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Protein kinase C activity of neutrophils in localized aggressive periodontitis Kim, Juliana J. 2008

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PROTEIN KINASE C ACTIVITY OF NEUTROPHILS IN LOCALIZED AGGRESSIVE PERIODONTITIS by JULIANAJ. KIM Diploma in Dental Hygiene, University of Alberta, 1992 BSDH, Old Dominion University, 1993 MS, Old Dominion University, 1995 MBA, INSEAD, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Dental Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2008 ©Juliana J. Kim, 2008 ABSTRACT Localized Aggressive Periodontitis (LAP) is clinically characterized as a rapid, early onset bone loss around the first molars and incisors. Polymorphonuclear neutrophils (PMN) in LAP patients are hyperactive, generating excessive superoxide anions, cytokines, and enzymes, leading to host tissue injury. This hyper-responsive phenotype of LAP neutrophils suggests that the cells are somehow pre-activated, or primed in LAP, implicating the signaling molecules in the pathway for NADPH oxidase activation. Protein Kinase C has been identified as key molecule in the production of superoxide in neutrophils. In the first study, the role of PKC in priming of neutrophils from LAP patients was investigated. PKC activity was found to be higher in the membranes of neutrophils from LAP patients than healthy subjects, suggesting translocation of the enzyme from the cytosol to the membrane. Analysis of five PKC isoforms indicated that at rest, phosphorylated PKC 131, , and from LAP subjects were expressed at higher levels than of those from healthy subjects. There were no differences in the expression of PKC isoform transcripts between healthy and LAP groups, indicating that the increase in PKC is not a result of increased transcription. Expression of 47Ph0x phosphorylation was enhanced in neutrophils from subjects with LAP, supporting our findings that the neutrophils are indeed primed. Inhibition studies, using PKC inhibitors, demonstrated a greater inhibition of superoxide production in neutrophils from healthy subjects than those subjects with LAP. Application of classical and zeta PKC inhibitors demonstrated greater superoxide inhibition than the use of beta and delta inhibitors. In the latter half of the study, whole cell neutrophil lysates from healthy and LAP groups pre treated with RvE1 were analyzed for phosphorylated PKC isoforms. In the healthy group, RvE1 did not inhibit phosphorylation of any PKC isoforms. However, in the LAP group, RvE1 reduced the phosphorylation of PKC ô and compared to PBS-treated neutrophils. Similarly, RvEI only reduced phosphorylation of p47PiX in neutrophils from LAP subjects. Pharmacologically induced dephosphorylation with RvE1 returns LAP PMN to a normal neutrophil phenotype. RvE1 is a potential therapeutic modality for the treatment of the periodontal lesions associated with LAP. III TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS x Acknowledgements xi Dedication xii CHAPTER ONE: INTRODUCTION I 1.1 Periodontal Disease: An Overview 2 1.2 Determinants and Risk Factors of Periodontitis 2 1.2.1 Age 2 1.2.2 Gender 3 1.2.3 Socioeconomic Status (SES) 3 1.2.4 Genetics 4 1.2.5 Plaque, Microbiota, and Oral Hygiene 4 1.2.6 Tobacco 5 1.3 Bacterial Etiology of Periodontology 5 1.4 Periodontal Disease Classifications 7 1.5 Aggressive Periodontal Disease 7 1.5.1 Localized Aggressive Periodontal Disease 8 1.6 Role of the Immune System in Periodontal Disease 11 1.7 The Polymorphonuclear Neutrophil 12 1.7.1 Differentiation 12 1.7.2 Function 13 1.7.3 Adherence 14 1.7.4 Chemotaxis 15 1.7.5 Neutrophil Priming 16 1.7.6 Phagocytosis 18 1.7.6.1 Recognition &Activation 18 1.7.6.2 Engulfment 19 1.7.6.3 Microbicidal Killing (intracellular/extracellular) 20 iv 1.8 Reactive Oxygen Species .20 1.8.1 Production of ROS 21 1.8.2 ROS Tissue Damage 23 1.8.3 Superoxide in the Pathogenesis of LAP 24 1.9 Protein KinaseC 24 1.9.1 PKC Structure & Isoforms 25 1.9.1.1 PKCa 30 1.9.1.2 PKC131 30 1.9.1.3 PKC2 31 1.9.1.4 PKCÔ 32 1.9.1.5 PKC 33 1.9.2 Regulation of Protein Kinase C 34 1.9.2.1 Phosphorylation 34 1.9.2.2 Co-factor Binding 34 1.9.2.3 Localization 35 1.9.3 ActivationofPKC 35 1.9.4 Protein Kinase C Intracellular Signaling 36 1.9.5 ProteinKinaseCinLAP 39 1.10 Resolution of Inflammation 40 1.10.1 Acute Inflammation 41 1.10.2 Chronic Inflammation 43 1.10.3 Resolution Phase of Inflammation 43 1.11 Anti-Inflammatory & Pro-resolving Mediators in Inflammatory Disease 44 1.11.1 Lipoxins and Endogenous Arachidonic Acid 44 1.11.2 Aspirin-Triggered Lipid Mediators 45 1.11.3 Resolvins — Exogenous 46 CHAPTER TWO: HYPOTHESIS AND AIMS 49 2.1 Hypotheses 50 2.2 Objectives 50 2.3 Rationale 51 CHAPTER THREE: METHODS AND MATERIALS 52 3.1 Patient Selection and Parameters of Diagnosis (Clinical & Radiographic) 53 3.2 Antisera 55 v 3.3 Isolation of Peripheral Blood Neutrophils .58 3.4 Stimulation of PMN 58 3.5 Cell Fractionation by Nitrogen Decompression 58 3.5.1 Rationale 58 3.5.2 Methodology 59 3.6 Assessment of Superoxide Generation 60 3.6.1 Superoxide Generation Assay 60 3.6.2 Superoxide Generation Assay with PKC Inhibitors 60 3.6.3 Superoxide Generation Assay with RvE1 60 3.7 RNA Extraction 61 3.8 Reverse Transcription 61 3.9 Quantitative Real-Time PCR 62 3.10 PKC Activity Assay 62 3.11 Protein Assay 63 3.12 Western Blotting 63 3.13 Statistical Analysis 64 CHAPTER FOUR: RESULTS 66 4.1 Superoxide Generation in Stimulated Neutrophils 67 4.2 Characterization of PKC Activity in Neutrophils 69 4.3 Kinetics of PKC Activity 73 4.4 Phosphorylation of PKC Isoforms in Whole Cell Lysates 76 4.4.1 Whole Cell Lysates 76 4.4.2 Cytosol and Membrane Fractions 84 4.5 Quantitative Assessment of PKC Expression in Neutrophils 91 4.6 Phosphorylation ofp47Phl0X in Healthy and LAP Neutrophils 95 4.7 Inhibition of PKC Isoforms 97 4.8 Inhibition of Superoxide in Neutrophils Incubated with RvEI 99 4.9 Phosphorylation of PKC isoforms in neutrophils incubated with RvEI 101 4.10 Phosphorylation of p47X in Neutrophils Incubated with RvE1 108 CHAPTER FIVE: DISCUSSION 110 CHAPTER SIX: CONCLUSIONS 127 vi REFERENCES .129 APPENDICES 160 A. Boston University Research Ethics Board Certificates 160 B. Ethical Approval for the Use of Human Subjects 161 C. Description of Subjects 162 VII LIST OF TABLES Table 1. PKC tissue distribution and specific findings 29 Table 2. Primary antibodies against phosphorylated antibodies of PKC isoforms 56 Table 3. Phosphorylation sites on the kinase of PKC isoforms 57 VIII LIST OF FIGURES Figure 1. Clinical manifestations of LAP 10 Figure 2. Neutrophil priming by inflammatory mediators 17 Figure 3. Structure of the PKC molecule 26 Figure 4. Signaling pathway of classical PKC isoforms 38 Figure 5. Lipid mediators in the resolution in acute inflammation 42 Figure 6. LAP patient selection criteria at Boston University 54 Figure 7. Superoxide release from healthy and LAP PMN 68 Figure 8. Total PKC activity in whole cell lysates in healthy and LAP PMN 70 Figure 9. PKC in cytosol and membrane from healthy and LAP PMN 72 Figure 10. PKC in cytosol from healthy and LAP PMN upon stimulation 74 Figure 11. PKC in membrane from healthy and LAP PMN upon stimulation 75 Figure 12. Phosphorylation of PKCa in whole cell lysates 79 Figure 13. Phosphorylation of PKCI31 in whole cell lysates 80 Figure 14. Phosphorylation of PKCI32 in whole cell lysates 81 Figure 15. Phosphorylation of PKCÔ in whole cell lysates 82 Figure 16. Phosphorylation of PKC in whole cell lysates 83 Figure 17. Phosphorylation of PKCa in cytosol and membrane 86 Figure 18. Phosphorylation of PKCI31 in membrane 87 Figure 19. Phosphorylation of PKCI32 in cytosol 88 Figure 20. Phosphorylation of PKCÔ in cytosol and membrane 89 Figure 21. Phosphorylation of PKC in membrane 90 Figure 22. Expression of PKC isoform transcripts in healthy and LAP PMN 92 Figure 23. Expression of PKC a, 131, 132, ô, and transcripts upon stimulation 94 Figure 24. fMLP-inducedp47P”O)< phosphorylation is enhanced in PMN from LAP patients 96 Figure 25. Inhibition of superoxide production with the use of PKC inhibitors 98 Figure 26. Inhibition of superoxide generation with RvE1 100 Figure 27. Phosphorylation of PKCa in PMN treated with RvE1 103 Figure 28. Phosphorylation of PKC131 in PMN treated with RvE1 104 Figure 29. Phosphorylation of PKC132 in PMN treated with RvE1 105 Figure 30. Phosphorylation of PKC in PMN treated with RvE1 106 Figure 31. Phosphorylation of PKC in PMN treated with RvE1 107 Figure 32. Inhibition of phosphorylation of 47Ph0X in PMN treated with RvE1 109 ix LIST OF ABBREVIATIONS Aa: Actinobacillus actinomycetemcomitans AA: Arachidonic Acid ATL: Aspirin-Triggered Lipoxin A4 ATP: Adenosine Tn-Phosphate CGD: Chronic Granulomatous Disease COX-2: Cyclooxygenase 2 DAG: Diacylglycerol DHA: Docosahexaenoic Acid (22:6) EPA: Eicosapentaenoic Acid (20:5) fMLP: N-formyl-meth ionyl-Ieucyl-phenylalanine GPCR: G Protein-Coupled Receptor H20: Hydrogen Peroxide HETE: Hydroxyeicosa tetraenoic acid I P3: I nositol 1,4, 5-trisphosphate LAP: Localized Aggressive Periodontitis LO: Lipoxygenase LPS: Lipopolysaccharide LTB4: Leukotriene B4 LX: Lipoxins LXA4: Lipoxin A4 LXB4: Lipoxin B4 MMP: Matrix Metalloproteinases NADPH: Nicotine Amide Dinucleotide Phosphate NSAI Ds: Non-Steroidal Anti-Inflammatory Drugs 02: Superoxide Anion PA: Phosphatidic Acid PAF: Platelet — Activating Factor P DK-1: Phosphatidylinositol-trisphosphate Dependent Kinase 1 P.g.: Porphyromonas gingivalis PGE2: Prostaglandin E2 P13-K: Phosphatidylinositol 3-Kinase PIP2: Phosphatidylinositol-4,5-bisphosphate PKC: Protein Kinase C PLC: Phospholipase C PMA: Phorbol 1 2-Myristate 13-Acetate PS: Phosphatidylserine PUFAs: Polyunsaturated Fatty Acids PVDF: Polyvinylidene Fluoride ROS: Reactive Oxygen Species RvE1: ResolvinEl SOD: Superoxide Dismutase x ACKNOWLEDGEMENTS This PhD would not have been possible without the expertise, generosity, and patience of my two supervisors, Dr. Thomas Van Dyke and Dr. J. Douglas Waterfield. I am deeply grateful for the opportunity to have worked with these two remarkable men whose support and kindness have left an indelible mark. I would also like to acknowledge my supervisory committee Dr. Don Brunette, Dr. Ed Putnins, and Dr. Clive Roberts for their sound advice and valuable contributions to my research. To the many members of the TVD Lab at Boston University, past and present, who have provided assistance over the years: Dr. Hatice Hasturk and the staff of the Clinical Research Center for the recruitment of human subjects and Dr. Alpdogan Kantarci, Ms. Martha Warbington, Ms. Jennifer Deady, and Ms. Amanda Blackwood for their expertise and laboratory assistance. My learning experience in the lab went beyond science to form international friendships. Special thanks to Dr. Yushi Uchida, my “sensai”, whose patience, knowledge, Japanese tutorials, and sense of humour made life in the lab truly memorable. I would also like to thank Dr. Kazuhiro Omori for his assistance in the lab and for reminding me what beautiful western blots looks like everyday; Dr. Eraldo Batista for his sarcastic wit and sense of humour; Dr. Elena Stan for her refreshing outspokenness and unexpected friendship; Dr. Justin Gee for his sweet disposition and thoughtfulness; and the very talented Ms. Gabby Fredman for her “seichel” and for making everything look so easy. My experience at UBC was enriched by the friendships formed along the way. Special thanks to Dr. Dean Hildebrand and Dr. lain Pretty, fellow POWs from the “Sweet” years at the BOLD Lab, where DNS orders were issued, organ donations were highly discouraged, and laughter was found in the most absurd DJS moments. My last summer at UBC was enjoyable thanks to newly formed friendships with three impressive perio residents: I would like to thank Dr. Mandana Nematollahi for her laughter and kindness; Dr. Dimitrios Karastathis for his colourful stories and his ability to be spontaneously “recruited” for projects; and Dr. Angela Demeter for her sweet and sassy energy. The future of periodontology is in good hands. Outside of the lab, I was very fortunate to have a supportive network of friends who gave me a great deal of personal encouragement via countless phone calls and kind emails. . .and the ability to “mooch” off them as a grad student. A BIG thank you to the following friends whose expectations demanded that I finish this PhD: Ms. Viki Koulouris Dr. Dean Wershler Fr. Jim Kruc Mrs. Barbara Van Dyke Dr. Kevin Bougher Ms. Victoria Bettis Dr. Paul and Patty Levi Dr. Frank Licari Ms. Virginia Vergara Dr. Michael and Gilia Reth man Dr. Mel Kantor Mr. Jeremy Abbate Dr. Thomas and Joanne Balshi Dr. Dan Nathanson Ms. Catherine Romero Dr. Jeffrey Burkes Dr. Juliana Ng Mr. Derrick Roney Most of all, I am forever indebted to my family for their endless support: my parents Kyu and Choo-Ja, my sister Liane and my brother-in-law Ian, and my brother Peter. I thank them for sustaining me everyday. xi To my wonderful parents Kyu and Choo-Ja Kim, for their unfailing support and profound love xii CO-AUTHORSHIP STATEMENT The chapters presented herein represent the work performed by the candidate with guidance from Dr. Thomas Van Dyke (Goldman School of Dental Medicine, Boston University). All data presented in this dissertation were acquired by the candidate with the exception of Figure 26, Inhibition of superoxide generation with RvE1. The candidate would like to acknowledge Dr. Hatice Hasturk and Dr. Alpdogan Kantarci for this contribution. xiii CHAPTER ONE: INTRODUCTION 1 1.1 Periodontal Disease: An Overview Several studies have shown that approximately 5-15% of any population suffers from severe generalized periodontitis (1-3). Moreover, the majority of adults is affected by moderate periodontitis (3-7) and the prevalence of milder forms of periodontitis is close to universal. Currently, periodontal disease is understood to be the result of a complex interaction between bacterial infection and host response (8). The host response plays a critical role in the clinical expression of periodontitis (9), of which 20% can be attributed to bacterial variance, 50% to genetic variance, and more than 20% to tobacco use (8). Associated with periodontal diseases are risk factors and determinants (risk factors that cannot be modified) that correlate the probability of disease with inherent characteristics, environmental exposure, or aspects of behavior. These include determinants such as age, gender, socioeconomic status, genetics and risk factors such as plaque, microbiota, oral hygiene, and tobacco. While these factors may or may not imply causality, there exists a correlation among the variables. 1.2 Determinants and Risk Factors of Periodontitis 1.2.1 Age Epidemiological studies demonstrated that age is directly related to the prevalence and severity of clinical attachment level (CAL) and pocket depth (PD). According to the 1985-1986 NIDR national survey of employed adults, more than 70% of adults aged 35 to 44 years and more than 90% of adults aged 55 to 64 had at least one loss of attachment site of 2 mm (1). For 4 mm CAL sites, the prevalence for 25 to 34 year olds and 55 to 64 year olds were 13.8% and 2 53.6% respectively. In this same study, periodontal pocket depths of 4 to 6 mm were present in 13.4% of all adults and were more prevalent in older age groups (10). Today, periodontal disease in older individuals is believed to be the result of accumulated destruction over time, not a disease of aging or an age-specific condition (11-14). Multiple studies have found that a relatively low prevalence of severe CAL existed in older adults populations (15-19). Additionally, periodontal disease occurs in children and young adult populations (20-30) and will be discussed later in this chapter under aggressive periodontitis. 1.2.2 Gender Prevalence of CAL has been consistently found to be greater in males than in females (1, 31, 32). This difference between the genders has not been clearly defined. However, they are thought to be related to variables such as poor oral hygiene (as defined as soft plaque deposits or calculus), less positive attitudes toward oral health, and less frequent visits for oral care than females (1, 32-34). 1.2.3 Socioeconomic Status (SES) Generally, individuals who are better educated, wealthier, and have a higher quality of life tend to be healthier than the less educated and affluent. Similarly, periodontal disease has been related to lower SES (31, 32, 35, 36). Socioeconomic status is multifaceted and involves several variables in a complex relationship. For example, in one study, researchers found that the prevalence of CAL at all levels of severity was not related to household income. However, CAL of 4 mm and 7 mm in at least one site were both closely correlated with educational levels (1). 3 The relationship between SES and periodontal health may be driven by the observation that more highly educated people have better oral hygiene, have more positive attitudes towards oral hygiene, and are able and willing to seek professional care because of dental insurance than less educated people. High SES individuals are most likely to be white-collar employees who have more education. 1.2.4 Genetics Genetics as a determinant of periodontal disease has been studied more recently. It was found that a specific genotype of the IL-i gene was associated with more severe periodontitis (37). Moreover, IL-i is a contributory cause of periodontitis in some patient groups (38-41). However, IL-i is unlikely the only genetic factor involved (42, 43). Further research is necessary to determine the strength of the relationship between genetic components and periodontitis. 1.2.5 Plaque, Microbiota, and Oral Hygiene Research conducted in multiple populations has established a causal relationship between poor oral hygiene and gingivitis (44-47). However, the relationship between poor oral hygiene and periodontal disease has been less clearly defined. While good oral hygiene can effectively remove plaque deposits in shallow to moderate pockets to reduce the microflora, it has little effect in removing bacteria in deep periodontal pockets (48). Studies confirm that plaque and supragingival calculus accumulations are poorly correlated with severe periodontitis (45-47, 49- 53) and that the amount of plaque accumulation was also of little significance to periodontitis (54-59). However, it was found that CAL can be arrested almost completely when meticulous oral hygiene was exercised concurrently with sub- and supragingival scaling and root planing three to six times per year (60, 61). 4 In addition to looking at the quantity of plaque accumulation, qualitative studies were conducted to examine the composition of plaque deposits and the effect on the periodontium. The identification and presence of specific microbiota were shown to result in CAL over the short term (62), but these were unable to predict the development of periodontitis in longitudinal studies for up to 3 years (63-65). Unsuccessful attempts to identify specific causative microorganisms have led to efforts that identify a broad group of Gram-negative pathogens that are consistently found at diseased sites. The etiology will be discussed later in this chapter. 1.2.6 Tobacco Numerous studies have shown that there is an association between smoking and periodontal diseases (independent of oral hygiene, age, or other factors) (66), that identify smoking as a risk factor for periodontal disease (55, 67-74), and that the prevalence of periodontitis is higher in smokers than nonsmokers in randomly selected patients (75-77). The risk of periodontitis among individuals who smoke is approximately 2.5 to 6.0 times higher than those who do not smoke (78). 1.3 Bacterial Etiology of Periodontology Periodontal disease results as a consequence of aberrant inflammation from products of microorganisms triggering the human inflammatory response. When considering the microorganisms periodontitis is associated with a change in microbial species in the gingival sulcus from gram-positive, facultative, fermentative microorganisms to predominantly gram negative, anaerobic, chemo-organotrophic and proteolytic organisms; the latter being strongly associated with periodontal tissues breakdown affecting the gingiva, the alveolar process, the connective tissue attachment of the tooth to the bone (periodontal ligament), and the cementum (79). 5 There are now over 600 recognized species that colonize the oral cavity (80). Although more than 415 species are estimated to be present in subgingival plaque (81) only a few are considered to be periodontopathic. The predominant pathogens include: Actinomyces actinomycetemcomitans (Aa), Bacteroides forsythus (Bf), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum, Campylobacter rectus, and Treponema denticola (82-89). These bacteria have different clonal types and it has not yet been determined whether all clonal types are pathogenic. However, some pathogens have been linked to certain types of periodontal disease. A. actinomycetemcomitans has been strongly implicated in localized aggressive periodontitis (90) and bacteria such as P. gingivalis, A. actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eikenella corrodens have been associated with chronic (adult) periodontitis (91, 92). However the data implicating specific pathogens remains weak, at the level of association with disease. As cited above, attempts to demonstrate cause and effect in longitudinal studies have universally failed. Although bacteria can have a direct effect on tissue destruction they can also cause tissue destruction indirectly by stimulating the host’s immune response. Host immune response involves: the recruitment of neutrophils, macrophages, T cells and B cells; the release of inflammatory cytokines; and the production of antibodies. Exaggeration of the host response (either in extent or duration) disturbs the homeostatic mechanisms and promotes the destruction of the extracellular matrix and resorption of the alveolar bone (93). 6 1.4 Periodontal Disease Classifications In 1999, the American Academy of Periodontology revised the periodontal disease classification system to reflect the most current evidence regarding the etiology and diagnosis of periodontal disease (94). The classifications are: - gingival diseases - chronic periodontitis - aggressive periodontitis - period ontitis as a manifestation of systemic diseases - necrotizing periodontal diseases - abcesses of the periodontium - periodontitis associated with endodontic lesions - developmental or acquired deformities and conditions Each classification has several subcategories delineating the extent and severity of each form. As this thesis concerns Aggressive Periodontal disease, I will limit the discussion to this disease. 1.5 Aggressive Periodontal Disease In 1999, the AAP published a Consensus Report written by a select group of members to discard the former term, “early-onset periodontitis” based on the premise that the nomenclature was too restrictive and that clinical manifestations of this form of periodontitis can occur at any age and not necessarily limited to individuals under the arbitrarily chosen age of 35 years (95). Therefore, the disease classification was based on clinical, radiographic, historical, and laboratory findings and the term Aggressive Periodontitis was decided upon by the AAP Workshop participants. 7 Aggressive Periodontitis is now defined as a specific type of periodontitis with clearly identifiable clinical and laboratory findings, which make it sufficiently different from Chronic Periodontitis to warrant a separate classification. The defining characteristics of localized and generalized forms of Aggressive Periodontitis are: - patients are otherwise clinically healthy (aside from the presence of periodontitis); - rapid attachment loss and bone destruction; - familial aggregation of the disease. Secondary features that are generally, but not always present include: - amounts of microbial deposits are inconsistent with the severity of periodontal tissue destruction; - elevated proportions of Actinobacillus actinomycetemcomitans and, in some populations, Porphyromonas gingivalis; - phagocyte abnormalities; - hyper-responsive macrophage phenotype, including elevated levels of PGE2 and lL-113; - progression of attachment loss and bone loss may be self-arresting For the disease to be classified as Aggressive Periodontitis, not all characteristics must be present. The diagnosis may be based on clinical, radiographic, and historical data. 1.5.1 Localized Aggressive Periodontal Disease Localized Aggressive Periodontal Disease, formerly called “localized juvenile periodontitis”, is a clinical classification of one of the aggressive periodontal diseases (96) and possesses distinct characteristics that differ from Chronic Periodontitis. LAP has a familial association and is characterized by the circumpubertal onset of periodontitis, rapid alveolar bone loss around only first permanent molar and incisor teeth, bleeding on probing at these local sites, and high levels 8 of A. actinomycetemcomitans and P. gingivalis in the periodontal pockets (Figure 1). With the exception of the periodontal disease, patients with LAP are otherwise in good general health. Unlike other adult forms of periodontitis, LAP has been associated with identifiable abnormalities of host cell function such as enhanced production of superoxide (97-99) and impaired chemotaxis of PMN (100). The observation of PMN abnormalities in the majority of patients and autosomal dominant inheritance (101) of both the clinical disease and PMN abnormality, appears most consistent in African American populations (97-99). 9 A. B. Figure 1. Clinical manifestations of LAP LAP is characterized by the circumpubertal onset of periodontitis, rapid alveolar bone loss around only first permanent molar and incisor teeth (A), bleeding on probing at these local sites, and high levels of A. actinomycetemcomitans and P. gingivalis in the periodontal pockets (B). 10 1.6 Role of the Immune System in Periodontal Disease As mentioned previously, periodontal tissue destruction is a result of the interaction between the host tissues and bacterial plaque or biofilm. While a number of periodontopathic organisms have been identified in sites of soft and hard tissue destruction, the mechanism of initial tissue destruction in healthy sites, where these bacteria either are not present or present in very low concentrations, remains unclear. Susceptibility to periodontopathic microorganisms varies amongst individuals and is the host response factor that plays a key role in the development of periodontal disease. The ability to protect or impede the progression of tissue destruction and inflammation contributes significantly in the overall outcome of maintaining periodontal health or determining periodontal disease. Various cell types are involved in the innate and acquired host response to bacterial challenges that result in the development of periodontal lesions. The neutrophil has been identified as the main protective host cell that plays a key role in the maintenance of periodontal health as well as limiting the progression of disease (102, 103). Histological observations made during the development of periodontal lesions linked the progression of the various lesions to the infiltration of different leukocyte subpopulations (104). Macrophages and T lymphocytes were predominant in early lesions in chronic periodontal disease. As the disease progresses to the point where connective tissue loss and bone resorption has occurred, the advanced lesions possess higher concentrations of B lymphocytes and plasma cells. The acquired immune lesion is the chronic lesion, It occurs on account of the failure of the host to resolve the earlier acute inflammatory lesion. Resolution of the acute lesion requires complete elimination of neutrophils from the tissues (105). The transformation of gingivitis to periodontitis correlates with the magnitude of chronicity, and lack of resolution of the neutrophil lesion (106). 11 1.7 The Polymorphonuclear Neutrophil Neutrophils play a significant role in the non-specific immune response and are recruited rapidly to sites of inflammation. They respond to injurious agents by phagocytosis, the release of matrix metalloproteinases (MMP) and preformed granular enzymes and proteins, and by the production of a range of potentially damaging, but transient, reactive oxygen species. These functions are critical for host defense against invading microorganisms as demonstrated by the propensity of patients with neutrophil deficiency syndromes such as neutropenia, chronic granulomatous disease and leukocyte adhesion deficiency to develop recurrent and often overwhelming infections (107, 108). Paradoxically, excessive activation of neutrophils may lead to inflammatory tissue injury in a variety of clinical conditions (e.g. the acute respiratory distress syndrome (ARDS), rheumatoid arthritis and ischaemia—reperfusion injury) (109-111). Therefore regulation of neutrophil activation is of paramount importance in determining the balance between defense and injury. 1.7.1 Differentiation Polymorphonuclear (PMN) neutrophils are important cells in the immune system involved in host defense and are the first cell type to arrive at inflamed sites. As the most abundant leukocyte in circulation, PMN arise in the bone marrow from CD34+ stem cells under the influence of regulatory cytokines (112). In vivo administration of G-CSF, GM-CSF, IL-3, stem cell factor, IL-i or IL-8 results in significant mobilization of PMN and hematopoietic progenitor cells. In the average adult human, approximately 10 million neutrophils are produced and released into circulation every minute, but the number of circulating cells remain constant (113). 12 Circulating neutrophils have a half-life of 6-10 hours after which they undergo apoptosis and are subsequently removed from circulation by phagocytic Kupifer cells of the liver (114, 115). PMN express many different cytokine and chemokine receptors either constitutively or in response to cytokines. Exposure to cytokines affects all aspects of neutrophil activation and recruitment and may result in the further release of cytokines, oxidative products, prostaglandins, and enzymes. Therefore, neutrophil phenotype and function are dependent on the microenvironment in which the cells were isolated, the age of the cells, and the previous exposure to cytokine stimulation. 1.7.2 Function In the host’s protective response to bacteria, the immune system is activated in an attempt to maintain periodontal health. However, the host’s competence to deal with these bacterial challenges and their virulence factors is a major factor in the prevention of periodontitis. This is particularly true when it comes to neutrophil dysfunction. There is a high prevalence of severe and early onset forms of periodontitis in individuals with conditions characterized by neutrophil dysfunction such as leukocyte adhesion deficiency, chronic neutropenia, and cyclic neutropenia (107, 108). In leukocyte adhesion deficiency, the neutrophil is unable to recognize inflamed endothelium due to the absence or decreased expression of the beta chain of CD18 (or alternately a defect in selectin ligand expression), thereby curtailing the endothelium transmigration of neutrophils. This deficiency results in a significantly reduced inflammatory response to the periodontopathic microorganisms and limits the host’s protective mechanisms to impede periodontal infection (116). Therefore, molecular defects in neutrophils have resulted in the acceleration of periodontitis. 13 A number of normal physiological functions that allow neutrophils to respond to microbial challenge are discussed below. 1.7.3 Adherence Mobilization of neutrophils out of general circulation and migration through the endothelial cell layer and extracellular matrix towards a bacterial insult involves a multi-step process. Upon activation by pathogens, resident macrophages in the affected tissue release cytokines such as IL-i, TNF-a and chemokines. These cytokines cause the endothelial cells of blood vessels near the site of infection to express cellular adhesion molecules, including P-selectins and integrins. Selectin ligands on circulating neutrophils bind the newly expressed selectins on the inner wall of the vessel, with low affinity. In a coordinate fashion L-selectins secreted by neutrophils bind their corresponding ligands on endothelial cells. These interactions cause the neutrophils to roll along the endothelial cells. During this rolling motion, temporary bonds are formed and broken between selectins and their ligands. The chemokines released by macrophages activate the rolling neutrophils and cause surtace integrin molecules to switch from a low-affinity state to a high-affinity state. This change is amplified by juxtacrine activation of integrins on neutrophils by chemokines produced by endothelial cells. In the activated state, integrins bind tightly to complementary receptors expressed on endothelial cells, with high affinity thereby immobilizing the neutrophils. The cytoskeletons of the neutrophils are then reorganized in such a way that the neutrophils spread out over the endothelial cells. The neutrophils then extend pseudopodia and pass through gaps between endothelial cells — a process known as diapedesis. Once in the interstitial fluid, leukocytes migrate along the chemotactic gradient towards the site of injury or infection (117-1 20). 14 1.7.4 Chemotaxis Chemotaxis is the process by which cells can detect and migrate in response to a chemotractant gradient, mediated by substances known as chemotactic factors that diffuse from an area of tissue damage. These factors, some of which are called chemokines, predominately regulate the activation and mobilization of leukocytes. There are currently 40 known chemokines and 16 chemokine receptors; with most chemokines binding to more than one receptor (121). Exposure of specific chemokines to their receptors on the neutrophil cell membrane not only induces cell migration, but also activates PMN adhesion, degranulation, and surface marker expression. Neutrophils migrate from the vessel lumen and enter sites of inflammation or infection in response to a shallow concentration gradient of chemoattractants such as C5a, LTB4, the chemokines CXCL1/CXCL8, and bacterial components (122-125). These chemotactic factors bind to the trimeric G-protein-coupled receptors on the surface of neutrophils and activate secondary messenger systems that regulate the reorganization of the actin cytoskeleton that underlies cell motility. In LAP, up to 80% of patients have a measurable decrease in chemotactic function compared to patients who are periodontally healthy (100, 126-131). Neutrophils from some patients with LAP migrate with approximately 50% or less of the velocity of neutrophils from patients who are considered healthy. Aside from the clinical manifestations of LAP, patients who are diagnosed with LAP and demonstrate neutrophil chemotactic dysfunction are otherwise healthy. However, not all patients with the clinical manifestations of LAP possess neutrophils with impaired chemotaxis. These findings have led researchers to wonder whether LAP is a result of neutrophil dysfunction, whether decreased chemotaxis is coincidental but unrelated to LAP, or 15 whether there are multiple etiologic subforms of LAP, only some of which are related to chemotactic dysfunction. 1.7.5 Neutrophil Priming Priming is said to occur when PMN, exposed to cytokines, demonstrate an enhanced responsiveness upon subsequent stimulation (Figure 2). For example, when normal circulating neutrophils are challenged with biological activating agents such as the bacterial formylated peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), they do not express anywhere near their full microbicidal capacity unless they have first been primed. Even at very high concentrations, these priming agents do not induce the effector functions on their own and must be presented to the cell for a variable period of time before the cell is exposed to the activating stimulus. Therefore, the neutrophil respiratory burst that occurs in response to an agonist (fMLP) and results in the release of superoxide anions (Oj) may be enhanced up to 20-fold by prior exposure to a priming agent such as lipopolysaccharide (132). Similarly, substantial priming of agonist-induced degranulation (133) and the generation of lipid mediators such as arachidonic acid (AA), leukotriene B4 (LTB4), and platelet-activating factor (PAF) (134, 135) have also been examined. Various priming agents have different signaling pathways and require varying pre-incubation times for maximal priming, which range from a few seconds (e.g. ATP) to over an hour [e.g. lipopolysaccharide (LPS), interferon-y] (136). TNF-a, a priming agent for PMN, causes PMN to secrete cytokines that inhibit phagocytosis, oxidative metabolism, and promote migration of freshly isolated PMN (137). Additionally, PMN treated with TNF-ct exhibit an enhanced oxidative burst upon subsequent treatment with fMLP, C5a, or zymosan, compared to unprimed PMN (138). Other agonists such as GM-CSF, G-CSF, IL-15, LTB4, PAF, and lL-8 have been shown to prime PMN for enhanced oxidative burst in 16 response to fMLP (138-140), indicating that the cytokine environment changes the responsiveness of PMN to subsequent stimulation. Although the exact mechanisms remain unknown, some of the priming effects in PMN function have been attributed to receptor upregulation following degranulation (141). Macrophage 1 GM-CSF, TNF-ci PAF, TNF-o Figure 2. Neutrophil priming by inflammatory mediators Exposure to cytokines primes neutrophils so that they exhibit enhanced oxidative burst upon stimulation. BacteriaEndothelial Cell LPS Unprimeci Neutrophil fMLP Primed Neutrophil C2- Q2- Q2- Q2- Q2- Q2- 17 1.7.6 Phagocytosis The cellular process of engulfing particles to remove foreign bodies, pathogens, and debris, known as phagocytosis, occurs in innate immune cells such as neutrophils, basophils, eosinophils, monocytes, macrophages, and dendritic cells. Stimulated by microbial contact, phagocytic cells are activated by intracellular signals that trigger the activation of functional processes, particularly the process of internalizing particles through a variety of distinct molecular and morphological developments. Phagocytosis is mediated simultaneously by a broad spectrum of receptors [Fcy-Receptors (FcyRs), complement receptor 3, scavenger receptors, lectins, and other integrins] for the recognition and internalization of microbes. Different signaling pathways can be activated, thereby increase the efficiency of internalization. [For review, see (142)]. 1.7.6.1 Recognition & Activation In neutrophils, different combinations of FcyRs are expressed and thus lgG-opsonized particles are recognized simultaneously through several receptors that bind to the Fc region of lgG (143, 144). There are two types of Fc’’Rs: FcyRs that contain ITAM motifs in their intracellular domains that recruit kinases and activate phosphorylation cascades, and Fc’yRs that contain ITIM motifs that recruit phosphatases that inhibit signaling (143, 144). Activating receptors with high affinity (Fcy RI) and low affinity (Fcy RIIA and Fcy RIIIA) bind lgG - opsonized particles and activate actin polymerization beneath the particle, membrane recruitment to the site of particle contact, membrane extension outward to surround the particle, leading to particle engulfment (145). Co-ligation of the inhibitory FcyRs (Fcy RllB) that recruits the phosphatase SHIP that blocks phosphoinositide signaling (144) regulates the efficiency of the process. Therefore, the relative expression of activating and inhibiting Fc’yRs determines the threshold for phagocytosis 18 and inflammatory responses to lgG - opsonized particles. Complement receptors are expressed in various cells and recognize and internalize foreign bodies by binding a broad spectrum of microbial opsonins including complement components Clq, C4b, and C3b, as well as mannan-binding lectin (146, 147). Common complement receptors expressed in neutrophils, such as CR1 and CR3, may require additional signals to mediate internalization of complement opsonized particles (148). While CR1 alone is unable to activate mechanisms of phagocytosis, ligated with Fc receptors, it enhances Fc receptor- mediated phagocytosis and can mediate phagocytosis of MBL-opsonized particles in fibronectin-treated PMN5 (146). Similarly, to trigger phagocytosis, CR3 requires a second activation step that increases the number of receptors expressed on the cell surface (149-151) or is activated by inflammatory cytokines (TNF a), microbial products (LPS), and adhesion (fibronectin) (152, 153). In vitro, this latter activation signal can be stimulated with phorbol esters and is thus likely to involve protein kinase C activation (145, 153). Therefore, complement receptors can activate phagocytosis independently or require the presence of different phagocytic receptors to produces synergistic effects (146, 154). 1.7.6.2 Engulfment Activation of many signaling pathways results in the rearrangement of the actin cytoskeleton, extension of the plasma membrane, and engulfment. Signaling molecules in phagocytosis such as phosphoinositide 3-kinase (P1 3-kinase), phospholipase C (PLC), Rho GTPases, and PKC are play a role in regulating the mechanics of particle ingestion, inflammatory responses, and microbial killing. P1 3-kinase and P1-PLC are not only required for the mechanical aspects of particle internalization, but also implicated in pro-inflammatory signaling (155). P1-PLC activity is required for microbe-induced pro-inflammatory signaling primarily due to its role in activating 19 PKC (156, 157). Rho GTPases are key regulators of the actin cytoskeleton in adhesion, membrane ruffling, and stress fiber formation and play a key role in regulating phagocytic efficiency due to their interactions with other signaling pathways (158, 159). These molecules activate multiple signaling pathways to the actin cytoskeleton and the nucleus and indicate that there are multiple levels of rGgulation of phagocytic efficiency (160, 161). 1.7.6.3 Microbicidal Killing (intracellularlextracellular) Phagocytic cells kill newly internalized microbes by the production of caustic reactive superoxide ions produced by assembly of the NADPH oxidase on phagosomal membranes (162). In unstimulated cells, components of the NADPH oxidase are distributed in the membrane (gp91PC and p22”<) and in the cytosol (p67ox p4oub0x, 47Ph0x, and Rac2). Phosphorylation of p47P by PKC, PKA, p21-activated kinase (PAK), and P1 3-kinase- stimulated kinases induces the translocation of the cytosolic components to the membrane, thereby assembling the NADPH oxidase complex. Electrons transferred from NADPH oxidase complex inside the cell can cross the membrane and couple to molecular oxygen to produce superoxide, which spontaneously recombines with other molecules to produce reactive free radicals. Superoxide is produced at the membrane thus can be deposited into phagosomes (which contain the engulfed microbe) or it can be produced outside of the cell. 1.8 Reactive Oxygen Species Reactive Oxygen Species (ROS) are chemically reactive molecules derived from molecular oxygen. These molecules are produced from cellular functions including mitochondrial metabolism (163, 164), PMN activation (165, 166), and other oxidation pathways. Additionally, ROS can be generated from numerous external sources including heat, UV light, therapeutic drugs, and X- and ‘- radiation (167-1 70). 20 1.8.1 Production of ROS The respiratory chain in the mitochondria is a source of ROS since mitochondria constantly metabolize oxygen to produce ROS (163, 171), primarily in the form of superoxide radical (02) and hydrogen peroxide (H20)(172, 173). Other ROS produced in the mitochondria include the hydroxyl radical (0H), nitric oxide (NO), and peroxynitrite (ONOO). The superoxide radical is produced by the transfer of one electron to molecular oxygen in the mitochondrial respiratory chain. This event occurs at complexes I (NADH dehydrogenase) and Ill (ubisemiquinone) of the electron transport chain, which reside in the inner mitochondrial membrane (174, 175). NADPH +202+ NADP + H + 2O2 The generation of the superoxide radical subsequently leads to its reaction with the mitochondrial enzyme manganese superoxide dismutase (MnSOD) to produce hydrogen peroxide. 202 + 2H+ H20 + 02 In the presence of reduced transition metals such as Fe2, hydrogen peroxide can produce the highly reactive hydroxyl radical (176). Fe2 + H20 + OH + 0H + Fe3 21 Nitric oxide may be derived from exogenous sources in the mitochondria. NO is a free radical produced from L-arginine through the action of isoenzyme NO synthases (177). Because nitric oxide is a free gas, it readily penetrates through biological membranes and can easily react with the superoxide radical, producing a new molecule peroxynitrite. L-arginine + NADPH ÷ 02 + NGhydroxyLarginine + H20 + NADP + H NGhydroxyLarginine + % NADPH + 02+ L-citrulline + NO + H20 N0÷0.ONOci+W.OH+N0 Inflammatory reactions, especially of a chronic nature, also serve as a major source of ROS due to the activated inflammatory cells such as macrophages and neutrophils. These cells release various ROS (H20, NO, 0H, and 02) and hypochlorite (HOCI) (178, 179) to produce antimicrobial effects that protect the host. 2Cr ÷ H20 + 2HOCI ROS production in inflammatory diseases can also lead to the destruction of connective tissue and extracellular matrix components (180, 181). In the mitochondria of the neutrophil, H20, NO, 0H, and O2are produced, resulting in increased numbers and activity of PMN. During the inflammatory response, HOCI is formed by the action of phagocyte (not macrophage) myeloperoxidase upon H2O and is released extracellularly (182). Such HOCI functions by inactivating essential enzymes (183), oxidizing membrane bound protein (184), disrupting some cellular membrane functions (185), decreasing the adhesive properties of some extracellular matrix components (186), and increasing endothelial permeability (187). 22 1.8.2 ROS Tissue Damage Excessive levels of ROS that possess high reactivity result in damage close to the original site of their generation. The damage to all cellular and extracellular components includes lipids of biological membranes, proteins, and DNA molecules. Lipids of biological cell membranes are prime targets for ROS (188). The polyunsaturated fatty acids located within these membranes possess multiple double bonds that are damaged by the hydroxyl radical. This process of lipid peroxidation results in the accumulation of hydroperoxides in cell membranes, affecting the activity of transmembrane enzymes, transporters, receptors, and other membrane proteins and changing the cell membrane permeability and selectivity (189, 190). Proteins are excellent targets of ROS, not only because they are the most abundant cell constituent, but also because the slightest structural change of a single protein molecule can significantly affect cell function (191) by modifying or weakening enzyme activity and/or denaturing proteins (192, 193). The hydroxyl radical has been shown to induce oxidative protein damage (191) resulting in altered function, turnover, and degradation. Secondary effects of ROS on proteins include protein fragmentation, cross-linking, and unfolding (194). Nitrosylation by peroxynitrite also modifies protein molecules (195, 196). For example, nitrosylation of tyrosine may not only compromise protein function, but also cellular regulation (197). DNA molecules in mammalian cells are damaged by ROS when oxidative modifications occur from exposure to environmental agents or endogenous metabolic processes (198, 199). Both nuclear and mitochondrial DNA are affected by ROS (200, 201). However, mitochondrial DNA is more susceptible to oxidative damage as it is located in the mitochondrial matrix, the site of 23 ROS generation. The hydroxyl radical is thought to be the most reactive ROS in attacking DNA molecules (202, 203) through ATP depletion, base hydroxylations and strand cleavage. Gene mutations can also occur which eventually lead to malignant transformation or cell death. 1.8.3 Superoxide in the Pathogenesis of LAP Superoxide and it’s products (peroxide, hydroxyl ions) are responsible for a great deal of the tissue destruction that occurs at sites of inflammation (204). PMN are abundant in inflamed periodontal tissue and are a major source of oxygen-derived free radicals (205). As mentioned above neutrophils are “hyperactive” in LAP. Gingival epithelial cells are highly susceptible to attack by oxidants. Human PMN can promote desquamation and lysis of gingival epithelial cells as a consequence of this activity (206). PMN can also promote damage to human periodontal ligament-derived fibroblasts when these cells are co-cultured in vitro and the PMN stimulated with N-formyl methionyl-leucylphenylalanine (fM LP) and endotoxin (207). Direct, destructive effects of active-oxygen species on hyaluran and other matrix proteins have also been reported (180, 208). 1.9 Protein Kinase C Discovered in the late 1970’s by Yasutomi Nishizuka and his colleagues at Kobe University, Protein Kinase C (PKC) has since gained notoriety for its molecular role in lipid hydrolysis. Further recognition was gained when it was discovered that PKC is the direct molecular target or receptor for phorbol esters, extremely potent tumor promoters. This discovery placed PKC at the center of signaling pathways that control normal cell function and carcinogenesis. 24 1.9.1 PKC Structure & Isoforms Protein Kinase C is a family of serine/threonine kinases that plays a pivotal role in cell signaling, transducing a multitude of signals that produce lipid second messengers. This 82kDa monomeric enzyme is comprised of a cysteine-rich N terminal regulatory domain and a C terminal catalytic domain (Figure 3). The regulatory domain contains the sites for phospholipid binding (Cl), calcium (C2), and an autoinhibitory pseudosubstrate sequence that maintains the enzyme in an inactive configuration. The catalytic domain consists of the kinase core, the target for PKC phosphorylation (209-211). 25 PROTEIN KINASE C ISOFORMS Classification Reaulatorv Domain tlvtfr Dnmin Classical cx 1, 2, y DAG calcium ATP pherbol ester binding binding binding 4 1 1 N-7---F-©43 kinase Novel ___ PS Cl b J—j C3 H kinase I—c N—--- PS pseudestbstraLe G glutamate Figure 3. Structure of the PKC molecule The family of PKC isoforms has an amino terminal regulatory moiety that contains the autoinhibitory pseudosubstrate (PS), a Cl domain which binds diacyiglycerol (DAG) and phorbol esters, and a C2 domain which binds calcium. The C2 domain in novel PKC isoforms and the Cl domain in atypical PKC isoforms are nonligand-binding variants. The carboxyl terminal catalytic moiety consists of the kinase core that has three phosphorylation sites: the activation loop, the turn motif, and the hydrophobic motif. Atypical LI c ____ 9 -IC3 I—I kinase [—C 26 The Cl domain is a Cys-rich region of approximately 50 amino acid residues that is present in all PKC isozymes. In conventional and novel PKC, it is present as a tandem repeat, named Cia and Cib. Atypical PKC contain a single copy of the domain, termed atypical because it does not bind phorbol esters. It is noteworthy that there is a growing family of proteins unrelated to PKC that contain either typical or atypical Cl domains. The structure of the Cia/b domain revealed a globular domain, with two-pulled apart beta sheets forming the ligand binding pocket. Two Zn2 atoms are coordinated by His and Cys residues at opposite ends of the primary sequence, helping to stabilize the domain. In atypical Cl domains, one face of the ligand-binding pocket is compromised and the module cannot bind phorbol esters or diacylglycerol. The Cl domain contains a Cys-rich motif that forms the diacylglyerol/phorbol ester binding and phosphotidylserine site and is immediately preceded by an autoinhibitory pseudosubstrate sequence. The pseudosubstrate is a sequence that resembles the naturally occurring substrate except an Ala occupies the phosphoacceptor position. Peptides based on this sequence are effective competitive inhibitors of PKC, and peptides modified to have a SER at the putative phosphoacceptor position are relatively good substrates for the enzyme. There is an autoinhibitory role, an antibody generated against the pseudosubstrate sequence is able to activate PKC in the absence of cofactors, presumably by withdrawing the pseudosubstrate from the active site. The pseudosubstrate occupies the substrate-binding cavity of PKC, thus maintaining the enzyme in an inactive conformation. PKC is allosterically regulated by its pseudosubstrate. Using proteases as conformational probes, it was established that activation of PKC is accompanied by release of the pseudosubstrate sequence from the kinase core. Specifically, the pseudosubstrate of PKC is resistant to proteolysis when the enzyme is inactive but highly susceptible to proteolysis at a 27 specific ARG in the pseudosubstrate sequence when the enzyme is active. This unmasking of the pseudosubstrate occurs independently of how PKC is activated; whether by binding its cofactors or by anomalous mechanisms such as by binding cofactor-independent substrates. The question then arises, how do cofactors cause release of the pseudosubstrate? Recent studies of energetics suggest that engaging of the membrane-targeting modules on the membrane provides the energy to release the pseudosubstrate from the kinase core. The C2 domain contains the recognition site for acidic lipids, and in some isoforms, the Ca2 binding site. The binding energy resulting from engaging these domains on the membrane contributes to the release of the pseudosubstrate from the substrate-binding cavity (212), thus activating the enzyme. The C3 and C4 domains form the ATP and substrate binding lobes of the kinase core. The PKC family consists of ten isozymes (in mammals) and are classified into three groups (classical, novel, and atypical) (Table 1) based on their molecular structure, mode of activation, and cofactor regulation (213-215). Classical or conventional PKCs (cL, 31, 132, and y) are activated by calcium (Ca2)via their C2 domains and diacylglycerol (DAG) and phorbol esters (TPA or PMA) through their cysteine-rich Cl domains and require phosphatidylserine (PS) (216). Novel PKCs (, , r1IL, and 0) are Ca2 independent but are still regulated by PS, DAG, and TPA. Atypical PKCs (, and t/?) are Ca2 independent and do not require DAG nor TPA for their activation (217, 218). This subclass contains a single membrane-targeting module, the Cl domain, but the ligand-binding pocket is compromised such that it is unable to bind diacylglycerol. All PKC isoforms are stimulated by phosphatidylserine, which anchors the Cl domain to the membrane. 28 Isoform Protein Protein Structure Putative Activated by Activates Location Functions or Inhibits DAG, Ca’ inhibits Ca2 - plasma MW: 76.869 kDa protecting actin membrane 673 amino acids, and Ca2 has 2 zinc- homeostasis; B dependent phorbol cell activation; B ester and DAG cell receptor binding domains and survival signaling 1 C2 domain cytosol MW: 76.8 kDa, 671 protecting actin amino acids and Ca2 homeostasis; B cell activation; B cell receptor survival signaling ô cytosol MW: 77.477 kDa, cell signaling, Rottlerin, PS, activates T 676 amino acids, differentiation, ‘! DAG, and p38-MAP has 2 zinc- proliferation, phorbol ester Kinase dependent phorbol secretion, ester and DAG apoptosis, and binding domains and growth 1 C2domain [ plasma MW: 67.718 kDa, insulin stimulated arachidonic inhibits membrane 592 amino acids, glucose acid, gamma- PKB has 1 zinc- transport; anti- lenolenic dependent phorbol apoptosis; acid, PS, TNF ester binding domain signaling oxidant a, etoposide, and 1 DAG binding generation in wortmannin, domain endothelial cells; and ceramide cell growth, proliferation, and differentiation Table 1. PKC tissue distribution and specific findings a plasma MW: 76.8 kDa, 672 membrane amino acids, has 2 zinc- dependent phorbol ester, DAG binding domains andi C2 domain migration, proliferation, communication, and motility dependent hINV promoter activation DAG, Ca’ - F DAG, Ca2 activates p57 MAPK Inhibited by Go697, HBDDE (2,2’,3,3’,4,4’- Hexahydroxy 1,1 -Biphenyl 6,6’- Dimethanoldim ethyl Ether) Caiphostin C, Ro 31-8220, GF109203X, Go 6976, Ro 31-7549, Go 6983 Calphostin C, Ro 31-8220, GF1 09203X, GO 6976, Ro 31-7549, GO 6983 Caiphostin C, GO 6983, Chelerythrine chloride, LY 333351, Balanol NO-induced p38 kinase 29 Although there are 10 PKC isoforms identified, several isoforms demonstrate tissue specificity and contribute to the regulation of diverse cellular responses in both normal and pathological functions including cell permeability, transcriptional regulation, contraction, migration, proliferation, hypertrophy, protein synthesis, apoptosis, and secretion. In human neutrophils, five PKC isoforms have been identified to date: a, 31, 32, and (219, 220). PKC isoforms undergo chronic activation/membrane translocation in healthy and LAP PMN and result in pre assembly/priming of NADPH oxidase. 1.9.1.1 PKCa: PKCa contains 672 amino acids and it has an approximate molecular weight of 76.8 kDa. It has two zinc-dependent phorbol ester or DAG binding domains and one C2 domain. Distributed ubiquitously in almost all tissues and located predominantly in the plasma membrane, this isoform is activated by diacyiglycerol, involved in various signal transductions, and plays an important role in cell proliferation, communication, and motility. 1.9.1.2 PKCf3I: PKC31 is a 76.869 kDa phospholipid-dependent, serine and threonine-specific protein containing 671 amino acids. It has two zinc-dependent phorbol esters and DAG binding domains and one 02 domain. Found in all cell types in the plasma membrane and activated by diacyiglycerol, PKCI3I plays an important role in novel biologic functions in protecting actin and Ca2 homeostasis, B cell activation, and in B cell receptor survival signaling. This isoform is associated with an immunodeficiency characterized by an impaired humoral immune response. It appears that PKC31 plays an important role in B cell activation and may 30 be functionally linked to Bruton’s Tyrosine Kinase in antigen receptor-mediated signal transduction (221). PKCI31 is abundant in naive cells and is essential to EGF (Epidermal Growth Factor) actin protection. Pretreatment with EGF or PKC activators activated PKCf31, enhanced Ca2 efflux, normalized Ca24, decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of actin. PKC inhibitors prevented these protective effects. EGF normalizes Ca24 by enhancing Ca24 efflux through PKCf31. It appears that PKC31 plays an important role in novel biologic functions in protecting actin and Ca24 homeostasis (222). 1.9.1.3 PKC 2: Protein Kinase C132 is an 80 kDa member of the conventional group (cPKCs: sensitive to diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine kinases that are involved in a wide range of physiological processes including mitogenic activation, cell survival, transcriptional regulation, and tumor promotion. PKC32 is phosphorylated on three sites, threonine 500 in the activation loop, threonine 641 in the turn loop, and serine 660 in the hydrophobic loop. Threonine 641 mediates PKC32 binding to Hsp7O, which regulates its stability and phosphorylation. Phosphorylation of threonine 641 is also crucial for PKCf32 proper subcellular localization and catalytic function as well as for its autophosphorylation on serine 660. It has been shown that isoforms PKC 131 and PKC f32 only differ in their C-terminal sequences and that these nucleotide sequences of the 50 or 52 amino acid residues of PKC 131 and 132 are within the D4 region (223). This region is the only location where the sequences diverged significantly between the two isoforms in human, rat, and rabbit tissues, thus implying that the 31 primary structure, including the D4 region, is important in the different functions of the PKC(3 isoforms. Expression of PKC2 is associated with multiple outcomes, such as tumor pathogenesis, particularly in B-cell malignancies (224) and the production of superoxide in neutrophils. The latter finding has uncovered that PKC32 is essential for ligand-initiated assembly of the NADPH oxidase for the production of superoxide and partly responsible for superoxide release in activated PMNs (225, 226). 1.9.1.4 PKC8: PKC contains 676 amino acids and has an approximate molecular weight of 77.477 kDa. With two zinc-dependent phorbol ester and DAG binding domains and one C2 domain, this isoform is expressed in the cytosol of almost all cell types. PKCÔ is dependent on the presence of phospholipids or activation by phosphatidylserine, diacylglycerol, and phorbol esters, while not requiring Ca2. The functional roles of PKCÔ include signaling for differentiation, proliferation, apoptosis, and growth. In terms of cytokine secretion, it plays a role in the induction of Type 1 INF (Interferon) biological responses. It is also important in effecting migration of human keratinocytes. Moreover, PKC works in concert with anti-cancer drugs to induce apoptosis. Intracellularly, PKCÔ activation is important for the phosphorylation of STAT1 on serine 727 as inhibition of this isoform attenuates the INF-a or lNF- dependent serine phosphorylation of STAT1. Rottlerin, a PKC inhibitor, inhibits INF-ct or INF-13 dependent gene transcription via ISRE or GAS elements. In addition, inhibition of PKCó also blocks activation of the p38 MAP 32 Kinase, the function of which is required for dependent INF-a transcriptional regulation, suggesting a dual mechanism by which this kinase functions in the generation of INF-a responses. Combined, these findings indicate that PKC serves as a serine kinase for STAT1 and an upstream regulator of the p38 MAP Kinase and plays an important role in the induction of INF biological responses (227, 228). 1.9.1.5 PKC: PKC plays an important role in multiple cell functions. It is involved in insulin stimulated glucose transport, inhibition of apoptosis, oxidant generation in endothelial cells, as well as cell growth, proliferation and differentiation (229-233). This isoform is a 67.718 kDa protein that has a 592 amino acid sequence, one zinc-dependent phorbol ester binding domain, and one DAG binding domain. It is located in the plasma membrane and is widely expressed among cell types and tissues, particularly high in lung and less in kidney and testis. PKC is activated by P13K and 3-Phosphoinositide-Dependent Kinase-1 (PDK-1), arachidonic acid, gamma-lenolenic acid, phosphatidylserine, TNF-a, etoposide, wortmannin, and ceramide and inhibited by NO- induced p38 Kinase. PKCt has been implicated in TN F-a (Tumor Necrosis Factor a) - induced oxidant generation in endothelial cells. Regulation of NADPH oxidase function by PKC was investigated by stimulating HPAE-cells (human pulmonary artery endothelial cell) with TNF-a. This stimulation resulted in the phosphorylation of p47PI0)< and its association with gp91I0)<. Inhibition of PKC prevented the phosphorylation of p47P10X and its translocation to the membrane, thereby preventing the interaction of p470X with gp910x. These results demonstrated a novel function of PKC? in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses (234). 33 1.9.2 Regulation of Protein Kinase C Intrinsic function of the PKC molecule is regulated by three sequential mechanisms: 1. phosphorylation triggered by 3-phosphioinositide-dependent kinase (PDK-1) which primes the enzyme for catalysis; 2. co-factor binding which allosterically activates the enzyme; and 3. localization at the correct intracellular site to interact with targeting proteins that position the enzyme near its substrates. Therefore, impairment of any of these mechanisms affects signaling by the kinase. 1.9.2.1 Phosphorylation Three sequential phosphorylations within the catalytic domain are required for PKC to be rendered catalytically competent (235, 236). Phosphorylations occur at three conserved positions at the kinase core, with the first one occurring at the entrance to the active site, referred to as the activation loop. Phosphoinositide-dependent kinase (PDK-1) phosphorylates the activation loops of conventional, novel, and atypical PKCs. Subsequently, this event triggers a rapid autophosphorylation on the carboxy terminus: a turn motif conserved among all PKCs and a hydrophobic phosphorylation motif conserved in conventional and novel PKCs. 1.9.2.2 Co-factor Binding All isoforms of PKC have a kinase core carboxyl terminal adjacent to a regulatory moiety that contains two key functions: an autoinhibitory sequence (pseudosubstrate) and one or two membrane-targeting modules. 34 1.9.2.3 Localization Subcellular location of PKC is typically in the cytosol where the molecule is phosphorylated and its substrate-binding cavity is occupied by the pseudosubstrate sequence. In conventional PKC isoforms, the binding of Ca2 to the C2 domain initiates the translocation of the enzyme to the membrane where it can bind to its membrane-embedded ligand, diacylglycerol. This generates sufficient binding energy to expel the autoinhibitory pseudosubstrate from the active site and renders the molecule active for substrate binding (237). 1.9.3 Activation of PKC Activation of conventional PKCs requires two regulatory switches. The first step in the activation of PKC is the phosphorylation on the activation loop by PDK-1. PDK-1 is released from the PKC, triggering the autophosphorylation of the two carboxyl terminal sites (turn motif and hydrophobic motif). This locks PKC in a catalytically competent conformation, i.e. capable of becoming activated (238, 239). The mature PKC molecule that is phosphorylated at three positions localizes to the cytosol where it is competent to respond to second messengers. PKC is activated when the second activation switch occurs: co-factor binding, which removes the autoinhibitory pseudosubstrate from the active site. Mobilization and binding of Ca2 to the C2 domain, causes mature PKC to translocate to the membrane, where it can better initiate contact with the membrane bound diacylglycerol (DAG). Here, the interaction of its two membrane targeting domains, CI and C2, with diacylglycerol and phosphatidylserine, provides the energy to release the pseudosubstrate from the active site, allowing substrate binding and catalysis. In addition to the regulation by phosphorylation and co-factors, anchoring/scaffold proteins play a key role in PKC function by positioning specific isozymes at particular intracellular locations. 35 Because novel PKCs have a Cl domain that binds DAG but an impaired C2 that does not bind Ca2, they do not have the advantage of pre-targeting by a Ca2-responsive 02 domain. As a result, conventional PKCs translocate faster than novel PKC isoforms. Activation of PKC? is different from the conventional and novel PKC isoforms and is activated through a different signaling pathway. PDK-l phosphorylates PKC on its activation loop, an event which serves as a molecular switch to turn on the catalytic activity of the kinase (239, 240). This phosphorylation is stimulated by Pl(3,4,5)P, and contrasts with that of the conventional PKC isoforms, which is independent of Pl(3,4,5)P. The direct activation of PKC resulting from phosphorylation of the activation loop by PDK-1 serves as a direct “on—off” switch for catalysis (241). 1.9.4 Protein Kinase C Intracellular Signaling The PKC signaling pathway can be triggered by ligands binding to G-protein coupled receptors that activate phospholipase C isoforms. Activated phospholipase C (PLC) subsequently cleaves membrane-bound phosphatidylinositol 4, 5 biphosphate (PIP2), releasing two fragments (second messengers), inositol 1, 4, 5-trisphosphate (1P3) and diacylglycerol (DAG) (242, 243). 1P3 binds to the receptors on the endoplasmic reticulum, causing a rapid release of calcium ions from intracellular stores. This molecule is short-lived and is rapidly dephosphorylated to inositol 1, 4-bisphosphate and inositol 1-phosphate, which are inactive as second messengers. The second fragment, DAG, recruits most conventional and novel protein kinase C isoforms from the cytosol, (where they are maintained in an inactive conformation), to the membrane, (where they become allosterically activated). Elevated levels of DAG results in increased activation of PKC in PMN by increasing the affinity of PKC for Ca2, which is required for maximum activity. In the absence of stimulation, PKC is present as a soluble cytosolic protein that is catalytically inactive. 36 The elevated levels of Ca2 result in the activation of classical PKC and the trans location of PKC from the cytosol to the membrane (244) (Figure 4). Novel and atypical PKC isoforms are activated differently as previously discussed. The PKC molecule is rendered fully active once the pseudosubstrate is released from the active site and substrate binding to PKC can occur. 37 NADPH OXPtDASE ASSEMBIX 77 GOP 02 Figure 4. Signaling pathway of classical PKC isoforms Binding of calcium to the 02 domains of the classical PKC isoforms initiates translocation from the cytosol to the membrane where it binds to the membrane-bound second messenger diacylglycerol on the Cl domain. The energy that is released from this binding results in the removal of the pseudosubstrate from the active site, thus allowing substrate-binding, phosphorylation, and downstream signaling. I I I $ I O€cPK. e e GOP K’IADP NADPH F I I I I .1 1 1 $ 38 Activated PKC phosphorylates serine and threonine residues on a variety of intracellular proteins. Many of the PKC target substrates are components of signal transduction pathways and include proteins that regulate ion channels, calcium- and calmodulin-binding proteins, growth factor receptors, structural and regulatory proteins of the cytoskeleton, as well as components of the transcriptional machinery and efflux pumps. In the case of superoxide production in PMNs, it has been identified that PKC phosphorylates p47PI0X, a molecule necessary for the assembly of the NADPH oxidase (245-247). Upon activation ofp47X, the oxidase is assembled on cytochrome b558 on the inner surface of the plasmalemma, thereby producing 02 (219, 248). 1.9.5 Protein Kinase C in LAP Recently, it has been demonstrated that one molecular lesion in LAP is likely at the level of DGKa (249). Investigation of signal transduction of G-protein linked receptors by oligonucleotide microarray analysis (Affimetrix) and Real Time PCR revealed an absence of DGKct expression in LAP patients. Diacylglycerol-kinase (DGK) activity was also monitored by DAG phosphorylation and individual DAG molecular species were quantified employing liquid chromatography and tandem mass spectrometry-based lipidomics. Using this methodology, diacylglycerol kinase activity was also significantly decreased and this decrease was closely correlated with increased accumulation of 1, 2-diacyl-sn-3-glycerol substrates. These results identify a molecular lesion in PMN signal transduction that may be ultimately responsible for their aberrant responses and tissue destruction observed in LAP. PMN5 exhibit a marked increase in diacyiglycerol (DAG) in both unstimulated and stimulated cells in LAP (250, 251). This increase in DAG is associated with increases in PKC activity as 39 DAG triggers activation of PKC by promoting the translocation/redistribution of this kinase from the cytosol to the membrane. Although PKC is a critical enzyme in triggering 02 production by PMN (245, 252), the role of each PKC isoform and its effect on superoxide generation remains unknown. 1.10 Resolution of Inflammation Inflammation is the body’s normal response to infection, mechanical irritation, or injury and thought to be a causative event for several of the most common human inflammatory diseases such as atherosclerosis, cancer, asthma, autoimmune disease, and various neuropathological disorders such as stroke, Alzheimer’s, and Parkinson diseases (253). Inflammatory diseases are believed to be the result of excessive inflammatory responses, or recurrent and chronic period of inflammation that fail to resolve (254). A major characteristic of the immune system is its functional redundancy: an insult triggers the synthesis and release of various pro- inflammatory mediators including the early lipid mediators, prostaglandins and leukotrienes. Further positive feedback loops are mediated by early infiltrating leukocytes and the release of cytokines and chemokines and lead to a full activation of both the innate and adaptive immune systems. The inflammatory response can be broken down into three components; acute inflammation, chronic inflammation and resolution of inflammation. Data indicate that catabasis (from inflammation to “normal” health) is not a passive termination of inflammation but rather an actively regulated program of resolution (255). Therefore it is important to identify key cellular events and molecular signals that determine the end of inflammation and the beginning of resolution in order to provide the molecular basis for treatment and prevention of inflammatory diseases (256-260). 40 1.10.1 Acute Inflammation Acute Inflammation is the initial response of the body to harmful stimuli such as microbial invader, tissue injury, and surgical trauma and involves the recruitment of leukocytes (particularly large numbers of neutrophils) (Figure 5). Extravasation of plasma proteins, prostaglandins, and leukotrienes floods the site of injury. In the early stages, arachidonic acid is converted to pro-inflammatory mediators leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) causing edema, fever, and pain. Later in the inflammatory response, neutrophils “switch” their phenotype to produce lipoxins (trihydroxytetraene-containing members of the eicosanoids family) and stop leukotriene biosynthesis and neutrophil recruitment (255, 261-263). Lipoxins are rapidly produced within vascular lumen via platelet-leukocyte interactions (264, 265) and serve as “braking signals” that are anti-inflammatory and pro-resolving. Concurrently, biosynthesis of resolvins and protectins transpires from omega-3 polyunsaturated fatty acids, which results in critically shortening the period of neutrophil infiltration by initiating apoptosis. Apoptotic neutrophils are phagocytosed by macrophages, resulting in neutrophil clearance and release of anti-inflammatory and reparative cytokines. 41 LX&ALTs stimulate uptake of dead and dying PMNs Mucosal surface UWAT Lxs stimulate limit PMN nonphloglst)c monocyte Infiltration recruitment — — — Figure 5. Lipid mediators in the resolution in acute inflammation Upon microbial invasion, leukotrienes such as LTB4 stimulate the recruitment of neutrophils to the site of injury/infection. During the temporal progression, neutrophils switch the lipid mediator classes that they biosynthesize and progress from generating LTs and prostaglandins to lipoxins (LXs). Lipoxins and Aspirin-triggered lipoxins (ATLs) serve as stop signals by stopping the further recruitment of neutrophils, stimulating the nonphlogistic recruitment of monocytes to the site, and stimulating the uptake of apoptotic neutrophils by macrophages to promote the clearance and resolution of leukocytes from the interstitium. Skirt cut tCocci Bacilli “ BactH PMN recruitment aposs LTB4 anplificaton PMN recruitment sina! Lipid_mediator classj. swiuching > _____ Macrophage ®LTB4 PGs regulate blood flow LXG4 telet - Lumen of PMNs poatcaplilary ___________________ vessel — — — — — — — — —-— — —-— — — y Monocyte 42 1.10.2 Chronic Inflammation Chronic inflammation occurs when the body fails to shut off its inflammatory response to infection resulting in tissue destruction. The same pro-inflammatory chemical mediators in the acute phase of inflammation, such as most of the classic prostaglandins and leukotrienes, do not terminate the inflammatory response (258). The switch from acute to chronic inflammation usually involves a progressive shift in the type of cells present at the site of inflammation and is characterized by both simultaneous destruction of tissue and attempts at repair. 1.10.3 Resolution Phase of Inflammation The resolution phase of inflammation has two possible outcomes. The first outcome is the complete restoration of the inflamed tissue back to a normal status. Inflammatory measures such as vasodilation, chemical production, and leukocyte infiltration cease and damaged parenchymal cells regenerate. In situations where limited or short-lived inflammation has occurred this is usually the outcome. The second outcome, fibrosis occurs if there is large amounts of tissue destruction or damage in tissues unable to regenerate (266). Fibrous scarring occurs in these areas of damage, forming a scar composed primarily of collagen. Although it was previously believed that during the resolution phase the inflammatory response gradually faded away, it is now thought that the immune system is under the active control of homeostatic resolution pathways that are essential in limiting the inflammatory process and restoring normal tissue architecture and function (267). The lipid mediators, which are involved in the initiation of acute inflammation are also thought to regulate chronic inflammation and the resolution of inflammation (255, 259, 260, 268). More recently, pro-resolving mediators generated from essential polyunsaturated fatty acids have been identified as possessing pro-resolving properties and serve as protective and anti 43 inflammatory agents. Pro-resolving chemical mediators function by targeting cell types and events involved in acute and chronic responses. For example, nonsteroidal anti-inflammatory drugs (e.g. naproxen, ibuprofen, etc.) block conversion of arachidonic acid into prostaglandins by acting on cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) (269). Elevated levels of PGE2 help resolve inflammation by stimulating production of enzymes that form anti- inflammatory lipoxins. 1.11 Anti-Inflammatory & Pro-resolving Mediators in Inflammatory Disease 1.11.1 Lipoxins and Endogenous Arachidonic Acid Lipoxins (LX), generated during the resolution of acute inflammation from arachidonic acid, are anti-inflammatory compounds that dampen the host response and orchestrate resolution (259, 270-272). This lipid mediator promotes resolution of inflammation by controlling the entry of new PMN to sites of inflammation (273) by reducing the vascular permeability (274) and promoting the return to homeostasis (275). Lipoxins stimulate the non-phlogistic recruitment of mononuclear cells and stimulate macrophage uptake of apoptotic PMN (276). These functions make lipoxins unique eicosanoids and the first recognized lipid mediators that can expedite resolution. Lipoxins are biosynthesized in human tissues via different pathways (275): mucosal and vascular cell — cell interactions, platelet — leukocyte interactions, and membrane phospholipid priming. Mucosal and Vascular Cell — Cell Interactions: Lipoxins are generated in cells enriched in 15- lipoxygenase (LO), such as airway epithelial cells, basophils, macrophages, and bronchial tissue. Oxygenation of arachidonic acid by 1 5-LO yields 15S-H(p)ETE, an epoxytetraene intermediate. Cell to cell interaction with neutrophils generating 5-LO results in the formation of Lipoxin A4 (LXA4)and Lipoxin B4 (LXB4)(277-279). 44 Platelet - Leukocyte Interactions: Lipoxins are generated via platelet-leukocyte interactions (280) by transcellular conversion of the leukocyte 5-lipoxygenase (5-LO) epoxide product LTA4. LTA4, released by activated leukocytes, can be converted by neighboring cell types (281, 282) during adherence and the platelet 12-LO converts LTA4 to LXA4 and LXB4 (265, 283), which serve as stop signals by restricting the further recruitment of neutrophils. Lipoxins stimulate the recruitment of monocytes and macrophages that phagocytose apoptotic neutrophils, reduce dendritic cell motility, reduce IL-12 (284), regulate T cell cytokines (285) and MMPs from fibroblasts (286, 287) that lead to anti-inflammation and pro-resolution in vivo. Membrane Phospholipid Priming: Membrane inositol-containing lipids are primed to produce lipoxins on activation of cells by releasing stored 15 - HETE. This is then transformed by neighboring leukocytes, resulting in a reduction in leukotriene biosynthesis, suggesting a reciprocal relationship between leukotrienes and lipoxin biosynthesis (253, 288). 1.11.2 Aspirin-Triggered Lipid Mediators Aspirin (ASA), a commonly used NSAID, triggers the endogenous generation of 15R-epimeric LX (specifically 15-epi-LXA4 and 15-epi-LXB4)that possess both anti-inflammatory and anti proliferative properties (271, 289, 290). This occurs via acetylation of cyclooxygenase-2 (CCX 2) in the presence of aspirin at sites of inflammation (291). Acetyl COX-2 inhibits the formation of prostaglandins (292), and produces 15R — HETE (293, 294) from AA that is converted by leukocytes to 15-epi-lipoxins. The aspirin-triggered lipoxin (ATL) has a longer duration because of their reduced catalytic activity for enzyme inactivation (273). Coupled with LXA4 as ligand, 45 this receptor rapidly regulates the phosphorylation of leukocyte-specific protein in PMNs and stops PMN migration and agonist-induced superoxide anion generation (295). Aspirin’s role in the generation of lipid mediators with anti-inflammatory pro-resolving properties presents a novel approach to therapeutic design and use. 1.11.3 Resolvins — Exogenous A new class of lipid mediators, called resolvins, has been found to play a key role in the active resolution of both acute and chronic inflammation. Resolvins are derived from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid and are endogenously biosynthesized via specific cell—cell interactions in murine inflammatory exudates in vivo, as well as during endothelial cell and leukocyte interactions in human vasculature (260, 268, 296, 297). The autacoids that require enzymatic generation from omega-3 EPA are termed resolvins of the E series, hence the name RvE1. Resolvin El is generated in the course of inflammation in vivo from EPA when aspirin is administered. At the local site of inflammation, aspirin treatment enhances EPA conversion to l8R — HEPE and RvE1, which carry potent anti-inflammatory signals. Resolvins reduce cellular inflammation by inhibiting the production and transportation of inflammatory cells and chemicals to the sites of inflammation. RvE1 (5S, 12R, 18R — trihydroxy — 6Z, 8E, iCE, 14Z, 16E — eicosapentaenoic acid) proved to dramatically reduce PMN transmigration and inflammation in vivo (260). Studies to date indicate a therapeutic potential for the EPA derived RvEl in managing both acute and chronic inflammation with particularly strong evidence in models of colitis, periodontitis, and arthritis including profound disease modifying properties (261, 298, 299). RvE1 has been shown to block PMN infiltration and inhibit bone loss in periodontal disease in a rabbit model (299) and protect against 2, 4, 6 — trinitrobenzene sulfonic acid (TNBS) — induced 46 colitis (298). In the latter study, RvE1 demonstrated protective actions through its receptor ChemR23, by reducing leukocyte infiltration, turning off pro-inflammatory gene expression, and decreasing the level of serum anti-TNBS lgG, suggesting attenuation of antigen presentation and B cell production of lgG. Additionally, mice treated with RvE1 showed significantly lower levels of myeloperoxidase compared to mice treated with the control vehicle. Thus the protective properties of RvE1 may serve as the basis for resolving inflammation. The regulation of neutrophil tissue destruction and resolution of inflammation using an aspirin- triggered lipoxin (LX) analog and RvE1 in localized aggressive periodontitis (LAP) were examined. Results indicated that neutrophils from LAP are refractory to anti-inflammatory molecules of the LX series, whereas LAP neutrophils responded to RvE1. It was also found that RvE1 specifically binds to human neutrophils at a site that is functionally distinct from the LX receptor. Consistent with these potent actions, topical application of RvE1 in rabbit periodontitis conferred dramatic protection against inflammation induced tissue and bone loss associated with periodontitis (299). EPA and DHA — derived chemical mediators and arachidonate — derived lipoxins demonstrate considerable potential for designing drug therapies to resolve inflammation, particularly, chronic inflammation associated with many diseases. Endogenous and exogenous anti-inflammatory and pro-resolving mediators such as lipoxins and resolvins serve as agonists for resolution and contribute to the key events in the regulation of inflammation and its natural resolution. Omega — 3 derived lipid mediators act as agonists for resolution at different stages of inflammation, particularly within the resolution phase (300, 301). These endogenous lipid mediators regulate the process of the resolution of inflammation (260) and are not solely pro-inflammatory, but are also anti-fibrotic (302, 303), underscoring the critical role of the PMN 5-LO in producing both pro-inflammatory leukotrienes in addition to lipoxins and resolvins (304, 305). Suppression of 5- 47 LO can have a negative impact on the timely resolution of inflammation by altering the generation of mediators that are pivotal to the active process of resolution. Therefore, the mechanisms of resolution and lipid mediators produced are potent in their regulation of inflammation and the resolution of inflammation. 48 CHAPTER TWO: HYPOTHESIS AND AIMS 49 2.1 Hypotheses 1. Excess generation of superoxide anion by neutrophils from LAP subjects is mediated by the pre-activation of specific PKC isoforms, leading to the pre-assembly of NADPH oxidase. 2. The endogenous lipid mediator Resolvin El (RvE1), derived from dietary omega-3 fatty acids, is an immunomodulatory compound that will reduce superoxide generation in neutrophils from LAP patients. 2.2 Objectives 1. To identify Protein Kinase C (PKC) isoforms in human neutrophils and characterize the differential expression of PKCs in neutrophils isolated from healthy and LAP subjects. 2. To elucidate the role of different PKC isoforms in superoxide generation and to characterize quantitative and kinetic differences in PKC subcellular distribution between LAP and healthy neutrophils. 3. To evaluate the actions of RvE1 treatment related to: a. PKC isoform phosphorylation and kinetics b. p47Phl°X phosphorylation and kinetics 50 2.3 Rationale The hyperactivity of PMNs in LAP patients is a major factor affecting the pathogenesis of the disease. The studies presented in this thesis will markedly increase our knowledge about PKC isoform involvement in this hyperactivity that, in turn, may aid development of therapeutic modalities targeting the relevant isoforms. My studies will also evaluate in vitro the new immmunoregulatory lipid mediators (RvE1) as one therapeutic modality to control the hyperactivity of neutrophils and in turn the progression of periodontal disease. 51 CHAPTER THREE: METHODS AND MATERIALS 52 3.1 Patient Selection and Parameters of Diagnosis (Clinical & Radiographic) LAP subjects and systemically healthy subjects were selected among patients that were recruited at the Clinical Research Center of Boston University’s Goldman School of Dental Medicine. Institutional Review Board approval and signed informed consent from individuals were obtained prior to the initiation of the study. Subjects enrolled in the study were advised about the benefits and risks involved in their participation. Potential subjects were assessed based on a comprehensive medical history to ensure absence of medical complications that could lead to misdiagnosis of LAP. Similarly, patients taking anti-inflammatory drugs were excluded from the study. LAP diagnosis was based on two criteria; bone loss in the form of periapical radiographs and clinical periodontal probing depth measurements (where local and angular defects in bone around incisors and first molars were considered diagnostic). Healthy donors with no radiographic and clinical evidence of periodontitis were used as controls. The patient selection criteria are summarized in Figure 6. Given the limitations of patient number age/gender/race matched studies could not be carried out. This will have an impact on the variability of the results. 53 Clinical Research Center Goldman School of Dental Medicine Boston University I New Patient I Demographic Background Clinical Evaluation Medical History I I Clinical Exam Radiographic Screening Periodontal Probing Measurements Periapical Radiographs Bleeding on Probing Interproximal Radiographs Attachment Loss Localized Attachment Loss 1st Molars Incisors I I I Absent I I Present (+) I I I Routine Periodontal Management Blood Withdrawal Oral Health Instruction PMN Isolation Superoxide Assay Chemotaxis Increased (+) Decreased (-) LAP Figure 6. LAP patient selection criteria at Boston University 54 3.2 Antisera Primary antibodies against phosphorylated PKC were acquired from Cell Signaling (Danvers, MA), Abcam (Cambridge, MA), and Epitomics (Burlingame, CA). Each of the antibodies used does not cross-react with other PKC isoforms. The secondary antibody, anti-rabbit lgG conjugated with horseradish peroxidase was also from Cell Signaling. The antisera used in these studies are listed in Table 2. 55 Anti body Manufacturer 4 Product Number Specifications Mouse monoclonal to PKCa Phospho PKC a Epitomics 1195-1 (phospho T638) Rabbit polyclonal to PKCf31 Phospho PKC 131 Abcam ab5782 (phospho T642) Rabbit polyclonal to PKCf32 Phospho PKC 132 Abcam ab5785 (phospho T641) Rabbit polyclonal to PKCÔ Phospho PKC ö Cell Signaling #9374 (phospho Thr505) Rabbit polyclonal to PKC Phospho PKC Cell Signaling #9378 (phospho Thr4l 0/403) Affinity purified goat anti-rabbit lgG (H&L) antibody is Anti-rabbit IgG, HRP Cell Signaling #70 cor ,ed to horseradish linked Antibody peroxidase. Affinity purified horse anti Anti-mouse lgG, HRP- mouse lgG (H&L) antibody is Cell Signaling #7076 linked Antibody conjugated to horseradish peroxidase Table 2. Primary antibodies against phosphorylated antibodies of PKC isoforms 56 These phosphorylated antibodies were selected based on specificity and sensitivity in the assay systems employed. There are three phosphorylation sites on the kinase thus it is not only important that the antibodies do not cross-react with the phosphorylated forms of other PKC isoforms, but also one that detects endogenous levels of the target PKC isoform only when phosphorylated at the identified threonine/serine site. Table 3 lists the target phosphorylation sites for the antibodies. Phosphorylation Sites PKC Isoform Activation Loop Turn Motif Hydrophobic Motif u Thr497 Thr638 Ser657 131 Thr500 Thr642 Ser662 132 Thr500 Thr641 Ser660 Thr505 Ser643 Ser662 Thr4lO Thr560 - Table 3. Phosphorylation sites on the kinase of PKC isoforms The bolded threonine/serine indicate the targeted phosphorylation sites by the antibodies used in this study. For PKC, a glutamate occupies the hydrophobic motif site. 57 3.3 Isolation of Peripheral Blood Neutrophils Approximately 50 ml of peripheral blood were obtained from subjects by venipuncture, collected in vacutainers containing 25 units/mI of heparin, and layered on top of a Ficoll-Hypaque discontinuous gradient system. Neutrophils were isolated via density gradient centrifugation for 15 minutes at 400 x g. Based on their position in the gradient (sedimentation properties) the neutrophil layer was collected with a Pasteur pipette and transferred to a separate tube. Contaminating red blood cells were subjected to isotonic lysis using standard technology (NH4CI, KHCO3,Na2EDTA, H20, pH was adjusted to 7.4 with NaOH). After two washes in Ca2/Mg - free PBS, PMN were evaluated for viability by Trypan blue exclusion. Changes in morphology were assessed visually, and PMN were counted and immediately processed for further experimentation. 3.4 Stimulation of PMN PMN were suspended in PBS, pre-incubated for 15 minutes at 37°C before stimulation with 1pM of N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 30, 60, 300, and 600 seconds. The reactions were stopped by the addition of ice cold PBS. Cells were collected as a pellet by centrifugation at 400 x g for 10 minutes! 4°C, and fractionated as described below. 3.5 Cell Fractionation by Nitrogen Decompression 3.5.1 Rationale Neutrophils were disrupted by nitrogen decompression. Decompression is the method of choice because it avoids rupture of granules containing proteolytic enzymes and thus minimizes protein degradation. Nitrogen gas is dissolved in the cell under high pressure within a suitable pressure vessel and when the pressure is rapidly released, the cell membrane ruptures. This method is 58 superior to ultrasonic and mechanical homogenizing methods as organelles and protein folding are preserved better and the chemical and physical stresses to the subcellular components are thought to be minimal. Since nitrogen bubbles are generated within each cell, the same disruptive force is applied uniformly throughout the sample, thus ensuring unusual uniformity in the homogenate. 3.5.2 Methodology Neutrophils were suspended in a disruption buffer (50 mM Tris-HCI, 50 mM 3-mercaptoethanol, 10 mM EGTA, 5 mM EDTA, 1 mM PMSF, 10 mM Benzamidine, pH 7.5) supplemented with protease inhibitor Complete Mini tablets (Roche Diagnostics, Indianapolis, IN) and placed in a nitrogen decompression bomb (Parr Instruments Stainless Steel Bomb — Model 4635, Moline, IL) at 450 psi for 15 minutes. The cavitate was collected and cell contents isolated via differential centrifugation. To remove unbroken cells and nuclei, samples were centrifuged first at 1500 x g for 30 minutes. The cavitate was then centrifuged at 10,000 x g for 45 minutes, resulting in a pellet of enzyme-rich granules. The supernatant of cytosolic and membrane fractions were subsequently centrifuged at 200,000 x g for 60 minutes at 4°C to separate the cell fractions, resulting in a membrane pellet and cytosolic supernatant. The cytosolic supernatant was then placed in Amicon® Ultra-4 Centrifugal Filter Devices (Millipore, Billerica, MA) to concentrate the samples, and centrifuged at 4000 x g for 15 minutes. The pellet, designated as the membrane fraction, was re-suspended in the cell disruption buffer with 1% Triton X-1 00. The plasma membrane marker, NaK-ATPase, was assessed in each fraction by Western Blotting and found in the membrane fraction preparations, but was absent in the cytosolic fractions (data not shown). 59 3.6 Assessment of Superoxide Generation 3.6.1 Superoxide Generation Assay Superoxide generation was analyzed using the superoxide dismutase inhibitable cytochrome C reduction assay (306). Peripheral Blood PMN were treated with PBS and were then added to a ferricytochrome C solution (300 tg/mol, 200 tL/well) in Gey’s Balanced Salt Solution (GBSS) (lnvitrogen, Carlsbad, CA). FMLP (1 M) was added to the reaction mixtures to trigger the respiratory burst. The assay was carried out in microplates, which were read at a wavelength of 550 nm by a Vmax microplate reader (Molecular Devices, Sunnyvale, CA). The amount of superoxide produced per well was calculated by the formula (307): nmole Cj = CD 550 nml[21 ,000M cm1 x 0.66cm x (1 000mL/1 L) x (5x1 x 20/1 000PMNs/mL)] 3.6.2 Superoxide Generation Assay with PKC Inhibitors Four PKC inhibitors were used: a classical PKC inhibitor Ro-31 -8425 (Calbiochem) (targeting a, 131, 132, y), a PKC 13 inhibitor (Calbiochem), a PKCÔ inhibitor Rottlerin (Calbiochem), and a PKC Pseudosubstrate Inhibitor, Myristoylated (Calbiochem, La Jolla, CA). The inhibitors were optimized for concentration and incubation times of 5, 10, and 15 minutes (based on cell viability tests using Trypan Blue) and the superoxide generation was assayed as described above. 3.6.3 Superoxide Generation Assay with RvEI The effect of RvE1 on superoxide generation was measured by treating PMNs with various concentrations of RvEI (0, 0.1, 1, and 10 nM) or PBS only for the control group for 15 minutes. The stock solution of RvE1 was originally dissolved in methyl ester, which was evaporated with nitrogen gas. PBS was subsequently added to the RvE1 to add to the PMNs. The cells were 60 stimulated with fMLP, harvested, incubated with RvE1, and superoxide generation was measured as described above. 3.7 RNA Extraction The RNA extraction and purification process was carried out using the RNeasy Mini Kit (Qiagen, Valencia, CA). The kit combines the selective binding properties of a silica-based membrane with the speed of microspin technology. A specialized high-salt buffer system allowed up to 100 tg of RNA longer than 200 bases to bind to a silica membrane. As per manufacturer’s instructions amples were first lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanate—containing buffer, which immediately inactivated RNases to ensure purification of intact RNA. Ethanol was added to provide appropriate binding conditions, and the sample was then applied to an RNeasy Mini spin column, where the total RNA bound to the membrane while contaminants passed through. The RNA was then eluted in 30 1iL of water. Purity and RNA concentration were spectrophotometrically determined at 260/280 nm using the NanoDrop® ND-i 000 Spectrophotometer (NanoDrop Technologies) and samples were stored at -80°C. 3.8 Reverse Transcription Synthesis of single-stranded cDNA from RNA was completed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) for subsequent Quantitative Real-Time PCR. This kit contained: lOX RT Buffer, 25X dNTP Mix, lox RT Random Primers, Multiscribe Reverse Transcriptase, RNase Inhibitor, and Nuclease-free H20. RNA samples were added to the Master Mix and placed in the GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA) for 10, 120, 5, and 10 minutes at 25, 37, 85, and 4 °C respectively to start the reverse transcription reactions. 61 3.9 Quantitative Real-Time PCR Fluorescence-based amplification was carried out using custom 5’-FAM labeled TaqMan probes for PKC c, f31, 132, , (Applied Biosystems, Foster City, CA). Primers were manufactured by Applied Biosystems (Foster City, CA) and to protect its proprietary rights, the company did not provide the primer sequences. 13-Actin was selected as an endogenous control and amplified using pre-formulated VIC-TAMRA labeled TaqMan probes (Applied Biosystems, Foster City, CA). A standard curve was used to obtain the concentration of the isoform(s) and the housekeeping gene. All measurements were conducted in triplicate. Quantification was performed in an automated thermalcycler (ABI Prism 7000 Sequence Detector, Applied Biosystems, Foster City, CA) and results analyzed through a software interface and Excel spreadsheet for calculation of relative expression (PKC isoform/13-Actin). The results from LAP patients were compared to non-diseased individuals. 3.10 PKC Activity Assay Activity of all PKC isoforms was measured using an enzyme-linked immunoabsorbent assay (ELISA) (Calbiochem, La Jolla, CA) based on the ability of endogenous PKC to phosphorylate the provided pseudosubstrate peptides on the serine residue. This assay was carried out according to manufacturers instructions. Briefly, a reaction mixture of 25 mM Tris-HCI, pH 7.0, 3 mM MgCI2, 0.1mM ATP, 2 mM CaCl2, 50 pg/mL phosphatidylserine, 0.5 mM EDTA, and 1 mM 13-mercaptoethanol was prepared and 108 L of this reaction mixture was added to an uncoated polyvinyl plate and pre-incubated at 25°C for 5 minutes. 12 1iL of the sample (whole cell lysates, membrane, or cytosolic fractions obtained as described above) and PKC standards were added to each well and mixed. Each sample was tested in duplicate. 100 tL of the 120 pL reaction mixture was transferred to a synthetic PKC pseudosubstrate-coated well with a multi-channel pipettor and incubated for 15 minutes at 25°C. The plate was washed five times 62 with PBS, treated with 100 tL of a biotinylated antibody, incubated for 60 minutes at 25°C, and washed again. The plate was then treated with 100 iL of peroxidase-conjugated streptavidin and incubated for one hour. The wash was repeated after incubation and 100 pL of a substrate solution (o-phenylenediamine) was added to react to the peroxidase. 100 pL of stop solution was added after 3-5 minutes and the plate was read at 492 nm by a microplate reader. The PKC activity was presented in units equivalent to ng of active PKC/mL adjusted to total protein, which was determined by the Bradford Assay (Bio-Rad Laboratories, Hercules, CA) as described below. 3.11 Protein Assay Protein concentration was determined by the Bradford Protein Assay. In a 96 well plate, 40 1tL of Bradford Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA) was added to each well. Standards were prepared using BSA (Bovine Serum Albumin I mg/mL) and 5 pL of sample were added to the wells. Water was added to the wells for a total volume of 160 1tL. The plate was read at 595 nm at 40X dilution by a microplate reader. 3.12 Western Blotting Purified whole cells, cytosolic and membrane fractions from healthy and LAP subjects were lysed by boiling with SDS-PAGE sample buffer (0.5 M Tris-HCI [pH 6.8], 10% glycerol, 10% SDS, 5% 3-mercaptoethanol, 0.1% bromophenol blue) for five minutes to denature the protein. The samples and a protein ladder (Bio-Rad Laboratories, Hercules, CA) were loaded and separated by SDS-PAGE on 9.0% polyacrylamide gels at 150 V for 85 minutes. The separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane utilizing TB Buffer (25 mM Tris, 192 mM glycine, 20% methanol; pH 8.4) at 100 V for one hour. 63 After protein transfer and blocking with TBST [20 mM Tris-HCI (pH 7.5), 250 mM NaCI, 0.10% Tween 20], supplemented with 5% skim milk for one hour at room temperature, the blocking solution was discarded and the membranes were incubated with the primary antibody against phosphorylated antibodies of PKC a, (31, (32, , and as listed earlier. The primary antibodies were diluted in TBST supplemented with 1% skim milk overnight at 4°C with different dilution factors [pPKC a (1 :20,000); pPKC (31 (1:1000); pPKC (32 (1:1000); pPKC ö (1:1000); pPKC (1:1000); 47Ph0x (1:1000); pPKC Substrate (1:1000)]. After incubating overnight, the membranes were washed three times (10 minutes/wash) with TBST and incubated with the secondary antibody, anti-rabbit lgG conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA), diluted in TBST for one hour at room temperature. Membranes were washed again three times (10 minutes/wash) with TBST. Detection of HRP activity was performed with SuperSignal® West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) by incubating the membranes for five minutes at room temperature followed by autoradiography. The amount of each form of phosphorylated PKC isozyme was quantified by densitometric scanning. In some experiments the effect of RvE1 on phosphorylation of PKC isoforms was determined. In these experiments PMNs were treated with 1 nM RvE1 and stimulated with fMLP. The cells were harvested and Western blotting carried out as described above. 3.13 Statistical Analysis All experiments were run in duplicate. Comparisons of the mean outcomes between groups were assessed using the two-sample t-test, with either the equal or unequal variance test depending on which was more appropriate for a particular outcome. Within group comparisons between time points were assessed using the paired t-test. Both raw and adjusted p-values were computed for each outcome; the adjusted p-values represent p-values adjusted for the multiple comparisons made for that particular outcome. Adjusted p-values were computed fro 64 multiple comparisons using the Hoim-Sidak Stepdown Method (308), which is similar to the Bonferroni correction, but less conservative. A type I error rate (significance level) of 0.05 has been used for all outcomes and for all comparisons 95% confidence intervals on the differences between groups or time points have been presented. 65 CHAPTER FOUR: RESULTS 66 4.1 Superoxide Generation in Stimulated Neutrophils Previous studies have demonstrated that PMNs from LAP patients exhibit a marked increase in diacyiglycerol in both stimulated and unstimulated cells when compared to PMNs from healthy individuals. This increase was associated with a decrease in DG kinase activity (309). As PKC is an important enzyme for triggering 02, the generation of O2 in PMNs from LAP patients and healthy controls was investigated. In order to characterize superoxide generation in response to stimuli (1 1iM fMLP), in the cohort being studied, superoxide anion production was measured in a subset of subjects using a SOD inhibitable cytochrome C reduction assay. Neutrophils treated with PBS alone were used to define the baseline level of 02 in the system. Neutrophils from 4 LAP patients and 4 healthy individuals were isolated and tested for their capacity to generate superoxide. Superoxide generation from unstimulated neutrophils was significantly higher in LAP subjects compared to healthy subjects (p<0.05, Figure 7), confirming earlier published work. This finding suggests that neutrophils from LAP patients are constitutively primed. These results confirm that neutrophils from LAP subjects exhibit greater Ojgeneration than neutrophils from healthy subjects. 67 5* Z 4 0 ‘— 3 2 .nofMLP - HfMLP E * Healthy LAP Figure 7. Superoxide release from healthy and LAP PMN PMN generation of superoxide before and after fMLP (1 pM) activation. The data represent the mean and standard deviation from 4 pairs of subjects (*p<O.05 for healthy vs. LAP comparison and between unstimulated and fMLP-stimulated PMN within healthy and LAP groups). 68 4.2 Characterization of PKC Activity in Neutrophils It has been well established in the literature that superoxide generation in neutrophils is regulated by the activity of NADPH oxidase. PKC is an important enzyme associated with the phosphorylation of p47PiX, a subunit of the NADPH oxidase complex, suggesting that the events associated with neutrophil activation require PKC. In order to determine whether increased superoxide generation is accompanied by PKC activation, an ELISA activity assay was performed on unstimulated neutrophils from healthy and LAP subjects. The PKC activity kit consisted of an immobilized pseudosubstrate affixed to the bottom of pre coated wells. PKC in the samples catalyzed the phosphorylation of a serine on the pseudosubstrate (samples was compared to a known standard, i.e. purified PKC, which created the standard curve). A biotinylated antibody bound to the phosphorylated substrate and the subsequent addition of a peroxidase-conjugated streptavidin bound to the antibody. A fluorogenic substrate was added to produce a visible signal and the optic density (OD) was read at 492 nm in a microplate reader. Neutrophils from 8 healthy and 8 LAP individuals were isolated and lysed and total PKC activity was measured in whole cell lysates. There was no statistically significant difference in total PKC activity between LAP and healthy neutrophils (Figure 8). However, it is possible that differences may be found when the cytosol and membrane fractions of neutrophils from healthy and LAP subjects are tested separately. 69 D100 a 0 0) 1 > C.) C) 0 80 60 40 20 0 Healthy • Healthy •LAP Figure 8. Total PKC activity in whole cell lysates in healthy and LAP PMN Whole cell lysates from 8 healthy and 8 LAP PMN were prepared. PKC activity was measured using an ELISA-based PKC kit. The O.D. was measured at 492 nm in a microplate reader. The results represent the mean and standard error of PKC activity4g protein. There was no statistically significant difference in PKC activity between healthy and LAP neutrophils. LAP 70 Since PKC was not different, the activation of PKC in the membrane and cytosol was investigated. To determine whether differences existed in PKC activity within the membrane and cytosol of healthy and LAP subjects, unstimulated neutrophils were lysed and cytosolic and membrane fractions were isolated and tested for PKC activity by ELISA. A total of 8 LAP patients and 8 healthy patients were evaluated. PKC activity was numerically greater in the membrane fraction of LAP neutrophils; conversely, PKC activity was numerically greater in the cytosol of healthy neutrophils (Figure 9). The PKC data for cytosolic and membrane fractions were consistent with the results from the whole cell lysates seen in Figure 8. 71 1.0 Figure 9. PKC in cytosol and membrane from healthy and LAP PMN Cytosolic and membrane fractions of PMN from 8 healthy and 8 LAP subjects were isolated. PKC activity was measured using a PKC activity kit based on an ELISA. The results represent the mean and standard error of PKC activity/rig protein. There was greater total PKC activity in the membrane of LAP PMN than healthy PMN. Conversely, higher PKC activity was detected in the cytosol of healthy PMN compared to those from healthy PMN. However, statistical analysis showed that these differences were not statistically significant. D C 0 0 > > C) 0 0 0.8 0.6 OA 0.2 0 • Healthy •LAP Cytosol Membrane 72 4.3 Kinetics of PKC Activity In order to determine whether there are differences in the kinetics of PKC activation, neutrophils from 4 LAP patients and 6 healthy controls were stimulated with 1 iM fMLP for 30, 60, 300, and 600 seconds. Reactions were stopped with ice cold PBS and cells were fractionated into cytosolic and membrane samples. PKC activity in the cytosol as assayed by ELISA decreased slightly within 10 minutes from t=0 (Figure 10). Additionally, neutrophils from healthy subjects exhibited numerically higher PKC activity than those found in LAP. In the membrane fraction (Figure 11), the LAP group demonstrated numerically higher levels of PKC activity than the healthy group, with both groups showing a slight increase in PKC activity upon stimulation. Differences between healthy and LAP groups in both the cytosolic and membrane fractions were not statistically significant. Although it could be assumed that stimulation caused PKC to translocate from the cytoplasm to the membrane, our results were not remarkable. 73 PKC Activity in Cytosolic Fractions 1.0 0.8 0 006 —— Healthy -, 0 100 200 300 400 500 600 700 Time (a) Figure 10. PKC in cytosol from healthy and LAP PMN upon stimulation Kinetics of PKC activation in 6 healthy and 4 LAP subjects was determined by stimulating PMN with fMLP (1 tM) for 30, 60, 300, and 600 seconds. Cell fractions were then isolated. PKC activity was measured using an ELISA kit. The results represent the mean and standard error of PKC activity/pg protein. At rest, PKC activity in cytosolic fractions of healthy subjects was numerically higher than those of LAP subjects and decreased with time. However the differences in PKC activity in the cytosol between healthy and LAP subjects were not statistically significant. 74 PKC Activity in Membrane Fractions 0.6 03 _____________________ Healthy 0.2 _ __ __ _ _ < 0.1 0 °- 0 0 100 200 300 400 500 600 700 Time(s) Figure 11. PKC in membrane from healthy and LAP PMN upon stimulation Kinetics of PKC activation in 6 healthy and 4 LAP subjects was determined by stimulating PMN with fMLP (1 1tM) for 30, 60, 300, and 600 seconds. Cell fractions were then isolated. PKC activity was measured using an ELISA kit. The results represent the mean and standard error of PKC activity/fig protein. At rest, PKC activity in membrane fractions of LAP subjects was numerically higher than those of healthy subjects. PKC activity increased over time and subsequently decreased. These differences in PKC activity in the membrane between healthy and LAP subjects were not statistically significant. 75 4.4 Phosphorylation of PKC Isoforms in Whole Cell Lysates The PKCs comprise a family of isoforms displaying differential cofactor requirements for activation. I tested the hypothesis that the changes in PKC activity occurred in one or more of the isoforms. Kinetic patterns of PKC isoforms in whole cell lysates were determined by stimulating neutrophils with fMLP (1 M) for 30, 60, 300 and 600 seconds, lysing the cells as above and immunoblotting to detect specific PKC isoforms. Antibodies recognizing the phosphorylated form of the various PKC isoforms were used to determine isoform activity in the samples, thus indicating isoform activation over time. Readings from the densitometric scanning of band intensities were normalized against the housekeeping gene 13-actin and expressed relative to the unstimulated healthy sample as a fold increase or decrease. 44.1 Whole Cell Lysates pPKCa: At rest, PMN from 5 LAP subjects exhibited numerically greater pPKCa compared to PMN from 6 healthy subjects. Upon stimulation, these levels increased slightly while pPKCa in healthy PMN remained approximately the same (Figure 12). The differences between the two groups were not statistically significant. jPKCI31: Numerically greater levels of pPKCI31 were observed in 4 LAP PMN from LAP patients than PMN from 6 healthy individuals at rest. This difference in PKC31 activity between the two 76 groups was statistically significant (p<0.05). However, there was no statistically significant difference between the two groups after stimulation (Figure 13). pPKCI32: Phosphorylation of PKCI32 in neutrophils from 6 LAP patients was found to be numerically higher at rest than neutrophils from 6 healthy individuals and increased or peaked at 30 seconds before decreasing at 10 minutes. While the same pPKCI32 in PMN from healthy subjects also increased, it appeared to be slower and peaked at 300 seconds before decreasing (Figure 14). The differences were not statistically significant. pPKCÔ: Statistically significant differences were found in the pPKCÔ levels at rest between PMN from 7 healthy and 4 LAP subjects (p<0.05). Numerically greater levels of pPKCÔ were found in PMN from LAP subjects than in PMN from healthy subjects after stimulation. These findings were not statistically significant (Figure 15). pPKC: While pPKC in samples from 6 LAP patients was numerically greater at rest than samples from 6 healthy individuals (p<0.05), upon stimulation, no difference was found between the groups (Figure 16). Results from immunoblotting with phosphorylated PKC isoform antibodies consistently showed that in whole cell lysates, levels of pPKC a, 131, 132, ô, and in LAP PMN were numerically higher than healthy PMN at rest. Statistically significant differences were found for phosphorylated PKC 131, ö, and , suggesting that the neutrophils are primed in subjects with 77 LAP. Stimulation with fMLP did not reveal any consistent translocation pattern within or between healthy and LAP PMN and no statistically significant differences were observed. 78 Healthy LAP pPKCct — -actin 0 30 60 300 600 0 30 60 300 600 15 —‘—Healthy 1.0 0 ______________ LL 0.5 0.0 I I I 0 100 200 300 400 500 600 700 Time (s) Figure 12. Phosphorylation of PKCa in whole cell lysates PMN from 6 healthy and 5 LAP subjects were stimulated with fMLP (1 1iM) for 30, 60, 300, and 600 seconds. Whole cell lysates were analyzed by SDS-PAGE, Western blotting with phosphorylated antibodies, and autoradiography. Antibodies against phospho-PKCa were used to detect the active PKC isoform in whole cell lysates of PMN. 13-actin was used to control and normalize for the amount of protein loaded on the gel. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. There was no statistical difference between the two groups. 79 pPKC 1 Healthy LAP (3-Actin — — 2.0 a) 1.5 ID 1.0 -o 0 0.5 0.0 0 30 603006000 30 60 300 600 2.5 0 100 200 300 400 500 800 700 Time (s) —- Healthy - LAP Figure 13. Phosphorylation of PKCI3I in whole cell lysates PMN from 6 healthy and 4 LAP subjects were stimulated with fMLP (1 1iM) for 30, 60, 300, and 600 seconds. Whole cell lysates were analyzed by SDS-PAGE, Western blotting with phosphorylated antibodies, and autoradiography. Antibodies against phospho-PKC31 were used to detect the active PKC isoform in whole cell lysates of PMN. 13-Actin was used to control and normalize for the amount of protein loaded on the gel. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t0. PKCI3I activity was higher at rest in PMN from LAP subjects than PMN from healthy subjects (p<0.05). 80 Healthy LAP pPKCI32 13—Acti n - — 0 30 60 300 600 0 30 60 300 600 i:: 0 100 200 300 400 500 600 700 Time (s) Figure 14. Phosphorylation of PKC2 in whole cell lysates PMN from 6 healthy and 6 LAP subjects were stimulated with fMLP (1 pM) for 30, 60, 300, and 600 seconds. Whole cell lysates were analyzed by SDS-PAGE, Western blotting with phosphorylated antibodies, and autoradiography. Antibodies against phospho-PKC32 were used to detect the active PKC isoform in whole cell lysates of PMN. 13-Actin was used to control and normalize for the amount of protein loaded on the gel. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. The findings for differences in pPKCf32 between PMN from healthy subjects and LAP subjects were not statistically significant. 81 30 2.5 0.5 0.0 Healthy LAP t3-Actin — 0 30 60 300 600 0 30 60 300 600 —‘.— Healthy —.— LAP Figure 15. Phosphorylation of PKCS in whole cell lysates PMN from 7 healthy and 4 LAP subjects were stimulated with fMLP (1 1iM) for 30, 60, 300, and 600 seconds. Whole cell lysates were analyzed by SDS-PAGE, Western blotting with phosphorylated antibodies, and autoradiography. Antibodies against phospho-PKCÔ were used to detect the active PKC isoform in whole cell lysates of PMN. 13-Actin was used to control and normalize for the amount of protein loaded on the gel. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. At rest, PKCÔ activity in PMN from LAP subjects was greater than in PMN from healthy subjects (p<0.05). pPKC ó a - € 4. a 2.0 0 100 200 300 400 500 600 700 Time (s) 82 Healthy LAP pPKC 13—Acti n — — — 0 30 60 300 600 0 30 60 300600 2.0 1.5 ___ 0 __ __ ________________________________________ Healthy —.—LAP 0 0LL 0.5 0.0 0 100 200 300 400 500 600 700 Time (s) Figure 16. Phosphorylation of PKCC in whole cell lysates PMN from 6 healthy and 6 LAP subjects were stimulated with fMLP (1 tM) for 30, 60, 300, and 600 seconds. Whole cell lysates were analyzed by SDS-PAGE, Western blotting with phosphorylated antibodies, and autoradiography. Antibodies against phospho-PKC were used to detect the active PKC isoform in whole cell lysates of PMN. 13-Actin was used to control and normalize for the amount of protein loaded on the gel. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. Results showed that at rest, there was statistical significance between PMN from LAP subjects and PMN from healthy subjects for pPKC (p<0.05). 83 To determine whether differences existed in PKC isoform activity within the membrane and cytosol of healthy and LAP subjects, a kinetic analysis was carried out. The kinetic patterns of PKC isoforms in cytosolic and membrane fractions were determined by stimulating neutrophils with fMLP (1 M) for 30, 60, 300 and 600 seconds, lysing and fractionating the cells, and using immunoblotting for the detection of specific PKC isoforms as above. Antibodies to the phosphorylated PKC isoforms were used to determine isoform activity in the samples, thus indicating the isoform translocation patterns over time. Numerical figures from the densitometric scanning of band intensities were normalized against the housekeeping gene f3-actin and expressed relative to the unstimulated healthy sample as a fold change. 4.4.2 Cytosol and Membrane Fractions pPKCa: In cytosol and membrane fractions, greater numerical values of pPKCcL were found at rest in PMN from 3 LAP subjects than PMN from 3 healthy subjects. However, no statistically significant differences in the activity of PKCa resulted within the cytosol and membrane fractions and between the two groups (Figure 17). rPKC61: Immunoblotting after the isolation of cytosol and membrane fractions revealed minimal or undetectable amounts of pPKCI3I in the cytosol of PMN of 7 healthy and 6 LAP subjects (data not shown). In the membrane fractions, there was approximately a 1.3 fold increase of pPKCI31 at rest in PMN from LAP patients compared to PMN from healthy individuals (p<0.05). Subsequent stimulation with fMLP did not reveal any statistically significant differences in the translocation patterns of pPKC31 (Figure 18). 84 pPKCI32: Phosphorylated PKCI32 was not found in the membrane of PMN from 10 healthy and 8 LAP individuals (data not shown). In the cytosol, while pPKCI32 at rest was detected at a 1.5 fold numerically higher level in PMN from LAP patients compared to PMN from healthy patient, these results were not statistically significant. Upon stimulation, there were no differences detected in the activity of PKC2 (Figure 19). pPKCÔ: Results for pPKCÔ revealed a 2.2 and 2.3 fold increase in isoform levels at rest in PMN from 3 LAP patients versus PMN from 3 healthy patients in cytosol and membrane fractions respectively (p<0.05). After stimulation with fMLP, there was no difference in the pattern of translocation of the isoform between healthy and LAP groups in the cytosol and membrane (Figure 20). pPKC: Tests of this isoform revealed that PMN from 9 healthy and 7 LAP individuals did not express pPKC in the cytosol (data not shown). However, in the membrane, there was a 2.2 fold increase of pPKC at rest in PMN from LAP patients (p<0.05). After stimulation with fMLP, there was no difference in the pattern of translocation of pPKC in the cytosol and membrane between PMN from healthy and LAP groups (Figure 21). The findings show that upon stimulation with fMLP, PMN from both healthy and LAP patients exhibited the same activation of PKC isoforms. However, the amount of phosphorylated PKC 131, ô, and at rest in the LAP group was greater than in the healthy group. These differences were statistically significant (p<0.05). 85 pPKCct Cytosol Membrane (3-Actin Healthy LAP — — — — — — — —— ——— — 0 30 60 300 600 0 30 60 300 600 pPKCci ir Cytosol — Healthy —.— LAP 0 100 200 300 400 500 600 700 Time (s) pPKCa in Membrane 12.0 10.0 D . 8.0 6.0 4.0 IL 2.0 0.0 3.5 3.0 D a 0 c 2.0 a 1.5 D 1.0 0LL 0.5 0.0 Time(s) Figure 17. Phosphorylation of PKCa in cytosol and membrane PMN were stimulated with fMLP for 30, 60, 300, and 600 seconds. Cells were lysed and cytosolic and membrane fractions isolated. Detection of pPKCct was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. Results demonstrate that there were higher levels of pPKCa in the cytosol and membrane fractions from PMN from 3 LAP patients than PMN from 3 healthy individuals. Results for pPKCcL were not statistically significant between the two groups. —.—- Healthy —.—LAP 0 100 200 300 400 500 600 700 86 Figure 18. Phosphorylation of PKC1 in membrane PMN were stimulated with fMLP for 30, 60, 300, and 600 seconds. Cells were lysed and cytosolic and membrane fractions isolated. Detection of pPKC31 was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. There were minimal levels of pPKC31 in the cytosol of PMN from 6 LAP and 7 healthy individuals. Consequently this data was not included. The majority of pPKCI31 was found in the membrane fraction from both groups. At rest, the levels of phosphorylation of PKC31 were found to be statistically higher in membranes of PMN from LAP patients than in membranes of PMN from healthy individuals (p<0.05). pPKC31 Healthy LAP Membrane 0 30 60 300 600 0 30 60 300 600 2.5 2.0 1.5 1.0 0.5 0.0 —.-— Healthy LAP 100 200 300 400 500 600 700 Time (a) 87 pPKCf32 Healthy LAP Cytosol — 3-Actin — 0 30 60 300 600 0 30 60 300 600 Healthy ‘- 0.5 0.0 0 100 200 300 400 500 600 700 Time(s) Figure 19. Phosphorylation of PKC2 in cytosol PMN were stimulated with fMLP for 30, 60, 300, and 600 seconds. Cells were lysed and cytosolic and membrane fractions isolated. Detection of pPKCj32 was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. There were minimal levels of pPKCI32 in the membrane of PMN from 8 LAP and 10 healthy individuals. Consequently the membrane data was not included. The majority of pPKCI32 was found in the cytosol fraction from both groups. Phosphorylation of PKCf32 was found to be numerically greater in the cytosol of PMN from LAP patients compared to PMN from healthy individuals. These results were not statistically significant. 88 pP KCÔ D C C 0 0 LL 4.0 C 0, 0 2.0 Healthy LAP Cytosol — — — — Membrane -. 13-Actin — — — — — 0 30 60 300 600 0 30 60 300 600 pPKC in Cytosot 2.5 2.0 1.5 1.0 0.5 0.0 6.0 Time (s) 0 100 200 300 400 500 600 700 pPKCi in Membrane Healthy —.—LAP Healthy —--LAP 0.0 700 Figure 20. Phosphorylation of PKC8 in cytosol and membrane PMN from LAP patients and healthy individuals were stimulated with fMLP for 30, 60, 300, and 600 seconds. Cells were lysed and cytosolic and membrane fractions isolated. Detection of pPKCÔ was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. There were higher levels of pPKCÔ in the cytosol and membrane fractions at rest in PMN from 3 LAP patients than in PMN from 3 healthy individuals (p<O.05). 0 100 200 300 400 500 600 Time Cs) 89 pPKCt D 1.5 C ‘1, -o 0 LJ Healthy LAP Membrane 0 30 60 300 600 0 30 60 300 600 2.5 2.0 0 100 200 300 400 500 600 700 —‘--Healthy —.—LAP 05 0.0 lime (s) Figure 21. Phosphorylation of PKC in membrane PMN were stimulated with fMLP for 30, 60, 300, and 600 seconds. Cells were lysed and cytosolic and membrane fractions isolated. Detection of pPKC was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from the healthy individuals at t=0. There were minimal levels of pPKC in the cytosol of PMN from LAP and healthy individuals. Consequently the cytosol data was not included. The majority of pPKC was found in the membrane fraction from both groups. At rest, numerically higher levels of pPKC were observed in membrane of PMN from 7 LAP patients than the membranes of PMN from 9 healthy individuals. These results were statistically significant (p<0.05). 90 4.5 Quantitative Assessment of PKC Expression in Neutrophils In LAP PMN, there is a select activation of certain PKC isoforms at rest. To further investigate this observation, PKC isoform expression was investigated at the transcription level. Cells were lysed, RNA was extracted, and cDNA was synthesized via reverse transcription. Real-time fluorescence-based PCR was used to quantitatively assess the expressed PKC isoforms in healthy and LAP neutrophils. The results presented in Figure 22 confirmed the expression of PKC isoforms a, 31, 32, ö, and in neutrophils from healthy and LAP subjects. The data also revealed that there were no differences in the expression of these isoforms between neutrophils from 8 healthy and 8 LAP individuals. Quantitation of results were expressed using the Relative CT Method, i.e. expressed as 2&CT This calculation method was applied as it analyzed changes in gene expression of PKC in a sample relative to another reference sample, i.e. the healthy sample at rest. The samples were normalized to the housekeeping gene, f-actin. In order to evaluate for possible variations in the expression of PKC isoforms upon stimulation with an agonist, this experiment was repeated with the modification of adding fMLP to the neutrophils. Cells were incubated with PBS or 1 pM fMLP for 30, 60, 300, and 600 seconds and lysed for RNA extraction. Real-time PCR showed that there were no significant differences in the levels of mRNA PKC isoforms in 5 healthy and 5 LAP groups after stimulation (Figure 23). 91 2 CT 1.6 1.4 1.2 0 10L 08 • Healthy •LAP 0.6 0.4 0.2 0.0 Figure 22. Expression of PKC isoform transcripts in healthy and LAP PMN The expression of PKC isoform transcripts from PMN of 8 healthy and 8 LAP subjects was assessed using RT-PCR. The results represent the mean and standard error of the 2T method for calculation of relative changes in gene expression. The results confirmed that five isoforms were expressed in human neutrophils, a, 131, 132, ö, and . No statistically significant differences were observed in the expression of PKC isoform transcripts between PMN from healthy and LAP subjects. Alpha Beta I Beta 2 Delta Zeta PKC Isoforms 92 PKCa5- , 1 .5 • Healthy C . 1.0 .LAP C-) -D 0.5 - 0.0 0 30 60 300 600 Time (s) PKC f31 D 1.5 o • Healthy 1.0 • LAP 0.5 0 C u.. 0 30 60 300 600 Time (s) PKC 2 1.5 1 0 • Healthy ‘LAP(13 05(_) . •6 LiLL 0 30 60 300 600 Time (s) 93 PKC D 1 5 • Healthy ) •LAP c 1.0 -c 0.5 0LL 0 30 60 300 600 Time (s) PKC D 1.5 (1.0 : Healthy LL 0 30 60 300 600 Time (s) Figure 23. Expression of PKC a, 31, 132, 8, and transcripts upon stimulation PMN from healthy and LAP patients were stimulated with fMLP (1 tM) for 30, 60, 300, and 600 seconds. Expression of PKC isoform transcripts was detected by RT-PCR. The graphs are a representative sample and express the fold change of the mean and standard error of the 2MCT determination of relative changes in gene expression. There was no difference in expression between healthy and LAP groups. 94 4.6 Phosphorylation of p47X in Healthy and LAP Neutrophils Characterization of p47P10)<, the initial protein of NADPH oxidase activation and the target of PKC, was assessed. The neutrophils of healthy and LAP individuals were stimulated with 1 pM of fMLP. The protein fraction was prepared as above, and immunoblotting performed with a phospho-specific antibody. The results revealed that the phosphorylation ofp47Ph10)< was greater in PMN from 5 LAP patients than in PMN from 5 healthy individuals upon stimulation (Figure 24). These differences were statistically significant (p<O.05). 95 >, c. >L — 0 G) 0. 0 Healthy LAP fMLP (—) (+) (—) (+) p47PWx pPKC(S) * 0.5 0.4 0.3 ‘Healthy ‘LAP 0.2 0.1 0 (—) Figure 24. fMLP-induced p47X phosphorylation is enhanced in PMN from LAP patients PMN from 4 healthy and 4 LAP subjects were stimulated with fMLP (0.1 1tM) for 1 mm. The results represent the mean and standard error of the densitometric readings of Western Blots from stimulated and unstimulated PMN. Samples stimulated with fMLP demonstrated greater levels of phosphorylated p47Ptl0X in PMN from 5 LAP patients than in PMN from 5 healthy subjects (p<0.05). fMLP, 100 nM, 1 mm 96 4.7 Inhibition of PKC Isoforms To further understand the relationship between PKC and superoxide generation, neutrophils were incubated with specific PKC inhibitors to determine the role of each isoform in the production of superoxide using the superoxide dismutase inhibitable cytochrome C reduction assay as described in Methods & Materials. Commercially available inhibitors of Classical (a, 131, and 132), Beta ((31 and 132), Delta, and Zeta PKC were used. Incubating neutrophils with the Classical PKC inhibitor resulted in the greatest inhibition of superoxide generation, followed by Zeta, Delta, and Beta in fMLP stimulated PMN5 (Figure 25). Comparisons between healthy and LAP groups, revealed that healthy neutrophils showed greater inhibition of superoxide production than LAP neutrophils. Statistically significant differences between healthy and LAP groups were found with the Classical PKC inhibitor and Zeta inhibitor, implying that these isoforms play a greater role in LAP PMN priming. Similarly, PKC may also influence the enhanced superoxide generation in LAP subjects. 97 601 * C 0 • Healthy •LAP Figure 25. Inhibition of superoxide production with the use of PKC inhibitors PMN from healthy and LAP subjects were incubated with PKC inhibitors targeted towards classical PKC isoforms, PKCf3, PKCÔ, and PKC and tested for superoxide production. The results represent the mean and standard error of the percent inhibition of superoxide production. Samples incubated with inhibitors resulted in greater inhibition of superoxide production (i.e. less superoxide generation) in PMN from healthy PMN5 compared to PMN from LAP patients. Statistically significant differences were found between groups, particularly with the classical PKC inhibitor and the PKC inhibitor (p<O.05). 40 20 classical beta delta zeta 98 4.8 Inhibition of Superoxide in Neutrophils Incubated with RvEI There is considerable interest in lipid mediators involved in the resolution of inflammation. One such mediator 1 8R-resolvin (RvE1) is of interest as a potential therapeutic modality for the treatment of periodontal disease. Previous work from the van Dyke lab established the relationship between RvE1 and superoxide generation in healthy and LAP neutrophils (data not shown). In this study, PMN from three healthy individuals and three LAP patients were incubated with various concentrations of RvE1 (1013 — 106 nM) and stimulated superoxide generation from these cells was assessed. Results revealed that RvE1 inhibited superoxide generation from both LAP and healthy cells by approximately 80 and 65% respectively (Figure 26). 99 0 .4-. I a) a) a, Figure 26. Inhibition of superoxide generation with RvEI PMN from healthy and LAP subjects were incubated with various concentrations of RvE1. Using the superoxide generation assay, levels of superoxide were measured against samples that were not incubated with RvE1. Data represent the means ± SEs of the percent inhibition of superoxide generation from neutrophils of three healthy individuals and three patients with LAP (299). RvE1 inhibited superoxide generation from both healthy and LAP cells by —80% at the highest concentration (p<0. 05). Healthy PMN o LAPPMN100 80 60 40 20 0 0 10-13 10-12 10-” 10-° 10-s 10-8 10-i 1O- RvEI (M) 100 4.9 Phosphorylation of PKC isoforms in neutrophils incubated with RvEI As RvE1 had such pronounced effect on superoxide generation, it was of interest to determine whether phosphorylation of PKC isoforms was altered by RvE1. Therefore, neutrophils from six healthy and LAP subjects were incubated with PBS or 1.0 nM RvE1 for 15 minutes and subsequently stimulated with 1 iM of fMLP for 30 and 60 seconds. The whole cell lysates were analyzed by SDS-PAGE, Western blotting with antibodies against phospho-PKC a, 131, 132, ö, and , autoradiography, and densitometric scanning. 13-actin was used to control and normalized for the amount of protein loaded on the gel. pPKCa: Neutrophils that were incubated with RvE1 did not exhibit a difference in the level of phosphorylation of PKCa from those that were incubated with PBS. This finding was observed in both healthy and LAP groups (Figure 27). rPKC61: Similar observations were found with the pPKCI31 in both healthy and LAP samples. There was no difference in PKCI31 activity between neutrophils that were incubated with RvE1 and those that were not (Figure 28). pPKCI32: In the healthy group, RvE1 did not appear to alter the phosphorylation of PKCI32. In contrast, neutrophils from the LAP group treated with RvE1 exhibited a decrease in the phosphorylation of PKC132 over time. This difference was not statistically significant (Figure 29). 101 pPKC& Healthy cells incubated with RvEI did not demonstrate any significant changes in phosphorylation of PKC ó from healthy cells that were incubated with PBS. However, neutrophils from LAP subjects showed a decrease in pPKCÔ when treated with RvE1 versus neutrophils that were not treated with RvE1 (0.73 and 1.5 from baseline respectively), suggesting that RvE1 inhibits the phosphorylation of PKC in neutrophils from subjects with LAP (Figure 30). The difference between these findings is statistically significant (p<0.05). pPKC: Consistent with the other isoforms, RvE1 had no effect on healthy neutrophils. There was no difference observed in the phosphorylation of PKC in cells that were and were not incubated with RvE1. In LAP neutrophils, RvEI appeared to suppress the phosphorylation of PKC by 0.51 fold of the baseline compared to 1.26 of the baseline from neutrophils that were not incubated with RvE1 (p<0.05). These results suggest that RvEI suppresses phosphorylation of PKC in LAP neutrophils (Figure 31). The results of these experiments involving RvEI revealed that RvE1 had no effect on the phosphorylation of PKC a, 131, 132, ,, and in healthy neutrophils. However, RvE1 inhibited the phosphorylation of PKC ô, and in LAP neutrophils by a fold of 0.73 and 0.51 from their respective baseline. 102 pPKCu in Healthy Subjects Healthy (-RvEI) Healthy (+RvE1) Figure 27. Phosphorylation of PKCa in PMN treated with RvEI PMN from 5 healthy and 5 LAP subjects were incubated with RvE1 (1 nM) for 15 minutes and stimulated with fMLP (1 M) for 30 and 60 seconds. Phosphorylated PKC a was determined by western blotting. The resufts represent the mean and standard error of the densitometric fold change from t=-l5min. No differences were observed in the amount of pPKCa in PMN in healthy and LAP groups incubated with and without RvE1. No statistically significant differences were found between groups. -15 mm ÷fMLP 30s 6Os 4.0 3.5 cr, 2.5 3) C -c o 1.5 C 0.5 0.0 3.0 __ 2.5 D 1) 0) C(U -c 0 LLO5 0.0 Ti me pPKCct in LAP Subjects a LAP(-RvE1) —i—LAP (-t-RvE1) -15 mm -‘-fMLP 30s Time 6Os 103 2.5 ..— 3.0 D 2.5 ci) 2.0 1.5 1.0 0 u-.5 0.0 pPKCI31 in Healthy Figure 28. Phosphorylation of PKCI3I in PMN treated with RvEI PMN from 4 healthy and 4 LAP subjects were incubated with RvE1 (1 nM) for 15 minutes and stimulated with fMLP (1 1iM) for 30 and 60 seconds. Phosphorylated PKC1 was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from t=-l5min. There were no differences in the amount of pPKC1 between PMN in both healthy and LAP groups incubated with and without RvE1. S2.0 ci) Cu o 1.0 0 0U- 0.5 0.0 3.5 -15mm +fM[P 30s 60s Time pPKC31 in LAP • Healthy (-RvE1) —•— Healthy (+RvEI) • LAP (-RvEI) —•— LAP (+RVE1) -15 mm +fMLP 30s 60s Time 104 pPKC12 in Healthy 3.0 - 0.5 0.0 Figure 29. Phosphorylation of PKC2 in PMN treated with RvEI PMN from 5 healthy and 4 LAP subjects were incubated with RvE1 (1 nM) for 15 minutes and stimulated with fMLP (1 1iM) for 30 and 60 seconds. Phosphorylated PKCI32 was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from t=-l5min. No statistical differences in phosphorylation of PKCI32 were observed between groups treated with and without RvE1. -l5miri +fMLP 30s 60s Time pPKC2 in LAP Healthy (-RvE1) Healthy (+RvEI) LAP(-RvE1) —•—LAP (-fRvEl) 1.6 1 .4 S1.2 1.0 0) C 0.6 0 0 IJ 0.2 0.0 -15 mm ÷fMLP 30s SOs lime 105 pPKC in Healthy Subjects 3.0 2.5 D 2.0 0 C C) 1.0 0.5 0.0 Figure 30. Phosphorylation of PKC8 in PMN treated with RvEI PMN from 5 healthy and 4 LAP subjects were incubated with RvEI (1 nM) for 15 minutes and stimulated with fMLP (1 1iM) for 30 and 60 seconds. Phosphorylated PKCó was determined by western blotting. The resufts represent the mean and standard error of the densitometric fold change from t=-l5min. There was no difference in phosphorylation of PKCÔ in PMN from healthy individuals untreated and treated with RvE1. However, in samples from LAP patients, RvE1 treated PMN exhibited decreased phosphoryation of PKCÔ compared to those not treated with RvE1, rendering statistical significance (p<0.05). - — — -15 mm +fMLP 30s Time 60s Healthy (-RvEI) —u— Healthy (+RvE1) • LAP (-RvE1) LAP (+RVEI) pPKCÔ in LAP Subjects 1.6 18 * 1.4 D 1.2 1.0 0.8 2 0.6 0 Li.. 0.2 0.0 -15mm +fMLp 30s SOs Time 106 pPKC in Healthy Subjects 1.8 1.6 1.4 <1.2 D) 1.0 a Healthy (-RvE1) —a— Healthy (+RvE1) —‘---LAP(-RVEl) —•—LAP (+RVE1) Figure 31. Phosphorylation of PKC in PMN treated with RvEI PMN from 5 heafthy and 5 LAP subjects were incubated with RvE1 (1 nM) for 15 minutes and stimulated with fMLP (1 riM) for 30 and 60 seconds. Phosphorylated PKC was determined by western blotting. The results represent the mean and standard error of the densitometric fold change from t=-l5min. There was no difference in phosphorylation of pPKC in PMN from healthy individuals untreated and treated with RvE1. However, in samples from LAP patients, RvE1 treated PMN exhibited decreased phosphorylation of pPKC compared to those not treated with RvE1, rendering statistical significance (p<0.05). --I-- -15mm 4fMLP 305 60s 0.4 0.2 0.0 1.6 1.4 1.2 0, 0.8 C.) . 0.6 0 ‘- 0.4 0.2 0.0 lime pPKCt in LAP Subjects -15 mm +fMLP 30s lime 107 4.10 Phosphorylation of p47X in Neutrophils Incubated with RvEI In this study, neutrophils from five healthy and five LAP subjects were incubated with PBS and various concentrations of RvE1 (0.1, 1, 10, and 100 nM) for 15 minutes. Upon stimulation with 1.0 p.M fMLP, samples of whole cell lysates were analyzed by SDS-PAGE, Western blotting with antibodies against phosphop47b0x, autoradiography, and densitometric scanning. 13-actin was used to control and normalize for the amount of protein loaded on the gel. Results revealed that in healthy subjects, various concentrations of RvE1 did not change the baseline phosphorylation ofp47P10)< in neutrophils. However, neutrophils from subjects with LAP that were incubated with various concentrations of RvE1 resulted in a greater percentage of inhibition of phosphorylation of p47X suggesting a reversal of the priming. At 100 nM, there was a 47% inhibition of p471)< phosphorylation (Figure 32). 108 0 Cu 0 0 Ci) a >< 0 1- 0 0 0 0 RvEI Does Not Inhibit p47phox Phosphorylation in Healthy Subjects 10 RvEI Concentration (nM) RvEI Inhibits p47phox Phosphorylation in LAP Subjects 80 60 40 20 0 -20 -40 80 0 Cu 160 0 0 20 -O 0 100 Figure 32. Inhibition of phosphorylation ofp47P10X in PMN treated with RvEI PMN from five healthy and five LAP subjects were incubated with RvE1 for 15 minutes and stimulated with 1 tM of fMLP. The results represent the mean and standard error of the densitometric readings of Western Blots from stimulated and unstimulated PMN. RvE1 did not suppress phosphorylation of p47Pubox in healthy PMN. In contrast, RvE1 suppressed phosphorylation of p47X in LAP PMN, with a 47% inhibition at a concentration of 100 nM (p<0.05). 0 0.1 1 10 RvEI Concentration (nM) 109 CHAPTER FIVE: DISCUSSION 110 In these studies, the role of PKC in priming of neutrophils from LAP was investigated. The major findings of these studies include the demonstration that specific isoforms of PKC are phosphorylated in resting LAP neutrophils, corresponding with increased translocation to the plasma membrane, phosphorylation of p471<, and increased superoxide generation. Priming of PMN from LAP patients at the level of PKC does not involve increased expression of PKC and upon complete activation of normal PMN; the PKC activity between the groups is indistinguishable suggesting that the priming is at the level of pre-activation of certain PKC isoforms in LAP. Investigation of potential therapeutic benefits of a new class of inflammation of resolution agonist revealed that the priming of LAP PMN at the PKC level can be reduced by RvE1 treatment of neutrophils in vitro. Localized aggressive periodontitis (LAP) is a clinical classification of one of the aggressive periodontal diseases (96). LAP is clinically characterized by the circumpubertal onset of periodontitis, rapid alveolar bone loss around only first permanent molar and incisor teeth and bleeding on probing at these local sites. Pathologically, LAP is a consequence of an inflammatory response to subgingival plaque. As this inflammatory response involves the recruitment of cells of both the innate and adaptive immune response,, production of inflammatory cytokines and activation of osteoclasts, the causes of the disease are probably multi-factorial. This has been well documented by a plethora of reports implicating abnormal and inappropriate modifications of the host’s inflammatory response in other diseases where periodontitis is expressed as a complication of the underlying disease. Reports have indicated problems with T regulatory cells, T helper cell bias, self and superantigen stimulated lymphocytes, and hyper-responsive neutrophils. These problems usually manifest themselves at the cellular level through disease conditions (i.e. diabetes), biased cytokine production, or production of enzymes (MMPs) and ROS species. These events almost always reflect an 111 inherent problem at the molecular level. As the neutrophil is both the earliest and the primary line of defense against bacterial and fungal challenges, it plays a significant role in initiation and progression of the inflammatory response. Upon activation, the neutrophil releases some of the contents of its cytoplasmic lysosomal granules into the extracellular environment. These granules contain many bioactive molecules that function in tissue destruction, replenishing cell surface receptors, and activating the complement pathway (310). The neutrophil is also capable of producing large quantities of ROS, causing oxidant-mediated injury (311). Finally, neutrophils amplify the inflammatory response by the production of pro-inflammatory cytokines (312, 313). The role of neutrophils in LAP is the focus of this investigation. PMN hyper-responsiveness in the generation of tissue damage in periodontitis has been recognized for over a decade. Specifically, studies have shown that LAP is associated with altered neutrophil function, such as reduced chemotaxis, reduced receptor expression, and enhanced production of superoxide (97- 99). Peripheral blood neutrophils from persons with chronic periodontitis generated significantly higher levels of ROS after in vitro Fcy receptor stimulation, compared with age- and gender- matched healthy controls (314). Similarly, unstimulated peripheral blood neutrophils of persons with LAP were hyper-responsive with respect to production of ROS generation, due to reduced expression of diacylglycerol kinase (DGK) (249). Consistent with the findings in the literature, our study confirmed that neutrophils from subjects with LAP generated greater levels of superoxide than neutrophils from healthy subjects, before and after stimulation with fMLP. This hyper-responsive phenotype of LAP neutrophils suggests that the cells are somehow pre-activated, or primed in LAP, implicating the signaling molecules 112 in the pathway for NADPH oxidase activation. Protein Kinase C has been identified as key molecule in the production of superoxide in neutrophils. Since the discovery of PKC in the late 1970s by Yasutomi Nishizuka and colleagues at Kobe University, PKC has emerged as an important enzyme in signal transduction. In addition to the finding that PKC transduces signals that cause lipid hydrolysis, it was also discovered to be the receptor for the potent tumor-promoting phorbol esters, thus placing PKC in the signaling pathway that regulates normal cell function and carcinogenesis. To date, ten mammalian isoforms of PKC have been identified in selective organ and tissues (315). The distribution of these isoforms often provides an initial indication of their functional roles. Several PKC isoforms have been implicated in the regulation of a variety of cellular processes such as proliferation, differentiation, and the release of hormones and neurotransmitters (316, 317). However, the presence of multiple isoforms in the same tissue or cell type creates a complexity in understanding isoform-specific signaling and function. In this study, an ELISA was used to characterize the PKC and its various isoforms at the protein level using a series of phospho-specific antibodies for the PKC isoforms. Results revealed that PKC activity levels were not changed in LAP. However, by lysing the neutrophil and isolating cytosolic and membrane fractions, it was demonstrated that PMN from LAP patients are pre activated as evidenced by the translocation of PKC to the plasma membrane. Using phospho specific isoform antibodies, it is clear that specific isoforms of PKC are responsible for the activation. This previously unreported observation implicates PKC 131, , and in the priming of LAP PMN. Interestingly, PMN from healthy individuals activate the same isoforms upon stimulation with fMLP implicating these isoforms in the normal activation pathway of the NADPH oxidase leading to superoxide generation. 113 The observed elevation in PKC activity in the membrane of neutrophils from LAP subjects may be explained by previously observations reporting elevated amounts of membrane bound DAG in PMN from LAP subjects. Previous research showed that impaired DGK activity in PMN from LAP subjects resulted in the upregulation of DAG in PMN (250, 251, 318). This finding has functional implications since classical and novel PKC isoforms are intracellular targets for the second messenger DAG, thereby activating PKC and phosphorylation of p47X (319-321). Activation of this pathway leads to NADPH oxidase complex assembly to produce a range of microbicidal reactive oxygen species, notably superoxide. This is consistent with the finding that LAP is associated with identifiable abnormalities of host cell function, including enhanced production of superoxide (97-99). Consistent to other studies, our research confirmed the presence of five PKC isoforms in human neutrophils: a, 131, 132, ö, and . However, upon isolating cytosolic and membrane fractions, certain isoforms were consistently detected in only one of the cell fractions. Specifically, PKCI31 was only found in the membrane, PKCI32 was in the cytosol, and PKC was in the membrane. PKC 131 and 132 originate by differential splicing and only differ in their 3’-ends with the PKC131 specific exon lying behind the PKC132 specific exon, separated by an intron of 4-5 kbp (322, 323). Our data conflicts with the assumption that if PKC 13 is expressed at all in a tissue or cell type, that one splice form is preferred. For our study, fMLP was selected as the agonist to activate PKC in healthy and LAP neutrophils. Using this G-protein coupled receptor agonist was important because PKC isoforms have different signaling pathways. For example, phorbol 12-myristate 13-acetate (PMA) as an agonist directly crosses the cell membrane and activates PKC by binding to the Cl 114 domains of classical and novel PKC isoforms; PMA does not activate atypical PKC isoforms (217, 218, 324, 325). Therefore, PKC isoforms were stimulated with 1 M IMLP for 30, 60, 300, and 600 seconds to examine the kinetic patterns in whole cells. Our data showed higher levels of PKC 131, , and in whole cells from LAP patients at rest (t=0) than in healthy subjects, suggesting that PKC 131, ö, and may play a relevant role for the primed state of the neutrophil in LAP. This was complemented by my subsequent finding that these same PKC isoforms were enhanced in the membrane of LAP neutrophils at rest. In addition, levels of pPKCa and pPKC in membrane and cytosol respectively were significantly greater in LAP subjects than in healthy subjects. Overexpression of PKC isoforms in other cell types has been observed in other inflammatory conditions or disease states. For example, alterations in PKC isoform expression and increased levels of DAG occur in psoriatic human epidermis suggesting that PKC plays a role in the pathophysiology of this disease (326, 327). Similarly, Wang et al found that overexpression of PKC a in the epidermis of transgenic mice resulted in striking alterations in phorbol ester- induced inflammation and COX-2, MIP-2 and TNF-cc expression (328), suggesting that PKCa in epidermal keratinocytes is important in the regulation of the expression of inflammatory mediators that result in edema and neutrophil infiltration. PKC activity via the DAG-PKC pathway is also associated with many vascular abnormalities in the retinal, renal, neural and cardiovascular tissues in diabetes mellitus (329-335). In vascular tissues from diabetic animals, importance has been placed on PKC isoforms, particularly PKC 13 and (333, 336). PKC 13 and ó were found to be pre-activated in the aorta (337, 338), kidney (339, 340), and heart of diabetic rats (337). Specifically, PKC a, 131, 132, and E isoforms were pre-activated in the retina (341) while PKC a, f31 and ó were pre-activated in the renal glomeruli 115 of diabetic rats (339, 340). In all the vascular tissues, PKC 13 expression was upregulated in the membrane compared to the other isoforms. Therefore, it is not unusual for the activity of PKC to be enhanced in some disease states. In LAP, we found PKC 131, ö, and to be significant isoforms and this finding warrants further investigation. Not only did the results show greater levels of PKC activity in the membrane of neutrophils from LAP subjects than healthy subjects, but also there were more PKC in the cytosol of the healthy group than the LAP group. To determine if this result was due to translocation or PKC synthesis, quantitative RT PCR was used to measure the PKC isoform transcripts. Quantitative RT PCR data concluded that there were no transcriptional differences between LAP and healthy PMN unless post-transcriptional efficiency is involved. In the synthesis of PKC isoforms, the observed PKC activity was a reflection of phosphorylation and translocation to the cell membrane. Any differences between LAP and healthy PMN were most likely due to priming of the cell and activation of the isoform itself. A similar finding was reported in another study examining expression of PKC isoforms ö, , , rj in normal and psoriatic adult human skin (326) where PKC was increased two-fold in psoriatic compared to normal skin, while levels of PKC ô, , and 1 remained unchanged. However, PCR analysis revealed that there were no significant differences in mRNA levels among all these PKC isoforms between normal and psoriatic skin. This supports our hypothesis that enhanced levels of PKC isoform activity observed in LAP neutrophils are a result of phosphorylation and translocation of pre-existing PKC. PKC is a multi-faceted enzyme family in which the regulation of the various isoforms is complex. Not surprisingly, our experiments examining the kinetic patterns of PKC isoforms when stimulated with IMLP were inconclusive in PMN from healthy and LAP subjects. There are 116 several possibilities to explain the wide variance in the data, not only between groups, but also within each group. Variables such as multiple cofactors, phosphorylation mechanisms, and interacting proteins are determinants that affect the signaling and phosphorylation of PKC isoforms. As discussed earlier in Chapter One, classical and novel PKC isoforms bind to DAG. This not only results in the strong affinity of PKC for phosphotidylserine (PS) (342, 343) but also gives rise to the selectivity of PKC for the PS head group among different phospholipids (237, 344, 345). Thus PKC is bound to the membrane through its cysteine-rich and C2 domains, anchored by DAG and PS respectively. However, studies now show that these domains are not the only regions involved in phosopholipid binding. There are regions on the pseudosubstrate domain (upon release of the pseudosubstrate from the active site) that may also contribute to membrane binding through its basic residues (237, 345-347). Murray et al discovered that phosphatidylglycerol stimulated PKC32 in the nuclear membrane, resulting in the phosphorylation of nuclear substrates (348). Additionally, other lipids have been found to stimulate PKC activity such as free fatty acids (349), short-chain phosphatidylcholine derivatives, lysophosphatidic acid, and phosphatidylinositol 3, 4, 5-triphosphate. These studies indicate that there is a possibility that individual PKC isoforms require different lipids for activation, increasing the complexity in the regulation of PKC activity. As previously discussed, regulation of PKC maturation and activity is controlled by phosphorylation mechanisms. Phosphorylation by PDK-1 within the activation loop of the kinase domain is followed by the autophosphorylation of two additional serine/threonine sites. However, there is controversial evidence about the role of tyrosine phosphorylation in PKC activation. It has been shown that PKCÔ is phosphorylated on tyrosine residues within the regulatory domain in response to different stimuli (350-353). However, recent studies have demonstrated that this tyrosine phosphorylation has either no effect (351), an increase (354), or 117 a decrease (355) on PKC activity. The possible role of tyrosine phosphorylation suggests that additional signaling pathways exist, inferring redundant or synergistic signaling of PKC. In addition to binding to lipids, the PKC molecule can also interact with different proteins, affecting localization and subsequent association with substrates. Scaffold proteins cluster signaling proteins for the tight control of cellular pathways and cross talk between different cascades. For normal PKC signaling to occur, PKC must be in the correct subcellular location, near its substrates, activators, and regulatory proteins (e.g. phosphatases). Various scaffold proteins tether PKC to specific intracellular sites such as caveolin, AKAPs, and INAD (356-359). In the Drosophila visual cascade, mutants lacking the protein kinase C-binding scaffold (INAD) are defective in visual transduction because the components of the signaling cascade are mis- localized (360). These findings may provide further insight into the primed state of neutrophils that may even be associated with scaffold proteins. Another interesting study looked at PDK-1 and the activation of PKC. As previously discussed in Chapter One, PDK-1 is the upstream activating kinase, which phosphorylates PKC at the activation loop Thr4lO residue in an on/off switch mechanism. Chou et. al observed that a pre existing complex of PDK-1 and PKC was present in unstimulated cells, both in vitro and in vivo (239). Therefore, they concluded that activation of PKC must occur at the membrane, the site of Ptdlns-3,4,5-P3synthesis and where PDK-1 is localized. Thus membrane targeting of PKC renders the enzyme constitutively active. Factors such as lipid cofactors, phosphorylation mechanisms, and scaffold proteins are only some of the variables that affect translocation of PKC from the cytosol to the membrane. Such complexity and may explain the high degree of variance between and within neutrophils from 118 healthy and LAP subjects when stimulated with IMLP. Understanding these regulatory mechanisms for each PKC isoform is a challenge that will require a full analysis of isoform specific signaling in different cellular systems to assess the involvement in cellular responses. It is noteworthy to add that while my data consistently shows that PKC activity is greater in neutrophils from LAP subjects than healthy subjects, the translocation of PKC from the cytosol to the membrane did not result in an established pattern. In addition to the regulatory factors discussed, the variances in PKC activity may be attributed to the differences in human subjects. When subjects are selected for a case-control study, a number of principles need to be followed (361). First, the LAP patients should constitute a unique group of affected individuals. In my study, standard criteria were used to diagnose the patients and each individual can be considered to have the disease. Second, the LAP patient should be newly diagnosed. However, in my study, the patients were under treatment, which could affect the priming indices. Third, the LAP patients should be available for study in order that necessary information can be collected. In my study, this was not an issue as the patients were on recall at the Clinical Research Center at Boston University. Finally, it is important that the LAP patient group should be representative of a defined eligible population. Herein lies the problem with my study. Due to a limited patient base, I was unable to match subjects according to age, gender, and race with healthy counterparts. As LAP is significantly more prevalent in the African American population than in the healthy Caucasian control subjects (362), I cannot consider my PMN findings truly matched and any comparisons will be diluted and bias the results of the study to the null value. That I found significant differences in my case-control study argues that the findings are real. The main goal of this research was to determine PKC’s role in the enhanced production of superoxide in neutrophils from LAP subjects. Our finding that PKC I1, ö, and are upregulated 119 in neutrophils from LAP subjects led us to examine the downstream molecule p47X and its association with superoxide production. The production of superoxide is the result of the activation of the neutrophils’ defense system and the activation of NADPH oxidase. In resting cells, the components of NADPH oxidase are dissociated within the neutrophil. The assembly of this multi-component enzyme is activated by phosphorylation of p47Ph0X and p67Pi0x, two of its major components (247, 363, 364) at the cell membrane (365) and PKC has been shown to play a key role in this activation (306, 366, 367). Experiments were conducted to examine p47Phb0x, a strong substrate of PKC and a major cytosolic component of the superoxide-generating NADPH oxidase (122). Greater levels of phosphorylated p471x were observed in fMLP-stimulated neutrophils from LAP subjects than healthy subjects. These differences were statistically significant and support our earlier findings that excess superoxide is generated in neutrophils from LAP subjects. Furthermore, the enhanced phosphorylation ofp47Phl0X in LAP neutrophils is consistent with our hypothesis that this excess superoxide generation by neutrophils from LAP subjects is mediated by the pre activation of PKC isoforms, thus priming the neutrophil, phosphorylating 47Phox, and leading to the pre-assembly of the NADPH oxidase. Our experiments suggest that regulatory mechanisms among PKC isoforms are different and that substrate activity may vary according to the PKC isoform. To identify the specific isoforms involved in this priming of the cell, PKC isoform inhibitors were used to treat neutrophils from healthy and LAP subjects. Results showed differences in the inhibition of superoxide generated between healthy and LAP groups when neutrophils were incubated with the classical PKC isoforms inhibitor (PKC a, 131, and 132) and PKC inhibitor. Greater inhibition of superoxide occurred in the healthy group than the LAP group. There were no statistically significant differences in the inhibition of superoxide generation when neutrophils from healthy and LAP 120 subjects were incubated with inhibitors for PKCI3 (PKC 131 and 132) and PKC& The results demonstrating that inhibition of superoxide in neutrophils from healthy subjects was greater than neutrophils from LAP subjects is most likely due to the primed state of neutrophils in LAP. At t=0, neutrophils from LAP subjects were already pre-activated and revealed enhanced superoxide production relative to neutrophils from healthy subjects. As a result, by treating the neutrophils with PKC inhibitors, a diminished inhibition level in LAP neutrophils was to be expected. These findings demonstrate the importance of the classical PKC isoforms and the PKC isoform in the generation of superoxide in PMN from LAP patients. While it is known that p47Pt0X is a substrate of PKC, the contribution of each isoform to the multi phosphorylation mechanism of p47P10X is not yet known. Majumdar et al examined substrate specificities of classical and novel PKC isoforms in neutrophils and found that PKCf3 phosphorylated p47PI0X but did not phosphorylate p67Phb0x. Conversely, PKCÔ phosphorylated p67P0x but did not phosphorylate 47Ph0x (368). Differences in the substrates of PKC 13 and ö imply that these isoforms play selective roles in the activation of neutrophils and the assembly or maintenance of an active NADPH oxidase. Similarly, PKCt selectively phosphorylated serine residues at position 303/304 and 315 of p47X and did not phosphorylate the other residues that were phosphorylated by classical PKC isoforms. This result suggested that PKC and PKC a, 131, and f32 may have discrete specificities forp47PI’O)< activation (369). Results from the first part of this study demonstrated that PKC is a complex signaling enzyme that is mediated by multiple molecular factors that influence cellular responses. The five PKC isoforms expressed in the neutrophil from healthy and LAP subjects demonstrated differences in the levels of phosphorylation and the amount of superoxide generated. At rest, neutrophils from LAP subjects expressed higher activation of PKC 131, , and than neutrophils from healthy 121 subjects, suggesting that the neutrophils from LAP subjects were primed. This enhanced PKC activation observed in LAP was in line with the enhanced phosphorylation of p47Pu10)< in LAP. Inhibition of superoxide generation using PKC inhibitors allowed for the isolation of PKC signaling pathways, implicating classical PKCs and PKC as relevant isoforms in the production of superoxide. Future studies in this area will allow us to understand how substrate specificity links to signal inductions of PKC isoforms and the downstream generation of superoxide in neutrophils from healthy and LAP subjects. It is possible that PKC isoforms act synergistically in driving the pathogenesis of LAP. The NADPH-oxidase complex assembled in the plasmalemma is a clear marker of neutrophil priming in LAP. Having characterized the role of PKC and its isoforms in LAP neutrophil priming, the question is raised of why this priming does not resolve. To further investigate regulation of neutrophil function in LAP, a series of studies looked at resolution of inflammation pathways and the possibility of reversing LAP PMN priming in vitro. The resolvins are a class of resolution agonist that is produced endogenously from dietary (omega-3) fatty acids. The actions of resolvins in mediating PKC pathways and LAP PMN priming were investigated. Neutrophils from healthy and LAP subjects were incubated with various concentrations of RvE1 (1013_106 M) and challenged with fMLP (1 tM). RvE1 inhibited superoxide generation from both healthy and LAP cells by —80% (p<0.05). Neutrophils from the LAP group responded well to RvE1 returning the superoxide generation levels to those of unprimed normal PMN. Investigation of RvE1 actions on the PKC pathways revealed in the LAP group that RvEI reduced the phosphorylation of PKC ô and compared to PBS treated neutrophils. There was 122 no statistically significant difference in the phosphorylation of PKC a, 131, and 132 when neutrophils of healthy and LAP subjects were treated with RvE1. RvE1 did not reduce phosphorylation of p47Phl0X in neutrophils from healthy subjects; RvEI returned phosphorylation ofp47Phl0X in neutrophils from LAP subjects to normal levels. The implication of these findings is that resolution agonists binding to their specific receptors drive a return to homeostasis through the down regulation of pro-inflammatory pathways in inflammatory cells. There are several possible molecular mechanisms of RvE1. RvE1 is synthesized during the spontaneous resolution phase and acts locally at sites of inflammation (260). Studies investigating peritonitis and colitis showed that RvE1 blocks neutrophil infiltration (296, 298) and reduces transendothelial migration and the release of superoxide (260, 299). RvE1 is a selective and potent agonist and specifically binds to the BLT1 receptor on human neutrophils (370). BLT1 is also a high-affinity receptor specific for LTB4. There are interesting implications for the sharing of receptors by both pro-inflammatory and pro-resolving molecules. The most direct is regulating the LTB4-dependent pro-inflammatory signals by competitive inhibition and differential agonist signaling. RvE1 selectively binds and activates BLT1 to transmit intracellular signals different from the LTB4-dependent pro-inflammatory signals (370). Only LAP neutrophils responded to RvE1 by dephosphorylation of specific isoforms of PKC suggesting an activation of specific phosphatases. Like all other periodontal diseases, LAP is a leukocyte-driven inflammatory disease characterized by soft-tissue destruction and osteoclast-mediated bone loss. The prevailing dogma is that PMN initiate the inflammatory response, which is followed by an established monocyte/macrophage lesion that becomes chronic and initiates the acquired immune response. Resolution of inflammation was believed to be a passive decay of pro-inflammatory signals that allowed the macrophage to “clean up” the lesion. It is now appreciated that 123 resolution of inflammation is an active process mediated in part by endogenously derived lipid mediators from cellular (arachidonate) or dietary (omega-3) sources. RvE1 initiates the resolution of inflammation by reducing the influx of neutrophils and initiating neutrophil apoptosis. It also stimulates the upregulation of the expression of CC-chemokine receptor 5 (CCR5) (371). CCR5 acts as a scavenger receptor removing chemokines from the environment. The same molecules (lipoxins and resolvins) are chemotactic for monocytes. However, pro-resolution mediators induce a phenotype change in the monocyte from pro inflammatory (Ml) to non-phlogistic (M2). Hence, in addition to actively being involved in clearance of pro-inflammatory mediators, resolvins, protectins, and lipoxins increase the clearance of bacteria and apoptotic PMN by macrophages in vitro and in vivo (372). Together, the nonphlogistic clearance of pro-inflammatory chemokines and the enhanced phagocytosis of microbes and apoptotic PMN by macrophages demonstrate how lipid mediators actively participate in dampening inflammation and driving resolution. More recent evidence showed that RvE1 functions as an anti-inflammatory and pro-resolution lipid mediator of allergic airway inflammation by decreasing the production of the pro-inflammatory cytokines lL-23, lL-6, and IL- 17 and increasing the production of the counter-regulatory mediators lFN-’ and LXA4 (373). Thus, RvEI both dampened the development and promoted the resolution of inflammation, indicating that it functions as a receptor agonist to promote homeostasis. In our study, PKCÔ, PKCt, and p47Ph0X demonstrated less phosphorylation when LAP PMN were treated with RvE1. Based on these results, three inferences can be made. First, because RvE1 was able to decrease the level of phosphorylation in PKC and 47Phox, RvE1 ‘s mechanism of action is upstream from PKC in the signaling pathway. 124 Second, RvEI’s molecular mechanisms are along a calcium independent pathway that reduces or de-phosphorylates PKC ô and . PKC is only active when it is bound to lipid second messengers and signaling is acutely terminated upon the removal of DAG. The active conformation of PKC is highly sensitive to the dephosphorylation of the kinase, which serves as a signal to promote the rapid degradation of the kinase and ultimately terminate the life cycle of PKC. Hence, phosphorylation protects PKC from degradation. The exact mechanisms of inactivating PKC are still unclear. However, the serine/threonine phosphatase PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) has been found to catalyze the dephosphorylation of the active phosphate site (the hydrophobic motif) and suppress the amplitude and duration of signaling by PKC (374). Hence, PHLPP chronically controls the levels of PKC in the cell as the dephosphorylated form is rapidly degraded. A depletion of PHLPP results in an increase in the levels of PKC. Conversely, activation of PHLPP leads to decreased levels of phosphorylated PKC. By specifically dephosphorylating the hydrophobic motif, PHLPP controls the degree of agonist-evoked signaling by PKC. PHLPP is a potential target for RvE1 in future research. Third, priming of LAP PMN is due to the phosphorylation and activation of PKC 6 and . The novel and atypical PKC isoforms are upregulated in the membrane at t=O, blocking these isoforms with inhibitors reduces the amount of superoxide produced, and RvE1 reduces their phosphorylation levels and normalizes the superoxide response. Based on these data, upregulation of PKC 6 and gives rise to the priming of LAP neutrophils and superoxide generation. To further support this hypothesis, PKC was found to be very different from other isoforms in the phosphorylation of 47Ph0x• While all PKC isoforms phosphorylated p47X, only PKC a, 13, and ,induced translocation and activation of NADPH. It is noteworthy that PKC phosphorylated fewer sites on p4710X and yet it induced higher levels of NADPH oxidase 125 activation (220, 369), while PKCÔ did not induce p47P[10X to translocate to the membrane (220). For activation of NADPH oxidase to occur, translocation of 67Ph0x to the membrane is essential. PKC was unable to phosphorylate p67Pub0x; however p67Pubo)< was activated by PKCÔ (375) RvE1 activates specific pathways in a variety of cells to promote a return to homeostasis and function. The hyperactivity of PMNs in LAP patients is one factor affecting the pathogenesis of the disease. The studies presented in this thesis have markedly increased our knowledge about PKC isoform involvement in this hyperactivity. My studies also evaluated one therapeutic modality (RvE1) that successfully dampened the hyperactivity of several isoforms of neutrophils from LAP patients. 126 CHAPTER SIX: CONCLUSIONS 127 1. Superoxide generation in PMN from LAP patients is elevated, suggesting priming of PMN. FMLP stimulation resulted in an enhanced superoxide production. 2. Polymorphonuclear neutrophils express five PKC isoforms (a, 131, 132, ó, and ) that are common to LAP and healthy individuals. 3. Priming of PMN from LAP patients is associated with pre-activation of Protein Kinase C (PKC), which is translocated to the membrane. PKC activity is higher in the membrane of neutrophils from LAP subjects than healthy subjects. 4. At rest, PKC 131, ó, and in neutrophils from LAP subjects are phosphorylated at higher levels than of those from healthy subjects. 5. There are no differences in the expression of PKC isoform transcripts between healthy and LAP groups, indicating that the increase in PKC activity is not a result of increased transcription and increased PKC protein. 6. RvE1 treatment of LAP PMN in vitro results in return of superoxide generation to normal PMN levels. 7. 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Boston University Research Ethics Board Certificates human Appiovist attN Page 1 of I ituD Univer;iI Med eel Center flltbtMtEfl $flhcLGF$SXJWDflK • tosWnsflhsflPrWT Mcml • DesTlwMmcflcvan fl Elk.waa atnuay .dIL’4S7a1 SPECIALIZED CENTER FOR ANTWNftAh$ TWiNl1e of marty AND RESOUJTTOI4 IN PERIODONTAl. 01SE4 ‘11± ROTOcCL#45O1B Protocoi Nuinboff Pt fl425 Cønflg vw Roilew Type: Pal Poad Aciloit Data ot Actloa 4128r100e Data at zp1rstIon; JflvZ000 Oar THOMAS VAN DVKE1 008, P1W: The Institutoral Review Board (IRS) has fevitiwed the thove mfersnao, protocol incItos deWmWiod hat 14 meats fbi raqiiromen*s set forth by the IRO ancfl approved. This protocol a valid through the dsfe bidicated above. ma study nay riot continua after the apDroval period without add itlahal 110 revIew aN approval for cmtirtuatinn. Yatr will recote ar oaaii rsnawsl rernrnder no cii prlot to sturty nplration; however. it is your responsibility to assure that this stud ii not cocioucted beyond the expiration date. Pense be swore that only IR5cproved thfnmied coneerti lamis may h used whon wricici irtnrmed cuneant is requfred, Mycrungee to the protocol in Uiknznsd consent flier be rewtewod and pjirond pict to rnpfl’iwtar sites the di.’gs te necessaly twine sa sly or subject In addiSon, you rent irtorn the IRS at seduce athene event nnsdd dtflQtiet ma at lbs stdy Ot OS any now cad signitcarl frirneton let niatit inpocto toss’ di Lt%P fl5iotaS lotahhin hyarshety. Pints flora inst ira appaseiw lbee NOT hicudeappru’M attn sccençpnfl HPM iunn(s)Iur tIe study. Once mved. vdsied HIPAAttrns mafle bc4id mtthht INSPifldthor a ttoa oroiduin* flthnwi*. hwssitots wereqt$d Iocnsan that 111PM rsciisma’fl ban been lila flit hlIl#tfl 11$ study. 9idy yumss. LOUIS VACHON iit3 CIoI’ ‘7lM36&iifle endi. di7oniflc 160 B. Ethical Approval for the Use of Human Subjects — $ J3un 1Jnjv1ty Midiai CnThr 3ISILW Th4 Medcar Cntr Liods a F dr! Wd Aurnc Ihe Expfraion Date for tit Federa’ Wrf Ass Otccos aptoiied by a-w of the three bac Jtare. TtP PR ccw1frrm with the r,qurernen e1 forth 21 CPR Fr-ti 50 arid 50 In adciiUon lu these ateguard5 thr rights nd wfI1are of riuman ‘ternnatkn regarding ?hlcal sanriardi kind rkheret rac aH tud ias ‘ sivw b.E& hi U AtySt.t i rnr, OOQOO3O1 racs 31212011 ‘e zt’ee Miry 9ank RN BS. SN Di t;:n’ I, Iii Whorti It My Concern: ‘The rjj, eview 3.n. to rtw Rot Jne ** %4e&I Ceri ‘ co4nLrIsed cf miø rvew piriaIs. TIer ar four tR rnecht5 per r’ionth. I? [UOUOtJ77 REOOO0O37 Hoslon 1) M€d Center IRR - Bk€ F3oston U Mc?ti Center IRO Green Boston U Md Center IRA 45 CFR Part 46 rd iandales, Die Board 7ubj4x15 by rnkIng ‘y eva1utitg * r -i 1hr “irflbei,, ol thr fl tiluEioiial Fevi. I Rnirri cin be fouil iiIcj burr.c.b 1u/www)bumciirb?PDFsf ord Menbrspr1f 161 C. Description of Subjects HealthyILAP Age Race Gender Hi 28 C M H2 28 AA •F H3 32 AA F H4 26 C F H5 25 C F H6 25 C M H7 27 H M H8 26 AA F H9 30 AA F H10 24 AA F Hil 22 H F H12 25. H F H13 25 C M H14 36 C M Hi5 32 C M H16 25 H F Hi7 28 C M Hi8 28 H Hi9 27 C M H20 30 H F H21 40 H F H22 26 AA M H23 26 AA F H24 28 C F H25 27 C F H26 27 C F H27 30 C M H28 27 AA F H29 36 A F H30 24 AA M H31 22 C F H32 26 C F H33 27 H F H34 29 A F H35 45 C F H36 37 C F H37 31 H F H38 22 AA F H39 25 AA M H40 28 C F 162 • LAPI LAP2 LAP3 LAP4 LAP5 LAP6 LAP7 LAP8 LAP9 LAP1O •LAPII V LAP12 V LAP13 LAP 14 LAPI5 LAP16 LAP17 LAP18 LAP19 LAP2O LAP2 1 LAP22 LAP23 LAP24 LAP25 LAP26 21 AA 29ftAA 31 V AA 25AA 28 H 21 MV. F M F M M F F M F F C: AA: H: A: M: F: Caucasian African American Hispanic Asian Male Female 28 25 22 [ 25 MI AA M A I18 AA 24 AA F 24 HIF 25 AA F 31 IIAAi F 28 C F 19 AA M 25 AA F 2441M M 27 VAA M [27IFAA M 25 AA M ‘I26 AA F 32 AA F 4fl2OAA F 26 AA F LEGEND 163

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