I't* ür, tù F UCL ¡ r. r lrr fll The Potential Immunomodulatory Effects of the Statins in Uveitis By Dr. Kenneth Gek-Jin Ooi Department of Surgery, Ophthalmology Division The Queen Elizabeth Hospital Faculty of Health Sciences University of Adelaide And Department of Clinical Ophthalmology Institute of Ophthalmology University College London A thesis submitted in the fulfillment of the requirement of the University of Adelaide for the degree of Master of Surgery The Potential Immunomodulatory Effects of the Statins in Uveitis Table of Contents Table of Contents 2 Table of Figures.. 10 List of Tables..... 15 Abstract. t6 Statement of Disclosure... 18 Acknowledgements t9 Abbreviations... ... 20 Chapterl 26 General Introduction... ... 27 l.l The Immune System 28 1.2 Thl lTh2 Balance... ., 30 3 lmmunologic Tolerance and Autoimmunity... 40 1.3.1 Mechanisms of Tolerance...... 40 1.3.1.1 Clonal Deletion and Anergy 4t I.3.I.2 Idiotype Regulation 4t 1.3.1.3 Suppressor or Regulatory T (Tr) cells.. 42 1.3.1.4 Inhibitory Cytokines... , 43 1.3. 1.5 Failed Tolerance. 43 1.3.2 Autoimmune Disease. 43 1.4 The Ocular Immune System. 44 1.4. I Immune Privilege...... 44 2 The Potential Immunomodulatory Effects of the Statins in Uveitis 1.4.1.1 The Blood-Retinal Barier (BRB) 47 1.4. l. 1.1 Anterior BRB. 47 1.4. 1. 1.2 Posterior BRB. 48 1.4.1.2 The Blood-Aqueous Barrier (BAB). 50 1,.4.1.3 Anterior Chamber Associated Immune Deviation (ACAID) 50 1.5 Intraocular Immune Disease. 52 1.5.1 Experimental Autoimmune Uveoretinitis (EAU) 53 I .5. l. I Retinal Autoantigens.......... 53 1.5.1.2 Induction of EAU. 54 1.5.2 Leukocyte Trafficking in Immune-mediated Diseases of the Posterior Segment. .55 1.5.3 Cytokines in Immune-mediated Diseases of the Posterior Segment.............58 1.s.3.1 rL-rp... 59 1.5.3.2 rL-2.. 59 1.s.3.3 tL-4...... 60 1.5.3.4 rL-5...... 6l 1.5.3.5 IL-6...... 62 1.5.3.6 rL-10 62 I{??IF'NT-¡I 64 1.5.3.8 TNF-o. 64 1.5.4 Chemokines 65 1.5.4.1 IL-8 66 1.5.5 Clinical Uveitis 66 J The Potential Immunomodulatory Effects of the Statins in Uveitis 1.5.5. I Idiopathic Uveitis. 69 1.5.5.2 Behçet's disease. 70 1.5.5.3 Sarcoidosis 7l 1.5.5.4 Vogt-Koyanagi-Harada disease. 72 1.5.5.5 Reiter's Syndrome. 73 1.5.5.6 Ankylosing Spondylitis 74 1.5.5.7 Fuch's Heterochromic Cyclitis 75 6 Therapies for Intraocular Autoimmune Disease...... 77 1.6.1 Established Therapies for Intraocular Autoimmune Disease 77 1.6.1.1 Glucocorticoids.. ,,77 1.6.1.2 Cyclosporin A (CsA). 79 1.6.1.3 Rapamycin 80 1.6.1.4 Mycophenolate mofetil .81 1.6.2 New Therapies for Intraocular Immune Disease.. 83 I.6.2.1 Anti-TNF-cr Therapies. 83 I.6.2.2 Other Monoclonal Antibody Therapies... 83 1.6.2.3 Other Novel Treatments 84 1.6.3 Potential Therapies in Uveitis.... .............85 I .6.3 .l Rheumatoid Arthritis-based Therapies ..85 1.6.3.2 Antibody Fragment Therapies. 85 1.6.3.3 Statin Therapies. 86 1.6.3.3.1 Statins in Experimental Inflammatory Models of Disease.......89 1.7 Aims.. .....92 4 The Potential Immunomodulatory Effects of the Statins in Uveitis Chapter 2... ... ...93 Materials and Methods ...94 2.1 Materials ...95 2.2 Antibodies. ..97 2.3 Donors ...99 2.3.1 Multiplex Cytokine Detection with Cytometric Bead Array .....99 2.3.2 T Cell Viability and Proliferation Inhibition . ......99 2.3.3Intracellular Cytokine Staining. ....100 2.3.4 Multiplex Cytokine Detection with Fluorescent Bead Immunoassay.........l00 2.4 Stock Solutions. ........101 2.4.1 2-Mercaptoethanol. .. ... l0l 2.4.2 Atorvastatin. ..........101 2.4.3 CFSE Stop Buffer....... .....101 2.4.4 Cyclosporin 4..... ...101 2.4.5 Dexamethasone. ......102 2.4.6 FACS Lysing Solution. t02 2.4.7 Lovastatin.. ....102 2.4.8 Mevalonate 102 2.4.9 Mycophenolate mofetil....... 1n? 2.4.10 Permwash Buffer. 103 z.4.llPhosphate Buffered Saline. .,......103 2.4.12 Propidium lodide. ..........103 2.4.13 Rapamycin 104 5 The Potential Immunomodulatory Effects of the Statins in Uveitis 2.4.14 Simvastatin ..........104 2.4.15 T Cell Medium. ....104 2.5 Multiplex Cytokine Detection with Cytometric Bead Aray. ..........105 2.5.1 Sampling Procedure.... .....105 2.5.2 Cytometric Bead Array (CBA). ...........105 2.5.2.1 Preparation of Thl/Th2 Cytokine Beads. ...105 2.5.2.2 Preparation of Thl/Th2 Cytokine Standards. ........106 2.5.2.3 ThllThz Cytokine CBA Assay Procedure. ..........106 2.5.2.4 Flow Cytometry. .........107 2.5.3 Enzyme-linked Imunosorbent Assay (ELISA). ..........107 2.5.4 Analysis.... ..............108 2.6 T Cell Viability and Proliferation Inhibition. .. 109 2.6.1 Sampling Procedure.... ......109 2.6.2 MNC Counts. 109 2.6.3 T Cell CuIture............ lll 2.6.4 Flow Cytometry. tt2 2.6.5 Analysis.... tt2 2.7 lnftacellular Cytokine Staining. rt4 2.7 .l Sampling Procedure... ... tt4 2.7 .2 Cell Culture. tt4 2.7.3 Staining for Intracellular Cytokines tt4 2.7 .4 Analysis.... 115 6 The Potential Immunomodulatory Effects of the Statins in Uveitis 2.8 Multiplex Cytokine Detection with Fluorescent Bead Immunoassay... .. .....117 2.8.1 Sampling procedure...... tt7 2.8.2 Cell Culture tt7 2.8.3 Multiplex Fluorescent Bead Immunoassay (FBI)... .tt7 2.8.3.1 Preparation of Assay Buffer. 118 2.8.3.2 Preparation of Biotin-Conjugate. 118 2.8.3.3 Preparation of Beads. I l8 2.8.3.4 Preparation of Th1/Th2 Standards. 2.8.3.5 Protocol. il9 2.8.3.6 Flow Cytometry.. 120 2.8.3.7 Analysis. t20 Chapter 3...... 122 Cytometric Bead Array t23 3.1 Introduction. 124 3 2 Aims .125 3.3 Results .t26 3.3.1 Optimising Cytokine Detection in AH by CBA and ELISA ...126 3.3.2 CRA Crrtnkine lìetectinn in llveitic and Cnnfrnls 129 3.3.3 Effect of Steroid Usage..... t32 3.3.4 Effect of Systemic Disease. t34 3.3.5 Cytokine Correlation in AH r35 7 The Potential Immunomodulatory Effects of the Statins in Uveitis 3.4 Discussion. 137 3.4.1, rL-2.... t37 3.4.2 TNF-o¿. 138 3.4.3 IFN-y.. 3.4.4 tL-4.... 139 3.4.5 IL-5.. 140 3.4.6 IL-10.. 142 Chapter 4.. ... t44 Viability and Proliferation Assays r45 4.1 Introduction. t46 4.2 Aims .148 4.3 Results 149 4.3.1 T Cell Viability.... ..149 4.3.2 T Cell Proliferation Inhibition 157 4.4 Discussion... 168 Chapter 5...... 172 Intracellular Cytokine Staining. t74 5.1 lntroduction... t75 5.2 Aims t76 5.3 Results 176 5.4 Discussion 178 8 The Potential Immunomodulatory Effects of the Statins in Uveitis Chapter 6 187 Fluorescent Bead Immunoassay 188 6.1 Introduction. 189 6.2 Aims 6.3 Results. 6.4 Discussion. .217 Chapter 7 .23r General Discussion. .232 Chapter 8. .. .243 Bibliography 244 9 The Potential Immunomodulatory Effects of the Statins in Uveitis Table of Figures Figure 1.1 The balance between Thl and Th2 cytokines... 32 Figure 1.2 Cytokines influencing the development of antigen-activated naiVe CD4* T cells into Thl and Th2 cells.. 3s Figure 1.3 Tight junctionproteins of the blood-retinal banier. ......49 Figure 1.4 Leukocyte transendothelial migration. .....57 Figure 1.5 Signs of anterior uveitis 67 Figure 1.6 Fundus fluorescein angiogram of posterior uveitis. ......67 Figure 1.7 Structures of statins 87 Figure 1.8 Pleiotropic effects of HMG-Co A reductase inhibitors...... .....88 Figure 1.9 Intracellular effects of HMG-CoA reductase (inhibition by statins)..... .....90 Figure 2.1 The principles of density gradient centrifugation.... , ...1 10 Figure 2.2 Winlist histogram plot series showing region definition of cell populations to analyze live stimulated cells in order to track their proliferation with CFSE labeling. ....1 13 Figure 2.3 Winlist histogram plot series showing quadrant analysis resulting in 4 region percentages enabling identification of cells double positive for CD3 and IFN-y. ...116 Figure 3.1 Cytokine levels (pglml) in AH of patients with uveitis as determined by CBA versus ELISA. 127 Figure 3.2 C5rtokine levels (pglml) in AH of patients with AU and panuveitis (PAN) versus controls. 130 l0 The Potential Immunomodulatory Effects of the Statins in Uveitis Figure 3.3 Cytokine levels (pglml) in AH of controls compared with AU either on or off steroids, and PAN taking steroids 133 Figure 3.4 Cytokine levels (pglml) in AH of controls compared to AU patients with or without systemic disease, and PAN with or without systemic disease. Figure 4.1 CD3lCD28 stimulated viable T cells (%) with increasing Atorvastatin concentrations. ...150 Figure 4.2 CD3|CD28 stimulated viable T cells (%) with different Lovastatin concentrations l5t Figure 4.3 CD31CD28 stimulated viable T cells (%) with different Simvastatin concentrations. 152 Figure 4.4 Unstimulated and CD3lCD28 stimulated viable T cells (%) with different Rapamycin and Mycophenolate mofetil concentrations... ... .. 154 Figure 4.5 Unstimulated and CD3 stimulated viable T cells (%) with different CsA concentrations. 155 Figure 4.6 Unstimulated and CD3/CD28 stimulated viable T cells (%) with different Dexamethasone concentrations 156 Figure 4.7 Inhibition of CD3/CD28 stimulated proliferation of T cells with i-nraqcin- Áfnn¡qcfqfin nnnncnlrqfinnc r¡¡ifh qnrl rr¡ifhnrrf l\ulcrrolnnqtp l5R Figure 4.8 Inhibition of CD3/CD28 stimulated proliferation of T cells with increasing lovastatin concentrations with and without Mevalonate..........l59 Figure 4.9 Inhibition of CD3 and CD28 stimulated proliferation of T cells with different Simvastatin concentrations.