Expression of CD13/Aminopeptidase N by Synovial Fibroblasts: Novel Roles in the Pathogenesis of Rheumatoid Arthritis by Rachel L. Morgan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Immunology) in the University of Michigan 2014 Doctoral Committee: Professor David A. Fox, Chair Professor Stephen W. Chensue Professor Jeffrey L. Curtis Professor Bethany B. Moore Professor Stephen J. Weiss © Rachel L. Morgan 2014 Dedication I would like to dedicate this to my mom. ii Acknowledgements I have had the wonderful support of so many people throughout my thesis work. First, thank you David for all of your help and support. You have been the best mentor I could ask for. You are always so patient, kind, and knowledgeable. I also have to thank all the members of the Fox lab past and present. Over the years you have all helped make my experiments and my life better. I would especially like to thank Judy; whether I needed help learning a new technique or I was just having a bad day, you always had the answer. I also have to thank my thesis committee members and numerous other collaborators at Michigan for giving me their time, knowledge, and reagents. It has been wonderful learning from and working with such generous scientists. Of course I also have to thank Zarinah, Beth, and Keith for making the Immunology Program such a great and supportive learning environment. Personally I’d like to thank my friends and family for supporting me through the years. Also, my doctors, especially Robert Ike, without whom I don’t think I would have been able to finish this work. Finally, a big thank you to all the tissue and blood donors. You are the ones who make work like this possible. iii Table of Contents Dedication ...................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii List of Figures ............................................................................................................................... vi Abstract ......................................................................................................................................... ix Chapter 1 Introduction ................................................................................................................ 1 Rheumatoid Arthritis .................................................................................................................. 1 Aminopeptidase N/CD13 .......................................................................................................... 11 Chemotaxis ............................................................................................................................... 25 Matrix Metalloproteinases and ADAMs ................................................................................... 30 Extracellular vesicles and exosomes ......................................................................................... 35 Potential roles of CD13 in rheumatoid arthritis ........................................................................ 39 Chapter 2 CD13 in Rheumatoid Arthritis ................................................................................ 41 Identification of CD13 as an IL-17-induced protein on FLS .................................................... 42 CD13 in vivo ............................................................................................................................. 46 CD13 on FLS from RA and OA ............................................................................................... 53 CD13 and aminopeptidase activity in the joint ......................................................................... 55 Discussion ................................................................................................................................. 59 Chapter 3 CD13 on Fibroblast Like Synoviocytes: Expression, Shedding, and Regulation 63 CD13 is present on extracellular vesicles including exosomes ................................................ 64 Metalloproteinases cleave CD13 from FLS .............................................................................. 69 MMP14/MT1-MMP cleaves CD13 from FLS ......................................................................... 71 Regulation of CD13 .................................................................................................................. 76 Discussion ................................................................................................................................. 80 Chapter 4 Functions of CD13 in Rheumatoid Arthritis .......................................................... 87 Recombinant human CD13 aids in the migration of cytokine activated T cells ...................... 88 CD13 contributes to the chemotactic activity of SF independent of its enzymatic activity ..... 91 iv CD13 initiates chemotaxis of T cells through a G-protein coupled receptor ........................... 93 CD13 aids in growth and migration of RA FLS ....................................................................... 95 Discussion ................................................................................................................................. 99 Chapter 5 Conclusions and Future Directions ....................................................................... 105 CD13 in RA: future directions ................................................................................................ 111 Regulation of CD13 ................................................................................................................ 122 CD13 receptor ......................................................................................................................... 125 Cleavage of CD13 ................................................................................................................... 129 CD13 in murine models .......................................................................................................... 131 CD13 and other diseases ......................................................................................................... 134 Appendix Materials and Methods ........................................................................................... 137 References .................................................................................................................................. 149 v List of Figures Figure 2.1 Identification of the protein recognized by mAb 591.1D7.34 and upregulated by IL-17 as CD13. ........................................................................................................................................ 44 Figure 2.2 Staining of different cell types with 591.1D7.34. ....................................................... 45 Figure 2.3 CD13 is found in vivo and is higher in RA than in OA synovial fluid. ....................... 49 Figure 2.4 CD13 staining is present in synovial tissue from normal, OA, and RA patients. CD13 staining more strongly co-localizes with FLS (cadherin-11) and monocytic lineage cells (CD11c) in RA tissue. .................................................................................................................................. 50 Figure 2.5 CD13 staining is present on neovasculature in RA synovium. ................................... 51 Figure 2.6 CD13 is significantly elevated in chronic graft-versus-host disease serum over CD13 in healthy control serum, and is significantly decreased in RA, myositis and lupus serum compared to healthy control serum. .............................................................................................. 52 Figure 2.7 Equivalent expression of CD13 by RA and OA FLS. ................................................. 54 Figure 2.8 CD13 accounts for most but not all of the aminopeptidase activity in synovial fluid. 57 Figure 2.9 1D7 efficiently depletes CD13 from synovial fluid and does not inhibit CD13 enzymatic activity. ........................................................................................................................ 58 Figure 3.1 CD13 was found as a soluble protein and on extracellular vesicles in FLS culture supernatant, normal human plasma, and RA synovial fluid. ........................................................ 66 vi Figure 3.2 CD13 was found as a soluble protein and in multiple fractions of a density gradient separation of extracellular vesicles of FLS culture supernatant, normal human plasma, and RA synovial fluid. ............................................................................................................................... 67 Figure 3.3 CD13 was found as a soluble protein and in multiple fractions of a density gradient separation of extracellular vesicles with distinct localization for FLS culture supernatant, normal human plasma, and RA synovial fluid. ......................................................................................... 68 Figure 3.4 Metalloproteinases cleave CD13 from the surface of FLS. ........................................ 69 Figure 3.5 MMP14 siRNA knocks down MMP14 in RA FLS..................................................... 73 Figure 3.6 MMP14 knockdown partially inhibits the shedding of CD13 from FLS. ................... 74 Figure 3.7 Fluorescent staining of FLS shows co-localization