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I h ‘lu- $41 ’7 A 3.3!. = l i. wan. data... .3. 3.}. _ \ .A 1...: . 5:32.... ”212;... i - . . a .. y; 3 5r}... if ... 22.} 2. r: «a. .3. r. i!.‘3l§}l| \fl. ‘.A.’.ifi\ay.7rncvl?|\i , VII...I£L1III.\.~ o)sln .4 AH ~M- 'QP‘F§AI;\I “Nguuau. .‘wate University This is to certify that the thesis entitled Bovine Blood Neutrophil Gene Expression and the Effects of Parturition presented by Sally Ann Madsen has been accepted towards fulfillment of the requirements for M.S. degree in Animal Science Major professor Date 1234,57; 63054 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE | DATE DUE DATE DUE D72~ 4 'JAN 1 M00 “A9611 izaoé’l 6/01 cJCIRCIDateDuo.p65-p.15 BOVINE BLOOD NEUTROPHIL GENE EXPRESSION AND THE EFFECTS OF PARTURITION By Sally Ann Madsen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 2001 ABSTRACT BOVINE BLOOD NEUTROPHIL GENE EXPRESSION AND THE EFFECTS OF PARTURITION By Sally Ann Madsen Neutrophil dysfunctions and increased occurrences of production diseases are often observed during periparturition. The objectives of the current study were to reveal and characterize differential gene expression in neutrophils from periparturient dairy cows, determine if altered gene expression relates to fluctuating concentrations of steroid hormones, and obtain putative identities of the difi‘erentially expressed genes. Altered gene expressions were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Fourteen cDNAs from DDRT-PCR were analyzed by dot blot hybridization for initial confirmation of altered mRNA abundance. Two of the most dramatically altered genes were quantitatively assessed by Northern and slot blot analyses using multiple pre- and post-parturition neutrophil RNA samples from four Holstein cows. Sera were also harvested from these animals to assay cortisol, progesterone, and estradiol by RIA. Results showed that the two pursued DDRT-PCR products had DNA sequences similar to genes for bovine mitochondrial cytochrome b and rig/ribosomal protein SIS involved in energy metabolism and translation regulation, respectively. Both genes showed significant (P 5 0.02) repression between parturition and approximately seven days into lactation, and significant positive correlations with serum progesterone and (or) estradiol concentrations. Thus, parturition induces changes in mRNA abundance of neutrophil genes consistent with known defects in cell function during the periparturient period. Copyright by SALLY ANN MADSEN 2001 ACKNOWLEDGEMENTS There are a nmnber of people who have made these past few years a successful and enjoyable experience. First, I would like to thank everyone in the Immunogenetics Laboratory for the constant support and especially for all the help with sample collections when we were all running on empty. I would not have made it through without you all. Thanks to the MSU dairy farm crew for notifying us when cows were calving and helping us out when needed. Thanks to Carolyn Cassar for instruction in the DDRT-PCR technique, Brian Tooker for insight on development of the dot blot protocol, Matt Coussens for assistance with sequencing, and members of Dr. Smith’s and Dr. Ireland’s laboratories for running the hormone assays. I would also like thank Dr. Cathy Ernst for the donation of DDRT-PCR primers and the use of laboratory space and equipment during all the experiments that required use of radioactivity. I would like to thank my committee members, Dr. Jeanne Burton (advisor), Dr. Ron Erskine, Dr. Jim Ireland, Dr. George Smith, and Dr. Rob Tempelman. Working with you has been enjoyable and I thank you for all of your comments, ideas, and support. Jennifer Wells, life in the basement would not have been the same without you. Thanks for taking me under your wing when I arrived. You are a constant supply of advice and support both in and out of the lab. I am lucky to have you as a colleague and a friend. A special thank you to Dr. Patty Weber. You are an amazing teacher and thinker who offered support whenever needed. Thank you for all of your advice and many hours of help, especially during the long hours of sample collection. iv Jeannie — thank you for taking a chance and bringing me to MSU. You have taught me how to “think outside of the box” and pushed me farther down the path to becoming the scientist I have always wanted to be. You are full of great ideas and I am looking forward to learning more from you as I start around the next bend in the road. Finally, I would like to thank that the rest of my support system. Thanks to Chad for being with me every step of the way. You kept my spirits up during the rough spots and always supported and stood behind me. Thanks for everything, you are truly my best friend. And last but not least, thank you to my parents Helge and Marilyn, my brother Toby, and my sister Mary for your support and encouragement. You are the greatest. Table of Contents LIST OF ABBREVIATIONS xm CHAPTERONE AReviewofLiterature.............................................................................6 II. NEUTROPHILS: THE FIRST LINE OF IMMUNE DEFENSE ................. 8 A. NeutrophilDevelopmentintheBoneMarrow.................................8 B. MatureNeutrophilFunctronle II I. PARTURITION: A TIME OF MANY CHANGES FOR THE A. Neutrophil Dysfunctions during Periparturition .............................. 17 B. Steroid Hormones of Parturition — Cortisol, Estradiol, and Progesterone ......22 C. Parturient Hormones may be Linked to Neutrophil Dysfunctions in Dairy Cows .....24 IV. MATURE NEUTROPHILS ARE RESPONSIVE TO THEIR ENVIRONMENT: EVIDENCE FOR ALTERED GENE EXPRESSION ....26 CHAPTER TWO Altered Blood Neutrophil Gene Expression in Periparturient Dairy Cows ........... 30 vi II. III. IV. MATERIALSANDMETHODS35 A. AnimalsandSampleCollection.................................................35 B. BloodLeukocytePurificationandRNAIsolation............................38 C. Primary Screening of Differential Gene Expression 1n Total Leukocytes ofPeriparturientCowsusingDDRT-PCRH 39 D. Secondary Screening of Differential Gene Expression in Neutrophils usingDotBlotHybridization.....................................................4l E. Northern Blot Analysis Characterization of Neutrophil Genes ............. 43 F. Characterization of Neutrophil mRNA Abundance Profiles from PenpartunentCows44 G. Radioimmunoassay of Serum Cortisol, Estradiol, and Progesterone . . . 44 H. Identification of Differentially Expressed Neutrophil Genes ............... 45 I. StatisticalAnalysis................................................................45 A. Primary Screening of Differential Gene Expression InducedbyParturitioninTotal Leukocytes....................................47 B. Secondary Screening of Differential Gene Expression InducedbyParturition.............................................................50 C. Characterization of Neutrophil mRNA Hybridized by 3-IOIa and 9-21 ...54 D. Characterization of 3-lOIa and 9-21 mRNA Abundance Profiles in NeutrophilsfromPeriparturientCows.........................................56 E. Correlations Between Neutrophil Gene Expression and Serum SteroidProfilesfromPeriparturientCows.....................................59 F. Putative Identifications of Genes Differentially Expressed in Neutrophils ofPeriparturient Cows ........63 vii VI. CONCLUSIONS .. CHAPTER THREE General Discussion and Conclusion CHAPTER FOUR Recommendations for Future Research APPENDIXONE.............. viii ....67 .70 7] 85 .95 LIST OF TABLES CHAPTER ONE Table 1.1 — Summary of blood neutrophil responses to parturient physiology: altered trafficking ofthe cells ......20 Table 1.2 — Summary of responses of blood neutrophils to parturient physiology: IQ Ix) altered phagocytic andkillingfunctionsofthe cells .. .. CHAPTER TWO Table 2.1 — Correlations between serum steroid hormone concentrations and neutrophil mRNA abundance of3-101a and 9-21 in periparturient dairy cows ....62 Table 2.2 — Summary of differentially expressed neutrophil genes .66 ix LIST OF FIGURES CHAPTER ONE Figure 1.1 — The immune system consists of two branches, innate immunity and adaptiveimmunity 7 Figure 1.2-Neutrophil development occurs in the bone marrow ....9 Figure 1.3 — Blood serum profiles of cortisol, estradiol, and progesterone in periparturient cows 24 CHAPTER TWO Figure 2.1 — Several methods were utilized to address our hypothesis that parturition induces changes 3......6 Figure 2.2 — Primary screening for difi'erential gene expression in total leukocytes from parturient and mid-gestation cows was performed using DDRT-PCR ...48 Figure 2.3 — Fourteen amplicons generated during primary screening of differential gene expression in total blood leukocytes were cloned and following PCR amplification ranged in sizes from 350 to 1100 base pairs ..5.....1 Figure 2.4 — Secondary screening for differential gene expression in neutrophils ofaparturienttest cow using cDNA dot blot hybridization Figure 2.5 — Characterization of size and number of transcripts complimentary to 3-101a and 9-21 in neutrophils from a periparturient test cow by Northern blot hybridization ......................................................................................................... Figure 2.6 — mRNA abundance profiles of genes encoded by amplicons 3-101a and 9-21 in neutrophils from periparturient cows were obtained using slot blot hybridization Figure 2.7 — Serum concentrations of three steroid hormones of bovine reproduction change significantly during parturition Figure 2.8 — The 3-101a amplicon was homologous to a region of the bovine gene for mitochondrial cytochrome b . .. .. Figure 2.9 - The 9-21 amplicon was homologous to a region of the human gene encoding rig/ribosomal protein 815 .. CHAPTER THREE .52 ..... 55 57 60 ....64 .65 Figure 3.1 — Mitochondrial cytochrome b is a key component of Complex 111 of the electron transport chain xi .75 Figure 3.2 - Ribosomal protein SIS is an integral component of the small ribosomalsubunit APPENDIX ONE Figure A.l — Purity of isolated neutrophils from d -14 and d 0.5 from which RNA wasreverse transcribed Figure A.2 — DDRT-PCR gel sections containing differentially expressed genes called 3-101aand9-21 ...... Figure A.3 — Autoradiographs of dot blots hybridized with radiolabeled cDNA created by reverse transcription ofneutrophil RNA Figure A.4 — Mean dot densities (:tSEM) of B—actin mRNA abundance in neutrophils before andafter parturition Figure A.5 - Scatter plots demonstrating relationships between neutrophil 3-101a and 9-21 mRNA abundance and steroid hormones xii ...79 .86 ...87 .88 89 ....90 LIST OF ABBREVIATIONS ACD = Acid Citrate Dextrose AML-l = (a transcription factor) BLASTn = Basic Local Alignment Search Tool — nucleotide bp = Base Pair C/EBP-a = (a transcription factor) CAP-18 = 18-kDa Cationic Antibacterial Protein CD = Cluster of Difl‘erentiation (e. g. CD11a, CD1 1b, CD14, CD15, CD16, CD18, CD68) CD62L = L-selectin cDNA = Complementary DNA cytb = Bovine Mitochondrial cytochrome b d = Day DNA = Deoxyribonucleic Acid dNTP = Deoxyribinucleoside Triphosphate DDRT-PCR = Differential Display Reverse Transcription Polymerase Chain Reaction EDTA = Ethylenediaminetetraacetic Acid fMLP = fonnyl-methionyl-leucyl-phenylalanine IL = Interleukin (e.g. IL-l B, IL-6, 1L-8) LFA-l = CD11a/CD18 Mac-l = CD1 1b/CD18 MHC = Major Histocompatibility Complex mRNA = Messenger Ribonucleic Acid xiii NADPH = Nicotinamide adenine dinucleotide phosphate - hydrogenated NaOH = Sodium Hydroxide NGAL = neutrophil gelatinase-associated lipocalin PBS = Phophate Buffered Saline PCR = Polymerase Chain Reaction rig/RPSIS = rig/Ribosomal Protein SIS ROS = Reactive Oxygen Species rRNA = Ribosomal Ribonucleic Acid RT = Reverse Transcription 505 = Sodium Dodecyl Sulfate SSC = Saline Sodium Citrate tRNA = Transfer Ribonucleic Acid xiv INTRODUCTION Parturition occurs multiple times in the life of an average dairy cow. This event is necessary to maintain a level of milk production beneficial to the dairy industry. Through the periparturient period, dairy cattle experience a multitude of changes including changes to the udder during the transition from non-lactating to lactating (Oliver and Sordillo, 1988), alterations in metabolism and intake (Ingvartsen and Andersen, 2000), as well as dramatic fluctuations of metabolic and steroid hormones (Smith et al., 1973; Goff and Horst, 1997). The periparturient period has also been associated with decreased immune function that has been postulated to be primarily responsible for the increased susceptibility to mastitis and other production diseases observed during this time (Cai et al., 1994; Mallard et al., 1998; Kehrli and Harp, 2001). Numerous investigators have hypothesized that the changes in steroid and metabolic hormones during periparturition are responsible for the decreased immune function leading to this disease susceptible state (Curtis et al., 1985; Goff et al., 1989; Goff and Horst, 1997; Lee and Kehrli, 1998; Kehrli and Harp, 2001; Weber etal., 2002). Several cell types of the immune system are affected during bovine parturition. For example, the effects of parturition have been studied on blood and milk neutrophils as well as various subsets of blood and milk lymphocytes (i.e., CD4+, CD8+, and y5 TCR+ T cells). In such studies, the T cells from parturient cows have been shown to decrease in numbers within the blood and milk; this is accompanied by decreased in vitro proliferative responses to several mitogens compared to T cells from cows in mid- lactation (Shafer—Weaver et al., 1996; Kimura et al., 1999a). However, the actual relevance of these changes in T cell numbers and in vitro functions to immunosuppression and periparturient disease susceptibility has not been established. In contrast, parturition’s negative effects on blood neutrophil counts and functions appear to be of considerable consequence to disease susceptibility of parturient cows. Under normal circumstances, blood derived neutrophils are the first line of innate immune defense against bacteria and other microorganisms that cause acute mastitis, metritis, and other common infectious diseases of parturient dairy cows. Neutrophils originate in the bone marrow and continuously enter the circulation with or without presence of peripheral tissue infections (Kuby, 1997; Janeway et al., 1999). Blood neutrophils spend much of their short life span (10 to 24 hours) adhering to and rolling on the endothelial cells that line the walls of post-capillary venules in peripheral tissues. They do this as a surveillance mechanism to determine the infection status of the underlying peripheral tissues. If rolling neutrophils detect endothelial and soluble signals such as cytokines and chemokines of inflammation and infection, the cells rapidly adhere to the endothelial cell wall and migrate from the blood compartment into the infected tissue (Kansas, 1996). The process of migration activates oxygen dependent and independent mechanisms in neutrophils enabling these cells to become highly efficient phagocytes that ingest and destroy infecting pathogens in tissues. Periparturition alters many of the functions of neutrophils, resulting in nearly complete dysfunction of the chain of events described above. For example, pronounced reductions have been observed in trafficking, random migration, chemotaxis, and myeloperoxidase activity of blood neutrophils from periparturient cows (Nagahata et al., 1988; Kehrli et al., 1989; Cai et al., 1994; Kimura et al., 1999b; Weber et al., 2002). 10 Although many of the neutrophils’ functions were decreased around parturition, neutrophilia, or an increase in the number of neutrophils in the blood, is generally observed during this time. Several researchers have shown that this could be related to decreased expression of adhesion molecules on the neutrophils’ surface (Lee and Kehrli, 1998; Kimura et al., 1999b; Weber et al., 2002). Such molecules are needed for normal interactions with endothelial cells that lead to neutrophil rolling and migration. The reduction in trafficking results in neutrophil accumulation in the circulation leaving the mammary gland and other tissues devoid of first line immunity against opportunistic bacteria. Despite the fact that researchers have documented observations of decreased neutrophil functions in periparturient dairy cows for several decades, little is known about how or why dysfunctions of these leukocytes occur. The work described in Chapter Two focuses on the possibility that neutrophils of parturient cows respond to the physiology of parturition with altered gene expression. It is widely believed by leukocyte biologists that circulating neutrophils are biosynthetically inactive phagocytes (Bainton et al., 1971). This is in large part due to the fact that these terminally differentiated white blood cells have highly condensed nuclei, store many preformed proteins in their multiple complex granules for rapid killing of pathogens in infected tissues, and have such a short half-life in blood. However, several recent studies challenge this dogma showing that neutrophils actively express hundreds of genes in the resting state and respond to multiple environmental stimuli, such as bacteria and cytokines, with altered expression of numerous genes (Lloyd and Oppenheim, 1992; Itoh et al., 1998; Newberger et al., 2000). These findings substantiate numerous observations from experiments in our laboratory (Weber et al., 2002), which have shown that bovine blood neutrophils respond to the stress of parturition and to administration of glucocorticoid hormones with inhibited expression of the gene for L-selectin. To our knowledge, however, no studies have been published that explore overall gene expression changes in bovine neutrophils around parturition, nor the potential factors responsible for these changes The goal of the present study was to begin exploring differences in overall gene expression in blood neutrophils as cows transition from the dry period through parturition. The objective of the first series of experiments in this work was to identify and fully confirm differential expression of at least two bovine neutrophil genes not previously reported from other periparturient dairy cow research. The objective of the second series of experiments was to determine if fluctuations in the three major steroid hormones of bovine parturition (cortisol, progesterone, and estradiol) correlate with changes in expression of the identified neutrophil genes. Steroid hormones were selected for this study as possible contributors to altered gene expression in blood neutrophils because these agents have direct and profound effects upon gene expression in other cell systems (Carson-Jurica et al., 1990; Beato et al., 1995). The final objective was to obtain putative identities of genes demonstrating differential expression in objective one. Results of the experiments reported in this thesis are novel and important because they clearly identify reduced mRNA abundance of two neutrophil genes shortly after parturition. One of these genes is required for energy metabolism in the cell, while the other gene may have a role in translation regulation. Expressions of both genes showed substantial relationships with serum steroid concentrations. Approximately ten additional mRNAs were identified as putatively reduced due to parturition, some of which appear to encode proteins involved in the citric acid cycle and DNA binding. Further experiments will be required to confirm and discern the contributions of the decreased mRNA abundance of all of these genes to neutrophil dysfunctions and mastitis susceptibility in periparturient cows. CHAPTER ONE A Review of Literature 1. THE IMMUNE SYSTEM The bovine immune system, like that found in all mammals, consists of two branches that work together to provide protection from invading pathogens. These branches are referred to as innate, or nonspecific immunity, and adaptive, or acquired immunity (See Figure 1.1) (Kuby, 1997; Janeway et al., 1999). The adaptive immune system is specific in its response to pathogens and is continually developing. This branch of the immune system has two components, cell-mediated and humoral immune responses. The humoral response is mediated through B lymphocytes and their ability to differentiate into antibody producing plasma cells that secrete large amounts of antibodies following exposure to foreign antigens (such as those on bacteria). Antigens presented by MHC molecules on macrophages, dendritic cells, and B lymphocytes are also capable of activating T lymphocytes, which subsequently facilitate the humoral immune responses of B lymphocytes or mediate cytotoxicity of T lymphocytes against virally-infected cells. Unlike the adaptive immune responses, innate immunity is present at birth, is immediately available through cellular and humoral factors present in the blood at all times, and provides the first line of defense against invading pathogens. It consists primarily of phagocytic leukocytes, called neutrophils and macrophages, and serum factors such as complement proteins, collectins, and acute phase proteins. In the event that the innate immune response is dysfunctional, or an infection overcomes the phagocytes’ abilities to clear it, adaptive immunity will become activated over subsequent days and weeks to provide highly specific and targeted defense against the invading pathogens. Immune System Innate Immunity Adaptive Immunity Phagocytes Lymphocytes Neutrophils Macrophages T lymphocytes B lymphocytes Figure 1.1 — The immune system consists of two branches, innate immunity and adaptive immunity. Phagocytic cells, such as neutrophils and macrophages, are key players in innate immunity. B and T lymphocytes are necessary for the proper function of adaptive immunity. Interactions between innate and adaptive immunity occur when macrophages present antigens to T lymphocytes for activation and when neutrophils phagocytose antigens opsonized by antibodies that were secreted from activated B lymphocytes. II. NEUTROPHILS: THE FIRST LINE OF IMMUNE DEFENSE Neutrophils play a key role in innate immunity in cattle. In particular, neutrophils are primarily responsible for clearing the bacteria that infect mammary quarters and cause clinical mastitis in dairy cows (Kehrli and Harp, 2001). The following summary of information about neutrophil development and the five main functions of this leukocyte subset is provided to set the stage for the context under which the current thesis research was performed. A. Neutrophil Development in the Bone Marrow Immune cells are produced in the bone marrow, which is also the site of neutrophil maturation. Approximately 60% of all marrow cells are neutrophils at various stages of development (Edwards, 1994). All granulocytes (neutrophils, basophils, and eosinophils) differentiate from a pool of pluripotent stem cells into myeloblasts and promyelocytes via the process of granulopoeisis. Neutrophilic cells then mature in stages into myelocytes, metamyelocytes, band cells, and finally the segmented (mature) neutrophils that are released into the circulating blood (Figure 1.2). The maturing cells were named according to appearance or disappearance of specific cellular markers (detected by various staining methods) and cell morphology, which is described below. Development from pluripotent stem cells into segmented neutrophils takes approximately 14 days, 7.5 days of which are spent in proliferation stages (myeloblast, promyelocyte, and myelocyte) and the remaining 6.5 days of which see maturation through metamyelocyte, band cell and segmented neutrophil stages (Bainton et al., 1971). ///I////Ill/I/////////////////////r/ I , . .. . . ‘////////1////////// 71/” ////.'//////// ‘//////////////// Proliferation Stages /; 2" - / ///////// // ”nu/”1 um” ////////////// // v ‘ I/ / /// « M I bl t a) ye o as ”MW/m , ,, , I ”my”, b) Promyelocyte //////////////////z,// ////////r / 7,, ///// / .- /// u ”xx ////////////"/'/l //.////'/////'l / u/ /, - - - Iz////////////////////////// , , ”71/ . . 2 Il/l/l/ll/lflfl//////////////II/l/I/I , - f) Segmented or d) Metamyelocyte Mature Neutrophil e) Band CC“ Figure 1.2 — Neutrophil development occurs in the bone marrow. There are a total of six developmental stages, three that are proliferative in which many granules are formed (mitotic; steps a, b, and c in shaded area) and three that mainly consist of changes in the nuclear morphology (steps d, e, and 1) resulting in mature cells with large, multi-lobed nucleus and abundant cytoplasmic granules. See text for details. Myeloblasts, cells in the first stage of granulopoeisis, are characterized by their positive staining for peroxidase in the rough endoplasmic reticulum and Golgi apparatus regions (Figure 1.2, step a) (Edwards, 1994). During the transition from myeloblast to promyelocyte, granules are formed within which the peroxidase is packaged (Figure 1.2, step b) (Edwards, 1994; Berliner, 1998). Promyelocytes contain a large number of these azurophil, or primary, granules that contain peroxidase and stain a red-purple color with azure dyes. The majority of the peroxidase within azurophil granules is myeloperoxidase, an enzyme important for the killing functions of all granulocytes. Cells in the next stage of development are called myelocytes (Figure 1.2, step c). This is the first cell type committed to become neutrophils (Berliner, 1998). The accumulation of numerous peroxidase-negative granules (named specific, or secondary granules) is characteristic of myelocytes. No additional azurophil granules are synthesized during this stage of development (Edwards, 1994). Myelocytes are the last stage of neutrophil development in which proliferation occurs. Metamyelocytes, band cells, and segmented neutrophils are distinguished by their granule protein content and nuclear morphology (Edwards, 1994; Figure 1.2, steps (I, e, 1' respectively). The mature neutrophil is distinct from other leukocytes in that it has a large, multi-lobed nucleus and abundant acidic and basic granules located in the cytoplasm that are packed full with myeloperoxidase, lytic enzymes, and other proteins required for surveillance, migration, phagocytosis, and killing functions of these leukocytes (Edwards, 1994; Kuby, 1997). Many additional membrane proteins, enzymes, and secretory proteins distinguish stages of neutrophil development in the bone marrow. In 1995, Borregaard et al. hypothesized that sorting of granule proteins is controlled by the timing of their synthesis, not by the targeting of proteins to the various granules after they have already formed. Since then, several groups have been able to support this hypothesis by showing that mRNA for granule contents was only produced and most likely processed during developmental stages in which those granules were formed. Most of the transcripts detected were for matrix proteins (proteinase-3, elastase, defensin, lysozyme, lactoferrin, gelatinase, NGAL, and CAP-18), membrane proteins (CD68), or cell surface markers 10 (CD1 lb, CD15, and CD16). For example, myeloblasts are shown to have both mRNA and protein for myeloperoxidase, proteinase-3, neutrophil elastase, CD68 (Cowland and Borregaard, 1999), and CD15 (Terstappen et al., 1990). Promyelocytes also exhibit large quantities of myeloperoxidase (Nagaoka et al., 1998), proteinase-3, neutrophil elastase, CD68, and CD15 as well as defensin (Nagaoka et al.; 1998). In myelocytes there is continued (CD15 and defensin) and decreased (myeloperoxidase, proteinase-3, neutrophil elastase, and CD68) expression of some mRNA although protein levels remain high. At this stage, when peroxidase-negative granules are being formed, expression of lactoferrin and gelatinase increase (Nagaoka et al., 1998) along with NGAL, CAP-l 8 (Cowland and Borregaard, 1999), and CD11b(Terstappen, et al., 1990). Metamyelocytes are characterized by continued expression of gelatinase (Nagaoka et al., 1998), CD15 and CD1 1b, and increases in expression of CD16 (Terstappen et al., 1990) and lysozyme (Cowland and Borregaard, 1999). In conjunction with timing of mRNA and protein production, Borregaard et al. (2001) has shown that certain transcription factors are present during specific developmental stages. They demonstrated that transcription factor AML-l was critical for primary granule protein expression while C/EBP-a was necessary for secondary granule protein expression. All of these data support the hypothesis that granule protein content is controlled by timing of synthesis. The research described above was performed using human neutrophils obtained from bone marrow. In addition to azurophil and specific granules, bovine neutrophils contain a third type of granule referred to as large granules. These large granules, named due to their relative size, constitute the main storage compartment of bovine neutrophils. They contain oxygen-independent bactericidal agents and lactoferrin but lack enzymes and II proteins typically found in azurophil and specific granules (Gennaro et al., 1983). Zanetti et al. (1990) demonstrated that the large granules are formed after the azurophil granules but before specific granules. Other than the additional large type granules in bovine neutrophils, azurophil and specific granule contents are relatively similar across species. The biggest difference in granule content across species lies in the amount of granule proteins and enzymes stored. In comparison to human neutrophil granules, bovine neutrophil granules contain 15-20% less peroxidase activity (Gennaro et al., 1983) as well as lowered lysozyme and catalase activities (Styrt, 1989). In contrast, concentration and/or activities of alkaline phosphatase, lactoferrin, vitamin Biz-binding protein and general protein are greater in bovine neutrophils (Gennaro et al., 1978). Comparisons described by these two groups demonstrate that although there are differences in concentrations of the various proteins, overall granule content and the functions of neutrophils are similar across species (Gennarro et al., 1983; Styrt, 1989). B. Mature Neutrophil Functions Upon release from the bone marrow, neutrophils spend 10 to 24 hours in the blood, followed by 1 to 2 days in peripheral tissues. As previously stated, mature neutrophils have large, multi-lobed, segmented nuclei in which the chromatin is coarsely clumped (Gennaro et al., 1978; Edwards, 1994). Other characteristics of circulating neutrophils include numerous cytoplasmic granules (azurophil, specific, and large granules in the bovine), small amounts of Golgi and endoplasmic reticulum, as well as a few mitochondria and ribosomes. This cellular morphology is not surprising because much of 12 what neutrophils require to function properly in innate immunity against infections is needed for rapid action and thus must be preformed and stored in cytoplasmic granules. Mature neutrophils must perform five main functions to effect immunity against invading pathogens - surveillance, recruitment, receptor-mediated phagocytosis, respiratory burst, and fusion of the phagosome (created by phagocytosis) with cytoplasmic granules containing lytic enzymes (i.e. lysosomes) to create phagolysosomes. Surveillance and recruitment occur while neutrophils are in the blood. Blood neutrophils are constantly surveying for infections in the peripheral tissue via a process called margination. If an infection is present, neutrophils become recruited to the infected area through the mechanisms of migration and chemotaxis. Margination and migration are known collectively as neutrophil trafficking. The remaining neutrophil functions take place once the cells have successfully migrated into tissues and result in the killing and clearance of invading pathogens. Researchers have shown that neutrophil trafficking is mediated by a variety of adhesion molecules, such as selectins and integrins, located on the cell’s surface. Under normal conditions, neutrophils attach lightly to the blood vessel endothelial cell wall via L-selectin adhesion molecules. The shear force of blood flow facilitates the rolling of L- selectin tethered neutrophils along the vessel wall (margination) (Jutila, 1992; Bargatze et al., 1994; Kansas, 1996), and on other neutrophils already arrested on the endotheliurn (Bargatze et al., 1994). High expression of integrin adhesion molecules is required for tight adhesions that arrest rolling neutrophils for migration into infected peripheral tissue (J utila, 1992). When rolling neutrophils detect infection in underlying tissues, the cells up-regulate protein expression of Bz-integrins, such as LFA-l (CD1 la/CD18) and Mac-1 l3 (CD1 1b/CD18), from preformed stores of these molecules in the peroxidase negative granules. Such up-regulation causes arrest of rolling neutrophils on the inflamed vessel and permits migration through the vessel wall into the infected tissue (J utila, 1992). Therefore, proper trafficking of neutrophils requires expression of both L-selectin and [32- integrins (Crockett-Torabi et al., 1995). However, while [32-integrin expression on activated neutrophils is increased, expression of L-selectin is rapidly shut down via proteolytic cleavage at a membrane proximal site of the molecule. Together, these processes halt the rolling phenotype of neutrophils and promote the arrested phenotype with subsequent migration (J utila, 1992; Soler-Rodriquez et al., 2000). While migration of arrested neutrophils into infected peripheral tissues requires Mac-l, it is also facilitated by heightened surface expression of chemokine receptors, especially the IL-8 receptor. Mi grated neutrophils continue to use Mac- 1 , IL-8, and other receptors to follow concentration gradients of chemokines, complement components, and multiple cytokines released at high concentration from the infection focus. This concentration-dependent movement of migrated neutrophils into the infection focus is called chemotaxis (Kuby, 1997). If margination, migration and chemotaxis are completed efficiently, neutrophils are properly placed and become highly activated for rapid clearance of the pathogen. Once in the infection focus, migrated neutrophils begin to clear pathogens by a process called phagocytosis (Kuby, 1997; J aneway et al., 1999). During phagocytosis, neutrophils mount a substantial respiratory burst that generates a variety of highly reactive oxygen species (ROS). The ROS begin to work on the phagocytosed pathogen, causing lipid peroxidation and oxidative damage to proteins, RNA, and DNA (Smith, 1994; Crockett-Torabi et al., 1995; Ward and Lentsch, 1999). The respiratory burst is signaled l4 when neutrophils bind to pathogens for phagocytosis. This is a receptor-mediated event in which a variety of cell surface receptors (e. g. CD14, CD18, F c receptors, and complement receptors) bind various bacterial cell wall components and (or) host immune proteins (antibody and complement) that have opsonized the pathogen for enhanced phagocytosis by neutrophils (Janeway et al., 1999). The receptors, located within the neutrophil plasma membrane, transduce signals to the cell interior that initiate respiratory burst activity and the formation of pseudopods that encircle the bound pathogen for internalization into the neutrophil in a vesicle known as a phagosome. Following phagocytosis, ROS-mediated oxidative degradation of the pathogens begins when incoming phagosomes fuse with outgoing lysosomes to form a phagolysosome. The various cytoplasmic granules, collectively called lysosomes, generate ROS by shuttling electrons across their membrane NADPH oxidase system resulting in the reduction of oxygen to superoxide anions, which is sometimes further converted to hydrogen peroxide. These lysosomes fuse with the incoming phagosome and release ROS into the phagolysosome to assist in the degradation of pathogens (Smith, 1994; Dahlgren and Karlsson, 1999). The various granules also contain myeloperoxidase and proteolytic enzymes (in azurophil granules), lactoferrin and lysozyme (in specific granules) (Smith, 1994), and bactenecins that are specific to the large granules of bovine neutrophils and have non-oxidative microbicidal activity (Zanetti et al., 1990). Myeloperoxidases aid in the oxidative damaging of pathogens. The other agents listed heighten damage already initiated by ROS and participate in complete digestion of the pathogen. For example, proteolytic enzymes aid in the digestion of bacterial structural proteins, lactoferrin sequesters numerous minerals and thus deprives the invading 15 pathogen of essential nutrients, and lysozyme and collagenase help destroy components of the bacterial envelope (Smith, 1994). In all, the functions of phagocytosis, respiratory burst, and generation of the phagolysosome are highly connected and necessary to mediate successful killing and clearance of invading pathogens by neutrophils. Work has been performed that demonstrates how several neutrophil fimctions are affected under various conditions. One in vitro study established that older neutrophils express lower levels of surface L-selectin as well as a decreased ability to change shape and chemotactically migrate following exposure to flVlLP, a bacterial protein that is commonly used to stimulate neutrophils during in vitro studies (Tanji-Matsuba etal., 1998). Aging neutrophils were also shown to degranulate more readily and release higher levels of reactive oxygen species than younger cells. A study in which cortisol and dexarnethasone were administered to cows in vivo showed that these glucocorticoids lead to complete down regulation of L-selectin and significant reductions in CD18 expression on the surface of blood neutrophils, demonstrating a possible mechanism for the potent anti-inflammatory actions of these steroid hormones (Burton et al., 1995 ). Glucocorticoid induced down regulation of neutrophil L-selectin also correlated well with neutrophilia and increased mastitis susceptibility in treated cows (Burton and Kehrli, 1995), highlighting a significant pitfall in the leukocyte’s sensitivity to steroidal anti- inflammatory agents. Others have shown that administration of epinephrine or cortisol to humans increased blood neutrophil counts (Hetherington and Quie, 1985; Steele et al., 1987), a phenomenon associated with the release of marginating neutrophils from the blood vessel wall. In addition, epinephrine and cortisol may cause premature release of neutrophils from bone marrow because up to 56% of circulating neutrophils have the band 16 nucleus morphology after administration of these hormones (Burton et al., 1995; Hetherington and Quie, 1985). Together, these studies demonstrate that neutrophils are sensitive to their environment and are capable of altering their phenotype and function under a variety of natural and artificial stimuli. This is in sharp contrast to a widely held but incorrect belief that neutrophils have little capacity to respond to the environment due to their cellular morphology and terminally differentiated status (Bainton et al., 1971). IH. PARTURITION: A TIME OF MANY CHANGES FOR THE BOVINE IMMUNE SYSTEM Parturition plays an important role in the life of a dairy cow. In order to continue producing milk, this event must occur approximately every thirteen months (to maintain production levels beneficial to producers) and is characterized by numerous dramatic physiological and metabolic changes. Not only does the cow go from the non-lactating to the lactating state, there is significantly reduced dry matter intake and pronounced fluctuations in blood concentrations of metabolic and steroid hormones. This section will present evidence that neutrophils respond to the physiology of parturition with altered phenotypes and functional capacities, possibly in response to integrated changes in blood concentrations of cortisol, estradiol, and progesterone. A. Neutrophil Dysfunctions During Periparturition Most metabolic diseases, including milk fever, ketosis, retained placenta, and displacement of the abomasum, occur during the first two weeks following parturition (Goff and Horst, 1997). This is also a time when infectious diseases become clinical, 17 particularly mastitis. Intramammary infections and clinical mastitis result in altered mammary function, decreased milk production, and altered milk composition costing the dairy industry over $2 billion annually (National Mastitis Council, 1996). Neutrophils are the main immunological defense against mastitis. Thus, several researchers have studied immune functions of periparturient dairy cows as a means to explain the heightened mastitis susceptibility in these animals (Tables 1.1 and 1.2). Oliver and Sordillo (1988) showed that clinical mastitis often results from mammary infections that occurred during the dry period. They also demonstrated that the rate of new infections during the dry period is substantially higher (6.25 times) than the rate of infections in the previous lactation. Some hypothesized that this phenomena is a result of immune system malfunction around dry off and during the periparturient period. Numerous investigators have tested this hypothesis and shown that blood leukocyte functions start to change three weeks prior to calving and continue to be dysfunctional through three weeks post partum (reviewed by Mallard et al., 1998). In particular, blood neutrophils of parturient cows have decreased respiratory burst activity when measured in vitro and this phenotype associates strongly with increased occurrence of clinical mastitis (Mallard et al., 1998). It has also been demonstrated that neutrophil recruitment into the mammary gland is reduced around parturition, a phenotype well correlated with increased severity of clinical coliforrn mastitis in periparturient dairy cows (Hill et al., 1979; Shuster et al., 1996; Kehlri and Harp, 2001). Thus, strong evidence supports that increased susceptibility to clinical mastitis during the peripartum period is a direct function of decreased abilities of blood neutrophils to migrate into infected mammary quarters and kill pathogens once the cells have migrated. l8 Other researchers examined specific aspects of neutrophil function during the periparturient period to discern why these dysfirnctions occur. For example, Guidry et al. (1976) found increases in circulating band neutrophils in blood of parturient cows, suggesting release of immature neutrophils from the bone marrow (Table 1.1). It was hypothesized by Guidry et al. that band neutrophilia occurs in response to the high levels of blood cortisol at parturition. Other researchers have also shown that the migration capacity of blood neutrophils, measured in vitro as random migration of the cells under agarose, is significantly reduced at parturition (Nagahata et aL, 1988; Kehrli et al., 1989; Detilleux et al., 1994). Reduced migration could also explain the pronounced increase in circulating mature neutrophils around parturition (Preisler et al., 2000a), possibly as a result of significantly reduced L-selectin and [32 integrin expression on the surface of blood neutrophils in response to the surge in cortisol (Lee and Kehrli, 1998; Kimura et al., 1999b; Weber at al., 2002). In any case, it is clear from these studies that blood and possibly bone marrow neutrophils respond to parturient physiology by altering the expression of their surface adhesion molecules, which changes the trafficking patterns of these cells in favor of reduced migration into infected mammary glands. This alone could explain the increased susceptibility to mastitis in parturient dairy cows. 19 Table 1.1 - Summary of blood neutrophil responses to parturient physiology: altered trafficking of the cells. Neutrophil Response Parturient Alteration Citation Migration Decreased Nagahata et al., 1988 Kehrli et al., 1989 Detilleux et al., 1994 Blood neutrophil numbers Increased Guidry et al., 1976 Preisler et al., 2000a Weber et al., 2002 L-selectin Decreased ' Lee and Kehrli, 1998 (surface expression) Kimura et al., 1999b Weber et al., 2002 [32 integrin Decreased Lee and Kehrli, 1998 (surface expression) Kimura et al., 1999b The functions of neutrophils known to be critical in bacterial clearance and killing in peripheral tissues have also been examined in vitro and shown to be markedly affected by parturition (Table 1.2). In one study, chemotaxis, detected as directed movement of neutrophils under agarose, was significantly reduced when the neutrophils were collected during the first week post partum versus several weeks prepartum (Nagahata et al., 1988). Cai et al. (1994) also observed decreased chemotaxis of parturient neutrophils and found it to be associated with occurrences of clinical mastitis, retained placenta, and metritis. Phagocytic ability of neutrophils from parturient cows has also been evaluated in vitro. Guidry et al. (1976) showed that the total phagocytic capacity of neutrophils increased at parturition, mainly due to the increased numbers of circulating neutrophils found at this time. However, these researchers also showed that the increase in total phagocytosis was followed by an overall decrease in phagocytosis during the first three weeks post partum. This may be explained by the findings of others who showed that while overall bacterial ingestion was increased around parturition, other aspects of phagocytic activity such as number of bacteria phagocytosed and killed were decreased in parturient cows (Kehrli and Goff, 1989; Kehrli et al., 1989; Cai et al., 1994). Superoxide anion production, which is critical for pathogen damage following the respiratory burst in phagocytosing neutrophils, was demonstrated as significantly reduced during periparturition (Kehrli and Goff, 1989; Detilleux et al., 1994). This was particularly evident in neutrophils from cows that exhibiting clinical mastitis, metritis, and retained placenta (Cai et al., 1994). Myeloperoxidase activity, another important factor in pathogen damage within the phagolysosome, was also deficient in neutrophils from periparturient cows (Kehrli and Goff, 1989; Kehrli et al., 1989; Cai et al., 1994; Detilleux et al., 1994; Kimura et al., 1999b), as was overall oxidative capacity of these phagocytes (Kehrli and Golf, 1989; Kehrli etal., 1989; Detilleux et al., 1994). In summary, researchers have clearly established that neutrophil trafficking (Table 1.1) and killing functions (Table 1.2) are significantly impaired in periparturient dairy cows. Not only does this show that neutrophils are highly sensitive to the physiology of parturition, results of past research easily explain the increased disease susceptibility to clinical mastitis and other common diseases of periparturient dairy cows. 21 Table 1.2 - Summary of responses of blood neutrophils to parturient physiology: altered phagocytic and killing functions of the cells. Neutrophil Response Parturient Alteration Citation Chemotaxis Decreased Nagahata et al., 1988 Cai et al., 1994 Bacterial Ingestion Increased Kehrli and Goff, 1989 Kehrli et al., 1989 Cai et al., 1994 Super Oxide Production Decreased Kehrli and Goff, 1989 Myeloperoxidase Activity Decreased Kehrli and Goff, 1989 Kehrli et al., 1989 Cai et al., 1994 Kimura et al., 199% Oxidative Capacity Decreased Kehrli and Goff, 1989 Kehrli et al., 1989 B. Steroid Hormones of Parturition — Cortisol, Estradiol, and Progesterone In order for parturition to occur, concentrations of cortisol, estradiol, and progesterone must fluctuate dramatically yet in a highly coordinated fashion. Several weeks prior to parturition, cortisol and estradiol are at low levels while progesterone concentrations are high (Figure 1.3) (Smith et al., 1973; Weber et al., 2002). Several days 22 prior to parturition, cortisol and estradiol begin to increase reaching peak levels at parturition. Progesterone concentrations drop off precipitously at parturition and stay low throughout the first week postpartum. Estradiol returns to prepartum levels within the first day after parturition and the concentration of cortisol returns to mid-gestation values by the second post partum day. The growing fetus initiates and coordinates these steroid hormone fluctuations in the following manner. As the fetus begins to experience space limitations in the uterus, it releases corticotropin-relcasing hormone from the hypothalamus, which stimulates the pituitary gland to secrete adrenocorticotropic hormone (ACTH) (Senger, 1997). The ACTH stimulates production of corticosteroids by the fetal adrenal glands. Fetal cortisol crosses the placenta into the dam’s blood stream, promoting enzymatic conversion of maternal progesterone into estradiol. This removes the “progesterone block” of pregnancy, increases blood estradiol concentrations, and together with the cortisol signals initiation of parturition and lactation. Approximately two to three days before parturition, estradiol concentrations begin to rise, peaking at parturition (Figure 1.3) (Smith et al., 1973; Weber et al., 2002). The estrogen dominance over progesterone increases contractility of the uterus and secretory activity of the cervix and vagina to aid in the expulsion of the fetus (Senger, 1997). This also causes the characteristic fall in progesterone immediately before parturition, a time when cortisol concentrations also peak. The cortisol spike at parturition is likely due to a combination of fetal and maternal hormone synthesis and plays an important role in the induction of parturition and the initiation of milk secretion and let down (Smith et al., 1973). Thus, the pronounced fluctuations in blood cortisol, estradiol, and progesterone concentrations around parturition are highly coordinated and lead to successful expulsion of the fetus and subsequent lactation. However, these fluctuating steroid concentrations may also be responsible for the immune dysfunctions of parturient dairy cows (see next section). E C . a 0 Cortisol o "’ Progesterone : . o o _ I ', Estradiol _ — — — I .4“ I at / O, \ ‘ o. 'i ‘ o . a o a I. I ' n IIIIIIVI-VIE; mt ...IIIIII ., '15: r ‘ A I I I I I L I H F ’1‘} in rt! 1 l I l l l l I I -10 -8 -6 -4 -3 -2 -l 0 1 2 3 4 5 Day Relative to Parturition Figure 1.3 — Blood serum profiles of cortisol, estradiol, and progesterone in periparturient dairy cows. Figure adapted from data presented in Smith et al. (1973) and Weber et al. (2002). C. Parturient Hormones May be Linked to Neutrophil Dysfunctions in Dairy Cows The hormone fluctuations at parturition have been hypothesized to cause immune dysfunctions and disease susceptibility in dairy cows. For example, elevated blood glucocorticoid concentrations, such as occurs when cortisol spikes at parturition or when animals are treated with the hormone, induce release of bone marrow neutrophils (Hetherington and Quie, 1985). Down-regulation of L-selectin expression on the surface of blood neutrophils (Burton et al., 1995; Lee and Kehrli, 1998; Nakagawa et al., 1999; 24 Weber et al., 2002) and pronounced neutrophilia (Preisler et al., 2000a; Weber et al., 2002) have also been noted under these conditions. Preisler et al. (2000a) demonstrated that bovine blood neutrophils bind labeled glucocorticoid in vitro and that binding is decreased during parturition most likely due to occupation of the receptor by endogenous cortisol. More recently, glucocorticoid receptor mRNA levels in neutrophils have been evaluated and shown to be at highest levels of expression in conjunction with the cortisol spike of parturition (Weber et al., 2002). These studies, combined with observations from other investigators that glucocorticoids suppress many of the functions of neutrophils (Guidry et al., 1976; Detilleux et al., 1994; Burton et al., 1995; Lee and Kehrli, 1998; Nakagawa et al., 1999), strongly argue that the cortisol spike at parturition is partly responsible for the heightened disease susceptibility of parturient dairy cows. However, it is not known how cortisol and its receptor mediate these suppressing effects on the bovine immune system. The two other steroid hormones that fluctuate dramatically at parturition, progesterone and estradiol, have also been shown to affect the functions of neutrophils in many species. Chemotaxis and migration of human neutrophils were enhanced by progesterone and reduced by estradiol in an in vitro study by Miyagi et al. (1992). Migration of neutrophils under agarose was enhanced when the cells were obtained from steers treated with progesterone (Roth et al., 1982). A study of cow neutrophils collected during various stages of the estrous cycle demonstrated increased in vitro migration when serum concentrations of progesterone or estradiol were high (Roth et al., 1983). In both studies, neutrophil myeloperoxidase activity, measured in vitro, was decreased during high serum progesterone concentrations. Although the presences of receptors for progesterone and estradiol in bovine neutrophils have not yet been described, biomedical researchers have used estrogen receptor antagonists to show that human neutrophils do express 1713- estradiol receptors (Ito et al., 1995; Stefano et al., 2000). In addition, the presence of progesterone receptors in murine neutrophils has been demonstrated through the generation of progesterone receptor knockout mice (Tibbetts et al., 1999). In this study, progesterone was shown to antagonize the degranulating effects of estrogen on neutrophils in normal mice but this did not occur in the progesterone receptor knockout mice, thus supporting a direct affect of progesterone on neutrophils. Furthermore, preliminary microarray data from our laboratory suggests that bovine leukocytes express progesterone receptor mRNA before parturition and down regulate expression of this receptor during parturition (Burton and Coussens, unpublished). Therefore, it is highly likely that bovine blood neutrophils express receptors for cortisol, progesterone, and estradiol and may have the capacity to respond to parturient physiology through these receptors. IV. MATURE N EUTROPHILS ARE RESPONSIVE TO THEIR ENVIRONMENT: EVIDENCE FOR ALTERED GENE EXPRESSION Based on the granule contents and morphological appearance of mature neutrophils, researchers have long believed that these phagocytic cells are biosynthetically inactive (Bainton et al., 1971 ). The presence of dense nuclei, coarsely clumped chromatin,'few ribosomes and endoplasmic reticulum, as well as numerous cytoplasmic granules packed full of oxidative and non-oxidative antimicrobial agents has been used to support this belief. However, difficulty accepting this view begins to occur when one considers the five functions (margination, migration, phagocytosis, respiratory burst, and 26 fusion of the phagolysosome) neutrophils must be able to perform to protect the host from infection In addition, dramatic alterations of these firnctions are clearly demonstrated in research performed on blood neutrophils from parturient dairy cows (Tables 1.1 and 1.2). Evidence indicates that the steroid hormone fluctuations during periparturition may play a role in altering the phenotypes and functions of bovine blood neutrophils (Guidry et al., 1976; Preisler et al., 2000a; Weber et al., 2002). On the other hand, little published research is available showing that the neutrophil dysfrmctions at parturition occur at the level of gene expression. For just over a decade, researchers have examined what mRNAs and proteins are synthesized in mature, circulating neutrophils. Some have chosen the candidate gene approach while others have used global gene expression analysis (i.e. analysis of all mRNA present in control versus treatment sample neutrophils). Using the candidate gene approach, mRNA for MHC class I and actin as well as proteins such as F c receptor, CD18, MHC class I, and actin were shown to be synthesized in human blood neutrophils (Jack and F earon, 1988). Others demonstrated that neutrophils are able to synthesize a wide variety of cytokines, including interleukin-13 (IL-13), IL-6, IL-8, and interferon-alpha (IFN-a) (review by Lloyd and Oppenhein, 1992; Cassatella, 1999). In 1998, Itoh et al. identified approximately 750 genes that are active in resting blood neutrophils. These genes encoded for DNA-binding proteins, cytokines, MHC proteins and receptors, and cell surface membrane proteins. More recently, global gene expression analysis approaches have been used to document altered gene expressions in blood neutrophils activated with cytokines (Cowling and Bimboim, 2000) or various pathogenic and non- pathogenic bacteria (Newberger et al., 2000). These studies have not only demonstrated that blood neutrophils are biosynthetically active but that global gene expression changes relate to the five functions key to neutrophil mediated immune defense against pathogens. In retrospect, it makes perfect sense that a cell so integral to host defense against infection has firll synthetic capacities. To date, altered gene expression in bovine neutrophils during the periparturient period has only been reported in one study (Weber et al., 2002). This study was performed to elucidate the mechanism behind altered neutrophil trafficking in parturient dairy cows (Table 1.1). Weber et al. (2002) demonstrated rapid down regulation of L- selectin mRNA abundance and more gradual down-regulation of glucocorticoid receptor mRNA abundance in blood neutrophils of periparturient Holstein cows. Furthermore, these investigators showed that repressed L-selectin gene expression was highly correlated with the surge in blood cortisol concentrations, down-regulation of surface L-selectin on the neutrophils, and neutrophilia. Weber et al. (2002) thus concluded that bovine neutrophils respond to parturient cortisol with altered gene expression that influenced the phenotype and trafficking functions of the cells. Thus, it is feasible that cortisol and the other steroids of bovine parturition have significant effects on expression of multiple genes in bovine blood neutrophils. If true, these altered gene expression profiles could certainly begin to explain why dairy cows become immunosuppressed and highly susceptible to mastitis and other diseases around parturition. The overall hypothesis of the current thesis research is that parturition induces changes in bovine blood neutrophil gene expression. The following three objectives were put forth to test this hypothesis: 28 Objective 1: Identify and characterize differential gene expression in bovine blood neutrophils from periparturient dairy cows; Objective 2: Determine relationships between altered mRNA abundance of neutrophil genes and concentrations of blood steroids (cortisol, estradiol, and progesterone) around parturition; Objective 3: Obtain putative identities and functions of differentially expressed genes discovered in objective 1. The research presented in Chapter Two of this thesis is novel because it demonstrates that multiple neutrophil genes respond to parturition with reduced mRN A abundance, potentially due to changes in serum steroid concentrations. These results may aid in the understanding of why dysfunction of normal neutrophil functions (including surveillance, recmitment, receptor-mediated phagocytosis, respiratory burst, and fusion of the phagolysosome) occur in periparturient dairy cows. The importance of these observations is that upon further research, the identified genes could become targets for new drug development or genetic selection strategies aimed at bolstering innate immune defenses and disease resistance in dairy cows during the periparturient period. CHAPTER TWO Altered Blood Neutrophil Gene Expression in Periparturient Dairy Cows 1. ABSTRACT The periparturient dairy cow undergoes a plethora of changes, including changes in the immune system, that lead to profound effects on animal health. Of the immune cells affected at parturition, the neutrophil has been of particular interest due to its primary role in innate immune defense against mastitis. Neutrophil function around parturition has been shown by assay of cellular functions and protein and/or enzymatic activities to be- reduced, but it has not been discerned at what level these altered functions occur, including the possibility that there are alterations in neutrophil gene expression during periparturition. The hypothesis of the present study was that global gene expression in circulating neutrophils is altered during periparturition. The main objectives of the study were to detect and characterize parturition-induced changes in neutrophil mRNA abundance, to determine if altered mRNA levels are associated with changes in the main steroid hormones of bovine parturition, and to obtain identities of the genes exhibiting altered expression during the periparturient period. Preliminary assessment of gene expression was done using differential display reverse transcription polymerase chain reaction (DDRT-PCR) followed by high throughput cDNA dot blot analysis. mRNA size and number of transcripts were determined using Northern blot analysis. Detailed mRNA profiles were then characterized using quantitative slot blot analysis. Putative identities for two cDNAs with pronounced repression of mRNA following parturition (P 5 0.02) had high DNA sequence homology with genes for 30 bovine mitochondrial cytochrome b (cytb) and rig/ribosomal protein SIS (rig/RPS15), which are genes of known importance in energy metabolism and translation of mRNA into protein (respectively). Cytb mRNA abundance profiles were significantly correlated with blood progesterone profiles (r = 0.44) and rig/RPSIS mRNA abundance with both progesterone and estradiol profiles (r = 0.35 and r = 0.36, respectively). Results of this study show for the first time that mRNA abundance of genes regulating basic functional machinery of bovine neutrophils is depressed by parturition, possibly due to the influences of steroid hormones on gene expression. 11. INTRODUCTION Parturition is a complex time for dairy cows, nutritionally, hormonally, and immunologically. It is during this time that cows experience an increased susceptibility to intrarnammary infections and clinical mastitis, quite possibly as a result of decreased immunocompetence observed during periparturition (Oliver and Sordillo, 1988; Cai et al., 1994, Mallard et al., 1998). Altered functions of blood neutrophils in periparturient cows are of particular interest to researchers due to their importance in innate mammary defense against mastitis (Guidry et al., 1976; Kehrli and Harp, 2001). In an attempt to explain immune dysfunctions of parturition, investigators have speculated that fluctuating metabolic and reproductive hormones are involved in disease susceptibility because immune dysfunction profiles tend to associate temporally with fluctuating hormone profiles (Goff and Horst, 1997; Ingvartsen and Andersen, 2000). As a part of the first line of immune defense, mature blood neutrophils perform five main functions to successfully clear pathogens that infect peripheral tissues. Circulating neutrophils marginate along blood vessel walls, after which they rapidly migrate into tissues if infection is detected Once in the tissue, neutrophils engage in the seeking out and ingestion of pathogens using energy-requiring, receptor mediated phagocytosis. Phagocytosis stimulates neutrophils to undergo massive respiratory burst, producing multiple reactive oxygen species and myeloperoxidase that initiate oxidative damage to ingested bacteria. Finally, vesicles containing the phagocytosed bacteria fuse with vesicles containing lysosome to complete pathogen killing via enzymatic lysis. Several of these functions are altered during parturition in dairy cows. For example, neutrophil margination and migration, known together as trafficking, are profoundly decreased due to reduced expression of key adhesion molecules on the surface of blood neutrophils (Cai et al., 1994; Lee and Kehrli, 1998; Weber et al., 2002). Similarly, neutrophil chemotaxis, myeloperoxidase activity, superoxide production, and general phagocytic ability are dramatically altered around parturition (Guidry et al., 1976; Nagahata et al., 1988; Kehrli et al., 1989; Cai et al., 1994; Detilleux et al., 1994; Lee and Kehrli, 1998; Kimura et al., 1999b). Each of these altered neutrophil functions could help explain the increased rates of mastitis that occur in periparturient cows. Although the studies above have clearly demonstrated the presence of multiple neutrophil dysfunctions around parturition, few report mechanisms describing why or at what level these dysfunctions occur. Indeed, most of the studies have focused on measuring altered protein levels and enzyme activities because leukocyte researchers have generally believed that blood neutrophils are terminal cells with little to no capacity to synthesize new molecules (Bainton et al., 1971). Recently, however, several studies using human neutrophils have begun to challenge this belief by showing that approximately 700 neutrophil genes are active during the resting state (Itoh et al., 1998). Others have begun to explore global gene expression changes in neutrophils presented with bacteria and cytokine stimuli (Lloyd and Oppenhein, 1992; Newberger et al., 2000). To date, only one study demonstrated gene expression changes in neutrophils in response to parturition (Weber et al., 2002). During bovine parturition, blood neutrophils are exposed to a rapidly changing hormonal environment to which they appear to be extremely sensitive. For example, observational studies have described relationships between changing corticosteroid levels and altered neutrophil phagocytic ability during parturition (Guidry et al., 1976), as well as generally altered neutrophil functions in association with changing estradiol and progesterone levels (Roth et al., 1982; Roth et al., 1983; Goff and Horst, 1997). Another study demonstrated that chemotaxis and migration increase while blood progesterone concentrations are high and decrease when neutrophils are exposed to high levels of blood estradiol (Miyagi et al., 1992). Still others have demonstrated that the dramatic increase in blood cortisol concentrations at parturition have pronounced down-regulating effects on neutrophil L-selectin expression (Lee and Kehrli, 1998; Weber et al., 2002), which correlates well with altered trafficking of these cells (Preisler et al., 2000a; Weber et al., 2002). In addition, Preisler et al. (2000a) began to address the issue of mechanism by showing that bovine neutrophils express glucocorticoid receptors that appear to become fully occupied by cortisol around parturition. This substantiates arguments by many researchers that bovine neutrophils are sensitive to hormones of parturition and respond to them with altered functional capacities. 33 To our knowledge, few studies have been published that describe alterations in the expression of genes that regulate neutrophil functions around parturition, nor whether such changes may relate to fluctuating blood steroid hormone concentrations. Our overall hypothesis is that parturition induces changes in bovine blood neutrophil gene expression. The current study was designed to begin testing this hypothesis. The objectives were to determine if parturition induces changes in blood neutrophil mRNA abundance of multiple genes, to determine if altered mRNA levels relate to blood concentrations of cortisol, estradiol, and (or) progesterone, and to obtain putative identities of neutrophil genes with altered mRNA abundance. Several experiments were used to complete the first objective. These included a primary screening for differential gene expression using differential display reverse transcription polymerase chain reaction (DDRT-PCR) of total leukocyte RNA, followed by secondary screening of altered neutrophil gene expression using high throughput cDNA dot blot analysis. Confirmation of differential mRNA abundance in neutrophils was performed using Northern blot analysis and characterization of changing mRNA levels by quantitative slot blot analysis (Figure 2.1). For objective 2, relationships between mRNA abundance profiles (from slot blot analysis) and steroid hormone profiles were determined statistically by correlation analysis. Finally, objective 3 was completed by sequencing the cDNAs encoding for mRNAs determined in objective 1 to be differentially expressed in neutrophils during parturition. These sequence data were subjected to BLASTn analysis to obtain putative identities of the differentially expressed neutrophil genes. 34 III. MATERIALS AND METHODS A. Animals and Sample Collection This work was performed using five primiparous, parturient Holstein cows as the test animals. Samples from several of these cows were utilized in multiple experiments (Figure 2.1). Two primiparous, mid-gestation cows were sampled to serve as controls for DDRT-PCR. All animals were fed and housed according to standard operating procedures at the Michigan State University Dairy Teaching and Research facility, a seven minute drive from our laboratory. Use of these animals for the experiments described below was approved by the All University Committee for Animal Use and Care of Michigan State University. Samples were collected at various times for use as described in Figure 2.1. Parturition is called day ((1) O. The control cows were sampled once each day that a periparturient test cow was sampled to provide ample amounts of control leukocyte RNA for DDRT-PCR. Test cows were sampled as needed on d -14, -12, -8, -4, -1, 0, 0.25, 0.5, l, 1.5, 2, and 7 relative to parturition for leukocyte and neutrophil test samples to complete all experiments shown in Figure 2.1. All samples were obtained as described in Weber et al. (2002). Briefly, 60 to 80 ml of blood from the coccygeal (tail) vein was collected into 6-ml evacuated tubes containing 1.0 ml of acid citrate dextrose (ACD) anti- coagulant using 20-gauge, 2.5-cm multi-sample needles (Fisher Scientific; Pittsburgh, PA). All samples were placed immediately on ice and transported back to the laboratory for processing. A lO-ml evacuated tube without anti-coagulant was also collected at each sampling for serum harvesting. 35 Figure 2.1 - Several methods were utilized to address our hypothesis that parturition induces changes in bovine blood neutrophil gene expression. Shown are the techniques, sample types, cows, and sample times we used to begin testing this hypothesis. Fourteen leukocyte genes were identified as differentially expressed upon primary screening, twelve of which were decreased in neutrophils of periparturient cows upon secondary screening. In the current study, two of these genes were pursued through the entire flow of experiments described in the figure. Figure 2.1 Flow of Experiments Primary Screening 2) DDRT-PCR Total leukocyte RNA Differential gene expression around parturition vs. mid-gestation 2 test cows“ (d -12, -l, 0, 0.25) 2 control cows (pooled samples from d -l 75 through -130) Secondary Screening 2 cDNA Dot Blot Hybridization Neutrophil RNA mRNA abundance before vs. just after parturition 1 test cow (d -14 and 0.5) Characterization :> Northern Blot Hybridization Neutrophil RNA mRNA abundance before, at, and just after parturition 1 test cow (d -8, 0, 0.25) Expression Profiles 2 Slot Blot Hybridization Neutrophil RNA mRNA abundance across multiple periparturient time points 4 test cows (d -8, -4, 0, 0.25, 0.5, 1, 1.5, 2, 7) ‘ I Correlation Analysis Between slot blot and sermn hormone profiles Cortisol, estradiol, and progesterone 4 test cows (d -8, -4, 0, 0.25, 0.5, 1, 1.5, 2, 7) Putative Gene Identification DNA sequence and BLASTn analysis ‘Test cows = Parturient Control cows = Mid-gestation Day (11) = Day relative to parturition B. Blood Leukocyte Purification and RNA Isolation Total leukocytes for preliminary DDRT-PCR screening of differential gene expression (Figure 2.1) were obtained from ACD-anticoagulated blood samples on d -12, -l, 0, and 0.25 from two test cows, and from the two control cows as described in Burton et al. (2001). Briefly, each 6-ml tube of whole blood was decanted into a 50-ml conical tube containing 24 ml cold hypotonic lysis solution (10.56 mM NazHPO4, 2.67 mM NaHzPO4, pH 7.3) and agitated for 90 seconds. A 12 ml volume of cold hypertonic restore solution (10.56 mM NazHPO4, 2.67 mM NaHzPO4, 0.43 M NaCl, pH 7.3) was added to restore isotonicity. Leukocytes were pelleted by centrifugation (1000 X g for 5 minutes at 4°C) followed by aspiration of the supematants. The cells were then washed in 15 ml ice cold PBS and centrifuged (1000 X g for 5 minutes at 4°C). Supematants were aspirated and discarded and cell pellets lysed in 4 ml TRIzol reagent (Life Technologies; Rockville, MD) for 10 minutes at room temperature. The time from blood sampling to lysis of leukocyte pellets in TRIzol was approximately 1.5 hours. Blood neutrophil samples for cDNA dot blot, Northern blot, and slot blot analyses (Figure 2.1) were obtained from test cows at (1 -l4, -8, -4, 0, 0.25, 0.5, 1, 1.5, 2, and 7 for gene expression analysis. Upon arrival in the laboratory, ACD blood tubes were centrifuged at 1000 X g for 20 minutes at 4°C to separate plasma and buffy coat from the red cell pack. Plasma, buffy coat, and approximately two-thirds of the red cell pack were discarded while the remaining red cell pack was transferred to a 50-ml conical tube containing 20 ml cold PBS. Additional cold PBS was added to a final volume of 35 m1. This was then under layered with 12 ml of 1.084 g/ml Percoll (Sigma Chemical Company; St. Louis, MO). Neutrophils were pelleted through the Percoll by 38 centrifugation at 400 X g for 40 minutes at 22°C. Supernatant, mononuclear layer, and Percoll were aspirated and discarded. Lysis of erythrocytes was performed as described above for total leukocyte lysis. Neutrophil pellets from a single cow and sample time were pooled for washing with 20 ml cold PBS. Following a final 5 minute centrifugation (1000 X g, 4°C), neutrophil pellets were lysed in 8 ml TRIzol reagent (Life Technologies; Rockville, MD) for 10 minutes at room temperature. Total processing time for neutrophil isolation from collection to TRIzol was less than three hours. Neutrophil purity was analyzed by flow cytometry (see Appendix One, Figure A.l). Prepartum samples were greater than 80% neutrophils while post partum samples exhibited greater than 90% neutrophils. All samples, total leukocytes and purified neutrophils, were stored at —80°C in TRIzol reagent. Total RNA was isolated from each cell preparation according to the manufacturer’s instructions. Following isolation, quantity and quality of RNA was evaluated in each sample using 260 and 280 nm spectrophotometry readings (DU-650 spectrophotometer; Beckrnan, Schaumberg, IL). C. Primary Screening of Differential Gene Expression in Total Leukocytes of Periparturient Cows using DDRT-PCR DDRT-PCR was utilized in initial screening for differences in gene expression in all blood leukocytes (Liang and Pardee, 1992). Total leukocyte RNA samples from two test cows on d -12, -1, 0, and 0.25, as well as samples from both control cows (pooled over multiple days), were treated with DNase 1 (Life Technologies, Rockville, MD) to remove contaminating genomic DNA prior to reverse transcription. A total of 18 39 differential display primer pairs were utilized in this study. Fourteen of the primer pairs were generated by the United States Pig Genome Coordination Program (provided by Dr. Max Rothschild, U.S. Pig Genome Coordinator, and donated for use in this project by Dr. Catherine Ernst; Department of Animal Science, Michigan State University). The remaining four primer pairs were obtained from the Heiroglyph mRNA kit Profile System for Differential Display Analysis (Beckrnan Coulter, Inc.; Fullerton, CA) and were donated by Dr. George Smith (Department of Animal Science, Michigan State University). To start, reverse transcription (RT) was performed on total leukocyte RNA (0.] ug/ul) using 1 unit Superscript II reverse transcriptase (Life Technologies; Rockville, MD), 1X First Strand Buffer, 0.1 M DTT, 250 M of each dNTP, and 2.0 M of various 3’ anchored primers. The cDNAs generated through RT were amplified by polymerase chain reaction (PCR) using Taq DNA Polymerase (Life Technologies; Rockville, MD), 10X PCR Buffer, 50 mM MgC12, 250 M of each dNTP, 2.0 uM of the 3’ anchored primer used in the RT reaction, and 2.0 M of various 5’ arbitrary primers. Also included in these PCR reactions was a-°°P-dATP for subsequent visualization of differentially expressed bands. All PCR products and a negative control (no RNA added) for each primer pair were electrophoresed on 5.2% polyacrylamide denaturing gels. Following electrophoresis, gels were dried to 3MM Chr Whatrnan chromatography paper (Fisher Scientific; Pittsburgh, PA) with a Slab Gel Dryer SDGZOOO (ThermoQuest Corp; Marietta, OH). Autoradiographs were produced for band visualization by 24 hours of exposure of the dried gels to BioMax MR X-ray film (Fisher Scientific; Pittsburgh, PA). Those differential display products (or amplicons) with putative changes in expression 40 were cut out and eluted from the gel matrix for probe generation and DNA sequence analysis. D. Secondary Screening of Differential Gene Expression in Neutrophils using Dot Blot Hybridization Excised amplicons were eluted from the gel matrix with Tris-EDTA (pH 7.4). Reamplification was performed by PCR using the same primer pairs and PCR conditions that created the amplicons. The reamplified amplicons were analyzed by agarose gel electrophoresis (1.2% gel) and visualized by ethidium bromide staining to confirm single bands. The amp] icons were then ligated into the pGEM-T Easy vector (Promega; Madison, WI) and resulting recombinant plasmids were transformed into JM109 competent cells (Promega; Madison, WI). Positive clones containing amplicons were selected by blue/white colony screening. Plasmids from white colonies were isolated using the Mini-prep Plasmid DNA Isolation kit (Promega; Madison, WI) and subjected to Eco RI restriction enzyme digestion (Life Technologies; Rockville, MD) to release the amplicons for confirmation of their presence by agarose gel electrophoreses and ethidium bromide staining. Finally, PCR was performed using M13 Forward and Sp6 sequencing primers (Promega; Madison, WI) to amplify a large quantity of each amplicon for validation of altered gene expression in neutrophils using cDNA dot blot hybridization. For cDNA dot blot hybridizations, PCR reamplified amplicons were denatured in a 0.4 M NaOH, 0.01 M EDTA solution for 10 minutes at 95°C and placed immediately on ice. Positively charged nylon membranes (Life Technologies; Rockville, MD) were pre-soaked in 6X SSC and each dot was rinsed with sterile water. The membranes were 41 then dotted in quadruplicate with 0.50 ug of each amplicon (determined by serial dilution and gel electrophoresis using a molecular ruler containing known concentrations of DNA at specific sizes) using a dot blot manifold (The CONVERTIBLE Filtration Manifold System, Life Technologies; Rockville, MD). The hybridization positive control was B- actin (described in Weber et al., 2002), 0.05 ug of which was also dotted in quadruplicate on both membranes. All dots were rinsed with 0.4 M NaOH followed by UV cross- linking of the amplicon to the membrane. The duplicate membranes were dotted on the same day and prehybridized overnight at 42°C using a solution containing 50% fonnamide, 5X Denhardt’s, 6X SSC, 0.1% SDS, 0.05 M phosphate buffer (pH 6.8), 1.0 mM EDTA, and 0.15 mg per ml yeast tRNA. Hybridizations were performed using or- 32P-dCTP (NEN Life Science Products, Inc.; Boston, MA) labeled cDNA created by reverse transcription of 5.0 pg neutrophil RNA from a single test animal sampled on d — 14 and 0.5 relative to parturition. Each labeled cDNA ((1 —14 versus (1 0.5) was hybridized for 18 hours with one of the two dotted membranes. Following hybridization, membranes were washed twice at room temperature in 2X SSC, 0.1% SDS for 15 minutes followed by twice at 65°C in 0.2X SSC, 0.1% SDS for 15 minutes prior to exposure to BioMax MS film (Fisher Scientific; Pittsburgh, PA) for 96 hours at -—80°C with an intensifying screen (Fisher Scientific; Pittsburgh, PA). Relative hybridizations of labeled cDNA from test samples to the spotted amplicons were estimated using a scanning densitometer (GS-710 Calibrated Imaging Densitometer and Multi-Analyst Software; BioRad; Hercules, CA) and recorded as dot density units. 42 E. Northern Blot Characterization of Neutrophil Genes Those neutrophil genes represented by our amplicons as being differentially expressed before and after parturition in the dot blot analysis were further analyzed by Northern blot hybridization to determine size and number of mRNA transcripts in blood neutrophils (Figure 2.1). A portion of the PCR amplified amplicon (described above) was gel purified to remove residual plasmid DNA using the Wizard PCR Prep kit (Promega; Madison, WI). These were then used as probes for Northern blot analyses. Approximately 100 ng of PCR products were used for dual labeling with 01-3 2P-dCTP and (1-3 2P-dATP (NEN Life Science Products, Inc.; Boston, MA) using the random prime method (F einberg and Vogelstein, 1983). A neutrophil Northern blot was generated using three RNA samples from a parturient test cow (d —8, 0, and 0.25) as described in Weber et al. (2002). Briefly, 10 ug of RNA from each time point was electrophoresed on a 1.2% denaturing agarose gel and transferred to a nylon membrane using a Turbo Blotter (Schleicher and Schuell; Keene, NH). RNA was UV cross-linked to the membrane. Prehybridization was carried out for 4 hours at 42°C in the same pre-hybridization cocktail described for the dot blots above. Hybridization was carried out for ~16 hours at 42°C in the same buffer using the purified, labeled amplicon. Membranes were washed once at room temperature with 2X SSC, 0.1% SDS for 15 minutes and 3 times with 0.1X SSC 0.1%SDS at 60°C prior to exposure using BioMax MS film (Fisher Scientific; Pittsburgh, PA). Time of exposure at —80°C varied from one to four days depending on the amplicon. Membranes were stripped between hybridizations with different amplicons and reprobed with a-32P-labeled B—actin cDNA, (obtained from Dr. L. Kedes; Stanford University School of Medicine; Palo Alto, CA), for normalization purposes (Weber et al., 43 2002). Approximate sizes of the mRNA transcripts hybridized by the various amplicons were estimated using the 28S and 188 rRNA bands (4718 and 1874 base pairs (bp), respectively) as markers from an ethidium bromide stained lane of the gel (Current Protocols in Molecular Biology, 1995). F. Characterization of Neutrophil mRNA Abundance Profiles from Periparturient Cows Neutrophil slot blots were used to quantify mRNA abundance of genes confirmed to be differentially expressed by Northern blot, as described in Weber et al. (2002). In brief, 5 ug of neutrophil RNA from 4 test cows on d -8, -4, 0, 0.25, 0.5, 1, 1.5, 2, and 7 (Figure 2.1) were spotted on a nylon membrane using a slot blot manifold (PR600 slot blot filtration manifold; Hoefer Scientific; San Francisco, CA) and cross-linked to the membrane using UV-light. Blots were then prehybridized, hybridized, and washed as outlined above for Northern blots. Autoradiographs were obtained after various hours of blot exposure to BioMax MS film (Fisher Scientific; Pittsburgh, PA) at -—80°C, depending on the hybridized amplicon probe. The slot blot was stripped between each hybridization with a new amplicon probe and, finally, with the B-actin probe. Relative mRNA abundance was quantitated using scanning densitometry units as described above, normalized to the relative abundance of B-actin mRNA, and recorded as ratio values. G. Radioimmunoassay of Serum Cortisol, Estradiol, and Progesterone Serum collections and assay of serum cortisol, estradiol, and progesterone levels (Figure 2.1) is described in Weber et al. (2002). Briefly, sera collected from the four test 44 cows sampled on d -8,-4, 0, 0.25, 0.5, 1, 1.5, 2, and 7 was harvested from clotted blood (IO-ml draw with no anti-coagulant) following centrifugation of the blood tubes at 1000 X g at 4°C for 20 minutes. Sera were stored at -20°C until assayed. Serum cortisol and progesterone concentrations were assayed in duplicate using commercially available Coat-A-Count RIA kits (Diagnostic Products Corporation; Los Angeles, CA). Sera for the estradiol RIA assay were extracted with diethyl ether before use in duplicate in the assay (Coat-A-Count RIA kit; Diagnostic Products Corporation; Los Angeles, CA). H. Identification of Differentially Expressed Neutrophil Genes Amplicon nucleotide sequences were obtained using fluorescent ABI dye- Tenninator cycle sequencing kits (Perkin-Elmer/ABI; Palo Alto, CA) in conjunction with M13 forward and Sp6 sequencing primers. Prepared samples were loaded onto a 4.75% polyacrylamide sequencing gel and analyzed using an ABI 373 Automated DNA Sequencer (Perkin-Elmer/ABI; Palo Alto, CA). Putative identities were obtained by comparing sequences to sequences available in DNA databanks using BLASTn (basic local alignment search tool - nucleotide), available through the National Center for Biotechnology Information (NCBI) web page (hgp://www.ncbi.nlm.nih.gov/BLAST) and The Institute for Genomic Research (http://wwwfigrorg/tdb/tgi.shtrnl). 1. Statistical Analysis Ratio data (mRNA abundance/B-actin) from the slot blots were analyzed using the PROC MIXED firnction of SAS (SAS Institute Inc., 1996) and the following statistical model: 45 Ya‘lltpi * “1+9"; where Y; is the observation of the i‘11 cow on the j‘h day relative to parturition, u is the overall mean, p. is the random effect of the im cow, a, is the fixed effect of the fh day relative to parturition, and e,- is the normal error if. Statistical analysis was performed on data collected from the slot blot analysis of neutrophil mRNA abundance from four parturient test cows (d -8, -4, 0, 0.25, 0.5, 1, 1.5, 2, and 7). The effect of day relative to parturition was tested first by analysis of variance, assuming a compound symmetry correlation of the residuals, and then by pair wise comparisons between least squares means of post-parturient samples and prepartum test samples (d -—8 and —4). Significance between these means was declared when P 5 0.05. Analysis assuming a first-order antedependence correlation of the residuals resulted in an even more significant effect of day relative to parturition (P < 0.0001; statistical model and data not shown). The cortisol, estradiol, and progesterone data were natural logarithm-transforrned prior to analysis of variance using the same statistical model presented above to test effect of day relative to parturition. The PROC CORR function of SAS (SAS Institute Inc, 1990) was used to determine Pearson Product Correlations between the transformed hormone data sets and normalized gene expression data sets from slot blot analyses. Statistical significance was acknowledged when P 5 0.05. 46 IV. RESULTS A. Primary Screening for Differential Gene Expression Induced by Parturition in Total Leukocytes DDRT-PCR identified putative differentially expressed leukocyte genes throughout the periparturient period as well as between parturition and mid-gestation samples. A total of eighteen 3'-5' primer pair combinations were used for DDRT-PCR, from which fourteen differentially expressed cDNA fragments (amplicons) were identified (some examples in Figure 2.2). These fourteen amplicons were named based on the 3’ and 5’ primer numbers used to generate them, and whether the cDNA appeared to be induced (I) or repressed (R) around parturition within test samples or between control and test samples. For example, if 3’ anchored primer number 5 and 5’ arbitrary primer number 15 yielded an amplicon that appeared to be induced its name would be 5- 151. If more than one amplicon was identified per primer pair, an additional letter (a, b, c, etc.) was added to indicate the relative position of the cDNA from the top of the gel (e. g. 5-151a, 5-15Ib, 5-151c, etc.). Figure 2.2 shows three such differentially expressed cDNAs. The first was repressed throughout all periparturient time points evaluated (2- 1 IR), the second was induced at parturition (2-1 11), and the third was induced throughout the entire periparturient period (9-71). Of the fourteen amplicons pursued, a total of eight appeared to be induced in leukocytes during parturition while the remaining six exhibited repressed expression during periparturition. 47 Figure 2.2 - Primary screening for differential gene expression in total leukocytes from parturient and mid-gestation cows was performed using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Shown are three examples of differentially expressed cDNAs observed when total leukocyte RNA from two mid- gestation control cows (M1 and M2; pooled samples from d -l 75 to -130 prepartum) and two parturient test cows (P1 and P2; sampled on d -12, -1, O, 0.25 relative to parturition) were electrophoresed on a 5.2% polyacrylamide gel. The sample in lane N was the DDRT—PCR negative control and received no RNA during reverse transcription. Amplicons were named based on 3’ and 5’ primer numbers and if they appeared induced (I) or repressed (R) by parturition. In the top panel, amplicon 2-11R was shown to be repressed in leukocytes throughout the periparturient period when compared to mid- gestation. The middle panel shows 2-111 induced at (d 0) and just after parturition (d 0.25) compared to prepartum (d -12, -l) and in mid-gestation. In the bottom panel, 9-7I showed strong induction throughout the entire periparturient period compared to mid- gestation. These amplicons, as well as eleven others, demonstrated differential expression in leukocytes of both parturient cows compared to both mid-gestation cows, thus they were excised from the DDRT-PCR gels and used for secondary screening of differential gene expression in periparturient neutrophils. Additional DDRT-PCR gel pictures in Appendix One, Figure A.2. 48 Figure 2.2 Representative DDRT-PCR Gels Cow: M1 P1 P2 P1 P2 P1P2 P1P2 M2N Sample d-12 d-l d0 d0.25 2-111 49 B. Secondary Screening of Differential Gene Expression Induced by Parturition The fourteen amplicons were successfully cloned and amplified by PCR into crisp, single bands (Figure 2.3). Comparison of the reamplified amplicons with a molecular marker showed that the cDNAs ranged from approximately 350 to 1100 bp in length. For secondary screening, confirmation, and characterization of differential gene expression, RNA from purified neutrophils was used instead of the total leukocyte RNA utilized for the primary screening. The reamplified amplicons were spotted in quadruplicate on duplicate nylon membranes, which were then interrogated with neutrophil RNA collected from a single test cow on d -—14 and d 0.5 relative to parturition (autoradiographs of dot blots in Appendix One, Figure A.3). Mean dot densities for all amplicons analyzed are shown in Figure 2.4 (separated into two graphs based on levels of dot intensity represented by mean density values). Upon initial observation, amount of labeled cDNA that hybridized to 12 of the 14 amplicons appeared altered between d -14 and d 0.5 neutrophil samples from this one parturient test cow. B-actin mRNA abundance exhibited very little change in pre- versus post partum samples (see Appendix One, Figure A.4) and has been shown to not be significantly affected by day relative to parturition by slot blot analysis (Weber et al., 2002). Thus, our secondary screening clearly demonstrated a decrease in mRNA abundance for several neutrophil genes in one parturient cow. Confirmation of these observations were pursued through the use of Northern and slot blot analyses on neutrophil RNA. 50 and ”m— 2:: .Hmd n: 25— .MONS "mm 2:: .GTK "a 2.x. .55. -m u: 2:: :3: Tm A: can. 4&on "a one. 4&on "a 2:: £07m K. 2:: £on 6 can. HSTN um 2:: .SE-N "v 2:: .m: -m ”n 2:: .2 2.. "N 2:: :23 338—02 3 2:5 ._om 05 t8 _ 28— E 550% 20 £23.53 638%: 9355 $32 830222 5.5.50.5 33 Nm 05 mi? 33.: 803 map—38.28%“ 05m .35 cosmoEEEmE m3“ 80¢ $58 03.5 .6536 3.36% Em wcioozo 808mm ..\e N; .mm_ @8535 .83 when owns 8: 8 Own 80¢ 8N5 E cow—EL :oceoEEEmn: mom wage—Be was coco—o 203 8:38—32 v83 38 E :23296 2% Bushfire mo 9:528 befits macaw BEEF—ow mace—9:“ :ootsom - n.~ 9...»...— 3 2: 32a ‘ I. .5 am it 3 2K .1 I 3 2:: m~3m-~=3a w b e m w m N fl 2:3 28:95“ 69:29 .3 m_mo..c__aeboo_o .ow omeawwe. 51 Figure 2.4 - Secondary screening for differential gene expression in neutrophils of a periparturient test cow using cDNA dot blot hybridization. Briefly, the fourteen amplicons identified in total leukocytes as either induced (I) or repressed (R) around parturition, were spotted on two dot blots in quadruplicate. Radiolabeled cDNA generated from neutrophil RNA sampled at d -14 (dark bars) and d 0.5 (light bars) were used to interrogate the blots. Shown are the mean densitometry units (4: SEM) of the quadruplicate dots for each amplicon spotted. Data in this figure demonstrates that post partum neutrophil mRNA hybridized to twelve of the fourteen amplicons at substantially lower levels than prepartum neutrophil mRN A. Genes represented by amplicons 3-101a and 9-21 were studied in greater detail in subsequent Northern and slot blot hybridization experiments (Figures 2.5 and 2.6). Figure 2.4 Mean Densitometry Values Mean Densitometry Values 0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 Neutrophil gene expressions — higher intensity dots 9-21 3-101a 3-101b 3-11Ra Amplicon Name I Before 1:] After Neutrophil gene expressions — lower intensity dots 2-111 2-11R 2-l4la 2-14lb 3—10Ra 3-10Rb 3-11Rb 7-161 7-2011 9-71 Amplicon Nam IBefone ElAfter 53 C. Characterization of Neutrophil mRNA Hybridized by 3-101a and 9-21 To date, two neutrophil genes have been followed through the entire scheme outlined in Figure 2.1. The genes encoded by amplicons 3-101a and 9-21 showed 57 % and 46 % less mRNA in neutrophils right after parturition compared to prepartum, respectively, as determined by cDNA dot blot analysis (Figure 2.4). They were selected for further characterization based on this pronounced repression after parturition in addition to the high level of expression prepartum. A Northern blot generated using RNA from purified neutrophils from one parturient test cow sampled on d -8, 0, and 0.25 was utilized to characterize size and number of transcripts for mRNA complimentary to 3-101a and 9-21 (Figure 2.5). Each of these amplicons hybridized to a single transcript, 3-101a at approximately 1100 bp and 9- 21 at approximately 500 bp. Transcripts hybridized by these two amplicons also demonstrated decreased mRNA abundance at and after parturition, an effect that did not appear to be due to unequal loading of the sample RNAs (see B-actin bands in Figure 2.5). This is the second parturient cow in which decreased mRNA abundance of 3-lOIa and 9-21 was observed (the first was the parturient cow sampled for dot blot hybridization). Size, number of transcripts, and differential expression of genes represented by the remaining amplicons will be confirmed in subsequent studies by our group. 54 Northern blot Sample Time: -8 0 0.25 Lane: 1 2 3 4'. .. : ‘f. ,‘t “ ' (~1,1 kb) 9-21 (~05 kb) B-actin (~ 2.6 kb) Figure 2.5 — Characterization of size and number of transcripts complimentary to 3-101a and 9-21 in neutrophils from a periparturient test cow using Northern blot hybridization. Neutrophil RNAs collected on d -8, 0, and 0.25 were subjected to electrophoresis and the resulting Northern blot was probed with amplicons 3-lOIa (top panel) and 9-21 (middle panel), as well as B-actin (a housekeeping gene used to verify even loading of the lanes; bottom panel). A single transcript was detected at approximately 1100 bp using the 3-101a probe while the transcript detected by the 9-21 probe was approximately 500 bp. Data in this figure show that parturition induced down regulation of 3-101a and 9-21, confirming results of our dot blot analysis shown in Figure 2.4. The greater intensity of the B-actin band in neutrophil RNA in lane 3 was from overloading of this lane relative to lanes 1 and 2. 55 D. Characterization of 3-101a and 9-21 mRNA Abundance Profiles in Neutrophils from Periparturient Cows Quantitative slot blot analysis was utilized to characterize mRNA abundance profiles of 3-101a and 9-21 in neutrophils from four periparturient dairy cows. Sample days included d -8, -4, O, 0.25, 0.5, 1, 1.5, 2, and 7, relative to parturition on d 0. Blots were probed with 3-101a, stripped, probed with B-actin, stripped, and probed with 9-21. Resulting mRNA abundance data were recorded as B-actin normalized abundances of transcripts hybridized by 3-IOIa and 9-21. Statistical analysis of these data with an assumption of compound symmetry correlation of the residuals showed that day of parturition significantly affected 3-101a (P = 0.018) and 9-21 (P = 0.003) mRNA abundance. When comparing daily least squares means for 3-101a mRNA abundance at post partum time points to the d -4 time point (Figure 2.6a), significant down-regulation was observed that began on d 0.25 (P 5 0.05) and continued through d 0.5, 1, 1.5, and 2 (P 5 0.01). Relative to d -8, there was significant down regulation of 3-101a on d 0.5, 1, 1.5, and 2 post partum (P g 0.05). Based on the least square means for 9-21, the gene corresponding to this amplicon appears to have slightly lower mRNA abundance in neutrophils than that corresponding to 3-101a (Figure 2.6b). However, the changes in mRNA abundance for 9-21 were similar to those for 3-101a, with mean decreases at d 0.25, 0.5, 1, 1.5, and 2 compared to d -4 (P g 0.01). Changes relative to d -8 were significant at P _<_ 0.05 on d 0.25 and 0.5 while d l, 1.5, and 2 were significantly different from d -8 at P 5 0.01. Therefore, mRNA abundance of neutrophil genes represented by 3-lOIa and 9-21 was repressed by parturition and remained low for at least the first two days of lactation. 56 Figure 2.6 - mRNA abundance profiles of genes encoded by amplicons 3-101a and 9-21 in neutrophils from periparturient cows were obtained using slot blot hybridizations. Shown are the B-actin normalized neutrophil mRNA abundances (i: SEM) from four cows at multiple parturient time points (relative to parturition on day 0). Parturition significantly repressed 3-101a mRNA (panel a, P = 0.018), with nadir being reached on day 2 postpartum. Neutrophil mRNA abundance of 9-21 was also significantly effected by parturition (panel b, P = 0.003), with lowest levels found on day l postpartum. For both genes, neutrophil mRNA abundance was significantly lower from day 0.25 through day 2 post partum (P 5 0.05) compared to prepartum abundance (indicated by 9 ). 57 ‘% Figure 2.6 a 5 Slot blot analysis of 3-101a E": 2'». 4 T , . . g ,_ l l T .. . i 1’ I I I I I I I l 0 ., 0. 25 0. 5 Day Relative to Parturition b Slot blot analysis of 9-21 mRNAAbundeoG—Zl) -8 -4 0 0.25 0.5 1 1.5 2 7 Day Relative to Parturition 58 E. Correlations between Neutrophil Gene Expression and Serum Steroid Profiles from Periparturient Cows Three steroid hormones, cortisol, estradiol, and progesterone, are known to be critical to bovine parturition and may have effects on neutrophil functions (Guidry et al., 1976; Roth et al., 1982; Roth et al., 1983; Goff and Horst, 1997). Sera from the four animals used for slot blot analysis of neutrophil mRNA abundance were analyzed by RIA to characterize steroid concentration profiles throughout the perime period. Actual hormone profiles were reported in Figure 7 of Weber et al. (2002) and are found in Figure 2.7 of this thesis. Briefly, cortisol increased significantly at parturition and continued to be significantly higher than basal levels for the first 24 hours post partum. Estradiol was significantly higher than basal levels at d -8, -4, 0, and 0.25 with a dramatic spike on d 0. Progesterone concentrations were high until d 0, at which time they plummeted to nearly undetectable levels (and remained so until d 7). Correlation analysis showed a clear association between neutrophil mRNA abundance of 3-lOIa and serum progesterone (r = 0.44, P = 0.0]; Table 2.1). In addition, serum estradiol and progesterone concentrations both correlated positively (r = 0.3595, P = 0.047 and r = 0.3495, P = 0.046 respectively) with 9-21 mRNA abundance in neutrophils (Table 2.1). No significant correlations were detected between serum cortisol concentrations and neutrophil mRNA abundance of 3-101a and 9-21 (Table 2.1). Scatter plots and regression analysis of these relationships between neutrophil 3-101a mRNA abundance and hormones (Figure A.5 panels a, b, c) and neutrophil 9-21 mRNA abundance and hormones (Figure A.5 panels d, e, f) appear in Appendix One. 59 Figure 2.7 - Senun concentrations of three steroid hormones of bovine reproduction change significantly during parturition. In panel (a), concentrations of serum cortisol rise sharply at parturition and return to basal by day 1.5 of lactation. 1n panel (b), serum progesterone concentrations are high before parturition but plummet at parturition and stay low throughout day 7 of lactation. In panel (c), serum estradiol concentrations begin to rise 8 days before parturition, peak at parturition, and rapidly return to basal concentration by day 0.5 of lactation. Data are presented as raw daily means ( : SEM). The solid black bar is the mean for mid-gestation control cows and the light gray bars are the daily means for parturient test cows. Bars without SEM had variation too low to be visualized on the graphs. Daily test cow means significantly different from the control cows mean are highlighted by asterisks ("P 5 0.01; *P 5 0.05). These data are presented in Weber et al., 2002 and are reprinted by permission from the editor of Veterinary Immunology and Immunopathology (Appendix Two). 60 Figure 2.7. (a) (b) Serum Estradiol (pglml) O\ A J Serum Cortisol (ngL) or: Cortisol I ' I -4 0 0. 25 0. 5 1 1.5 2 7 Days Relative to Parturition a l N Serum Progesterone (ngl‘ml) .3 I C l Progesterone -4 0 0.25 0.5 1 1.5 2 7 Days Relative to Parturition 800 _ ** Estradiol 600 - 400 — 200 - *9: a: 0 _J'T'i '.' -8 .I... :.I. . . I.. -4 0 0.25 0.5 l 1.5 2 7 Days Relative to Parturition 61 Table 2.1 - Correlations‘ between serum steroid hormone concentrations and neutrophil mRNA abundance of 3-101a and 9-21 in periparturient dairy cows. DD Product Amplicon Cortisol Estradiol Progesterone 3-101a -0.2614 0.2354 0.4430“ 9-21 -0.0389 0.3595* 0.3495* " Pearson Correlation Coefficient "' P_<_ 0.05; ** P < 0.01 F. Putative Identifications of Genes Differentially Expressed in Neutrophils of Periparturient Cows Putative identities of 3-101a and 9-21, as well as the other amplicons screened by cDNA dot blot hybridization, were obtained through DNA sequence and BLASTn analyses. Each amplicon contained approximately 225 bp of plasmid DNA, which was trimmed prior to BLASTn analysis. Significant homology was considered when an expectation value (E-value) was less than 104. This value describes “the number of hits one can expect to see by chance when searching a database of a particular size” (from the National Center for Biotechnology web page BLAST Program Frequently Asked Questions ht_tp://www.ncbi.nlm.nih.gov/BLAST/blast FAQshtml). The closer the E- value is to zero, the more significant the homology is to the sequence identified in the database. The 3-101a amplicon, which was approximately 375 bp in length after vector trimming, was homologous (E-value of 10"”) to the bovine mitochondrial gene for cytochrome b (cytb) (Figure 2.8). The 9-21amplicon displayed high homology (E-value of 1052) to the human insulinoma rig-analog mRNA encoding DNA-binding protein, which has been shown to encode ribosomal protein SIS (rig/RPSIS) (Figure 2.9). The full-length cDNAs for cytb (1 l40bp) and rig/RPSIS (498 bp) are comparable in size to the mRNA species detected on Northern blots probed with 3-101a and 9-21 (Figure 2.5) respectively, further supporting that the identities of these genes were accurate. Putative identities of the remaining twelve amplicons shown by dot blot analysis to be differentially expressed in periparturient cow neutrophils (Figure 2.4) are listed in Table 2.2. Identities were not currently available in the public databases for all amplicons, but some appear to represent genes for DNA binding proteins or factors that have a role in 63 the citric acid cycle. Thus, the putative identities of amplicons obtained in this study demonstrate that we have identified cellular genes that are important to general life functions of neutrophils. 95 IctcagantagacaaaatcccattccacccctactataccaCtaaggacatcttaggqgc 154 IIIIIIIIIIIIlllllllllllllllllllllllllllll llllllllllllllllll 636 ctcagacgtagacaaaatcccattccacccctactataccattaaggacatcttaggggc 695 155 cctcttactaattctagctctaatactactagtactattcgcacccgacctcctcggaga 214 lllllllllllllllllllllllllllllllllllllll II III lllllllllllll 696 cctcttactaattctagctctaatactactagtactatttgcgcccaacctcctcggaga 755 215 cccagataactacaccccagccaatccactcaacacaCCQGthacatcaaacccgagtg 274 lllllllllllllllllllllllllllllllllllllll llllllllllllllllllll 756 cccagataactacaccccagccaatccactcaacacacctcctcacatcaaacccgagtg 815 275 atacttcttatttgcatacgcaatcttacqatcaaeccccaacaagctagqaggagtact 334 Illlll IIIIIIIIII IIIII llllllllllllllll ll IIIIIIIIIII II 816 gtacttcctatttgcatatgcaattctacgatcaatccccaataaactaggaggagtcct 875 335 aqccctagccttctctatcctaattcttgctctaatCGCcctaCtacadac 385 llllllll llllll llllllll III III II III Illlllllll 876 agccctagtcttctccatcctaatccttattctcattcccttactacacac 926 Figure 2.8 — The 3-101a amplicon was homologous (E-value 10"”) to a region of the bovine gene for mitochondrial cytochrome b (See Table 2.2 for Genbank Accession number). Shown are the aligned sequences for 3-lOIa (top line, highlighted in gray) and bovine mitochondrial cytochrome b (bottom line). 64 118 467 177 407 237 347 Figure 2.9 — The 9-21 amplicon was homologous (E-value 1052) to a region of the human ttacttqagaggqatgaaacgg-aggaatggqtqgccccqataccqgcccggccatQCtt 176 l|||||||l Illlllll III III! llllllllllllll lllllllllllllllll ttacttgagagggatgaagcgggaggagtgggtggccccgatgccgggccggccatgctt 408 cacgggcttgttattgatggagaactcccctacgtagtgchgatcatcccacgcttgat 236 llllllllll IllllllllllllllIlllllllllllllllll lllllll tacgggcttgtaggtgatggagaactcgcccaggtagtggccgatcatctcgggcttgat 348 ttccacCtggttgaaggtcttgccgttatanacgccaaccatgct 281 Illlllllllllllllllllllllll ll Illll llllllll ctccacctggttgaaggtcttgccgttgtagacgcccaccatgct 303 gene encoding rig/ribosomal protein SIS (See Table 2.2 for Genbank Accession number). Shown are the aligned sequences for 9-21 (top line, highlighted in gray) and rig/ribosomal protein 815 (bottom line). 65 A _ mmmonmv E268 <29: :8 ..5w05 5550.050 5.5505095 m...00 0... 5.. 5.5. .0 50.55.050w 000550. 05.5 5300 50.5555 50... 25.00550: 5. 5.5500 59.0.5. 055.55 3555050055 0:. 50.500. 050.05. 5053 50550. 0.. 50.55% 055 50.55% 50.0... 5 5.55050 005% 0555505505. 0:. 0. 55.55 555050055 0... 50.... 550.0... ..0 55.52. 05. 5.. 3.550. 50.5.? 500555.. 505.020 55.5 .0 05050030 0. .. 0035.. .055 0 0555000 50... 50500.0 55 5.50005 .: 0.0.0500 0555505 .5.5550_.00..5 .055. 0... 5. 50.500. 5.550 50055.. 50500.0 0.... ..0 =. 00.0500 ..0 50500500 .00. 5 m. 35.0. 5 05050058 5.5505055. - fin 055m...— 75 5.53). 3.5050505). — 50.0800 + E 4.... =— 50.0800 « + L. 00550 0555505525. ..m 2:0: 76 While still to be substantiated in future studies, reduced cytb mRN A abundance in parturient neutrophils may cause reduced expression of the cytb protein in mitochondria. If cytb proteins were absent or decreased, the ability of the cells to form complex 111 would be reduced resulting in disruption of the electron transport chain and deficient ATP production by the cells. Certainly, such problems have been recorded in numerous human diseases where cytb gene mutations disrupt cellular respiratory metabolism (reviewed by Fisher and Meunier, 2001). Recall fi'om the literature review that only few mitochondria are present in mature blood neutrophils. If this integral portion of the electron transport chain were missing in these phagocytes, as may be the case when cytb mRNA abundance is reduced during parturition, general energy metabolism would be predicted to be in jeopardy. Because all immune functions of neutrophils require energy in the form of ATP, inhibited cytb expression could also be predicted to affect the surveillance, migration, phagocytosis, oxidative burst, and phagolysosome killing by bovine neutrophils. If true, the cytb results of the current study may begin to explain why neutrophils of periparturient cows are dysfunctional and thus why the animals are so susceptible to mastitis and other infectious diseases. The down regulation of neutrophil rig/RPSIS mRNA observed in this study may also have negative consequences for the immune functions of these cells. Originally identified in a rat insulinoma cDNA library, the rig gene has been shown to encode for mammalian ribosomal protein 815 (RPSlS) (Kitagawa et al., 1991). RPSIS was recently found to be a key component in the assembly of the small ribosomal subunit (Figure 3.2) and in prokaryotes is one of the first to bind with 168 rRNA during assembly of the small subunit (Agalarov et al., 2000; Nikulin et al., 2000). This protein 77 is required for recnritrnent of additional proteins to ensure that proper formation of the small subunit occurs. If the small subunit is not formed, the ability of the affected cells to translate mRNA into proteins may be generally inhibited. In this regard, it was interesting that three major proteins (CD62L, CD18, and G1) of bovine neutrophils are down regulated during periparturition (Lee and Kehrli, 1998; Kimura et al., 1999b; Weber et al., 2002; Burton and Weber, Appendix One, Figure A.l ). Deficient expression of these and other proteins could signal neutrophils to become inactive or even apoptotic. Indeed, apoptosis could be an important mechanism for clearing aging neutrophils from the circulation, especially during times of neutrophilia (as occurs during parturition; Preisler et al., 2000a; Weber et al., 2002). Combined with repressed cytb expression in blood neutrophils of parturient cows, reduced expression of RPSlS could be a hallmark of aging (and dysfunctional) neutrophils. Clearly, these possibilities must be substantiated through additional research. 78 Ribosome RPSlS Figure 3.2 - Ribosomal protein SIS (RPSlS) is an integral component of the small ribosomal subunit. Researchers have demonstrated in prokaryotes that RPSIS is one of the first ribosomal proteins to bind 16$ rRNA, and induces binding of additional proteins for small subunit formation. When cells are deficient in RPSIS, the small subunit may not form correctly and overall translational capacity of the cells may be inhibited Preliminary evidence from our laboratory (Chapter Two; Weber et al., 2002; Burton and Weber, unpublished) and others (Lee and Kehrli, 1998; Kimura et al., 1999b) suggest that bovine blood neutrophils deficient in RPSlS mRNA also have deficient expression of key membrane proteins such as L-selectin, CD18, and G1 (examples of L-selectin and G1 in Appendix One, Figure A.l). 79 Finally, the putative identities of eleven additional genes observed in the current study as modestly to moderately down regulated in bovine blood neutrophils during parturition are noteworthy (Chapter Two, Table 2.2). The cDNA sequences of these genes had high homology with genes known to be involved in DNA binding (eg. by steroid and vitamin D receptors) and in the citric acid cycle. Again, therefore, we identified basic cellular genes as being affected in neutrophils during parturition. Several of our differentially expressed neutrophil genes had high homology with ESTs in the TIGR database, but with no known identities and (or) functions. These cDNAs, and ESTs along with the cytb and rig/RPSIS cDNAs have been included on our groups cDNA micorarrays (Burton et al., 2001) for future studies of parturition effects on gene expression in neutrophils. In conclusion, the research presented in Chapter Two demonstrates clearly that parturition induces reductions in mRNA abundance of several key genes important for the basic cellular functions of neutrophils. Data presented in Chapter Two also implicates the reproductive steroid hormones as possible factors that influence neutrophil gene expression. Although the genes identified through this research are not considered classical immune response genes, their protein products are intimately involved in the basic functions of cells including respiratory metabolism, the citric acid cycle, transcription, and translation. Therefore, lowered mRNA abundance of these genes at parturition may help explain the general dysfunctions of blood neutrophils (surveillance, recruitment, phagocytosis, respiratory burst, and phagolysosome killing) that have been reported by bovine leukocyte biologists for several decades. In turn, these neutrophil dysfunctions are clearly associated with the heightened disease susceptibility of parturient 8O dairy cows. The work of this thesis has thus provided a launch point for future studies aimed at explaining the causes and consequences of gene repression in bovine neutrophils around parturition. 8] CHAPTER FOUR Recommendations for Future Research As mentioned in previous chapters, resting blood neutrophils actively express at least 750 genes. While this is a relatively small number of genes compared to expression in other cell systems, it is not surprising given that mature neutrophils are terminally differentiated cells with few mitochondria, Golgi apparatus, and excessive cytoplasmic granules packed full of proteins required for many of the cell’s innate immune functions. Interestingly, researchers are beginning to publish data showing that exposure of neutrophils to various stimuli significantly alter global gene expression (Cowling and Bimboim, 2000; Newberger et al., 2000). Until our work, however, no one had demonstrated that bovine blood neutrophils respond to parturition with globally altered gene expression. We have now shown that the cortisol spike of parturition inhibits neutrophil expression of the L-selectin gene, preventing these leukocytes from marginating and causing neutrophilia (Weber et al., 2002). Furthermore, the work of the current study shows that mRNA abundances of at least eleven other neutrophil genes are repressed around parturition, two of which appear to associate with serum concentrations of progesterone and estradiol (cytb and rig/RPSIS. However, much more work is required to fully confirm the identities and causes of the decreased mRNA levels of the neutrophil genes shown through this study. The tools are now in place to carry on such research. For example, the fourteen amplicons generated by DDRT-PCR and used on dot blots could now be used to screen a bovine leukocyte cDNA library to obtain full-length cDNA sequences. The full-length cDNAs would then be sequenced to confirm the 82 identities of cytb, rig/RPSIS, and the other genes putatively identified. This may also help identify genes represented by those amplicons that demonstrated no significant homology to DNA sequences currently available in the public databases. Neutrophils from periparturient cows accumulate in the blood resulting in pronounced neutrophilia (Nagahata et al., 1988; Kehrli et al,, 1989; Preisler et al., 20003; Weber et al., 2002). Although many functional aspects of these phagocytes are altered, these cells’ immediate fate is still unknown. The present study demonstrated mRNA of multiple neutrophil genes to be down regulated shortly after parturition when neutrophil numbers are at their highest in the blood. We have hypothesized that the decreased mRNA abundance is in response to fluctuating hormone levels but there is also the possibility that neutrophilia may be inducing other changes in the cell such as apoptosis. With apoptosis, cell death would result in an overall decrease in genes that could be expressed, mimicking a regulated decrease in mRNA abundance of numerous genes. Several methods can be utilized to determine if blood neutrophils from parturient cows are apoptotic. These include the use of fluorescent dyes, cell surface markers specific for apoptosis, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), all methods that can be evaluated by flow cytometry which is readily available in our laboratory. In spring 2002, we plan to implement a few of these methods to determine the relative contributions of altered gene expression and apoptosis to the phenomena described in this thesis and our companion paper (Burton et al., 2001; Madsen et al., submitted). Both scenarios are unique and could potentially provide interesting explanations for the increased disease susceptibility of periparturient dairy COWS. 83 In addition to confirmation of differential gene expression in neutrophils from periparturient cows, it would be of interest to observe if other situations result in similar changes in gene expression. This will be done by adding the 14 amplicons identified by DDRT-PCR to the bovine total leukocyte cDNA library created by the Center for Animal Functional Genomics at Michigan State University. Microarrays generated fi'om this expanded cDNA library will be used for even broader studies of blood neutrophil gene expression in periparturient cows, including in vitro studies to observe individual effects of cortisol, estradiol, and progesterone on neutrophil gene expression. Future studies could also evaluate differences in bone marrow, blood, and tissue neutrophil gene expression under these scenarios. The results of these studies will help us gain a better understanding of gene expression in relation to the dysfunctional neutrophil biology observed during the periparturient period. They may also result in discovering which of these steroid hormones have the greatest affect on neutrophil function leading to increased disease susceptibility. 84 APPENDIX ONE 85 (a) 14 days before (b) 14 days before g ‘ g T'“ ‘-:...€ § .8. 3 a. § § '. §§ ' 8 O 5% 38 § § 0 O FL-Z Channel Fluorescence FL-Z Channel Fluorescence Gl Mean Fluorescence Intensity = 155.39 CDGZL Mean Fluorescence Intensity = 74.20 (C) 0.5 days after (d) 0.5 days after 8 . ' -v- ...— - .r 8 L 2 ' ° - ' O 8 § as 3% <§ 8 o g§ gs O O 8 8 o O r‘rrnm, . . n.-. . um... 1 . " 10° 10' 102 103 10" 10° to‘ 102 103 to4 FL-2 Channel Fluomcence FL-Z Channel Fluorescence G1 Mean Fluorescence Intensity = 26.22 CD62L Mean Fluorescence Intensity = 7.04 Figure A.1 — Purity of isolated neutrophils from d —14 and d 0.5 from which RNA was reverse transcribed and used in dot blot hybridization. An aliquot of purified neutrophils was obtained prior to the TRIzol step and fluorescently immuno-stained for the cell surface markers G1 and L-selectin (CD62L). G1 is neutrophil specific and indicates the purity of the cell population (panels a and c). Overall, our cell populations had a purity of 80% or greater. The decreased CD62L intensity (panels b and d) is well documented and characteristic of neutrophils from parturient dairy cows (Lee and Kehrli, 1998; Weber et al., 2002). 86 Representative DDRT-PCR Gels Cow: P1 P2 P1 P2 M] P1 P2 N Sample Time: d-12 d0 d0.25 , a <-*-'- 3-101a Cow: Ml P1 P2 P1 P2 P1 P2 P1 P2 M2 N Sample Timezd-IZ d-l d0 d0.25 Figure A.2 — DDRT-PCR gel sections containing differentially expressed genes called 3-101a and 9-21. Expression of 3-IOIa and 9-21 in neutrophils was followed through the entire flow of experiments outlined in Chapter Two, Figure 2.]. Letters above the lanes indicate the following samples and time points: mid-gestation control cows M1 and M2; pooled samples from d -175 to -130 prepartum; parturient test cows P1 and P2; sampled on d -12, -l, 0, 0.25 relative to parturition; N is the negative control (no RNA in reverse transcription reaction). 87 Figure A.3 — Autoradiographs of dot blots hybridized with radiolabeled cDNA created by reverse transcription of neutrophil RNA from pre- (day -14) and post partum (d 0.5) time points. All 14 amplicons were spotted in quadruplicate (example of 3-101a in boxes). B-actin was also spotted in quadruplicate as a control (dark circled spots) and four spots containing no DNA for negative control are indicated by the arrows (one replicate per blot). 88 1.2 0.8 0.6 0.4 0.2 Mean Densitometry Values B—actin I Before [I Alter Figure A.4 — Mean dot densities (i SEM) of B-actin mRNA abundance in neutrophils before (d —14) and afier (d 0.5) parturition. B-actin mRNA abundance was not significantly different between before and afier parturition (P > 0.10 by t- test). Data in this graph were obtained from a shortened autoradiograph exposure compared to the data shown in Chapter Two, Figu re 2.4, to achieve dot intensities that were measurable by the seaming densitometer. 89 .360 .33 20.5.8th 5 .o. .8380 98 2.38.9.0 5.... -m gouge: 2:82.52 o: .3: .3. 3.62.8 No .... 28.6.8on :5 00:35.3 SAME a... .-m 5038: 3382.28 0328a 33m San. 62.53.. E .5295 92?... 8.888: 2803 98 3.8-... 005655.. SAME «....-m .5255: 5230: 8528.522 338.2882. 32.. 538m - ma... «.5»...— 5595:0230 .2223”.— .5 ..2..” 2...... ......6 ........m 2...... .......n .......N 2...... ..2............ T o 9.. 8.; o 83. . c 32... .. "m 8...? 8:. _ + ”NNNN. ..u N .2.... e r 9:32.20.— a—c..m I .2358 ......i 5:238:09 .8539 .5 ....mN .......N ....m. — 2....— ..em.... 2......oem. 64.2....— ....m._. .. O O. O Z786." O O gecél I Ny— O O. O O 0 SN. N _+. NNNNN. ...- u N _ 9:28:29. an ...-n. I 392.3 .9. 2...... “a... ..2... m. gash—.350 o:e..o.8we._m .... 2.... n 2.... N ..2... . 2...... ..2.. T 2.... IN- 2.... n- ..2... v- ..2.. m- 2...... Ioiol .9. o o u .I. 9 82K." 0 .25.. % 99 ---...nmfi .....m._ 1°80" 9 22... n N: ,.,,...»NNNN.N.N.NN£..N..."...m-.- , 2:22.59. 3......” I 32882....— .... 90 .C. .8080 828 5.3 88.2 8.. 8: .8. 8.08.8 ...... .... 0.88.88... 88% :85 88.2 30328.. N. 8588:“ <73... ..Nd 8... .808th ... .823... $08-... 8888: .885 .2... .88-... 8:88.... <22... .N-o .2858: 8038: 8328.522 2.58.2882. m8... 858m .....2.......8 m.< 08w... 8.8.5.8280 ......auam .... 2...; ..2... ........0.......m ..2..... ........n ........N......... ................... .I 8.8.5.8980 .8580 ..I. ....m. N ..2... N ....m. . ..2.. — ....Lm. .. 2..... .. 2.0.. ..I 2..... .I ....m. .I ‘0. co 8...... ...... —. ’ 2.0.. . ‘l o 83 88... n ... .NNWHNHMNNNWWN- 8m... N It 9:28.539. ...N.&. I .8589 .... 8.8.5.8980 288.88.... .1. 2..... n 2..... N 2..... N 2...... ..2.. . 2..... N- 2.... m- ..2.. ..- ..2.. m- 2...... 00 d O In 8:... 09 a Q o a g... o m ...—...L O .......N . O o W o o e O .83 o . 2......“ m 8.. N NNNN. ... + 1...... .....n N 2.8.... N 2:82.29. .NIa I .2858 .0. o 88. :3. ..I I ... . ., ., .. . 8.80..-. 2:82.29... .NIa I 28.8.8.8... .... m v o o o W o 83.. ...v...0.. N; + n+0...” ..u n , N. m .... 91 APPENDIX TWO November 16, 2001 Dr. C.L. Baldwin Editor-in-Chief for the Americas Veterinary Immunology and Immunopathology Department of Veterinary & Animal Sciences Paige Laboratory Holdsworth Way University of Massachusetts Amherst, MA 01003 Dear Dr. Baldwin: I am writing to you in regards to the article “Pre-translational Regulation of Neutrophil L- selectin in Glucocorticoid-Challenged Cattle” by Weber et al. that has been accepted for publication in Veterinary Immunology and Immunopathology (revised manuscript VII #01-26). I am currently a Master’s student at Michigan State University and am scheduled to defend my thesis on December 4, 2001. I request permission to include Figure 7 (Serum concentrations of three steroid hormones of bovine reproduction change significantly during parturition.) in Chapter Two of my thesis as this data was utilized in statistical analyses of my results. Thank you for your prompt consideration of this manner! Sincerely, .3 ”WI/m4..- Sally A. Madsen Veterinary immunology and immunopathology an international journal of comparative immunology . . , Editors-in-Chiet‘ II}.‘.;§‘.’1'I‘.‘.';I'$§,- Dr C.L. Baldwin Dr. CJ. Howard Dr. J. Naessens r ll irrumnmlmllmlogy Dr C.L. Baldwin Department of Veterinary 8:. Animal Science Paige Laboratory University of Massachusetts, Amherst, MA 01003 Tel: 4l3-545-3l67 Fax: 413-545-6326 E-mail: Cbaldwin@vasci.umass.edu Ms. Sally A. Madsen Dept animal Science Anthony Hall East Lansing, Michigan 48824-1225 Fax 517-353-1699 Dear Sally, It is acceptable for you to include the figure in your thesis. Technically a thesis or dissertation is a publication and copyrighted by the university or individual since it is a publicly accessible document. However the practice of publishing your graduate degree research results that appear in those documents in peer-reviewed journals as well is not only long-standing but highly encouraged by the scientific community. This is because the data is usually more readily accessed through this medium and second it has been validated by the peer-review process. Good luck with your defense. / Cynt Ia L. Baldwin Edit r 94 REFERENCES 95 Agalarov, S.C., Prasad, G.S., Funke, P.M., Stout, C.D., Williamson, J .R., 2000. Structure of the S15, S6, SIS-rRNA complex: assembly of the 30S ribosome central domain. Science. 288, 107-112. Bainton, D.F., Ullyot, J .L., F arquhar, MG, 1971. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J. Exp. Med. 134, 907-934. Bargatze, R.F., Kurk, S., Butcher, E.C., Jutila, MA, 1994. 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