the mannose receptor without an accompanying respiratory burst and it may be that this  47  receptor is a better candidate for the silent entry of mycobacteria into Mfys (104). There are a multitude of receptors accompanying various signaling mechanisms to account for the production of the respiratory burst in the absence of CR3. An interesting observation made when examining the role of ROI on the survival of Mtb in the host M(j) was the toxicity of some of the most common antioxidants used to assess the role of ROI in host defenses. I found, with both DPI and NAC, that M(|> cell death in the monolayer was evident at 24 hours after infection. In the presence of DPI the monolayers had completely deteriorated and the Mtb CFU counts were almost undetectable by Day 4 of growth in vitro. The toxicity of these compounds to the M<> | s means that previous information on bacterial growth, in relation to ROI, obtained by examining growth in the presence of these compounds, is now subject to questioning. With Mtb in particular, the presence of an intact, live M§ is necessary to facilitate intracellular growth of the bacteria. If the antioxidant being used to examine the role of ROI is toxic to the M<j> one cannot be sure if any results obtained were due strictly to the inhibition of ROI or if it is simply the death of the M<|) that is responsible for the outcome. For example, if I did not consider the role of cell death due to the presence of the antioxidant, I could conclude from my results that ROI were essential for the survival of Mtb as, in the absence of ROI, rapid mycobacterial clearance was observed. It is imperative that future examinations on the role of ROI on intracellular mycobacterial survival refrainfromusing compounds that are toxic to the M(|> to ensure that only the role of ROI is determined. It has been concluded that NO is essential for the control of mycobacterial growth in the host, based on studies using both iNOS KO mice and the use of iNOS inhibitors. I performed two sets of experiments related to this phenomenon: the examination of NO production in relation to the presence of CR3 and the role NO plays in the control of intracellular Mtb growth.  48  Treatment of M(|>s with IFN-y alone prior to infection has been shown to result in a significant increase in production of NOfromthe cells (105), which is potentiated by infection with Mtb (77) or the addition of LPS (106). I found that NO production from Mfys, in response to Mtb, was significantly enhanced by pre-treatment of the cells with IFN-y prior to infection. In Itgam BMM(|>, the production of NO was further increased by the addition of LPS to the IFN-y activated monolayers at infection, confirming that LPS and IFN-y can act synergistically to induce iNOS (107). Mtys synthesize large amounts of IL-1 and TNF-a in response to LPS (108) which in turn, upregulate the activity of iNOS and the production of NO (109, 110). IFN-y treatment and the subsequent TNF-a induction induces the production of NOfromLPS-treated murine Mfys (111, 112). CR3 can also interact with GPI-anchored proteins such as CD 14, the receptor for LPS, providing the signal transduction machinery (31) for transmembrane signalling capabilities. It may be via this interaction, and through the transcriptional regulator N F K B , that IFN-y and LPS in conjunction with Mtb provides such a significant production of NO after infection (113). In contrast to the Itgam BMM(|> populations, addition of LPS to IFN-y activated Glaxo BMM<|> monolayers did not result in a significant increase in NO productionfromthese cells. This difference in NO response to Mtb indicated that the Glaxo BMM<j> did not respond to LPS, perhaps due to a defective CD 14 or Toll-4 receptor or because of a deficiency in downstream receptor signaling pathways. By Day 7 after infection, I saw very little NO in the supernatantsfrominfected resident M(|> populations and significant production of NOfromIFN-y activated M<> | s corresponding to the inhibition of Mtb growth in these populations. The levels of NO from the supernatants from IFN-y activated Itgam KO Mcjis infected at an MOI of 20:1 were significantly lower than NO  49  levelsfromWT 20:1 on Day 7 indicating that CR3 may play a role in the production of NO from M<|>s. The decrease in NO production, however, was annulled by the addition of more Mtb, indicating that, much like the loss of binding due to the absence of CR3, the loss of NO production in CR3 KO M<j)s could be overcome through alternative surface receptors. Even though there was a significant difference in the levels of NO produced between WT and KO monolayers infected at the same MOI, there was no indication that this difference had any effect on the growth of Mtb, as DT between WT and KO IFN-y activated M$s were not found to be significantly different. This may be because the mycobactericidal effect of NO has a threshold effect, i.e. only a certain level is needed to achieve growth inhibition and anything more does not significantly affect the end point. Alternatively, it may be that NO is not directly responsible for the killing of mycobacteria in IFN-y activated Mfys and so no difference in Mtb growth was observed. Therefore, I conclude that although CR3 may play a small role in the production of NOfromhost M(|)s, it played no significant role in the control of Mtb replication. The addition of LPS to the Itgam monolayers resulted in a large increase in the levels of NO in IFN-y activated supernatants, but did not show a corresponding increase in inhibition of Mtb growth in the host. This result further strengthened the above conclusion that NO may either have had an effective threshold that is needed to control mycobacterial growth or played no role in controlling growth in M<> |. The production of NOfromthe M(|) has been shown to play an important role in the inhibition of intracellular growth of mycobacteria both in vivo and in vitro. Work with inhibitors of the production of NO has shown that NO plays an important role in the antimycobacterial effect of M(j)s (62, 114) and that the use of chemical inhibitors of NO is an effective tool to examine the role of NO in biological systems by preventing its production. The addition of L-  50  NMMA or other iNOS inhibitors to MfJ) populations inhibits the production of NO from the cell (115). It has been shown that L-NMMA accelerated the growth of Mtb H37Rv inside IFN-y activated murine peritoneal Mfys (116) and of Mycobacterium bovis in IFN-y activated BMM<j) (91) indicating an essential role of NO production in the inhibition of mycobacterial replication. I therefore expected that the addition of L-NMMA to Mtb infected IFN-y activated M<> | s would abrogate the inhibition of Mtb growth in these M<))S (117). However, I did not observe any effect of the NO-inhibition on the survival of intracellular Mtb. The data available on the contribution of NO to the control of mycobacterial growth is currently diverse and contradictory. Whereas many researchers have established the link between the production of NO and killing of mycobacteria, other groups have also found that NO may not play the key role it has been assigned. Inhibition of NO with L-NMMA has no effect on the growth of M. bovis (BCG) within human monocytes and M<))s, suggesting that NO does not play a role in growth in these cells (118). Inhibition of mycobacterial growth in an IFN-y plus LPS activated M(J) cell line could not be completely abrogated in the presence of L-NMMA (119). The growth of M. leprae was not increased in iNOS KO mice and growth could be controlled in the absence of NO (120). Other groups have found that iNOS KO mice as well as IFN-y activated M<|)s from these animals are still able to control growth of M. avium, underscoring the lack of a role for RNI in the control of this organism as well (121, 122). It has been suggested that under stringent conditions, RNI can kill Mtb, but that under less harsh, more physiological conditions, the effects of RNI range from partial to negligible (123). An interesting possibility to explain these differences is that the susceptibility of Mtb to RNI and the ability of L-NMMA to reverse the suppression of growth of Mtb in M<j)s may be a phenomenon related to the strain of Mtb being used for experimentation. It is known that strains  51  of Mtb vary in their susceptibility to RNI generated in vitro (124) and in IFN-y activated Mfys (123). I used Mtb strain Erdman for the current study and so it may be that the observations I made were limited specifically to this strain, though Erdman is neither particularly resistant or susceptible to RNI generated in vitro (123). I also consider that the methodology used to examine the role of RNI on mycobacterial growth in M<>| influences the results obtained. In the current study I infected M<>| monolayers with Mtb and monitored the growth of the bacteria and NO productionfromthe same well. In addition, the enumeration of Mtb in infected M<J> included the contents of the entire well; i.e. the monolayer plus the cells that have lifted over the course of the experiment into the culture medium. Other studies have reported Mtb growth only in the intact monolayer that is remaining after the reported period of intracellular growth. Previous work has shown that, over time, M(|> infected with Mtb detachfromthe coverslip yet still contain viable bacteria (38). These bacteria still remain part of the closed in vitro system and should be included in any quantitation of CFU present in that experimental group. Quantitating bacteria in just the monolayer does not reflect the true consequences of any treatment added to the well and may result in under- or overestimating the CFU present in the well. For instance, I observed that L-NMMA treatment greatly reduced the loss of M<j>fromthe monolayer when compared to IFN-y treated M<j>. Enumeration of CFU in the monolayers alone could have erroneously led to the conclusion that L-NMMA resulted in enhanced viability of the Mtb. However, by assessing the viable bacteria present in both adherent and non-adherent M<j) I found that there was no significant difference in the CFU present in M<|) treated with IFN-y alone or IFN-y plus L-NMMA. Pathogenic mycobacteria have been shown to be able to inhibit the production of NO from IFN-y activated M(|>s (125, 126) as well as have the capacity to resist toxic nitrogen radicals  52  (127) and it may be in this capacity that virulent mycobacteria evade the M<> | immune response. The ability of other intracellular pathogens, such as Salmonella typhimurium and Candida albicans, to suppress the NO response from IFN-y activated M<))s has also been reported (128, 129). Furthermore, virulent mycobacteria have also been shown to be resistant to RNI in vitro (130) and have evolved potential virulence factors that protect themselvesfromRNI (131). The presence of phthiocerol dimycocerosates in the mycobacterial membrane have been shown to protect the bacteriafromthe cidal activity of RNI released by activated M<J>s (132). Recently, it has been shown that iNOS does not colocalize to the mycobacterial phagosome, suggesting that the mycobacteria may inhibit iNOS recruitment to phagosomes during M<> | infection as a means to protect themselvesfromintracellular NO exposure (133). While my results showed that RNI produced by M<))s infected with Mtb was not directly mycobactericidal, my data does not contradict the observations that iNOS mice are more susceptible to Mtb. An alternative explanation for why RNI may be essential for optimal control of mycobacterial infections independent of enhanced M(|) killing lies in the ability of the host to develop RNI-expressing granulomas (49, 134).  iNOS KO mice have large, bacteria-filled  granulomas at time of death instead of the small compact granulomas that are normally seen in WT mice. Adams et al. were able to abrogate the inhibition of mycobacterial growth in IFN-y activated peritoneal M(|>s by the addition of the iNOS inhibitor AG but also found that in the lungs of iNOS knockout mice, growth of Mtb was the same compared to WT controls early in infection (up to day 28) and only after this time point was a role of NO evident in these mice (62). In iNOS KO mice infected with Mtb via low-dose aerosol infection (mimics the path by which humans are infected) bacterial growth is also not different in early infection than in WT mice infected via the same route (135). Results such as these may indicate that, under certain  53  conditions and with certain M§ types in an artificial in vitro system, RNI can play a direct role in the control of mycobacterial growth. However, in vivo, the role that RNI play may be limited to the effects they have on the formation of a tight granuloma to control mycobacterial growth. Evidence suggests that NO production may be a double-edged sword in the fight against mycobacteria.  On one hand the molecule may be able to directly inhibit the growth of  mycobacteria, but on the other, its detrimental role in inflammation/granuloma formation and the T-cell response may be what allows mycobacteria to survive in the host. As suggested by work with a NOS2 KO mouse, it is probably not the direct role of NO that plays in the killing of mycobacteria but the important role it plays in the development of the immune response (135). NO still plays a important role in the cellular response to infection, and it is the cellular response that is important in regulating granuloma formation and the dissemination of mycobacteria throughout the tissues. Without NO, granulomas do not form properly and bacterial spread is not controlled, the bacteria can spread throughout the body where they grow more readily in the absence of NO, especially in the liver (135). In addition to poor granuloma formation, the lack of NO allows tissue infiltration of T-cells and other lymphocytes that would not normally happen in the presence of NO, resulting in damage to the lung tissues. It is possible that the increased murine death rate in NOS KO mice is not seen simply because NO is needed to kill the bacteria but is due to increased lymphocyte infiltration and damage to the lungs, followed by the inability to control bacterial growth once the pathogen spreads to the surrounding tissues. NO may still play a role in the control of mycobacterial growth however, based on my observations, this role cannot be as a direct toxicity to the bacteria inside the M§. It is more likely that NO plays a role in regulating other immune events that are responsible for the control of mycobacterial growth in vivo. Perhaps most interestingly, NO has been shown to have both  54  pro-and anti-apoptotic effects related to the concentration of NO elicited under physiological and pathophysiological conditions: high concentrations tend to trigger apoptosis and low, inhibits apoptosis (136). NO has been shown to play a role in inducing T-cell apoptosis (137) as well as having the ability to protect cells from TNF-a induced apoptosis (138). As apoptosis has been shown to be a mechanism by which the host controls the replication of Mtb (139, 140), this presents the possibility that NO is inhibiting the growth of Mtb via the apoptotic killing of infected cells. Support for the suggestion that NO plays an important role in the induction of apoptosis was seen in BMMfj) monolayers with and without L-NMMA. In BMM<|> monolayers treated with IFN-y, which induces NO production and consequently apoptosis, I observed a progressive detachment of Mtb infected M<J>s from the coverslip into the supernatant over the course of infection. In the presence of L-NMMA, however, the Mcj) monolayers remained attached to the coverslip throughout the infection, indicating that the elimination of NO prevents the induction of M(j> apoptosis. The absence of gross abnormalities or lack of immune compromisation in CDllb deficient mice also supports my conclusions that CR3 does not play a specific role in the success of Mtb infection (16). In addition, no discernable effect of the loss of CR3 on the disease course of Mtb in either resistant or susceptible mouse strains has been observed (10, 141). Data from experiments using M.avium and CD18-deficient mice also support the indication that mycobacteria can enter tissue Mcj)s through receptors other than CR3 and that CR3 may not play as important a role in the course of TB infection as previously thought (142). The mannose receptor has been named as a receptor that may play an important role in mediating Mcj) adherence of virulent strains of Mtb (3, 143) and CD14 has been suggested via its interactions with mycobacterial lipoarabinomannan (144). Looking at the role of CR3-mediated uptake from  55  the point of view of the bacilli, also supports the idea that CR3 does not play a role in the control of intracellular growth.  It has also been found, using selective receptor blockade, that  intracellular survival and replication of mycobacteria were the same no matter which cell surface receptor was used for binding and phagocytosis, indicating that the mechanisms used by mycobacteria to evade killing are independentfromthe receptor route of entry (6). The initial association of Mtb with M(|>s in the relatively serum-free lung would be an important step in the infection of the host. However, as resident alveolar M$s poorly express CR3 and do not efficiently bind mycobacteria in the absence or presence of serum (39) this suggests that the role CR3 plays in Mtb pathogenicity is, at best, limited to the later stages of infection. My current results further demonstrate that the role of CR3 in the pathogenesis of Mtb is minimal.  56  C H A P T E R 5. CONCLUSION  Together, the data presented indicated that although CR3 played a definitive role in the non-opsonic and opsonic binding and uptake of Mtb by murine BMM<|) in vitro, the presence of this cell surface receptor was not essential for the successful establishment of host infection. Furthermore, entry via CR3 did not contribute to the intracellular survival of Mtb in either resident or IFN-y activated M<)>s. IFN-y activation of the host Mfys significantly inhibited intracellular mycobacterial growth in addition to increasing the induction of the respiratory burst and the production of RNI, however, CR3 was not shown to be essential for any of these Mfj> functions. Of particular interest is the finding that NO could not be shown to play a direct role in the control of intracellular Mtb growth. Finally, whereas differences were found between BMM(|) derived from mice with distinct genetic knockouts of the same gene, the utilization of different KO backgrounds did not change the conclusions made with respect to the function of CR3 in the pathogenesis of Mtb and the M^> defense mechanisms examined. Because a stronger role for Mtb uptake via CR3 was evident in the presence of serum opsonins, a role for CR3 in the pathogenesis of Mtb may still be found upon further examination of both WT and KO Mfys under opsonic conditions. Whereas a role for CR3 could not be shown in resident Mfys under opsonic and non-opsonic conditions, or in IFN-y activated M<))S under nonopsonic conditions, it is still of interest to look at intracellular Mtb growth, the induction of the respiratory burst and the production of RNI under opsonic conditions in IFN-y activated M^s. It may be under these conditions that a role for CR3 can be established. Most important is further examination of the role of NO in the control of Mtb. While the use of iNOS KO mice have shown a definitive role for the production of NO in host control of  57  Mtb, I have shown that its effects cannot be attributed to direct toxicity of NO to the bacteria. It would be of interest to examine if the difference between my results and those reported by other groups, in terms of direct toxicity of NO, is due simply to examination of whole culture wells versus only the intact monolayer. Alternatively, in vitro examination of the growth of Mtb in M<> | s in the presence of alternative NO inhibitors, such as AG, could be performed to confirm that the observation that NO does not play a direct role in mycobacterial growth inhibition is not limited to the effects of L-NMMA. Also, comparison of the induction of apoptosis in resident and IFN-y activated M<j)s with and without the presence of L-NMMA, in vitro, would help to determine if the production of NO by M<> | s triggers the induction of apoptosis as a means to control intracellular Mtb growth.  Finally, future experiments should focus on how NO  contributes to the host control of Mtb in vivo, specifically, the role that NO plays in the induction of host MfJ) apoptosis and granuloma formation and how these relate to mycobacterial killing.  58  REFERENCES  1.  World Health Organization. Tuberculosis Fact Sheet Number 104. August 2002.  2.  World Health Organization. Tuberculosis Fact Sheet Number 104. March 2004.  3.  Schlesinger, L.S. 1993. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. The Journal of Immunology 150:2920-2930.  4.  Means, T.K., E. Lien, A. Yoshimura, S. Wang, D.T. Golenbock, and M.J. Fenton. 1999. The CD 14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors. The Journal of Immunology. 163:6748-6755.  5.  Gaynor, CD., F.X. McCormack, D.R. Voelker, S.E. Mcgowan, and L.S. Schlesinger. 1995. Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. The Journal of Immunology 155:5343-5351.  6.  Zimmerli, S., S. Edwards, and J.D. Ernst. 1996. Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages. American Journal of Respiratory Cell and Molecular Biology  15:760-770. 7.  Hirsch, C.S., J.J. Ellner, D.G. Russell, and E.A. Rich. 1994. Complement receptormediated uptake and tumor necrosis factor-oc-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. The Journal of Immunology 152:743-753.  8.  Stokes, R.W., I.D. Haidl, W.A. Jefferies, and D.P. Speert. 1993. Mycobacteriamacrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages. The Journal of Immunology  151:7067-7076. 9.  Schlesinger, L.S. 1996. Entry of Mycobacterium tuberculosis into mononuclear phagocytes. Current Topics in Microbiology and Immunology 215:71-96.  10.  Melo, M.D., I.R. Catchpole, G. Haggar, and R.W. Stokes. 2000. Utilization of CD1 lb knockout mice to characterize the role of complement receptor 3 (CR3, CD1 lb/CD 18) in the growth of Mycobacterium tuberculosis'm macrophages. Cellular Immunology.  205:13-23. 11.  Schlesinger, L.S., and M.A. Horwitz. 1991. Phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages is mediated by complement receptors CRI (CD35), CR3 (CD1 lb/CD 18), and CR4 (CD1 lc/CD18) and IFN-gamma activation  59  inhibits complement receptor function and phagocytosis of this bacterium. The Journal of Immunology 147:1983-1994. 12.  Ross, G.D., and M.E. Medof. 1985. Membrane complement receptors specific for bound fragments of C3. Advances in Immunology 37:217-267.  13.  Arnaout, M.A. 1990. Structure and function of the leukocyte adhesion molecules CD 11 /CD 18. Blood 75:1037-1050.  14.  Rosenkranz, A.R., A. Coxon, M. Maurer, M.F. Gurish, K.F. Austen, D.S. Friend, S.J. Galli, and T.N. Mayadas. 1998. Impaired mast cell development and innate immunity in MAC-1 (CDllb/CD18, CR3)-deficient mice. The Journal of Immunology 161:64636467.  15.  Muto, S., V. Vetvicka, and G.D. Ross. 1993. CR3 (CD 11 b/CD 18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents. Journal of Clinical Immunology 13:175-184.  16.  Coxon, A., P. Rieu, F.J. Barkalow, S. Askari, A.H. Sharpe, U.H. von Andrian, M.A. Arnaout, and T.N. Mayadas. 1996. A novel role for the 02 integrin CD 1 lb/CD 18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653-666.  17.  Hughes, D.A., and S. Gordon. 1998. Expression and function of the type 3 complement receptor in tissues of the developing mouse. The Journal of Immunology 160:4543-4552.  18.  Ross, G.D., and V. Vetvicka. 1993. CR3 (CD1 lb, CD18) - a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions. Clinical and Experimental Immunology 92:181 -184.  19.  Xia, Y., V. Vetvicka, J. Yan, M. Hanikyrova, T. Mayadas, and G.D. Ross. 1999. The pglucan-binding lectin site of mouse CR3 (CD1 lb/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3bopsonized target cells. The Journal of Immunology 162:2281-2290.  20.  Xiong, Y.M., J. Chen, and L. Zhang. 2003. Modulation of CD 11 b/CD 18 adhesive activity by its extracellular, membrane-proximal regions. The Journal ofImmunology 171:1042-1050.  21.  Brown, E., and N. Hogg. 1996. Where the outside meets the inside - integrins as activators and targets of signal transduction cascades. Immunology Letters 54:189-193.  22.  Xie, J., R. Li, P. Kotovuori, C. Vermot-Desroches, J. Wijdenes, M.A. Arnaout, P. Nortamo, and C.G. Gahmberg. 1995. Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD1 lb/CD 18 through the A domain. The Journal of Immunology 155:3619-3628.  60  23.  Diamond, M.S., D.E. Staunton, A.R. de Fougerolles, S.A. Stacker, J. Garcia-Aguilar, M.L. Hibbs, and T.A. Springer. 1990. ICAM-1 (CD54): a counter-receptor for MAC-1 (CD 11 b/CD 18). The Journal of Cell Biology 111:3129-3139.  24.  Altieri, D.C., and T.S. Edgington. 1988. The saturable high affinity association of factor X to ADP-stimulated monocytes defines a novel function of the Mac-1 receptor. The Journal of Biological Chemistry 263:7007-7015.  25.  Relman, D., E. Tuomanen, S. Falkow, T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Recognition of a bacterial adhesin by an integrin: Macrophage CR3 (OLM$2, CD1 lb/CD18) binds filamentous hemagglutinin of Bordetellapertussis. Cell 61:13751382.  26.  Wright, S.D., J.I. Weitz, A.J. Huang, S.M. Levin, S.C. Silverstein, and J.D. Loike. 1988. Complement receptor type three (CD1 lb/CD 18) of human polymorphonuclear leukocytes recognizes fibrinogen. Proceedings of the National Academy of Sciences of the United States of America 85:7734-773 8.  27.  Wright, S.D., and M.T. Jong. 1986. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. The Journal of Experimental Medicine 164:1876-1888.  28.  Wright, S.D., S.M. Levin, M.T.C. Jong, Z. Chad, and L.G. Kabbash. 1989. CR3 (CD1 lb/CD 18) expresses one binding site for Arg-Gly-Asp-Containing peptides and a second site for bacterial lipopolysaccharide. The Journal of Experimental Medicine 169:175-183.  29.  Rieu, P., T. Ueda, I. Haruta, CP. Sharma, and M.A. Arnaout. 1994. The A-domain of beta 2 integrin CR3 (CD1 lb/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF. The Journal of Cell Biology 127:2081-2091.  30.  Drevets, D.A., P.J.M. Leenen, and P.A. Campbell. 1993. Complement receptor type-3 (CD11B/CD18) involvement is essential for killing of Listeria monocytogenes by mouse macrophages. The Journal of Immunology 151:5431 -5439.  31.  Agramonte-Hevia, J., A. Gonzalez-Arenas, D. Barrera, and M. Velasco-Velazquez. 2002. Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3. FEMS Immunology and Medical Microbiology 34:255-266.  32.  Diamond, M.S., J. Garci-Aguilar, J.K. Bickford, A.L. Corbi, and T.A. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD1 lb/CD 18) for four distinct adhesion ligands. The Journal of Cell Biology 120:1031-1043.  61  33.  Balsam, L.B., T.W. Liang, and CA. Parkos. 1998. Functional mapping of CD1 lb/CD18 epitopes important in neutrophil-epithelial interactions: a central role of the I domain. The Journal of Immunology 160:5058-5065.  34.  Thornton, B.P., V. Vetvicka, M. Pitman, R.C. Goldman, and G.D. Ross. 1996. Analysis of the sugar specificity and molecular location of the p-glucan-binding lectin site of complement receptor type 3 (CD1 lb/CD18). The Journal of Immunology 156:1235-1246.  35.  Ross, G.D., J.A. Cain, and P.J. Lachmann. 1985. Membrane complement receptor type 3 (CR3) has lectin-like properties analogous to bovine conglutin and functions as a receptor for zymosan and rabbit erythrocytes as well as a receptor for iC3b. The Journal of Immunology 134:3307-3315.  36.  Le Cabec, V., S. Carreno, A. Moisand, C. Bordier, and I. Maridonneau-Parini. 2002. Complement receptor 3 (CD1 lb/CDl 8) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis, respectively. The Journal ofImmunology 169:2003-2009.  37.  Wright, S.D., P.A. Detmers, M.T.C. Jong, and B.C. Meyer. 1986. Interferon-y depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin. The Journal of Experimental Medicine 163:1245-1259.  38.  Stokes, R.W., and D. Doxsee. 1999. The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophage-like cell line THP-1: A comparison with human monocyte-derived macrophages. Cellular Immunology. 197:1-9.  39.  Stokes, R.W., L.M. Thorson, and D.P. Speert. 1998. Nonopsonic and opsonic association of Mycobacterium tuberculosis with resident alveolar macrophages is inefficient. The Journal of Immunology 160:5514-5521.  40.  Schlesinger, L.S., C.G. Bellinger-Kawahara, N.R. Payne, and M.A. Horwitz. 1990. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. The Journal ofImmunology 144:2771-2780.  41.  Cywes, C , N.L. Godenir, H.C. Hoppe, R.R. Scholle, L.M. Steyn, R.E. Kirsch, and M.R.W. Ehlers. 1996. Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells. Infection and Immunity 64:5373-5383.  42.  Cywes, C , H.C. Hoppe, M. Daffe, and M.R.W. Ehlers. 1997. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent. Infection and Immunity 65:4258-4266.  43.  Ernst, J.D. 2000. Bacterial inhibition of phagocytosis. Cellular Microbiology. 2:379-386.  62  44.  Jackett, P.S., P.W. Andrew, V.R. Aber, and D.B. Lowrie. 1983. Guinea pig alveolar macrophages probably kill M. tuberculosis H37Rv and H37Ra in vivo by producing hydrogen peroxide. Advances in Experimental Medicine and Biology 162:99-104.  45.  Flesch, I.E., S.H. Kaufmann, G. Schwamberger, I. Flesch, and E. Ferber. 1991. Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates. Infection and Immunity 59:3213-3218.  46.  Ng, V.H., J.S. Cox, A.O. Sousa, J.D. MacMicking, and J.D. McKinney. 2004. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Molecular Microbiology 52:1291-1302.  47.  Jung, Y.J., R. LaCourse, L. Ryan, and R.J. North. 2002. Virulent but not avirulent mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1dependent, nitric oxide synthase 2-independent defense in mice. The Journal of Experimental Medicine 196:991 -998.  48.  Boom, W.H. 1996. The role of T-cell subsets in Mycobacterium tuberculosis infection. Infectious Agents and Disease 5:73-81.  49.  MacMicking, J.D., RJ. North, R. LaCourse, J.S. Mudgett, S.K. Shah, and C.F. Nathan. 1997. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proceedings of the National Academy of Sciences of the United States ofAmerica  94:5243-5248. 50.  Scanga, C.A., V.P. Mohan, K. Tanaka, D. Alland, J.L. Flynn, and J. Chan. 2001. The inducible nitric oxide synthase locus confers protection against aerogenic challenge of both clinical and laboratory strains of Mycobacterium tuberculosis in mice. Infection and Immunity 69:7711-7717.  51.  Chan, J., K. Tanaka, D. Carroll, J. Flynn, and B.R. Bloom. 1995. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis. Infection and Immunity 63:736-740.  52.  Flynn, J.L., CA. Scanga, K.E. Tanaka, and J. Chan. 1998. Effects of aminoguanidine on latent murine tuberculosis. The Journal of Immunology 160:1796-1803.  53.  Nozaki, Y., Y. Hasegawa, S. Ichiyama, I. Nakashima, and K. Shimokata. 1997. Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in human alveolar macrophages. Infection and Immunity 65:3644-3647.  54.  Firmani, M.A., and L.W. Riley. 2002. Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. Journal of Clinical  Microbiology 40:3162-3166.  63  55.  Eriksson, S., BJ. Chambers, and M. Rhen. 2003. Nitric oxide produced by murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv. Typhimurium. Scandinavian Journal of Immunology 58:493-502.  56.  Gazzinelli, R.T., I.P. Oswald, S. Hieny, S.L. James, and A. Sher. 1992. The microbicidal activity of interferon-y-treated macrophages against Trypanosoma cruzi involves an Larginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-p. European Journal of Immunology 22:2501-2506.  57.  Jeon, Y.J., S.B. Han, K.S. Ahn, and H.M. Kim. 2000. Differential activation of murine macrophages by angelan and LPS. Immunopharmacology 49:275-284.  58.  Pendino, K.J., CR. Gardner, S. Quinones, arid D.L. Laskin. 1996. Stimulation of nitric oxide production in rat lung lavage cells by anti-Mac-1 beta antibody: effects of ozone inhalation. American Journal of Respiratory Cell and Molecular Biology 14:327-333.  59.  Goodwin, J.L., M.E. Kehrli, Jr., and E. Uemura. 1997. Integrin Mac-1 and beta-amyloid in microglial release of nitric oxide. Brain Research 768:279-286.  60.  Goodrum, K.J., L.L. McCormick, and B. Schneider. 1994. Group B streptococcusinduced nitric oxide production in murine macrophages is CR3 (CD1 Vol CD 18) dependent. Infection and Immunity 62:3102-3107.  61.  Chan, J., Y. Xing, R.S. Magliozzo, and B.R. Bloom. 1992. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. The Journal of Experimental Medicine 175:1111-1122.  62.  Adams, L.B., M.C Dinauer, D.E. Morgenstern, and J.L. Krahenbuhl. 1997. Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to Mycobacterium tuberculosis using transgenic mice. Tubercle and Lung Disease 78:237-  246. 63.  Cooper, A.M., B.H. Segal, A.A. Frank, S.M. Holland, and I.M. Orme. 2000. Transient loss of resistance to pulmonary tuberculosis in p47(phox-/-) Mice. Infection and Immunity 68:1231-1234.  64.  Wright, S.D., and S.C. Silverstein. 1983. Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygenfromhuriian phagocytes. The Journal of Experimental Medicine 158:2016-2023.  65.  Berton, G., C. Laudanna, C. Sorio, and F. Rossi. 1992. Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gpl50, 95 but not CR3, are able to stimulate the respiratory burst of human neutrophils. The Journal of Cell Biology 116:1007-1017.  64  66.  Zhou, M.J., and E.J. Brown. 1994. CR3 (Mac-1, alpha M beta 2, CD1 lb/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation. The Journal of Cell Biology 125:1407-1416.  67.  Greenberg, S.S., J. Xie, J.J. Spitzer, J.F. Wang, J. Lancaster, M.B. Grisham, D.R. Powers, and T.D. Giles. 1995. Nitro containing L-arginine analogs interfere with assays for nitrate and nitrite. Life Sciences 57:1949-1961.  68.  Rook, G.A.W., J. Steele, S. Umar, and H.M. Dockrell. 1985. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma interferon. Journal of Immunological Methods 82:161-167.  69.  Hibbs, J.B., Jr., Z. Vavrin, and R.R. Taintor. 1987. L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. The Journal of Immunology 138:550-565.  70.  Reif, D.W., and S.A. McCreedy. 1995. N-nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation toward the three nitric oxide synthases. Archives of Biochemistry and Biophysics 320:170-176.  71.  Olken, N.M., and M.A. Marietta. 1993. NG-methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase. Biochemistry 32:96779685.  72.  Mogues, T., M.E. Goodrich, L. Ryan, R. LaCourse, and R.J. North. 2001. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. The Journal of Experimental Medicine.  193:271-280. 73.  Ehlers, M.R.W., and M. Daffe. 1998. Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key? Trends in Microbiology 6:328-335.  74.  Rothlein, R., and T.A. Springer. 1985. Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages. The Journal of Immunology 135:2668-2672.  75.  Reynolds, H.Y., and H.N. Newball. 1974. Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage. The Journal of Laboratory and Clinical Medicine 84:559-573.  76.  Flesch, I., and S.H.E. Kaufmann. 1987. Mycobacterial growth inhibition by interferongamma-activated bone marrow macrophages and differential susceptibility among strains ofMycobacterium tuberculosis. The Journal of Immunology 138:4408-4413.  77.  Sato, K., T. Akaki, and H. Tomioka. 1998. Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal  65  macrophages activated by interferon-gamma (IFN-y) and tumour necrosis factor-alpha (TNF-a). Clinical and Experimental Immunology 112:63-68.  78.  Murray, H.W. 1988. Interferon-gamma, the activated macrophage, and host defense against microbial challenge. Annals of Internal Medicine 108:595-608.  79.  Jungi, T.W., P.G. Lerch, and M. Brcic. 1987. The effect of recombinant interferongamma on human monocyte-derived macrophages. European Journal of Immunology 17:735-738.  80.  Esparza, I., R.I. Fox, and R.D. Schreiber. 1986. Interferon-gamma-dependent modulation of C3b receptors (CRI) on human peripheral blood monocytes. The Journal of Immunology 136:1360-1365.  81.  Speert, D.P., and L. Thorson. 1991. Suppression by human recombinant gamma interferon of in vitro macrophage nonopsonic and opsonic phagocytosis and killing. Infection and Immunity 59:1893-1898.  82.  Sundaram, R., M. Oconnor, M. Cicero, A. Ghaffar, J.D. Gangemi, and E.P. Mayer. 1993. Lipopolysaccharide-induced suppression of erythrocyte binding and phagocytosis via FcyRI, FCyRII, FCyRIII, and CR3 receptors in murine macrophages. Journal of Leukocyte Biology 54:81-88.  83.  van Lenten, B.J., and A.M. Fogelman. 1992. Lipopolysaccharide-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through tumor necrosis factor-alpha. The Journal of Immunology 148:112-116.  84.  Ross, G.D., J.A. Cain, B.L. Myones, S.L. Newman, and P.J. Lachmann. 1987. Specificity of membrane complement receptor type three (CR3) forfi-glucans.Complement 4:61-74.  85.  Ross, G.D., T. R.A., M.J. Walport, T.A. Springer, J.V. Watson, R.H.R. Ward, J. Lida, S.L. Newman, R.A. Harrison, and P.J. Lachmann. 1985. Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leucocyte membrane complement receptor type 3 and the related membrane antigen LFA-1. Blood 66:882-890.  86.  Manca, C , S. Paul, C E . Barry, V.H. Freedman, and G. Kaplan. 1999. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infection and Immunity. 67:74-79.  87.  Piddington, D.L., F.C. Fang, T. Laessig, A.M. Cooper, I.M. Orme, and N.A. Buchmeier. 2001. Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst. Infection and Immunity 69:4980-4987.  66  88.  Chan, J., X. Fan, S.W. Hunter, P.J. Brennan, and B.R. Bloom. 1991. Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infection and Immunity 59:1755-1761.  89.  Sibley, L.D., S.W. Hunter, P.J. Brennan, and J.L. Krahenbuhl. 1988. Mycobacterial lipoarabinomannan inhibits gamma interferon-mediated activation of macrophages. Infection and Immunity 56:1232-1236.  90.  Jackett, P.S., V.R. Aber, and D.B. Lowrie. 1978. Virulence and resistance to superoxide, low pH and hydrogen peroxide among strains of Mycobacterium tuberculosis. Journal of General Microbiology 104:37-45.  91.  Flesch, I.E.A., and S.H.E. Kaufmann. 1988. Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages. Infection and Immunity 56:1464-1469.  92.  Holzer, T.J., K.E. Nelson, V. Schauf, R.G. Crispen, and B.R. Andersen. 1986. Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation. Infection and Immunity 51:514-520.  93.  Mor, N., M.B. Goren, and M.J. Pabst. 1988. Mycobacterium lepraemurium activates macrophages but fails to trigger release of superoxide anion. The Journal ofImmunology 140:3956-3961.  94.  Le Cabec, V., C. Cols, and I. Maridonneau-Parini. 2000. Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD1 lb/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation. Infection and Immunity 68:4736-4745.  95.  Vetvicka, V., B.P. Thornton, and G.D. Ross. 1996. Soluble p-Glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD1 lb/CD 18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. The Journal of Clinical Investigation 98:50-61.  96.  Speert, D.P., and S.C. Silverstein. 1985. Phagocytosis of unopsonised zymosan by human monocyte-derived macrophages: maturation and inhibition by mannan. Journal of Leukocyte Biology 38:655-658.  97.  Giaimis, J., Y. Lombard, P. Fonteneau, CD. Muller, R. Levy, M. Makaya-Kumba, J. Lazdins, and P. Poindron. 1993. Both mannose and beta-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages. Journal of Leukocyte Biology 54:564-571.  98.  Brown, G.D., P.R. Taylor, D.M. Reid, J.A. Willment, D.L. Williams, L. MartinezPomares, S.Y.C. Wong, and S. Gordon. 2002. Dectin-1 is a major P-glucan receptor on macrophages. The Journal of Experimental Medicine 196:407-412.  67  99.  Wright, S.D., and B.C. Meyer. 1986. Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes. The Journal of Immunology 136:1759-1764.  100.  Altieri, D.C, and T.S. Edgington. 1988. A monoclonal antibody reacting with distinct adhesion molecules defines a transition in the functional state of the receptor CD1 lb/CD18 (Mac-1). The Journal of Immunology 141:2656-2660.  101.  Xia, Y., G. Borland, J. Huang, I.F. Mizukami, H.R. Petty, R.F. Todd, 3rd, and G.D. Ross. 2002. Function of the lectin domain of Mac-l/complement receptor type 3 (CD1 lb/CD 18) in regulating neutrophil adhesion. The Journal of Immunology 169:64176426.  102.  Tang, T., A. Rosenkranz, K.J.M. Assmann, MJ. Goodman, J.C Gutierrez-Ramos, M.C Carroll, R.S. Cotran, and T.N. Mayadas. 1997. A role for Mac-1 (CDllb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis. The Journal of Experimental Medicine 186:18531863.  103.  Van Strijp, J.A.G., D.G. Russell, E. Tuomanen, E.J. Brown, and S.D. Wright. 1993. Ligand specificity of purified complement receptor type-3 (CD1 lb/ CD18, a (32, Mac-1) - indirect effects of an ARG-GLY-ASP (RGD) sequence. The Journal ofImmunology 151:3324-3336. m  104.  Astarie-Dequeker, C , E.N. N'Diaye, V. Le Cabec, M.G. Rittig, J. Prandi, and I. Maridonneau-Parini. 1999. The mannose receptor mediates uptake of pathogenic and nonpathogenic mycobacteria and bypasses bactericidal responses in human macrophages. Infection and Immunity. 67:469-477.  105.  Hanano, R., and S.H. Kaufmann. 1995. Nitric oxide production and mycobacterial growth inhibition by murine alveolar macrophages: the sequence of rIFN-gamma stimulation and Mycobacterium bovis BCG infection determines macrophage activation. Immunology Letters 45:23-27.  106.  Ding, A., C. Nathan, and D. Stuehr. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. The Journal of Immunology 141:2407-2412.  107.  Bogdan, C , Y. Vodovotz, J. Paik, Q.W. Xie, and C. Nathan. 1993. Traces of bacterial lipopolysaccharide suppress IFN-gamma-induced nitric oxide synthase gene expression in primary mouse macrophages. The Journal of Immunology 151:301-309.  68  108.  Meng, F., and CA. Lowell. 1997. Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn. The Journal ofExperimental Medicine 185:1661-1670.  109.  Lee, E.S., H.K. Ju, T.C Moon, E. Lee, Y. Jahng, S.H. Lee, J.K. Son, S.H. Baek, and H.W. Chang. 2004. Inhibition of nitric oxide and tumor necrosis factor-alpha (TNFalpha) production by propenone compound through blockade of nuclear factor (NF)kappaB activation in cultured murine macrophages. Biological and Pharmaceutical Bulletin 21':617-620.  110.  Zeidler, P.C, L.M. Millecchia, and V. Castranova. 2004. Role of inducible nitric oxide synthase-derived nitric oxide in lipopolysaccharide plus interferon-gamma-induced pulmonary inflammation. Toxicology and Applied Pharmacology 195:45-54.  111.  Drapier, J.C, J. Wietzerbin, and J.B. Hibbs, Jr. 1988. Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages. European Journal ofImmunology 18:1587-1592.  112.  Frankova, D., and Z. Zidek. 1998. IFN-gamma-induced TNF-alpha is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFNgamma. European Journal ofImmunology 28:838-843.  113.  Chan, E.D., and D.W. Riches. 1998. Potential role of the JNK/SAPK signal transduction pathway in the induction of iNOS by TNF-alpha. Biochemical and Biophysical Research Communications 253:790-796.  114.  Jagannath, C , E. Sepulveda, J.K. Actor, F. Luxem, M.R. Emanuele, and R.L. Hunter. 2000. Effect of poloxamer CRL-1072 on drug uptake and nitric-oxide-mediated killing of Mycobacterium avium by macrophages. Immunopharmacology 48:185-197.  115.  Kilbourn, R.G., and P. Belloni. 1990. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. Journal of the National Cancer Institute 82:772-776.  116.  Akaki, T., H. Tomioka, T. Shimizu, S. Dekio, and K. Sato. 2000. Comparative roles of free fatty acids with reactive nitrogen intermediates and reactive oxygen intermediates in expression of the anti-microbial activity of macrophages against Mycobacterium tuberculosis. Clinical and Experimental Immunology 121:302-310.  117.  Denis, M. 1991. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cellular Immunology 132:150-157.  118.  Fazal, N. 1996. The effect of NG-monomethyl-L-arginine(LNMMA), an NO-synthase blocker on the survival of intracellular BCG within human monocyte-derived macrophages. Biochemistry and Molecular Biology International 40:1033-1046.  69  119.  Majumdar, S., R. Gupta, and N. Dogra. 2000. Interferon-gamma- and lipopolysaccharideinduced tumor necrosis factor-alpha is required for nitric oxide production: tumor necrosis factor-alpha and nitric oxide are independently involved in the killing of Mycobacterium microti in interferon-gamma- and lipopolysaccharide-treated J774A.1 cells. Folia Microbiologica (Praha) 45:457-463.  120.  Adams, L.B., C.K. Job, and J.L. Krahenbuhl. 2000. Role of inducible nitric oxide synthase in resistance to Mycobacterium leprae in mice. Infection and Immunity 68:54625465.  121.  Bermudez, L.E. 1993. Differential mechanisms of intracellular killing of Mycobacterium avium and Listeria monocytogenes by activated human and murine macrophages - the role of nitric oxide. Clinical and Experimental Immunology 91:277-281.  122.  Gomes, M.S., M. Florido, T.F. Pais, and R. Appelberg. 1999. Improved clearance of Mycobacterium avium upon disruption of the inducible nitric oxide synthase gene. The Journal ofImmunology 162:6734-6739.  123.  Rhoades, E.R., and I.M. Orme. 1997. Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infection and Immunity  65:1189-1195. 124.  O'Brien, L., J. Carmichael, D.B. Lowrie, and P.W. Andrew. 1994. Strains of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen intermediates in vitro. Infection and Immunity 62:5187-5590.  125.  Kawakami, K., T. Zhang, M.H. Qureshi, and A. Saito. 1997. Cryptococcus neoformans inhibits nitric oxide production by murine peritoneal macrophages stimulated with interferon-gamma and lipopolysaccharide. Cellular Immunology 180:47-54.  126.  Da Silva, T.R., J.R. De Freitas, Q.C. Silva, CP. Figueira, E. Roxo, S.C Leao, L.A. De Freitas, and P.S. Veras. 2002. Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosome-lysosome fusion. Infection and Immunity 70:5628-5634.  127.  Doi, T., M. Ando, T. Akaike, M. Suga, K. Sato, and H. Maeda. 1993. Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis. Infection and Immunity 61:1980-1989.  128.  Eriksson, S., J. Bjorkman, S. Borg, A. Syk, S. Pettersson, D.I. Andersson, and M. Rhen. 2000. Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production. Cellular Microbiology 2:239-250.  129.  Chinen, T., M.H. Qureshi, Y. Koguchi, and K. Kawakami. 1999. Candida albicans suppresses nitric oxide (NO) production by interferon-gamma (IFN-gamma) and  70  lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. Clinical and Experimental Immunology 115:491-497.  130.  Firmani, M.A., and L.W. Riley. 2002. Mycobacterium tuberculosis CDC 1551 Is Resistant to Reactive Nitrogen and Oxygen Intermediates In Vitro. Infection and Immunity 70:3965-3968.  131.  Garbe, T.R., N.S. Hibler, and V. Deretic. 1999. Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alphacrystallin homolog by exposure to nitric oxide donors. Infection and Immunity. 67:460465.  132.  Rousseau, C , N. Winter, E. Pivert, Y. Bordat, O. Neyrolles, P. Ave, M. Huerre, B. Gicquel, and M. Jackson. 2004. Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosisfromthe cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection. Cellular Microbiology 6:277-287.  133.  Miller, B.H., R.A. Fratti, J.F. Poschet, G.S. Timmins, S.S. Master, M. Burgos, M.A. Marietta, and V. Deretic. 2004. Mycobacteria inhibit nitric oxide synthase recruitment to phagosomes during macrophage infection. Infection and Immunity 2872-2878.  134.  Zahrt, T.C., and V. Deretic. 2002. Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis. Antioxidants and Redox Signaling 4:141-159.  135.  Cooper, A.M., J.E. Pearl, J.V. Brooks, S. Ehlers, and I.M. Orme. 2000. Expression of the nitric oxide synthase 2 gene is not essential for early control of Mycobacterium tuberculosis in the murine lung. Infection and Immunity 68:6879-6882.  136.  Dimmeler, S., and A.M. Zeiher. 1997. Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide. Nitric Oxide 1:275-281.  137.  Dalton, D.K., L. Haynes, CQ. Chu, S.L. Swain, and S. Wittmer. 2000. Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. The Journal of Experimental Medicine 192:117-122.  138.  Bulotta, S., R. Barsacchi, D. Rotiroti, N. Borgese, and E. Clementi. 2001. Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha. A novel feedback mechanism regulating cell death. The Journal of Biological Chemistry 276:6529-6536.  139.  Fratazzi, C , R.D. Arbeit, C. Carini, M.K. Balcewicz-Sablinska, J. Keane, H. Kornfeld, and H.G. Remold. 1999. Macrophage apoptosis in mycobacterial infections. Journal of Leukocyte Biology 66:763-764.  71  140.  Sly, L.M., S.M. Hingley-Wilson, N.E. Reiner, and W.R. McMaster. 2003. Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1. The Journal of Immunology 170:430-437.  141.  Hu, C , T. Mayadas-Norton, K. Tanaka, J. Chan, and P. Salgame. 2000. Mycobacterium tuberculosis infection in complement receptor 3-deficient mice. The Journal of Immunology 165:2596-2602.  142.  Bermudez, L.E., J. Goodman, and M. Petrofsky. 1999. Role of complement receptors in uptake of Mycobacterium avium by macrophages in vivo: evidence from studies using CD18-deficient mice. Infection and Immunity 67:4912-4916.  143.  Schlesinger, L.S., S.R. Hull, and T.M. Kaufman. 1994. Binding of the terminal mannosyl units of lipoarabinomannanfroma virulent strain of Mycobacterium tuberculosis to human macrophages. The Journal of Immunology 152:4070-4079.  144.  Pugin, J., D. Heumann, A. Tomasz, V.V. Kravchenko, Y. Akamatsu, M. Nishijima, M.P. Glauser, P.S. Tobias, and R.J. Ulevitch. 1994. CD14 is a pattern recognition receptor. Immunity 1:509-516.  72  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items