Mechanisms and functions of lymphangiogenesis in regulating the immune response and inflammation resolution 1

  MECHANISMS AND FUNCTIONS OF LYMPHANGIOGENESIS IN REGULATING THE IMMUNE RESPONSE AND INFLAMMATION RESOLUTION TAN KAR WAI (B.Sc (Pharm), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2011   Acknowledgements I want to thank my supervisor, Dr Veronique Angeli for her guidance in the last years. You have been generous in support, immovable in faith and a beacon in stormy waters. We had our differences, I am very grateful that you were magnanimous enough to allow me the freedom and space to pursue my research interests. I would also like to thank Dr Jean-Pierre Abastado for his invaluable advice and discussions on the projects. A heartfelt shout of thanks also goes out to Dr Anne-Laure Puaux and Dr Jo Keeble for their help and suggestions at various points in the years. I am thankful to the various friends who I have met in the last years, without whom the journey would not have been filled with great memories. Thank you Shuzhen, for the support and corridor memories. Thank you Fiona, for being my long-suffering accomplice in crime and food. Thank you Fei Chuin, for being putting up with my rantings. And understanding. Thank you Hazel, for being my first friend in the program. Thank you Victoria, for showing me that courage comes in many forms. Thank you Jocelyn, you are the stone that I missed and that turned out to be a diamond. I wish to thank my colleagues and friends in the VA lab, Angeline, Kim, Jun Xiang, Michael, Serena, Lawrence, Ivan and Sandra for the help and meaningful discussions in the years. Yes, thanks JX, SY and MT for the clowning and the insanity. I want to thank my friends outside of science for their tremendous support. You may not understand what I am doing but you guys have not dismissed me as a geek. Lastly, I want to dedicate this journey and thesis to my family. I want to thank my parents who have been loving, supportive and understanding in their usual understated and unwavering manner. And to my siblings, thank you for being different and yet the same.   i   Table of Contents   ACKNOWLEDGEMENTS .i TABLE OF CONTENTS ii ABSTRACT .ix PUBLICATIONS xi LIST OF TABLES .xii LIST OF FIGURES .xii LIST OF ABBREVIATIONS xvi     CHAPTER 1. INTRODUCTION   1.1 The lymphatic vasculature and its functions……………………… .… 1.2 Lymphatic vessels during development .……………………………….4 1.2.1 Morphogenesis of lymphatics during development…………………………… .4 1.3 Lymphatic vessels during inflammation……………………… …… 1.3.1 Molecular control of inflammation-associated lymphangiogenesis…………… 1.3.2 Cellular mediators of inflammatory lymphangiogenesis……………………….10 1.3.3 Biological roles of lymphatics during inflammation……………………………11 1.3.3.1 Lymphatics and immune cells trafficking……………………………………… 11 1.3.3.2 Lymphatics and inflammation resolution……………………………………… .13 1.3.3.3 Lymphatics and immune tolerance………………………………………………15 1.4 The biology of VEGFs and their receptors…………………………… .16 1.4.1 Introduction……………………………………………………………………….16 1.4.2 Molecular and functional diversity of VEGFs………………………………… 17 1.4.2.1 VEGF-A…………………………………………………………………………17 1.4.2.2 VEGF-C………………………………………………………………………….21 1.5 The microanatomy of the lymph node………………………………… 22 1.5.1 The building blocks of the lymph node………………………………………….22 1.5.2 Blood endothelial cells……………………………………………………………24 1.5.3 Fibroblastic reticular cells……………………………………………………… 24 1.6 Lymph node remodeling during inflammation……………………… .26 1.7 Neutrophils and immunity ……………………… .27 1.7.1 Regulation of neutrophil homeostasis during basal conditions …………………………………………………………………………27 1.7.2 Mobilization of neutrophils from BM during stress……………………………28 1.7.3 Activation of neutrophils…………………………………………………………30 1.7.4 Neutrophils and their protein cargoes ………………….………………………30 1.7.4.1 Granules and granule proteins………………………………………………… 17 1.7.4.2 Cytokines, chemokines and angiogeneic factors……………………………… 31 1.8 Aims and rationale ……………………… 32   CHAPTER 2. MATERIALS AND METHODS 33 2.1 Mice……………………… .………………………………………… 33 2.2 Immunization of mice with complete Freund’s adjuvant/ keyhole limpet hemocyanin…………………… .……………………………………33 2.3 Elicitation of chronic cutaneous hypersensitivity in mice……… .… .33 2.4 Cells isolation………………………………………………… .… .34 2.4.1 Isolation of stromal cells from lymph nodes…………………………………….34 2.4.2 Isolation of dendritic cells from lymph nodes…………………………….…….35 2.4.3 Isolation of cells from spleen and lymph nodes………….…………………… 35 2.4.4 Isolation of neutrophils from bone marrow……………………………… .… 35 2.4.5 Isolation of peritoneal macrophages……………………………………… .… 36 2.5 Neutrophil and macrophage cultures and stimulation ……… .37   iii   2.6 Labelling of cells with carboxyfluorescein diacetate succinimidyl ester (CFSE)……… 38 2.7 Adoptive cell transfers ……… .39 2.8 Culture of hybridoma cells and purification of antibodies… .39 2.9 Treatment of mice with antibodies and FTY720… 40 2.10 Dendritic cell migration assay ……… .41 2.11 Disruption of lymphatic flow through the LN ……… .41 2.12 Flow cytometry ……… .42 2.13 Immunofluorescence analysis………… .44 2.14 Hematoxylin and Eosin (H &E) staining………… …… .47 2.15 Enzyme-Linked Immunoabsorbent Assay (ELISA) .… … 47 2.16 Real time-PCR……………………………… … .……… 48 2.17 Western Blot …………… .………………… …… …… .50 2.17 Statistical analysis …………… .……………… … .… .52   CHAPTER 3. LYMPH NODE LYMPHATICS UNDERGO DIFFERENTIAL REMODELING DURING THE COURSE OF INFLAMMATION 53 3.1 Introduction…………………… .……………………….……… … 53 3.2 Results…………………………………………………….………… .…55 3.2.1 Immunization induces prolonged inflammation and lymphatic vessel expansion in draining lymph nodes……………………………………………………… 55 3.2.2 Lymph node lymphangiogenesis is initiated and sustained by lymphangiogenic factors derived from the inflamed footpad and lymph node .………… 60 3.2.3 Lymph node lymphatics undergo differential remodeling during the course of inflammation……………………………………………………………………69 3.3 Summary……………………………………….………………… ……76   iv   CHAPTER 4. REMODELING DIFFERENTIAL MODULATES LYMPHATICS IMMUNE CELL TRAFFICKING THROUGH INFLAMED LYMPH NODES 78 4.1 Introduction…………………… .……………………………… .… 78 4.2 Results……………………………………………………………….…79 4.2.1 Expansion of the subcapsular sinuses augmented dendritic cell migration into draining lymph nodes during early inflammation…………………… 79 4.2.2 Expansion of cortical and medullary sinuses during prolonged inflammation supported naïve lymphocyte egress out of draining lymph node…… .…… 81 4.2.3 Expansion of cortical and medullary sinuses supports egress of antigenactivated and naïve T cells from stimulated lymph nodes……………….…96 4.3 Summary ……………………………………………………………101 CHAPTER 5. GROWTH OF CORTICAL AND MEDULLARY SINUSES DURING LATE INFLAMMATION IS DRIVEN BY A DISTINCTIVE SPATIAL-TEMPORAL DISTRIBUTION OF VEGF-A WITHING ACTIVATED LYMPH NODES 103 5.1 Introduction…………………… .……………………………… .103 5.2 Results…………………………………………………………… .…104 5.2.1 Spatial differences in VEGF-A distribution accompany the remodeling of cortical and medullary sinuses during prolonged inflammation .…… .… 104 5.2.2 Interstitial flow is required for the differential distribution of VEGF-A in lymph node during inflammation………………………………………….… 111 5.3 Summary …………………………………………………………….116   v   CHAPTER 6. NEUTROPHILS CAN DRIVE LYMPH NODE LYMPHANGIOGENESIS IN MICE LACKING B CELLS 118 6.1 Introduction…………………… .……………………………… .118 6.2 Results…………………………………………………………… .…119 6.2.1 Lymph node lymphangiogenesis in µMT mice is greater than WT mice during later phases of inflammation……………………………………… .…… .….119 6.2.2 Lymph node lymphangiogenesis in µMT mice is accompanied by accumulation of neutrophils and monocytes… ………………………………………….… 122 6.2.3 Neutrophils are critical for lymph node lymphangiogenesis in the absence of B cells … ………………………………………………………………… ….… 127 6.2.4 Increased accumulation of neutrophils within µMT lymph nodes is mediated by increased expression of neutrophil chemoattractants… ……………… ….… 131 6.2.5 Neutrophils and non-granulocytic myeloid cells cooperate to drive lymphangiogenesis in the absence of B cells… ……………… ….… 134 6.3 Summary …………………………………………………………….137   CHAPTER 7. NEUTROPHILS DRIVE LYMPHANGIOGENESIS IN THE INFLAMED PERIPHERY OF WILD TYPE MICE 139 7.1 Introduction…………………… .……………………………… .139 7.2 Results…………………………………………………………… .…140 7.2.1 Neutrophils are required for lymphangiogenesis in the inflamed periphery of WT mice…………………….……………………………………… .…… .….140 7.2.2 Neutrophils are required for lymphangiogenesis and inflammation resolution in chronic skin inflammation…………… .……………………… .…… .….153 7.3 Summary …………………………………………………………….157   vi   CHAPTER 8. NEUTROPHILS MEDIATE LYMPHANGIOGENESIS BY SECRETION OF VEGF-C AND MMP9 158 8.1 Introduction…………………… .……………………………… .158 8.2 Results…………………………………………………………… .…160 8.2.1 VEGF-A and VEGF-C are present in similar levels in immunized footpads of NIMP-R14 and control rat IgG-treated mice …………………… .…… .….140 8.2.2 Neutrophils are the main source of matrix metalloproteinase-9 (MMP-9) within inflamed footpads and MMP-9 expression is decreased in the absence of neutrophils …………… .………………………………………… .…… .….163 8.2.3 Neutrophils secrete TIMP-1 free MMP-9 and VEGF-C in vitro following stimulation…… .………………………………………… .………….… .….168 8.3 Summary …………………………………………………………….174 CHAPTER 9. DISCUSSION 176 9.1 Differential lymphatics remodeling regulates immune cell trafficking through the inflamed lymph node ……………………………… 176 9.1.1 Counter-regulating lymph node expansion during inflammation.…… .….177 9.1.2 Mechanisms driving differential lymphatics remodeling in the lymph node during inflammation.…… .….179 9.1.3 Cross-talk between lymph node stromal cells and immune cells during inflammation may modulate lymph node remodeling.………………….….181 9.1.4 Why should we understand lymph node lymphangiogenesis? .…… ….….184 9.1.5 Future Work.…… ….185 9.2 Neutrophils as a novel and important player in lymphangiogenesis………… ……………………………… .186   vii   9.2.1 Neutrophils regulate lymphangiogenesis in inflamed sites by modulating bioavailability of VEGF-A and producing VEGF-C.………………….….187 9.2.2 Neutrophils act as a regulator to balance angiogenesis and lymphangiogenesis.…………………………………………………… ….191 9.2.3 Why should we understand the role that neutrophils play in driving lymphangiogenesis.…………………………………………………… ….193 9.2.4 Future Work .…………………………………………………… ……… 196 REFERENCES 198 APPENDICES 231 Appendix 1. Buffers and media 231 Appendix 2. List of antibodies used for flow cytometry 233 Appendix 3. List of antibodies used for immunofluorescence 234 Appendix 4. List of antibodies used for western blots 234 Appendix 5. List of primers used for RT-PCR 235   viii   Abstract Research on inflammation-induced lymphangiogenesis in lymph nodes (LNs) has been largely centered on the early phases of inflammation, subcapsular lymphatics remodeling on the afferent side and its attendant effects on the immune response. Consequently, how cortical and medullary sinuses, which serve as exits for lymphocytes, are remodeled during inflammation and how this impacts the immune response and inflammation resolution remain unexplored questions. When we induced inflammation by footpad immunization, expansion of lymphatics within draining LNs persisted beyond 90 days. Analysis of the lymphatic vessel network within inflamed LNs revealed that the subcapsular sinuses and medullary and cortical sinuses were differentially expanded during the early and late phases of inflammation, respectively. Expansion of the subcapsular sinuses during early inflammation augmented dendritic cell migration into inflamed LNs. During later and prolonged inflammation, the more predominant expansion of medullary and cortical sinuses supported lymphocyte egress, restoring it to steady state levels following an initial phase of retention. In addition, such restoration of lymphocyte egress from inflamed LNs was demonstrated to be similar for both antigenactivated and naïve T cells. Preferential expansion of cortical and medullary sinuses during prolonged inflammation was a dynamic process dependent on crosstalk and synergy between fibroblastic reticular cells (FRCs), interstitial flow and vascular endothelial growth factor-A (VEGF-A). Our data sheds new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscores the collaborative roles of lymphatics and FRCs in modulating LN plasticity and function. In an earlier study, we showed that B cells were critical for initiating lymphangiogenesis within the draining LN during early inflammation induced by footpad immunization.   ix   down-regulation in the expression of these chemokines. Consistent with previous reports (Angeli et al., 2006), blocking VEGFR2 and VEGFR3 signaling had no effect on the preexisting lymphatic and blood vessels in control DLNs (not shown).   90   Figure 4.5. T cell egress during later phases of inflammation occurs through cortical and medullary sinuses in the DLNs. (A) Confocal images of LN sections at T20 confirmed that numerous CD45.1 T cells transversed the abundant cortical and medullary sinuses present at day 14 post-immunization. (B) Compared to their untreated counterparts, the lumen of cortical and medullary sinuses in FTY720-treated immunized mice were emptied of CD45.1 T cells. Images are representative of independent experiments (n = mice). Scale bar represent 50µm.   91   Figure 4.6. Blocking VEGFR2 and VEGFR3 signaling abrogates lymphangiogenesis. (A and B) Anti-VEGFR2 and anti-VEGFR3 blockade markedly reduced inflammation-induced angiogenesis (A) and lymphangiogenesis (A and B) compared to control rat IgG-treated mice. Expansion of FRCs is not affected. Results are pooled from independent experiments with mice per group in each experiment. Student’s t test, *: p[...]... differential remodeling of cortical and medullary sinuses during prolonged inflammation 10 5, 10 6 Figure 5.2 Association of VEGF-A with the FRCs lining lymphatics 10 9, 11 0 Figure 5.3 LV surgery to disrupt lymph flow to the popliteal lymph node 11 2 Figure 5.4 Disrupting interstitial flow through draining LNs affected VEGF-A localization 11 4, 11 5 Figure 6 .1 LN remodeling in WT and µMT mice following CFA/ KLH... was primarily attributed to the inhibition of lymphangiogenesis (Dietrich et al., 2 010 ) On the other hand, lymphangiogenesis might be beneficial for the resolution of chronic   13   inflammation since lymphatic vessels drain and remove accumulated fluid, immune cells and inflammatory cytokines from the sites of inflammation Indeed, inhibition of VEGFR-3 signaling and hence lymphangiogenesis, has been... molecule -1 (ICAM -1) , vascular adhesion molecule -1 (VCAM -1) , and E-selectin (Johnson et al., 2006) and CCL 21 (Johnson and Jackson, 2 010 ) These molecules were shown to be important for facilitating the entry of DCs and lymphocytes into afferent lymphatic vessels during inflammation (Johnson et al., 2006; Johnson and Jackson, 2 010 ) Interestingly, a recent study suggests expression of inflammatory chemokines in. .. containing activated antigen presenting cells (APCs), inflammatory cytokines or antigens to drain from the periphery into LNs where they prime the LNs for an impending immunological response Expansion of lymphatic vessels within the immunized LNs and peripheral tissues have   11   been shown to support a more robust migration of DCs from the periphery into the draining LNs Interestingly, such increased... filtrate continuously from the capillaries into the interstitial space Approximately 90% of the extravasated fluid is reabsorbed at the venous side of the capillary bed, where the colloid osmotic pressure of the blood exceeds the blood pressure The main function of the lymphatic vasculature is to return the remaining 10 % of this fluid back to the circulating blood Fluid, macromolecules and cells, such... et al., 2 011 )   5   Table 1. 1 (continued) Knockout or mutant mouse models and their phenotypes according to stages in lymphatic vessel morphogenesis Adapted from (Schulte-Merker et al., 2 011 )   6   1. 3 .1 Molecular control of inflammation- associated lymphangiogenesis Although extensive evidence supports the link between inflammation and lymphangiogenesis, the molecular mechanisms underlying this association... to induce de novo lymphangiogenesis when recruited into murine thyroids over-expressing chemokine (C-C motif) ligand 21 (CCL 21) (Furtado et al., 2007) Interestingly, T cells have recently been described to play a counter-regulatory role in inhibiting lymphangiogenesis during inflammation through a paracrine secretion of interferon-γ (Kataru et al., 2 011 ) T cells are therefore proposed to maintain a... occurring early after immunization and sensitization Although neutrophils have been described to express VEGF-C in a murine model of chronic airway inflammation (Baluk et al., 2005), the observation was casual and a direct involvement in lymphangiogenesis has not been carefully examined 1. 3.3 Biological roles of lymphatics during inflammation 1. 3.3 .1 Lymphatics and immune cells trafficking During inflammation, ...   List of tables Table 1. 1 Knockout or mutant mouse models and their phenotypes according to stages in lymphatic vessel morphogenesis 5, 6 List of figures Figure 1. 1 Schematic overview of the structure and function of the lymphatic vasculature 3 Figure 1. 2 VEGF receptor-binding properties and signalling complexes 19 Figure 1. 3 DNA, RNA and protein products of human VEGF-A families 20 Figure 1. 4 Organization... during later phases of inflammation, neutrophils accumulate in inflamed B cell deficient (µMT) LNs and can substitute for B cells in driving lymphangiogenesis Neutrophils do not migrate to wild type LNs during inflammation and hence did not play a role in mediating LN lymphangiogenesis in these mice However neutrophils accumulate in immunized WT footpads and in sensitized skin and were found in these . lymphangiogenesis …………………… .10 1. 3.3 Biological roles of lymphatics during inflammation ……………………… 11 1. 3.3 .1 Lymphatics and immune cells trafficking……………………………………… 11 1. 3.3.2 Lymphatics and inflammation resolution ……………………………………. ! MECHANISMS AND FUNCTIONS OF LYMPHANGIOGENESIS IN REGULATING THE IMMUNE RESPONSE AND INFLAMMATION RESOLUTION TAN KAR WAI (B.Sc (Pharm), NUS) A THESIS SUBMITTED FOR THE DEGREE. during inflammation. …… … .17 7 9 .1. 2 Mechanisms driving differential lymphatics remodeling in the lymph node during inflammation. …… … .17 9 9 .1. 3 Cross-talk between lymph node stromal cells and immune