Characterisation of Hmgb1 in Inflammation Characterisation of Hmgb1 in Inflammation

Thesis for doctoral degree (Ph.D.) 2008 Thesis for doctoral degree (Ph.D.) 2008

CHARACTERISATION OF HMGB1 IN INFLAMMATION CHARACTERISATION OF HMGB1 IN INFLAMMATION

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

To my family

The way I see it, if you want the rainbow, you gotta put up with the rain.

Dolly Parton

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

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

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

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

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.

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 the immune system is expected to tolerate self. This will be discussed further in next chapter. The proposed theory is that cells that are stressed or injured send signals called danger or alarm signals. These signals activate antigen presenting cells which in turn respond

10 through up-regulation of co-stimulatory signals and take action. Thus sterile and microbial-induced cell damage gives rise to a similar, adequate immune response. The molecules serving as signs of damage and death are of two main types, hydrophobic and nucleic acids such as DNA and RNA. These stimuli are normally not exposed in healthy living cells and both endogenous and exogenous danger signals fit in this description and bind and signal via cell surface TLRs (reviewed in (2)) (3).

DAMPs, PAMPs and Alarmins The immune system is alerted to pathogens, cell damage or trauma by damage- associated molecular patterns (DAMPs). Depending of their origin they are classified either as pathogen-associated molecular patterns (PAMPs) or endogenous alarmins. PAMPs are a diverse set of small molecular motifs present on pathogens. These exogenous motifs are recognized and alert the immune system primarily through TLRs and PRRs. Included in the PAMP family are for example lipopolysaccaride (LPS), flagellin, lipoteichoic acid, peptidoglycan, and nucleic variants such as double-stranded RNA or unmethylated CpG motifs. Corresponding to the exogenous PAMPs are the endogenous alarmins. The term alarmin was proposed by Joost Oppenheim to identify molecules signalling tissue and cell injury. Alarmins are characterized by their rapid release following accidental cell death (necrosis) but are not released during primary programmed cell death (apoptosis); they can also be released from cells of the immune system, without connection to cell death, via non-conventional secretory pathways. Cells of the innate immune system will then be recruited and activated and some of the alarmins may promote tissue regeneration (reviewed in (4). Multiple molecules have been suggested to be alarmins, such as several heat shock proteins(5), uric acid (6), S100 (7), IL-1α (8) and High mobility group box protein 1 (HMGB1) (9), which will be discussed in detail in chapter 4.

Cytokines Cytokines are 15-30 kD proteins involved in nearly every biological event taking place in the body. During immunological processes they regulate the immune response. A part of the innate immune reaction is a cytokine storm as a response to pathogens or tissue damage. An important task of the cytokines is to allow communication between the innate and adaptive immune systems. Cytokines are secreted by one cell and act as a messenger

11 for another cell or the same cell itself, thereby altering its behaviour or properties. Essentially all cells, apart from red blood cells, can produce and respond to a cytokine. Cytokines can be divided into functional classes depending on the actions taken of the responding cell, for example pro-inflammatory and anti-inflammatory. Yet another categorization is the Th1/Th2 paradigm. Type 1 cytokines such as IFN-γ, IL-12 and TNF stimulate cell-mediated immunity and cytotoxic T cells, whereas type 2 cytokines such as IL-4 and IL-13 activate B cells and induce humoral immunity. These properties have been beneficial to researchers trying to understand the pathogenesis of autoimmune diseases and are used when it comes to finding new therapy targets. Even though cytokines are important in diseases it may appear that they are not needed for health. Mice deficient in single pro-inflammatory cytokine genes such as IL- 1α, IL-1β, IL-6 and TNF are fertile and do not develop spontaneous diseases, indicating a redundant role for these cytokines. Their cytokine deficiency only seems to be important when challenged with tissue-damaging events. However, mice deficient in anti- inflammatory cytokines such as IL-1RA, IL-10 or IL-2 do develop spontaneous inflammatory diseases.

Inflammation Inflammation is the body’s response to infection or tissue injury. Using different mechanisms inflammation achieves three different tasks; it enables the delivery of effector molecules, the killing of invading pathogen and it provides a physical encapsulated barrier preventing spread of the infection, and lastly promotes tissue repair. The classical manifestations of tissue injury and inflammation are rubor (redness), tumor (swelling), calor (heat), dolor (pain), and functio laesa (impaired function). These signs reflect the central events of an inflammatory response; cytokines induce local dilation in capillaries which gives rise to increased blood flow, making the skin red and swollen. Fluid starts to leak through the gaps between endothelial cells leading to oedema which in turn puts pressure on nerve endings causing pain. An influx of cells, mainly macrophages and neutrophils, from the blood into the tissue and their simultaneous production of proinflammatory cytokines also triggers receptor-induced pain. These actions of inflammation are induced and regulated by a large number of chemical mediators, such as complement factors, kinins, histamines and cytokines. These various mediators of the immune response must be regulated precisely, since deficient

12 responses can result in immunodeficiency diseases and excessive responses can cause morbidity and mortality.

Adaptive immunity This system is in contrast to innate immunity antigen-specific, having the capacity to distinguish subtle changes in molecular conformation. It also possesses the ability to create and sustain immunological memory, enabling an accelerated response to recall antigens. The adaptive immune system can be divided into humoral immunity mediated by antibody-producing B lymphocytes and cell-mediated immunity driven by T lymphocytes.

Chapter two

The immune system expresses a vast capacity to eradicate pathogens. To avoid attacks on self-antigens this powerful machinery has evolved mechanisms to regulate its actions called tolerance. However, instead of only reacting against foreign antigens, the immune system can sometimes be deviated and focuses its attack on self-antigens. This attack of the immune system against self-components is called autoimmunity. Paul Ehrlich described autoimmunity in the early 1900s when he established the term “Horror autotoxicus” (reviewed in (10). This chapter will briefly review the mechanisms of immunological tolerance and the results of its loss.

Tolerance Mechanisms acting during several stages of the lymphocyte lifespan have developed to discriminate self from non-self, thereby protecting normal tissue from autoimmune reactions. Each stage is partly effective in preventing anti-self responses and all of them work synergistically. The first and most important mechanism is central tolerance which takes place in the thymus or bone marrow for T cells and B cells respectively, when autoreactive cells are deleted. Even tissue-restricted self-antigens are presented in the thymus by a subset of dendritic-like cells, medullary epithelial cells, and the transcription factor AIRE is thought to be responsible for transcription of these genes (11, 12). All T cells are screened for reactivity against self-antigen. The second step in the process of tolerance takes place in the periphery, complementing the central tolerance mechanisms since self-reactive lymphocytes might escape thymic selection (13). The principal mechanisms of this step are functional unresponsiveness (anergy) (14, 15), deletion and suppression by regulatory T cells.

Autoimmunity When an autoimmune reaction leads to an inflammatory response and results in clinical pathology an autoimmune disease has evolved. Autoimmune diseases affect about 4-5% of the Western World population (16) and have a higher incidence in females than in males (17). Autoimmune diseases can be divided into two broad categories: organ-specific and systemic autoimmune diseases. In the first, the immune response is often directed against an organ- specific antigen, leading to an organ-specific reaction. Nearly every organ in the body can be affected. Examples of such diseases are Grave’s disease (thyroid gland), multiple sclerosis (CNS) and insulin-dependent diabetes (pancreatic islets β cells). In the latter category the immune response is directed towards a broad range of target self- antigens; SLE is an example of such a disease. There are also diseases not falling into either of these two categories, being intermediates by having organ-specific targets and additional systemic manifestations, such as Rheumatoid arthritis (RA) which affects joints. How immunological tolerance is broken is unclear, but both genes and the environment are involved. Most autoimmune diseases have been demonstrated to have MHC-linkage and MHC is the strongest genetic susceptibility association (reviewed in

(18). The disease could also in rare cases be due to a single mutation, as with the AIRE (autoimmune regulator) gene. Mutations in AIRE result in a syndrome called autoimmune polyendocrinopathy-candidasis ectodermal dystrophy (APECED) (19). Patients with APECED suffer from autoimmune diseases in multiple organs. Autoimmunity is most likely not only initiated by genetic factors, indicated by epidemiological studies, but also by environmental factors. The immune system can be activated in a non-specific polyclonal manner by products from microorganisms such as lipopolysaccaride (LPS), bacteria DNA or viruses. By binding to PPRs these products can act as adjuvants, enhancing the immune systems, response to self-antigens; eventually leading to autoimmunity. Hormonal factors, exposure of normally hidden antigens or modifications of autoantigens have also been suggested to be inducers of autoimmunity. However, in most cases of autoimmune diseases the disease-inducing antigen unknown. In an autoimmune condition autoreactive T cells and/or B cells are activated. However, all individuals possess lymphocytes in their blood that can respond to self- antigen (20, 21). Additionally, low levels of autoantibodies can be demonstrated in healthy individuals (reviewed in (22, 23), indicating an importance of some self-reactivity for maintaining the function of the immune system.

Chapter three

The inflammatory joint disease Rheumatoid Arthritis (RA) received its name in 1859 by the British rheumatologist Sir Alfred Baring Garrod. The name is based on the term “rheumatic fever”, an illness which includes joint pain and is derived from the Greek word for flowing, rheumatos. The suffix -oid meaning resembling indicates a translation as a joint inflammation that resembles rheumatic fever. But the disease has been around long before that; it was described by Hippocrates in the 4th century and signs of the disease have been found in skeletons from Native Americans. Extensive research has focused on the aetiology and pathogenesis of RA, but still we lack a cure or pathogenetic explanation for this disease which affects about 1% of the western population (24). In this chapter I will review some characteristics of RA and its experimental models.

Rheumatoid Arthritis Etiological features The aetiology of RA is incompletely understood, but there is strong evidence that both genes and environment do play a part. The strongest evidence for this is provided by twin studies (25). The most important genetic contribution to RA is contained within the MHC class II locus. The haplotypes DR1 and DR4 share an epitope (the shared epitope), which is expressed in more than 80% of Caucasian RA patients (reviewed in(26)). This genetic association implicates T cells in the pathogenesis of RA, since the only known function of MHC class II is to present peptides to CD4+ T cells. Since RA mostly affects women, ratio 3:1, hormonal factors are suggested to play part (26). During pregnancy many women report remission while flare is common postpartum and during breastfeeding (27). Environmental factors are of importance for the development of RA. Inconclusive studies of viral influence on the development of RA have been performed; Epstein-Barr virus and cytomegalovirus have been detected in the synovial membrane of patients with RA (28-30). There are several reports showing a connection between smoking and RA, indicating smoking as the strongest environmental factor for increased risk of developing RA (31- 33). Strikingly, a combination of smoking and shared epitope alleles further increases the risk, suggesting interaction between genetic and environmental factors (34).

Clinical features RA is a chronically, disabling disease characterised by synovitis, destruction of cartilage and bone and, ultimately, loss of joint function. Furthermore, hematological, cardiovascular and respiratory systems also are frequently affected. Diagnostic criteria were developed by The American College of Rheumatology (ACR) (35) (Table 1). These criteria are used by physicians to set a diagnosis for the patient; four of the seven criteria must be fulfilled for the diagnosis. Furthermore, criteria 1-4 must have been experienced for at least six weeks. Rheumatoid factor (RF) is the only serological marker included in the ACR criteria. RF is an antibody, mainly of IgM isotype, reactive with the Fc region of IgG or IgM. RF is not exclusive to RA but can be detected in the blood of 70% of the patients with RA, and is used as a diagnostic tool to estimate the progress and the severity of the disease

(36). A more specific serological marker for RA is anti-CCP antibody, with a specificity of 95% and sensitivity of 55-80% (37). The anti-CCP antibody is directed to proteins in which arginine has undergone posttranslational modification to citrulline through action of the converting enzyme peptidylarginine deiminases (PAD).

Table 1. ACR Criteria for RA

1. Morning stiffness > one hour

2. Arthritis of three or more joint areas

3. Arthritis of hand joints 4. Symmetric arthritis 5. Rheumatic nodules 6. Serum rheumatic factor 7. Radiographic changes

Pathology of RA Normal synovial tissue is a thin membrane comprising one-to-three cell layers that are loosely associated. The membrane consists of two major cell types, macrophage-like and fibroblast-like synoviocytes. During RA synoviocytes proliferate and inflammatory cells are recruited from the blood. This results in a thickened membrane with a depth of 6-8 layers and more. A massive accumulation of macrophages is apparent during rheumatoid synovitis (Figure 1). Macrophages appear to play a pivotal role in RA; they are numerous in the inflamed synovial membrane and the cartilage-pannus junction. Activated macrophages produce prostaglandins and other inflammatory mediators and enzymes, in particular IL-1 and TNF. These cytokines are active in destruction of bone and cartilage in the pathogenesis of RA. The activated macrophages over-express proinflammatory or regulatory cytokines, growth factors, chemokines and upregulate MHC class II in response to stimuli. Plasma cells in the inflamed synovial membrane produce large quantities of immunoglobulins including autoantibodies such as RF. T lymphocytes are present within the deeper layers of the synovium. Activated T lymphocytes produce certain cytokines including IL-17, which can be found in both synovial fluid and synovial membrane. Dendritic cells and activated fibroblasts also occur in the inflamed synovium.

Cytokines are important key players in the pathogenesis of RA, synovial fluid containing large quantities of them secreted by macrophages, dendritic cells, neutrophils and synovial fibroblasts. However, T cell-derived cytokines are rare, with the exception of IL-17. Some of these cytokines, such as TNF, IL-1 and IL-17, stimulate the production of destructive proteases which in turn enhance cartilage destruction. Matrix metalloproteinases (MMPs) breakdown collagen and have been implicated in this damage. The inflamed synovium produces a chronic inflammatory pannus tissue, which grows into the the articular cartilage and leads to cartilage and bone destruction. This bone destruction usually first appears as a marginal erosion at the site of synovial proliferation, where bone is unprotected by hyaline cartilage. Subsequent bone destruction leads to subluxation and deformity.

Fig. 1: Schematic illustration of normal (left) and arthritic (right) joint. Adapted from Feldmann.

Treatment of RA Traditionally, treatments for RA patients have been NSAID drugs, systemic administration or intra-articular injections of corticosteroids, disease- modifying antirheumatic drugs such as methotrexate, administration of gold salt compounds or antimalarias (Table 2). Lately, specific antagonists against TNF or IL-1 have provided beneficial treatment results in many, but not all RA patients. This demonstrates the central importance of cytokines in the pathogenesis of RA and stimulates a search for additional cytokine blocking therapies.

Table 2. RA treatment Biologicals Antagonists including mAb and soluble receptors against endogenous proinflammatory mediators. TNF blockers (infliximab, etanercept, adalimumab), rIL-1 receptor antagonist, IL-6 R blocking mAb (tocilizumab), anti-CD20 mAb (B cells Rituximab), CTLA4-Ig fusion protein (T cells, Abatacept).

Corticosteroids Anti-inflammatory mechanisms. Beneficial effects on joint damage.Discovery awarded Nobel Prize 1950.

DMARDs Disease-modifying anti-rheumatic drugs. Halt or retard progression of disease. Prevent or reduce joint damage, preserve joint integrity and function. Methotrexate, sulphasalazine, gold salts, chloroquine and cyclosporine A.

NSAIDs Non-steroidal anti-inflammatory drugs. Analgesic and mild anti- inflammatory effect. Do not alter the disease course or prevent joint destruction. Inhibit cyclooxygenase enzyme leading to decrease in prostaglandin synthesis.

Juvenile Idiopathic Arthritis Chronic arthritis starting before 16 years of age is classified as juvenile idiopathic arthritis (JIA). These multiple diseases are of unknown cause but seem to include both genetic and environmental components (reviewed in (38)); JIA is classified into seven disease categories based on the features present during the first 6 months of illness (Table 3) (39). JIA is the most common chronic rheumatic disease in children; studies have reported a prevalence of 16-150 per 100 000. The inflammatory synovitis is similar to that seen in rheumatoid arthritis (reviewed in(38)). The synovitis displays distinct hyperplasia with an infiltration of T cells, B cells, macrophages and dendritic cells (40, 41). The inflammatory process leads to pannus formation, cartilage and bone erosions.

Experimental models of arthritis As previously mentioned, RA is a disease with a complex interaction of environmental influences and genetic background. Many pathogenic mechanisms can be studied by using cells, tissue and peripheral blood from patients and healthy volunteers. However, some studies require larger study groups to elucidate the specific characteristics of RA or the ability to test a new treatment approach. Animal models offer researchers a tool to further investigate rheumatic diseases, the ability to reduce the heterogeneity by controlling the environment, genes and onset of disease. The experimental models also provide an

23 opportunity to study early events leading to disease, events that have already taken place in patients arriving at the clinic. Today a variety of models exists, both spontaneous and inducible, each presenting a unique opportunity to study different aspects of arthritis. However, no animal model recapitulates RA in great detail.

Table 3. Categories of JIA Category Frequency* Onset age Sex ratio

Systemic arthritis 4–17% Throughout childhood F=M

Oligoarthritis 27–56% Early childhood; peak at 2–4 F>>>M years Rheumatoid-factor- 2–7% Late childhood or adolescence F>>M positive polyarthritis Rheumatoid-factor- 11–28% Biphasic distribution; early peak F>>M negative at polyarthritis 2–4 years and later peak at 6–12 years

Enthesitis-related 3–11% Late childhood or adolescence M>>F arthritis Psoriatic arthritis 2–11% Biphasic distribution; early peak F>M at 2–4 years and later peak at 9–11 years

Undifferentiated arthritis 11–21%

*Reported frequencies refer to percentage of all juvenile idiopathic arthritis.

Inducible arthritis models Arthritis can be induced by immunisation with antigen together with adjuvant or with adjuvant alone. An adjuvant is a substance used to enhance the immune systems response to an antigen. The experimental disease collagen-induced arthritis (CIA) is the most used model of arthritis. This arthritis model is achieved by injecting collagen type II, the main component of cartilage, with either Freund´s complete adjuvant (CFA, inactivated mycobacteria suspended in mineral oil) or Freund´s incomplete adjuvant (IFA, mineral oil alone) (42). It was first developed in rats in 1977 by Trentham et al (43), and has over the years also been established in mice (44) and primates (45). Being symmetric and affecting peripheral joints, forming a synovial pannus and leading to erosions of cartilage and bone,

CIA has many similarities to human rheumatoid arthritis. Both cellular and humoral immune responses are also involved in the disease. However, in contrast to RA no lymphoid aggregates are formed; there is no difference in sex predilection; and no RF can be demonstrated in CIA (46, 47). The susceptibility of CIA has been related to certain MHC class II alleles, in mice, strains bearing MHC I-Aq, I-Ar or I-Ab developing arthritis after challenge with CII (48). Transfer of spleen and lymph node cells and also serum from arthritic animals to naive animals leads to disease in the recipient (49, 50), indicating the importance of both cellular and humoral immune responses towards CII in order to develop CIA. There are also models of arthritis in which induction only uses adjuvants. The first one described was adjuvant-induced arthritis (AIA) (51). Synovitis is induced by injecting rats with CFA resulting in aggressive disease with extra-articular manifestations. Later on it was demonstrated that mineral oil alone could also induce arthritis in certain highly susceptible rat strains. Oil-induced arthritis (OIA) is accomplished by immunisation of IFA (52) or with injections with the plant oil pristane (53) or the cholesterol precursor squalene (54).

Spontaneous arthritis models Nagata et al described in 2006 a new model for spontaneous, destructive polyarthritis, revealing the greatest resemblance of RA of all models to date. Mice rendered gene double-deficient for DNase type II and IFN-IR develop symmetrical polyarthritis at around 8 weeks of age starting in digital joints (55). DNase type II is an enzyme in macrophages that digests the chromosomal DNA of apoptotic cells and nuclei which are mainly expelled from erythroid precursor cells following macrophage engulfment (56, 57). IFN-IR mediates the type I interferons (IFN-β and IFN-α) signals, with have strong cytotoxic effects in newborn mice (58, 59). Crossing the mice with Rag-/- mice did not change the clinical outcome, indicating that the adaptive immune system is not involved in the phenotype in these mice (personal communication Dr Nagata). Conversely, this might be a model in which autoinflammation leads to autoimmunity. Deletion of the TLR9 gene had no apparent effect on the development of arthritis in DNase-/- IRF-IR-/- mice, indicating that DNA in the serum might not contribute to the pathogenesis directly. In contrast, deletion of the MyD88 gene, encoding for an intracellular adaptor protein utilized by most TLRs and IL-1 type 1 receptor for intracellular signaling, prevented the development of arthritis (personal communication Dr Nagata). Both clinically and with

25 regards to several immunological and histological parameters, arthritis in the DNase II-/- x IFN-IR-/- mouse share many features with human RA. For instance the mice express high serum levels of anti-CCP antibodies, RF and matrix metalloproteinase-3. Furthermore, administration of anti-TNF antibody prevented the development of arthritis in these mice. Spontaneous severe arthritis develops in K/BxN mice, an intercross between K/B TCR transgenic mice and NOD mice, due to the autoreactivity of the transgenic TCR and subsequent induction of autoantibodies directed against glucose-6-phosphate isomerase (60). These autoantibodies induce arthritis upon transfer to most naïve syngeneic and allogenic hosts (61). The importance of cytokines in the pathogenesis of arthritis can be observed in TNF transgenic mice overexpressing TNF systemically (62, 63) and IL-1Ra deficient mice (64), both of which develop spontaneous arthritis. Not only genetically modified mice develop spontaneous arthritis but aging male DBA/1 mouse can as well (65). However, castration prevents disease, indicating not only a genetic importance but also a hormonal influence (66).

Chapter four

High mobility group box chromosomal protein 1 (HMGB1) was described over three decades ago as a nuclear protein. The protein was given its name because of its ability to migrate rapidly in agarose gels during electrophoresis (67). HMGB1 is an abundant protein and is distributed in all mammalian nucleated cells. More than one million molecules per nucleus can be found in the thymus (68). Intracellulary, HMGB1 is more concentrated in the cytoplasm of cells in the liver and brain and is concentrated in the nuclei of most other tissues (69). Over the years HMGB1 has been studied and additional properties besides its originally described nuclear functions have been revealed. Extracellular HMGB1 induces migration, recruits stem cells, possesses antibacterial functions and complexed HMGB1 induces cytokine production. In this section the different properties of HMGB1 will be discussed.

HMGB1 has over the years been studied under several names, the “differentiation enhancing factor” was released by murine erythroleukemic cells (70, 71) and “sulfoglucuronyl carbohydrate binding protein” localised in neuronal nuclei (72) proved to be identical to HMGB1. Moreover, for many years HMGB1 was known as “amphoterin” when involved in neurite outgrowth (73).

Structure HMGB1 is highly conserved between species with a sequence homology of 99% between the rodent and human forms, and is present in all mammalian tissues. HMGB1 comprises a single polypeptide chain of 214 amino acids (the gene encodes for 215 amino acids residues but the initial methionine is not expressed). HMGB1 is a member of the high-mobility group (HMG) protein superfamily which includes HMGB1, HMGB2, HMGB3 and SP100HMG. The amino acid sequences within the HMGB family are highly conserved and all members consist of three distinct domains. The two DNA-binding elements of HMGB1, denoted the A and B boxes, respectively, are made up of approximately 80 amino acid residues arranged in three alpha helices (Figure 2). The A and B boxes are strongly positively charged. These DNA-binding boxes are followed by a highly acidic 30 amino acid tail containing only aspartic and glutamic acids which can interact with and fold over the HMG boxes and may thereby interfere with their intermolecular activity (74). The positively charged amino acids may be the reason for its migration as a 30kDa molecule in SDS-PAGE gels even though HMGB1 has a molecular weight of approximately 25kDa. The large number of charged residues (43 lysines and 9 arginines in the N-terminal DNA binding domains and 36 glutamic acids and 20 aspartic acids in the C-terminal acidic tail) gives the protein strong bipolar properties. These bipolar features may promote binding to endogenous and exogenous components. HMGB1 has an uneven number of cysteines, a feature shared with IL-1β and IL-18, and should theoretically be able to complex with other proteins.

DNA Binding domain 1DNA Binding domain 2 NLS1 NLS2 9 79 89 162 214 N A box B box C 89 108 150 183 Proinflammatory RAGE binding domain cytokine domain

Figure 2. Structure of HMGB1.

Post-translational modifications Besides its native form HMGB1 can exist in several different conformations generated through post-translational modifications. HMGB1 derived from necrotic cells can be acetylated on lysines at positions 2 and 11. While the whole protein actively secreted from monocytes can be acetylated in all positions, segments 27-43 and 178-184 are particulary affected, yielding at least 10 isoforms of the protein (75). These two positively charged segments act as nuclear localization signals (NLSs) and neutralization of these NLSs results in relocation of HMGB1 from the nucleus into the cytoplasm. Moreover, phosphorylated HMGB1 is also translocated into the cytoplasm and prevented from re- entering the nucleus. However, phosphorylated HMGB1 has not been demonstrated extracellularly (76). Lastly, mono-methylation of HMGB1 on lysine 42 can occur in neutrophils. This methylated HMGB1 can be both relocated into the cytoplasm and secreted (77). All these modifications diminish HMGB1-chromatin interactions but it is not clear whether they are required for lysosomal and plasma membrane passage. HMGB1 from metabolically stressed cells can also be translocated to the cytoplasm without undergoing acetylation or any other known modification (78).

Intranuclear In the nucleus HMGB1 plays an important role in transcription regulation, modifying the structure of DNA and stabilizing nucleosomes (79) (74). HMGB1 binds the minor groove of DNA without sequence specificity and induces bends, but has a preference for binding sharply bent structures (80). This binding of HMGB1 to DNA facilitates physical interactions between DNA and transcription factors, including p53, homeobox-containing proteins, steroid hormone receptors and recombination activating gene 1/2 (RAG1/2) proteins which are needed for VDJ recombination in T and B lymphocytes (81). There is

29 one intriguing report about HMGB1 transactivating the human IL1-β gene promoter through association with an Ets transcription factor (82). There is also one publication demonstrating that HMGB1 may directly bind to the RRS sequence in the TNF promoter in osteoclasts to activate TNF transcription (83). Binding of HMGB1 to undamaged DNA is a rapid and transient process, while HMGB1 binds tightly to sites of distorted DNA (84). HMGB1 constantly shuttles within the nucleus and between the nuclear and cytoplasmic compartments (85). HMGB1 has two nuclear localisation signals that direct the protein to the nucleus and can in addition bind to calmodulin which also can target HMGB1 to the nucleus (75, 86). Export of HMGB1 from the nucleus to the cytoplasm is independent of protein synthesis and is mediated by the chromosome region maintenance 1 protein (CRM1) (76, 87). The nuclear function of HMGB1 has been demonstrated to be essential to life since HMGB1 knockout mice die 24-48 hours after birth due to hypoglycemia (88). Phenotypic features include small size, ruffled and disorganised fur, long hind paws and absence of fat. Cells lines deficient in HMGB1 display a normal growth but have an impaired gene expression of different factors including the glucocorticoid receptors (88).

Extracellular Release HMGB1 can be secreted from cells in two ways, either passively or actively (Figure 3). Cells made necrotic release their HMGB1 passively whereas the original observation was that cells undergoing apoptotic cell death would not release HMGB1 due to sequestration of HMGB1 to the condensed chromatin (89, 90). However, it has recently been demonstrated that certain apoptotic cells undergoing secondary necrosis and may passively leak HMGB1, indicating that the originally described dichotomy between necrosis and apoptosis may not actually be so distinct (91). Furthermore, it has also recently been reported that cells exposed to metabolic stress (including hypoxemia) may translocate nuclear HMGB1 to the cytosol (78). If such a cell would undergo apoptotic cell death there would not be any HMGB1-DNA interaction that would prevent its release. In response to proinflammatory stimuli such as LPS, IFN-γ, IFN-α/β and nitric oxide HMGB1 can actively be released from a number of different cell types including macrophages, pituicytes, mature dendritic cells, NK cells and fibroblasts (92-94) (Wähämaa, personal communication) and protein synthesis is not required for to secretion (95). The mechanism underlying the active secretion is incompletely understood since HMGB1 lacks a classical signal sequence. Studies suggest HMGB1 release to be mediated

30 via a non-classical pathway by Ca2+ regulated endosome-like organelles termed secretory lysosomes (96). This route shares features with the IL-1β secretory pathway (97, 98). However, IL-1β release displays a somewhat faster kinetic response than does HMGB1 release. The secretion of IL-1β is induced by autocrinally released ATP while HMGB1 secretion is triggered by lysophosphatidylcholine (LPC) which is produced later than ATP (96). Secretory lysosomes are expressed in hemopoietic cells which do play an important role in immune and inflammatory events (99). The role of transporters in nonclassical protein secretion has been widely studied. One suggested transporter for HMGB1 is the ATP-binding cassette transporter 1 (100, 101). Inhibition of this transporter inhibits HMGB1 release from monocytes/macrophages (102). Furthermore, the ABC transporter, ABCC1 (aka multi-drug resistance protein-1 (MRP-1)) has been described to mediate HMGB1 secretion from macrophages (101).

Active release Passive release

DC Any cell Endothelial cells Fibroblasts Macrophages LPS NK cells IFN-γ Pituicytes IFN-α/β Smooth muscle cells NO Apoptosis Necrosis

Inflammation

Figure 3. Release of HMGB1.

Receptors The first described receptor for HMGB1 was the receptor for advanced glycation end products (RAGE) (103). However, since RAGE-deficient cells were shown to still be able to respond to HMGB1 stimulation and anti-RAGE antibodies only partially suppressed the activity of HMGB1, RAGE is not believed to be the only receptor for HMGB1 (I) (104,

105). Recently, toll-like receptors (TLR) 2 and 4 were suggested to interact with HMGB1 (Figure 4) (106-108). RAGE belongs to the immunoglobulin (Ig) superfamily and comprises of three extracellular Ig domains, a single transmembrane segment and a short cytoplasmic tail. RAGE- deficient mice are viable and less susceptible to sepsis (109). As a pattern recognition receptor it interacts with several ligands such as multiple members of the S100 protein family, amyloid-β and advanced glycation end products (reviewed in (110). RAGE is only highly constitutively expressed in lung and skin tissues, but can be up-regulated in almost every tissue (111). Binding of HMGB1 to RAGE has two main consequences: activation of intracellular signal transduction through mechanisms involving Cdc42 and Rac, guanosine triphosphatases that regulate cell motility and neurite outgrowth, and the other pathway activates mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) (112, 113). In macrophages, neutrophils and Caco-2 epithelial cells the MAPKs activated by HMGB1-RAGE interaction are p38 and p42/44 kinases, stress-activated protein kinase/c-Jun N-terminal kinase and ERK1/2 (114-116). Smooth muscle cell migration mediated by HMGB1 involves MAPK pathways and a G-protein-coupled receptor (117). The only known adaptor molecules binding to the cytoplasmic tail of RAGE are ERK 1/2 and diaphanous-1 (118). A soluble isotype splice form called sRAGE exists. The function of this isoform is incompletely understood but does provide protective effects in many diseases (as discussed later in this chapter). An HMGB1-dependent activation and recruitment of neutrophils has been demonstrated to require interplay between RAGE and Mac-1 (119), indicating the ability of HMGB1 to interact with other immunostimulatory molecules in order to amplify their activity. HMGB1 has been described, as mentioned above, to be a ligand of TLRs. There are currently ten different TLRs identified in humans (120). The cellular innate immune response is initiated by a limited number of pattern recognition receptors present in several various immune cell types. The TLRs are typical representatives of those receptors. TLRs are type I transmembrane glycoproteins composed of an extracellular domain, a single transmembrane domain, and an intracellular Toll-IL-1 Receptor domain (TIR). The extracellular domains contain leucine-rich repeats and are responsible for recognition of common structural patterns in diverse microbial molecules, but also unexpectedly of certain endogenous ligands. The TIR domain may engage adaptor molecules such as MyD88, TRIF and TRAM, activating downstream signalling cascades

32 ultimately leading to NFκB nuclear translocation leading to e.g. transcription of inflammatory genes and DC maturation (121).

TLR2 RAGE TLR4

HMGB1

ligand

p38 MyD88 p42/44 MyD88 TRAM MAL/TIR Cdc42 ERK1/2 MAL/TIR TRIF Rac SAPK/JNK

NF‐κB Inflammation Migration

Figure 4. Signalling receptors for HMGB1.

Cytoplasmic role HMGB1 plays an important role in migration, interaction with RAGE mediating cellular neurite outgrowth and tumor formation (73, 112-114). HMGB1 added to normal and dystrophic mouse muscles attracts mesoangioblasts, further supporting a role for HMGB1 as a chemoattractant (122, 123). In addition, HMGB1 promotes angiogenesis, the process leading to formation of new blood vessels during development, growth, tissue repair and tumor growth (124-126). Studies have placed HMGB1 in the antibacterial barrier defence system, HMGB1 purified from human adenoid glands eliminating bacteria within a few minutes in cultures (127).

Regenerative function Extracellular HMGB1 can function as a mitogen by attracting and activating stem cells to damaged tissue for repair. Supporting this theory is the finding that special beads coated with HMGB1 injected into murine normal healthy muscles recruit mesoangioblasts to the site (122). In another experiment colonization of mesoangioblasts in drystrophic muscles were observed after HMGB1 administration (123). In addition, in a mouse model of myocardial infarction local HMGB1 injection resulted in the accumulation of myocytes within the infarcted area, leading to a significant recovery of cardiac performance (128).

Cytokine activity The first report of a proinflammatory role for HMGB1 came in 1999 from Kevin Tracey’s research group in New York. They reported HMGB1 being a late mediator of sepsis, being released from monocytes following stimulation with LPS (93) (which represents a well established gram-negative sepsis model). Moreover, injections of HMGB1 caused toxic shock in mice, particularly when combined with endotoxin (129). Supporting the proinflammatory role of HMGB1 in sepsis was the finding of HMGB1 being a strong activator of human monocytes (130). HMGB1 stimulation of PBMCs led to release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β. Moreover, a dose-dependent increase in ICAM, VCAM and RAGE expression on endothelial cells following HMGB1 stimulation has also been observed. HMGB1 can act as a mediator to initiate adaptive immune responses as the protein promotes maturation of dendritic cells (DCs), which are specialized in antigen presentation to T cells (131-134). The HMGB1 secreted by DCs was also required for the clonal expansion, survival, and functional polarization of naive T cells (135). Furthermore, DCs can stimulate natural killer (NK) cells through release of IL-18; the activated NK cell in turn releases HMGB1 which will induce DC maturation and protect DCs from lysis (92). However, recent studies indicate that highly purified HMGB1 may not by itself be active as a pro-inflammatory mediator. Several recent reports indicate that HMGB1 needs to form a complex with proinflammatory ligands to exert its synergistic influence. Reported ligands for HMGB1 to date include LPS, class A CpG oligodeoxynucleotides (ODN), CpG-DNA and IL-1β (136-139)(II). Futhermore, HMGB1 selectively forms complexes with its ligands since no associations were observed with TNF, RANKL, IL-18

34 or Poly I:C (II). The ability of HMGB1 to form complexes with ligands might illustrate a key function of this protein, as an enhancer of inflammatory responses by binding to other co-existing danger molecules.

Table 4. Cells targeted by HMGB1 Target cell Effect of HMGB1 Astrocytes Proinflammatory activity, including chemotactic signals and MMP formation. Cardiac myocytes Negative inotropic effects. Involved in repair. Dendritic cells Maturation and capacity to home to lymph nodes. B cells Potentiates activation by DNA-IgG complexes. Endothelial cells Proangiogenic. Upregulation of adhesion molecules. Epithelial cells Increased enterocyte permeability causing barrier dysfunction. Bactericidal effects. Monocytes/macrophages Potential proinflammatory cytokine production in collaboration with PAMP or DAMP molecules. Promotes transendothelial migrations of monocytes. Neurons Induces neurite outgrowth. Neutrophils Enhances chemotaxis. Prevents apoptotis by binding to surface expressed phosphatidylserine (“eat me signal”). Osteoclasts Enhances osteoclast formation and TNF release by direct binding to the TNF promoter. Platelets Expressed on the cell surface by activated platelets. Smooth muscle cells Causes migration and proliferation. Stem cells Chemotaxis. Promotes differentiation. T cells Proliferation and polarisation toward TH1. Tumor cells Enhances invasiveness.

Diseases Several studies implicate HMGB1 in the pathogenesis of various inflammatory conditions and diseases (Table 5). Important characteristics of chronic synovitis are necrotic cell death, apoptosis and activation of macrophages. All these processes lead to release of HMGB1, which can in turn contribute to chronic arthritis inflammation. Extracellular and cytoplasmic expression of HMGB1 has been observed in tissue sections from synovitis in both human arthritis and in experimental models. In addition, the synovial fluid of RA patients contains high levels of HMGB1 (140, 141). Intra-articular injections of recombinant HMGB1 into murine knee joints induce a local, erosive arthritis (142). Longitudinal studies of synovial tissues from rats with CIA indicate a similar kinetic pattern of extracellular expression of HMGB1, IL-1β and TNF without a discriminative sequential order (143, 144). However the tissue localisation of these mediators appears to differ. Maximal HMGB1 expression at both mRNA and protein levels occurs in areas where the pannus tissue invades bone and articular cartilage. TNF expression peaks in the lining layer as well as in the sublining areas of the synovial membrane, whereas HMGB1 and IL-1β are also present in chondrocytes. HMGB1 can be detected in sera and synovia from DNase-/- x IFN-IR-/- mice, which spontaneously develop a disease sharing many features with human RA. Taken together these findings indicate a functional role of HMGB1 during the course of arthritis (III)(55).

Therapy Ever since the first report of the pathogenic role of HMGB1 in sepsis, the search for an HMGB1-targeted therapy has been vigorous. The search has so far generated promising results in multiple pre-clinical models but no HMGB1-specific therapy is yet available for patients. Neutralizing polyclonal antibodies ameliorate CIA in rodents, reducing clinical signs of established arthritis and attenuating TNF and IL-1β expression in joint tissues (143). Even though the results from polyclonal antibody treatment were very promising the sequential outcome when using monoclonal antibodies has been disappointing (Östberg unpublished data). Use of such antibodies in other diseases (endotoxemia, sepsis, acute lung injury and stroke) has also been shown to be beneficial (93, 129, 145-147). Different HMGB1-specific mAbs have provided excellent therapeutic results in experimental stroke (147) and sepsis (148).

Analysis of the structure-function of HMGB1 revealed that the proinflammatory properties of HMGB1 reside in the B box of the protein, while the DNA-binding A box acts in a antagonistic fashion, inhibiting the activities mediated by full-length HMGB1 (105). When applied therapeutically in CIA, the A box diminished arthritic score, synovitis, IL-1β expression in synovia and cartilage destruction (143). Treatment with A box has also been successful in endotoxemia, sepsis (105, 129), hepatitis (149) and in myocardial and cerebral ischemia models (manuscripts in press). A novel way to therapeutically target HMGB1 is to induce its nuclear retention in order to inhibit the extracellular release. Oxaliplatin, a platinating anti-tumor compound, induces distorted DNA to which HMGB1 binds avidly. Administration of oxaliplatin reduces clinical signs and structural damage of murine CIA as long as extracellular HMGB1 expression is low due to therapy (IV). However, articular inflammation is drastically aggravated and disease flares when oxaliplatin treatment is interrupted. Ghrelin is an endogenous potent orexigenic peptide which unexpectedly also has strong anti- inflammatory activity. Ghrelin decreases secretion of HMGB1 by blocking its cytoplasmic translocation. Therapeutic administration of ghrelin rescues mice from lethal sepsis and attenuates arthritis in mice (150). Certain therapies already used in clinical practice also inhibit HMGB1 release. In vitro studies have shown that gold salts can inhibit the nuclear export of HMGB1 in activated macrophages (V). In addition, chloroquine and dexamethasone restrain HMGB1 release (Schierbeck, unpublished data). Administration of sRAGE, the soluble splice isoform of RAGE, prevents HMGB1 interaction with RAGE and ameliorates arthritis and decreases growth and invasiveness of tumors (113, 151). Furthermore, anti-RAGE mAb can also block experimental models of sepsis and EAE (109, 152) Thrombomodulin is a transmembrane protein which regulates enzymatic activities of thrombin and its anticoagulant properties by activating protein C. The N-terminal lectin- like domain binds to HMGB1 and inhibits its proinflammatory activities (153). Administration of thrombomodulin prevents LPS-induced lethality and ameliorates experimental arthritis (153, 154). Ethyl pyruvate derived from pyruvic acid improves survival and ameliorates organ system dysfunction in mice, even when treatment is started as late as 12-24 hours after the onset of sepsis. Among several effects this compound blocks secretion of HMGB1 (155).

Table 5. diseases with HMGB1 involvement Diseases with HMGB1 involvement Successful anti-HMGB1 therapy References Acute lung injury + (145, 146, 156-163) Anorexia (164) Arthritis + (9, 140-144, 159, 165-173) Atherosclerosis (174-179) Cancer + (78, 113, 180-186) Endotoxemia + (93, 129, 139, 187-191) Hepatic injury + (192-195) Hepatitis + (149, 196, 197) Hemorrhagic shock + (193, 198-200) HIV (201-204) Inflammatory bowel disease + (116, 183, 200, 205, 206) Malaria (183, 207-210) Myocardial ischemia + (128, 211-215) Myositis (187, 216, 217) Pancreatitis + (218-221) Sepsis + (93, 95, 129, 148, 150, 158, 189, 190, 222-228) Stroke + (147, 213, 229-234) SLE and Sjögren’s syndrome (138, 235-239) Thermal injury (burn) + (240, 241)

Assays A conventional assay for measuring soluble mediators is enzyme-linked immunosorbent assay (ELISA). However, for HMGB1 the usefulness of this assay has been limited since other factors in biological fluids interfere with proper detection of HMGB1 (242). Instead, most of the studies of HMGB1 expression levels are based on other antibody-based assays such as Western blotting and microscopy. These semi- quantitative methods enable studies of HMGB1 in biological fluids, cells and tissues. The detection of HMGB1 release at a single cell level requires more sensitive methods and today there is only one such assay available. The Elispot method is sensitive enough to detect individual cells secreting HMGB1 and provides a complement to other methods (243). Further, the Elispot enables kinetic studies of HMGB1 release. A new promising technique for HMGB1 detection was very recently described by a French research group;

38 the assay takes advantage of the high affinity binding between distorted DNA loops (hemicatenated) and HMGB1 and is analysed using a band-shift assay (244). Furthermore, the inventors of this assay also took advantage of the fact that HMGB1 has a unique quality among serum proteins to stand exposure to low pH and remain in solution without precipitating. They thus enriched and purified HMGB1 from serum samples through perchloric acid treatment. The recovered HMGB1 was not well recognized by HMGB1- specific mAb, but did demonstrate unaltered DNA-binding capacity.

AIMS OF THE THESIS

The general objective of my thesis work was to characterise the role of HMGB1 in inflammation and how it can be targeted. Specific aims were to:

• Determine the surface receptors used by HMGB1 for its proinflammatory properties.

• Analyse the enhancing actions of HMGB1 to alert the immune system to danger by forming complexes with immunocompetent molecules.

• Study the role of HMGB1 in the pathogenesis of a novel spontaneous experimental arthritis model.

• Investigate the therapeutic potential in collagen-induced arthritis of induced nuclear retention of HMGB1.

• Scrutinize the effects of gold salts, an established anti-rheumatic therapy already used in clinical practice, on HMGB1 release.

METHOLOGICAL CONSIDERATIONS

This section will illuminate a number of the methods used in the studies. Details are described in the separate papers.

Investigation of complexed HMGB1 HMGB1 is suggested to promote inflammation in complex with other molecules. To investigate this possibility we used highly purified HMGB1 from different sources, produced in E. coli (our own production and HMGBiotech, Milano, Italy), baculovirus (kind gift from H. Rauvala, Helsinki) and native bovine thymus HMGB1 (kind gift from M. Bustin, NIH). Each of the different HMGB1s was incubated with either LPS, Pam3CSK4, CpG DNA or IL- 1β at 4°C overnight before added to cell cultures. The incubation was crucial for the cytokine- inducing properties of the complex. After 24-48h of HMGB1-ligand complex stimulation supernatants were harvested, cell debris was removed by centrifugation and stored at -20°C until IL-6 assessment was performed by ELISA.

Evaluation of arthritis Clinical evaluation of experimental arthritis can be conducted in different ways. The system I have used is based on observations of erythema and swelling of joints and is the scoring system routinely used in our laboratory. Mice were observed for clinical signs of arthritis and were scored as follows: the intraphalangeal joints of digits, metacarpal joints and wrist in the forepaw and metatarsal and ankle joint in hind paw were each considered as one category of joint. Individual paws were evaluated using a scale ranging from 0–3 in which 0 = no signs of arthritis, 1 = one type of joint affected, 2 = two types of joints affected, and 3 = the entire paw affected, including signs of ankylosis. Thus the maximal score for each animal was 12. At several time points evaluation of arthritis was performed by staff members blinded to the identity of the animals. In contrast to other scoring systems such as radiography and measurements of paw swelling this method is easily performed. Furthermore, it has a high consistency and corresponds to the scoring assessment of RA in patients.

Preparation of tissue samples Immunohistochemistry is a good method for evaluation of the localization of immunological mediators in tissues. Reproducible, reliable results require standardized tissue

42 handling and carefully selected reagents. We used two different methods to preserve tissues for further analysis. In the first, paws were collected and fixed in Zamboni solution (paraformaldehyde and picric acid) for 24 hours and thereafter washed in PBS until clear of picric acid. Demineralization was conducted for four weeks in 4% EDTA containing sodium cacodylate followed by 8 days in 20% sucrose solution. Subsequently, the tissues were frozen at –80°C until sectioning. In the second method, paws and spleen were collected, dissected and fixed in 4% formaldehyde at 4oC overnight. Thereafter paws were decalcified in EDTA buffer for 3-4 weeks before dehydration in 70% EtOH and paraffin embedding. Sections from paraffin-embedded material can be stained with hematoxylin/eosin after deparaffinization which visualizes cell nuclei and extracellular matrix, and for cytokines following antigen retrieval.

Detection of HMGB1 Examples of the methods used in this thesis for detection of HMGB1 are: immunocytochemistry, immunohistochemistry and an Elispot assay. All of these methods require HMGB1-specific antibodies. During our early studies of HMGB1 we used a polyclonal rabbit anti-HMGB1 from Pharmingen (San Diego, CA, US). However, the production of this antibody was unfortunately discontinued. In collaboration with Critical Therapeutics Inc. (Boston, MA, US) I then screened a panel of anti-HMGB1-specific mAbs (approximately 60 different monoclonal antibodies) and identified one antibody (2G7) that performed well in all our assays. The recently published in-house developed HMGB1 Elispot assay is a highly sensitive method for detecting HMGB1 released from individual cells and allows performance of kinetic studies (243). Multiscreen, 96-well plates were coated with mouse mAb, 2G7, at 4°C overnight. Following washing, cells were added (1000 –3000/well). Oxaliplatin or Myocrisin were added to the plate 24h prior to stimulation with LPS and IFN-γ. After washing the plates, polyclonal antigen affinity purified rabbit anti-HMGB1 antibodies (BD Biosciences PharMingen, San Diego, CA, USA) were added, and the plates were incubated overnight at 4°C. Thereafter followed incubation with biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Lab, West Grove, PA, USA) and Streptavidin-alkaline phosphatase (Mabtech AB, Stockholm, Sweden). Colour development was via 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/nitroblue tetrazolium chloride chromogen liquide substrate (Sigma- Aldrich Chemie, Schnelldorf, Germany). The reaction was stopped with water, and the plates were dried at room temperature overnight.

RESULTS AND DISCUSSION

HMGB1 promotes inflammation in synergy with endogenous and exogenous danger signals and acts via multiple receptors (Paper I and II) In the first study we investigated possible candidate receptors for HMGB1 on macrophages (Mφ) and defined pathways activated by HMGB1 ligation. Bone marrow-derived Mφ were stimulated with recombinant HMGB1. Proinflammatory mediators such as TNF and NO were up-regulated as a consequence of this stimulation. In addition, MHC class II expression was up-regulated on Mφ as a response to HMGB1 stimulation, indicating enhancement of a proinflammatory phenotype. Furthermore, three different MAP kinases, p38 MAPK, p44/42 MAPK and SAPK/JNK were phosphorylated and NF-κB was translocated from the cytoplasm to the nucleus following HMGB1 stimulation. We and most other groups in our research field have for many years used carefully purified recombinant HMGB1 produced in E.coli in our in vitro experimentation. However, since it is an inherent biological property of HMGB1 to bind LPS avidly it has not been possible to completely eliminate all contaminating endotoxin without causing unacceptable losses of HMGB1. We have had to accept HMGB1 preparations containing 1-20 pg LPS per µg HMGB1 and have added polymyxin B to the cell cultures in quantities sufficient to quench ng amounts of LPS. Several research groups have fortunately recently refined the production and purification protocols for HMGB1 providing highly purified native and recombinant protein preparations. These purified HMGB1 batches have demonstrated low or no cytokine- inducing capacity (245-247), in contrast to the previously used preparations. In the second paper we therefore investigated if the proinflammatory role of HMGB1 in innate immunity could be to act in synergy with endogenous and exogenous danger signals. Highly purified HMGB1 from different prokaryotic and eukaryotic sources were added to cell cultures of PBMC or synovial fibroblasts and IL-6 release was measured as a read-out for HMGB1 activity. HMGB1 on its own could not induce detectable levels of IL-6 production. However, preformed HMGB1-complexes with distinct proinflammatory molecules including IL-1β, the TLR4-ligand LPS, the TLR9-ligand CpG-ODN or

TLR1/TLR2-ligand Pam3CSK4 demonstrated strong enhancing effects on IL-6 production when added to cell cultures. No synergistic effects were observed when HMGB1 was co-

44 cultured with TNF, RANKL, or IL-18, indicating proinflammatory HMGB1 complex formation to be a selective phenomenon. The ability of HMGB1 to form complexes and to act in synergy with other molecules has also recently been reported by other groups (136- 139). Our study extends the validity of these observations by demonstrating the functional importance of HMGB1 complex formation with chemically distinctly different molecules. Furthermore, we studied multiple different sources of highly purified HMGB1 preparations with highly reproducible results. The role of RAGE as a signalling receptor for HMGB1 on Mφ was investigated in the first paper. Previous studies had reported RAGE being a receptor for HMGB1 in endothelial cells, neurons and certain tumor cells, ligation resulting in activation, neurite outgrowth and invasiveness, respectively. Bone marrow-derived Mφ from wild type mice and RAGE gene- deficient mice were challenged with HMGB1 and proinflammatory activities were measured. Mφ from RAGE-deficient mice produced significantly lower amounts than controls of TNF, IL-1β and IL-6 following HMGB1 stimulation. Interestingly, RAGE-deficient Mφ still produced noteworthy amounts of TNF, IL-1β and IL-6, indicating a role for additional receptors in HMGB1-mediated signaling. While IL- 1RI (127)and TLR2 (108) have been reported as receptors for HMGB1, we could not in this system detect any reduced cytokine production in Mφ derived from respective IL- 1RI- and TLR2 gene deficient mice. A functional collaboration between RAGE and other receptors to selectively enhance inflammation is an attractive possibility to explain the divergent biological effects mediated by the RAGE-HMGB1 interaction. Recently, two separate studies have been published supporting this concept when HMGB1-CpG-ODN complexes were demonstrated to utilize combined RAGE-TLR9 activation in follicular DCs to induce IFN-γ production. The second report demonstrated a functional cell surface interplay between the beta 2-integrin Mac-1 and RAGE on neutrophils (119, 138). In conclusion, HMGB1 complexed with certain endogenous and exogenous molecules expresses proinflammatory properties and these complexes may act via several receptors including RAGE.

A novel model for the involvement of HMGB1 in arthritis (Paper III) In the third study we investigated the role of HMGB1 in a newly described model of arthritis. In late October 2006, Professor Shigekazu Nagata and his research group described the DNase II-/- x IFN-IR-/- mice in Nature (55). Shortly after, in Paris at the Death, Danger and Immunity meeting, we met Professor Nagata and initiated

45 collaboration and subsequently had the mice imported to Stockholm. We hypothesized that HMGB1 released from accumulated undigested apoptotic bodies or secreted from activated macrophages may contribute to the progression of arthritis in this model. Mice with combined gene deficiency for DNase II-/- x IFN-IR-/- spontaneously develop a disease with remarkably strong resemblance to human RA. The disease had a somewhat faster kinetics in our animal facility compared to the original publication, with a disease onset of about two months. The mice display symmetric polyarthritis, initially affecting the digits, followed by arthritis in larger joints. Histology of affected joints reveals synovitis with villus proliferation accompanied by pannus formation and eroded joint anatomy. As described earlier, sera from DNase II x IFN-IR deficient mice contain high levels of anti-CCP, TNF, IL-1β, IL-6, IL-10, RF and MMP-3. The involvement of HMGB1 in this model was studied in DNase II-/-xIFN-IR-/- mice and compared to heterozygous and wild type controls. HMGB1 expression in articular tissue was analysed by immunohistochemistry. Sections from non-arthritic mice of all genotypes displayed a HMGB1 expression restricted to the nucleus. No influx of inflammatory cells or abnormal macrophages could be visualised in non-arthritic DNase II-/- x IFN-IR-/- mice. In contrast, DNase II-/-x IFN-IR-/- mice that had developed symmetric polyarthritis with destructive synovitis demonstrated strong aberrant cytosolic and extracellular HMGB1 expression in synovial tissue. Furthermore, destruction of cartilage and bone was observed and a clear correlation between severity of disease and extracellular HMGB1 could be demonstrated. Mice with a deficiency in DNase II suffer from splenomegaly, originating from an accumulation of engulfed undigested chromosomal DNA from apoptotic cells or erythoid precursor cells in their macrophages. Since HMGB1 is known to bind DNA we investigated whether this engulfed DNA was associated with HMGB1. However, specific staining of splenic tissue sections revealed that vacuoles with undigested DNA were devoid of HMGB1. Macrophages harvested from the peritoneum displayed a similar pattern, with occasional exceptions when HMGB1 was demonstrated to co-localise with engulfed DNA. However, both cytoplasmic HMGB1 and IL-1β were observed in peritoneal macrophages from both arthritic and non-arthritic DNase II-/-x IFN-IR-/- mice, indicating that these cells were activated. The absence of HMGB1 in compartments with engulfed DNA could have different explanations. HMGB1 is normally only weakly adherent to DNA and chromatin and may have leaked out from these DNA-filled cells, either together with DNA or on its own. DNA has previously been demonstrated as a

46 component present at µg/ml levels in sera from these mice. An active secretion of HMGB1 is also feasible, since the presence of intracellular IL-1β indicates macrophage activation. There is also a possibility of proteolytic degradation of the HMGB1 associated with the engulfed DNA to explain the absence of HMGB1 in the phagozytosed apoptotic material. Furthermore, HMGB1 associated to DNA might have undergone modifications which changed the structure of the epitope recognized by the anti-HMGB1 antibody used. HMGB1 and anti-HMGB1 antibodies (Abs) in the DNase II-/-xIFN-IR-/- mice serum samples were collected at three different time points, before onset, at onset and during established arthritis in order to study kinetic expression patterns. HMGB1 was already detected at the earliest time point, two weeks before onset of disease, peaked at onset of disease and declined during established arthritis. This finding is in concordance with previous reports of fluctuations of TNF and DNA content in sera from DNase II-/-x IFN- IR-/- mice (55). Anti-HMGB1 Abs have been reported in systemic rheumatic diseases, with HMGB1 being a target for pANCA (171). The presence of anti-HMGB1 Abs was therefore analysed and detected in sera at all time points investigated. Notably, high levels of HMGB1 in sera before onset of arthritis correlated significantly with high levels of anti- HMGB1 Abs during established arthritis in the same individual animals, when serum HMGB1 had declined. Free HMGB1 at this late time point in established arthritis may have formed complexes with the anti-HMGB1 Abs or other serological factors; these complexes may possibly have been removed during the filtration step prior to the Western blot assay and have thereby been eliminated from detection. It is also possible that serum HMGB1, with or without anti-HMGB1 Abs, had bound to cellular receptors. In conclusion, these results implicate HMGB1 in the pathogenesis of the RA-like disease occurring spontaneously in DNase II-/-x IFN-IR-/- mice, further supporting HMGB1 as a central arthritogenic mediator.

Nuclear retention, a novel way to therapeutically target HMGB1 (Paper IV and V) Targeting HMG1 in arthritis and other inflammatory diseases is an appealing therapeutic strategy. Therapies based on HMGB1 antagonists such as polyclonal anti- HMGB1 antibodies, A box, sRAGE and thrombomodulin have yielded encouraging results in studies of experimental arthritis (143, 151, 154). However, despite our long search for a potent monoclonal anti-HMGB1 antibody that can ameliorate arthritis no such

47 candidate has yet fulfilled our expectations. Thus in the fourth and fifth studies we investigated whether there was any novel approach to target HMGB1, including established anti-rheumatic drugs already used in clinical practice.

HMGB1 is known to bind undamaged DNA in a transient manner. HMGB1 constantly shuttles within the nucleus and between the cytoplasmic and nuclear compartments. In contrast, HMGB1 binds distorted DNA avidly (84). Oxaliplatin and other anti-tumor platinated compounds generate adducts in DNA, leading to nuclear sequestration of HMGB1 (248, 249). In the fourth study we used this knowledge to investigate if oxaliplatin had any beneficial effects on collagen-induced arthritis (CIA) and if those might be HMGB1-dependent. DBA/1 mice were challenged with collagen to induce CIA. At expected onset of disease the mice received one intraperitoneal injection of oxaliplatin and therapy was evaluated in CIA by clinical scoring. The administration of oxaliplatin in early arthritis ameliorated the clinical score and delayed onset of disease with an effect lasting one week. The studies were repeated with an additional injection of oxaliplatin to investigate if the effect could be prolonged. Duplicated administration of oxaliplatin only affected the clinical score but not the onset of arthritis. In both settings an aggressive flare was observed one week after the last dose of oxaliplatin was given. Analysis of articular HMGB1 demonstrated that beneficial results were obtained with oxaliplatin treatment as long as HMGB1 was mainly retained intranuclearally and unable to reach the extracellular compartment. During later stages of the observation period, when clinical relapse occurred, an extensive cytoplasmic and extracellular localisation of HMGB1 was apparent. We have no defined mechanism for this dramatic flare but suggest the nuclear entrapment of HMGB1 to be a reversible process leading to massive release at a later stage. The doses of oxaliplatin used in the in vivo experiment correlated with those used in the in vitro experiments and determined not to affect cell viability. However, one could not exclude toxicity followed by non-programmed cell death, leading to passive release of HMGB1 which in turn would recruit inflammatory cells and other mediators. An alternative explanation could be that the origin of the proinflammatory extracellular HMGB1 came from novel, oxaliplatin-unexposed cells that were recruited to the articular compartment. There is one study reporting that cells dying by apoptosis after treatment with oxaliplatin release HMGB1 after caspase activation (106).

To evaluate the effect mediated by oxaliplatin on localisation and secretion of HMGB1 we performed two assays, Elispot and immunoflourescent staining. Co-culturing of RAW 264.7 cells and oxaliplatin resulted in a strong nuclear retention of HMGB1. Furthermore, a dose- dependent extracellular reduction of HMGB1 release in cultures containing oxaliplatin was observed. In contrast, parallel in vitro studies demonstrated that TNF secretion was unaffected by oxaliplatin treatment. Cultures with lymph node cells challenged with ovalbumin were strongly suppressed by oxaliplatin, indicating an inhibitory role of oxaliplatin in antigen-induced proliferation. This is an important finding, since induction of CIA is a T lymphocyte-dependent process. It is presently unknown to what extent the impaired proliferative capacity of T cells is an HMGB1- dependent process.

Gold salt compounds were the first widely used disease-modifying anti-rheumatic drug (DMARD) to treat RA. Although the clinical efficacy of gold salts (GST) in certain RA patients is well established, their mechanisms of action are still poorly understood. In vitro studies demonstrate suppression of formation of proinflammatory mediators such as IL-1β, NO, inhibition of PGEs and increased production of IL-10 (250). In the fifth study we explored whether GST could affect HMGB1 release as a mode of action. To address this issue, Myocrisin, the most commonly used therapeutic gold- containing compound, was incubated with macrophage-like cells (RAW 264.7 and THP- 1). GST caused a dose-dependent inhibition of extracellular HMGB1 translocation from unstimulated and activated RAW 264.7 cells and from activated THP-1 cells as evaluated by Elispot. Parallel experiments were performed to specify the inhibition, to determine whether GST also modulates TNF secretion from these cells. The results indicate a divergent effect of GST on TNF or HMGB1 secretion. GST neither affected spontaneous TNF release nor LPS-IFN-γ-induced release from RAW 246.7 cells or THP-1 cells. Poly(I:C), a ligand of TLR-3, induced HMGB1, IFN-β and NO release from RAW 246.7 cells, but the responses were strongly diminished in cultures supplemented with GST. In contrast, TNF release as a response to Poly(I:C) was not affected. AuCl3 administration to cells gave similar results to Myocrisin regarding NO release, indicating this is a general mechanism of action for gold salt compounds. It has been shown that both NO and IFN-β can induce HMGB1 release (251). However, neither NO or IFN-β was able to induce HMGB1 release from RAW246.7 cells treated with GST, indicating that GST acts atb more than one level than release.

To verify the results observed in the Elispot assay and to elucidate HMGB1 location immunocytochemical staining of intracellular HMGB1 was performed. Activated cells incubated with GST displayed an intense nuclear and faint cytoplasmic HMGB1 staining in contrast to stimulated cells untreated with GST which demonstrated the reversed pattern.

We have validated the concept of HMGB1 sequestration in an experimental model of RA and in vitro assays for the platinum-based compound oxaliplatin and for gold salt compounds, drugs which are already in clinical use. However, therapeutic treatment with oxaliplatin in arthritis gave rise to rebound phenomena which coincided with extracellular HMGB1 release at late time-points following drug administration. Despite this, nuclear retention may provide a novel pathway to inhibit HMGB1 release.

GENERAL DISCUSSION AND FUTURE PERSPECTIVES

During my thesis work I have studied the endogenous, ubiquitously expressed protein HMGB1 and its role in inflammation. I have demonstrated the following:

• The proinflammatory activity of HMGB1 is to a major part, but not solely, facilitated by ligation to the receptor RAGE. • To be able to initiate inflammatory responses HMGB1 needs to be complexed with distinct endogenous and exogenous pro-inflammatory mediators. • HMGB1 may be involved in the pathogenesis of a novel spontaneous arthritis model. • Experimental arthritis can be ameliorated by inducing nuclear retention of HMGB1 through oxaliplatin treatment. Nuclear retention is thus a valid strategy for development of HMGB1-targeting anti-rheumatic drugs. • Gold salt, a well-established anti-rheumatic drug in clinical practice, can inhibit HMGB1 secretion in vitro and functions in part via nuclear retention.

We and others have demonstrated that RAGE is a receptor of major importance for HMGB1 signalling. The results from our studies with RAGE-deficient cells indicate that HMGB1 may also interact with other receptor(s) than RAGE as RAGE deficiency did not completely suppress HMGB1-induced cytokine production. Suggested additional receptors for this interaction are TLR2 and TLR4 (106-108). However, TLR2 deficiency did not affect HMGB1-induced cytokine production in our system. In order to evaluate the interplay of these receptors with HMGB1 we generated double and triple gene deficient mice (RAGExTLR2, RAGExTLR4 and RAGExTLR2xTLR4). These mice will be used to study both HMGB1- and HMGB1-complex responses using in vitro cell culture systems but also to study the role of HMGB1 signalling in arthritis pathogenesis. In collaboration with KJ Tracey’s group in New York, the mice will also be used to study the role of HMGB1 in sepsis. An important issue for future studies is to clarify how HMGB1 can induce migration and stem cell differentiation without inflammation in certain systems, while in other systems it can induce strong proinflammatory cytokine production. My data presented in this thesis have contributed to the emerging view that the biological function of HMGB1

51 are dependent on its formation of a complex with other active mediators, its ability (or possibly the HMGB1-complexes’ ability) to simultaneously interact with multiple receptors as well as its redox status and modification status. The different receptor- deficient mice we have bred will provide a tool to elucidate these questions. An appealing thought is that complexed HMGB1 will cause dual recognition by bringing two kinds of receptors in close proximity and thereby making them interact and enhance a proinflammatory response. In contrast, uncomplexed HMGB1 might bind RAGE solely without engaging any other type of receptor. The resulting outcome of such signalling might be migration/stem cell differentiation only without cytokine production. Another fascinating question is why HMGB1 forms complexes with some endogenous and exogenous mediators and not others. For example, why does HMGB1 bind to IL-1β but not to IL-18 or TNF? At present we do not know why, but the question deserves further thought. A third and intriguing aspect is that RAGE is evolutionarily a novel receptor present only in opossums and placental mammals. In contrast, HMGB1 is an evolutionarily ancient protein. It is thus a distinct possibility that there are as yet unidentified HMGB1 receptors to be discovered.

DNase II-/- x IFN-IR-/- mice develop spontaneous arthritis around 8 weeks of age. We have demonstrated the involvement of HMGB1 through our findings of cytoplasmic and extracellular HMGB1 expression in articular joints from arthritic mice. HMGB1 and anti- HMGB1 antibodies were even detected before disease was established, suggesting a role for HMGB1 in disease induction. We do not yet know the role of the anti-HMGB1 antibodies demonstrated in the sera in these mice. To investigate if they have neutralising capacity these antibodies could be purified and administrated to arthritic mice. This model provides a unique opportunity to evaluate new therapies directed against HMGB1 and this under current investigation. Crossing of them onto different TLR-deficient mouse strains did not change the phenotype. In contrast, deletion of the MyD88 gene prevented the development of arthritis (personal communication Dr Nagata). Immunising RAGE- deficient mice with collagen gave rise to a milder disease with delayed onset compared to arthritis in wild type mice (Östberg, unpublished data). However, in this model it is difficult to know if the evident amelioration of disease was due to the absent HMGB1- RAGE interaction or if other factors also contributed. To address this matter I have

52 initiated crossings between DNase II-/- x IFN-IR-/- mice and RAGE- deficient mice and will in the future follow development of clinical arthritic course.

A number of HMGB1 blocking therapies have been tested in several experimental models of different diseases to date. Despite enormous efforts none of these therapies have yet been translated to clinical practice. The outcome of pre-clinical trials in different experimental models has so far been most successful and consistent using A box-based therapy. I have demonstrated that oxaliplatin ameliorated experimental arthritis through nuclear retention of HMGB1. However, an aggressive relapse was observed one week later. This may not make oxaliplatin a suitable therapy for patients with RA but our study can be considered a proof-of-concept. We have also shown that an established therapy in RA, gold salts, act by targeting HMGB1 and inhibiting nuclear release. We are therefore currently investigating several other therapies used to treat RA patients to study their HMGB1 blocking capacities. So far, chloroquine and dexamethasone have been shown to restrain HMGB1 release (Schierbeck, personal communication). During my years as a graduate student I have also evaluated numerous monoclonal antibodies in experimental models of arthritis, but unfortunately none of the antibodies tested have provided the right properties to be able to significantly ameliorate arthritis.

Finally, because of the work presented in this thesis and of contemporary work published by other groups, it is my opinion that HMGB1 is a prototypic member of a new class of molecules: endogenous molecules that besides their non-immune function(s) also have been evolutionarily selected to enhance and strengthen various aspects of an inflammatory response. This is performed by autonomous actions or by interaction with other immunocompetent molecules. I believe that HMGB1 will be as abundantly detected in and associated with inflammatory conditions as TNF and IL-1 are today and that the future will include HMGB1-specific therapy as a treatment option for several inflammatory conditions.

ACKNOWLEDGEMENTS

This thesis work was carried out at the Rheumatology unit, Center for Molecular Medicine, Karolinska Institutet, Sweden. I would like to express my gratitude to all colleagues and friends who have helped me during my PhD studies. I would especially like to express my gratitude to the following:

Ulf Andersson, my supervisor, a brilliant researcher, a humble, generous person with an amazing sense of humour. Taking this scientific journey with you has been incredible. Thank you for always believing in me, letting me travel the world (not only the scientific one) and having the time of my life.

Helena Erlandsson Harris, my co-supervisor, a living proof of the fact that science and beauty do go together. Always ready to answer my questions, sharing your knowledge regarding immunology and life, supporting me, giving me inspiration and guidance. I sure have enjoyed being your student! Thank you for raising me as one of your chickens teaching me how to become a swan.

Lars Klareskog, thank you for providing a unique scientific environment, with excellent research opportunities for all of us in your lab.

Marie Wahren-Herlenius, thank you for being my informal mentor, for always having time for a chat, and for giving me very useful advises regarding science and life. From wherever you got your extra hours per day, I’m sending my application..

The HMGB1 group, I have really enjoyed working with you all! Lotta Aveberger, for always knowing, always helping. Lena Klevenvall, for all the help with the genotyping and for all the fun, Karin Palmblad, our queen in immunohistology, thank you for your support and for pushing for the DNase-paper. Lars Ottosson, for all the computer-related help, gossip during experiments and fun at meetings. Hulda Hreggvidsdottir, for fun at meetings and parties and fruitful collaborations. Heidi Wähämaa, for being such a caring person, always bringing fika to JC and meetings. Hanna Schierbeck, our baby chicken, for being so sweet and sparkling. Erik Sundberg, for always answering my clinical questions, good luck with your own book! Cecilia Zetterström, for nice company in the lab, at meetings and at Vårruset. Rikka Söderling, Rüdiger Weiss and Tomas Ernemar.

Ann-Christine Eklöf, for taking such good care of me and the rest of the members in Ulfs group. Susanne Karlfeldt and Gunnel Bremerfeldt, what would Rheuma be without you taking care of us?! Astrid Häggblad, for invaluable help with alla blanketter och sånt.

All the co-authors, Riikka Kokkola, Åsa Andersson, Gail Mullins, Carl Johan Treutiger, Berndt Arnold, Peter Nawroth, Robert A Harris, Hulda S Hreggvidsdottir, Heidi Wähämaa, Hanna Schierbeck, Ann-Charlotte Aveberger, Lena Klevenvall, Lars Ottosson, Shigekazu Nagata, Kohki Kawane, Huan Yang, Sangeeta Chavan, Norimasa Ito, Pernilla Stridh, Maria Shoshan, Michael T Lotze, Cecilia Zetterström, Weiwen Jiang, David Pisetsky, Karin Palmblad, Helena Erlandsson Harris and Ulf Andersson. Thank you for sharing your knowledge and experience!

Bob Harris, for the very much appreciated linguistic advice!

Angerika Bierhaus, the loveliest person. Always, regardless of how busy, replying to my e-mails sometimes even before I realise they’re being sent.

The amazingly entertaining gang I occasionally tend to meet with; Leverknall, Slirbeck, Fulda, Rusberger, Heiniken, Packblad, Helan, Fulf and lilla Nasse.

All present and former colleagues at the Rheumatology lab, in particular, Cissi, for being my friend, both at the lab and outside the lab. Thank you for the fika, movies, travel and crazy party nights. I miss you! Alex, thank you for great company worldwide, it was a blast! Margaritas, vitaminas.. Karin, for your friendship, I think you’ve been in London long enough now. Jian, my roommate, thank you for nice chats and for answering my endless questions regarding China. Emeli, Marcus and Patrick for great company, training is so much more fun with you guys. Andreas, for fun in NY and a super collaboration within SWIMM. Mona for being such a lovely person and for fun in Vermont. Vivi, for always having time and patience to answer questions. Gustavo, for bringing some fresh wind to the lab. Carina, for the funniest jokes. Lena, for giving helpful advises regarding careers and stuff. Dimitris for being so cool. Nånnis, for your energy and enthusiasm spreading to all of us. Omri, for Mac related help. Wei, for happy company in the lab regardless the time of day or night. Dzung, for your sweet personality. Therese V, T1, for all massages and advises regarding training. Vijole, for the best pies ever and nice discussions. Monika, for nice lab company. Sevim, for being so frank. Leonid, for always inviting to badminton matches, one day I’ll be there. Malin, Marina, Stina, Ulrika, Anca, Robert, Ewa, Duojia, Anna. Esbjörn, Ingela, Marie, Vilja, Aurelie, Maria, Tina, Jay, Shankar, Hiba, Mai, Liselotte and Linn.

All our lab technicians, without you the lab would just not function. Eva Lindros, your kindness goes above and beyond! Eva Jemseby, for always providing us all with PBS, Marianne, for helpful advice and delicious cakes, Åse, for always being so sweet and caring, Gull-Britt and Doina.

All present and former collegues in the Neuroimmunology group, in particular Pernilla, thanks for friendship, company in China and an incredible patients when it comes to teaching me the art of statistics. An art I prefer more is shopping, which we together take to a totally new level! Ruxandra, former roommate and training partner, thank you for being such a sweet person. Ame, for being just fabulous. Åsa, for inspiring discussions. Alan, Margarita, Karin, Johan, Melanie, Mohsen, Maja, Olle, Mikael, Rikard, Britt, Mimmi, Ami, Rita, Anna, Maria, Maja, Judit, for being such a fun crowd.

The people at AKM, Anna-Lena, thank you so much for the help with scoring weekends and nights. Sandra, Nathalie, Jessica, Kicki, Magnus, Linda, Pauline and Cissi, you have all contributed so much to this thesis.

My dear friends, Annika, Anna, Andreas for being true friends, always caring for me, for all the times we have shared, for all the fun we have had, for all the long talks regarding things that are important in life and some things maybe not so important. Veronika, for being my friend and for company at yoga classes, Janne for talks and runs and for being my friend. Annika, Åsa, Pinglan, Marie, Madde, Lusse, Ann-Sofie, Jenny, Vicky, Tobbe and Jörgen, growing up with you meant the world to me, we sure did have fun!

My wonderful family, all of you make my world go around. Pappa, thank you for everything! Your support and love is invaluable for me. Mamma, I miss you every day. I wish we could have experienced this together. Carita, my favourite storacykel, even though we do live quite distant you always feel close. Micke, my favourite lillesnor, your support and care means a lot to me. Hanna, the best svägis ever, I’m so happy to have you in the family. Filippa, darling angel Flipps, you are a true joy. A couple of minutes with you and all my troubles are gone. Agneta, thank you for joining the family. Tjockis, you crazy thing, thanks for the company during all days and nights spent on this book. Pelle, for your support and love, the future belongs to us.

REFERENCES

1. Matzinger, P. 1994. Tolerance, danger, and the extended family. Annu Rev Immunol 12:991-1045. 2. Matzinger, P. 2007. Friendly and dangerous signals: is the tissue in control? Nat Immunol 8:11-13. 3. Seong, S.Y., and P. Matzinger. 2004. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 4:469-478. 4. Bianchi, M.E. 2007. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 81:1-5. 5. Schmitt, E., M. Gehrmann, M. Brunet, G. Multhoff, and C. Garrido. 2007. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol 81:15-27. 6. Shi, Y., J.E. Evans, and K.L. Rock. 2003. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425:516-521. 7. Foell, D., H. Wittkowski, T. Vogl, and J. Roth. 2007. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 81:28-37. 8. Werman, A., R. Werman-Venkert, R. White, J.K. Lee, B. Werman, Y. Krelin, E. Voronov, C.A. Dinarello, and R.N. Apte. 2004. The precursor form of IL-1alpha is an intracrine proinflammatory activator of transcription. Proc Natl Acad Sci U S A 101:2434-2439. 9. Pisetsky, D.S., H. Erlandsson-Harris, and U. Andersson. 2008. High-mobility group box protein 1 (HMGB1): an alarmin mediating the pathogenesis of rheumatic disease. Arthritis Res Ther 10:209. 10. Silverstein, A.M. 2001. Autoimmunity versus horror autotoxicus: the struggle for recognition. Nat Immunol 2:279-281. 11. Anderson, M.S., E.S. Venanzi, L. Klein, Z. Chen, S.P. Berzins, S.J. Turley, H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, and D. Mathis. 2002. Projection of an immunological self shadow within the thymus by the aire protein. Science 298:1395-1401. 12. Derbinski, J., A. Schulte, B. Kyewski, and L. Klein. 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat Immunol 2:1032-1039. 13. Danke, N.A., D.M. Koelle, C. Yee, S. Beheray, and W.W. Kwok. 2004. Autoreactive T cells in healthy individuals. J Immunol 172:5967-5972. 14. Jenkins, M.K., D.M. Pardoll, J. Mizuguchi, T.M. Chused, and R.H. Schwartz. 1987. Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones. Proc Natl Acad Sci U S A 84:5409-5413. 15. Jenkins, M.K., and R.H. Schwartz. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 165:302-319. 16. Wandstrat, A., and E. Wakeland. 2001. The genetics of complex autoimmune diseases: non-MHC susceptibility genes. Nat Immunol 2:802-809. 17. Whitacre, C.C. 2001. Sex differences in autoimmune disease. Nat Immunol 2:777-780. 18. Klein, J., and A. Sato. 2000. The HLA system. Second of two parts. N Engl J Med 343:782-786. 19. Nagamine, K., P. Peterson, H.S. Scott, J. Kudoh, S. Minoshima, M. Heino, K.J. Krohn, M.D. Lalioti, P.E. Mullis, S.E. Antonarakis, K. Kawasaki, S. Asakawa, F. Ito, and N. Shimizu. 1997. Positional cloning of the APECED gene. Nat Genet 17:393-398. 20. Liblau, R., E. Tournier-Lasserve, J. Maciazek, G. Dumas, O. Siffert, G. Hashim, and M.A. Bach. 1991. T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects. Eur J Immunol 21:1391-1395. 21. Sun, J.B., T. Olsson, W.Z. Wang, B.G. Xiao, V. Kostulas, S. Fredrikson, H.P. Ekre, and H. Link. 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur J Immunol 21:1461-1468. 22. Dighiero, G., and N.R. Rose. 1999. Critical self-epitopes are key to the understanding of self-tolerance and autoimmunity. Immunol Today 20:423-428. 23. Goldrath, A.W., and M.J. Bevan. 1999. Selecting and maintaining a diverse T-cell repertoire. Nature 402:255-262. 24. Lee, D.M., and M.E. Weinblatt. 2001. Rheumatoid arthritis. Lancet 358:903-911. 25. MacGregor, A.J., H. Snieder, A.S. Rigby, M. Koskenvuo, J. Kaprio, K. Aho, and A.J. Silman. 2000. Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins. Arthritis Rheum 43:30-37. 26. Feldmann, M., F.M. Brennan, and R.N. Maini. 1996. Rheumatoid arthritis. Cell 85:307-310. 27. Olsen, N.J., and W.J. Kovacs. 2002. Hormones, pregnancy, and rheumatoid arthritis. J Gend Specif Med 5:28-37. 28. Balandraud, N., J.B. Meynard, I. Auger, H. Sovran, B. Mugnier, D. Reviron, J. Roudier, and C. Roudier. 2003. Epstein-Barr virus load in the peripheral blood of patients with rheumatoid arthritis: accurate quantification using real-time polymerase chain reaction. Arthritis Rheum 48:1223-1228.

29. Mehraein, Y., C. Lennerz, S. Ehlhardt, K. Remberger, A. Ojak, and K.D. Zang. 2004. Latent Epstein- Barr virus (EBV) infection and cytomegalovirus (CMV) infection in synovial tissue of autoimmune chronic arthritis determined by RNA- and DNA-in situ hybridization. Mod Pathol 17:781-789. 30. Takeda, T., Y. Mizugaki, L. Matsubara, S. Imai, T. Koike, and K. Takada. 2000. Lytic Epstein-Barr virus infection in the synovial tissue of patients with rheumatoid arthritis. Arthritis Rheum 43:1218- 1225. 31. Silman, A.J., J. Newman, and A.J. MacGregor. 1996. Cigarette smoking increases the risk of rheumatoid arthritis. Results from a nationwide study of disease-discordant twins. Arthritis Rheum 39:732-735. 32. Uhlig, T., K.B. Hagen, and T.K. Kvien. 1999. Current tobacco smoking, formal education, and the risk of rheumatoid arthritis. J Rheumatol 26:47-54. 33. Vessey, M.P., L. Villard-Mackintosh, and D. Yeates. 1987. Oral contraceptives, cigarette smoking and other factors in relation to arthritis. Contraception 35:457-464. 34. Padyukov, L., C. Silva, P. Stolt, L. Alfredsson, and L. Klareskog. 2004. A gene-environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum 50:3085-3092. 35. Arnett, F.C., S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper, L.A. Healey, S.R. Kaplan, M.H. Liang, H.S. Luthra, and et al. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324. 36. Agrawal, S., R. Misra, and A. Aggarwal. 2007. Autoantibodies in rheumatoid arthritis: association with severity of disease in established RA. Clin Rheumatol 26:201-204. 37. Mewar, D., and A.G. Wilson. 2006. Autoantibodies in rheumatoid arthritis: a review. Biomed Pharmacother 60:648-655. 38. Ravelli, A., and A. Martini. 2007. Juvenile idiopathic arthritis. Lancet 369:767-778. 39. Petty, R.E., T.R. Southwood, P. Manners, J. Baum, D.N. Glass, J. Goldenberg, X. He, J. Maldonado- Cocco, J. Orozco-Alcala, A.M. Prieur, M.E. Suarez-Almazor, and P. Woo. 2004. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 31:390-392. 40. Murray, K.J., L. Luyrink, A.A. Grom, M.H. Passo, H. Emery, D. Witte, and D.N. Glass. 1996. Immunohistological characteristics of T cell infiltrates in different forms of childhood onset chronic arthritis. J Rheumatol 23:2116-2124. 41. Gregorio, A., C. Gambini, V. Gerloni, A. Parafioriti, M.P. Sormani, S. Gregorio, G. De Marco, F. Rossi, A. Martini, and M. Gattorno. 2007. Lymphoid neogenesis in juvenile idiopathic arthritis correlates with ANA positivity and plasma cells infiltration. Rheumatology (Oxford) 46:308-313. 42. Holmdahl, R., M. Andersson, T.J. Goldschmidt, K. Gustafsson, L. Jansson, and J.A. Mo. 1990. Type II collagen autoimmunity in animals and provocations leading to arthritis. Immunol Rev 118:193-232. 43. Trentham, D.E., A.S. Townes, and A.H. Kang. 1977. Autoimmunity to type II collagen an experimental model of arthritis. J Exp Med 146:857-868. 44. Courtenay, J.S., M.J. Dallman, A.D. Dayan, A. Martin, and B. Mosedale. 1980. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283:666-668. 45. Yoo, T.J., S.Y. Kim, J.M. Stuart, R.A. Floyd, G.A. Olson, M.A. Cremer, and A.H. Kang. 1988. Induction of arthritis in monkeys by immunization with type II collagen. J Exp Med 168:777-782. 46. Billingham, M.E. 1983. Models of arthritis and the search for anti-arthritic drugs. Pharmacol Ther 21:389-428. 47. Erlandsson Harris, H., M. Liljestrom, and L. Klareskog. 1997. Characteristics of synovial fluid effusion in collagen-induced arthritis (CIA) in the DA rat; a comparison of histology and antibody reactivities in an experimental chronic arthritis model and rheumatoid arthritis (RA). Clin Exp Immunol 107:480-484. 48. Campbell, I.K., J.A. Hamilton, and I.P. Wicks. 2000. Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur J Immunol 30:1568- 1575. 49. Kerwar, S.S., M.E. Englert, R.A. McReynolds, M.J. Landes, J.M. Lloyd, A.L. Oronsky, and F.J. Wilson. 1983. Type II collagen-induced arthritis. Studies with purified anticollagen immunoglobulin. Arthritis Rheum 26:1120-1131. 50. Trentham, D.E., R.A. Dynesius, and J.R. David. 1978. Passive transfer by cells of type II collagen- induced arthritis in rats. J Clin Invest 62:359-366. 51. Pearson, C.M. 1956. Development of arthritis, periarthritis and periostitis in rats given adjuvants. Proc Soc Exp Biol Med 91:95-101. 52. Kleinau, S., H. Erlandsson, R. Holmdahl, and L. Klareskog. 1991. Adjuvant oils induce arthritis in the DA rat. I. Characterization of the disease and evidence for an immunological involvement. J Autoimmun 4:871-880.

53. Vingsbo, C., P. Sahlstrand, J.G. Brun, R. Jonsson, T. Saxne, and R. Holmdahl. 1996. Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major histocompatibility complex and non-major histocompatibility complex genes. Am J Pathol 149:1675-1683. 54. Carlson, B.C., A.M. Jansson, A. Larsson, A. Bucht, and J.C. Lorentzen. 2000. The endogenous adjuvant squalene can induce a chronic T-cell-mediated arthritis in rats. Am J Pathol 156:2057-2065. 55. Kawane, K., M. Ohtani, K. Miwa, T. Kizawa, Y. Kanbara, Y. Yoshioka, H. Yoshikawa, and S. Nagata. 2006. Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages. Nature 443:998-1002. 56. Evans, C.J., and R.J. Aguilera. 2003. DNase II: genes, enzymes and function. Gene 322:1-15. 57. Nagata, S. 2005. DNA degradation in development and programmed cell death. Annu Rev Immunol 23:853-875. 58. Gresser, I., M. Aguet, L. Morel-Maroger, D. Woodrow, F. Puvion-Dutilleul, J.C. Guillon, and C. Maury. 1981. Electrophoretically pure mouse interferon inhibits growth, induces liver and kidney lesions, and kills suckling mice. Am J Pathol 102:396-402. 59. Gresser, I., M.G. Tovey, C. Maury, and I. Chouroulinkov. 1975. Lethality of interferon preparations for newborn mice. Nature 258:76-78. 60. Korganow, A.S., H. Ji, S. Mangialaio, V. Duchatelle, R. Pelanda, T. Martin, C. Degott, H. Kikutani, K. Rajewsky, J.L. Pasquali, C. Benoist, and D. Mathis. 1999. From systemic T cell self-reactivity to organ- specific autoimmune disease via immunoglobulins. Immunity 10:451-461. 61. Kouskoff, V., A.S. Korganow, V. Duchatelle, C. Degott, C. Benoist, and D. Mathis. 1996. Organ- specific disease provoked by systemic autoimmunity. Cell 87:811-822. 62. Butler, D.M., A.M. Malfait, L.J. Mason, P.J. Warden, G. Kollias, R.N. Maini, M. Feldmann, and F.M. Brennan. 1997. DBA/1 mice expressing the human TNF-alpha transgene develop a severe, erosive arthritis: characterization of the cytokine cascade and cellular composition. J Immunol 159:2867-2876. 63. Keffer, J., L. Probert, H. Cazlaris, S. Georgopoulos, E. Kaslaris, D. Kioussis, and G. Kollias. 1991. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 10:4025-4031. 64. Horai, R., S. Saijo, H. Tanioka, S. Nakae, K. Sudo, A. Okahara, T. Ikuse, M. Asano, and Y. Iwakura. 2000. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med 191:313-320. 65. Nordling, C., A. Karlsson-Parra, L. Jansson, R. Holmdahl, and L. Klareskog. 1992. Characterization of a spontaneously occurring arthritis in male DBA/1 mice. Arthritis Rheum 35:717-722. 66. Holmdahl, R., L. Jansson, M. Andersson, and R. Jonsson. 1992. Genetic, hormonal and behavioural influence on spontaneously developing arthritis in normal mice. Clin Exp Immunol 88:467-472. 67. Goodwin, G.H., and E.W. Johns. 1973. Isolation and characterisation of two calf-thymus chromatin non-histone proteins with high contents of acidic and basic amino acids. Eur J Biochem 40:215-219. 68. Muller, S., L. Ronfani, and M.E. Bianchi. 2004. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med 255:332-343. 69. Mosevitsky, M.I., V.A. Novitskaya, M.G. Iogannsen, and M.A. Zabezhinsky. 1989. Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions. Eur J Biochem 185:303-310. 70. Melloni, E., B. Sparatore, M. Patrone, A. Pessino, M. Passalacqua, and S. Pontremoli. 1995. Identity in molecular structure between "differentiation enhancing factor" of murine erythroleukemia cells and the 30 kD heparin-binding protein of developing rat brain. Biochem Biophys Res Commun 210:82-89. 71. Sparatore, B., M. Patrone, M. Passalacqua, A. Pessino, R. Falchetto, E. Melloni, and S. Pontremoli. 1993. Characterization of the biological role of murine erythroleukemia cells "differentiation enhancing factor" using antisense oligodeoxynucleotides. Biochem Biophys Res Commun 193:941-947. 72. Chou, D.K., J.E. Evans, and F.B. Jungalwala. 2001. Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain. J Neurochem 77:120-131. 73. Rauvala, H., and R. Pihlaskari. 1987. Isolation and some characteristics of an adhesive factor of brain that enhances neurite outgrowth in central neurons. J Biol Chem 262:16625-16635. 74. Bustin, M. 1999. Regulation of DNA-dependent activities by the functional motifs of the high-mobility- group chromosomal proteins. Mol Cell Biol 19:5237-5246. 75. Bonaldi, T., F. Talamo, P. Scaffidi, D. Ferrera, A. Porto, A. Bachi, A. Rubartelli, A. Agresti, and M.E. Bianchi. 2003. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J 22:5551-5560. 76. Youn, J.H., and J.S. Shin. 2006. Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J Immunol 177:7889-7897.

77. Ito, I., J. Fukazawa, and M. Yoshida. 2007. Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils. J Biol Chem 282:16336-16344. 78. Hamada, T., M. Torikai, A. Kuwazuru, M. Tanaka, N. Horai, T. Fukuda, S. Yamada, S. Nagayama, K. Hashiguchi, N. Sunahara, K. Fukuzaki, R. Nagata, S. Komiya, I. Maruyama, and K. Abeyama. 2008. Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis. Arthritis Rheum 58:2675-2685. 79. Bianchi, M.E., and M. Beltrame. 2000. Upwardly mobile proteins. Workshop: the role of HMG proteins in chromatin structure, gene expression and neoplasia. EMBO Rep 1:109-114. 80. Bianchi, M.E., M. Beltrame, and G. Paonessa. 1989. Specific recognition of cruciform DNA by nuclear protein HMG1. Science 243:1056-1059. 81. Brickman, J.M., M. Adam, and M. Ptashne. 1999. Interactions between an HMG-1 protein and members of the Rel family. Proc Natl Acad Sci U S A 96:10679-10683. 82. Mouri, F., J. Tsukada, T. Mizobe, T. Higashi, Y. Yoshida, Y. Minami, H. Izumi, Y. Kominato, K. Kohno, and Y. Tanaka. 2008. Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1. Eur J Haematol 80:10-19. 83. Yamoah, K., A. Brebene, R. Baliram, K. Inagaki, G. Dolios, A. Arabi, R. Majeed, H. Amano, R. Wang, R. Yanagisawa, and E. Abe. 2008. High-mobility group box proteins modulate tumor necrosis factor- alpha expression in osteoclastogenesis via a novel deoxyribonucleic acid sequence. Mol Endocrinol 22:1141-1153. 84. Agresti, A., R. Lupo, M.E. Bianchi, and S. Muller. 2003. HMGB1 interacts differentially with members of the Rel family of transcription factors. Biochem Biophys Res Commun 302:421-426. 85. Bustin, M., and N.K. Neihart. 1979. Antibodies against chromosomal HMG proteins stain the cytoplasm of mammalian cells. Cell 16:181-189. 86. Hanover, J.A., D.C. Love, N. DeAngelis, M.E. O'Kane, R. Lima-Miranda, T. Schulz, Y.M. Yen, R.C. Johnson, and W.A. Prinz. 2007. The High Mobility Group Box Transcription Factor Nhp6Ap enters the nucleus by a calmodulin-dependent, Ran-independent pathway. J Biol Chem 282:33743-33751. 87. Tang, D., R. Kang, W. Xiao, H. Wang, S.K. Calderwood, and X. Xiao. 2007. The anti-inflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages. J Immunol 179:1236-1244. 88. Calogero, S., F. Grassi, A. Aguzzi, T. Voigtlander, P. Ferrier, S. Ferrari, and M.E. Bianchi. 1999. The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice. Nat Genet 22:276-280. 89. Rovere-Querini, P., A. Capobianco, P. Scaffidi, B. Valentinis, F. Catalanotti, M. Giazzon, I.E. Dumitriu, S. Muller, M. Iannacone, C. Traversari, M.E. Bianchi, and A.A. Manfredi. 2004. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep 5:825-830. 90. Scaffidi, P., T. Misteli, and M.E. Bianchi. 2002. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191-195. 91. Bell, C.W., W. Jiang, C.F. Reich, 3rd, and D.S. Pisetsky. 2006. The extracellular release of HMGB1 during apoptotic cell death. Am J Physiol Cell Physiol 291:C1318-1325. 92. Semino, C., G. Angelini, A. Poggi, and A. Rubartelli. 2005. NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1. Blood 106:609-616. 93. Wang, H., O. Bloom, M. Zhang, J.M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, K.R. Manogue, E. Faist, E. Abraham, J. Andersson, U. Andersson, P.E. Molina, N.N. Abumrad, A. Sama, and K.J. Tracey. 1999. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285:248-251. 94. Wang, H., J.M. Vishnubhakat, O. Bloom, M. Zhang, M. Ombrellino, A. Sama, and K.J. Tracey. 1999. Proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes. Surgery 126:389-392. 95. Wang, H., H. Liao, M. Ochani, M. Justiniani, X. Lin, L. Yang, Y. Al-Abed, C. Metz, E.J. Miller, K.J. Tracey, and L. Ulloa. 2004. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 10:1216-1221. 96. Gardella, S., C. Andrei, D. Ferrera, L.V. Lotti, M.R. Torrisi, M.E. Bianchi, and A. Rubartelli. 2002. The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep 3:995-1001. 97. Andrei, C., C. Dazzi, L. Lotti, M.R. Torrisi, G. Chimini, and A. Rubartelli. 1999. The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol Biol Cell 10:1463-1475. 98. Rubartelli, A., F. Cozzolino, M. Talio, and R. Sitia. 1990. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 9:1503-1510.

99. Blott, E.J., and G.M. Griffiths. 2002. Secretory lysosomes. Nat Rev Mol Cell Biol 3:122-131. 100. Dean, M., A. Rzhetsky, and R. Allikmets. 2001. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11:1156-1166. 101. Harris, H.E., and A. Raucci. 2006. Alarmin(g) news about danger: workshop on innate danger signals and HMGB1. EMBO Rep 7:774-778. 102. Rouhiainen, A., J. Kuja-Panula, E. Wilkman, J. Pakkanen, J. Stenfors, R.K. Tuominen, M. Lepantalo, O. Carpen, J. Parkkinen, and H. Rauvala. 2004. Regulation of monocyte migration by amphoterin (HMGB1). Blood 104:1174-1182. 103. Parkkinen, J., E. Raulo, J. Merenmies, R. Nolo, E.O. Kajander, M. Baumann, and H. Rauvala. 1993. Amphoterin, the 30-kDa protein in a family of HMG1-type polypeptides. Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation. J Biol Chem 268:19726-19738. 104. Czura, C.J., H. Yang, C.A. Amella, and K.J. Tracey. 2004. HMGB1 in the immunology of sepsis (not septic shock) and arthritis. Adv Immunol 84:181-200. 105. Li, J., R. Kokkola, S. Tabibzadeh, R. Yang, M. Ochani, X. Qiang, H.E. Harris, C.J. Czura, H. Wang, L. Ulloa, H.S. Warren, L.L. Moldawer, M.P. Fink, U. Andersson, K.J. Tracey, and H. Yang. 2003. Structural basis for the proinflammatory cytokine activity of high mobility group box 1. Mol Med 9:37- 45. 106. Apetoh, L., F. Ghiringhelli, A. Tesniere, M. Obeid, C. Ortiz, A. Criollo, G. Mignot, M.C. Maiuri, E. Ullrich, P. Saulnier, H. Yang, S. Amigorena, B. Ryffel, F.J. Barrat, P. Saftig, F. Levi, R. Lidereau, C. Nogues, J.P. Mira, A. Chompret, V. Joulin, F. Clavel-Chapelon, J. Bourhis, F. Andre, S. Delaloge, T. Tursz, G. Kroemer, and L. Zitvogel. 2007. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13:1050-1059. 107. Park, J.S., F. Gamboni-Robertson, Q. He, D. Svetkauskaite, J.Y. Kim, D. Strassheim, J.W. Sohn, S. Yamada, I. Maruyama, A. Banerjee, A. Ishizaka, and E. Abraham. 2006. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 290:C917-924. 108. Park, J.S., D. Svetkauskaite, Q. He, J.Y. Kim, D. Strassheim, A. Ishizaka, and E. Abraham. 2004. Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J Biol Chem 279:7370-7377. 109. Liliensiek, B., M.A. Weigand, A. Bierhaus, W. Nicklas, M. Kasper, S. Hofer, J. Plachky, H.J. Grone, F.C. Kurschus, A.M. Schmidt, S.D. Yan, E. Martin, E. Schleicher, D.M. Stern, G.G. Hammerling, P.P. Nawroth, and B. Arnold. 2004. Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response. J Clin Invest 113:1641-1650. 110. Bierhaus, A., P.M. Humpert, M. Morcos, T. Wendt, T. Chavakis, B. Arnold, D.M. Stern, and P.P. Nawroth. 2005. Understanding RAGE, the receptor for advanced glycation end products. J Mol Med 83:876-886. 111. Shirasawa, M., N. Fujiwara, S. Hirabayashi, H. Ohno, J. Iida, K. Makita, and Y. Hata. 2004. Receptor for advanced glycation end-products is a marker of type I lung alveolar cells. Genes Cells 9:165-174. 112. Merenmies, J., R. Pihlaskari, J. Laitinen, J. Wartiovaara, and H. Rauvala. 1991. 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane. J Biol Chem 266:16722-16729. 113. Taguchi, A., D.C. Blood, G. del Toro, A. Canet, D.C. Lee, W. Qu, N. Tanji, Y. Lu, E. Lalla, C. Fu, M.A. Hofmann, T. Kislinger, M. Ingram, A. Lu, H. Tanaka, O. Hori, S. Ogawa, D.M. Stern, and A.M. Schmidt. 2000. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 405:354-360. 114. Huttunen, H.J., C. Fages, J. Kuja-Panula, A.J. Ridley, and H. Rauvala. 2002. Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis. Cancer Res 62:4805-4811. 115. Park, J.S., J. Arcaroli, H.K. Yum, H. Yang, H. Wang, K.Y. Yang, K.H. Choe, D. Strassheim, T.M. Pitts, K.J. Tracey, and E. Abraham. 2003. Activation of gene expression in human neutrophils by high mobility group box 1 protein. Am J Physiol Cell Physiol 284:C870-879. 116. Sappington, P.L., R. Yang, H. Yang, K.J. Tracey, R.L. Delude, and M.P. Fink. 2002. HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology 123:790-802. 117. Degryse, B., T. Bonaldi, P. Scaffidi, S. Muller, M. Resnati, F. Sanvito, G. Arrigoni, and M.E. Bianchi. 2001. The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. J Cell Biol 152:1197-1206. 118. Ishihara, K., K. Tsutsumi, S. Kawane, M. Nakajima, and T. Kasaoka. 2003. The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site. FEBS Lett 550:107-113.

119. Orlova, V.V., E.Y. Choi, C. Xie, E. Chavakis, A. Bierhaus, E. Ihanus, C.M. Ballantyne, C.G. Gahmberg, M.E. Bianchi, P.P. Nawroth, and T. Chavakis. 2007. A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin. EMBO J 26:1129-1139. 120. Beutler, B., K. Hoebe, and L. Shamel. 2004. Forward genetic dissection of afferent immunity: the role of TIR adapter proteins in innate and adaptive immune responses. C R Biol 327:571-580. 121. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu Rev Immunol 21:335-376. 122. Palumbo, R., M. Sampaolesi, F. De Marchis, R. Tonlorenzi, S. Colombetti, A. Mondino, G. Cossu, and M.E. Bianchi. 2004. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J Cell Biol 164:441-449. 123. Galvez, B.G., M. Sampaolesi, S. Brunelli, D. Covarello, M. Gavina, B. Rossi, G. Constantin, Y. Torrente, and G. Cossu. 2006. Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. J Cell Biol 174:231-243. 124. Chavakis, E., A. Hain, M. Vinci, G. Carmona, M.E. Bianchi, P. Vajkoczy, A.M. Zeiher, T. Chavakis, and S. Dimmeler. 2007. High-mobility group box 1 activates integrin-dependent homing of endothelial progenitor cells. Circ Res 100:204-212. 125. Mitola, S., M. Belleri, C. Urbinati, D. Coltrini, B. Sparatore, M. Pedrazzi, E. Melloni, and M. Presta. 2006. Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine. J Immunol 176:12-15. 126. Schlueter, C., H. Weber, B. Meyer, P. Rogalla, K. Roser, S. Hauke, and J. Bullerdiek. 2005. Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule. Am J Pathol 166:1259-1263. 127. Zetterstrom, C.K., T. Bergman, B. Rynnel-Dagoo, H. Erlandsson Harris, O. Soder, U. Andersson, and H.G. Boman. 2002. High mobility group box chromosomal protein 1 (HMGB1) is an antibacterial factor produced by the human adenoid. Pediatr Res 52:148-154. 128. Limana, F., A. Germani, A. Zacheo, J. Kajstura, A. Di Carlo, G. Borsellino, O. Leoni, R. Palumbo, L. Battistini, R. Rastaldo, S. Muller, G. Pompilio, P. Anversa, M.E. Bianchi, and M.C. Capogrossi. 2005. Exogenous high-mobility group box 1 protein induces myocardial regeneration after infarction via enhanced cardiac C-kit+ cell proliferation and differentiation. Circ Res 97:e73-83. 129. Yang, H., M. Ochani, J. Li, X. Qiang, M. Tanovic, H.E. Harris, S.M. Susarla, L. Ulloa, H. Wang, R. DiRaimo, C.J. Czura, J. Roth, H.S. Warren, M.P. Fink, M.J. Fenton, U. Andersson, and K.J. Tracey. 2004. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci U S A 101:296-301. 130. Andersson, U., H. Wang, K. Palmblad, A.C. Aveberger, O. Bloom, H. Erlandsson-Harris, A. Janson, R. Kokkola, M. Zhang, H. Yang, and K.J. Tracey. 2000. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 192:565-570. 131. Dumitriu, I.E., P. Baruah, M.E. Bianchi, A.A. Manfredi, and P. Rovere-Querini. 2005. Requirement of HMGB1 and RAGE for the maturation of human plasmacytoid dendritic cells. Eur J Immunol 35:2184- 2190. 132. Manfredi, A.A., A. Capobianco, A. Esposito, F. De Cobelli, T. Canu, A. Monno, A. Raucci, F. Sanvito, C. Doglioni, P.P. Nawroth, A. Bierhaus, M.E. Bianchi, P. Rovere-Querini, and A. Del Maschio. 2008. Maturing dendritic cells depend on RAGE for in vivo homing to lymph nodes. J Immunol 180:2270- 2275. 133. Messmer, D., H. Yang, G. Telusma, F. Knoll, J. Li, B. Messmer, K.J. Tracey, and N. Chiorazzi. 2004. High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization. J Immunol 173:307-313. 134. Yang, D., Q. Chen, H. Yang, K.J. Tracey, M. Bustin, and J.J. Oppenheim. 2007. High mobility group box-1 protein induces the migration and activation of human dendritic cells and acts as an alarmin. J Leukoc Biol 81:59-66. 135. Dumitriu, I.E., P. Baruah, B. Valentinis, R.E. Voll, M. Herrmann, P.P. Nawroth, B. Arnold, M.E. Bianchi, A.A. Manfredi, and P. Rovere-Querini. 2005. Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products. J Immunol 174:7506-7515. 136. Ivanov, S., A.M. Dragoi, X. Wang, C. Dallacosta, J. Louten, G. Musco, G. Sitia, G.S. Yap, Y. Wan, C.A. Biron, M.E. Bianchi, H. Wang, and W.M. Chu. 2007. A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA. Blood 110:1970-1981. 137. Sha, Y., J. Zmijewski, Z. Xu, and E. Abraham. 2008. HMGB1 develops enhanced proinflammatory activity by binding to cytokines. J Immunol 180:2531-2537. 138. Tian, J., A.M. Avalos, S.Y. Mao, B. Chen, K. Senthil, H. Wu, P. Parroche, S. Drabic, D. Golenbock, C. Sirois, J. Hua, L.L. An, L. Audoly, G. La Rosa, A. Bierhaus, P. Naworth, A. Marshak-Rothstein, M.K. Crow, K.A. Fitzgerald, E. Latz, P.A. Kiener, and A.J. Coyle. 2007. Toll-like receptor 9-dependent

activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8:487-496. 139. Youn, J.H., Y.J. Oh, E.S. Kim, J.E. Choi, and J.S. Shin. 2008. High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes. J Immunol 180:5067-5074. 140. Kokkola, R., E. Sundberg, A.K. Ulfgren, K. Palmblad, J. Li, H. Wang, L. Ulloa, H. Yang, X.J. Yan, R. Furie, N. Chiorazzi, K.J. Tracey, U. Andersson, and H.E. Harris. 2002. High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis. Arthritis Rheum 46:2598-2603. 141. Taniguchi, N., K. Kawahara, K. Yone, T. Hashiguchi, M. Yamakuchi, M. Goto, K. Inoue, S. Yamada, K. Ijiri, S. Matsunaga, T. Nakajima, S. Komiya, and I. Maruyama. 2003. High mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine. Arthritis Rheum 48:971-981. 142. Pullerits, R., I.M. Jonsson, M. Verdrengh, M. Bokarewa, U. Andersson, H. Erlandsson-Harris, and A. Tarkowski. 2003. High mobility group box chromosomal protein 1, a DNA binding cytokine, induces arthritis. Arthritis Rheum 48:1693-1700. 143. Kokkola, R., J. Li, E. Sundberg, A.C. Aveberger, K. Palmblad, H. Yang, K.J. Tracey, U. Andersson, and H.E. Harris. 2003. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity. Arthritis Rheum 48:2052-2058. 144. Palmblad, K., E. Sundberg, M. Diez, R. Soderling, A.C. Aveberger, U. Andersson, and H.E. Harris. 2007. Morphological characterization of intra-articular HMGB1 expression during the course of collagen-induced arthritis. Arthritis Res Ther 9:R35. 145. Abraham, E., J. Arcaroli, A. Carmody, H. Wang, and K.J. Tracey. 2000. HMG-1 as a mediator of acute lung inflammation. J Immunol 165:2950-2954. 146. Kim, J.Y., J.S. Park, D. Strassheim, I. Douglas, F. Diaz del Valle, K. Asehnoune, S. Mitra, S.H. Kwak, S. Yamada, I. Maruyama, A. Ishizaka, and E. Abraham. 2005. HMGB1 contributes to the development of acute lung injury after hemorrhage. Am J Physiol Lung Cell Mol Physiol 288:L958-965. 147. Liu, K., S. Mori, H.K. Takahashi, Y. Tomono, H. Wake, T. Kanke, Y. Sato, N. Hiraga, N. Adachi, T. Yoshino, and M. Nishibori. 2007. Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J 21:3904-3916. 148. Qin, S., H. Wang, R. Yuan, H. Li, M. Ochani, K. Ochani, M. Rosas-Ballina, C.J. Czura, J.M. Huston, E. Miller, X. Lin, B. Sherry, A. Kumar, G. Larosa, W. Newman, K.J. Tracey, and H. Yang. 2006. Role of HMGB1 in apoptosis-mediated sepsis lethality. J Exp Med 203:1637-1642. 149. Sitia, G., M. Iannacone, S. Muller, M.E. Bianchi, and L.G. Guidotti. 2007. Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice. J Leukoc Biol 81:100-107. 150. Chorny, A., P. Anderson, E. Gonzalez-Rey, and M. Delgado. 2008. Ghrelin protects against experimental sepsis by inhibiting high-mobility group box 1 release and by killing bacteria. J Immunol 180:8369-8377. 151. Hofmann, M.A., S. Drury, B.I. Hudson, M.R. Gleason, W. Qu, Y. Lu, E. Lalla, S. Chitnis, J. Monteiro, M.H. Stickland, L.G. Bucciarelli, B. Moser, G. Moxley, S. Itescu, P.J. Grant, P.K. Gregersen, D.M. Stern, and A.M. Schmidt. 2002. RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response. Genes Immun 3:123-135. 152. Lutterloh, E.C., S.M. Opal, D.D. Pittman, J.C. Keith, Jr., X.Y. Tan, B.M. Clancy, H. Palmer, K. Milarski, Y. Sun, J.E. Palardy, N.A. Parejo, and N. Kessimian. 2007. Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection. Crit Care 11:R122. 153. Abeyama, K., D.M. Stern, Y. Ito, K. Kawahara, Y. Yoshimoto, M. Tanaka, T. Uchimura, N. Ida, Y. Yamazaki, S. Yamada, Y. Yamamoto, H. Yamamoto, S. Iino, N. Taniguchi, and I. Maruyama. 2005. The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest 115:1267-1274. 154. Van de Wouwer, M., S. Plaisance, A. De Vriese, E. Waelkens, D. Collen, J. Persson, M.R. Daha, and E.M. Conway. 2006. The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis. J Thromb Haemost 4:1813-1824. 155. Fink, M.P. 2007. Ethyl pyruvate: a novel treatment for sepsis. Curr Drug Targets 8:515-518. 156. Bartling, B., C. Fuchs, V. Somoza, B. Niemann, R.E. Silber, and A. Simm. 2007. Lung level of HMBG1 is elevated in response to advanced glycation end product-enriched food in vivo. Mol Nutr Food Res 51:479-487. 157. Goodman, R.B., J. Pugin, J.S. Lee, and M.A. Matthay. 2003. Cytokine-mediated inflammation in acute lung injury. Cytokine Growth Factor Rev 14:523-535. 158. Hagiwara, S., H. Iwasaka, K. Togo, and T. Noguchi. 2008. A neutrophil elastase inhibitor, sivelestat, reduces lung injury following endotoxin-induced shock in rats by inhibiting HMGB1. Inflammation 31:227-234.

159. Hamada, N., T. Maeyama, T. Kawaguchi, M. Yoshimi, J. Fukumoto, M. Yamada, S. Yamada, K. Kuwano, and Y. Nakanishi. 2008. The role of high mobility group box1 in pulmonary fibrosis. Am J Respir Cell Mol Biol 39:440-447. 160. Ogawa, E.N., A. Ishizaka, S. Tasaka, H. Koh, H. Ueno, F. Amaya, M. Ebina, S. Yamada, Y. Funakoshi, J. Soejima, K. Moriyama, T. Kotani, S. Hashimoto, H. Morisaki, E. Abraham, and J. Takeda. 2006. Contribution of high-mobility group box-1 to the development of ventilator-induced lung injury. Am J Respir Crit Care Med 174:400-407. 161. Rowe, S.M., P.L. Jackson, G. Liu, M. Hardison, A. Livraghi, G.M. Solomon, D.B. McQuaid, B.D. Noerager, A. Gaggar, J. Clancy, W. O'Neal, E.J. Sorscher, E. Abraham, and J.E. Blalock. 2008. Potential Role of High Mobility Group Box 1 in Cystic Fibrosis Airway Disease. Am J Respir Crit Care Med 162. Silva, E., J. Arcaroli, Q. He, D. Svetkauskaite, C. Coldren, J.A. Nick, K. Poch, J.S. Park, A. Banerjee, and E. Abraham. 2007. HMGB1 and LPS induce distinct patterns of gene expression and activation in neutrophils from patients with sepsis-induced acute lung injury. Intensive Care Med 33:1829-1839. 163. Ueno, H., T. Matsuda, S. Hashimoto, F. Amaya, Y. Kitamura, M. Tanaka, A. Kobayashi, I. Maruyama, S. Yamada, N. Hasegawa, J. Soejima, H. Koh, and A. Ishizaka. 2004. Contributions of high mobility group box protein in experimental and clinical acute lung injury. Am J Respir Crit Care Med 170:1310- 1316. 164. Yasuhara, D., T. Hashiguchi, K. Kawahara, T. Nakahara, T. Harada, H. Taguchi, S. Yamada, I. Maruyama, and A. Inui. 2007. High mobility group box 1 and refeeding-resistance in anorexia nervosa. Mol Psychiatry 12:976-977. 165. af Klint, E., C. Grundtman, M. Engstrom, A.I. Catrina, D. Makrygiannakis, L. Klareskog, U. Andersson, and A.K. Ulfgren. 2005. Intraarticular glucocorticoid treatment reduces inflammation in synovial cell infiltrations more efficiently than in synovial blood vessels. Arthritis Rheum 52:3880- 3889. 166. Andersson, U., and K.J. Tracey. 2004. HMGB1 as a mediator of necrosis-induced inflammation and a therapeutic target in arthritis. Rheum Dis Clin North Am 30:627-637, xi. 167. Goldstein, R.S., A. Bruchfeld, L. Yang, A.R. Qureshi, M. Gallowitsch-Puerta, N.B. Patel, B.J. Huston, S. Chavan, M. Rosas-Ballina, P.K. Gregersen, C.J. Czura, R.P. Sloan, A.E. Sama, and K.J. Tracey. 2007. Cholinergic anti-inflammatory pathway activity and High Mobility Group Box-1 (HMGB1) serum levels in patients with rheumatoid arthritis. Mol Med 13:210-215. 168. Gonzalez-Rey, E., A. Chorny, N. Varela, F. O'Valle, and M. Delgado. 2007. Therapeutic effect of urocortin on collagen-induced arthritis by down-regulation of inflammatory and Th1 responses and induction of regulatory T cells. Arthritis Rheum 56:531-543. 169. Ostberg, T., H. Wahamaa, K. Palmblad, N. Ito, P. Stridh, M. Shoshan, M.T. Lotze, H.E. Harris, and U. Andersson. 2008. Oxaliplatin retains HMGB1 intranuclearly and ameliorates collagen type II-induced arthritis. Arthritis Res Ther 10:R1. 170. Sundberg, E., C. Grundtman, E. Af Klint, J. Lindberg, S. Ernestam, A.K. Ulfgren, H.E. Harris, and U. Andersson. 2008. Systemic TNF blockade does not modulate synovial expression of the proinflammatory mediator HMGB1 in rheumatoid arthritis patients--a prospective clinical study. Arthritis Res Ther 10:R33. 171. Uesugi, H., S. Ozaki, J. Sobajima, F. Osakada, H. Shirakawa, M. Yoshida, and K. Nakao. 1998. Prevalence and characterization of novel pANCA, antibodies to the high mobility group non-histone chromosomal proteins HMG1 and HMG2, in systemic rheumatic diseases. J Rheumatol 25:703-709. 172. Voll, R.E., V. Urbonaviciute, M. Herrmann, and J.R. Kalden. 2008. High mobility group box 1 in the pathogenesis of inflammatory and autoimmune diseases. Isr Med Assoc J 10:26-28. 173. Zetterstrom, C.K., W. Jiang, H. Wahamaa, T. Ostberg, A.C. Aveberger, H. Schierbeck, M.T. Lotze, U. Andersson, D.S. Pisetsky, and H. Erlandsson Harris. 2008. Pivotal advance: inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate. J Leukoc Biol 83:31-38. 174. Inoue, K., K. Kawahara, K.K. Biswas, K. Ando, K. Mitsudo, M. Nobuyoshi, and I. Maruyama. 2007. HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques. Cardiovasc Pathol 16:136-143. 175. Jaulmes, A., S. Thierry, B. Janvier, M. Raymondjean, and V. Marechal. 2006. Activation of sPLA2-IIA and PGE2 production by high mobility group protein B1 in vascular smooth muscle cells sensitized by IL-1beta. FASEB J 20:1727-1729. 176. Kalinina, N., A. Agrotis, Y. Antropova, G. DiVitto, P. Kanellakis, G. Kostolias, O. Ilyinskaya, E. Tararak, and A. Bobik. 2004. Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines. Arterioscler Thromb Vasc Biol 24:2320-2325.

177. Li, W., A.E. Sama, and H. Wang. 2006. Role of HMGB1 in cardiovascular diseases. Curr Opin Pharmacol 6:130-135. 178. Porto, A., R. Palumbo, M. Pieroni, G. Aprigliano, R. Chiesa, F. Sanvito, A. Maseri, and M.E. Bianchi. 2006. Smooth muscle cells in human atherosclerotic plaques secrete and proliferate in response to high mobility group box 1 protein. FASEB J 20:2565-2566. 179. Soro-Paavonen, A., A.M. Watson, J. Li, K. Paavonen, A. Koitka, A.C. Calkin, D. Barit, M.T. Coughlan, B.G. Drew, G.I. Lancaster, M. Thomas, J.M. Forbes, P.P. Nawroth, A. Bierhaus, M.E. Cooper, and K.A. Jandeleit-Dahm. 2008. Receptor for advanced glycation end products (RAGE) deficiency attenuates the development of atherosclerosis in diabetes. Diabetes 57:2461-2469. 180. Apetoh, L., F. Ghiringhelli, A. Tesniere, A. Criollo, C. Ortiz, R. Lidereau, C. Mariette, N. Chaput, J.P. Mira, S. Delaloge, F. Andre, T. Tursz, G. Kroemer, and L. Zitvogel. 2007. The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy. Immunol Rev 220:47-59. 181. Campana, L., L. Bosurgi, and P. Rovere-Querini. 2008. HMGB1: a two-headed signal regulating tumor progression and immunity. Curr Opin Immunol 20:518-523. 182. Lotze, M.T., and R.A. DeMarco. 2003. Dealing with death: HMGB1 as a novel target for cancer therapy. Curr Opin Investig Drugs 4:1405-1409. 183. Maeda, S., Y. Hikiba, W. Shibata, T. Ohmae, A. Yanai, K. Ogura, S. Yamada, and M. Omata. 2007. Essential roles of high-mobility group box 1 in the development of murine colitis and colitis-associated cancer. Biochem Biophys Res Commun 360:394-400. 184. Oue, N., P.P. Aung, Y. Mitani, H. Kuniyasu, H. Nakayama, and W. Yasui. 2005. Genes involved in invasion and metastasis of gastric cancer identified by array-based hybridization and serial analysis of gene expression. Oncology 69 Suppl 1:17-22. 185. Poser, I., M. Golob, R. Buettner, and A.K. Bosserhoff. 2003. Upregulation of HMG1 leads to melanoma inhibitory activity expression in malignant melanoma cells and contributes to their malignancy phenotype. Mol Cell Biol 23:2991-2998. 186. Zhang, C.L., M.G. Shu, H.W. Qi, and L.W. Li. 2008. Inhibition of tumor angiogenesis by HMGB1 A box peptide. Med Hypotheses 70:343-345. 187. Lang, C.H., C. Silvis, N. Deshpande, G. Nystrom, and R.A. Frost. 2003. Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high- mobility-group protein-1 in skeletal muscle. Shock 19:538-546. 188. Pavlov, V.A., M. Ochani, L.H. Yang, M. Gallowitsch-Puerta, K. Ochani, X. Lin, J. Levi, W.R. Parrish, M. Rosas-Ballina, C.J. Czura, G.J. Larosa, E.J. Miller, K.J. Tracey, and Y. Al-Abed. 2007. Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis. Crit Care Med 35:1139-1144. 189. Suda, K., Y. Kitagawa, S. Ozawa, Y. Saikawa, M. Ueda, M. Ebina, S. Yamada, S. Hashimoto, S. Fukata, E. Abraham, I. Maruyama, M. Kitajima, and A. Ishizaka. 2006. Anti-high-mobility group box chromosomal protein 1 antibodies improve survival of rats with sepsis. World J Surg 30:1755-1762. 190. van Zoelen, M.A., P.F. Laterre, S.Q. van Veen, J.W. van Till, X. Wittebole, P. Bresser, M.W. Tanck, T. Dugernier, A. Ishizaka, M.A. Boermeester, and T. van der Poll. 2007. Systemic and local high mobility group box 1 concentrations during severe infection. Crit Care Med 35:2799-2804. 191. Zamora, R., A. Grishin, C. Wong, P. Boyle, J. Wang, D. Hackam, J.S. Upperman, K.J. Tracey, and H.R. Ford. 2005. High-mobility group box 1 protein is an inflammatory mediator in necrotizing enterocolitis: protective effect of the macrophage deactivator semapimod. Am J Physiol Gastrointest Liver Physiol 289:G643-652. 192. Jeyabalan, G., A. Tsung, and T.R. Billiar. 2006. Linking proximal and downstream signalling events in hepatic ischaemia/reperfusion injury. Biochem Soc Trans 34:957-959. 193. Levy, R.M., K.P. Mollen, J.M. Prince, D.J. Kaczorowski, R. Vallabhaneni, S. Liu, K.J. Tracey, M.T. Lotze, D.J. Hackam, M.P. Fink, Y. Vodovotz, and T.R. Billiar. 2007. Systemic inflammation and remote organ injury following trauma require HMGB1. Am J Physiol Regul Integr Comp Physiol 293:R1538-1544. 194. Tsung, A., R. Sahai, H. Tanaka, A. Nakao, M.P. Fink, M.T. Lotze, H. Yang, J. Li, K.J. Tracey, D.A. Geller, and T.R. Billiar. 2005. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med 201:1135-1143. 195. Watanabe, T., S. Kubota, M. Nagaya, S. Ozaki, H. Nagafuchi, K. Akashi, Y. Taira, S. Tsukikawa, S. Oowada, and S. Nakano. 2005. The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice. J Surg Res 124:59-66. 196. Mollica, L., F. De Marchis, A. Spitaleri, C. Dallacosta, D. Pennacchini, M. Zamai, A. Agresti, L. Trisciuoglio, G. Musco, and M.E. Bianchi. 2007. Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities. Chem Biol 14:431-441.

197. Sass, G., S. Heinlein, A. Agli, R. Bang, J. Schumann, and G. Tiegs. 2002. Cytokine expression in three mouse models of experimental hepatitis. Cytokine 19:115-120. 198. Fan, J., Y. Li, R.M. Levy, J.J. Fan, D.J. Hackam, Y. Vodovotz, H. Yang, K.J. Tracey, T.R. Billiar, and M.A. Wilson. 2007. Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling. J Immunol 178:6573-6580. 199. Ombrellino, M., H. Wang, M.S. Ajemian, A. Talhouk, L.A. Scher, S.G. Friedman, and K.J. Tracey. 1999. Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet 354:1446-1447. 200. Raman, K.G., P.L. Sappington, R. Yang, R.M. Levy, J.M. Prince, S. Liu, S.K. Watkins, A.M. Schmidt, T.R. Billiar, and M.P. Fink. 2006. The role of RAGE in the pathogenesis of intestinal barrier dysfunction after hemorrhagic shock. Am J Physiol Gastrointest Liver Physiol 291:G556-565. 201. Naghavi, M.H., P. Nowak, J. Andersson, A. Sonnerborg, H. Yang, K.J. Tracey, and A. Vahlne. 2003. Intracellular high mobility group B1 protein (HMGB1) represses HIV-1 LTR-directed transcription in a promoter- and cell-specific manner. Virology 314:179-189. 202. Nowak, P., B. Barqasho, and A. Sonnerborg. 2007. Elevated plasma levels of high mobility group box protein 1 in patients with HIV-1 infection. AIDS 21:869-871. 203. Nowak, P., B. Barqasho, C.J. Treutiger, H.E. Harris, K.J. Tracey, J. Andersson, and A. Sonnerborg. 2006. HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages. Cytokine 34:17-23. 204. Thierry, S., J. Gozlan, A. Jaulmes, R. Boniface, N. Nasreddine, F. Strauss, and V. Marechal. 2007. High-mobility group box 1 protein induces HIV-1 expression from persistently infected cells. AIDS 21:283-292. 205. Sobajima, J., S. Ozaki, H. Uesugi, F. Osakada, H. Shirakawa, M. Yoshida, and K. Nakao. 1998. Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) directed against HMG1 and HMG2 in ulcerative colitis (UC). Clin Exp Immunol 111:402-407. 206. Yang, R., T. Harada, K.P. Mollen, J.M. Prince, R.M. Levy, J.A. Englert, M. Gallowitsch-Puerta, L. Yang, H. Yang, K.J. Tracey, B.G. Harbrecht, T.R. Billiar, and M.P. Fink. 2006. Anti-HMGB1 neutralizing antibody ameliorates gut barrier dysfunction and improves survival after hemorrhagic shock. Mol Med 12:105-114. 207. Alleva, L.M., A.C. Budd, and I.A. Clark. 2008. Systemic release of high mobility group box 1 protein during severe murine influenza. J Immunol 181:1454-1459. 208. Alleva, L.M., H. Yang, K.J. Tracey, and I.A. Clark. 2005. High mobility group box 1 (HMGB1) protein: possible amplification signal in the pathogenesis of falciparum malaria. Trans R Soc Trop Med Hyg 99:171-174. 209. Clark, I.A., L.M. Alleva, A.C. Mills, and W.B. Cowden. 2004. Pathogenesis of malaria and clinically similar conditions. Clin Microbiol Rev 17:509-539, table of contents. 210. Kumar, K., A. Singal, M.M. Rizvi, and V.S. Chauhan. 2008. High mobility group box (HMGB) proteins of Plasmodium falciparum: DNA binding proteins with pro-inflammatory activity. Parasitol Int 57:150-157. 211. Andrassy, M., H.C. Volz, J.C. Igwe, B. Funke, S.N. Eichberger, Z. Kaya, S. Buss, F. Autschbach, S.T. Pleger, I.K. Lukic, F. Bea, S.E. Hardt, P.M. Humpert, M.E. Bianchi, H. Mairbaurl, P.P. Nawroth, A. Remppis, H.A. Katus, and A. Bierhaus. 2008. High-mobility group box-1 in ischemia-reperfusion injury of the heart. Circulation 117:3216-3226. 212. Germani, A., F. Limana, and M.C. Capogrossi. 2007. Pivotal advances: high-mobility group box 1 protein--a cytokine with a role in cardiac repair. J Leukoc Biol 81:41-45. 213. Goldstein, R.S., M. Gallowitsch-Puerta, L. Yang, M. Rosas-Ballina, J.M. Huston, C.J. Czura, D.C. Lee, M.F. Ward, A.N. Bruchfeld, H. Wang, M.L. Lesser, A.L. Church, A.H. Litroff, A.E. Sama, and K.J. Tracey. 2006. Elevated high-mobility group box 1 levels in patients with cerebral and myocardial ischemia. Shock 25:571-574. 214. Hagiwara, S., H. Iwasaka, T. Uchino, and T. Noguchi. 2008. High mobility group box 1 induces a negative inotropic effect on the left ventricle in an isolated rat heart model of septic shock: a pilot study. Circ J 72:1012-1017. 215. Kitahara, T., Y. Takeishi, M. Harada, T. Niizeki, S. Suzuki, T. Sasaki, M. Ishino, O. Bilim, O. Nakajima, and I. Kubota. 2008. High-mobility group box 1 restores cardiac function after myocardial infarction in transgenic mice. Cardiovasc Res 80:40-46. 216. De Mori, R., S. Straino, A. Di Carlo, A. Mangoni, G. Pompilio, R. Palumbo, M.E. Bianchi, M.C. Capogrossi, and A. Germani. 2007. Multiple effects of high mobility group box protein 1 in skeletal muscle regeneration. Arterioscler Thromb Vasc Biol 27:2377-2383. 217. Ulfgren, A.K., C. Grundtman, K. Borg, H. Alexanderson, U. Andersson, H.E. Harris, and I.E. Lundberg. 2004. Down-regulation of the aberrant expression of the inflammation mediator high

mobility group box chromosomal protein 1 in muscle tissue of patients with polymyositis and dermatomyositis treated with corticosteroids. Arthritis Rheum 50:1586-1594. 218. Cheng, B.Q., C.T. Liu, W.J. Li, W. Fan, N. Zhong, Y. Zhang, X.Q. Jia, and S.Z. Zhang. 2007. Ethyl pyruvate improves survival and ameliorates distant organ injury in rats with severe acute pancreatitis. Pancreas 35:256-261. 219. Sawa, H., T. Ueda, Y. Takeyama, T. Yasuda, M. Shinzeki, T. Nakajima, and Y. Kuroda. 2006. Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis. World J Gastroenterol 12:7666-7670. 220. Yang, Z.Y., Y. Ling, T. Yin, J. Tao, J.X. Xiong, H.S. Wu, and C.Y. Wang. 2008. Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats. World J Gastroenterol 14:4546-4550. 221. Yuan, H., X. Jin, J. Sun, F. Li, Q. Feng, C. Zhang, Y. Cao, and Y. Wang. 2008. Protective Effect of HMGB1 A Box on Organ Injury of Acute Pancreatitis in Mice. Pancreas 222. Andersson, U., and K.J. Tracey. 2003. HMGB1 in sepsis. Scand J Infect Dis 35:577-584. 223. Angus, D.C., L. Yang, L. Kong, J.A. Kellum, R.L. Delude, K.J. Tracey, and L. Weissfeld. 2007. Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated pneumonia and pneumonia with severe sepsis. Crit Care Med 35:1061-1067. 224. Gaini, S., S.S. Pedersen, O.G. Koldkjaer, C. Pedersen, and H.J. Moller. 2007. High mobility group box- 1 protein in patients with suspected community-acquired infections and sepsis: a prospective study. Crit Care 11:R32. 225. Gibot, S., F. Massin, A. Cravoisy, D. Barraud, L. Nace, B. Levy, and P.E. Bollaert. 2007. High-mobility group box 1 protein plasma concentrations during septic shock. Intensive Care Med 33:1347-1353. 226. Huston, J.M., H. Wang, M. Ochani, K. Ochani, M. Rosas-Ballina, M. Gallowitsch-Puerta, M. Ashok, L. Yang, K.J. Tracey, and H. Yang. 2008. Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels. J Immunol 181:3535-3539. 227. Sunden-Cullberg, J., A. Norrby-Teglund, A. Rouhiainen, H. Rauvala, G. Herman, K.J. Tracey, M.L. Lee, J. Andersson, L. Tokics, and C.J. Treutiger. 2005. Persistent elevation of high mobility group box- 1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 33:564-573. 228. Ulloa, L., M. Ochani, H. Yang, M. Tanovic, D. Halperin, R. Yang, C.J. Czura, M.P. Fink, and K.J. Tracey. 2002. Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci U S A 99:12351-12356. 229. Faraco, G., S. Fossati, M.E. Bianchi, M. Patrone, M. Pedrazzi, B. Sparatore, F. Moroni, and A. Chiarugi. 2007. High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo. J Neurochem 103:590-603. 230. Hayakawa, K., K. Mishima, M. Nozako, M. Hazekawa, S. Mishima, M. Fujioka, K. Orito, N. Egashira, K. Iwasaki, and M. Fujiwara. 2008. Delayed treatment with minocycline ameliorates neurologic impairment through activated microglia expressing a high-mobility group box1-inhibiting mechanism. Stroke 39:951-958. 231. Kim, J.B., C.M. Lim, Y.M. Yu, and J.K. Lee. 2008. Induction and subcellular localization of high- mobility group box-1 (HMGB1) in the postischemic rat brain. J Neurosci Res 86:1125-1131. 232. Kim, J.B., J. Sig Choi, Y.M. Yu, K. Nam, C.S. Piao, S.W. Kim, M.H. Lee, P.L. Han, J.S. Park, and J.K. Lee. 2006. HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain. J Neurosci 26:6413-6421. 233. Li, W., J. Li, M. Ashok, R. Wu, D. Chen, L. Yang, H. Yang, K.J. Tracey, P. Wang, A.E. Sama, and H. Wang. 2007. A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late- acting proinflammatory mediator, high mobility group box 1. J Immunol 178:3856-3864. 234. Qiu, J., M. Nishimura, Y. Wang, J.R. Sims, S. Qiu, S.I. Savitz, S. Salomone, and M.A. Moskowitz. 2008. Early release of HMGB-1 from neurons after the onset of brain ischemia. J Cereb Blood Flow Metab 28:927-938. 235. Barkauskaite, V., M. Ek, K. Popovic, H.E. Harris, M. Wahren-Herlenius, and F. Nyberg. 2007. Translocation of the novel cytokine HMGB1 to the cytoplasm and extracellular space coincides with the peak of clinical activity in experimentally UV-induced lesions of cutaneous lupus erythematosus. Lupus 16:794-802. 236. Ek, M., K. Popovic, H.E. Harris, C.S. Naucler, and M. Wahren-Herlenius. 2006. Increased extracellular levels of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 in minor salivary glands of patients with Sjogren's syndrome. Arthritis Rheum 54:2289-2294. 237. Espinosa, A., W. Zhou, M. Ek, M. Hedlund, S. Brauner, K. Popovic, L. Horvath, T. Wallerskog, M. Oukka, F. Nyberg, V.K. Kuchroo, and M. Wahren-Herlenius. 2006. The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death. J Immunol 176:6277-6285.

238. Lafyatis, R., and A. Marshak-Rothstein. 2007. Toll-like receptors and innate immune responses in systemic lupus erythematosus. Arthritis Res Ther 9:222. 239. Popovic, K., M. Ek, A. Espinosa, L. Padyukov, H.E. Harris, M. Wahren-Herlenius, and F. Nyberg. 2005. Increased expression of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 in skin lesions of patients with lupus erythematosus. Arthritis Rheum 52:3639- 3645. 240. Huang, L.F., Y.M. Yao, L.T. Zhang, N. Dong, Y. Yu, and Z.Y. Sheng. 2008. The Effect of High- Mobility Group Box 1 Protein on Activity of Regulatory T Cells after Thermal Injury in Rats. Shock 241. Singer, A.J., S.A. McClain, B.R. Taira, J.L. Guerriero, and W. Zong. 2008. Apoptosis and necrosis in the ischemic zone adjacent to third degree burns. Acad Emerg Med 15:549-554. 242. Urbonaviciute, V., B.G. Furnrohr, C. Weber, M. Haslbeck, S. Wilhelm, M. Herrmann, and R.E. Voll. 2007. Factors masking HMGB1 in human serum and plasma. J Leukoc Biol 81:67-74. 243. Wahamaa, H., T. Vallerskog, S. Qin, C. Lunderius, G. LaRosa, U. Andersson, and H.E. Harris. 2007. HMGB1-secreting capacity of multiple cell lineages revealed by a novel HMGB1 ELISPOT assay. J Leukoc Biol 81:129-136. 244. Gaillard, C., C. Borde, J. Gozlan, V. Marechal, and F. Strauss. 2008. A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes. PLoS ONE 3:e2855. 245. Li, J., H. Wang, J.M. Mason, J. Levine, M. Yu, L. Ulloa, C.J. Czura, K.J. Tracey, and H. Yang. 2004. Recombinant HMGB1 with cytokine-stimulating activity. J Immunol Methods 289:211-223. 246. Rouhiainen, A., S. Tumova, L. Valmu, N. Kalkkinen, and H. Rauvala. 2007. Pivotal advance: analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin). J Leukoc Biol 81:49-58. 247. Zimmermann, K., D. Volkel, S. Pable, T. Lindner, F. Kramberger, S. Bahrami, and F. Scheiflinger. 2004. Native versus recombinant high-mobility group B1 proteins: functional activity in vitro. Inflammation 28:221-229. 248. Dong Xda, E., N. Ito, M.T. Lotze, R.A. Demarco, P. Popovic, S.H. Shand, S. Watkins, S. Winikoff, C.K. Brown, D.L. Bartlett, and H.J. Zeh, 3rd. 2007. High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy. J Immunother 30:596-606. 249. Vaisman, A., S.E. Lim, S.M. Patrick, W.C. Copeland, D.C. Hinkle, J.J. Turchi, and S.G. Chaney. 1999. Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts. Biochemistry 38:11026-11039. 250. Yamashita, M., G. Ichinowatari, K. Yamaki, and K. Ohuchi. 1999. Inhibition by auranofin of the production of prostaglandin E2 and nitric oxide in rat peritoneal macrophages. Eur J Pharmacol 368:251-258. 251. Jiang, W., and D.S. Pisetsky. 2006. The role of IFN-alpha and nitric oxide in the release of HMGB1 by RAW 264.7 cells stimulated with polyinosinic-polycytidylic acid or lipopolysaccharide. J Immunol 177:3337-3343.