STUDIES OF GUT-ASSOCIATED LYMPHOID TISSUES AND OTHER SECONDARY LYMPHOID TISSUES IN 12 WEEK OLD NEW ZEALAND WHITE SPECIFIC PATHOGEN FREE RABBITS A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Rebeccah Urbiztondo, DVM Graduate Program in Veterinary Biosciences The Ohio State University 2010 Master’s Examination Committee: Professor Michael Lairmore, Advisor Associate Professor Tracey Papenfuss Associate Professor Mary Jo Burkhard Copyright by Rebeccah Urbiztondo 2010 ABSTRACT Rabbits serve as valuable animal models to study the immunopathogenesis of human diseases that gain entry through the gastrointestinal (GI) tract. Rabbits possess abundant mucosa-associated lymphoid tissue (MALT), both in the GALT and at other mucosal sites. This makes them uniquely suited to studying diseases which are transmitted across these surfaces. This study looks at the gut-associated lymphoid tissues (GALT), as well as, the lymphoid populations in the spleen, mesenteric lymph nodes, and peripheral blood of twelve week old New Zealand White rabbits. Herein, we used flow cytometric and immunohistochemical methods to phenotypically characterize lymphoid populations. Results obtained via flow cytometric analysis were comparable to the distribution of leukocyte subsets in other animal species, including humans, both in the GALT inductive and effector sites and in mesenteric lymph nodes, spleen, and peripheral blood. Immunohistochemical analyses of tissues were comparable to results obtained via flow cytometry. Our data collectively indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues. Furthermore, these data will support future studies that utilize the rabbit model to study human gut-associated disease or infectious agents that gain entry via the oral route. ii ACKNOWLEDGMENTS I would like to thank Krissy and my lab mates for their help with this project. Special thanks goes to Sha who got me started on the right foot and showed me some valuable tricks regarding sample processing. Without his guidance I never would have been able to process over 100 flow samples in one day. I would also like to extend my gratitude to Robyn who has gone through this project with me. Without her help and meticulous organizational skills, things would not have run as smoothly as they did. Though I did not work directly with Raj and Nadine, their singing in the lab provided constant amusement. I would also like to acknowledge Sarah Leavell for her critical role in helping me ease into a research setting. She was an excellent mentor to me regarding good laboratory techniques. I am so appreciative of the patience she showed me when I was transitioning from clinical pathologist in training to graduate student. Last but not least, I would like to thank Dr. Lairmore. His “leave no stone unturned” attitude is very refreshing in a day in age when “good enough” is the norm. I thank him for letting me be a part of his laboratory. The lessons I learned in his lab, both research and non-research related, will help me in the future regardless of my endeavors. iii VITA March 9, 1982…………………………......................Born - San Juan, Puerto Rico 2008……………………………………………………DVM, Iowa State University College of Veterinary Medicine 2008-Present………………………………………….Graduate Research Associate/ Clinical Pathology Resident The Ohio State University Department of Veterinary Biosciences PUBLICATION Urbiztondo R, Chapman S, Benjamino K. Meseneteric root osteosacoma in a dog. Vet. Clin Path. In press. FIELDS OF STUDY Major Field: Veterinary Biosciences Minor Field: Veterinary Clinical Pathology iv TABLE OF CONTENTS Page Abstract ………………………………………………………………………………....ii Acknowledgments…………………………………………………………………..….iii Vita……………………………………………………………………………..………..iv List of Tables…………………………………………………………………………...vii List of Figures……………………………………………………………………....….viii 1. Literature Review: Mucosal immunology with the primary emphasis on gastrointestinal-associated lymphoid tissues across all animal species and in the rabbit Introduction…………………………………………………....….1 Mucosa-Associated Lymphoid Tissue.………………………...2 Gastrointestinal-Associated Lymphoid Tissue..…………..…..3 GALT Inductive Site………………………….. ……..................4 GALT Effector Sites……………………………….....................7 Intraepithelial Leukocytes……………………………………….8 Lamina Propria Leukocytes…………………………..............11 Mesenteric Lymph Nodes…………………………………......13 Comparative Organization of GALT in humans and other animals……………………………………………………….....14 Rabbits: A Comparative Animal Model in Investigations of Mucosal immunity And Infectious Disease Studies………...16 Conclusions…………………………………….......................19 2. Isolation and Phenotypic Characterization of New Zealand White Rabbit Gut-associated lymphoid tissues and other secondary lymphoid tissues Introduction……………………………………………………..23 Material and Methods……………………………………….…27 Results…………………………………………………………..32 Discussion……………………………………………………....38 v 3. FUTURE DIRECTIONS: Summary…………………………………………………………..56 List of References………………………………………………………………...64 vi LIST OF TABLES Table Page 1.1 MALT and MALT-related abbreviation………………………………………21 1.2 Summary of Lymphocyte Phenotypes and T cell ratios in Percentages in selected lymphoid compartment across Animal species……………….22 2.1 Rabbit Monoclonal Antibody Table…………………………………………..44 2.2 White blood cell differentials of cytospin preparations from each anatomic location evaluated in percentages………………………………..45 2.3 CD4:CD8 ratios in lymphoid tissues………………………….……………...46 2.4 B:T cell ratios in lymphoid tissues……………………………………………47 vii LIST OF FIGURES Figures Pages 2.1 Gross and microscopic appearance of rabbit gastrointestinal inductive sites……………………………………………………………………………..48 2.2 H&E sections of small intestine highlighting intraepithelial and lamina propria leukocyte isolation……………………………………………………49 2.3 Cytospin preparations of isolated leukocytes from each of the nine tissues………………………………………………………………………….50 2.4 Flow cytometric scatter plots highlighting gating patterns for each of the nine tissues ……………………………………………………………………51 2.5 Flow cytometric staining patterns highlighting T cell and CD8 surface marker expression for each of the nine tissues…………………………….52 2.6 IHC images of mesenteric lymph node highlighting various T and B cell antibody markers………………………………………………………………53 2.7 IHC images of cecal tonsil highlighting various T and B cell antibody markers…………………………………………………………………………54 2.8 Bar charts for each of the nine tissues evaluated summarizing flow cytometric surface marker expression in percentages…………………….55 viii CHAPTER 1 LITERATURE REVIEW MUCOSAL IMMUNOLOGY WITH THE PRIMARY EMPHASIS ON GASTROINTESTINAL LYMPHOID TISSUES ACROSS ALL ANIMAL SPECIES AND IN THE RABBIT Introduction All mammals have both primitive and highly sophisticated mechanisms to keep harmful pathogens from invading the interior of the body. These mechanisms include mechanical barriers, cellular and chemical defenses. Among these defenses is the indispensable branch of the immune system, known as, the innate immune system. The innate immune system includes physical barriers, antimicrobial peptides, and phagocytic cells that respond very quickly to invading bacteria, which are recognized as distinct from normal cells. The second major branch of the immune system is known as the adaptive immune system. As the name implies, it “adapts” its response to the offending agent and acquires memory that will protect the body from any future attacks from the same pathogen. The adaptive immune system is characterized by specificity and memory 1, 2. There are tissue compartments throughout the body which house lymphocytes and professional antigen presenting cells that work together to create adaptive immunity. These cells originate from primary 1 lymphoid organs, mainly thymus and bone marrow, and migrate to secondary lymphoid organs 3. Secondary lymphoid tissues include the spleen, lymph nodes, and organized cellular aggregates scattered throughout mucosal surfaces 1, 2. This latter group is commonly referred to as mucosa-associated lymphoid tissue (MALT). Table 1.1 lists commonly used abbreviations used for describing MALT structures and functional characteristics. MUCOSA-ASSOCIATED LYMPHOID TISSUE Mucosa-associated lymphoid tissue comprises a large and important part of the entire immune system in mammals and contains approximately half of the lymphocytes in the entire body 4, 5. Regardless of anatomic location, MALT has similar cellular arrangements and function. This is mainly due to the comparable roles these mucosal surfaces have, which is to produce and secrete IgA across the surface epithelium, as well as, participate in cytotoxic T cell-mediated responses 4. Organized MALT compartments are present in the gastrointestinal tract, urogenital tract, conjunctiva, and upper respiratory system including the nasopharynx and upper airways. These compartments can be further subdivided into inductive and effector sites. Inductive sites are made-up of organized aggregates of lymphocytes, analogous to systemic lymph node architecture 6. However, MALT inductive sites do not have afferent lymphatics, unlike systemic lymph nodes 5. In these inductive sites, antigen specific T-cell activation results in clonal expansion of B cells and IgA class switching 4. Antigen is sampled directly across the mucosal surface either by