T CELL INTERACTIONS IN THE FOREIGN BODY RESPONSE TO BIOMATERIALS by ANALIZ RODRIGUEZ Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: James M. Anderson, M.D., Ph.D. Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. A mi mamá, Angela. Desde el primer momento en que supe que tú… Condemn no man and consider nothing impossible, for there is no man who does not have a future and there is nothing that does not have its hour ~The Talmud~ Table of Contents List of Tables……………………………………………………………………………..2 List of Figures……………………………………………………………………………3 Abstract…………………………………………………………………………………..6 Chapter 1: Introduction………………………………………………………………….8 Chapter 2: T Cell Subset Distributions following Primary and Secondary Implantation at Subcutaneous Biomaterial Implant Sites………………………………………………...29 Chapter 3: Quantitave in vivo Cytokine Analysis at Biomaterial Implant Sites………...56 Chapter 4: Evaluation of Biomaterial Surface Effects on T cell Activation in vitro……85 Chapter 5: The Foreign Body Response in T Cell Deficient Mice…………………….113 Chapter 6: Effects of in vivo IL-4 and IL-13 Neutralization on Foreign Body Giant Cell Formation……………………………………………………………………………….135 Chapter 7: Conclusions and Future Directions…………………………………………153 References………………………………………………………………………………165 1 List of Tables Table 1.1: Relative Hydrophobicity of Materials of Study Table 2.1: Antibodies used for identification of exudate cells by flow cytometry. Table 2.2: Total Exudate Leukocyte concentrations Table 3.1: Cytokines of Interest Table 3.2: Sensitivity of Cytokine Multiplex Detection Assay Table 3.3: In vivo Exudate IL-2 Concentrations as Detected by Multiplex Immunoassay Table 4.1: CD69 and CD25 Expression on CD4+ T Lymphocytes Table 4.2: IL-2 and IFNγ Concentrations from Cell Culture Supernatants Table 5.1: Summary of Immunodeficient Models Table 5.2: Total Exudate Leukocyte Concentrations Table 6.1: Summaryof Future Studies for the Elucidation of Macrophage Fusion in vivo 2 List of Figures Figure 1.1: Inflammatory and Wound Healing Events following material implantation Figure 1.2: A schematic of possible macrophage/T cell interactions at the biomaterial interface. Figure 1.3: Chemical Structures of Poly(ethylene terephthalate), Elasthane 80A, and Silastic® Figure 2.1: Alterations in the phases of the T cell response that leads to inadequate memory. Figure 2.2: Cage Implant Scheme for Investigation of the Primary and Secondary Host Response Figure 2.3: Flow Cytometry Scattergram of exudate sample depicting identification of leukocyte subpopulations by forward and side scatter. Figure 2.4: Quantification of exudate T cells, granulocytes, and macrophages by flow cytometry. Exudates were collected on days 4 (A), 7 (B), and 14 (C) following primary implantation. Figure 2.5: Percent Composition of exudate total T cells as determined by flow cytometric staining. CD8+, CD4+, and CD4+/CD25+ T subsets were quantified. Exudates were collected on days 4 (A), 7 (B), and 14 (C) following primary implantation Figure 2.6: Flow Cytometry scattergrams of CD4+/CD25+ cells from the control group (A), the PEU group (B), the SR group (C), and the PET group (D) at day 4. Figure 2.7: Quantification of exudate T cells, granulocytes, and macrophages by flow cytometry. Exudates were collected on days 4 (A), 7 (B), and 14 (C) following secondary implantation. Figure 2.8: Percent Composition of exudate total T cells as determined by flow cytometric staining. CD8+, CD4+, and CD4+/CD25+ T subsets were quantified. Exudates were collected on days 4 (A), 7 (B), and 14 (C) following secondary implantation. Figure 2.9: Comparison of T cell concentration (A), and CD8+ (B), CD4+ (C),CD4+/CD25+ (D) percent T cell composition at day 14 following primary and secondary implantation. Figure 2.10: Macrophage adherent cell density at day 14 after primary and secondary exposure on PEU surfaces (A), SR surfaces (B), and PET surfaces (C). 3 Figure 2.11: Percent fusion at day 14 after primary and secondary exposure on PEU surfaces (A), SR surfaces (B), and PET surfaces (C). Figure 3.1: IL-1B, IL-6, and TNFa concentrations at days 4, 7, and 14 post implantation. Figure 3.2: IL-4 and IL-13 concentrations at days 4, 7, and 14 post implantation. Figure 3.3: IL-10 and TGF-B concentrations at days 4, 7, and 14 post implantation. Figure 3.4: GRO/KC and MCP-1 concentrations at days 4, 7, and 14 post implantation. Figure 3.5: Adherent cell density and percent fusion at days 4, 7, and 14. Figure 3.6: Optical photomicrographs of adherent cells on PEU (A), SR (B), and PET (C) surfaces following 14 days of implantation (Magnification: 20X). Figure 4.1: CFSE histograms of lymphocytes isolated from mononuclear cells cultured on biomaterial surfaces for 3 days from one representative donor. Figure 4.2: CFSE histograms of lymphocytes isolated from mononuclear cells cultured on biomaterial surfaces with PHA stimulation for 3 days from one representative donor. Figure 4.3: CFSE histograms of lymphocytes isolated from mononuclear cells cultured on biomaterial surfaces for 7 days from one representative donor. Figure 4.4: CFSE histograms of lymphocytes isolated from mononuclear cells cultured on biomaterial surfaces for 3 days with PHA stimulation from one representative donor. Figure 4.5: IL-2 concentrations of cell culture supernatants from positive control cultures. Figure 4.6: IFNγ concentrations of cell culture supernatants from positive control cultures. Figure 4.7: Adherent cell density at days 3 and 7. Figure 4.8: Optical micrographs of adherent cells at day 7. Mononuclear cells were cultured on TCPS, PEU, SR, and PET. Figure 4.9: Percent fusion at days 3 and 7 on PET surfaces. Figure 4.10: Optical Micrograph of FBGCs on a PET surface at day 7. Figure 4.11: Optical micrographs of adherent cells at day 7 from PHA stimulated cultures. Mononuclear cells were cultured on TCPS, PEU, SR, and PET. 4 Figure 5.1: Quantification of Exudate T cells, granulocytes, and macrophages by flow cytometry. Exudates were collected from BALB/c mice and nude BALB/c mice at days 7 (A) and days 14 (B). Figure 5.2: IL-13 levels in exudate supernatant at days 7 (A), 14 (B), and 21 (C) post- implantation in BALB/c and nude BALB/c mice. Figure 5.3: Macrophage adherent cell density at days 7 (A), 14 (B), and 21 (C) post- implantation in BALB/c and nude BALB/c mice. Figure 5.4: Percent Fusion at days 14 (A) and 21 (B) post-implantation in BALB/c and nude BALB/c mice. Figure 5.5: Average nuclei per foreign body giant cell (FBGC) at days 14 (A) and 21 (B) post-implantation in BALB/c and nude BALB/c mice. Figure 5.6: Optical Micrographs of adherent macrophages following 21 days of implantation in BALB/c (A, B, C) and nude BALB/c (D, E, F) mice. Figure 6.1: Adherent cell density at day 14 on PET surfaces. Figure 6.2: Percent fusion at day 14 on PET surfaces after treatment with neutralizing IL-4 or IL-13 antibody. Figure 6.3: Optical micrographs of adherent cells on PET surfaces after treatment with goat IgG (A), IL-4 neutralizing antibody (B), or IL-13 neutralizing antibody (C). Figure 6.4: Percent fusion at day 14 on PET surfaces after treatment with both neutralizing IL-4 and IL-13 antibodies. Figure 6.5: Optical micrographs of adherent cells on PET surfaces after treatment with goat IgG (A) or IL-4 and IL-13 neutralizing antibodies (B). Figure 6.6: Schematic of macrophage fusion process on biomaterial surfaces in vitro and in vivo 5 T Cell Interactions in the Foreign Body Response to Biomaterials Abstract by ANALIZ RODRIGUEZ The role of T cells in the foreign body response to biomaterials has not been elucidated. Clinically, however patients implanted with a left ventricular assist device, a polyurethane, exhibit biomaterial induced T cell activation. Our laboratory has demonstrated that lymphocytes enhance macrophage adhesion and fusion in vitro, although it is not known if these effects are mediated by T cells. The goal of this research was to investigate potential T cell interactions in the foreign body response to three clinically relevant non-biodegradable synthetic biomedical polymers: Elasthane 80A (PEU), silicone rubber (SR), and polyethylene terephthalate (PET). Chapter 2 addresses the role that adaptive immunity, specifically memory, may play by evaluating the primary and secondary host response. We observed quantitative increases in T cells following secondary biomaterial exposure without alterations in T cell subset distributions. In Chapter 3, further characterization of the biomaterial implant site was carried out by analysis of cytokine profiles. The presence of a polymer implant did affect cytokine profiles. Most notably, IL-6 and TNFα levels were significantly greater in those animals implanted with PEU and SR, materials that do not promote fusion. In Chapter 4, in vitro T cell activation was investigated using