Thesis for doctoral degree (Ph.D.) 2008 Thesis for doctoral degree (Ph.D.) 2008 CHARACTERISATION OF HMGB1 IN INFLAMMATION CHARACTERISATION OF HMGB1 IN INFLAMMATION CHARACTERISATION Therese Östberg Therese Östberg From the Rheumatology unit, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden CHARACTERISATION OF HMGB1 IN INFLAMMATION Therese Östberg Stockholm 2008 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Larserics Digital Print AB, Sweden © Therese Östberg, 2008 ISBN 978‐91‐7409‐121‐2 2 To my family 3 The way I see it, if you want the rainbow, you gotta put up with the rain. Dolly Parton 4 SUMMARY High mobility group box chromosomal protein 1 (HMGB1) was discovered over three decades ago as a nuclear protein which is present in all mammalian nucleated cells. Subsequent studies have revealed additional properties of HMGB1 besides its originally described nuclear functions. Extracellular HMGB1 induces cellular migration, recruits stem cells, possesses antibacterial functions and somewhat surprisingly is involved in proinflammatory responses. HMGB1 can be released from certain cells in two distinct ways, either passively by dying cells or through active release from multiple cell types such as myeloid cells. The active secretion of HMGB1 is mediated via a non-classical pathway involving secretory lysosomes, a route sharing many features with the IL-1β secretion pathway. My studies of macrophages from RAGE gene-deficient mice indicate that RAGE is the major functional receptor for HMGB1 on these cells. The results also show that HMGB1 interacts with additional receptor(s), since the absence of RAGE molecules did not completely abolish HMGB1-induced cytokine production. HMGB1 needed to form complexes with selected endogenous and exogenous danger signals in order to promote inflammation, as highly purified HMGB1 on its own did not induce cytokine production. I have demonstrated the potential involvement of HMGB1 in the pathogenesis of a novel spontaneous experimental arthritis model, DNase II x Interferon type I receptor double gene-deficient mice. Marked, aberrant cytoplasmic and extracellular HMGB1 expression was evident in joint tissues from arthritic mice. HMGB1 and anti-HMGB1 antibodies could be detected in serum long before established disease, suggesting a role for HMGB1 in the initiation phase of the disease. Finally, I have used a novel approach to inhibit extracellular HMGB1 release by inducing its nuclear retention. Chromatin sequestration of HMGB1 by oxaliplatin ameliorated collagen-induced arthritis in mice. Nuclear retention of HMGB1 was also demonstrated to be a potential mechanism for the therapeutic effects of gold salts which are commonly used in rheumatic diseases. In conclusion, these studies demonstrate that HMGB1 when complexed with distinct molecules potentiates inflammation, provide further evidence of a role of extracellular HMGB1 in inflammatory arthritis, and that targeting HMGB1 is therapeutically beneficial. ISBN 978-91-7409-121-2 5 LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages Riikka Kokkola, Åsa Andersson, Gail Mullins, Therese Östberg, Carl Johan Treutiger, Berndt Arnold, Peter Nawroth, Ulf Andersson, Robert A Harris, Helena Erlandsson Harris Scandinavian Journal of Immunology. 2005 Jan;61(1):1-9 II. The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote inflammation Hulda S Hreggvidsdottir*, Therese Östberg*, Heidi Wähämaa, Hanna Schierbeck, Ann-Charlotte Aveberger, Lena Klevenvall, Karin Palmblad, Lars Ottosson, Ulf Andersson, Helena Erlandsson Harris Submitted. III. HMGB1 participates in the pathogenesis of a novel experimental arthritis model that recapitulates rheumatoid arthritis Therese Östberg, Shigekazu Nagata, Kohki Kawane, Huan Yang, Sangeeta Chavan, Lena Klevenvall, Ann-Charlotte Aveberger, Ulf Andersson, Helena Erlandssson Harris, Karin Palmblad Submitted. IV. Oxaliplatin Retains HMGB1 Intranuclearly and Ameliorates Collagen Type II-induced Arthritis Therese Östberg, Heidi Wähamaa, Karin Palmblad, Norimasa Ito, Pernilla Stridh, Maria Shoshan, Michael T Lotze, Helena Erlandsson Harris, Ulf Andersson Arthritis Research and Therapy. 2008;10(1):R1. V. Pivotal Advance: Inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate Cecilia K Zetterström, Weiwen Jiang, Heidi Wähämaa, Therese Östberg, Ann-Charlotte Aveberger, Hanna Schierbeck, Michael T Lotze, Ulf Andersson, David S Pisetsky, Helena Erlandsson Harris Journal of Leukocyte Biology. 2008 Jan;83(1):31-8 *These authors contributed equally 6 TABLE OF CONTENTS Part I Chapter one 9 Innate immunity DAMPs, PAMPs and Alarmins Cytokines Inflammation Adaptive immunity Chapter two 15 Tolerance Autoimmunity Chapter three 19 Rheumatoid arthritis Etiological features Clinical features Pathology of RA Treatment of RA Juvenile Idiopathic Arthritis Experimental models of arthritis Inducible arthritis models Spontaneous arthritis models Chapter four 27 HMGB1 Structure Post-translational modifications Intranuclear Extracellular release Receptors Cytoplasmic role Regenerative function Cytokine activity Diseases Assays 7 Part II Aims 41 Methological considerations 42 Results and Discussion 44 HMGB1 promotes inflammation in synergy with endogenous and exogenous danger signals and acts via multiple receptors (Paper I and II) A novel model for the involvement of HMGB1 in arthritis (Paper III) Nuclear retention, a novel way to therapeutically target HMGB1 (Paper IV and V) General discussion and future perspectives 51 Acknowledgements 54 References 57 Part III Publications RAGE is the major receptor for the proinflammatory activity of I HMGB1 in rodent macrophages The alarmin HMGB1 acts in synergy with endogenous and II exogenous danger signals to promote inflammation HMGB1 participates in the pathogenesis of an experimental III arthritis model that recapitulates rheumatoid arthritis Oxaliplatin Retains HMGB1 Intranuclearly and Ameliorates IV Collagen Type II-induced Arthritis Pivotal Advance: Inhibition of HMGB1 nuclear translocation as V a mechanism for the anti-rheumatic effects of gold sodium thiomalate 8 Chapter one Protection against infectious agents is mediated by the immune system. As a first line of defence the innate immune system with its physiological, anatomical, phagocytic and inflammatory barriers protects the host. The innate immune system acts rapidly, without antigen specificity and repeatedly with the purpose of eliminating pathogens. If this first clearance fails, a slower but antigen-specific system will be activated, the adaptive immune system. Antigen-specific T and B lymphocytes will then clear the infection and also give rise to immunological memory. The term immunity derives from the Latin word immunitas, meaning exemption from military service, tax payments or other public services or exemption from diseases. This chapter will give an overview of the magnificent immune system and its range of immunological players and processes. 9 Innate immunity Innate immunity provides the front line of host defence against pathogens. Regardless of what kind of danger is threatening our health it responds immediately and effectively. The innate immune system does not possess a memory nor does it discriminate between different invading pathogens. Many different barriers protect the host from the entry of pathogens. Physiological barriers consist of epithelial cells in skin, gut, lungs and eyes; chemical barriers are fatty acids, low pH, enzymes and antibacterial peptides; and finally there are microbiological flora. However, if a microorganism capable of causing damage is able to cross these barriers and enters the body it is most likely to be recognised and phagocyosed by a macrophage, dendritic cell or neutrophil granulocyte. The engulfed pathogen is then killed by a variety of toxic products produced within the phagosome. The interaction between the engulfing cell and pathogen also leads to production of cytokines and chemokines important for cell recruitment and initiation of inflammation. Several different receptors, called pattern-recognition receptors (PRRs) are used by the innate immune system to recognise and signal presence of pathogens. This recognition can lead to different events, such as stimulation of phagocytotosis when the macrophage mannose receptor is engaged. Signals through the evolutionarily conserved Toll-like receptors (TLRs) can upregulate co-stimulatory molecules on macrophages and dendritic cells, enabeling these to initiate an adaptive immune response. Thus TLRs are an important bridge between innate and adaptive immunity. Danger model Established by Matzinger, the Danger model is based on the idea that the crucial controlling signals of the immune system are actually endogenous and not exogenous. They are alarm signals that come from stressed or injured cells. The model suggests that an evolutionary useful immune system concentrates on challenges that are dangerous and cause cell damage and not necessarily the ones that are foreign (non-self). That is the critical element in the decision to initiate an immune response or not (reviewed in (1)). This hypothesis separates the Danger model from the self-nonself discrimination model in which