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High density human neutrophils produce a transferable factor that delays apoptosis Ahmad, Sharon Ameena 1998

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H I G H D E N S I T Y H U M A N N E U T R O P H I L S P R O D U C E A T R A N S F E R A B L E F A C T O R T H A T D E L A Y S A P O P T O S I S by S H A R O N A M E E N A A H M A D B . S c , The University o f Alberta, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S Department of Pathology and Laboratory Medicine We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A September 1998 © Sharon Ameena Ahmad, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pa-Hnrittfr.) * I 7A)^D \AtA~XUf\L The University of British Columbia Vancouver, Canada •ate (kp\.an(ng • DE-6 (2/88) ABSTRACT Pilot experiments performed in this laboratory showed that there was a delay in neutrophil apoptosis in culture at high cell densities. Increasing cell density resulted in a logarithmic decline in the percentage o f apoptotic neutrophils when cells were cultured at densities between 3 x l 0 3 and l x l O 7 cells/cm 2 (p<0.003, n=3). Investigation of this phenomenon resulted in the detection of a transferable factor produced by high density neutrophils, that was able to delay apoptosis in low density neutrophils when co-cultured. Various soluble mediators were studied to determine the identity of the transferable factor. E L I S A assays performed on high density neutrophil-conditioned medium indicated that, of the cytokines tested ( G M - C S F , IL-8, IL-6, IL-113, and TNF-a ) , neutrophils were producing only IL-8 in significant quantities. Monoclonal neutralizing antibodies to the cytokines G M -C S F , G - C S F , and IL-8 added to neutrophil cultures were not successful in inhibiting the high density delay in neutrophil apoptosis, nor was the platelet-activating factor inhibitor, W E B 2170. The role of cell-cell contact and adhesion molecules CD18, C D l l b , C D l l a , and L -selectin, were also investigated as high neutrophil density suggests increased cell-cell contact. Blocking antibodies to these cell adhesion molecules added to neutrophil cultures had no effect on the density-dependent delay in neutrophil apoptosis. The C D 1 8 / C D l l b upregulator, f M L P , added at either high or low concentrations, also had no effect on neutrophil apoptosis at any density. These data show that there is a delay in neutrophil apoptosis at high cell densities, potentially involving a transferable factor produced by the high density neutrophils, and unlikely to involve intercellular interaction of C D 18, C D l l b , C D l l a , or L-selectin. These findings have important implications for the study of neutrophils both in vitro and in vivo. i i TABLE OF CONTENTS Abstract 1 1 Table of Contents i i i List o f Tables v i List o f Figures v i i List o f Abbreviations v i i i Preface ix Acknowledgements x CHAPTER I INTRODUCTION 1 1.1 Neutrophils and Acute Inflammation 1 1.2 Mediators of Acute Inflammation 3 1.3 Neutrophils and Apoptosis 5 1.4 Neutrophil Apoptosis and Acute Inflammation 9 1.4.1 Soluble mediators of acute inflammation 9 1.4.2 Neutrophil functional responses 10 1.4.3 Neutrophil adhesion 13 1.5 Hypothesis and Objectivives 14 CHAPTER II METHODS AND MATERIALS 20 2.1 Monoclonal Antibodies 20 2.2 Monoclonal Antibody Working Concentrations 20 2.3 Basic Methods 21 2.3.1 Neutrophil isolation 21 2.3.2 Assessment of apoptosis 22 i n 2.4 Standard Conditions and Experimental Protocols 22 2.4.1 Time course of neutrophil apoptosis 23 2.4.2 Assessment of serum source in neutrophil cultures 23 2.4.3 Neutrophil-conditioned medium transfer experiments 24 2.4.4 Identification of a transferable factor 24 2.4.5 Measurement of cytokine levels in supernatants 26 2.4.6 Cytokine neutralizing experiments 27 2.4.7 Investigation o f platelet-activating factor antagonist W E B 2170 27 2.4.8 Cel l adhesion molecule blocking experiments 28 2.4.9 Investigation of f M L P in neutrophil apoptosis 28 2.5 Statistical Analyses 28 CHAPTER III RESULTS 29 3.1 Time Course of Neutrophil Apoptosis in Culture 29 3.2 Assessment of Serum Sources in Neutrophil Cultures 29 3.3 Investigation of a Transferable Factor in Neutrophil-Conditioned Medium 32 3.3.1 Neutrophil-conditioned medium does not delay apoptosis in fresh neutrophil cultures 32 3.3.2 Neutrophils produce a transferable factor that delays high density neutrophil apoptosis 32 3.3.3 Cultured neutrophils produce IL-8 in supernatants 36 3.3.4 Cytokine neutralizing antibodies do not inhibit the delay in apoptosis in high density neutrophils 37 iv 3.3.5 Platelet-activating factor is not involved in the high density delay in neutrophil apoptosis 39 3.4 Investigation of Neutrophil Adhesion and Apoptosis 39 3.4.1 Blocking antibodies to cell adhesion molecules do not inhibit the delay in apoptosis at high neutrophil densities 39 3.4.2 Neutrophil incubation with f M L P does not inhibit apoptosis at high cell densities 45 CHAPTER IV DISCUSSION 49 4.1 Neutrophil Adhesion 51 4.2 Transferable Factor 52 4.3 Summary and Conclusions 55 Bibliography 58 v LIST OF TABLES Soluble Mediators of Neutrophil Functional Responses in Acute Inflammation: Effects on Neutrophil Apoptosis Specifications of E L I S A Assay Cytokine Production by High Density Cultured Neutrophils A s Measured by E L I S A v i LIST OF FIGURES 1. Photograph o f cytocentrifuged preparation of cultured neutrophils stained with Wright's stain 7 2. Photograph of cultured neutrophils at densities of 1x10 (A), 3x10 (B), l x l O 4 (C), 3 x l 0 4 (D), l x l O 5 (E), 3 x l 0 5 (F), l x l O 6 (G), 3 x l 0 6 (H) cells/well 16 3. Effect of neutrophil density on apoptosis 18 4. Co-culture of donor and reporter neutrophils to detect the presence of a transferable factor produced by high density donor cells 25 5. Time course of neutrophil apoptosis 30 6. Evaluation o f the effect o f human A B serum versus fetal calf serum (FCS) in neutrophil cultures 31 7 A , B Effect of neutrophil-conditioned medium on neutrophil apoptosis 33-34 8. Production of a transferable factor by high density "donor" neutrophils 35 9. Effect of cytokine neutralizing antibodies on neutrophil apoptosis in high and low density neutrophil cultures 38 10. Effect o f W E B 2170 on the density-dependent delay in neutrophil apoptosis 40 11 A , B Effect o f monoclonal blocking antibodies to cell adhesion molecules on neutrophil apoptosis at high and low neutrophil densities 42-44 12A,B Effect of f M L P on the density-dependent delay in neutrophil apoptosis 46-48 v i i LIST OF ABBREVIATIONS B S A Bovine Serum Albumin E L I S A Enzyme-Linked Immunosorbent Assay FasL Fas-ligand F C S Fetal Ca l f Serum f M L P Formyl-Methionyl-Leucyl-Phenylalanine G - C S F Granulocyte Colony-Stimulating Factor G M - C S F Granulocyte-Macrophage Colony-Stimulating Factor H 2 0 2 Hydrogen Peroxide H N S H E P E S Normal Saline L P S Lipopolysaccharide P B S Phosphate-buffered Saline T G F Transforming Growth Factor T N F Tumor Necrosis Factor v i i i PREFACE Neutrophil apoptosis plays a critical role in the process of acute inflammation. Neutrophil infiltration is a key occurrence in acute inflammation, and the actions of the neutrophils can be both beneficial and harmful. Neutrophils are necessary to ingest and k i l l offending agents, however they often prolong inflammation and induce damage to surrounding tissues by releasing enzymes and toxic reactive oxygen species. Under normal circumstances, neutrophils undergo apoptosis, a type of programmed cell death that allows for their removal by marcrophages without further release o f tissue-toxic products. In pilot experiments we have shown that when neutrophils were cultured at high densities, similar to the number of neutrophils in an inflammatory focus, apoptosis was delayed. Thus the longer neutrophils remain alive in an inflammatory focus, the longer they have to cause damage to surrounding tissues. We have examined the density-dependent delay in neutrophil apoptosis, and some potential mechanisms of action, including cytokines and cell adhesion molecules. Alternative modes of action and areas for further investigation are suggested, and both in vitro and in vivo implications are discussed. ix ACKNOWLEDGEMENTS I would like to thank Dr. Blair Walker for his guidance, support, and enthusiasm throughout my graduate studies. I greatly appreciate the invaluable research skills I learned under his guidance, and thank him for his patience, his time, and his commitment which enabled me to complete this work. I would also like to thank Corinne Rocchini for the assistance she gave me with the experimental work, and for her patience when I was beginning. Thank you to Rob Boone, who completed the pilot studies for this thesis. Finally, I thank my family for their constant support and encouragement. In particular, thank you, Sarah, for knowing me so well . Chapter I INTRODUCTION 1.1 Neutrophils and Acute Inflammation Acu te in f l ammat ion is the immediate response that occurs as a result o f invas ion by , most common l y , bacteria and other microbes, but also i n response to mechan ica l trauma, neoplasms, and toxins (1,2). It is general ly o f short durat ion, last ing f r om just a few minutes to one to two days (2). Edema , due to changes i n vascular f l o w and permeabi l i ty , and the emigrat ion o f leukocytes characterize acute in f lammat ion . D u r i n g the first 6-24 hours, neutrophils are the predominant ce l ls , be ing replaced b y monocytes i n 24-48 hours (2). The in i t ia l insult causes the release o f chemica l mediators f r om loca l tissues and ce l ls , w h i c h stimulates vasodi la t ion and s low ing o f the c i rcu lat ion, and eventual ly leads to stasis (2). The c i rcu lat ing neutrophils s low suff ic ient ly to a l l ow marginat ion a long the vascular endothel ium, i n preparation for extravasation and migrat ion to the inf lammatory focus (2). L-selectin mediates the ro l l i ng o f the neutrophils a long the surface o f the endothe l ium w i t h weak adhesive bonds (3-6). Th is ro l l i ng activates integrins on the neutrophi ls b y induc ing a conformat ional change i n the extracel lular b ind ing doma in on these molecu les . U p o n act ivat ion, L-selectin is rapid ly shed and the ro l l i ng process ends, leading to a f i rm attachment o f the neutrophils to the endothel ium, us ing ma in l y the C D 1 8 / C D l l b comp lex (7). C D 1 8 / C D l l b also mediates the movement o f the neutrophils through the endothe l ium and into the extravascular space (diapedesis) (7). The migrat ion o f neutrophi ls toward the inf lammatory focus is k n o w n as chemotaxis , meaning movement a long a chemica l gradient. Bacter ia l products, the C 5 a component o f the complement system, and cytokines such as IL-8 and transforming growth factor-13 (TGF -P) , are some o f the k n o w n neutrophi l chemoattractants (2,8-10). 1 Once at the acute inflammatory site, neutrophils adhere to the extracellular matrix and prepare to engulf the foreign material and k i l l it through oxygen-dependent mechanisms and degranulation (2). After eliminating the source of the acute inflammation, resolution must occur in the form of removal of the neutrophils as they are unable to migrate out of an inflammatory focus (11,12). Disintegration of these cells with subsequent release of granule contents (necrosis) would cause further tissue injury and amplification of the inflammatory response (13). Therefore, effective resolution involves a process called apoptosis. Apoptosis is a process which invokes endogenous biochemical process, resulting in the death o f the cell without further release of tissue-toxic products (14-18). This reduces the cytotoxic action of neutrophils and terminates the neutrophil response by allowing the recognition and removal by inflammatory macrophages, thereby avoiding injury to surrounding tissues (19-30). It is well established that macrophages use integrin a v P3 (vitronectin receptor, CD51/CD61) and CD36 (thrombospondin receptor) on the macrophage surface cooperatively (23-26,31-33), as well as thrombospondin in the recognition and removal of apoptotic cells. However, the changes on the neutrophil membrane that allow for macrophage recognition have not yet been defined. There is speculation that surface carbohydrate composition may be altered as treatment with proteases does not affect recognition (34). Surface phospholipids are also suspect, as apoptosis induces the acquisition of these annexin V binding sites on neutrophils (35). Annexin V is a calcium-dependent protein that binds phospholipids and has a particularly high affinity for phosphatidylserine. Phosphatidylserine residues are normally found only on the inner surface of the plasma membrane of cells, but are expressed on the outer surface of the membrane on apoptotic cells. Phosphatidylserine expression allows for recognition and binding by annexin V , and providing another means of detection of apoptotic cells (36). More importantly, however, the phosphatidylserine residues are essential for macrophage recognition and phagocytosis (37). Another possible change on neutrophils is an RGD-containing ligand expressed by neutrophils undergoing apoptosis (38). This ligand's 2 receptor appears to be the macrophage integrin a v P3, and would thus allow for macrophage recognition and removal of these neutrophils (39,40). The focus of this research is apoptosis, an important part of the resolution phase of inflammation. Studying the factors that delay apoptosis in neutrophils may lead to novel therapeutic strategies in which neutrophils are forced into apoptosis at inflammatory sites, thus preventing or minimizing their destructive potential. 1.2 Mediators of Acute Inflammation Neutrophils were originally considered to be terminally differentiated cells, without the ability to synthesize and secrete proteins (41,42). It is now recognized that neutrophils are capable of synthesizing and secreting cytokines and other factors, possibly including interferon- (IFN-), platelet activating factor (PAF) , leukotriene B 4 , EL-1, IL-6, IL-8, tumour necrosis factor- (TNF-), granulocyte colony-stimulating factor (G-CSF) , and granulocyte-macrophage colony-stimulating factor ( G M - C S F ) (43-62). The most important cytokines present in an acute inflammatory environment are EL-1, T N F - a , IL-8, G M - C S F , and G - C S F , and their main source is activated macrophages and endothelial cells (2). Other factors with important roles in acute inflammation are EL-6, P A F , reactive oxygen species, and bactericidal enzymes. Both EL-1 and T N F - a are often cited as being the key mediators of biological responses to inflammatory stimulants and infection, and share many of their biologic properties (2,63,64). EL-1 has several pro-inflammatory roles and can induce the production of CSFs and other cytokines, including T N F (64). T N F is also a pro-inflammatory cytokine and in addition can cause neutrophil aggregation and priming, leading to heightened responses to other mediators (2,61,65,66). EL-8 is most commonly known as a neutrophil activating factor and a chemoattractant (8,9,67,68). Both endothelial cells and neutrophils produce EL-8 in the acute inflammatory environment, keeping the neutrophils exposed to high quantities of this cytokine (66,69,70). 3 The most important inducers of IL-8 m R N A expression and secretion are I L - l a , J X - l p , and T N F - a (8,71). The many actions IL-8 has on neutrophils are mainly identified on the basis of chemotaxis and the release of granule enzymes from the neutrophils (8,72). IL-8 also upregulates surface expression of the cell adhesion molecules CD18/CD1 l b (73). G M - C S F and G-CSF are pro-inflammatory cytokines which act to enhance the function of neutrophils both directly and indirectly (74-85), however G - C S F has weaker effects (86-89). IL-6 is a pleiotropic cytokine possessing a broad range of biologic functions (70). It has many inducers, however, production generally requires cell activation. IL-6 is important in mediating acute inflammatory reactions. In particular it induces acute phase proteins (91-93), and enhances cytotoxic functions of neutrophils (94,95), suggesting that IL-6 plays a role in neutrophil-mediated tissue injury. P A F is a phospholipid derivative with biological activity that mediates interactions between cells, and is a known activator of neutrophils (96-100). P A F has many proinflammatory roles (97,99,101-106), including stimulating both functional and non-functional upregulation of C D 1 8 / C D l l b and C D 1 8 / C D l l a adhesion protein complexes on neutrophils leading to enhanced binding to endothelium (105,107-109). IL-6 has been shown to stimulate the production of P A F in neutrophils (94). A s well , one study reports that IL-6-stimulated neutrophils are primed for 0{ release by lower doses of P A F than unstimulated neutrophils (110). ; 1 In addition to the cytokines and other soluble mediators secreted by neutrophils, reactive oxygen species and bactericidal enzymes are also important mediators of acute inflammation. Neutrophil exposure to bacteria, chemoattractants, immune complexes, or microcrystals of calcium oxalate or calcium pyrophosphate activates the N A D P H oxidase system located in the neutrophil plasma membrane, leading to the generation o f superoxide molecules and subsequently, hydrogen peroxide (111-116). Released into the acute inflammatory environment, the reactive oxygen species are non-discriminatory, ki l l ing host 4 tissue, microbes, and even other neutrophils (113,117-121). Neutrophils also produce bactericidal enzymes such as myeloperoxidase and proteases which, like reactive oxygen species, can be secreted into the extracellular environment damaging surrounding tissues and amplifying the inflammatory response (111-113). In this way, neutrophils themselves become the injurious agent if they are not removed after resolution of the infectious challenge. 1.3 Apoptosis Apoptosis can be defined as an intrinsic mechanism of a cell involving a specific series of cellular events that culminates in the death of that cell (14-18). Apoptosis can result from a variety of sources, however in the immune system a mechanism allowing for direct self-destruction of individual cells is arguably the most important. In this type of apoptosis, cell surface receptors (death receptors) transmit an apoptosis signal initiated by specific ligands (122). The death receptors themselves possess domains that categorize them as belonging to the tumor necrosis factor (TNF) receptor gene superfamily, however they also possess a "death domain" which typically allows them to engage the cell's apoptotic machinery (123,124). The best characterized of the receptors are Fas (CD95 or Apol) and TNFR1 (p55 or CD120a) (125). Ligation of a death receptor generally leads to clustering of the receptors' death domains, and leads to binding of an adaptor protein (122). Adaptor proteins possess "death effector domains" which, directly or indirectly, can trigger the activation of a family of proteases called caspases (122). These proteases are thought to be responsible for driving all the structural nuclear changes that accompany apoptosis (126,127). Caspase induction of apoptosis can also be activated from the mitochondria (128). Mitochondria can be triggered by multiple stimuli such as oxidants, C a 2 + overload, active caspases, and possibly ceramide, to release caspase-activating proteins such as cytochrome c (128). 5 Ultimately, through either signaling pathways or mitochondria, the activation of caspases results in apoptosis (129). Caspases are very specific proteases which have an absolute requirement for cleavage after aspartic acid residues (126,127). They are synthesized as precursors that undergo proteolytic maturation (126). The relationship between caspase cleavage and cell death is only well understood for a small number of the known caspases, but it is thought that a subset is responsible for effecting the cellular changes that occur in apoptosis through disassembly of the cell (129). Caspases can inactivate proteins that inhibit apoptosis (130). A n example would be cleavage of the nuclease C A D (caspase-activated deoxyribonuclease), which is responsible for D N A fragmentation (130). Caspases can also disassemble cell structures directly, such as the nuclear lamina which provides structure to the nuclear membrane (131), or indirectly by cleaving proteins involved in cytoskeleton regulation (132). Finally, caspases can deregulate protein activity, inactivating necessary functions such as D N A repair. Although there has been extensive investigation into caspases and apoptosis in some cells, the role caspases play in neutrophil apoptosis remains unknown. There is little information available regarding their activation or regulation. Neutrophil apoptosis is a process involving an orderly resorption of the neutrophil and its contents, and is distinct from necrosis, which involves disintegration of the cell through osmotic lysis following irreversible injury. Apoptosis is characterized by distinct morphological and biochemical changes. Using light or electron microscopy, the classic morphological features of apoptotic cells are compaction and margination of chromatin, a reduction in cytoplasmic volume and cell size, and blebbing of both nuclear and cytoplasmic membranes. This latter process can lead to fragmentation of the cell and the formation of apoptotic bodies that may contain nuclear material. Cytocentrifuged preparations of neutrophils stained with Wright's stain are useful in distinguishing apoptotic and non-apoptotic neutrophils. Apoptotic neutrophils appear small and rounded, with dark, evenly stained nuclei, and may show evidence of chromatin margination and karyorhexis (Fig. 1). 6 Fig . 1. Photograph of cytocentrifuged preparation of cultured neutrophils stained with Wright's stain. Broken arrow indicates normal neutrophil showing large size, with lobated, unevenly stained nucleus. Solid arrow indicates apoptotic neutrophil, which is rounded, smaller in size, with a dark, evenly stained nucleus, and shows evidence of chromatin margination. 7 Normal, non-apoptotic neutrophils are larger in size, with lobated, unevenly stained nuclei. Apoptotic neutrophils show characteristic evidence of D N A fragmentation (133-136), which is due to the action of activated endogenous endonucleases that cleave double-stranded D N A at linker regions between nucleosomes resulting in D N A lengths of integer multiples o f 180 to 200 base pairs (137,138). This nucleosomal chromatin cleavage results in a characteristic "ladder pattern" seen on gel electrophoresis, and is exploited in assays for apoptosis, such as flow cytometry, in which normal and apoptotic cells can easily be distinguished (139-141). Other changes that occur in apoptotic neutrophils include the loss of both L-selectin (CD62L) and C D 16 (Fc receptor III) (142,143), and a reduced ability to undergo respiratory burst, stimulated shape change, chemotaxis, degranulation and adhesion (144-148). A s mentioned previously, little is known about internal cell regulation o f neutrophil apoptosis. Two areas of interest to researchers are the Bcl-2 gene family, and the Fas antigen. The Bcl-2 gene family is a family of cell death regulators possessing either anti- or pro-apoptotic activity (149,150). bcl-2 and A l are both members of the Bcl-2 gene family, bcl-2 is a human proto-oncogene product originally described in human B cell lymphomas at the chromosomal breakpoint of t( 14; 18) (151-154). bcl-2 is unique among proto-oncogenes, as it localizes to inner mitochondrial membranes in cells (155). Overexpression of bcl-2 prevents apoptosis by inhibiting the release of cytochrome c from the mitochondria, prolonging the survival of these cells (142,143,156-159). Early myeloid cells of the bone marrow are the only cells that express bcl-2 endogenously, and it is usually absent in blood neutrophils (160-162). However, bcl-2 does inhibit apoptosis of mature neutrophils in transgenic mice expressing this protein (163). A l is a hemopoietic tissue-specific early response gene expressed in murine T helper lymphocytes, macrophages, and neutrophils (164). It is inducible by G M - C S F , and other proinflammatory cytokines in endothelial cells (165), and its peptide sequence shows similarity to bcl-2 and a related gene in the same family, M C L 1 (164). The human bfl-1 8 protein shows the highest homology with murine A l protein of all the bcl-2 family members, and has been shown to suppress apoptosis induced by p53 tumour suppresser protein (150). Fas antigen is a cell-surface protein, of the tumor necrosis factor/nerve growth factor receptor family (166-168). Both Fas and its natural ligand, Fas-ligand (FasL) are believed to be expressed on the surface of mature human neutrophils (169,170), and the interaction of Fas/FasL or Fas/anti-Fas monoclonal antibody leads to apoptosis in these cells, suggesting that this system may play an integral role in regulating apoptosis (166,168,171-173). However, one group was unable to detect Fas antigen on human neutrophils, nor show acceleration of apoptosis by anti-Fas antibody (174). 1.4 Neutrophil Apoptosis and Acute Inflammation In the circulation, neutrophils have a half-life of five to six hours and after this time they are sequestered, most likely, in the macrophages of the spleen and liver (21). Neutrophils that migrate to inflammatory tissue sites have a greatly extended half-life o f one to two days, suggesting that the neutrophils respond to their inflammatory environment in such a way that the induction of apoptotic neutrophil death and recognition by macrophages is delayed. There are many factors to consider in the study of the regulation of neutrophil apoptosis. Those relevant to acute inflammation can be grouped into three categories: soluble mediators, neutrophil functional responses, and neutrophil adhesion. , , , ., , 1.4.1 Soluble mediators of acute inflammation The cytokines best known for delaying apoptosis in neutrophils are G M - C S F and G -C S F (20,64,175-190). A s with its other actions, G M - C S F has a more profound effect than G -C S F (86). One study even reports that G - C S F has no effect on neutrophil apoptosis (176). J L - i p is well known as an inhibitor of neutrophil apoptosis (178,191). Although T N F is generally considered a pro-apoptotic cytokine (174,192,193), there have been some 9 conflicting reports showing that it delays apoptosis in neutrophils in culture (178,194). Murray et al (195) also describe a decrease in neutrophil apoptosis upon prolonged incubation with T N F - a , while earlier incubation times seem to induce apoptosis. The effect of EL-8 on neutrophil apoptosis has not been resolved. Earlier studies reported that EL-8 has no effect on neutrophil apoptosis (176,178,187,196), while more recent findings report that the cytokine may delay (197) or promote (198) apoptosis in neutrophils. Conflicting evidence exists as to whether or not EL-6 can cause a delay in neutrophil apoptosis. EL-6 has been shown to delay neutrophil apoptosis (199,200), and it has been suggested that the delay caused by EL-6 may be dependent on the density of the neutrophils in culture (201). Other groups, however, have described a variety of neutrophil responses to EL-6, including no delay in neutrophil apoptosis upon addition of EL-6 (176), and even induction of neutrophil apoptosis by EL-6 (202). However, Bi f f l et al (199) have also suggested that P A F , which is implicated in postinjury inflammation (203-205), may play a role in EL-6-mediated inhibition of neutrophil apoptosis. Evidence for this theory shows that production of P A F is stimulated by EL-6 (94), and P A F itself can inhibit T N F from inducing neutrophil apoptosis (195). Furthermore, Bi f f l et al (199) have shown that neutrophil pretreatment with a P A F antagonist ( W E B 2170) abrogates the anti-apoptotic effects of both EL-6 and P A F . Interestingly, however, one study reports that the production and release of P A F from neutrophils decreases on a per well basis at high total cell concentrations (206). , . ,„ ; . , 1.4.2 Neutrophil functional responses It is generally believed that reactive oxygen species are involved in the process of apoptosis, and may act to promote programmed cell death (207). Hannah et al (208) show that hypoxia profoundly inhibits neutrophil apoptosis in vitro, and hydrogen peroxide (H2O2), one of the metabolites of superoxide, induces a concentration-dependent increase in the rate of neutrophil apoptosis. A more recent study reports that both superoxide anion and H2O2 can accelerate apoptosis (209). Additional work in this area seems to support the idea that 10 reactive oxygen species may help to regulate neutrophil apoptosis. It also has been reported that high neutrophil density greatly inhibits the production of reactive oxygen species, increasing neutrophil survival in culture (210,211), and that the addition of extracellular antioxidants can inhibit spontaneous neutrophil apoptosis (212). The ingestion of bacteria by neutrophils may also be involved in the regulation of neutrophil apoptosis, although the type of bacteria may determine the manner of involvement (213,214). One study reports that neutrophil ingestion of Escherichia coli (gram-negative bacteria) accelerates inducible apoptosis through oxygen-dependent mechanisms (213). Two other studies show prolonged survival of neutrophils following ingestion of Staphylococcus aureus (gram-positive bacteria) (190,214). Bacterial endotoxin is a lipopolysaccharide (LPS) that is a structural component in the outer cell wall o f gram negative bacteria (2). Neutrophils primed by L P S show enhanced secretory responses to secretagogues like formyl-methionyl-leucyl-phenylalanine ( fMLP) (186,215,216). L P S markedly inhibits neutrophil apoptosis, possibly in a concentration-dependent fashion, and prolongs neutrophil functional ability (84,178,186,188). 11 Table 1. Soluble Mediators and Neutrophil Functional Responses in Acute Inflammation: Effects on Neutrophil Apoptosis (numbers in brackets refer to references) A G E N T I N C R E A S E S A P O P T O S I S N o E F F E C T O N A P O P T O S I S D E C R E A S E S A P O P T O S I S G M - C S F (20), (64), (175), (176), (178-180), (183-187), (189) G - C S F (176) (175), (177), (178), (181-183), (187-190) IL-10 (178), (191) T N F - a (174), (192), (193), (195) (178), (194), (195) IL-8 (198) (176), (178), (187), (196) (197) IL-6 (202) (176) (199-201) , P A F (195 a) Reactive oxygen species (207), (208 b), (209), (210 c), (21 l d ) , (212 e) Ingestion of gram — bacteria (166) Ingestion of gram + bacteria (214) L P S (178), (186), (188) a P A F inhibits T N F from inducing apoptosis i Hypoxia inhibits apoptosis i ' 1 . ' > c ' d High neutrophil density inhibits reactive oxygen species production, increasing neutrophil survival ? Addit ion of anti-oxidants to neutrophil cultures inhibits apoptosis 12 1.4.2 Neutrophil adhesion The role of neutrophil-endothelial adhesion in acute inflammation has been studied extensively and is essential in the recognition of inflammatory sites and migration through the endothelium and extracellular matrix (217). L-selectin is constitutively expressed on all leukocytes, excluding a subpopulation of B lymphocytes (6,218) and, as mentioned, is used by neutrophils to roll along the endothelium in the post-capillary venules (3-6). L-selectin is expressed until the leukocytes become activated, and is then shed (219,220). The C D 1 8 / C D l l b complex stops the rolling and allows the neutrophils to adhere firmly to the endothelium, then helps them to migrate through to the extravascular space where they are in constant contact with the extracellular matrix (7). Neutrophils express many cell adhesion molecules on their outer surfaces, including the P2 integrins (most notably CD18/CD1 l b and C D 1 8 / C D l l a ) , L-selectin, CD31 ( P E C A M - 1 ) , I C A M - 3 , and sialyl-Lewis x (221-224), and while the relationship between these adhesion proteins and apoptosis has been well examined in many cell types (225-231), it has not been in neutrophils. There is no consensus on exactly how cell adhesion molecules contribute to neutrophil apoptosis. Neutrophil adhesion to extracellular matrix proteins, such as fibronectin, vitronectin, or collagens, has been shown to promote apoptosis (232), or to have no effect (239). Conflicting evidence also exists as to the role played by P2 integrins, with some studies reporting that expression of CD18, C D l l a , and C D l l b are reduced on apoptotic neutrophils (233), while others show that increased levels of the C D 1 8 / C D l l b and CD18/CD1 l a complexes are expressed on apoptotic cells (234,235). Crosslinking of CD1 l a or C D l l b has been shown to delay neutrophil apoptosis in culture (236), although others have reported that the P2 integrin C D 1 8 / C D l l b accelerates apoptosis (237,238), or is not involved at all (239). 13 1.5 Hypothesis and Objectives Hypothesis Neutrophil apoptosis is delayed by intercellular contact between neutrophils which is dependent on CD18/CDllb activation and leads to the release of anti-apoptotic cytokines. Objectives 1. To demonstrate the presence of a soluble factor(s) in neutrophil-conditioned medium that delays apoptosis. 2. To demonstrate the role of neutrophil cell adhesion molecules in delaying apoptosis at high cell densities. The relationship between neutrophil density and various aspects of neutrophil physiology has been studied by several groups. These studies indicate that neutrophils cultured at high densities in vitro show greatly reduced in function (201,210,211,239,240) Peters et al (211) have investigated the functional aspects of the neutrophil activation response at varied cell densities. They found that when adherent, high density neutrophils (20x10 6 cells/ml) are activated, the activity of N A D P H oxidase is approximately 10-fold less per cell than when low density neutrophils are activated. They also show a three- to seven-fold decrease in superoxide anion release in high density neutrophils, and report that the production of H2O2 by neutrophils at high density is markedly attenuated on a per cell basis. In addition, Peters et al (211) report attenuation of arachidonic acid mobilization, phospholipid metabolism, and possibly phosphotidylinositol turnover when high density neutrophils are activated. Tanigawa et al (210) also report that the amount of superoxide generated is inversely related to cell density, and that this is not due to the release of nitric oxide or adenosine by 14 the high density neutrophils. Additionally, this group found that survival rates of neutrophils after stimulation are increased when neutrophils are cultured at higher densities (approximately 3x10 6/ml). Neutrophil density and cytokines have been studied by two groups. Jobin et al (232) Q cultured extremely high densities of neutrophils in 96 well tissue culture plates (up to 1x10 cells/ml) and found that these high density conditions led to the induction of the EL-1(3 gene at both R N A and protein levels. Bi f f l et al (201) investigated neutrophil density in relation to the cytokine EL-6. They report that there is an inhibition of neutrophil apoptosis by EL-6 that is dependent on the concentration of neutrophils, or that there was more inhibition of neutrophil apoptosis by EL-6 when neutrophils were at a density of 2 0 x l 0 6 / m l versus 1-5 x l 0 6 / m l . They also mention that the concentration of neutrophils in culture does not appear to affect the apoptotic process in IL-6-untreated cultures. However, close examination of their data indicates that in untreated cells, there is at least at 20% increase in survival in neutrophils at a density of 20x l0 6 /ml as compared to those at l x l 0 6 / m l . Very recently, Hannah et al (239) published a study showing that cell density modulates neutrophil apoptosis in culture, and that this is not dependent on P2 integrin-mediated adhesion, nor influenced by adhesion to certain extracellular matrix components. They clearly show that the rate of apoptosis is inversely proportional to the cell density, or the number o f cells per unit surface area. The hypothesis and specific aims of this thesis were developed from pilot studies in which it was observed that increasing cell density of neutrophils in culture directly correlated with a decrease in apoptosis. Initially, photographs were taken of neutrophils settled on the surface of tissue culture plates at various densities (Fig. 2). A t the lower densities ( l x l 0 3 and 15 16 3x10 3 cells/well) there was very little contact between neutrophils in the wells. A s the density of neutrophils was increased, however, greater numbers of neutrophils came in to contact with each other, so that at the highest densities ( l x l O 6 and 3 x l 0 6 cells/well), virtually all neutrophils in the wells were touching. Figure 3 shows that after 16 hours of culture, increasing cell density resulted in a logarithmic decline in apoptosis (p<0.003, n=3). This experiment led to the hypothesis that high neutrophil density results in a high degree of contact between the cells which activates the CD18/CD1 l b complex, stimulating the neutrophils to produce anti-apoptotic cytokines. The cell-cell contact experienced by neutrophils in this system is similar to a phenomenon known as homotypic aggregation. Neutrophils are typically found in the circulation as single cells in the resting state. In response to various soluble stimuli, such as P A F or f M L P , neutrophils bind transiently to one another, both in vivo and in stirred cell suspensions. This phenomenon is known as homotypic aggregation (241-248) and is mediated mainly by the CD18/CD1 l b complex, and to a lesser extent, L-selectin (5,242,247-250). Although neutrophil homotypic aggregation has been well studied, neutrophil-neutrophil adhesion under static conditions, as might be found in both the tissues and the model system being used in this thesis, have not. Because of the critical roles the C D 1 8 / C D l l b complex and L-selectin play in homotypic aggregation, and the delay in neutrophil apoptosis seen with cross-linking of C D l l a or C D l l b (236), these cell adhesion molecules were postulated to be the most likely to be involved in delaying neutrophil apoptosis at high cell densities. A s mentioned, neutrophils are a known source of cytokines, many of which delay apoptosis (43-61). Neutrophils produce varying amounts of cytokines, depending on the 17 100 + + OH " 1 1 1 10 3 10 4 10 s 10 6 10 7 Neutrophil Density (cells/well) Fig . 3. Effect of neutrophil density on apoptosis. Neutrophils were cultured under standard conditions and assessed for apoptosis by morphology. Results are expressed as means ± S E M for three separate experiments. * p<0.003 as compared to the lowest density "Apoptotic" value ++ p<0.003 as compared to the lowest density "Normal" value 18 stimulus they receive. IL-8 seems to be produced in the greatest quantities, ranging from 50 g/ml in unstimulated cells, to 5000 pg/ml in stimulated (61,251), or adherent (53) cells. G M -C S F , a well known cause of delay in neutrophil apoptosis (20,64,175-190), is reported to be produced at 100 pg/ml by ionomycin-stimulated neutrophils (57), but no other sources show neutrophil production of this cytokine. T N F - a and H - i p are produced in very small quantities in unstimulated neutrophils; usually less than 5 pg/ml for either cytokine (53,59,61), and stimulated neutrophils are reported to produce up to 1000 pg/ml of I L - i p (53,59). Unstimulated neutrophils are not known to produce much, i f any, IL-6 (58,252), although IL-6 levels have been measured at 240 pg/ml in stimulated cells (252). The most recent study published investigating neutrophil cytokine production, however, states that only IL-8 is produced by neutrophils (251). These cytokines were included in the hypothesis because all are prominent in acute inflammatory foci, and there is no consistent data stating they are not produced by neutrophils, or cannot delay neutrophil apoptosis. 19 Chapter II METHODS AND MATERIALS 2.1 Monoclonal Antibodies Murine I g G l blocking antibodies against human C D 18 ( M H M 2 3 ) and C D l l b (2LPM19c), as well as FITC-conjugated F(ab !) fragments of rabbit anti-mouse immunoglobulins and rabbit anti-human beta-2-microglobulin immunoglobulins were purchased from D A K O (Mississauga, ON) . Murine blocking antibodies against human CD18 (IgG2a, clone EB4), C D l l b ( IgGl , clone ICRF44), and C D l l a (IgG2a, clone 38) were purchased from ID Labs (London, ON) . Mouse isotype controls for I g G l (MOPC-21) and IgG2a (UPC-10) were from Sigma Chemical Co. (St. Louis, M O ) . Azide-free, low endotoxin anti-human neutralizing antibodies to L-selectin (mouse I g G l , clone DREG-56) , G - C S F (rat I g G l , clone B V D 1 3 - 3 A 5 ) , and G M - C S F (rat IgG2a, clone BVD2-23B6) , and rat isotype controls for I g G l (R3-34) and IgG2a (R35-95) were purchased from PharMingen (Mississauga, ON) . Lyophilized murine neutralizing antibody against human IL-8 ( IgGl , clone 6217.11) was purchased from R & D Systems (Minneapolis, M N ) . Those antibodies used for cell culture and not specified above as azide-free were dialyzed against sterile HEPES-buffered normal saline (HNS, see below) using Slide-A-Lyzer cassettes (Pierce, Rockford, IL) to remove the azide preservative. 2.1 Monoclonal Antibody Working Concentrations The working concentrations for the blocking antibodies M H M 2 3 , EB4, 2 L P M 1 9 C , ICRF44, 38, and D R E G - 5 6 were calculated from receptor saturation points, as determined by flow cytometry. The manufacturer-recommended doses of the antibodies 23B6, 3A5 , and 20 6217.111 were tested to determine their ability to neutralize the appropriate cytokines at levels greater than that produced by neutrophils and measured in cell supernatants (detailed below). 2.3 Basic Methods A l l reagents used were tissue-culture grade, all buffers were pyrogen-free, and all other fluids were found to contain <0.01 ng/ml endotoxin, as assessed by the Limulus Amoebocyte Lysate assay (BioWhittaker, Walkersville, M D ) . 2.3.1 Neutrophil isolation Peripheral blood samples were drawn by venipuncture from healthy donors into syringes containing the anticoagulant acid-citrate-dextrose ( A C D ) (Formula A , Baxter, Deerfield, JL). The blood was diluted 1:1 with H N S [10 m M H E P E S (Sigma), in 0.9% N a C l solution (Baxter), adjusted to p H 7.36], layered onto 10 ml of Histopaque 1077 (Sigma), and centrifuged at 400 x g for 30 minutes (at 4°C). The upper layers containing the Histopaque 1077, lymphocytes, and monocytes were aspirated, leaving the granulocytes and the erythrocytes. A s hypotonic lysis may induce loss of neutrophils by clumping and damage to membrane integrity (216), ammonium chloride lysis (0.8% N H 4 C 1 , 0.08% N a H C 0 3 , and 0.037% E D T A ) at 4°C was used to remove the erythrocytes. This method yields greater than 98%) neutrophils, as assessed by microscopic examination of cytocentrifuged preparations fixed in methanol and stained with modified Wright's stain (Sigma). Neutrophil viability was greater than 99% as assessed by trypan blue (0.4%, Life Technologies) dye-exclusion. After washing once in H N S , the neutrophils were then suspended at required concentrations 21 in R P M I 1640 (Life Technologies, Gaithersburg, M D ) , penicillin (5 U/ml) and streptomycin (5 mg/ml) (Life Technologies), and 10% heat-inactivated non-autologous human blood.type A B serum. The serum was obtained from a single human source to maintain consistency amongst experiments. The serum donor was of blood type A B , meaning no antibodies to either A or B antigens were present, and therefore reactivity between the serum, and donor blood, was minimized. 2.3.2 Assessment of apoptosis Neutrophils were cytocentrifuged using a Shandon Cytospin (Pittsburgh, P A ) , air dried, fixed in methanol, and stained with modified Wright's stain (Sigma). The cytocentrifuged cells were examined using light microscopy to determine the percentage of apoptotic, non-apoptotic (normal), and other neutrophils. Cells classified as "other" were those that had lost cellular integrity. They generally comprised fewer than 5% of all cells and so were not included in the counts. Apoptotic neutrophils were identified by the dark, homogenous staining of the chromatin, loss of lobulation in the nuclei, and cell shrinkage (18,12,13,15) (see Fig . 1). Three hundred cells per slide were assessed for apoptosis per slide. The slides were randomized several times before microscopic examination, and were numbered in the order in which they were read. After microscopy, the slide labels were matched with the randomized numbers for identification. 2.4 Standard Conditions and Experimental Protocols Freshly isolated neutrophils were suspended at 3 x l 0 7 / m l in R P M I 1640, penicill in and streptomycin, and 10% non-autologous human blood type A B serum. Neutrophils were then 22 serially diluted and from each density, 100 ul aliquots were added to separate wells o f a flat-bottomed, polypropylene 96 well tissue culture plate (Nunc, Roskilde, Denmark), each well having a surface area of 0.32 cm 2 . The final number of neutrophils per well (actual density in brackets) was l x l 0 3 / w e l l (3xl0 3 /cm 2 ) , 3x l0 3 /we l l ( l x l 0 4 / c m 2 ) , l x l 0 4 / w e l l (3x l0 4 / cm 2 ) , 3x l0 4 /we l l ( l x l 0 5 / c m 2 ) , l x l 0 5 / w e l l (3xl0 5 /cm 2 ) , 3x l0 5 /we l l ( l x l 0 6 / c m 2 ) , l x l 0 6 / w e l l (3xl0 6 /cm 2 ) , 3x l0 6 /we l l ( l x l 0 7 / c m 2 ) . The plates were then incubated for 16 hours at 37°C, in a humidified atmosphere containing 5% CO2 and 95% air. These standard conditions were used for the density experiments, and the following experiments, with the changes noted. Neutrophil apoptosis in all experiments was assessed morphologically. 2.4.1 Time course of neutrophil apoptosis Freshly isolated neutrophils were suspended at l x l 0 6 / m l . Neutrophils were plated in seven 96 well plates at l x l O 5 cells/well, with each plate representing a different time point. The percentage of apoptotic neutrophils was then determined after 0, 5, 16, 18, 20, 22, and 24 hours. 2.4.2 Assessment of serum source in neutrophil cultures Freshly isolated human neutrophils were suspended at 3 x l 0 7 / m l in media supplemented with 10% human A B serum (negative controls), or in media supplemented with 10%o heat-inactivated fetal calf serum (FCS) (Life Technologies). . The cells were serially diluted and cultured for 16 hours. 23 2.4.3 Neutrophil-conditioned medium transfer experiments 7 6 Neutrophils were suspended at 3x10 /ml , serially diluted and added to plates at 3x10 neutrophils/well, 3x l0 5 /we l l , 3x l0 4 /we l l , and 3x l0 3 /we l l and cultured for 16 hours. G M -C S F (50 U/ml) was used as a positive control. After culture, 50 ul of the neutrophil-conditioned medium was collected from each well , centrifuged to remove any remaining cells, and added to wells containing fresh neutrophils at l x l 0 4 cells/well in 50 ul of fresh, supplemented media, at the appropriate densities. The neutrophils were cultured for 16 hours. 2.4.4 Identification of a transferable factor A volume of 1.5 ml of neutrophils in media (donor cells) was added to six well polypropylene plates (9.6 cm , Costar, Cambridge, M A ) to give 1x10 neutrophils/well, l x l 0 7 / w e l l , l x l 0 6 / w e l l , and l x l 0 5 / w e l l , equivalent to the densities (neutrophils/cm 2) used in 96 well plates. The equivalent number of cells/well in a 96 well plate would be 3x10 3 , 3x10 4 , 3x10 5 , and 3x10 6 . Controls included reporter cells in media alone or media with 50 U / m l G M - C S F , and media cultured without cells. To each well , a Transwell® tissue culture insert was added (0.4 urn pore size, 24 mm diameter, 4.7 c m 2 surface area, Costar) containing 1.5xl0 5 cells in 1.5 ml (reporter cells) (Fig. 4). These plates and inserts were then incubated for 16 hours. 24 Fig. 4. Co-culture of donor and reporter neutrophils to detect the presence of a transferable factor produced by high density donor cells. Reporter cells, at a constant, low density (lxl0 4 'cells/well) were cultured in tissue culture inserts over top of varied densities of donor cells. A transferable factor, produced by high density donor cells, could move through the permeable membrane on the bottom surface of the insert and cause a delay in apoptosis in the low density reporter cells. 25 2.4.5 Measurement of cytokine levels in supernatants From the six well plates in the transferable factor studies, supernatants were obtained from the highest density wells ( l x l O 8 donor cells/well). Control wells included those with only reporter cells in media, and media alone. The same supernatant samples were taken from three separate experiments. The supernatants were frozen at -70°C. Cytokine levels in the supernatants were measured by two-antibody enzyme-linked immunosorbent assay (ELISA) using biotin-strepavidin-peroxidase detection, by the U M A B Cytokine Core Laboratory (Baltimore, M D ) . Their method is as follows. "Polystyrene plates (Maxisorb, Nunc) were coated with capture antibody in P B S overnight at 25°C. The plates were washed 4 times with 50 n M Tris, 0.2% Tween-20, p H 7.0-7.5 and then blocked for 90 minutes at 25°C with assay buffer (PBS containing 4% B S A (Sigma) and 0.01% Thimerosal, p H 7.2-7.4). The plates were washed 4 times and 50 ul of sample or standard prepared in assay buffer and incubated at 37°C for 2 hours. The plates were washed 4 times and 100 u.1 o f biotinylated detecting antibody in assay buffer was added and incubated for 1 hour at 25°C for approximately 10-30 minutes. The reaction was stopped with 100 ul 2 N HC1 and the A450 (minus A650) was read on a microplate reader (Molecular Dynamics). A curve was fit to the standards using a computer program (SoftPro, Molecular Dynamics) and cytokine concentration in each sample was calculated from the standard curve equation." The specifics for each human cytokine assay are listed below in Table 2. 26 Table 2. Specifications of ELISA Assay1 C Y T O K I N E A S S A Y R A N G E (pg/ml) C A P T U R E A N T I B O D Y D E T E C T I N G A N T I B O D Y S T A N D A R D Endogen mAb- Endogen m A b - Endogen R-T N F - a 15.625 to 1000 M303 M302B T N F A - 1 0 Endogen Endogen Endogen I L - i p 3.125 to 200 M421B M 4 2 0 B - B R-IL1B-5 Endogen Endogen G M - C S F 9.375 to 300 M - 5 0 0 A M-501 R - G M C S F - 2 Endogen Endogen Endogen IL-8 6.25 to 400 M-620 M-621B R-IL-6-10 Biosource Biosource Biosource IL-6 7.8125 to 500 A H C 0 9 8 2 A H C 0 7 8 9 58.130.10 1 as per Cytokine Core Laboratory, University of Maryland at Baltimore 2.4.6 Cytokine neutralization experiments Neutrophils were suspended at high density ( l x l O 6 cells/well) and low density ( l x l O 4 cells/well) and added to 96 well plates containing either H N S (negative control) or test antibody, and cultured for 16 hours. The neutralizing antibodies and concentrations were G M - C S F (23B6, 20 pg/ml), G-CSF (3A5, 20 pg/ml), and IL-8 (6217.111, 10 pg/ml). Negative antibody controls included MOPC-21 for the 6217.111 antibody, R3-34 and R35-95 for the 3A5 and 23B6 antibodies, respectively. Antibody to 02 microglobulin, a small, noncovalently associated polypeptide chain found on major histocompatibility complex class I and II molecules, was included to control for the nonspecific effects o f antibody-coated cells. 2.4.7 Investigation of platelet-activating factor antagonist WEB 2170 Neutrophils at each density were added to plates containing the P A F antagonist W E B 2170 at 1 u M , or with H N S alone (negative control), and cultured for 16 hours. 27 2.4.8 Cell adhesion molecule blocking experiments Neutrophils in suspension were added to 96 well plates at either high density ( l x l 0 6 neutrophils/well) or low density ( l x l 0 4 neutrophils/well) and cultured for 16 hours. The wells contained either H N S (negative control), or test antibody. The blocking antibodies and concentrations were: CD18 (IB4), 5 ng/ml, and ( M H M 2 3 ) , 15 ng/ml; CD1 l b (2LPM19C) , 5 I4g/ml, and (ICRF44) 20 ng/ml; C D l l a (clone 38), 5 ng/ml; and L-selectin (DREG-56) 20 ng/ml. Antibody controls included mouse isotype-identical non-specific I g G l (MOPC-21) , and IgG2a (UPC-10), and antibodies to an unrelated surface antigen (p 2 microglobulin) to control for the effects of antibody coated cells. 2.4.9 Investigation of fMLP in neutrophil apoptosis Neutrophils at densities between l x l 0 4 cells/well and 3x10 6 cells/well were added to plates containing f M L P in H N S at 100 n M , 10 p M , or with H N S alone (negative control) and cultured for 16 hours. 2.5 Statistical Analyses Statistical analysis was performed using the Student's two-tailed paired t test. A l l the data are presented as means + S E M . Differences between group means were considered statistically significant i f p <0.05. 28 Chapter III RESULTS 3.1 Time Course of Neutrophil Apoptosis in Culture Neutrophils were cultured in standard conditions (with human A B serum) at the mid-range density ( l x l 0 5 cells/well) for up to 24 hours, with the percentage of apoptotic neutrophils being assessed at various time points. Neutrophil apoptosis increased by 77% between 0 and 24 hours (Fig. 5). This increase was found to be statistically significant (p<0.002, n=3). The half-life of the cultured neutrophils was determined from each experiment, with the mean at 13.8 + 1 hours. 3.2 Assessment of Serum Sources in Neutrophil Cultures Typically, neutrophils are cultured in F C S , but since human serum would be more like in vivo conditions, we compared the effects the two serum types have on neutrophil apoptosis. Figure 6 shows that neutrophils cultured in A B serum generally live longer than those cultured in F C S . Five of the eight density points show a significant decrease in the percentage of apoptosis for neutrophils cultured in A B serum as opposed to F C S (p<0.05, n=3). In A B serum, the decrease in neutrophil apoptosis from the lowest to the highest cell density ( l x l 0 3 vs 3 x l 0 6 cells/well) is approximately 64%, while in F C S serum it is approximately 52%. 29 Time (hours) Fig . 5. Time course of neutrophil apoptosis. Neutrophils were cultured under standard conditions for 0, 5, 16, 18, 20, 22, and 24 hours, then assessed for apoptosis by morphology. Results are expressed as mean ± S E M for three separate experiments. * p<0.002 compared to lowest time point 30 100 i OH 1 1 ' ' 10 3 10 4 10 s 10 6 10 7 Neutrophil Density (cells/well) Fig . 6. Evaluation of the effect of human A B serum versus F C S in neutrophil cultures. Neutrophils were cultured under standard conditions, with the addition of either 10% A B serum, or 10% F C S , and assessed for apoptosis morphologically. Results are expressed as means ± S E M for three separate experiments. * p<0.05 as compared to corresponding A B Serum value 31 3.3 Investigation of a transferable factor in neutrophil-conditioned medium 3.3.1 Neutrophil-conditioned medium does not delay apoptosis in fresh neutrophil cultures Neutrophil-conditioned medium ( N C M ) was obtained from donor cells after they were cultured at various densities under standard conditions. The N C M was added to fresh, low density ( l x l 0 4 cells/well) "reporter" neutrophils, which were then cultured for 16 hours. A s expected, higher densities resulted in decreased apoptosis in donor neutrophils (73% decrease, /K0 .003 , n=3) (Fig. 7A). The reporter neutrophils, however, showed no change in percentage apoptosis in any well when incubated in high density N C M (Fig. 7B). 3.3.2 Neutrophils produce a transferable factor that delays high density neutrophil apoptosis One of the possible explanations for the results detailed above is that the factor delaying apoptosis in the neutrophil-conditioned medium is labile and inactive after 16 hours culture. Experiments were performed to co-culture donor and reporter cells. Donor neutrophils were incubated at varied densities in the bottom of tissue culture plates, and reporter neutrophils at a constant density were placed over top in tissue culture inserts. Donor neutrophils showed the typical high cell density decrease in apoptosis, comparable to that seen in Figure 3 (Fig. 8). There was a 63% decrease in apoptosis from the -a c lowest density to the highest density (equivalent to 3x10 vs 3x10 cells/well), and this decrease was statistically significant (p<0.0006, n=5). The reporter neutrophils exhibited a comparable decrease in the percentage of apoptosis. Thus the reporter cells cultured over top higher density donor cells exhibited less apoptosis than reporter cells cultured over top low 32 Fig . 7 Effect of neutrophil conditioned medium on neutrophil apoptosis. A : Neutrophils were cultured at various densities under standard conditions. After culture, neutrophil conditioned medium was removed from wells containing 3 x l 0 3 , 3 x l 0 4 , 3 x l 0 5 , and 3 x l 0 6 cells/well. B : Neutrophil conditioned medium removed from wells was added to low density ( l x l O 4 cells/well) neutrophils, and cultured for 16 hours. Results are expressed as mean j ^ S E M (n=3). * ;?<0.003 as compared to lowest density 33 7B. Apoptos is i n Reporter Ce l l s wi th Donor N C M a 1001 I 80-A 20" o-l 1 1 1 1 10 3 10 4 10 s 10 6 10 7 Neutrophil Density (cells/well) of Donor N C M 34 Fig . 8. Production of a transferable factor by high density "donor" neutrophils. Constant density (equivalent to lxl0 4 cel l /well) "reporter" cells were cultured over top of "donor" neutrophils, which were at varied densities, under standard conditions. ' Results are presented as means ± S E M for five separate experiments. *p<0.006 as compared to the value for reporter cells cultured over top lowest density donor cells ++ p<0.0006 as compared to lowest density donor cells value 35 density donor cells. The decrease in apoptosis seen in the reporter cells was 48%, and was statistically significant (p<0.006, n=5). Furthermore, those controls in which only reporter cells were cultured i n tissue culture inserts over top wells containing only media (no donor cells), showed that without donor cell influence, reporter cell apoptosis was 81 + 5 % (results not shown). Therefore, at lower donor cell densities ( l x l O 5 and l x l 0 6 cells/well), reporter cell apoptosis was not significantly different from negative control reporter cell apoptosis, while at the higher donor cell densities (1x10 and 1x10 cells/well) reporter cell apoptosis was statistically different from the negative control (p<0.002 and p<0.0006, respectively, n=5). 3.3.3 Cultured neutrophils produce IL-8 in supernatants The conditioned media in which the reporter cells and the highest density donor cells Q (1x10 cells/well) were cultured together, described in the above experiment, were collected from three separate experiments along with conditioned media from the positive (reporter cells in media) and negative (media alone) control wells. E L I S A assays for G M - C S F , T N F -a, I L - i p , IL-6, and IL-8 were performed on the conditioned media samples. The results showed that of the cytokines tested, only IL-8 was produced at a detectable level (Table 3). The remaining cytokines were below the levels of detection for these E L I S A assays. 36 Table 3. Cytokine Production by High Density Cultured Neutrophils As Measured by ELISA C Y T O K I N E D E T E C T I O N R A N G E (pg/ml) E L I S A R E S U L T (pg/ml) G M - C S F 9.4-600 8.4 ± 0 . 4 EL-8 7.8-500 184 + 24 EL-6 6.25-400 0.43 ± 0 . 1 2 EL- ip 3.1-200 2.2 + 0.04 T N F - a 15.6-1000 0.73 ± 0.06 Results are presented as means + S E M of three separate experiments. 3.3.4 Cytokine neutralizing antibodies do not inhibit the delay in apoptosis in high density neutrophils To further investigate the cause of the transferable factor found in high density neutrophil-conditioned media, neutralizing monoclonal antibodies to EL-8 (6217.111), G M -C S F (23B6), and G - C S F (3A5) were added to cultures of both high and low density neutrophils ( l x l O 4 and l x l O 6 cells/well). EL-8 was chosen because it was shown to be present in the neutrophil-conditioned media (see Table 2), and G M - C S F and G - C S F were chosen because they of their highly potent anti-apoptotic properties (20,64,175-190). Figure 9 shows that in each set of cultures, containing either FINS (control) or monoclonal neutralizing antibody, the mean difference in the percentage of apoptosis between high and low density was 28+1% and was statistically significant (p<0.00006, n=5). Comparing the high density neutrophil apoptosis values shows that although the ability of 37 80 Low Density • High Density Fig . 9. Effect of cytokine neutralizing antibodies on neutrophil apoptosis in high and low density neutrophil cultures. Neutrophils at high ( lxlO b cel ls /wel l) and low (1x10* cells/well) densities were cultured under standard conditions in the presence of H N S (control) or monoclonal neutralizing antibody to G M - C S F (23B6, 20 pig/ml), G - C S F (3A5, 20 U-g/ml), or IL-8 (6217.111, 10 Ug/ml). Results are presented as means ± S E M . * p<0.007 as compared to high density value in pair (n=5) 38 these antibodies to neutralize their respective cytokines was confirmed, their addition to cultures did not increase the percentage of apoptosis, so was unable to inhibit the density-dependent delay typically seen. Neither was there any statistically significant difference in the percentage of apoptosis seen in low density neutrophils as compared to the control value. Nonspecific antibody control-treated cells did not show a change in apoptosis at either high or low density (results not shown). 3.3.5 Platelet-activating factor is not involved in the high density delay in neutrophil apoptosis Although our results did not show EL-6 production by high density neutrophils, EL-6 has been linked to a delay in neutrophil apoptosis through P A F (199). We investigated the role P A F may play in delaying neutrophil apoptosis at high densities by incubating neutrophils at various densities with W E B 2170, a P A F antagonist. With increasing densities ( l x l 0 3 to 3 x l 0 6 cells/well), the control and W E B 2170-treated cells showed significant decreases in apoptosis by 59 and 61%, respectively (p<0.006, n=3) (Fig. 10). A t no individual density was the percentage of apoptosis significantly different from the corresponding density in the control wells. 3.4 Investigation of Neutrophil Adhesion and Apoptosis 3.4.1 Blocking receptors to cell adhesion molecules does not inhibit the delay in apoptosis at high neutrophil densities A s increasing density may allow greater opportunity for cell-cell interactions, we investigated the potential role of the more important cell adhesion molecules in the density-39 80 Fig . 10. Effect of W E B 2170 on the density-dependent delay in neutrophil apoptosis. Neutrophils were cultured under standard conditions in the presence of H N S (control), or W E B 2170 (1 \iM), and assessed for apoptosis morphologically. Results are presented as means ± S E M for three separate experiments. ++ p<0.006 as compared to lowest density +WEB 2170 value * p<0.006 as compared to lowest density control value 40 dependent delay in neutrophil apoptosis. Monoclonal blocking antibodies to the cell adhesion molecules C D I 8 , C D I lb , C D I l a , and L-selectin were added to both high and low density neutrophil cultures (Fig. 11). Additional clones of C D 18 and C D I l b monoclonal antibodies were included to confirm that the results for the two most likely cell adhesion molecules to be involved in the density-dependent delay were not specific to the antibody clone type. Comparing the low and high densities in each set of cultures, the statistically significant mean reduction in apoptosis was 23+2% (p<0.00003, n=5). The changes in percentage of apoptosis in the low density neutrophils cultured with cell adhesion molecule blocking antibodies were not statistically significant compared to the control value, nor were the changes in percentage apoptosis for the high density neutrophils. To account for the possibility of redundancy between cell adhesion receptors on the neutrophils, in which the blocking of one class of receptors would be compensated for by another, all blocking antibody clones were added together to neutrophils at both high and low densities. Comparing the high density " A l l Abs" wells and high density control wells shows that the addition of multiple cell adhesion molecule blocking antibodies did not result in inhibition of the delay in neutrophil apoptosis seen at high densities (n=5) (Fig. T I B ) . Neither was there was a significant change in the low density wells compared to their corresponding control. For both sets of experiments, nonspecific antibody controls added to neutrophil cultures did not affect apoptosis at either high or low densities (results not shown). 41 Fig . 11. Effect of monoclonal blocking antibodies to cell adhesion molecules on neutrophil apoptosis at high and low neutrophil densities. Neutrophils at high ( l x l O 6 cells/well) and low ( l x l 0 4 cells/well) densities were cultured under standard conditions in the presence o f H N S (control) or (A) single blocking monoclonal antibody clones to C D 18 (clone 1 [IB4], 5 Hg/ml; clone 2 [ M H M 2 3 ] , 15 ng/ml), C D l l b (clone 1 [ICRF44], 20 ng/ml; clone 2 [2LPM19C] , 5 ng/ml), C D l l a (38, 5 ng/ml), or L-selectin (DREG-56, 20 ng/ml), or (B) all blocking antibody clones. Apoptosis was assessed morphologically. Results are presented as means + S E M for five separate experiments. F ig . 11 A : * /K0.03 as compared to paired high density value F ig . 1 I B : * /j<0.0008 as compared to high density " A l l A b s " value 42 Low Density • High Density (clone 1) (clone 2) (clone 1) (clone 2) L-selectin 43 44 3.4.2 Neutrophil incubation with fMLP does not inhibit apoptosis at high cell densities Because f M L P can function to upregulate the cell adhesion molecules C D 18 and C D l l b on the neutrophil surface (253-255), its effect on the density-dependent delay in neutrophil apoptosis was investigated. Both a high (10 pM) and low (100 n M ) of f M L P were used. Figs. 12A and B show that at either concentration of f M L P , comparing the percentage o f apoptosis at each density to its respective control, no significant change was observed. Figure 12A shows a significant decrease of 35% apoptosis between the lowest ( l x l 0 4 cells/well) and highest (3x10 6 cells/well) cell densities when 100 n M f M L P was added to the cultures (p<0.003, n=6). When the f M L P concentration was reduced to 10 p M , the decrease in apoptosis from lowest to highest density was also significant, at 43% (p<0.001, n=5) (Fig. 12B). 45 Fig . 12. Effect of f M L P on the density-dependent delay in neutrophil apoptosis. Neutrophils cultured under standard conditions in the presence of H N S (control), 100 n M f M L P (A) , or 10 p M f M L P (B) and assessed for apoptosis morphologically. Results are presented as means + S E M . F i g . l 2 A (n=5): * /K0.001 as compared to lowest density f M L P value, ++ /K0.0002 as compared to lowest density control value F i g . l 2 B (n=5): * /K0.002 as compared to lowest density f M L P value, ++ /?<0.00002 as compared to lowest density control value 46 F i g . 12 A Fig. 12B Chapter IV DISCUSSION The hypothesis of this thesis was that high neutrophil density resulted in a delay in apoptosis due to increased contact between neutrophils leading to activation of CD18/CD1 l b and subsequent release of anti-apoptotic cytokines. We formulated this hypothesis based on the observation that when neutrophils were cultured at high densities there was a delay in apoptosis. Our results indicated that there was a density-dependent delay in neutrophil apoptosis, and at high densities, neutrophils did produce a transferable factor that was able to significantly delay apoptosis in low density neutrophils. We showed that the cell adhesion molecules C D 18, C D l l a , C D l l b , and L-selectin were not involved, in normal or f M L P -activated neutrophils, nor was platelet-activating factor (PAF) , the cytokines G M - C S F , G -C S F , I L - l p , EL-6, IL-8, or T N F - a . A study by Doerschuck et al (256) was used to compare the densities used in this thesis with neutrophil densities in vivo. The mid-range density used in the current body of work was l x l 0 5 neutrophils/well (96 well plate), however under these culture conditions neutrophils settle on the surface of the culture plate in a fluid layer assumed to be approximately 10 um deep. Therefore, the effective neutrophil concentration would be l O x l O 6 neutrophils/ml. Doerschuk et al (256) have shown that in a four hour pneumonic rabbit lung, the number of migrated neutrophils is approximately 1 7 x l 0 6 /ml , comparable to the mid-range density used in this thesis. In addition, the number of marginated neutrophils in capillary blood is approximately l l x l 0 8 / m l (256), well in excess of the highest effective density used here (30x10/ml). Most papers examining neutrophil apoptosis document the use of media supplemented with fetal calf serum (FCS) to incubate neutrophils (176,178,186, 187,213,232). N o rationale for this is given, but these conditions are analogous to those used 49 for standard tissue culture. Hannah et al (239) recently investigated the effects of different serum types and concentrations on constitutive neutrophil apoptosis after 20 hours of culture. They examined both autologous human serum and bovine serum albumin ( B S A ) , and found that while lower concentrations of B S A (0.1-1% w/v) rescue significant numbers of neutrophils from necrosis compared to serum-free cultures, at high concentrations (10% w/v) there is an increase in neutrophil apoptosis. Conversely, neutrophils cultured in autologous serum at concentrations from 0.1-3% (w/v) show profound inhibition of apoptosis. Hannah et al (239) suggest it may be due to the presence of additional "survival factors", and conclude that neutrophil survival factors require the presence of protein but not specifically serum in vitro. A different study, however, found similar levels of apoptosis in neutrophils after 18 hours in culture in either a protein-free medium or in 0.1 % B S A (257). They also found similar rates of apoptosis in neutrophils cultured in F C S or autologous human serum. Because of these conflicting reports, we chose to investigate the different serum sources. A s outlined in the methods and materials section, we chose to use allogeneic A B serum over autologous serum to minimize a potential variable between donors. We did not use F C S as we found that neutrophils had a longer half-life in A B serum, and because A B serum would provide a more physiologically accurate environment. To address the question of inter-experimental variation, the results were grouped by similarity of protocol, to assess consistency between experiments. In those experiments in which apoptosis was assessed between 1x10 and 3x10 cells/well, the mean decrease in apoptosis was highly consistent at 63+3% (n=5), and the mean half-maximal response density (for our particular density range) was Ix l0 5 +0 .6x l0 5 cells/well. Correspondingly, this was the mid-point o f the densities used. 50 4.1 Neutrophil Adhesion We were not able to prove that increased contact between cells led to activation of the C D 1 8 / C D l l b complex Mac-1, which is the main mediator involved in homotypic aggregation (242,247-250). Our results in these adhesion experiments are in agreement with several other studies reporting that P2 integrins are not involved in delaying neutrophil apoptosis (237-239). Watson et al (236), in a conflicting report, describe a correlation between C D I l a or C D I l b and neutrophil apoptosis. However, their study involved cross-linking of C D I l a or C D I lb , rather than blocking these cell adhesion molecules. Cross-linking of P2 integrins initiates intracellular signaling resulting in induction of Ca signaling (258-261). Increases in intracellular Ca2+ have been shown to significantly delay neutrophil apoptosis (262). However, direct engagement of CD18/CD1 l b has been shown to be unable to mediate changes in cytosolic free calcium (263,264). Engagement of L-selectin is also known to initiate a cascade of intracellular processes that include release of intracellular Ca inside the cell (265), however no link has been shown between this cell adhesion molecule and neutrophil apoptosis. Indeed, L-selectin is shed from the surface of neutrophils before the cells even arrive at a site of acute inflammation (7), and is markedly down regulated in apoptotic cells (234). Neutrophil adhesion in general still cannot be completely ruled out as a contributing factor in delaying apoptosis at high neutrophil densities. There are other cell adhesion molecules present on neutrophils, such as CD31 ( P E C A M - 1 ) , I C A M - 3 , and sialyl-Lewis x (221-224), that have yet to be explored in terms of a potential relationship with neutrophil apoptosis. Another possibility is the presence of high numbers of platelets in the neutrophil isolates. A recent study by Andonegui et al (266) reports that neutrophils cultured in the presence of platelets at ratios as low as 1:10 (neutrophil :platelet) show a delay in neutrophil apoptosis. Although we did not observe any considerable platelet contamination in the isolated neutrophils, exact numbers were not determined. 51 Neutrophil adherence to tissue culture polystyrene plastic is said to prolong the survival of neutrophils (232). The polystyrene surface has been described as an activating surface since adherent neutrophils show induction of respiratory burst activity and an influx of extracellular C a 2 + (232,267,268). Although neutrophil apoptosis may be delayed by neutrophil adherence to tissue culture plastic, this does not provide an explanation for the density-dependent delay in apoptosis we observed in our cultures. We also investigated f M L P , a representative of bacterial polypeptides, to determine i f upregulation of cell adhesion molecules was involved in the density-dependent delay, and because there are conflicting reports regarding the effects of f M L P on neutrophil apoptosis (30,176,178). We found no change in the rate of neutrophil apoptosis with either high (100 n M ) or low (10 p M ) concentrations of f M L P . Similarly, we found no change in neutrophil apoptosis upon the addition of neutralizing antibodies to the other classic chemoattractant, EL-8. Our negative results in these two experiments correlate with other similar studies (176,178). A possible explanation is provided by Colotta et al (178) who group the agents affecting neutrophil apoptosis into two categories based on the different functions exerted in the inflammatory process: those having chemotaxis as a primary function, and "activating" cytokines. They postulate that those agents having chemotactic primary functions act rapidly, and are required for only a short duration, so therefore do not need to affect neutrophil survival. However, those agents that induce critical changes in neutrophil functional status by possibly altering gene expression, such as cytokines, require considerably more time, and therefore are more likely to be involved in affecting neutrophil apoptosis. , . i 4.2 Transferable Factor It has been suggested by Colotta et al (178) that neutrophils in culture do produce a soluble anti-apoptotic factor. They describe preliminary experiments in which conditioned medium from activated neutrophils sustained the survival of fresh neutrophils for more than 72 hours. We found that in our unstimulated neutrophils, conditioned medium was not able 52 to delay apoptosis of low density neutrophils. But when the high and low density neutrophils were co-cultured, there was a significant delay in apoptosis in the low density neutrophils, suggesting a potentially labile transferable factor may be produced by high density neutrophils in culture. In our investigation of this transferable factor, we did not find evidence that P A P or the cytokines G M - C S F , G - C S F , EL-6, EL-8, T N F - a , or EL- lp were involved. The E L I S A assay results, in fact, showed that of the cytokines we tested, only EL-8 was produced by the neutrophils at a significant level. The assay range of detection for each cytokine encompassed concentrations shown to stimulate biological activity in neutrophils (73,178,202,269,270). Our negative findings with the P A F inhibitor W E B 2170 contradict those of Bi f f l et al (199). They found that the addition of either EL-6 or P A F to neutrophil cultures resulted in prolonged neutrophil survival, and that this survival could be abrogated, in either case, by the addition of the P A F inhibitor, W E B 2170. We did not detect significant levels of EL-6 in our neutrophil cultures, but more importantly, we found that W E B 2170 was not able to inhibit the delay in neutrophil apoptosis observed at high cell densities. From these data we potentially ruled out the EL-6/PAF system suggested by Bi f f l et al (199) as being the transferable factor in our high density cultures. There are other cytokines and systems to consider in identifying the transferable factor. One possibility is TGF-p\ This cytokine can induce chemotaxis in neutrophils (10), and is an important mediator in both the initiation and the resolution of inflammation (271,272). Human neutrophils are able to secrete an active form of this cytokine, and they possess specific T G F - p receptors (273). Recently Ward et al (179), in contrast to a previously published study (179), showed that TGF -P can inhibit neutrophil apoptosis in culture. They mention that TGF -P can regulate its own production, which can lead to further autocrine secretion of this cytokine. In our system then, increased cell-cell contact between neutrophils at high densities could provide a signal to the neutrophils to secrete increased amounts of TGF -P , which would prolong the survival of these cells by delaying apoptosis 53 itself, or through an alternative internal mechanism. Monoclonal antibodies to TGF -P would be useful in investigating this hypothesis. Although we have not yet identified the transferable, anti-apoptotic factor produced by high density neutrophils, it is still important to consider the mechanism o f its action. One possibility is the involvement of Fas and Fas ligand (Fas/FasL), which are co-expressed on the neutrophil surface (169,170). The interaction of Fas/FasL induces neutrophil apoptosis (166,167,169-171), but it has been shown that incubation of neutrophils with the proinflammatory cytokines G - C S F , G M - C S F , EFN-y, or T N F - a suppresses Fas-induced apoptosis in normal neutrophils (169). Although it would seem likely that at high neutrophil densities there would be a greater likelihood of Fas/FasL engagement, and an increase in apoptosis, we have shown that this is not the case. High density neutrophils, then, may produce a transferable factor, possibly a cytokine we have not yet investigated, that would downregulate the cell surface expression of Fas. A t lower densities, without the transferable factor, neutrophils would be unable to prevent the engagement of Fas/FasL, leading to higher rates of apoptosis. Fas has also been connected with the bcl-2 gene in that bcl-2 has been shown to suppress Fas-induced death in certain cell types (274,275), and to protect certain cell types from oxidant-induced apoptosis (276,277). A s mentioned by Liles et al (278), this raises the possibility that oxidant-based cell death programs may be influenced by Fas. The superoxide anion has been shown to be a natural, inducible inhibitor of Fas-mediated apoptosis in tumour cells (279). In neutrophils adherent to microtitre plates, high cell density results in significantly attenuated superoxide anion release, phospholipid metabolism, apparent N A D P H oxidase activity, and H2O2 production (211). These effects are not dependent on protein synthesis, cell adherence to a surface or other neutrophils, or to the upregulation of C D 1 8 / C D l l b molecules. Peters et al (211) suggest that high density causes a general inhibition of neutrophil activation. The addition of anti-oxidants to neutrophils in culture also delays apoptosis (212). In our cultures then, reactive oxygen 54 species cou ld be the intracel lular l i nk i n the process: the transferable factor produced at h igh densities acts to inhib i t ce l l act ivat ion leading to decreased product ion o f reactive oxygen species, and a delay i n neutrophi l apoptosis. Another opt ion is detai led b y Hannah et al (239) who found that hypox i c condi t ions cause a s ignif icant delay i n apoptosis. Th is w o u l d seem to suggest that reactive oxygen species induce neutrophi l apoptosis. However , they also found that act ivat ion o f respiratory burst by f M L P or EL-8 does not increase neutrophi l apoptosis. In fact, on ly supraphys io logica l concentrations o f H2O2 are able to increase neutrophi l apoptosis i n culture. Th i s , they suggest, impl ies that reactive oxygen species product ion may not be a major mediator o f neutrophi l apoptosis. O u r data seems to support this idea, as we found no change i n the leve l o f apoptosis w i t h the addit ion o f f M L P . S ince hypox i a does exist i n in f lammatory foc i (280,281), h igh neutrophi l densities cou ld induce hypox i c condi t ions and p H changes (cel l ac id i f i cat ion has been shown to induce apoptosis [190]) w h i c h w o u l d pro long the surv iva l o f the neutrophils b y de lay ing apoptosis. The transferable factor we observed i n h igh density neutrophi l condi t ioned m e d i u m may also contribute to delay ing apoptosis by affect ing regulat ion o f R N A or prote in synthesis. S ince the inh ib i t ion o f R N A synthesis by act inomycin-D, or o f prote in synthesis b y cyc lohexamide , s igni f icant ly increases the rate o f apoptosis (136,176,282), the delay i n apoptosis we see at h igh neutrophi l densities seems to require R N A and/or prote in synthesis. W e speculate, then, that the transferable factor produced by the neutrophi ls may be direct ly st imulat ing cont inued R N A and/or protein synthesis 4.3 Summary and Conclusions In summary, we have demonstrated that h igh density neutrophils showed pro longed surv iva l due to inh ib i t ion o f apoptosis. A s per our first objective, we showed that at h igh densit ies, the neutrophils produced a transferable anti-apoptotic factor. Th i s transferable factor has not yet been ident i f ied, but G M - C S F , G-CSF , EL-8, EL-6, EL-lp\ T N F - a , and P A F 55 are unlikely. In investigating our second objective, to demonstrate the role of neutrophil cell adhesion molecules in the delay of apoptosis at high cell densities, we showed that this delay in apoptosis was most likely not regulated by the p 2 integrins CD18/CD1 l b or CD18/CD1 l a , or by L-selectin. Further investigation into this phenomenon could focus on other cell adhesion molecules present on neutrophils, or the possibility of platelet-neutrophil contact by high numbers of platelets. In identifying the transferable factor, other cytokines, such as T G F - p , could be examined. The mode of action of the transferable factor is also important to identify. Fas/FasL, hypoxic conditions, and changes in protein synthesis regulation are three avenues warranting further investigation. The work entailed in this thesis has important implications for studies of neutrophil apoptosis both in vitro and in vivo. We have shown that the density at which neutrophils are cultured had a profound effect on the survival these cells. Neutrophil density has also been shown to affect several biochemical aspects of neutrophil activation (211). It is important, therefore, to take these factors into consideration when designing protocols for the study of neutrophil apoptosis and physiology. In terms of in vivo implications, we have shown that neutrophils at high densities lived longer than neutrophils at low densities. Acute inflammation is characterized by a large influx of neutrophils to the site (2). A n example we would anticipate is acute respiratory distress syndrome. Because the neutrophil response is nonspecific, large amounts of bactericidal enzymes and reactive oxygen species are released into the inflammatory focus. Ordinarily, the neutrophils would become apoptotic, and would be subsequently removed by macrophages. However the high density of the neutrophils, as we have shown, means that they w i l l probably stay alive longer, giving them more opportunity to release their products into the lung tissue and cause increased tissue damage. 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