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Role of Bcl-2 proteins in neutrophil activation and delayed apoptosis in crystal-induced arthritis Higo, Tobi T. 2008

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Role of BCL-2 Proteins in Neutrophil Activation and Delayed Apoptosis in Crystal-Induced Arthritis  by TOBI T. HIGO B.Sc., Simon Fraser University, 2001.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2008 © Tobi T. Higo, 2008  ABSTRACT The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases.  ii  TABLE of CONTENTS  ABSTRACT.................................................................................................................. ii TABLE of CONTENTS .............................................................................................. iii LIST of TABLES........................................................................................................ vii LIST of FIGURES ..................................................................................................... viii LIST of ABBREVIATIONS........................................................................................ ix ACKNOWLEDGEMENTS.......................................................................................... x 1  INTRODUCTION ................................................................................................ 1  2  BACKGROUND .................................................................................................. 3 2.1  Crystal-Induced Arthritis .............................................................................. 3  2.1.1  Calcium Pyrophosphate Dihydrate Deposition Disease ....................... 4  2.1.2  Mechanism of CPPD Crystal Deposition ............................................. 6  2.1.3  Proposed Mechanisms of CPPD-Induced Inflammation ...................... 6  2.1.4  Neutrophil-Mediated Inflammation ...................................................... 7  2.2  Neutrophil-Crystal Interactions .................................................................... 8  2.2.1  Crystal-Induced Neutrophil Responses................................................. 9  2.2.1.1  Phagocytosis ..................................................................................... 9  2.2.1.2  Generation of Reactive Oxygen Species (Respiratory Burst)......... 10  2.2.1.3  Lysosomal Release of Proteolytic Enzymes (Degranulation) ........ 11  2.2.1.4  Effect of Opsonization/Protein Adsorption on Crystal Surfaces .... 11  2.2.2  Neutrophil Activation Signalling........................................................ 12  2.2.2.1  Involvement of PI3-Kinase Pathway .............................................. 16  2.2.2.2  Involvement of the ERK 1/2 and p38 MAP Kinase Pathways ....... 18 iii  2.2.2.3 2.3  Apoptosis .................................................................................................... 21  2.3.1  Constitutive Neutrophil Apoptosis ..................................................... 25  2.3.2  Delay of Neutrophil Apoptosis Signalling.......................................... 27  2.3.2.1  Involvement of the PI3-Kinase Pathway ........................................ 30  2.3.2.2  Involvement of the ERK 1/2 Pathway ............................................ 30  2.3.2.3  Involvement of the p38 MAP Kinase Pathway............................... 31  2.3.2.4  Involvement of NF-κB Transcription Factor Signalling ................ 32  2.3.3  3  Involvement of Ca2+ Secondary Signalling ................................... 19  Bcl-2 Family Proteins ......................................................................... 33  2.3.3.1  Bcl-2 Protein Function in Other Cell Types ................................... 35  2.3.3.2  Bcl-2 Protein Function in Neutrophils............................................ 38  2.3.3.3  Proposed Mechanism of Bcl-2 Protein Balance in Neutrophils ..... 43  EXPERIMENTAL.............................................................................................. 44 3.1  Materials ..................................................................................................... 44  3.1.1  Chemicals and Solvents ...................................................................... 46  3.1.2  Stock Solutions ................................................................................... 47  3.1.3  Labware............................................................................................... 48  3.2  Equipment ................................................................................................... 49  3.2.1  X-Ray Powder Diffractometer............................................................ 49  3.2.2  Light Microscopy................................................................................ 49  3.2.3  Hemacytometer ................................................................................... 49  3.2.4  UV-Vis Spectrophotometer ................................................................ 50  3.2.5  Fluorometer......................................................................................... 50  3.2.6  SDS-PAGE/Electrotransfer................................................................. 50 iv  3.2.7  ECL Detection/Autoradiography ........................................................ 50  3.2.8  Band Densitometry ............................................................................. 51  3.3  Preparation of Calcium Pyrophosphate Dihydrate Crystals ....................... 51  3.3.1  Synthesis of Calcium Dihydrogen Pyrophosphate (CDPP)................ 51  3.3.2  Synthesis of Calcium Pyrophosphate Dihydrate (CPPD - triclinic) ... 52  3.3.3  X-Ray Powder Diffraction Analysis ................................................... 53  3.4  In Vitro Neutrophil Studies......................................................................... 53  3.4.1  Opsonization of CPPD Crystals.......................................................... 53  3.4.2  Isolation of Neutrophils from Peripheral Blood ................................. 54  3.4.3  In Vitro Treatments............................................................................. 54  3.4.4  Cellular Apoptosis Assays .................................................................. 55  3.4.4.1  DNA Fragmentation Assay............................................................. 55  3.4.4.2  Caspase-3 Activation Assay ........................................................... 56  3.5  4  Western Analysis ........................................................................................ 58  3.5.1  Preparation of Whole Cell Lysates for Western Analysis .................. 58  3.5.2  Protein Determination......................................................................... 59  3.5.3  Gel Preparation ................................................................................... 59  3.5.4  SDS-PAGE/Electrotransfer................................................................. 60  3.5.5  Immunoblotting................................................................................... 61  3.5.6  Enhanced Chemiluminescence/Autoradiography............................... 62  3.6  Band Densitometry Analysis ...................................................................... 63  3.7  Statistical Analysis...................................................................................... 63  RESULTS ........................................................................................................... 64 4.1  Purity of Synthesized CPPD Crystals ......................................................... 64 v  4.2 4.2.1  DNA Fragmentation Assay................................................................. 65  4.2.2  Caspase-3 Activation Assay ............................................................... 67  4.3  5  Cellular Apoptosis Assays .......................................................................... 65  Western Analysis of Bcl-2 Proteins............................................................ 68  4.3.1  Expression of BCL-XL ....................................................................... 71  4.3.2  Expression of MCL-1 ......................................................................... 73  4.3.3  Expression of BIM-EL........................................................................ 74  4.3.4  Expression of BAX-α ......................................................................... 79  4.3.5  Expression of A1/BAD ....................................................................... 80  DISCUSSION ..................................................................................................... 82 5.1  Purity of Synthesized CPPD Crystals ......................................................... 82  5.2  Cellular Apoptosis Assays .......................................................................... 83  5.2.1  DNA Fragmentation............................................................................ 83  5.2.2  Caspase-3 Activation Assay ............................................................... 84  5.3  Western Analysis of Bcl-2 Proteins............................................................ 85  5.3.1  Expression of BCL-XL ....................................................................... 86  5.3.2  Expression of MCL-1 ......................................................................... 87  5.3.3  Expression of Bim-EL ........................................................................ 88  5.3.4  Expression of BAX-α ......................................................................... 89  5.3.5  Expression of A1/BAD ....................................................................... 90  6  SUMMARY AND CONCLUSIONS ................................................................. 92  7  REFERENCES ................................................................................................... 96  8  APPENDIX....................................................................................................... 120  vi  LIST of TABLES  Table 1. List of XRPD D-Spacing values for CPDD and CPPD…………………….65 Table 2. List of Bcl-2 Protein Targets Examined……………………………………69 Table 3. Summary of Statistical Analysis on Western Blot Experiments…………...77  vii  LIST of FIGURES  Figure 1. Summary of Anti-Microbial Reagents Produced by Neutrophils…………10 Figure 2. Summary of the Death Receptor-Mediated Apoptosis Pathway…………..22 Figure 3. Summary of the Mitochondrial-Mediated Apoptosis Pathway……………24 Figure 4. Members of the Bcl-2 Family Proteins……………………………………35 Figure 5. DNA Fragmentation Assay: Representative Data…………………………67 Figure 6. Caspase-3 Fluorometric Assay: Representative Data……………………...68 Figure 7. Representative Data for Bcl-XL Western Analysis Experiments…………71 Figure 8. Representative Data for Mcl-1 Western Analysis Experiments…………...73 Figure 9. Representative Data for Bim-EL Western Analysis Experiments………...74 Figure 10. Representative Data for Bim (Isoforms) Western Analysis Experiments..75 Figure 11. Representative Data for Bax-α Western Analysis Experiments…………79  viii  LIST of ABBREVIATIONS 2θ  2 theta, X-ray diffraction angle  °C  Degrees Celsius  CDPP  Calcium Dihydrogen Pyrophosphate  CPPD  Calcium Pyrophosphate Dihydrate  fMLP  Formyl-methionyl-leucyl-phenylalanine  GM-CSF  Granulocyte Macrophage-Colony Stimulating Factor  LPS  Lipopolysaccharide  M-CSF  Macrophage-Colony Stimulating Factor  MSUM  Monosodium Urate Monohydrate  ROI  Reactive Oxygen Intermediates  SOD  Superoxide Dismutase  TBS/TBST  Tris Buffered Saline / Tris Buffered Saline w/ Tween  TNF-α  Tumor Necrosis Factor – alpha  XRPD  X-ray powder diffraction  ix  ACKNOWLEDGEMENTS  I would like to thank my supervisor, Dr. Helen Burt, for her patient wisdom and guidance over the course of this project. I would also like to thank my committee members for their direction and influence: Drs. Vincent Duronio and Alice Mui. I am grateful to Drs. Christopher Tudan and Steven Pelech and the staff at Kinexus for providing training, proteomic screening and technical assistance. Special thanks to Dr. Horton Middletoe for helping me to keep it real – especially when everything goes wrong. Thank you to the members of the Burt lab: Kevin Letchford, Chiming Yang, Karen Long, Melanie ter Borg, Wes Wong, Chris Springate, Jason Zastre, Linda Liang, Katherine Haxton, Mable Shi and John Lu for almost believing that I really was at Jack Bell. And many thanks to the members of the Duronio lab for allowing my many trespasses and borrowing of things big and small. Thanks to my family and friends for providing an even keel of focus and distraction to keep me sane for the past 4 years… ok, 5 years… but who’s counting???  x  1  INTRODUCTION  The interaction of neutrophils with crystals of calcium pyrophosphate dihydrate (CPPD) results in a cascade of signalling and morphological events associated with the inflammatory condition known as pseudogout. CPPD deposition disease afflicts between 3-6% of the population, but is far more common in the elderly. The injury to joint tissues as a result of this inflammatory state is due mainly to the recruitment and activation of peripheral blood neutrophils. Neutrophils are professional phagocytes and serve as essential mediators of systemic and local inflammation, as early activation of these cells results in oxidase activation and degranulation responses. Normally, as part of the resolution of inflammation, infiltrating neutrophils undergo spontaneous apoptosis and are cleared by resident macrophages. In the case of CPPD crystal deposition disease, the interaction of neutrophils with crystals serves to delay apoptosis leading to prolonged inflammation and further tissue damage. It has been shown that this prosurvival mechanism is mediated through the ERK1/2 and PI3-K kinases upstream of caspase 3 and p38 MAP kinase. It has also been shown through inhibition of protein synthesis (by topoisomerase inhibitors) that this prosurvival signal is dependent (at least in part) on upregulation of anti-apoptotic proteins. Bcl-2 family member proteins are known to control the apoptotic process in all known cell types. To date, the exact interplay between these Bcl-2 proteins in neutrophils is not well known. The goal of the study was therefore to examine the role of select Bcl-2 family member proteins in the crystal-induced repression of neutrophil apoptosis. Via cellular apoptosis assays and Western analysis, we examined the expression of neutrophil Bcl-2 proteins in vitro in the presence of CPPD crystals.  1  Significance: An elucidation of the signalling mechanisms involved in the crystalassociated delay of neutrophil apoptosis and the identification of the key Bcl-2 proteins involved in the delay would possibly offer new specific targets for drug therapies in the future. Of added interest, several other prominent types of inflammatory disease (diabetes mellitus type II, inflammatory bowel disorder, other arthritic diseases, etc.) are also known to be largely neutrophil mediated. Thus, a better understanding of neutrophil apoptosis might aid in the advancement of treatments for these diseases as well.  2  2 2.1  BACKGROUND Crystal-Induced Arthritis  Arthritis is one of the oldest, well-known and most common of human ailments affecting millions of people world-wide, but due to the fact that it is not usually a fatal disease, very little of the medical research community works on curing this crippling disease (1). However, the impact of arthritis cannot be overlooked; as reported by the Arthritis Foundation in 2005 it is estimated that 66 million people in the United States (nearly 1 out of 3 adults) suffered from arthritis or joint pain. It is the nation’s leading cause of disability in people over the age of 15 and is second only to heart disease as a cause of work disability. Arthritis results in more than 39 million visits to physicians and over a half-million hospitalizations each year as a cost exceeding $86.2 billion US annually. Crystal-induced arthropathies are a specific subset of the many different types of rheumatic conditions that plague the population. They are generally classified by the deposition of an agonistic crystalline agent in the articular or periarticular tissues that leads to an acute inflammatory state and subsequent tissue/joint damage (2). These articular diseases are associated with various factors both genetic and environmental, however, they are generally considered as metabolic or endocrine syndromes associated with rheumatic states (3). Of the crystal-induced arthropathies, those caused by microcrystals of monosodium urate monohydrate (gout) and calcium pyrophosphate dihydrate (pseudogout) have been studied the most. Historically, gout is one of the most well characterized diseases in all of medicine. In the mid-17th century, Antoni van Leeuwenhoek, the inventor of the microscope, was the first to document the presence of MSUM crystals taken from the draining tophus (a crystalline 3  deposit of sodium urate found in gouty joints) of an arthritic subject (4). However, he was unaware of the chemical composition of these crystals, as uric acid was not discovered until 1776 by Scheele (5). As early as 1876, A.B. Garrod remarked on the consistent presence of MSUM crystal deposition in hand-cut tissue samples taken from patients thought to be suffering from gout (6). And in 1897, Viennese dermatologist Gustav Riehl observed the phagocytosis of MSUM crystals by polymorphonuclear leukocytes and monocytes extracted from recently ruptured human skin tophi via polarized light microscopy (7). At the dawn of the 20th century, German scientists Wilhelm His, Jr. and Max Freudweiler discovered that injection of synthetic MSUM and other crystals into humans and several other species resulted in immediate inflammation and subsequent tophi that were histologically indistinguishable from those observed in natural gout (8, 9). Other known crystal-induced arthropathies include inflammatory states caused by microcrystals of various calcium phosphates, adrenocorticosteroid esters, cysteines, tyrosines, and cholesterol (1). Of these non-MSUM crystals, inflammation due to microcrystals of calcium pyrophosphate dihydrate has been studied the most.  2.1.1  Calcium Pyrophosphate Dihydrate Deposition Disease  In 1961, McCarty and colleagues discovered crystals of calcium pyrophosphate dihydrate (CPPD) in wet preparations made from the synovial fluids of suspected gout patients under compensated polarized light microscopy. These crystals showed some similarity to MSUM crystals existing as both monoclinic (unit cell with one obtuse angle and two right angles) and triclinic (unit cell with three obtuse angles) isoforms as well as  4  displaying “twinning” or pairing properties. However, they were morphologically distinct from MSUM crystals with respect to bifringence and extinction patterns. Hence, the term “pseudogout” was coined (10) but subsequent studies have shown that many patients do not experience the acute arthritic episodes that are the trademark of true gout. Other terms such as “chondrocalcinosis” (suggested by Zitnan and Sitaj based on the radiological characteristics of CPPD-associated arthritis) (11) and “pyrophosphate arthropathy” (coined by Currey) (12) have been used in the past. However, these terms are overgeneralized and do not sufficiently describe the disease state. Thus, the term “calcium pyrophosphate dihydrate deposition disease”, being far more specific, is currently the most widely accepted idiom. Calcium pyrophosphate dihydrate deposition disease is marked by acute or sub-acute arthritic episodes caused by the inflammation triggered by the formation and contact of CPPD crystals with infiltrating neutrophils in the synovial fluid of appendicular joints. These attacks are self-limiting, with the resolution of inflammation occurring between one day to four weeks, and typical episodes can be as severe as true gout, however, they usually take longer to reach peak intensity and are not as painful and disabling (1). These attacks occur primarily in the knee joint of pseudogout patients, although inflammation can spread to nearby “daughter” joints resulting in a “cluster” attack common in crystal deposition diseases. Overall, the end result of pseudogout attacks is two-tiered: one, the pain and disability associated with each acute attack and two, the cumulative effect of these acute attacks and/or chronic inflammation leading to tophii formation, permanent bone and cartilage damage and degenerative joint disease resulting in the incapacity or dysfunction of the affected joint (13).  5  2.1.2  Mechanism of CPPD Crystal Deposition  The accepted mechanism of CPPD crystal deposition is as follows. CPPD microcrystals form in the synovial fluid of appendicular joints through various modes of regulatory dysfunction. In either the setting of systemic dysfunction (ie: kidney or liver dysfunction) leading to an increase in inorganic pyrophosphate-containing metabolic wastes in the blood and the subsequent increase in localized concentration of inorganic pyrophosphate into the joint space. Or, in the setting of localized deregulation of inorganic pyrophosphate production in the articular chondrocytes comprising the cellular component of the hyaline cartilage in the joint (14). In either case, the presence of inorganic pyrophosphate is necessary for CPPD crystal formation as it is the anionic component of the crystals. The overproduction of inorganic pyrophosphate may occur due to several different factors. Under favourable conditions, articular cartilage vesicles may act as sites of CPPD crystal formation (15). Hereditary cartilage matrix abnormalities may precede CPPD crystal formation and deposition in these regions is common (16). Also, abnormalities in growth factors and cytokines have been shown in CPPD deposition disease and induce changes in chondrocytes and matrix that favour crystal nucleation and growth (17).  2.1.3  Proposed Mechanisms of CPPD-Induced Inflammation  The inflammatory response observed in CPPD deposition disease is thought to arise from the formation or shedding of crystals into the joint space where they are opsonized by  6  plasma proteins due to the net negative charge present at the crystal surface. Such proteins as IgG, complement factors and Hagemann’s factor (a blood clotting protein) readily bind to the surface of CPPD crystals thereby enhancing their interactions with cells (18). Following protein binding, opsonized crystals interact with pro-inflammatory cells such as neutrophils and macrophages leading to the generation and release of cytokines and chemoattractants such as Tumor Necrosis Factor-α, Interleukin-8 and GMCSF that attract large numbers of neutrophils into the joint space to remove the crystals (19). It has been shown that the interaction of CPPD crystals with the neutrophil cell membrane leads to activation of cellular inflammatory responses, such as respiratory burst and phagocytosis, and prosurvival signalling within the cells, thereby exacerbating and prolonging inflammation. Typically, the CPPD crystals, which are highly insoluble at physiological pH levels, are not destroyed by neutrophils, leading to eventual neutrophil cytolysis and complete release of their inflammatory components (19).  2.1.4  Neutrophil-Mediated Inflammation  Neutrophils, or polymorphonuclear leukocytes, are a class of short-lived white blood cells that play a key role in the early stages of the inflammatory response to infection, particularly infections of rapidly dividing bacteria, yeast and fungi. Human neutrophils are produced at a rate of 1-2 x 1011 cells/day and survive for approximately 24-36 hours before undergoing spontaneous apoptosis (20). This rapid turnover and efficient clearance of such a large number of effete (terminally differentiated) cells is a remarkable homeostatic action designed to seek out, neutralize and remove infection quickly without prolonged, sustained inflammation which could be deleterious to host tissue and function. 7  Neutrophil production is escalated during infection by the action of cytokines such as GM-CSF and Interleukins leading to significant neutrophilia. Peripheral blood neutrophils leave the circulation under the influence of chemotactic factors such as complement components (C5a) and chemokines (IL-8) released by local tissue leukocytes at the site of infection (21). Upon reaching the infected tissue, they begin to phagocytose and kill ingested pathogens. To optimize their microbicidal function, neutrophil lifespan is extended by a range of inflammatory mediators such as cytokines and bacterial cell components (22). Following microbe killing, the neutrophil dies by apoptosis and is phagocytosed by macrophages, preventing the loss of toxic neutrophil contents and tissue damage. It should be noted that the correct regulation of the spontaneous apoptotic programme of neutrophils is essential to the maintenance of neutrophil numbers in circulation, efficient removal of pathogens and resolution of the inflammatory response (23).  2.2  Neutrophil-Crystal Interactions  To date, not much is known about the exact mechanism by which crystals activate neutrophil inflammatory and pro-survival signalling. Some authors have suggested that it may be by non-specific charge-related interactions at the cell surface. However, due to the fact that CPPD crystals are opsonized by plasma proteins in vivo, it is more likely that the interactions are more specific and that signalling might be achieved via IgG-Fcgamma receptor binding (18). Interestingly, Dr. Burt and colleagues have shown that neutrophils are activated by “naked” CPPD crystals, however, the activation of neutrophils by plasma-opsonized crystals is far greater, indicating that the role of plasma 8  proteins on the crystal surface is rather important to the nature of neutrophil-crystal interactions.  2.2.1  Crystal-Induced Neutrophil Responses  The physical aggregation of CPPD crystals with the cellular membrane of peripheral neutrophils leads to several modes of neutrophil activation. In vivo, neutrophils that encounter foreign bodies will identify the bodies as a potentially pathogen via cell surface receptor activation that begins a cascade of event within the cell. The neutrophil initiates phagocytosis to engulf the pathogen at which point it is brought into contact with vesicles containing a variety of anti-microbial agents such as lysozyme (degranulation) and reactive oxygen species (respiratory burst) in an effort to destroy the pathogen within the phagolysosome (24). In the case of CPPD, neutrophils follow their regular anti-microbial path, but are unable to dissolve the crystals leading to an acute and sustained inflammation. Eventually, the neutrophils suffer cytolysis due crystal-associated disruption of the membrane spilling the reactive inflammatory components into the extracellular space exacerbating the inflammatory condition even further.  2.2.1.1 Phagocytosis Phagocytosis is the process in which neutrophils, and other phagocytes, internalize foreign matter. Contact of microbial surface markers and or opsonized IgG with the Fc-γ receptor on the neutrophil membrane initiate the action. The engulfment results in the compartmentalization of the foreign matter within a specialized vesicle called the 9  phagosome. It is in this vesicle that the neutrophil introduces microbicidal agents to the microbe in order to destroy it via fusion of lysosome-containing vesicles to create a phagolysosome. In the case of CPPD crystals, due to their size, often they cannot be completely phagocytosed, resulting in only partial engulfment and eventual cytolysis of the neutrophil (25). Although it is not known exactly by which means CPPD crystals activate neutrophils to initiate phagocytosis, it presumably is mediated by opsonized proteins such as complement and IgG found adsorbed to the crystal surface in vivo. Phagocytosis may also be mediated by non-specific receptor rafting in the neutrophil membrane via charge interaction with crystal surfaces.  2.2.1.2 Generation of Reactive Oxygen Species (Respiratory Burst) Another mechanism utilized by neutrophils to destroy microbes is the generation and release of toxic reactive oxygen molecules. This process, known as respiratory burst, is activated by the binding of aggregated antibodies on the microbe surface to neutrophil Fcγ receptors. Upon activation, the neutrophil abruptly increases its oxygen uptake in preparation for producing these molecules. The key species of toxic oxygen molecules that neutrophils create are hydrogen peroxide (H2O2), superoxide anion (O2-), and nitric oxide (NO) which are directly damaging to bacteria. The biochemical mechanisms involved in the production of these species are summarized in Figure 1.  10  (Figure 1 was removed due to copyright restriction. The removed figure was: Summary of Anti-Microbial Reagents Produced by Neutrophils. Refer to website URL: www.uni-leipzig.de).  2.2.1.3 Lysosomal Release of Proteolytic Enzymes (Degranulation) In addition to microbicidal oxygen species, neutrophils also produce a range of enzymes to destroy bacteria. Lysozyme is a 14.4 kDa enzyme that damages the cell walls of  Gram-positive bacteria by catalyzing the hydrolysis of 1,4-beta-linkages between Nacetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycans and between N-acetyl-D-glucosamine residues in chitodextrins. It is also found in abundance in a number of secretions, such as tears, saliva and mucus. In neutrophils, lysozyme is kept safely housed in specialized vesicle compartments called lysosomes. Upon phagocytosis of foreign bodies such as bacteria, the lysosomes mobilize within the neutrophil and fuse with the bacteria-containing phagosome, releasing their contents to aid in the destruction of the pathogen (26).  2.2.1.4 Effect of Opsonization/Protein Adsorption on Crystal Surfaces It has been shown that, in vivo, crystals do indeed become coated by many plasma proteins including the immunoglobulins IgG and IgM (18). The effect of protein binding on the surface of inflammatory crystals such as calcium pyrophosphate dihydrate has been well documented by Dr. Burt’s research group. In in vitro studies, crystals of CPPD 11  or MSUM pre-coated by plasma proteins showed enhanced activation of neutrophil inflammatory responses when compared to activation by naked crystals. Respiratory burst, degranulation, and intracellular signalling activation all showed an increased response to plasma-opsonized crystals (19, 24, 25, 27). Such observations would suggest that, in vivo, the effect of protein binding on CPPD and other inflammatory crystals, serves to increase the inflammatory reaction by immune cells.  2.2.2  Neutrophil Activation Signalling  Plasma opsonized CPPD crystals have been shown to activate neutrophils and lead to the generation of superoxide anions and the release of proteolytic enzymes via degranulation. (1). The full mechanism by which these crystals activate neutrophils and elicit these responses is unknown but there are numerous studies identifying the contribution of key signaling enzymes in neutrophils and some elucidation of links between these enzymes (see following sections). The role of signaling enzymes in cell activation is usually determined by studying the time course of the activation state of the enzymes following challenge of the cells with the potential activating agent. For neutrophils, these agents might be fMLP, PMA, platelet activating factor (PAF), opsonized zymozan or crystals (such as MSUM or CPPD), immunoglobulin complexes or growth factors and cytokines. More specific analysis may then determine the exact identity of the protein band following molecular weight analysis and subsequent immunoblotting with antibodies specific for the particular protein (Western blotting).  Usually, specific inhibitors of enzymes may be used to  12  determine the involvement of the particular enzyme in neutrophil activation by the activating agent. Signal transduction cascades may then be deduced by using the specific inhibitor to block the activity of one enzyme with Western blotting to study the activation state of a second enzyme. For crystal induced neutrophil activation, the current state of knowledge proposes that following G protein coupling to phospholipase C (19), tyrosine phosphorylation enzymes are turned on (28), and a cascade of signalling systems are activated. Phospholipase C activation (19) causes intracellular calcium concentration increases (29) and the activation of protein kinase C (PKC) (30). Also activated are the central signaling enzymes, PI3kinase (31), p38 MAP kinase (27), Erk1/2 (32), and p70 S6 kinase (33). Although specific inhibitors to all the enzymes may strongly inhibit CPPD-induced neutrophil activation, the exact signal transduction pathways linking these enzymes remains unknown. All these pathways are discussed in more detail in the following sections. Approximately 30% of all proteins in mammalian cells are in a state of phosphorylation, often at more than one site. The phosphorylation and dephosphorylation of tyrosine residues on proteins by kinases and phosphatases (respectively) is associated with numerous aspects of the control of protein function in cells (34). These functions may include cell cycle control, general homeostasis and repair, proliferation, differentiation or functional activation (34, 35, 36). The usual site for protein phosphorylation is on either a serine/threonine or tyrosine residue.  Often the phosphorylation enzyme itself is  activated by a kinase so that for example, the activation enzyme MAP kinase occurs via the phosphorylation of both threonine and tyrosine residues by MAP kinase kinase which 13  itself has to first be phosphorylated on serine residues by Map kinase kinase kinase (36). These inter-linked and dependent processes may act as signal transduction pathways for specific cellular responses. Antibodies are available that bind to tyrosine phosphorylated residues in proteins and the use of these antibodies in Western blotting techniques allows for the non-specific identification of the tyrosine phosphorylation of numerous proteins that separate during polyacrylamide gel electrophoresis protein analytical methods. This allows for evaluation of the general phosphorylation effect of agents that might activate cells. The tyrosine phosphorylation of numerous proteins in neutrophils has been previously described.  The chemotactic peptide fMLP has been shown to tyrosine  phosphorylate numerous proteins that normally lead to activation in many cells, including ERK1/2, p38 MAPK, Akt/PKB and JNK (37, 38, 39). In particular the SRC family of kinases Hck and Fgr have been reported to be the likely kinases responsible for these phosphorylation events since a specific inhibitor (or the absence of these kinases) reduced the fMLP-induced phosphorylation of certain target proteins. Furthermore, inhibition (or the absence of) Hck or Fgr in neutrophils results in the full inhibition of degranulation and respiratory burst processes, demonstrating the critical importance of tyrosine phosphorylation activation pathways in these cells (37). Since neutrophils are critically involved in inflammatory processes in crystal-induced arthritis, the role of tyrosine phosphorylation has been extensively investigated in crystalinduced activation of these cells (28, 38, 40-44). Both MSUM and CPPD crystals induce the tyrosine phosphorylation of numerous neutrophil proteins with similar patterns of phosphorylation but patterns that are quite different from those induced by fMLP, opsonized zymozan C5A or LTB4 (44). Interestingly, anti-gout drugs that target  14  microtubule function in cells such as colchicines, inhibited these crystal-induced patterns of tyrosine phosphorylation whereas more classic anti-inflammatory drugs such as indomethacin do not (43). MSUM crystal induced changes are more pronounced than those induced by CPPD (44) so these crystals have been studied more frequently in tyrosine phosphorylation studies. MSUM crystals have been shown to be particularly effective in activating the tyrosine kinase Syk in neutrophils (40, 42) although the specific role of this kinase in regulating neutrophil function is unknown. The soluble neutrophil activators, fMLP, PAF and LTB4 are all known to interact directly with specific receptors on the plasma membrane of neutrophils. These agents have been shown to achieve peak tyrosine phosphorylation effects in neutrophils within 2 minutes, a time course that is rapid enough to match the needs of the physiological response function of these cells, namely, respiratory burst, superoxide anion generation and degranulation responses (45, 46). Indeed, inhibition of tyrosine kinases by agents such as ST639, genistein, erbstain or dihydroxycinnamide have been shown to inhibit superoxide anion generation in neutrophils (47-51). Similarly, the importance of tyrosine phosphorylation in controlling crystal-induced neutrophil activation has been demonstrated in studies that used inhibitors of tyrosine kinases (28, 48). The tyrosine kinase inhibitors erbstatin, methyl-2,5-hydroxycinnamate and lavendustin C have all been shown to inhibit crystal induced tyrosine phosphorylation in neutrophils and to inhibit inflammatory superoxide anion generation and degranulation responses. These various studies have demonstrated the central role of tyrosine phosphorylation in the signal transduction cascades leading to the inflammatory responses of neutrophils to 15  crystals. However, they also show that tyrosine kinases may offer an effective target for agents that might have therapeutic effects as anti-inflammatory agents (rather than disease modifying agents such as allopurinol) in the treatment of acute and chronic crystal-induced arthritis. 2.2.2.1 Involvement of PI3-Kinase Pathway Although this enzyme has been extensively studied as an important component of the signal transduction mechanisms involved in apoptosis regulation, PI3-Kinase is also a central signalling enzyme in neutrophil activation. There are two well-characterized specific inhibitors of this enzyme, wortmannin and LY294002. These compounds work in the nanomolar concentration range to inhibit PI3-Kinase and they have been used extensively to block enzyme function in order to look at potential downstream enzyme activation levels. PI3-Kinase has been implicated as one of the major signalling enzymes involved in neutrophil responses to cytokines and crystals (31, 52). The cytokines Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Tumor Necrosis Factor (TNF) are present at very low concentrations in the body and are usually thought to prime neutrophils only. Such priming has been shown to result in a larger respiratory burst/superoxide anion release and degranulation response in neutrophils subsequently activated by crystals (53). However these agents can activate neutrophils at high concentrations to cause superoxide anion release.  This cellular  activation has been shown to require the activation PI3-Kinase and the inhibitors wortmannin or LY294002 are able to inhibit both the activation of the enzyme and the physiological response (superoxide anion release) (54). Interestingly, the same study identified Akt/PKB and ERK activation as being dependent on PI3-Kinase activation, 16  directly linking these enzymes in the signal transduction pathway elicited by GM-CSF and TNF. However, p38 MAPK activation in neutrophils was shown to not be dependent on PI3-Kinase activation (54). PI3-Kinase has also been implicated in neutrophil migration into inflamed tissues (trafficking) (55, 56). Trafficking occurs when neutrophils are exposed to chemoattractant molecules and involves selectin/intergrin mediated binding to capillary walls followed by migration into the tissue. These neutrophil responses require considerable structural changes within the neutrophil, usually without superoxide anion and degranulation responses, and activation pathways are probably similar to those encountered in priming scenarios. Both PAF and the chemoattractant fMLP have been shown to activate ERK and p38 MAPK, to cause intracellular calcium concentration changes and to subsequently activate PKC (57). However, although both PAF and fMLP activated PI3-Kinase, the effect of wortmannin and LY294002 on the activation of ERK, p38 and MAPK by these two agents was quite different (57). The role of PI3-Kinase in crystal-induced neutrophil activation has been clearly defined. (30, 31, 33). The enzyme is strongly activated by both MSUM and CPPD crystals and wortmannin and LY294002 inhibit the activation of PI3-Kinase as well as the superoxide anion release and the degranulation responses of neutrophils (31).  Crystal-induced  Akt/PKB activation but not p70S6K activation in neutrophils has been shown to be dependent on PI3-Kinase (33). There is strong evidence that the enzymatic products of PI3-Kinase (PIP2 and PIP3) can directly bind to p47 and p40- phox, which are the assembly blocks of the NADPH oxidase that generates superoxide anion at the neutrophil plasma membrane (58). 17  2.2.2.2 Involvement of the ERK 1/2 and p38 MAP Kinase Pathways Mitogen activated protein kinases (MAPK’s) have been shown to be essential components of signal transduction pathways in numerous cell types. There are three main sub groups of MAPK’s, p44/42 (previously known as extracellular-signal-regulated kinase ERK1/2), p38 MAPK and Stress Activated Protein Kinase (SAPK). These kinases are activated by many agonists such as growth factors, hormones, UV or heat shock, inflammatory cytokines (e.g. TNF) and osmolarity changes.  These kinases usually  transmit signals to the nucleus via activation factors such as AP-1 to regulate cell function following activation of both G-protein and non-G protein coupled plasma membrane receptors (59). These kinases are strongly associated with cell growth, differentiation and apoptosis. However they are also involved in non-nuclear mediated events such as neutrophil activation (superoxide anion generation and degranulation) and migratory responses to chemoattractants (27, 37, 60-64). In neutrophils the activation of p38 MAPK by soluble agents such as PAF, fMLP, TNF and PMA has been previously described (37, 57, 60, 61, 62, 64, 65, 66). Using the specific inhibitor of p38 MAPK, SB20358, Zu et al demonstrated a strong inhibition of TNF-induced neutrophil superoxide anion release but no inhibition of PMA-induced responses. Coxon et al showed that SB20358 inhibited fMLP-induced superoxide anion generation. Opsonized zymozan or CPPD crystals have also been shown to activate p38 MAPK in neutrophils (27, 66) and treatment with SB20358 inhibited chemiluminescence, superoxide anion release and degranulation responses. Collectively, these data suggest that most agents that activate neutrophils also activate p38MAPK. Furthermore, the clear 18  involvement of this enzyme in neutrophil inflammatory responses to opsonized CPPD crystals has been established. The water-soluble agents, lipopolysaccharide (LPS), GM-CSF, PMA and fMLP have been shown to activate p44/42 (ERK1/2) in neutrophils (60, 65, 67, 68). Currently, there is no available specific inhibitor for ERK1/2 but PD98059 indirectly blocks this enzyme and may be used to study downstream effects. Whereas inhibition of fMLP-activated p38 MAPK abolishes neutrophil superoxide production, inhibition of ERK1/2 has no effect on this response (60). Similarly, Coffer et al (65) found that inhibition of GMCSF, PAF or TNF-induced activation of ERK1/2 had no effect on neutrophil superoxide anion generation. Both uncoated and plasma opsonized CPPD crystals were shown to strongly activate ERK1/2 in neutrophils (30, 32) and indirect inhibition of ERK1/2 with Taxol strongly inhibited neutrophil superoxide anion production and degranulation responses to CPPD crystals and opsonized zymosan (30).  2.2.2.3 Involvement of Ca2+ Secondary Signalling Calcium concentration changes in cells are associated with surface receptor mediated signal transduction events leading to both nuclear and non-nuclear-mediated responses. These changes are easily observed using intracellular fluorescence probes such as Fura 2, which bind calcium, causing changes in fluorescence wavelength and intensity. In neutrophils calcium concentration increases are the result of the activation of phospholipase C. Chemoattractant signaling is relatively well described so that, for example, fMLP binds to receptors and causes G protein uncoupling (pertussis toxin 19  sensitive) and binding of subunits to membrane bound phospholipase C. This enzyme then cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), to form inositol-3-phosphate (IP3) and diacylglycerol.  IP3 mobilizes calcium from intracellular stores, which in  combination with DAG activates the central signaling enzyme PKC and other calcium sensitive kinases. This is followed by downstream effects including cytoskeletal changes (for chemotaxis) and NADPH oxidase assembly for superoxide anion generation (69-73). The specific inhibitor of PLC, U73122, is commonly used to block this enzyme in order to investigate the modulation of downstream enzymes and so unravel signal transduction pathways. Inhibition of fMLP induced PLC activation with U73122 results in inhibition of IP3 generation, calcium mobilization, oxidase activation and degranulation (74-76). Both MSUM and CPPD crystals induce calcium concentration increases in neutrophils (29, 40, 77-79). It is now well established that upon activation of neutrophils by fMLP, cytochalasin, PMA and opsonized zymosan, the phox protein components of the NADPH oxidase system are phosphorylated by PKC to allow for translocation and assembly at the plasma membrane (80-84). Tudan et al (30) demonstrated that CPPD crystals, fMLP and PMA induced a strong activation of PKC in neutrophils.  The same study showed that  inhibition of PKC, using the specific inhibitors BIM and compound 3 as well as the nonspecific inhibitor AGM-1470 resulted in almost full inhibition of NADPH oxidase activation.  Overall these studies indicate that chemoattractant and crystal-induced  neutrophil oxidase activation both share a common dependence on the PLC/calcium mobilization/PKC activation pathway.  20  2.3  Apoptosis  Apoptosis, or programmed cell death, is an essential physiological process that plays a key part in controlling the number of cells in development and throughout an organism’s life by removal of cells in a controlled and directive fashion. With respect to biochemical and morphological aspects, it is distinct from other forms of cell death such as necrosis or cytolysis. Apoptosis is most apparent in proliferating cells during development and in proliferating lymphocytes during immune responses. It is a natural process, however, it has been implemented in a variety of pathological conditions including acute neurological injuries, neurodegenerative, cardiovascular and immunological diseases, acquired immuno-deficiency disorder and cancer. It is therefore currently a hotbed of therapeutic research. Apoptosis is typically characterized by the activation of caspase-class enzymes, cleavage of internucleosomal-DNA, mitochondrial release of cytochrome c, externalization of phosphatidylserine on the cell surface and proteolytic cleavage of a number of intracellular substrates and cytoskeletal components. An apoptotic cell becomes visually distinct as it undergoes membrane blebbing, chromatin condensation, nuclear fragmentation, loss of adhesion and rounding (in adherent cells), and cell shrinkage. The contained cellular debris is then removed by macrophages via phagocytosis without the release of cellular components into the extracellular space and subsequent inflammatory response (85, 86). There are many various known initiators of apoptosis including Fas-ligand binding, external damage to the cell membrane by physical stress or by chemicals, DNA damage  21  due to ultraviolet or gamma irradiation, growth factor withdrawal, actinomycin D, staurosporine, glucocorticoids, and DNA poisons such as etoposide. (87-90). Death initiators exact their action typically through one of two independent pathways. The first pathway is a receptor-mediated system involving the binding of a death receptor with its ligand (such as Fas-FasL) to activate procaspase-8 (91). This pathway allows for endocrine or autocrine signalling to initiate apoptosis in a specific circumstance, for example, during developmental stages in which timely apoptosis of unwanted cells plays a paramount role in producing a normal cellular phenotype. Figure 2 shows a schematic diagram of the death receptor-mediated pathway.  (Figure 2 was removed due to copyright restriction. The removed figure was: Summary of the Death Receptor-Mediated Apoptosis Pathway (Refer to Cell Signaling Technologies website: www.cellsignal.com).  Fas, a glycosylated Type I transmembrane receptor ~ 45-52kDa (which can also exist in soluble form via alternative splicing), is expressed on several different cell types. However, it is mainly expressed in the thymus, activated T and B lymphocytes, macrophages, liver, spleen, lung, testis, brain, intestines, heart and ovaries. Its expression is also augmented by cytokines such as interferon-γ and TNF-α as well as by lymphocyte activation. In contrast, Fas ligand is more tightly regulated, often only inducible under certain conditions. FasL expression is restricted to immune cells, including T and B lymphocytes, macrophages, and natural killer cells, and to non-immune sites such as the 22  testis, kidney, lung, intestine and eye (92-94). Ligation of death receptors causes the rapid formation of a death-inducing signalling complex via the receptor’s DD (death domain). This domain is responsible for coupling the death receptor either to a cascade of caspases (via recruiting Fas-associated DD protein (FADD) and subsequent auto-activation of procaspase-8), leading to an induction of apoptosis, or to the activation of kinase signalling pathways, resulting in gene expression via nuclear factor-κB (NF-κB) and/or activating protein-1 (AP-1) (91). The active caspase-8 can then go on to trigger the apoptotic caspase cascade. The second death pathway is dependent on the involvement of the mitochondria (receptor-independent) and is regulated by pro- and anti-apoptotic members of the Bcl-2 family of proteins (95). In this case, cellular stress induces pro-apoptotic Bcl-2 members to translocate from the cytosol to the mitochondrial membrane where they induce the release of cytochrome c, disrupting oxidative phosphorylation and electron transfer (96, 97). Anti-apoptotic Bcl-2 family members work to inhibit this release in an attempt to preserve cell survival. The presence of cytochrome c in the cytosol leads to its binding with apoptotic protease activating factor-1 and subsequent activation of procaspase-9 (98). Figure 3 shows a schematic representation of this Bcl-2-mediated pathway. Both the activation of procaspase-8 and –9 lead to the cascade activation of executioner caspases (caspases-3, -6 & -7).  23  Figure 3 was removed due to copyright restriction. The removed figure was: Summary of the Mitochondrial-Mediated Apoptosis Pathway. (Refer to Cell Signaling Technologie website: www.cellsignal.com).  Procaspase-3 is the predominant executioner caspase and acts as the central hub and main effector in the late-stage cleavage events of cellular substrates by activating the caspasedependent nucleases caspase-activated Dnase and DNA fragmentation factor 40 (99, 100), as well as the cleavage and activation of gelsolin (101) and p21-activated kinase-2 (102) to dissociate the plasma membrane from the cytoskeleton (blebbing). Caspases are a tightly regulated family of aspartate-specifc cysteine proteases containing 14 mammalian members, of which, 11 human enzymes are known. Phylogenetic analysis indicates that this gene family is composed of two major subfamilies that are related either to ICE (caspase-1; inflammation group) or to the mammalian counterparts of ced-3 in C. elegans (apoptosis group). Caspases share similarities in amino acid sequence, structure, and substrate specificity (103). These molecules are expressed as non-active procaspase enzymes that contain three domains: an N-terminal prodomain, a large subunit containing the active site cysteine within a conserved QACXG motif, and a Cterminal small subunit. Caspases are among the most specific of proteases, with an unusual and absolute requirement for cleavage after aspartic acid (Asp) residues (104). An aspartate cleavage site separates the prodomain from the large subunit, and an interdomain linker containing one or two aspartate cleavage sites separates the large and small subunits. The presence of these cleavage sites is consistent with the ability of caspases to auto-activate or to be activated by other caspases as part of an amplification 24  cascade. Procaspases are activated to fully functional proteases by two cleavage events. The first proteolytic cleavage divides the chain into large and small subunits, and a second cleavage removes the N-terminal prodomain. The active caspase is a tetramer of two large and two small subunits, with two active sites (105).  Individual caspase  prodomains vary in length and sequence. Long prodomain caspases serve as signal integrators for apoptotic or pro-inflammatory signals. The apoptotic initiators (caspases – 2, -8, -9, and -10) generally act upstream of the small prodomain executioners (caspases – 3, -6, and –7) (91, 103, 106).  2.3.1  Constitutive Neutrophil Apoptosis  Neutrophils differ from most cell types in the body with respect to apoptosis. Constitutively programmed to undergo cell death 24-36 hours once in the peripheral blood stream, they exist only to destroy invading microbes and succumb to death themselves shortly thereafter. The reasons for this are unclear, but it can be hypothesized that it is related to their potential toxicity and hence their capacity to inflict possibly lifethreatening tissue damage against the host when activated inappropriately (107). Indeed, such damaging error in neutrophil activation is seen in several human diseases such as rheumatoid arthritis, diabetes mellitus, and irritable bowel syndrome. It stands to reason that such a constitutive program serves to control these cells with such a rapid turnover without the need for a positive death signal in order to protect against the possibility of large numbers of activated, highly toxic cells persisting in the blood stream for extended periods of time.  25  In contrast to the short half-life of circulating cells, neutrophils recruited into tissues have a much longer survival time because they become rescued from this constitutive apoptotic pathway. Presumably, this effect exists to increase the time that activated neutrophils are in contact with pathogens thereby increasing their efficacy in neutralizing infectious threats. Indeed, apoptotic neutrophils are incapable of mounting their normal cellular responses such as chemotaxis, degranulation, adherence, phagocytosis and activation of respiratory (108, 109). Also, the expression of many cell surface receptors is severely decreased via receptor shedding or internalization (110, 111). A variety of proinflammatory cytokines and other agents have been shown to readily delay neutrophil apoptosis. In vitro, such agents include IL-1β, IL-2, IL-4, IL-15, INF-γ, G-CSF, GM-CSF and LPS (112-118). The reported effects of TNF-α are conflicting, and its effects are extremely dependent upon the concentrations used and the duration of exposure (119). TNF-α rapidly induced apoptosis in a subpopulation of blood neutrophils, but delays apoptosis in the surviving cells (120). These opposing dual effects may be related to the ability of TNF-α to stimulate either the death receptor pathway or a cell survival NFκBmediated pathway in the different subpopulations of neutrophils. Although they are preconditioned to undergo apoptosis, neutrophils are also susceptible to Fas-mediated apoptosis (121). They express significant levels of Fas and may or may not also express Fas ligand (122, 123). Co-expression of Fas and Fas ligand may indicate an autocrine method for constitutive apoptosis. Triggering of cell adhesion and endothelial transmigration has also been shown to induce enhanced cell survival (124, 125). In contrast to findings in other cell types, hypoxia can delay neutrophil apoptosis (126, 127). Similarly, anti-oxidants such as catalase (but not SOD) have been shown to delay  26  neutrophil apoptosis in vitro suggesting that local oxygen conditions may play a role in neutrophil survival (128). Since infected or inflamed tissues are relatively low in oxygen tension compared to that found in circulation (approximately 2% in contrast to the 1113% present in blood), this suggests that oxygen conditions may play a pivotal role in extending neutrophil survival at sites of infection or tissue damage. Interestingly, there is some evidence that the combinatorial balance of pro- and antiapoptotic Bcl-2 proteins most involved in neutrophil survival is a key factor in the constitutive apoptotic programming present.  2.3.2  Delay of Neutrophil Apoptosis Signalling  Neutrophils are normally safely cleared from the bloodstream by spontaneous apoptosis within approx 24 hours of release from the bone marrow. However, chemoattractant molecules may cause circulating neutrophils to bind to blood vessel walls and migrate (through the walls) into tissues (chemotaxis). The purpose of this process is to allow the neutrophils to potentially engage and destroy the pathogens responsible for the release of the chemoattractant (s). Since the non-stimulated neutrophil has only a short lifetime it is important for the chemoattractant-activated neutrophil to extend it’s lifetime to enable a reasonable chance of engaging the pathogen before the cell undergoes apoptosis. (129131). Therefore, most chemoattractants including, GMCSF, LTB4, LPS, PAF, fMLP and TNF (extended exposure) stimulate a prosurvival process to occur in neutrophils without actually activating the oxidase and degranulation systems within the cells. (120, 130). Interestingly, TNF actually activates proapoptotic signalling during the first few hours of  27  incubation with neutrophil, but this changes to a strong prosurvival signal at longer time points (120, 132). It has been shown that synovial fluid from Rheumatoid Arthritis patients (presumably containing proinflammatory cytokines and chemoattractants) has a strong prosurvival effect on human neutrophils indicating that this effect has a pathophysiological relevance (133). Studies in Dr Burt’s laboratory have established that both CPPD and MSUM crystals also engage strong prosurvival machinery in neutrophils that is even able to overcome the proapoptotic signalling caused by TNF (27, 134-137). For bacteria, once the neutrophil reaches the pathogen, the full activation of neutrophils and the phagocytosis of bacteria is reported to re-engage the apoptotic process (138-141). This proapoptotic, phagocytotic process has been reported to result from oxidase activation and alteration of the internal oxidative state of neutrophils (139-141). However, Lundquist et al (142) have reported that only phagocytosis of living S. aureus bacteria (not heat-treated bacteria) results in pro-apoptotic signalling, indicating that water soluble molecules released from living bacteria cause this resumption of neutrophil apoptosis. Certainly, this phagocytosisinitiated resumption of neutrophil apoptosis is an important aspect of reducing unnecessary neutrophilic activity in disease sites.  However, for crystal-induced  neutrophil activation and phagocytosis there is no evidence that such self-limiting inflammatory control exists in synovial sites. Indeed, the acute (extended) nature of neutrophil derived inflammation might argue against phagocytosis-induced override of the crystal-induced pro-survival signaling. Clearly, there is a strong interest in understanding the intracellular signaling processes involved in the control of apoptosis in neutrophils. In fact, in some disease states (such 28  as Rheumatoid Arthritis), chemoattractant-derived accumulation of neutrophils in normal (non-pathogenic) tissues the process may be central to a therapeutic treatment of the disease. The role of the central signal transduction enzymes, PI3-kinase, p38MAPK, and ERK1/2 in chemoattractant- and crystal-induced neutrophil pro-survival signaling is discussed below. There are three or four main processes involved in neutrophil responses to inflammatory stimuli: A. Chemoattractant binding to membrane receptors followed by selectin/intergrin mediated binding to blood vessel walls and cytoskeletal changes which allow chemotaxis into the source tissues, B. Suppression of the spontaneous/intrinsic apoptotic pathway to extend the lifetime of the cells, C. Full oxidase/degranulation activation and phagocytosis of the pathogen and (potentially) D. Reactivation of apoptosis to clear the site of potentially inflammatory debris. All these systems involve many of the central signaling enzymes such as PI3-Kinase, p38 MAPK and ERK1/2, identified in the neutrophil activation pathways. To investigate the role of these enzymes in the prosurvival reaction to chemoattractants or crystals, the same methods were used as for oxidase activation and degranulation. Fresh neutrophils were incubated with low concentrations of the chemoattractant (to avoid full oxidase activation and degranulation responses) or crystals and the activation status of the kinases were studied in the presence or absence of inhibitors including Wortmannin/LY294002 (PI3-Kinase), PD98059 (ERK1/2) or SB203580 (p38MAPK) using SDS PAGE/Western blotting methods. Furthermore, the physiological response of the neutrophils (level of apoptosis) was compared in the presence or absence of the inhibitors. Of the various inflammatory microcrystals, only CPPD induced neutrophil prosurvival signaling has been studied by any investigators. In  29  Dr Burt’s laboratory, three methods of measuring apoptosis have been employed for studies with crystals. These include DNA laddering (using agarose gel electrophoresis), quantitative DNA fragmentation determinations and caspase 3 activity determinations. 2.3.2.1 Involvement of the PI3-Kinase Pathway A strong activation of this enzyme has been reported for all chemoattractant stimuli studied including fMLP, PAF, GM-CSF, LTB4, TNF, VCAM, LPS (132, 143-152). By studying both the activation status of PI3-Kinase and the level of apoptosis in neutrophils in the presence/absence of wortmannin/LY294002, the majority of reports demonstrate an absolute dependence of the prosurvival effect of chemoattractants on activation of this enzyme (132, 137, 144, 145, 146, 148, 150).  Both MSUM and CPPD crystals inhibit  neutrophil apoptosis (134). Both types of crystals were able to overcome the strong proapoptotic signal from TNF.  CPPD crystal-induced prosurvival was PI3-Kinase  dependent, witnessed by strong suppression of DNA fragmentation and caspase 3 activation elicited by these crystals.  2.3.2.2 Involvement of the ERK 1/2 Pathway Using the inhibitor PD98059, numerous studies have shown that chemoattractant- or CPPD crystal-induced inhibition of apoptosis depends on activation of ERK1/2. PD98059 does not directly inhibit ERK1/2 but rather the upstream controlling kinase MAPKK or MEK. Activation of ERK1/2 has been shown to directly depend on MEK activation, validating the use of PD98059 to indirectly inhibit ERK1/2. The critical prosurvival role for ERK1/2 in neutrophils has been established for the chemoattractants 30  TNF (143), LTB4 (145, 151), C5A (153), GM-CSF (150), LPS (151) and IL-15 (154). Similarly, CPPD crystals were shown to induce a strong activation of ERK1/2 (32) and pre-treatment of cells with PD98059 was demonstrated to overcome the prosurvival effect of CPPD crystals alone or in the presence of TNF (short exposure; proapoptotic). It has been noted that, in vivo, the role of the MAPK’s is complex so that, for example, in an inflammatory milieu, neutrophils may be under the prosurvival influence of agents such as TNF and GM-CSF whilst phagocytosing complement-opsonized pathogens. This phagocytosis can lead to the generation of reactive oxygen species and proapoptotic signalling at the same time MAPK initiated prosurvival signalling is occurring (141). In carageenen-induced inflammation, inhibition of ERK1/2 with PD98059 was shown to reduce the level of inflammation by increasing the rate of neutrophil apoptosis (155).  2.3.2.3 Involvement of the p38 MAP Kinase Pathway Many chemoattractants and the stress stimuli Fas have been shown to activate p38 MAPK (140, 145, 149, 151, 154, 156) in neutrophils. There are only a few reports that p38 may be involved in the prosurvival signal caused by these chemoattractants. Alvarado-Kristensson et al (149) speculated that p38 activity represents a pro-survival signal that is inactivated during Fas-mediated or spontaneous apoptosis but did not identify a direct activation of p38 with survival. This activity of p38 was later shown to result from phosphorylation (inhibition) of caspase-3 and -8 but these studies did not involve pro- survival agonists (156). Interleukin-15 has been shown to be a strong prosurvival neutrophil agonist, which causes the strong activation of p38 MAPK (154).  31  Inhibition of p38 MAPK with SB20350 resulted in the inhibition of the pro-survival effect indicating the involvement of this enzyme. However, there are many studies reporting that inhibition of chemoattractant-induced p38 activity had no effect on the prosurvival signal caused by LTB4 (145, 151), TNF (143) or LPS (151).  CPPD crystals were reported to induce a mild activation (2-fold) of  p38MAPK whereas TNF (short exposure= apoptotic signal) induced a 6-fold activation of p38 MAPK (27). However, in the same study, CPPD crystals reduced the TNF signal to basal (CPPD alone) levels. Since CPPD crystals were able to overcome TNF-induced apoptosis in neutrophils and maintain the prosurvival effect it is likely that p38 activation in this case represents a prosurvival signal. However, in these studies a direct link between CPPD induced p38 MAPK activation and inhibition of apoptosis was not established. Overall, for chemoattractants, only one study indicated a signal transduction pathway connecting p38 MAPK activation and a prosurvival effect. For CPPD crystals, any p38 MAPK-mediated apoptotic control was confined to overcoming a TNF induced proapoptotic signal. In light of the many studies describing no chemoattractant-induced prosurvival signal facilitated by p38 MAPK, any significant role of this enzyme in agonist-induced inhibition of apoptosis in neutrophils seems unlikely.  2.3.2.4 Involvement of NF-κ κB Transcription Factor Signalling Generally, a group of proteins known as the Bcl-2 family are responsible for proapoptotic or prosurvival control in all cells. The expression or activation level of these proteins is 32  thought to affect the manner in which they interact with mitochondrial-derived apoptosis processes. In neutrophils the prosurvival signal initiated by chemoattractants is likely to involve an increase in the expression of some of these proteins such as the prosurvival Bcl-2 members Mcl-1, A1, Bcl-XL and/or inhibition of the expression of proapoptotic Bcl-2 members Bad, Bax, or Bim (this is discussed in more detail below). However, any concerted modulation of protein expression generally requires nuclear transcription events to occur. The transcription factor NF-kB is considered the primary transcription factor involved in mitogen-initiated stress signalling in cells and may be activated by the various MAPK systems including p38MAPK, ERK1/2 and JNK. Therefore, the role of NF-κB in chemoattractant or CPPD crystal-induced prosurvival signalling is of great interest (131). The involvement of NF-κB in cellular responses is generally demonstrated by investigating NF-κB activation levels in cells and how they correspond to PI3-kinase, ERK1/2 or p38 MAPK activation levels (and inhibition of these kinases) and apoptotic/survival events. Using such methods, TNF (132, 143), LPS (146), VCAM (144) induced neutrophil survival has been linked to increased NF-κB activation.  2.3.3  Bcl-2 Family Proteins  The family of Bcl-2 proteins plays a central role in the early life/death decisions of all known cell types (157-159). The first member of this group, Bcl-2, was discovered as a proto-oncogene in the follicular B-cell lymphoma and was subsequently identified as a mammalian homologue to the apoptosis repressor ced-9 in C. elegans. Since then, at least  33  19 Bcl-2 family members have been identified in mammalian cells based upon the presence of at least one of four highly conserved sequence motifs or BH domains (Bcl-2 homology domains) within their structures (160, 161). The Bcl-2 family members can be divided into three classes according to their function and structure (Figure 4).  (Figure 4 was removed due to copyright restrictions. The removed figure was: Members  of  the  Bcl-2  Protein  Family.  Refer  to  website:  www.nature.com/nrm/journal/v9/n3/box/nrm2312_BX1.html)  The first class, the anti-apoptotic members, such as Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1, all exert pro-survival activity and contain at least the BH1 and BH2 domains (with those members most similar to Bcl-2 containing all four BH domains). The second class of proteins, the pro-apoptotic members, such as Bax, Bak, and Bok, carry out pro-death activities and share sequence homology in the BH1, BH2, and BH3 domains, but not in BH4. The final class of Bcl-2 proteins, the “BH3-only” pro-apoptotic members, which include Bid, Bad, Bim, Bik, Bim, and Noxa, also produce pro-death effects and possess only the central short BH3 domain (98). The hallmark feature of the Bcl-2 proteins is their ability to form hetero- or homodimers (162-164). The BH domains are the key to this property and form α-helical structures that serve as protein-protein interaction motifs. For example, the BH4 domain found in most of the anti-apoptotic members may be responsible for molecular interactions with such downstream pro-survival effectors like calcineurin and Raf-1 (165, 166). In addition, 34  studies of the BH domains of Bcl-XL and the pro-apoptotic protein, Bak, have shown that the BH1-3 domains of Bcl-XL form a hydrophobic pocket into which the BH3 domain of Bak could fit suggesting a mode of action for how Bcl-XL may counter the pro-death effect of Bak within a cell (167). However, molecular modelling indicates that in some pro-apoptotic proteins, the BH3 domain is normally contained within the core of the protein suggesting that at least some proteins may require posttranslational modification to become activated. For example, Bad has been shown to require a dephosphorylation event to dissociate from 14-3-3 protein and become activated (168) and Bid requires caspase-mediated cleavage to aid the dimerization and insertion of Bax into the mitochondrial membrane (169).  2.3.3.1 Bcl-2 Protein Function in Other Cell Types To date, the major function of Bcl-2 family member proteins is to regulate cytochrome c release through interaction with the outer mitochondrial membrane.  Subcellular  localization studies have shown that Bcl-2, Bcl-XL and Mcl-1 reside on the mitochondrial outer membrane, while the pro-apoptotic members may either be cytosolic or present on the mitochondrial membrane. Although they are found on the endoplasmic reticulum and nuclear envelope, their major effect appears to be on the mitochondria. During apoptosis, the pro-apoptotic family members are activated, possibly undergo conformational change (170) leading to the exposure of their BH3 domain and then translocate to the mitochondria (168, 171-174). Bax translocation to the mitochondria requires homo-dimerization (174). Bim is normally localized within the microtubules via association with LC8 dynein light chains, but relocates to the mitochondria upon 35  initiation of apoptosis (168). The translocation of pro-apoptotic members to the mitochondria can then induce release of mitochondrial proteins, including cytochrome c, into the cytosol. Pro-apoptotic proteins with BH1-3 domains (eg. Bax) may insert into the membrane thereby destabilizing it and directly promoting mitochondrial leakage, whereas proteins containing only the BH3 domain (eg. Bid, Bim and Bad) may interact with antiapoptotic members (eg. Bcl-2 and Bcl-XL) to induce conformational changes that neutralize their cytochrome c release-inhibiting action (175). Oddly, cytochrome c is encoded by a nuclear gene, but when it is imported into the mitochondria, it is coupled with a heme group to form holocytochrome c, which is the only form that functions to induce caspase activation (97). While the anti-apoptotic proteins Bcl-XL and Bcl-2 work to prevent cytochrome c release from mitochondria to preserve cell survival (96, 97), the pro-apoptotic members attempt to initiate cytochrome c release in certain settings. For example, Bid has been implicated in Fas-mediated apoptosis, Bax in DNA damage-induced apoptosis and neutrophin deprivation-induced death, and Bad in cytokine deprivation-induced cells death of certain neuronal cells. In addition, time-lapse confocal microscopy using HeLa cells expressing cytochrome c-GFP (green fluorescent protein) have revealed that cytochrome c release is an early event during apoptosis, occurring hours before phosphatidylserine (PS) exposure and loss of plasma membrane integrity (176). These studies also showed that cytochrome c release did not occur until hours after the initial apoptosis-inducing stimulus. However, once initiated, all mitochondria release their cytochrome c-GFP within a 5 minute time frame, regardless of the nature of the initial apoptosis signal, suggesting that the pathways of cytochrome c release are either universal or remarkably similar.  36  Following the release of cytochrome c, it binds to the cytosolic protein apoptotic protease activating factor-1 (Apaf-1) in an ATP/dATP-dependent manner and induces it to oligomerize (177, 178). In turn, the oligomerized Apaf-1 complex recruits procaspase-9 to form an “apoptosome” (169, 178). Of interest, unlike other caspases, procaspase-9 does not appear to be activated by simple cleavage, and instead, it must be bound to Apaf-1 to become activated (104, 178). The final action of the completed apoptosome is to then recruit procaspase-3 and cleave it via the activated caspase-9 and then release the active caspase-3 to begin mediating downstream apoptotic events. Another mitochondrial protein that is released co-ordinately with cytochrome c is Smac/DIABLO (180, 181). This protein functions to promote caspase activation by associating with the apoptosome and by inhibiting IAPs (inhibitors of apoptosis proteins). While cytochrome c activates Apaf-1, Smac/DIABLO relieves the inhibition on caspases by binding to the IAPs and disrupting their association with activated caspase-9, thus allowing caspase-9 to activate caspase-3 (182). Mcl-1 and A1 differ from other anti-apoptotic Bcl-2 proteins in that they lack the BH4 domain. As this domain is thought to be required for the molecular interactions with other proteins, their absence in Mcl-1 and A1 suggests that these members interact with a different set of proteins than Bcl-2 and Bcl-XL. Also exclusive to these proteins are PEST sequences and Arg:Arg domains that are often found in proteins that are subject to rapid turnover (183). Indeed, Mcl-1 has a relatively short half-life (1-5 h) and has the fastest turnover rate of any of the Bcl-2 family members (184). It has recently been shown that this unique N-terminus of Mcl-1 is responsible for its localization and function in both inhibiting apoptosis and cell proliferation (185). Mcl-1 has been shown  37  to be expressed in a variety of cells and tissues. In many cell types, its expression is transient, often occurring at specific stages of development or differentiation (186-188). Mcl-1 expression is often observed as being independent of Bcl-2 expression, suggesting that Mcl-1 plays a distinct role in cell survival. It has been shown that enhanced Mcl-1 expression can confer a malignant phenotype in normal cells (189) and its overexpression is thought to be responsible for the impaired apoptosis and resistance to chemotherapy of malignant myeloma cells (190). Expression of Mcl-1 is highly inducible by a range of cytokines in various cell types typically via PI3-K/Akt, ERK, and NF-κB-dependent pathways (191, 192). Also inducible by cytokines is the anti-apoptotic Bcl-2 protein A1. Like Mcl-1, it lacks the BH4 domain suggesting it also has different binding partners than Bcl-2 and Bcl-XL. It is also expressed in a variety of cell types and tissues where it has been shown to be vital to the inhibition of apoptosis (193). Until recently, no reliable commercial antibody has existed for the detection of human A1 protein, so most studies have been only able to assess its expression at the mRNA level. In such studies, it has been shown that A1 also has a short half-life comparable to Mcl-1 and that the expression of A1 is cytokine-regulated (157, 194). Recent studies have shown that other agents such as glucocorticoids, LPS and peptidoglycan can induce increased expression of A1 in other cell types (146, 195).  2.3.3.2 Bcl-2 Protein Function in Neutrophils The Bcl-2 family proteins present in neutrophils, and their exact functions have not been completely elucidated. To date, there remains much ambiguity and confusion as to which Bcl-2 proteins are present and which are predominant in vivo. However, based on 38  references showing the existence of some Bcl-2 family proteins being present in neutrophils, and what is known of the paramount importance of Bcl-2 family proteins to the survival of all other cell types, it stands to reason that Bcl-2 proteins play a pivotal role in the constitutive apoptotic system observed in these specialized cells. Although it has been reported in the literature that neutrophils express a range of death receptors including TNFR1 and CD95 as well as their respective ligands (120, 196, 197), it is widely considered that they do not contribute greatly to the spontaneous apoptosis observed in neutrophils. Studies using antagonistic anti-Fas, anti-TRAIL receptor and anti-TNF-α antibodies have shown that they did not prevent spontaneous apoptosis from occurring (196). In addition, neutrophils from Fas or Fas ligand deficient mice show a normal rate of spontaneous apoptosis (198). Such observations would suggest that the mitochondrial pathway, and thus, Bcl-2 family protein involvement, is predominant during spontaneous apoptosis in neutrophils. Several studies have reported that freshly isolated human neutrophils do not express the anti-apoptotic proteins Bcl-2 or Bcl-XL (114, 199, 200, 201), although they do have detectable levels of the Bcl-2 homologues Mcl-1 and A1 (114, 152, 193, 194, 202, 203, 204). It should be noted that early studies by Weinmann et al reported detecting both BclXL mRNA and protein in neutrophils (159) and since then, others have also reported BclXL presence in neutrophils (157, 205). With respect to Bcl-2 itself, interestingly, it may be its lack of presence that lends to the constitutive nature of neutrophil apoptosis. In a study by Iwai et al (201), it was confirmed that mature blood neutrophils do not express Bcl-2 and that when comparing neutrophil, monocyte and leukocyte apoptosis, Bcl-2 expression was inversely related to the degree of spontaneous apoptosis observed 39  between these cell types. It has also been reported that the pro-apoptotic proteins Bak, Bax, Bad, Bik and Bid are present in neutrophils (114, 152, 159, 199, 204, 206). These proteins are expressed at fairly high levels and they have a relatively long half-life. In most reports, their cellular levels in neutrophils remain fairly constant as the cells undergo spontaneous or induced apoptosis. Based on studies (145, 159, 207) that reported a decrease in Bax levels neutrophils stimulated by cytokines (such as GM-CSF, G-CSF, LTB4), and other reports in the literature citing no change in overall Bax levels, it would seem that cytokine regulation of Bax (and perhaps other pro-apoptotic members) may be two-fold: working to alter both the function and/or the absolute protein levels of such proteins to inhibit their pro-death effects (153, 208, 209, 210). Likewise, there appears to be some reports that have shown both an increase in Bax expression in response to known pro-apoptotic stimuli such as live and attenuated Mycobacterium tuberculosis, LPS, and γ-irradiation (205, 211, 212), as well as functional changes in Bax (dephosphorylation/cleavage) in delayed, spontaneous and induced apoptotic states via PI3-K/Akt/calpain-1-depedent pathways (208, 210). Indeed, Maianski et al (213) showed that Bax translocates from the cytosol to the mitochondria to induce caspase-3 activation during spontaneous apoptosis in neutrophils without significant changes in overall Bax protein levels and that G-CSF blocked the translocation of Bax to the mitochondria and subsequent caspase-3 activation. However, they also showed that neutrophil-derived cytoplasts lacking mitochondria undergo caspase-3-dependent spontaneous apoptosis suggesting that Bax does play a role in the control of apoptosis in neutrophils, however its influence is neither absolute nor necessary. As for other pro-apoptotic Bcl-2 family members present in neutrophils, such as Bak, Bim and Bad that act upon the  40  mitochondrial apoptotic pathway and are expressed at relatively stable levels, such findings suggests a similar degree of apoptotic control for these proteins. Bak expression is constitutive and not modulated by many of the known apoptosis-mediating agents such as complement c5a, IFN-γ, TNF-α, (153, 206), with some exceptions such as IL-8:antiIL-8 complexes and glucocorticoids which may modulate Bak expression via PI3-K and ERK pathways (195, 214). Bad expression would also appear to be constitutive, however, its activational status has been shown to be abrogated via PI3-K/Akt and/or ERK phosphorylation by such factors as GM-CSF, TNF-α, heme (NADPH-oxidase dependent), LPS, peptidoglycan, c5a, and platelet-activating factor (146, 148, 153, 215, 216). Upon phosphorylation, inactive Bad is typically sequestered by 14-3-3 protein and subsequently degraded within the proteosome, contributing to a delay of neutrophil apoptosis. The novel expression of Bim in neutrophils found in Dr. Burt’s lab has been recently reported by others. Studies on neutrophils isolated from rats have shown that sepsisinduced survival signals via the PI3-K and ERK pathways downregulates the expression of Bim (217). However, in general, there is a lack of information on the role of Bim in neutrophil apoptosis. Based on studies in other cell types, Bim expression can be upregulated via FOXO transcription factors by reactive oxygen species, paclitaxol and removal of growth factors (218-220) and downregulated by such agents as phorbol esters (PMA) via PI3-K/Akt and ERK1/2-mediated phosphorylation and subsequent ubiquitinylation (221-223). Interestingly, Han et al have proposed that in type II cell lines, under non-apoptotic conditions, Bim is sequestered by the anti-apoptotic protein  41  Mcl-1. Following Mcl-1 degradation by TRAIL-activated caspase-8 or caspase-3, Bim is released and then mediates a Bax-dependent apoptotic cascade (224). The role of Mcl-1 in neutrophils has been well established relative to other Bcl-2 family members in this cell type. Bloodstream neutrophils express both Mcl-1 mRNA and protein (114, 127, 152, 207). Agents that accelerate neutrophil apoptosis (such as cyclohexamide) decrease Mcl-1 levels, whereas agents that delay apoptosis (such as GMCSF, Il-1β, TNF-α) all increase or sustain Mcl-1 levels. Furthermore, in experiments where Mcl-1 is depleted by antisense, neutrophil apoptosis is accelerated, regardless of GM-CSF stimulation (225). Mcl-1 mRNA is cytokine-regulated in neutrophils, thus perhaps providing a mechanism for the rapid increase in expression levels for this prosurvival protein (159). Having such a rapid turnover rate and short half-life, when this cytokine-induced upregulation is ceased, levels of Mcl-1 immediately begin to decline below the threshold level necessary for protection against apoptosis. Such molecular properties make Mcl-1 an ideal protein to function as the key regulator of neutrophil survival. Another anti-apoptotic Bcl-2 family member that has been recently implicated in the survival of neutrophils is A1 (bfl-1). As mentioned before, like Mcl-1, it lacks a BH4 domain, suggesting that it also interacts with a different set of proteins than Bcl-XL and Bcl-2. The overall amount of information on A1 expression in neutrophils is rather limited, again, due to the lack of a reliable commercial antibody for human A1 until only recently. At the mRNA level, it has been shown that the A1 transcript is abundant in human neutrophils with a half-life similar to that of Mcl-1 (152, 194). It has also been shown that A1 expression is regulated by cytokines and other agents such as 42  glucocorticoids, LPS and peptidoglycan (146, 152). In the latter study, TLR agonists (LPS, peptidoglycan) were shown to upregulate A1 (and Mcl-1) expression in a PI3K/Akt- and NF-κB-dependent manner.  2.3.3.3 Proposed Mechanism of Bcl-2 Protein Balance in Neutrophils Based on the information on the Bcl-2 family proteins and their currently known roles in neutrophil apoptosis as well as in other cell types, we proposed a balance model of the key Bcl-2 proteins involved in the cell survival/death decision within neutrophils. The proposed objectives for this study were as follows: 1) To determine if the delay of spontaneous apoptosis observed in CPPD-treated neutrophils was due to an upregulation in the expression of the pro-survival Bcl-2 proteins Bcl-XL, Mcl-1 and A1. 2) To determine if the delay of spontaneous apoptosis observed in CPPD-treated neutrophils was due to a downregulation or deactivation of the pro-apoptotic Bcl-2 proteins Bad, Bax-α and Bim.  43  3 3.1  EXPERIMENTAL Materials  Antibodies (primary): Anti-A1 antibody, polyclonal. Abgent, San Diego, CA, USA. #AP1300a. Anti-Bad antibodies, Phospho-Ser112/136, polyclonal. Millipore (Upstate), Billerica, MA, USA. #AB3571. Cell Signaling Technology, Boston, MA, USA. #9295S. Anti-Bax-α 6A7 antibodies, monoclonal. Trevigen, Gaithersburg, MD, USA. #2281-MC-100. Biosource International, Inc., Camarillo, CA, USA. #AHO0232. Anti-Bax-α NT antibody, polyclonal. Millipore (Upstate), Billerica, MA, USA. #06-499. Anti-Bcl-XL antibody, monoclonal. Biosource International, Inc., Camarillo, CA, USA. #AHO0222. Anti-Bcl-XL antibody, polyclonal. Transduction Laboratories (BD Biosciences), San Jose, CA, USA. #B22630. Anti-Bim-EL antibody, monoclonal. Affinity Bioreagents, Golden, CO, USA. #OPA1-01021. Anti-Bim antibody, polyclonal. Oncogene, San Diego, CA, USA. Anti-Bim antibody, polyclonal. Cell Signaling Technology, Boston, MA, USA. #4582. Anti-Bim-EL antibody, phospho-specific, polyclonal. Millipore (Upstate), Billerica, MA, USA. Anti-Mcl-1 antibodies, monoclonal. Transduction Laboratories (BD Biosciences), San Jose, CA, USA. #M54020, #M559027. Biosource International, Inc., Camarillo, CA, USA. #AHO0102. Anti-Mcl-1 antibody, polyclonal. Santa Cruz Biotech, Santa Cruz, CA, USA. #sc819.  44  Anti-p85 antibodies, polyclonal. Santa Cruz Biotech, Santa Cruz, CA, USA. #sc423. Millipore (Upstate), Billerica, MA, USA #06-195.  Antibodies (secondary): Goat-anti-mouse antibody, HRP-conjugated. Dako, Kyoto, Japan. #P0447. Goat-anti-rabbit antibodies, HRP-conjugated. Cell Signaling Technology, Boston, MA, USA. #9240. Dako, Kyoto, Japan. #P0448. Bio-Rad Protein Assay. Bio-Rad, Hercules, CA, USA. #500-0002. Calcium Pyrophosphate Dihydrate. Synthesized on-site. CaspACE Assay System, Fluorometric. Promega Corporation, Madison, WI, USA. #G3540. Cell Death Detection ELISA Plus. Roche Molecular Biochemicals, Mannheim, Germany. #1 774 425. Complete-Mini Protease Inhibitor Tablets. Roche Molecular Biochemicals, Mannheim, Germany. #1 836 153. Enhanced Chemiluminescence Super Signal West Femto. Pierce, Rockford, IL, USA. #34095. Ficoll-Paque Plus. Amersham Pharmacia Biotech AB, Uppsala, Sweden. #17-1440-02. Filter Paper, Mini-Trans Blot. Bio-Rad Laboratories, Hercules, CA, USA. #1703932. Fresh whole blood was drawn from various donors and used the same day to prepare neutrophil lysates. Blood was collected into 15mL Vacutainers vials containing 1mL of Acid Citrate Dextrose for neutrophil isolation or Heparin-sulphate salt for plasma collection. Formyl-methionyl-leucyl-phenylalanine, fMLP. Amersham Pharmacia Biotech AB, Uppsala, Sweden. Nitrocellulose, Biotrace-NT, 0.45µm. Pall-Gelman Lab Corporation, Pensacola, FL, USA. #66485.  45  Plasma, 50% in HBSS. Prepared from heparinised fresh whole blood. Ponceau S Stain. Sigma, St. Louis, MO, USA. #P-7170. Prestained SDS-PAGE Standards, Low Range. Bio-Rad Laboratories, Hercules, CA, USA. #161-0305. Prestained SDS-PAGE Standards, Kaleidoscope. Bio-Rad Laboratories, Hercules, CA, USA. #161-0324. Protease Inhibitor Cocktail. Sigma, St. Louis, MO, USA. #P8340. Tumor necrosis factor-alpha (TNF-α), human, recombinant, E. Coli. Calbiochem Biosciences, La Jolla, CA, USA. #654205.  3.1.1  Chemicals and Solvents  Acetic Acid (Glacial). Fisher Scientific, Napean, ON, Canada. #A38-4. Acetone, HPLC grade. Fisher Scientific, Fair Lawn, NJ, USA. #A-949. Β-Glycerophosphate, Disodium Salt, Hydrate. Sigma, St. Louis, MO, USA. #G-6251. Calcium acetate, Ca(C2H3O2)2.H2O. Fisher Scientific, Fair Lawn, NJ, USA. #C-46. Calcium tetrahydrogen di-orthophosphate, CaH4(PO4)2.H2O. Sigma, St. Louis, MO, USA. C-8017. Citric Acid, Anhydrous. Fisher Scientific, Fair Lawn, NJ, USA. #A-940-500. Dextran T-500. Fisher Scientific, Fair Lawn, NJ, USA. #BP-1580-100. Dextrose, Anhydrous. EM Science, Gibbstown, NJ, USA. #DX0145-1. Disodium Ethylenediamine-tetraacetate (EDTA). Fisher Scientific, Fair Lawn, NJ, USA. #S-311. Distilled, deionized water (ddH2O). Supplied by on-site facilities. Dimethyl Sulfoxide, DMSO. Fisher Scientific, Fair Lawn, NJ, USA. #D-128. Dithiothreitol, 99%. (DTT). Fisher Scientific, Fair Lawn, NJ, USA. #AC16568.  46  Ethanol (70%). Supplied by on-site facilities. Ethylenebis-(oxytheylenenitrilo)tetraacetic acid (EGTA). Fisher Scientific, Fair Lawn, NJ, USA. #O2783. Glycine. Omnipur. EMD Chemicals, Gibbstown, NJ, USA. #4810. Hydrochloric acid (concentrate, 37%). Fisher Scientific, Fair Lawn, NJ, USA. #SA55. Isobutanol. ACS. Fisher Scientific, Fair Lawn, NJ, USA. #A397. Ortho-phosphoric acid, 85%. Fisher Scientific, Napean, ON, Canada. #A242-P212. N,N,N’,N’-Tetramethylethylenediamine. TEMED. Bio-Rad. #161-0800. Sodium Dodecyl Sulfate (SDS). JT Baker, Phillipsburg, NJ, USA. #4095-04. Trizma Base (Tris-Base). Sigma, St. Louis, MO, USA. #T-6066. Trizma Hydrochloride (Tris-HCl). Sigma, St. Louis, MO, USA. #T-3253.  3.1.2  Stock Solutions  Acrylamide Solution, 30% in water (Resolving). Acrylamide:bis-acrylamide = 150:1. Acrylamide Solution, 30% in water (Stacking). Acrylamide:bis-acrylamide = 37.5:1. Ammonium persulfate. APS 20% w/v in water. Antibody Working Solutions. Antibodies were added to either TBS-T (with 0.02% Sodium Azide) or to Blocking Buffer (with 0.02% Sodium Azide). Blocking Buffer. 5% Skim Milk in TBS-T (0.05% Tween-20). Gel Buffer (Resolving). 1.5M Tris-HCl, 4% SDS, pH 8.8. Gel Buffer (Stacking). 0.5M Tris-HCl, 0.4% SDS, pH 6.8. Hank’s Balanced Salts Solution (HBSS) (without sodium bicarbonate and phenol red) was prepared from powder (Sigma #H-1387) into 1L of ddH20. Sodium bicarbonate (0.35g) was then dissolved into solution; pH to 7.4 and sterilized through a 0.2 µm filter.  47  KPKS Lysis Buffer, pH 7.0, 0.2µm filter-sterilized. Kinexus protocols. 20mM HEPES pH 7.5, 30mM NaF, 5mM EDTA, 2mM EGTA, 40mM β-Glycerophosphate, 2mM Na3VO4, 0.5% NP-40, 10mM Sodium pyrophosphate, 1mM PMSF, 5µM Pepstatin A, 10µM Leupeptin. RPMI 1640 Media, without L-Glutamine. Invitrogen Canada, Inc., Burlington, ON, Canada. #11879-020. Running Buffer (1x), Tris/SDS. Glycine 14.42g/L, Tris-Base 3.0g/L, SDS 1.0g/L in water. Sample Loading Buffer (5x). 250mM Tris-HCl, pH 6.8, 10% SDS, 25% Glycerol, 0.7M β-mercaptoethanol, 0.02% Bromophenol Blue in water. Tris-Buffered Saline Solution, pH 7.5. (TBS). Tris-Base 3.03g/L, NaCl 18.26g/L in water. Tris-Buffered Saline Solution with Tween, pH 7.5. (TBS-T). Tris-Base 3.03g/L, NaCl 18.26g/L, 0.05% Tween-20 in water. Transfer Buffer (1x), pH 8.3. 39mM Glycine, 48mM Tris-Base, 0.037% SDS, 20% Methanol in water.  3.1.3  Labware  Beakers, 50ml, 250ml, 600ml, 1L, Pyrex. Fisher Scientific, Fair Lawn, NJ, USA. Screw cap conical tubes, 15mL, 50mL. Sarstedt Inc. Newton, NC, USA. Sintered glass funnel, 3L, medium-pore glass frit. Fisher Scientific, Fair Lawn, NJ, USA. 48  Vacutainers, 15mL. No additive. BD, Franklin Lakes, NJ, USA. Vacutainers, 10mL. Heparin salt. BD, Franklin Lakes, NJ, USA.  3.2 3.2.1  Equipment X-Ray Powder Diffractometer  The purity of synthesized calcium pyrophosphate dihydrate crystals (triclinic) was determined using a Rigaku Geigerflex Diffractometer System Cat. # 4037A1 (Rigaku Denki Corporation, Tokyo, Japan) equipped with a biplanar goniometer and D/max-B controller unit.  3.2.2  Light Microscopy  Visual characterization of triclinic CPPD crystals and neutrophil suspension cell counts were performed by light microscopy on a Fisher Scientific Micromaster (Fisher Scientific, Fair Lawn, NJ, USA. #12-561-4B).  3.2.3  Hemacytometer  Neutrophil suspension cell counts were performed using a Hausser Scientific Bright-Line Hemacytometer with a 10µL loading capacity (Sigma # Z35,962-9; Sigma Chemical Co. St. Louis Mo, USA).  49  3.2.4  UV-Vis Spectrophotometer  Colormetric measurements for Bradford Assays and DNA Fragmentation Assays were performed in 96-well microtitre plates using a Labsystems 354 Multiskan Ascent Spectrometer (Cat. # 1507 540, Labsystems Oy, Helsinki, Finland).  3.2.5  Fluorometer  Fluorometric measurements for the Caspase-3 Activation Assays was performed in 96well microtitre plates using an Applied Biosystems CytoFluor Series 4000 Fluorescence Multi-Well Plate Reader with temperature control (Applied Biosystems Model # MIFS0C2TC, Framingham, MA, USA).  3.2.6  SDS-PAGE/Electrotransfer  Ten-well SDS-Polyacrylamide gels of 1.5mm thickness were hand-cast and run in a Mini-PROTEAN 3 Electrophoresis Cell System (Bio-Rad #165-3302; 165-3312; 1653365). Current was provided by a PowerPac 300 System 100/120 V power supply (BioRad #165-3314). Electrotransfer of gels to nitrocellulose were performed in a Mini Trans-Blot Cell System (Bio-Rad #170-3930). Bio-Rad, Hercules, CA, USA. 3.2.7  ECL Detection/Autoradiography  Enhanced Chemiluminescence detection of immunoblots was performed either by digital light capture CCD camera (Alpha Innotech Luminescence Detection Systems, Alpha Innotech, San Leandro, CA, USA) or by exposure to KODAK BioMax MS film (Cat. #  50  829 4985; Eastman Kodak Company, Rochester, NY, USA) in a FisherBiotech Autoradiography Cassette (Cat. # FBXC 810; Fisher Scientific, Pittsburgh, PA, USA) with an KODAK BioMax MS Intensifying Screen (Cat # 851 8706) for enhanced detection.  3.2.8  Band Densitometry  For all blot images/films, band densitometry was performed using FluorChem V2.0 software (Alpha Innotech Corporation, San Leandro, CA, USA).  3.3 3.3.1  Preparation of Calcium Pyrophosphate Dihydrate Crystals Synthesis of Calcium Dihydrogen Pyrophosphate (CDPP)  The first step in synthesizing CPPD crystals was to synthesize calcium dihydrogen pyrophosphate intermediate. This was performed as per J.R. Leher et al (226). Briefly, 250 mL of 85% H3PO4 was heated to 210oC in a 600 mL tinfoil-jacketed beaker with a Teflon-coated stir-bar and mercury thermometer. Once the acid was brought up to temperature, heat was reduced to maintain a constant temperature. Fifty grams of calcium phosphate (Ca(H2PO4)2.H2O) was then added at a rate of 2-4 g/min (allowing the temperature to stabilize between additions). Meanwhile, 50 mL of H3PO4 was heated in a 100 mL covered beaker to be used to preheat the fritted funnel. Another 40 g of calcium phosphate was then added to the 600 mL beaker at a rate of 1 g/min. A final few grams of calcium phosphate were added very slowly, waiting 3-5 minutes between additions to monitor changes in cloudiness and viscosity. At this point, scratching the side of the 51  beaker with a glass rod may aid in starting precipitation. Once the system was at approximately 25% precipitation, the 50 mL of hot H3PO4 was used to heat the mediumfritted glass funnel, and the precipitate was immediately filtered through using pump suction. After allowing the precipitate and funnel to cool, the precipitate was washed with copious amounts of acetone under suction and scraped out of the funnel onto watch glasses to air-dry overnight.  3.3.2  Synthesis of Calcium Pyrophosphate Dihydrate (CPPD - triclinic)  The second step in synthesizing CPPD crystals was to convert the calcium dihydrogen pyrophosphate (CDPP) intermediate into CPPD. This step was performed as per DJ McCarty et al (227). Briefly, 103 mL of ddH2O, 0.71 mL of conc. HCl, and 0.32 mL of glacial acetic acid were added to a 250 mL beaker with a Teflon stirbar. Twenty mL of ddH2O was also added to a separate 100 mL beaker and both beakers were placed into a 60oC water bath. Two 0.6 g aliquots of calcium acetate (Ca(C2H3O2)2.H2O) weighed out and one aliquot was added to each beaker. Once the beakers reached temperature, 2.0 g of CDPP intermediate was rapidly added to the 250 mL beaker and allowed to dissolve completely. The 20mL of calcium acetate solution (in the 100 mL beaker) was then added drop-wise into the large beaker. The congealing mixture was then removed from stirring and heat, covered and allowed to cool to room temperature. After 1-5 days, the white gel in the bottom of the beaker formed into crystals of CPPD. Excess liquid was decanted off and the crystals were then washed 3 times in ddH2O, once in ethanol, and once in acetone and allowed to air dry.  52  3.3.3  X-Ray Powder Diffraction Analysis  Determination of CPPD crystal purity was performed using X-Ray Powder Diffraction (XRPD) analysis as per C.I. Winternitz (228). Crystal samples were loaded flat into an aluminum sample tray and scanned using a Kα-emitting copper source/nickel filtered Xray tube (at 40kV potential; 20mA current). Scans were performed at 5-40 degrees 2θ at a scan rate of 3o/min and scan step of 0.05 degrees 2θ. XRPD scans of samples were then compared to published D-spacings for calcium dihydrogen pyrophosphate and calcium pyrophosphate dihydrate (Geological Sciences Library, Room 208 QC 481 A1 A51, UBC) to establish purity.  3.4 3.4.1  In Vitro Neutrophil Studies Opsonization of CPPD Crystals  Prior to use in the in vitro neutrophil experiments, CPPD crystals were opsonized in 50% plasma in HBSS as in (19, 24, 25, 27-33). One vacuum tube of whole blood containing heparin sulphate was centrifuged at 300x g for 15 minutes to separate plasma from cellular components. The plasma portion was removed and mixed 1:1 with HBSS and 400µL was added to each 1.5mL Eppendorf tube containing 30mg of CPPD crystals. Tubes were then vortexed and incubated at 37oC for 30 minutes with occasional mixing. The plasma was then aspirated off and the crystals were washed in 500µL of HBSS.  53  3.4.2  Isolation of Neutrophils from Peripheral Blood  Preparation of neutrophils from whole blood was performed as described in (19, 24, 25, 27-33). Ten 15 mL vacutainer tubes preloaded with 1mL acid citrate dextrose solution were drawn from a healthy donor. The blood was transferred into 2-3 50 ml Falcon tubes and 5mL of 5% Dextran T-500/RPMI 1640 media was added to each tube. Tubes were mixed by inversion and left to sediment for 30-40 minutes. The leukocyte-containing plasma partition was then removed by pipette, gently layered onto 10mL of Ficoll-Paque Plus in fresh 50mL Falcon tubes and centrifuged at 500x g for 30 minutes. The plasma layer, monocyte interface and Ficoll layers were then aspirated off leaving only the neutrophil-containing pellet in the bottom of the tubes. Cotton swab applicators were then used to wipe away remnants of the monocyte interface and any excess Ficoll. The neutrophil pellets were then further purified by mixing with 1ml ice-cold ddH2O for 10 seconds (hypotonic shock) to lyse any remaining erythrocytes. All cell suspensions were then quickly transferred to 46-48mL of ice cold RPMI 1640 to a total volume of 50mL and vortexed to mix homogenously. A 10µL aliquot was removed for neutrophil cell count via hemacytometer. Cells were then centrifuged at 250x g for 10 mins and resuspended in the appropriate volume of RPMI 1640 for the number of samples required and desired cell titre and placed on ice until needed. 3.4.3  In Vitro Treatments  In vitro treatments of neutrophils were performed as in (19, 24, 25, 27-33). The stock suspension of neutrophils in RPMI 1640 were preheated to 37oC for 10 mins prior to beginning treatment incubations. One mL of cells (15-20 x 106/mL) were incubated in 1.5mL Eppendorf tubes at 37oC either alone (spontaneous control), +/- formyl-methionyl54  leucyl phenylalanine (fMLP 1µM final conc.), or +/- CPPD (30mg/mL final) for 0, 0.5, 1.5 and 3 hours with occasional inverting of tubes to resuspend the cells. At the end of each time point, the designated tubes were removed from the water bath and placed on ice.  3.4.4  Cellular Apoptosis Assays  Quantification of neutrophil apoptosis was performed using two different cellular assays that looked at early and late apoptotic events. 3.4.4.1 DNA Fragmentation Assay The quantification of apoptotic DNA fragmentation in the various neutrophil samples was performed using the Cell Death Detection ELISA-Plus System (Roche Diagnostics GmbH, Mannhein, Germany). Kit components were prepared as per the manufacturer’s manual. Small aliquots (3x 2.5x105 cells) were taken from each in vitro neutrophil sample prior to spinning down the final pellet. These aliquots were placed into fresh 1.5 mL Eppendorf tubes and centrifuged at 5200x g for 1 minute. The media was then aspirated off and 200µL of lysis buffer (supplied in the kit) was added to each pellet, vortexed to mix and left at room temperature to lyse for 30 minutes. The lysates were then centrifuged and 20µL/well was loaded into the streptavidin-coated microtitre plate (supplied in the kit). To each well, 80µL of Immunoreagent solution (composed of antiDNA-peroxidase and anti-histone-biotin antibody conjugates in an incubation buffer – all supplied by the kit) was added and allowed to incubate at room temperature (23oC) for 2 hours on a plate shaker. Following antibody incubations, the lysate/immunoreagent 55  mixtures were decanted out and the wells were washed with Incubation buffer (3x 200µL). To each well, 100µL of ABTS (2,2’-azino-di (3-ethyl-benzthiazoline-6sulfonate)) substrate solution was added and the microtitre plate was read in a spectrophotometer at a wavelength of 405nm over 15 minutes. A negative and positive DNA control (supplied in the kit) was also run in each experiment.  3.4.4.2 Caspase-3 Activation Assay Early events in neutrophil apoptosis were detected using the CaspACE Assay System – Fluorometric (Promega, Madison, WI) to measure the amount of cleaved (activated) Caspase-3 in neutrophil samples. The assay was carried out as per manufacturer’s specifications. Preparation of cellular samples was modified from the manufacturer’s general methods. Briefly, following time course incubations of treated and non-treated neutrophils, the cells (6-8 x 106) were centrifuged at 5200x g for 1 minute and the medium was aspirated off. The pellet was washed once in cold HBSS and then resuspended in 100µL of hypotonic lysis buffer containing a protease inhibitor cocktail. Cell samples were typically snap frozen in liquid nitrogen and stored at –70oC. On the day of the assay, the samples were lysed via 3 cycles of freeze/thawing from –70oC to 30oC with vortexing between each cycle. The lysates were then centrifuged at 5200x g for 15 minutes to remove cellular debris and the supernatants removed and held on ice. Assay reaction buffers were prepared according to the manufacturer’s protocol and 15µL of each lysate was loaded into a 96-well plate in triplicate. Concomitantly, 2µL of each lysate was aliquoted into the wells of a separate plate for protein concentration  56  determination via Bradford Assay. To the caspase-3 assay plate, substrate and/or inhibitor was added to the designated wells and the reaction was monitored by fluorometer in seven 15 minute cycles with an excitation wavelength of 360nm and an emission wavelength of 460nm. Fluorometric data collected for each sample was then normalized by protein content.  57  3.5  Western Analysis  In an attempt to elucidate which Bcl-2 family proteins may be involved in the delay of neutrophil apoptosis observed in the cellular assays, Western analysis was performed on treated and non-treated neutrophil samples.  3.5.1  Preparation of Whole Cell Lysates for Western Analysis  Following in vitro time course incubations, neutrophil whole cell lysates were prepared as per protocols relating to the Kinexus KPKS apoptosis screen system. Briefly, neutrophil samples were centrifuged 5200x g for 30 seconds and the media was aspirated off. To each pellet, 450µL of KPKS Lysis Buffer was added and then vortexed to resuspend the cells. Samples were then placed on ice for 10 minutes. Each sample was then lysed by sonication (3 x 8 second pulses) on ice and then centrifuged at 5200x g for 1 minute at 4oC to remove cellular debris. Four hundred microliters of supernatant was then transferred to a new tube and 3 x 2µL of excess lysate was aliquoted into a 96-well microtitre plate for protein determination via Bradford assay. To the 400µL of lysate, 100µL of 5x Sample Buffer (containing β-mercaptoethanol) was added and the samples were vortexed and then boiled at 100oC in a water bath for 10 minutes. Samples were then allowed to cool before loading into gels. Loading volumes were calculated based on protein concentration to assure equal loading.  58  3.5.2  Protein Determination  In all experiments where protein determination was required, the Bradford-based BioRad Protein Assay Kit was used. A modified version of the manufacturer’s microtitre plate protocol was applied. Briefly, 2µl aliquots were taken from samples in triplicate and loaded into a 96-well microtitre plate. A set of serially diluted bovine serum albumin standards (0 - 5.76 mg/mL) were also included in each assay. To each well, 200µL of 1x Bio-Rad Protein Assay reagent was added. The plates were shaken and left to stand for a minimum of 10 minutes at room temperature before spectrophotometric measurement at 595nm.  3.5.3  Gel Preparation  All gels used in Western Blot analysis were prepared in house as 1.5mm-thick, 10-well, 13% Resolving/4% Stacking dual-layer SDS-polyacrylamide gels using the Bio-Rad Mini-Protean III Casting Module. General gel-making practices were taken from Dr. Steven Pelech’s lab – Faculty of Medicine, UBC. Briefly, glass plates were cleaned thoroughly with 70% ethanol and Kimwipes and allowed to dry. The plates were then locked into the casting apparatus and tested for leakage using ddH2O. The water was then removed from the plates and excess water was removed by blotting. Stacking and Resolving gel solutions were prepared with the TEMED added just before casting. The resolving gel solution was degassed in a sonic water bath briefly and then added between the plates by transfer pipette and filled to a height of 5.5 cm from the base of the plates. Approximately 1mL of saturated isobutanol was layered gently to the surface of the  59  resolving gel to remove menisci and ensure a flat edge to the top of the gel. The gel was then allowed to polymerize at room temperature for 30-40 minutes. Following polymerization, the isobutanol was poured off and the top of the gel was washed 3-4 times with ddH2O. The stacking gel solution was then mixed and added on top of the resolving gel and a ten-well gel comb was inserted to complete the casting. The stacking gel was typically polymerized within 10 minutes. The gel plates were then removed from the casting module and the well combs pulled. The wells were washed free of gel debris with ddH2O and the plates were mounted into the electrophoresis chamber as directed by the manufacturer.  3.5.4  SDS-PAGE/Electrotransfer  Following gel mounting in the electrophoresis chamber, the inner reservoir was filled with 1x Running Buffer past the top of the short plates so that the buffer was in direct contact with the wells of the stacking gel. The wells were loaded equally with 70-100µg of protein/well (amount depending on the concentration of the samples) and the gel chamber was placed in the outer reservoir (tank) where 1x Running Buffer was added to a minimum height of 4cm (to ensure the bottoms of the gels were fully submerged in buffer offering good conductivity). Gels were run at 70mA/gel for 60-70 minutes (until the dye-front reached the bottom of the gel) at room temperature under constant current. Following gel electrophoresis, the gel chamber was disassembled and the gels were removed from the glass plates and placed in a plastic container containing cold 1x Transfer Buffer for 5 minutes to allow for equilibration. Also in the container, were 6 x 9  60  cm nitrocellulose blots pre-soaked for a minimum of 30 minutes. The gels and blots were loaded into the Electrotransfer cassette and inserted in the transfer module. The transfer module was then placed into the running tank and filled with 1x Transfer Buffer and an ice-pack. Electrotransfer was run at 300mA for 45 minutes under constant current. The blots were then removed from the cassettes and Ponceau S staining for 5 minutes was then used to visually verify the transfer of proteins. The stain was then removed with multiple washings with ddH2O and the blots were allowed to dry at room temperature in the dark.  3.5.5  Immunoblotting  Prior to incubation with antibodies, the stored blots were first rehydrated in 1x TBS-T (Tris Buffered Saline with 0.05% Tween 20) for 20-30 minutes and then cut horizontally between the 45 and 80 kDa markers to separate the blot into high and low molecular weight sections. The high molecular weight halves (uppers) were used to detect the housekeeping protein p85 PI3-Kinase for total protein normalization and the low molecular weight halves (lowers) were used to detect the Bcl-2 protein of interest. For all halves, the blots were then blocked in 5% Skim Milk/TBS-T for 45 minutes on a shaker at room temperature and then washed 3 x 10 minutes with 1x TBS-T to remove excess blocking agent. The blot halves were then separated into different containers and the Uppers were incubated in rabbit-anti-human p85 PI3-Kinase primary antibody in 1x TBS-T at a 1:2500 dilution. The Lower halves were incubated with one of the selected antibodies specific to the Bcl-2 proteins of interest under various dilutions ranging from 1:200 – 1:500 depending on the relative affinities and strength of signal of the given 61  antibody. All primary antibody incubations were performed overnight at 4oC on a rotary shaker. On the following day, the primary antibody solutions were removed from the blots, recycled, and stored at 4oC being reused no more than 4-5 times. The blots were washed 3 x 10 minutes in 1x TBS-T and then incubated with the appropriate secondary antibody solution: Goat-anti-Rabbit- or Goat-anti-Mouse-Horseradish Peroxidase 1:2500 in 1x TBS-T for 1 hour at room temperature on a rotary shaker. Following secondary antibody incubation, the antibody solutions were removed and the blots were washed 3 x 10 minutes with 1x TBS-T. Enhanced Chemiluminescence was then performed on the blots soon thereafter.  3.5.6  Enhanced Chemiluminescence/Autoradiography  Detection of antibody-specific target proteins was performed using the Pierce Super Signal West Femto ECL kit (Pierce #34095) as per manufacturer instructions. Briefly, following the washing off of secondary antibodies, the blots were then removed from the wash solution and any access TBS-T was dabbed off. The blots were then laid out on Saran wrap and 2 mL of ECL working solution (300µL each of reagent A and B with 1.4mL ddH2O) was dispersed across the blot surface evenly by pipette and then further circulated across the entire blot surface by cradling the Saran wrap in a rotary fashion for 1 minute. Light capture from target protein bands was performed either by CCD camera with typical exposure times ranging from 1 – 6 minutes, or by direct exposure to Kodak Biomax film in an autoradiography cassette for times ranging from 20 seconds to 30 minutes. Stored digital image files from the CCD camera unit and scanned images of the developed film were saved in the highest resolution TIFF format possible. 62  3.6  Band Densitometry Analysis  Band densitometry was performed using Alpha Innotech’s ChemFluor software as per manufacturer’s instructions. Briefly, images were imported into ChemFluor and by use of the 1-Dimensional Multi-lane densitometry feature, bands of interest were measured for density and their integral areas calculated. Target Bcl-2 protein band areas were first divided by their associated housekeeping gene (p85 PI3-Kinase/hcIgG) band areas in order to normalize for loading differences. These normalized areas were then made relative by dividing each normalized area by the greatest normalized area recorded for each blot. These normalized relative areas were expressed as Relative % Areas in order to compare bands from blot to blot since not all blots were incubated or exposed under the same conditions. For each Bcl-2 protein target, the total Relative % Areas were compiled and averaged to give Average Relative % Expression for each treatment condition. Standard deviations for each average were also calculated and reported. In cases where individual relative normalized areas did not fall within 2 standard deviations of the mean, all data from that blot was removed from the data set as outlier data.  3.7  Statistical Analysis  Statistical analysis of the inter-treatment differences between the calculated average relative % expression levels of target Bcl-2 proteins was performed using an unpaired Student’s t-Test, one-tailed, with equal variance in an Excel spreadsheet. The confidence interval was set at 95%.  63  4 4.1  RESULTS Purity of Synthesized CPPD Crystals  Crystals of Calcium Pyrophosphate Dihydrate were produced as described by J.R. Leher et al and D.J. McCarty et al (226, 227). Analysis of the crystals was required to ensure that the proper agonist was created for use with the cellular apoptosis assays and Bcl-2 Western analysis of in vitro neutrophil preparations. X-Ray Powder Diffraction analysis indicated that the crystals produced were indeed the triclinic form of calcium pyrophosphate dihydrate. Based on relative peak intensities (I) and D-spacings (distances between crystal planes) for CPPD (Geological Sciences Library, Room 208 QC 481 A1 A51, UBC), it was determined that the synthesized triclinic CPPD crystals were of high purity. Table 1 shows the recorded peak intensities and D-spacings from the literature as well as those measured for the synthesized crystals.  Table 1. List of X-Ray Powder Diffraction D-spacings for CDPP and CPPD. Compound  D-Spacings  CaH2P2O7 (CDPP)  3.35 3.19 3.74 100 60 45 9-354  Ca2P2O2.2H2O (CPPD Triclinic) 8.01 6.95 3.21 100 100 80 12-27 Synthesized CPPD (T)  8.01 6.95 3.21 100 90  78  To further mimic the pathophysiological conditions of pseudogout, the size of the synthesized crystals was also measured by light microscopy. Naturally occurring crystals are typically 5-20µm in length and 1-5µm in width (4). Synthesized crystals were first 64  measured and then selectively separated by a 20µm sieve. Only crystals fitting the physiological size requirements were used in neutrophil experiments.  4.2  Cellular Apoptosis Assays  The effect of opsonized CPPD(t) crystals on neutrophil apoptosis was determined by two different cellular methods. The DNA Fragmentation assay measures the late-events of apoptosis as endogenous Caspase-dependent DNAases cleave the nuclear DNA into specific histone-associated fragments prior to dissolution of the nuclear membrane and final blebbing of the cytosolic membrane. The Caspase-3 assay measures the early events of apoptosis as the upstream Caspase-3 is activated via cleavage of its inhibitory domain. This is the first event in the Caspase cascade that subsequently leads to the activation of several apoptotic proteases and DNAases involved in the controlled dismantling of the function and structure of the cell. These assays, in combination with the preparation of neutrophils for Western analysis served to offer a measure of overall apoptosis occurring with respect to the effect of crystals observed on individual Bcl-2 family members.  4.2.1  DNA Fragmentation Assay  As previously reported by Burt et al (19, 24, 25, 27-33, 53), the data collected here in this study showed that both spontaneous and TNF-α-induced neutrophil DNA fragmentation was reduced in the presence of opsonized CPPD crystals. When neutrophils were incubated with TNF-alpha for 3 hours there was a statistically significant increase in the level of DNA fragmentation over spontaneous apoptosis observed in control cells.  65  Incubation of neutrophils with CPPD crystals caused a significant reduction in DNA fragmentation levels over control cells levels. The large TNF-alpha induced increases in DNA fragmentation (apoptosis) were significantly reduced when the cells were co incubated with CPPD crystals.  These experiments were repeated several times and  representative data are shown in Figure 5.  2.500  Absorbance (A595)  2.000 1.500 1.000 0.500 0.000 Spontaneous t=0  Spontaneous t=3h  TNF-alpha t=3h Treatments (n=5)  CPPD t=3h  TNFalpha/CPPD t=3h  Figure 5. DNA Fragmentation Assay: Representative Data. Quantitation of apoptosis levels (via DNA fragmentation) in treated and non-treated neutrophils as measured by relative absorbance at 595nm.  66  4.2.2  Caspase-3 Activation Assay  As with the DNA Fragmentation assay, the data compiled for this study coincide with those previously done by Burt et al. Figure 6 shows the representative data observed in this study. Opsonized crystals of CPPD appeared to reduce the amount of Caspase-3 activation observed in both spontaneous and TNF-α-induced apoptosis in neutrophils. The caspACE assay measures the amount of cleaved (activated) caspase-3 in neutrophil samples and this assay demonstrated excellent sensitivity in measuring background apoptosis occurring in untreated neutrophils. When neutrophils were incubated with TNF-alpha for 3 hours there was a significant increase in the level of caspase-3 activity over the spontaneous apoptosis observed in control cells as shown in Figure 6. Incubation of neutrophils with opsonized CPPD crystals caused a reduction in caspase-3 activity over control cells levels (Figure 6). The large TNF-alpha induced increases in caspase-3 activity were significantly reduced when the cells were co incubated with CPPD crystals. These experiments were several many times and representative data are shown in Figure 6.  67  Caspase-3 Activation (FU/mg protein)  12000 10000 8000 6000 4000 2000 0 Spontaneous Spontaneous t=0 t=3h  TNF-alpha t=3h  CPPD t=3h  TNFalpha/CPPD t=3h  Treatments (n=5)  Figure 6. Caspase-3 Fluorometric Assay: Representative Data. Quantitation of apoptosis levels (via caspase-3 activation) in treated and non-treated neutrophils as measured by relative fluorescent units per mg of protein.  4.3  Western Analysis of Bcl-2 Proteins  To further our knowledge of how opsonized CPPD crystal exact their apoptosis-delaying effect on neutrophils, Western analysis was performed, specifically targeting several Bcl2 protein family members to determine if the crystals modulate the levels of expression of any of these targets. Table 2 outlines which Bcl-2 targets were examined. The major intracellular proteins controlling apoptosis in all cells are the Bcl-2 family of proteins. Some of the proapoptotic proteins in this family are Bim, Bax and Bad and the prosurvival proteins are Bcl-2, Bcl-XL, A1 and Mcl-1.  68  Table 2. List of Bcl-2 Family Proteins Targeted by Western Analysis. Pro-Apoptotic Bcl-2 Targets  Anti-Apoptotic Bcl-2 Targets  Bax-α  Bcl-XL  Bim  Mcl-1  Bad  A1 (Bfl-1)  These proteins are thought to work in concert by assembling or disassembling apoptosisinducing protein clusters on the mitochondria. The composition and activity of the cluster determines the amount of each family member available (protein expression) at a particular time or the activation state of a particular protein which may affect the clustering activity of the particular protein. For example, the prosurvival protein Mcl-1 is reported to be turned over rapidly in cells (200, 202, 204) so that expression of this protein might tip the balance in favour of survival instead of apoptosis. On the other hand the phosphorylation state of Bad controls the contribution of this Bcl-2 family member so that the protein tends to be deactivated (less apoptotic potential) by phosphorylation. In neutrophils, the Bcl-2 family members reported to be present in these cells are (prosurvival) Mcl-1, A1 and Bcl-XL and (proapoptotic) Bim, Bax and Bad. Successful Western blot analysis requires effective antibody binding to target proteins. Many of the antibodies commercially available for this purpose had not been optimized for use with neutrophils. 69  Objective 1: The initial objective of the Western analysis section of this project was to screen neutrophils for the presence of each of the Bcl-2 family members considered most relevant. Antibodies to Mcl-1, A1, Bcl-XL, Bim, Bax and Bad from many sources were used in separate Western blot analysis of neutrophil proteins to try to initially establish whether the protein was present in significant quantities in the control cells. Objective 2: To investigate the expression or phosphorylation state of these Bcl-2 family members in neutrophils treated with either fMLP (prosurvival chemoattractant) or opsonized CPPD (t) crystals (also a prosurvival stimulant). These experiments were performed following incubation with fMLP or CPPD crystals for times, t= 0, 1 hour, 2 hours and 3 hours, with untreated cells incubated for the same periods for comparison. An increased or decreased expression of the proteins might therefore be visualized by either a significant increase in the protein band density of stimulated cells vs. control cells at the same time point. Such density was quantitated using densitometry analysis of individual bands. Any change in the activation state of proteins might be visualized by the appearance or disappearance of a phosphorylated protein band close to the original band (the protein specific antibodies might still bind to the phosphorylated forms of the protein  but  show a slightly increased  molecular weight  compared to the  unphosphorylated form). Again, the relative levels of the bands were determined using densitometry techniques. Briefly, the density of bands were normalized against housekeeping p85 protein and converted into % of the densest band per each blot so that data from each individual blot could be compared to one another.  70  4.3.1  Expression of BCL-XL  Data collected in this study indicates that opsonized CPPD had no effect on the level of Bcl-XL expression over the course of 3 hours. No measurable difference in expression was observed between spontaneous controls and crystal-treated neutrophils. Figure 7 shows the representative Western blot and the cumulative data for BCL-XL expression observed. Using 10-20 million cells per sample and a loading concentration of protein of 30-40µg in 20µL, the protein Bcl-XL was easily identified using Western blot analysis at an approximate molecular weight of 27 kDa using a variety of antibodies from different sources. Although nearly all antibodies tested gave a reasonable signal, the polyclonal antibody from Transduction Laboratories consistently gave excellent band densities and was used in all further studies to determine Bcl-XL levels in neutrophils incubated with fMLP or CPPD crystals.  71  100.0 80.0 60.0 40.0 20.0  t= 3h  t= 2h  PP D C  t= 1h  PP D C  t= 0h  PP D C  t= 3h  PP D C  t= 2h  LP fM  fM  LP  t= 1h  t= 0h  LP fM  LP  fM  lt  =3 h  =2 h  ro  lt C  on t  ro  lt C  on t  ro  lt  on t  ro C  on t C  =1 h  0.0 =0 h  Relative Expression (% of Max)  120.0  Treatments (n=6)  Figure 7. Representative data for Bcl-XL Western Analysis Experiments. Relative expression of Bcl-XL protein in treated and non-treated human neutrophils at 0, 1, 2 and 3 hours via Western analysis.  The band density for Bcl-XL in all studies was high and no apparent difference was observed between Bcl-XL expression in neutrophils incubated with CPPD crystals for 1, 2 or 3 hours and control neutrophils. Figure 7 shows both cumulative band density data for the time course of Bcl-XL expression along with bands taken from a representative Western blot.  72  4.3.2  Expression of MCL-1  From the data collected in this study, there appeared to be no differential expression of Mcl-1 between spontaneous controls and CPPD-agonized neutrophils over the course of 3 hours. Figure 8 shows the representative Western blot and the cumulative data for Mcl1 expression observed. Using 10-20 million cells per sample and a loading concentration of protein of 30-40µg in 20µL, the protein Mcl-1 was easily identified using Western blot analysis at an approximate molecular weight of 42 kDa using a variety of antibodies from different sources. Although nearly all antibodies tested gave a reasonable signal, the polyclonal antibody from Santa Cruz Biotech consistently gave excellent band densities and was used in all further studies to determine Mcl-1 levels in neutrophils incubated with fMLP or CPPD crystals.  Relative Expression (%)  120.0 100.0 80.0 60.0 40.0 20.0 0.0 Control Control Control Control fMLP fMLP fMLP CPPD CPPD CPPD t=0h t=0.5h t=1.5h t=3.0h t=0.5h t=1.5h t=3.0h t=0.5h t=1.5h t=3.0h Treatm ents (n=10)  Figure 8. Representative Data from Mcl-1 Western Analysis Experiments. Relative expression of Mcl-1 protein in treated and non-treated human neutrophils at 0.5, 1.5 and 3.0 hours via Western analysis. 73  The band corresponding to Mcl-1 in all studies was consistently detected and no apparent difference was observed between Mcl-1 expression in neutrophils incubated with CPPD crystals for 0.5, 1.5 or 3 hours and control neutrophils. Figure 8 shows both cumulative band density data for the time course of Mcl-1 expression along with bands taken from a representative Western blot.  4.3.3  Expression of BIM-EL  The Western analysis of Bim-EL expression was of particular interest in this study, as it has not been previously reported by any group in the literature. From the data collected in this study, it appears that opsonized CPPD crystals may have initially reduced the amount of activated native Bim-EL expressed in neutrophils when compared to the spontaneous controls as measured at the 0.5 hour time point. However, by 1.5 hours, any survival effect illicited by CPPD was absolved and the levels of BIM matched those seen in the control samples. On an added note, there was a trend of increasing native Bim-EL observed in CPPD-agonized neutrophils over the time course of 3 hours, but also an inversely related decrease of phosphorylated (inactive) Bim-EL was detected during the same time period. Figure 9 shows the representative Western blot and the cumulative expression data for Bim-EL in neutrophils. Since there have not been other reports of the presence of this Bcl-2 family protein in neutrophils, an appropriate antibody for use in neutrophils had not been previously described.  74  Relative Protein Expression  120.0  BIM-P  100.0  BIM 80.0 60.0 40.0 20.0 0.0 Control t=0.5h  Control t=1.5h  Control t=3.0h  fMLP t=0.5h  fMLP t=1.5h  fMLP t=3.0h  CPPD t=0.5h  CPPD t=1.5h  CPPD t=3.0h  Tre atm e nts (n=4)  Figure 9. Representative Data from Bim-EL Western Analysis Experiments. Relative expression of native BIM-EL protein and phosphorylated BIM-EL protein in treated and non-treated human neutrophils at 0.5, 1.5 and 3.0 hours via Western analysis.  However, the monoclonal antibody from Affinity Bioreagents proved to provide adequate detection of this protein in neutrophils. With this antibody, all three isoforms of Bim protein: Bim-EL (23 kDa), Bim-L (16 kDa) and Bim-S (13 kDa) were detected as shown in Fig. 10. The function of these three isoforms is not known, however, it has been shown that the EL isoform has the greatest apoptotic effect compared to its truncated counterparts (218, 222). The band densities for all isoforms of the protein in neutrophils were consistently strong lending the protein to detailed band density analysis following 7 repeated experiments with fresh samples used each time. 75  120.0 BIM-EL BIM-L  Relative Expression (%)  100.0  BIM-S  80.0 60.0 40.0 20.0 0.0 Control t=0.5h  Control t=1.5h  Control t=3.0h  fMLP t=0.5h  fMLP t=1.5h  Treatments (n=3)  fMLP t=3.0h  CPPD t=0.5h  CPPD t=1.5h  CPPD t=3.0h  Figure 10. Representative Data for the Isoforms of Bim in Western Analysis Experiments. Relative expression of the –S, -L, and –EL isoforms of BIM protein in treated and nontreated human neutrophils at 0.5, 1.5, and 3.0 hours via Western analysis.  In control cells at 0.5 hours, 1.5 hours and 3 hours incubation, there was no detectable change in the band densities of the three isoforms of Bim (Figure 10). Similar results were found for fMLP stimulated neutrophils with an approximately equal distribution of the three isoforms of Bim and no apparent difference in the expression compared to those of control cells at the same time points.  76  In CPPD crystal stimulated neutrophils, the expression level of Bim-EL was similar to that found in both control and fMLP stimulated cells. The level of Bim-EL expression was slightly higher than that found in control cells at 0.5, 1.5 and 3 hours but not significantly so. The expression level of the Bim-EL isoform was reduced at 1.5 hours relative to both CPPD at 0.5 hours and control cell levels at 1.5 hours. However the variation in levels was large as witnessed by large error bars on the values at 1.5 hours and there was no statistical differences between values. The expression levels of all three isoforms of Bim were further reduced at 3 hours for CPPD stimulated neutrophils relative to those for CPPD stimulated cells at both 0.5 hours and 1.5 hours and compared to control values at 3 hours (Figure 10). Due to the large variations in expression levels, these reduced expression levels were statistically significant for Bim-S only. The Bim-EL isoform may be phosphorylated to an inactive state (221-223). In cells, this phosphorylated form may be detected using the same antibody but at a molecular weight position slightly shifted up from the native Bim-EL form of the protein due to the increased net negative charge caused by the added phosphate group. The split protein band at a molecular weight of 23 kDa indicates that the antibody is measuring both the native and phosphorylated form of this protein. In control cells at 0.5 hours, most of the Bim protein was found in the non-phosphorylated form as seen in Figure 9. At 1.5 and 3 hours, this disparity had largely disappeared in control cells. In CPPD crystal stimulated neutrophils at 0.5 hours, most of the Bim protein was in the phosphorylated, inactive form with only a minor amount of the protein in the non-phosphorylated, native form. This disparity slowly reversed itself over the next 2.5 hours so that at 3 hours there was approximately twice as much Bim in the native form (Figure 9). The concomminant  77  decrease in the phosphorylated form of Bim in CPPD treated cells was however, not significantly different from levels observed in Control cells (Table 3). Table 3. Summary of Statistical Analysis on Band Densitometry (Western) Data. Comparison: Control vs. CPPD (3h) Target  Experimental Hypotheses:  p-value:  BCL-XL 0: no difference in expression A: increased expression in CPPD  MCL-1  Statistical Inference: 0.027 Reject Null Hypothesis  (negative t-value!)  0: no difference in expression  Type I Error?  0.465 Cannot Reject Null Hypothesis  A: increased expression in CPPD  BAXalpha  0: no difference in expression  0.00003 Reject Null Hypothesis  A: decreased expression in CPPD  BIM-EL 0: no difference in expression  0.184 Cannot Reject Null Hypothesis  A: decreased expression in CPPD  P-BIM  0: no difference in expression  0.408 Cannot Reject Null Hypothesis  A: increased expression in CPPD  A1  N/A  P-BAD  N/A  78  Comparison: CPPD (0.5h) vs. CPPD (3h) Target  Experimental Hypotheses:  p-value:  BCL-XL 0: no difference in expression A: increased expression in CPPD  MCL-1  Statistical Inference: 0.040 Reject Null Hypothesis  (Negative t-value!)  0: no difference in expression  Possible Type I Error  0.004 Reject Null Hypothesis  A: increased expression in CPPD  BAXalpha  0: no difference in expression  0.001 Reject Null Hypothesis  A: decreased expression in CPPD  BIM-EL 0: no difference in expression  0.004 Reject Null Hypothesis  A: decreased expression in CPPD  P-BIM  0: no difference in expression  0.021 Reject Null Hypothesis  A: increased expression in CPPD  A1  N/A  P-BAD  N/A  4.3.4  Expression of BAX-α α  The Bax-α protein was difficult to probe using commercially available antibodies. The monoclonal 6A7 clone and polyclonal NT antibodies from Biosource International and Upstate gave inconsistent and variable results on Western blots. However, the 6A7 monoclonal antibody from Trevigen gave consistent, but low levels of detection and was used for all quantitative western blotting described in this study.  79  Using this antibody, the expression of Bax-α in control cells remained unchanged at 0.5, 1.5 and 3 hours as shown in Figure 11. The level of Bax-α at 0.5 hours in cells treated with CPPD crystals was approximately the same as that found in control cells at all time points. However, the level of Bax-α reduced over time in CPPD-treated cells and at 3 hours this reduction was statistically different (Table 3).  Relative Expression (%)  120 100 80 60 40 20  .0 h t= 3  .5 h t= 1  C PP D  t= 0  C PP D  3. 0 t=  C PP D  fM LP  t=  .5 h  h  h 1. 5  h 0. 5 t=  fM LP  t= ro l  fM LP  3. 0h  1. 5h t= C on t  ro l C on t  C on t  ro l  t=  0. 5h  0  Treatm ents (n=7)  Figure 11. Representative Data of Bax-alpha Western Analysis Experiments. Relative expression of Bax-α protein in treated and non-treated human neutrophils at 0.5, 1.5, and 3.0 hours as measured via Western analysis.  4.3.5  Expression of A1/BAD  During the course of this study, the detection of the Bcl-2 proteins A1 and BAD via Western Analysis was attempted several times. However, due to technical difficulties, 80  neither of these proteins was detected at measurable levels. In the case of A1, no reliable commercial antibodies were available at the time of these experiments. An antibody produced by Abgent was used in several Western blot experiments, but was unable to detect any A1 protein. As for the detection of Bad protein, different antibodies from Upstate and Cell Signaling Technologies detecting various phosphorylated species of Bad were tried several times at increasingly higher concentrations but with no success. After further futile adjustments to Western protocols in an attempt to optimize the detection of Bad, the search for this protein was abandoned.  81  5  DISCUSSION  5.1  Purity of Synthesized CPPD Crystals  Calcium pyrophosphate crystals found in the synovial joint of people suffering from pseudogout have been reported to be in both triclinic and monoclinic polymorphic forms (18). However, the majority of the precipitated CPPD is found in the triclinic form (18, 228).  Therefore, in these studies the triclinic form of CPPD was synthesized,  characterized and used in all cellular studies. This synthesis was a two-step process whereby an intermediate crystal form (Calcium Dihydrogen Pyrophosphate (CDPP)) was manufactured under rather extreme conditions (by the addition of calcium phosphate to phosphoric acid at 210oC) with precipitant clean up in acetone. This was followed by a slow crystallization process whereby the triclinic form of CPPD crystallized from a supersaturated gel of the CDPP and calcium acetate under acidic conditions. The initial synthesis of CDPP allowed for the manufacture of one large batch of material (perhaps 40-60 grams). The second slow crystallization of CPPD was limited to the production of small batches (perhaps 2-4 grams). The purity of the CDPP was established using X ray diffraction. The d spacings for this intermediate have been previously described (228) and the manufactured intermediate product gave strong X ray diffraction peaks with the correct d spacings for this salt. The formation of amorphous non-crystalline by-products may be identified by increases in the baseline signal from the X-ray detector across all angles, but the baseline for the CDPP was effectively zero for all the various batches manufactured during these studies. The addition of excess non-dissolved calcium phosphate would be identified by the presence  82  of additional x-ray diffraction peaks overlapping the CDPP peaks but these were also not observed in these studies. Under optical microscopy, the CPPD samples showed an elongated prismatic habit with dimensions of between 2-20uM, typical of CPPD triclinic crystals found in joints. There was no other material present in the samples. The three most intense X-ray diffraction peaks are usually used as unique identifiers in X ray crystallography and, in these studies, these peaks translated to d-spacings of 8.01 6.95 3.21 Angstroms. For all CPPD samples studied by X ray, the baseline (non-diffracted X rays) was effectively zero. The Joint Committee of Powder Diffraction Standards (JCPDS) values for CPPD triclinic form are 8.01, 6.95 and 3.21. Therefore, it was established that the crystals manufactured in these studies were indeed the triclinic form of CPPD.  5.2 5.2.1  Cellular Apoptosis Assays DNA Fragmentation  The DNA Fragmentation assay by Roche is based on a two-step, dual-antibody sandwich ELISA reaction that provides reliable, reproducible quantitative analysis of late-stage apoptotic events from cellular lysates. It has been utilized by many groups studying apoptosis, including this lab. Dr. Burt’s lab has previously used this assay in neutrophil apoptosis studies where the assay showed correlation with the results of caspase-3 activation and trypan blue staining of dead cells (27, 30, 33). In these experiments, the DNA Fragmentation assay was used in conjunction with the CaspACE cellular assay and Western analysis of Bcl-2 proteins to show the effect of 83  CPPD crystals on the delay of neutrophil apoptosis and the effect on individual Bcl-2 protein expression levels. In all cases, the observed reduction of DNA fragmentation levels coincided with the delay of apoptosis observed in CPPD-treated neutrophil versus control cells. It should be noted that the results of the caspase-3 activation assay also showed a decrease in caspase-3 activity in CPPD-treated cells compared to control cells, suggesting that any differences in Bcl-2 protein expression observed may be associated with this delay of apoptosis.  5.2.2  Caspase-3 Activation Assay  The CaspACE Assay System by Promega is an enzymatic-based, quantitative, fluorometric assay for detecting one of the pivotal early events in cellular apoptosis. The cleavage and activation of procaspase-3 by upstream apoptotic initiators (namely caspase-8 and -9) is a major step in the amplification of the intracellular apoptotic signalling pathway (98). This assay allows for quick, reliable detection of the amount of activated caspase-3 from cellular lysates and has been used extensively by several groups including this one. Previously, Dr. Burt’s group has used this assay to measure apoptosis in CPPD-treated neutrophils in order to elucidate upstream molecules involved in apoptosis signalling (27, 30, 33). In these experiments, the CaspACE cellular assay was used in conjunction with the DNA fragmentation assay and Western analysis of Bcl-2 proteins to show the effect of CPPD crystals on the delay of neutrophil apoptosis and the effect on individual Bcl-2 protein expression levels. Again, in all cases, the reduction of caspase-3 activation observed coincided with the delay of apoptosis observed in CPPD-treated neutrophil versus control 84  cells suggesting that any observed differences in Bcl-2 protein expression may be associated with this delay effect. It should be noted that the results of the DNA fragmentation assay also showed a decrease in caspase-3 activity in CPPD-treated cells compared to control cells.  5.3  Western Analysis of Bcl-2 Proteins  Previous studies in Dr. Burt’s lab on other neutrophil proteins (eg. PI3-kinase, PLCγ, ERK1/2) have used extracts from approx 107 cells to give very strong signals by western analysis. However, Bcl-2 family protein Western analysis was complicated by poor signal strength at these cell concentrations. Therefore the number of cells used had to be increased, as well as the concentration of antibody.  Also, the enhanced  chemiluminescence exposure times were required to be longer, sometimes resulting in excessive background interference. These low detection levels may reflect an overall small concentration of the Bcl-2 family members present, generally. To compound this problem, neutrophils contain extremely high levels of various proteases as part of their antimicrobial arsenal. Under normal conditions, these destructive enzymes are safely compartmentalized into specialized vesicles and delivered to their intended target under the precisely controlled fusion of such vesicles with the bacteria-containing phagosome. However, when preparing whole cell lysates for western analysis, the disruption of these cells by detergent or sonication results in the release of these proteases into the lysate. Cellular proteins are immediately exposed and become easy targets for proteolytic degradation. For that reason, much time and effort was spent  85  optimizing the protocol for preparing these lysates. Factors such as time, temperature and protease inhibitor combinations were exhaustibly tested to create the best conditions to reclaim as much native protein from the neutrophils as possible. Previously, this group had tried using such inhibitor combinations including Sigma’s Protease Inhibitor cocktail (for mammalian cells) in conjunction with the addition of PMSF, STI, pepstatin A, leupeptin, DFP, and others. The use of Roche’s Complete-Mini Tablet protease inhibitors with additional PMSF proved to give the best inhibition of these resident proteases. All the following experiments in this study exclusively used this combination.  5.3.1  Expression of BCL-XL  The protein Bcl-XL has been reported to be associated with survival signalling in numerous cells and is one of the most common Bcl-2 family members reported to be present in apoptosis studies. Indeed, antisense oligonucleotides to this protein have been developed in an attempt to promote drug-induced apoptosis in proliferating cells in cancer and angiogenesis (229). However, the presence of this protein in neutrophils has been contentious in that a number of studies failed to detect Bcl-XL protein by Western analysis (114, 199-201). On the other hand, some studies reported detecting both Bcl-XL mRNA and protein in neutrophils (157, 159, 205). Western blotting techniques used in these studies clearly identified Bcl-XL in neutrophils. The presence of the protein was visualized using antibodies from many sources as a strong band at a molecular weight of approximately 27 kDa. These new data conclusively demonstrate that Bcl-XL is present in neutrophils.  86  Interestingly, there were no detectable differences in Bcl-XL expression in neutrophils in any of these studies, irrespective of whether the cells were untreated or activated by crystals or fMLP. Like other prosurvival proteins, this Bcl-2 member is located on the mitochondrial membrane and the role of the protein is thought to depend on an inhibitory interaction with proapoptotic proteins. For example, it has been suggested that the BH13 domains of Bcl-XL form a hydrophobic pocket into which the BH3 domain of Bak could fit suggesting a mode of action for how Bcl-XL may bind and counter the prodeath effect of Bak within a cell (167). If Bcl-XL was involved in the prosurvival process in neutrophils then an increased expression might be anticipated whereby more protein was available at the mitochondrial membrane to inhibit the action of proapoptotic members.  5.3.2  Expression of MCL-1  The Mcl-1 protein was detected in both control and CPPD-treated neutrophils using an antibody from Santa Cruz BioTech. The band density of this protein was weak but this may reflect the binding strength of the particular antibody for the amino acid sequence on the electrophoresis transfer rather than the amount of Mcl-1 present. A number of antibodies were evaluated for use in these studies and only the Santa Cruz Biotech antibody gave reasonable levels of detection. Several studies have reported that freshly isolated human neutrophils do have detectable levels of the Bcl-2 homologues Mcl-1 and A1 (114, 152, 184, 193, 194, 202, 203). The results described in this study support these previous reports. However, there was no  87  change in Mcl-1 expression observed in these studies, potentially suggesting that Mcl-1 is not involved in the CPPD crystal - prosurvival signal pathway. Like the prosurvival protein Bcl-XL, Mcl-1 is thought to be associated with the mitochondrial membrane where it may interact with proapoptotic proteins to inhibit the cytochrome c releasing potential of these proteins. It has been reported that Mcl-1 has a rapid turnover in cells, rather than a long residence time on the mitochondrial membrane (152). One mechanism for Mcl-1 control of apoptosis may be via apoptosis-stimuli directed degradation of this protein by caspases allowing the release of Mcl-1sequestered apoptosis-inducing Bcl-2 family members such as Bim (224).  5.3.3  Expression of Bim-EL  All three Bim isoforms were found to be expressed in neutrophils. The signal strengths from commercially available antibodies were strong, reinforcing the positive identification of the protein in these cells. To date, there is little information about the role of Bim in neutrophil apoptosis. There has been one recent report of the expression of Bim in neutrophils where sepsis-induced survival signals were shown to downregulate Bim. Interestingly, this downregulation was shown to be dependent on the PI3-K and ERK pathways (217). The prosurvival chemoattractant fMLP had no significant effect on Bim expression but CPPD crystal-activation of cells induced a significant decrease in the expression of all three forms after three hours incubation (Fig. 10). Unfortunately, the phosphorylated form (inactive) of Bim-EL was not found to increase in CPPD activated  88  neutrophils over 3 hours in contrast to the native (active) form of Bim-EL as would have been expected (Fig. 9). In other cell types, apoptosis inducing agents such as reactive oxygen species, drugs or growth factor removal have been shown to increase Bim expression (218-220). These previous studies support the well-accepted biochemical relationship linking an increased expression of a proapoptotic Bcl-2 family member with cell death. Of interest, more complex mechanisms for Bim involvement in apoptosis have been proposed involving the sequestration of Bim by the prosurvival protein Mcl-1 so that caspase cleavage of this protein may release functional Bim. This functional form of Bim may then work in concert with another pro-apoptotic protein Bax to accelerate apoptosis. (224). It would have been expected to observe a CPPD crystal-induced reduction in Bim-EL (active form) and an increase in the phosphorylated form (inactive) in these studies, which would fit the recognized biochemical model linking Bim expression with the apoptotic state. However, this was not the case and no associative link between posttranslational modification of Bim and crystal-induced survival in neutrophils was found.  5.3.4  Expression of BAX-α α  The proapoptotic protein Bax-alpha was clearly identified in neutrophils in these studies. The expression levels of this protein were found to remain fairly constant in control cells. Of all the Bcl-2 family members, Bax has been quite extensively studied in neutrophils. The presence of the protein has been reported by many workers (145, 153, 159, 207-210). In neutrophils, the expression of proapoptotic proteins Bak, Bax, Bad, Bik and Bid has 89  generally been thought to remain fairly constant through the apoptotic process with a relatively long half life for each of these proteins (152, 159, 184, 199, 206). In this study the expression of Bax-α in CPPD crystal activated neutrophils was found to decrease over time (Fig. 11). This reduction in Bax-α expression may mirror the results for Bim-EL whereby a reduction in expression of the proapoptotic protein equates to a prosurvival signal in the cells. The involvement of Bax-α in pro-survival signalling in neutrophils has been previously described in support of these results. The chemoattractant cytokines GM-CSF, G-CSF, LTB4 were all reported to decrease the expression levels of Bax (145, 159, 207). It should be noted that, like Bim and Bad, the phosphorylation state of Bax-α is related to the functional role of this protein in apoptotic control (208, 210). Furthermore, Maianski et al (130) demonstrated mitochondrial Bax translocation during spontaneous apoptosis in neutrophils without significant changes in overall Bax protein levels and that G-CSF blocked the translocation of Bax to the mitochondria and subsequent caspase-3 activation. These previous studies indicate an alternative or parallel scenario whereby the expression, location and phosphorylation state of Bax may all affect the apoptotic potential of this protein. These other aspects of Bax were beyond the scope of these studies and not investigated.  5.3.5  Expression of A1/BAD  The identification of A1 in neutrophils using “in-house” antibodies or nucleic acid tags has been previously reported (114, 152, 184, 193, 194, 202, 203). However, well90  described, commercially available antibodies to this protein were not available at the time of these studies. Numerous attempts were made to detect A1 using a polyclonal antibody from Abgent, but no trace of protein could be found. A similar picture evolved for Bad analysis in neutrophils with no protein detected using a variety of antibodies. This apparent absence of BAD in neutrophils was confusing since BAD expression has been previously reported. Indeed, the phosphorylation of BAD to it’s inactive form by cellular activation with such prosurvival factors as GM-CSF, TNF-α, heme (NADPH-oxidase dependent), LPS, peptidoglycan, c5a, and platelet-activating factor has been described in detail (148, 153, 215, 216). The reason for the lack of detection of this protein in our neutrophil samples is unknown, but the most likely candidate would be the resident proteases present in abundance in neutrophils. There may be some unforeseen sensitivity of Bad, or at least the epitopes on Bad utilized by the antibodies tested, to be degraded more quickly than other Bcl-2 proteins. In any event, since this protein could not be detected in neutrophils, efforts were directed to the study of the Bcl-2 family members that showed good detection sensitivity namely: Bim, Bax, Bcl-XL and Mcl-1.  91  6  SUMMARY AND CONCLUSIONS  Although it seems that all cells contain the biochemical machinery for apoptosis, the study of cellular apoptosis over the last few decades has been largely restricted to cancer cell processes.  Normal resting cells do not experience damaging environmental  circumstances where apoptosis might be of physiological relevance. For example, if cells were subjected to extremes of temperature, radiation or chemical challenge the integrity of the cells would be so compromised so that cytolysis or necrosis would occur before sophisticated apoptosis mechanisms might engage. In cancer, however, cells that would not normally proliferate may undergo phenotypical changes that cause disruption to normal protein homeostasis and allow the proliferation processes to begin.  Normally, such radical biochemical changes inside cells and  unwanted proliferation would be identified and countered by the apoptotic processes inside the cells. However, the intracellular balance of Bcl-2 family members that might promote apoptosis in such cancer cells may also be compromised phenotypically so that the cells may survive and proliferate. Accordingly, small molecule anticancer drugs target the DNA of these cells to cause nuclear damage that overrides the prosurvival state of the cells and allows apoptosis to proceed.  Alternatively, modern anticancer  therapeutics use gene therapy agents (antisense oligonucleotides, ribozymes or siRNA) to target the upregulated prosurvival Bcl-2 family member. Therefore, immortal cancer cell lines offer a unique opportunity to study apoptotic control as the relationship between Bcl-2 composition, balance and activation state and apoptotic rates may be more easily understood. These studies in cancer cells have proved invaluable in the construction of a  92  rough biochemical roadmap linking signal transduction cascades with Bcl-2 family balance and function. Neutrophils offer a similar intriguing cellular prospect since these cells are almost unique in having a constitutive apoptotic process on board as part of normal cell function. They are destined to die by apoptosis within a day of entering the bloodstream. These cells may be compared to the Japanese Kamikaze pilots in the second world war who would take off from the mainland knowing they did not have enough fuel to return home. If they flew over empty ocean then they would simply run out of fuel and pitch into the ocean without explosion. If they saw enemy boats they would dive in and explode. For neutrophils, the safe disposal of cells that have not engaged bacteria ensures that no background damage is caused by the onboard proteolytic “munitions”. However, neutrophil apoptosis is intriguingly frustrating to study because in comparison to cancer, seemingly diametrically opposite circumstances are encountered. Cancer is characterized by processes that see abnormal expressions of Bcl-2 family members occurring which prevent apoptosis and the therapeutic objective of anticancer drugs may be to reverse such an imbalance. Conversely, neutrophils have an onboard constitutive apoptosis program engaged (with no apparent disease implications) but this program is inhibited or stopped if the cells are stimulated by pathogenic agonists. Subsequent phagocytic responses of neutrophils in such a prosurvival state may be associated with some unwanted background inflammation but such collateral damage is tolerable with respect to the elimination of bacteria. However, the prosurvival state encountered in numerous disease states (such as arthritis, psoriasis, inflammatory lung disease, etc.) may lead to chronic pathological and degradative processes. 93  Therefore, as in cancer, the  therapeutic objective of treating such diseases may be to counter the prosurvival state of these neutrophils to reduce cell numbers safely in inflamed sites. Clearly most studies in cancer cells describe a complex and highly controlled apoptotic machinery. The sophistication of this machinery derives from normal cell biochemistry and originates in the need to prevent the elimination of healthy cells in the body. This need is essential to the overall survival of the organism so that only when critical safety control checks have been breached does the cell then begin the process of assembling the on board “gallows” for the final self execution. It seems likely that in neutrophils, such fine control systems may be redundant. Excluding necrosis or cytolytic death, whatever situation is encountered by a neutrophil will not prevent final death by apoptosis. Encountering pathogenic material or chemoattractants may delay the execution but non-engagement or even the phagocytic process halts the delay process and apoptosis proceeds.  Since a physiological  requirement for apoptosis under any condition occurs in these cells it makes sense that the cells should have a Bcl-2 composition and activation state primed in a proapoptotic state. This might equate to a pre-assembled gallows (perhaps constructed from Bcl-2 family members on the mitochondrial membrane) requiring, for example, either a positive/negative input from the hangman for the execution to proceed/delay. In other words, the only required circuitry for the control of neutrophil apoptosis may involve a simple delay signal. In support of this concept, the multi-lobed nucleus of neutrophils is known to have very low transcriptional activity so the ability to direct apoptotic events from the nucleus may be limited (108). In other words, when a neutrophil encounters pathogenic stimuli, it is unlikely that the prosurvival response arises from complex 94  nuclear control of Bcl-2 family members but rather slight modulation of existing Bcl-2 protein expression or activation status. In this study, it was expected that an observed decrease in the expression of active Bim, and increased expression of the phosphorylated form (inactive) may represent a simple prosurvival switch. Unfortunately, the results of our studies on Bim yielded conflicting data. Still, Han et al (224) have described a relationship between Bim and Bax, suggesting a pro-apoptotic collaboration in neutrophils.  Other workers have  demonstrated that the phosphorylation state of Bim depends on PI-3 Kinase and ERK1/2 (221-223). Similarly, we have previously demonstrated that the CPPD prosurvival signal also depends on these signalling enzymes. Despite the results of this study, there may yet be a relation between CPPD crystal-induced PI-3 Kinase and ERK1/2 activation and an increase in the phosphorylation state of Bim, decreasing the proapoptotic potential of this protein to induce a prosurvival state. 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