Thesis for doctoral degree (Ph.D.)2010

Fredrik Wermeling

Thesis for doctoral degree (Ph.D.) 2010

Fredrik Werm

eling

INNATE MECHANISMSREGULATING PERIPHERAL

B CELL TOLERANCE

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From the Department of Medicine, Solna

Clinical Allergy Research Unit Karolinska Institutet, Stockholm, Sweden

INNATE MECHANISMS REGULATING PERIPHERAL

B CELL TOLERANCE

Fredrik Wermeling

Stockholm 2010

2010

Gårdsvägen 4, 169 70 Solna

Printed by

All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by REPROPRINT AB. © Fredrik Wermeling, 2010 ISBN 978-91-7409-864-8

While I breathe, I hope

To Eva

ABSTRACT

Our immune system protects us from invading pathogens such as bacteria, viruses, fungi and parasites. However, it can also cause disease by being improperly activated against our own tissues (autoimmune diseases) and harmless environmental factors (allergy). The immune system is built up by multiple, often overlapping, layers represented by cells and soluble factors working together to stop invading pathogens. The immune system is commonly described as being divided into a fast, more primitive, innate and a slower, more refined, adaptive part. Most often are both genetic and environmental factors affecting several parts of the immune system involved in autoimmune disease development. Thus, tolerance (related to tolerating our own tissues) is broken in patients with these diseases. The pathology associated with autoimmune disease is in general linked to the presence and activation of autoreactive B and T cells. The aim of the work presented in this thesis has been related to how certain innate mechanisms regulate autoreactive B cells. The two first papers [I, II] mainly address how increased levels of circulating apoptotic cells, evident in patients with the autoimmune disease SLE, activate autoreactive B cells. The two last papers [III, IV] relate, in a broad sense, to the splenic marginal zone (MZ) macrophage population and its function. Paper I identifies an important regulatory role for invariant natural killer T (iNKT) cells, controlling the activation of autoreactive B cells. This is dependent on a direct iNKT–B cell interaction taking place before the autoreactive B cell enters a germinal center (GC). Of significance, the GC entry and subsequent autoantibody production in iNKT cell deficient mice can be limited by increasing iNKT cell levels, thus being relevant for SLE patients who have reduced levels of iNKT cells. Paper II identifies that splenic MZ macrophages with importance for tolerance bind apoptotic cells present in the circulation and that this binding involves the scavenger receptor MARCO. Furthermore, autoantibodies binding the MARCO receptor are found in SLE patients and before onset of disease in mice that develop spontaneous SLE-like disease. Paper III further addresses the role of the MARCO (macrophage) receptor and autoantibodies that bind it. The injection of an anti-MARCO antibody affected B cells and increased the production of disease-related autoantibodies following both in vivo and in vitro stimulation. The antibody-mediated MARCO engagement results in the release of soluble proinflammatory factors such as TNF-α. Paper IV identifies an important role for SIGN-R1, expressed on MARCO+ MZ macrophages, in the anti-inflammatory affect mediated by IVIG treatment in a model of rheumatoid arthritis. In summary, these studies identify iNKT cells as a promising therapeutic target in SLE patients, MARCO as a novel autoantigen and describe a multifaceted role for the splenic MZ macrophage involved in removing apoptotic cells and (dependent on the type of stimuli) the ability to both activate and dampen the immune system. Furthermore, this thesis discusses the conflicting view of apoptotic cells as inducers of tolerance but also, during certain conditions, as activators of the immune system.

LIST OF PUBLICATIONS

I. Wermeling F, Lind S. M, Domange Jordö E., Cardell S, and Karlsson MCI. Invariant NKT cells limit activation of autoreactive CD1d positive B cells. J. Exp. Med. [Accepted for publication March 2010]

II. Wermeling F, Chen Y, Pikkarainen T, Scheynius A, Winqvist O, Izui S, Ravetch JV, Tryggvason K, Karlsson MC. Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus. J. Exp. Med. 2007 Oct 1;204(10):2259-65. (Highlighted in Nat. Clin. Pract. Rheumatology, 2008, 4:7)

III. Wermeling F, Prokopec K, Grönlund H and Karlsson MCI. MARCO engagement triggers a sterile inflammatory response with implication for autoimmune B cell activation. Manuscript

IV. Anthony RM, Wermeling F, Karlsson MCI and Ravetch JV. Identification of a receptor required for the anti-inflammatory activity of IVIG. Proc Natl Acad Sci U S A. 2008 Dec 16;105(50):19571-8.

Additional publications not included in the thesis: Sakthivel P, Wermeling F, Elmgren A, Hulthe J, Kakoulidou M, Kari Lefvert A, Lind L. Circulating soluble CTLA-4 is related to inflammatory markers in the 70 year old population. Scand J Clin Lab Invest. 2010 Mar 24. [Epub ahead of print] Chen Y, Wermeling F, Sundqvist J, Jonsson AB, Tryggvason K, Pikkarainen T and Karlsson MCI. A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses. Eur J Immunol. 2010 Feb 16. [Epub ahead of print] Wermeling F, Karlsson MCI, McGaha TL. An anatomical view on macrophages in tolerance. Autoimmun Rev. 2009 Sep;9(1):49-52. Epub 2009 Mar 10. Review. Thunberg S, Neimert-Andersson T, Cheng Q, Wermeling F, Bergström U, Swedin L, Dahlén SE, Arnér E, Scheynius A, Karlsson MCI, Gafvelin G, van Hage M, Grönlund H. Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice. Allergy. 2009 Jun;64(6):919-26. Epub 2009 Jan 31. Westerberg L, A. de la Fuente M, Wermeling F, Ochs HD, Karlsson MCI, Snapper SB and Notarangelo LD. WASP Confers Selective Advantage for Specific Hematopoietic Cell Populations and Serves a Unique Role in Marginal Zone B cell Development and Function. Blood. 2008 Nov 15;112(10):4139-47

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CONTENTS

1 Introduction .......................................................................................................... 3 1.1 The immune system .................................................................................. 4

1.1.1 Innate and adaptive immune system ........................................ 4 1.1.2 Lymphoid organs.......................................................................... 6 1.1.3 The spleen...................................................................................... 6

1.2 B cells............................................................................................................ 7 1.2.1 The germinal center (GC)........................................................... 8 1.2.2 Antibodies (or Immunoglobulins (Ig)) .................................. 10

1.3 NKT cells ................................................................................................... 11 1.4 Macrophages............................................................................................. 13

1.4.1 Scavenger receptors.................................................................... 13 1.4.2 SIGN-R1....................................................................................... 14

1.5 Cell death................................................................................................... 14 1.5.1 Clearance of dying cells ............................................................. 15

1.6 Autoimmune disease............................................................................... 16 1.6.1 Etiology of autoimmune diseases ............................................ 17 1.6.2 Tolerance ..................................................................................... 18 1.6.3 Systemic lupus erythematosus (SLE) ...................................... 19 1.6.4 Cell death and SLE ..................................................................... 19 1.6.5 Rheumatoid Arthritis (RA)....................................................... 20

2 The present investigation .................................................................................. 22 2.1 Methodology............................................................................................. 23 2.2 Results and discussion............................................................................. 26

2.2.1 Invariant NKT cells limit activation of autoreactive CD1d positive B cells. ................................................................ 26

2.2.2 Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus. .............................................. 28

2.2.3 MARCO engagement triggers an inflammatory response with implication for autoreactive B cell activation................ 30

2.2.4 Identification of a receptor required for the anti-inflammatory activity of IVIG. ........................................ 32

2.3 Final reflections and future perspectives.............................................. 33 3 Populärvetenskaplig sammanfattning............................................................. 35 4 Acknowledgements ............................................................................................ 38 5 References ............................................................................................................ 40

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LIST OF ABBREVIATIONS

αGalCer APC Asn BCR C. Elegans CIA CR DAMP DC Fab Fc FcR and FcγR FDC FoB GC HIV Ig iNKT IVIG LDL LPS LysoPC M-CSF MHC MMM MZ MZB MZM NKT PAMP PRR PS RA RF SHM SLE TCR TD TI (1 and 2) TLR KILFAR

Alpha-galactosylceramide Antigen presenting cell Asparagine B cell receptor Caenorhabditis elegans Collagen-induced arthritis Complement receptor Danger-associated molecular pattern Dendritic cell Fragment, antigen binding Fragment, crystallizable Fc (gamma) receptor Follicular dendritic cell Follicular B cell Germinal center Human immunodeficiency virus Immunoglobulin Invariant (type 1) natural killer T cell Intravenous immunoglobulin Low-density lipoprotein Lipopolysaccharide Lysophosphatidylcholine Macrophage colony-stimulating factor Major histocompatibility complex Marginal metallophilic macrophages Marginal zone Marginal zone B cell Marginal zone macrophage Natural killer T cell Pathogen-associated molecular patterns Pattern recognition receptor Phosphatidylserine Rheumatoid arthritis Rheumatoid factor Somatic hypermutation Systemic lupus erythematosus T cell receptor Thymus/T cell dependent (antigen) Thymus/T cell independent (antigen) type I and 2 Toll-like receptor

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1 INTRODUCTION

All living organisms have systems to protect themselves from harmful encounters with other organisms. From the most primitive unicellular organism that release substances that target rival organisms and thereby impart self-survival benefits, to the developed multilayered mammalian immune system involving multiple cell types and soluble factors. In the jawed vertebrates that appeared about 450 million years ago, the adaptive immune system started to evolve with the ability to generate specificity and memory in the response to invading infectious agents. The adaptive immune system complements the non-specific, non-memory generating innate immune system that is the base for defense in more primitive organisms. The power of the immune system is manifested in patients with autoimmune and allergic diseases, in which inappropriate activation of the immune system damages the host. Despite these examples, the importance of the immune system for the development of higher organisms can hardly be stressed enough. As exemplified by individuals with immunodeficiencies that make them susceptible to fatal infection and dependent on different types of prophylactic treatments. The targets that the adaptive immune system is activated against are called antigens (antibody generators), a term often used to describe the target during an infection, but also self-structures targeted during autoimmune disease (autoantigens) and antigens during allergic reactions (allergens). Although a limited number of processes are completely conserved through evolution, a lot of relevant knowledge can be gained by establishing models for how different organisms approach tasks, such as combating an infection or taking care of dying cells. This thesis deals with the regulation of autoreactive B cell by different innate mechanisms.

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1.1 THE IMMUNE SYSTEM

Infection and inflammation are two terms closely linked to the immune system. An infection is caused by invading microorganisms or pathogens, i.e. virus, bacteria, fungi and parasites, which leads to the activation of the immune system and thus inflammation. Infection always results in an inflammatory response, but inflammation can also occur in the complete absence of pathogens. This sterile or non-infectious type of inflammation is evident in autoimmune diseases, even though infections can also be involved in the etiology of these diseases (1). Here follows a classical example of an immune response to put this introduction in a context: Via an accidental cut in the skin a bacterium enters the body. Local cells belonging to the innate immune system (e.g. macrophages, dendritic cells (DCs), mast cells) recognize this and produce soluble factors promoting a general inflammatory milieu as well as the recruitment of more immune cells (e.g. neutrophils and monocytes). The area becomes swollen, red and painful. Local dendritic cells engulf bacteria and migrate towards a lymph node, which are small structures spread all over the body optimized for the activation of B and T cells. Bacterial components also reach the lymph node by passive diffusion via the lymph fluid. The activation of T cells involves the presentation of small pieces of the bacteria in an MHC context, e.g. expressed on the DCs. In contrast, B cells are mainly activated by non-digested antigens. After expansion the activated B and T cells target the bacteria by producing soluble factors that enhance the attack by innate immune cells. B cells produce specific antibodies that directly bind the bacteria. After the bacteria is defeated, the involved B and T cells that specifically recognize the bacteria remain in the body for a long time with the ability to be rapidly activated, thus representing a memory.

Figure 1. A classical example of an immune response. 1.1.1 Innate and adaptive immune system

The innate or non-specific immune system is found in all living organisms. This system is connected to the immediate response to invading pathogens. It includes several different types of functions ranging from the physical barrier of the skin, anti-microbial proteins or peptides released on surfaces where pathogens enter the body (e.g. skin, airways), soluble and cell bound proteins binding to pathogens, cells that ingest

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(phagocytose) pathogens and cells that recognize and kill stressed and infected cells. The non-specificity of the system should not be interpreted as that it is activated randomly; instead the innate immune system is activated by structures or patterns that are associated to pathogens. The basis of this is that pathogens often express certain signatures, or pathogen-associated molecular patterns (PAMPs), which are recognized by pattern recognition receptors (PRRs). The most studied, but far from only, PRR family is the toll-like receptor (TLR) family, which has very important functions in activating the immune system (2). Different pathogens express different PAMPs and thus activate the immune system differently, most often via multiple interactions with the innate immune system. An example of these differences is seen comparing gram-negative and gram-positive bacteria, where the first bacteria expresses lipopolysaccharide (LPS) that activates TLR4 and the second lipoteichoic acid (LTA) that activates TLR2 (3), in combination with other PRR ligands. Bacteria and viruses are more different from humans than are fungi and parasites, something that is of relevance for the innate system (which is based on the recognition of differences). A recognition system for parasites has in this regard been suggested to be based on that the parasite releases enzymes in order to invade tissues, which generate degradation products that could be recognized. Thus, the response does not only need to be directed to structures found on the parasite (4). The immune system can furthermore be activated by danger-associated molecular patterns (DAMPs). These are mainly intracellular components such as ATP and HMGB1, which are released from damaged tissues. The effect of DAMPs on the immune system relates to the activation of receptors, that they sometimes share with PAMPs, leading to a ‘sterile’ inflammatory response (5, 6). This gives the body another recognition system to rapidly activate the immune system and to combat potential infection. Relating to the activating features, these tissue-derived signals are also referred to as alarmins. Components of the innate immune system are also highly involved in homeostatic processes, for example in the removal of dying cells and in lipid metabolism (7-9). The adaptive or specific immune system is focused around B and T cells with their specific antigen recognition receptors: the B cell receptor (BCR) and the T cell receptor (TCR) which are both expressed on the surface. B and T cells are lymphocytes, a family that also includes the innate NK cell population and several subpopulations of unconventional B, T and NK cells, including the NKT cell population. These cells share common progenitor cells in the bone marrow but develop into separate lines (10). The antigen receptors of B and T cells are generated as the cells develop through the activity of several enzymes, together referred to as the V(D)J recombinase. These enzymes rearrange the genes coding the BCR/TCR, located in the Ig and Tcr loci, respectively (11). If the rearrangement is successful the result is a cell expressing an antigen recognition receptor encoded by a DNA sequence unique to that cell. This system allows a comparably small region of the DNA to generate a very high number of possible reactivities. This is crucial for the specific and potent response mediated by B and T cells (12). As the initial B or T cell will divide and thereby multiply, the term clone is often used to describe the cell, referring to the unique encoded specificity they express and not the number of cells. A clone that has generated a functional BCR/TCR and gone through all other needed developmental stages is referred to as being mature, in contrast to developing cells that are immature. The term naïve is also used for a mature cell that has not encountered its antigen, i.e. has not been activated.

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Although the antigen receptors of B and T cells are similar in many aspects they are used very differently. T cell activation depends on other cells presenting antigens by MHC (major histocompatibility complex) molecules, a surface molecule expressed from a genetic region originally identified as being important for the survival (and thus compatibility) of transplanted grafts (13). Through their TCR, conventional T cells recognize peptides presented in either MHC class I or II, depending on if they belong to the MHC class II-restricted CD4+ (helper) or MHC class I-restricted CD8+ (killer) T cell population. B cells, in contrast, directly interact with antigens via the BCR without the need for the involvement of other cells. B cell activation can, however, involve other cells presenting or transporting the antigen to the B cell in different (non-MHC related) ways (14, 15). Another difference is that while the TCR only serves in antigen recognition, the BCR also is a direct template for the subsequently produced antibody. B and T cells respond much more slowly to pathogens than the innate mechanisms. The active involvement of the B and T cells takes several days to get started, compared to the innate system that only takes minutes or hours. This lag phase is caused by the time it takes for the few clones with specificity for the antigen to be selected and expanded. Although often conceptually divided, the two parts of the immune system are closely connected; the activation of the innate immune system is essential for the activation of the adaptive immune system that, once activated, potentiates innate effector mechanisms. 1.1.2 Lymphoid organs

The lymphoid organs are related to development and activation of the adaptive lymphocytes. Several hierarchically different lymphoid organs/structures exist. The primary lymphoid organs are the thymus and the bone marrow that in this regard are related to the development of these lymphocytes. Lymph nodes and the spleen are secondary lymphoid organs, essential to the adaptive immune system, containing structures and cells needed for the coordinated activation of B and T cells (16). This activation is related to pathogens that, with a few exceptions, enter the body via epithelial barriers. The cells in the secondary organs encounter the pathogen by indirect or direct transportation of antigens to the organ. Especially DCs are described to be active in this process and therefore often seen as a coordinator of the activation of the adaptive immune system (17). 1.1.3 The spleen

The spleen is an organ located in the abdomen important for activating the immune system against pathogens present in the blood. The presence of bacteria (or fungi) in the blood, called sepsis, is a potentially fatal condition as the pathogen rapidly gets access to the whole body via the circulation. Sepsis represents a substantial burden for the society and is the tenth leading cause of death (18). Absence of the spleen, most often due to removal after trauma, does not generally affect quality of life, but leads to an increased susceptibility to sepsis (19, 20) and other, both infectious and non-infectious, diseases (21). The spleen receives a lot of blood from the splenic artery, something that is evident during trauma, which leads to heavy bleeding and the need for immediate surgery. The structure of the spleen is rather complicated and differs somewhat between mice and humans, although retaining similar general functionality (22).

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CD1d (MZB) / CD169 (MMM) SIGN-R1 (MZM) / B220 (FoB)

T cells

Red Pulp

MZ

Follicle

Follicle

MZ

The blood that enters the spleen through the hilum branches into smaller vessels inside the spleen. Surrounding these vessels are lymphoid or white pulp tissues where B and T cells are activated. The blood is then released into the marginal sinus compartment that opens up. Thereby the flow rate is lowered, increasing the possibility for the cells resident in the marginal zone (MZ) area, e.g. macrophages, B cells and DCs, to interact with potential pathogens, but also other components of the blood that could need attention (23). The blood continues into the red pulp area that, like the MZ, contains macrophage populations involved in removing old red blood cells, another important function of the spleen. Then the blood is gathered up in the red pulp and leaves the spleen via venous drainage. No afferent lymph vessels lead to the spleen, defining that activation of splenic lymphocytes is dependent on antigen transportation via the blood (24). The spleen has several other functions besides supporting lymphocyte activation, which is mainly localized to the white pulp. These include the phagocytic network of several phagocytic populations in the MZ and red pulp. Furthermore, B cells have a developmental phase at least partly dependent on the spleen (25). The spleen is also enervated by the splenic nerve that, interestingly, has implications for the generation of immune responses in the organ (26).

Figure 2. A large part of this thesis is focused around the splenic MZ that is localized in between the white pulp (or follicle), where conventional B and T cells reside, and the red pulp (dominated by F4/80+

macrophages). In the MZ, the blood is scanned for pathogens and waste by the MARCO+/ SIGN-R1+ MZ macrophages (MZM), but also by CD169+/ MOMA+ marginal metallophilic macrophages (MMM), CD21hi/ CD1dhi MZ B cells as well as 33D1+ DCs (not depicted in the

figure). The MZ B cells also mediate transport of antigens from the MZ to follicular dendritic cells inside the follicle (27). 1.2 B CELLS

Several phenotypically different mature naïve B cell populations have been described. The bulk of these belong to the follicular B cell population (FoB) that recirculates through the body trying to locate their antigen. FoBs represent the typical B cell population that represents more than 90% of all mature naïve B cells (28). The resident splenic MZ B cells and the peritoneal B1 B cells are the other major mature naïve populations and are described as innate-like, due to their rapid production of (low affinity) antibodies and involvement in the production of germline encoded natural antibodies (29, 30). The B1 and MZ B cell populations have also been suggested to be

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connected to autoreactive responses due to an activated phenotype and an enrichment in autoreactive clones (31, 32). Several different types of B cell activation exist, mainly depending on properties of the antigen. These are categorized as T-dependent (thymus-dependent and thus T cell-dependent; TD) or T-independent (TI). As the name implies, this is related to if the B cell activation needs T cell help or not. However, the fact that an antigen is classified as being independent of T cells does not mean that T cells are not involved during the response. Instead, although not essential, they probably influence it (33). The classical TD antigen is a protein that a B cell clone recognizes through its specific BCR. Following recognition the antigen is engulfed by the B cell, broken down into peptides and presented on MHC class II. If CD4+ T helper cell clones with affinity for the TD-derived antigen encounter the activated B cell they will provide help for the subsequent B cell activation. The T cell is also activated in this process and emerging knowledge indicates that B cells are potent antigen presenting cells (APCs), which might be relevant in several pathophysiological conditions such as multiple sclerosis (MS) (34). An interesting aspect of B cells as APCs is that the antigen-specific nature of the antigen uptake, via the BCR, can take place at very low antigen concentration due to the high affinity interaction (35). The TI responses can be further divided into type I and II, where the former is independent on the BCR, while the latter is dependent on it (36). TI-I antigens are most often exemplified by polyclonal activation with LPS via activation of TLR4 on the B cell. TI-II antigens are often long polysaccharides that contain multiple antigenic sites, thus being able to bind and tether numerous BCRs and thereby cause an intensive activation signal. B1 and MZ B cells are often connected to the TI responses while FoB cells are mainly related to TD responses. An important point to make is that assumptions regarding antigens are mainly based on prototypic and artificial antigens. During a real infection a combination of all these three types of antigens are probably most often involved. One of the types of activation might, however, be more important for the host survival. This is suggested by the survival related to sepsis caused by encapsulated bacteria, being largely dependent on a TI-2 type of response (37). Nonetheless, reducing the complexity in experimental systems can aid in focusing on important features of the system. Also, vaccination development clearly gains from the knowledge of the response to model antigens. 1.2.1 The germinal center (GC)

When the B cell encounters its antigen via the BCR it receives activating signals. For full activation the B cell generally also needs non-specific activators such as TLR ligands, pro-inflammatory factors and cytokines as well as later T cell interaction. Notably the binding (opsonization) of an antigen with complement makes it a lot more immunogenic, illustrated by the need for a 10 000 times higher dose of antigen in the absence of complement in order to trigger a comparable antibody response (38). Before the newly activated B cell differentiates into an antibody-secreting plasma cell it generates additional genetic modification in the locus coding the BCR/antibody. Naïve T cells, in contrast, do not change their tcr locus after activation. The B cell will upon activation migrate towards an area making up the GC in the secondary lymphoid organ, located near the T cell zone and thereby enabling B–T cell interactions. In the GC, affinity maturation and class switching takes place. A specialized cell population

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called follicular dendritic cells (FDC) is found in the GC. The FDCs are, despite their name, not DCs but rather a type of stromal cell. Via several receptors the FDC binds antigens that enter the body, thus most probably including the antigen that activated the B cell. These receptors include Fc receptors (FcR) and complement receptors (CR)

that bind antigens opsonized with antibodies and/or complement, respectively. This feature is critical for the generation of a high quality antibody response (39). Figure 3. A GC in the splenic white pulp area. Once activated a B cell clone proliferates and starts to induce changes in the Ig locus while located to the GC. Part of this process, called somatic hypermutation (SHM), involves the

activation of a deaminase enzyme called AID (40). The activation of AID, and other SHM related enzymes, results in the introduction of mutations in the BCR and thereby a modified BCR reactivity that is continuously, or perhaps better repeatedly, tested on the antigens bound to the FDCs. An increased affinity for the antigen leads to survival signals and expansion of the clone. However, most of the mutations that are introduced do not increase the affinity and the cell dies by apoptosis. In this way high affinity clones are selected. The germinal center is an open structure that also allows entry for non-activated B cells, something that can have importance for the activation of rare antigen-specific B cell clones (41). The enzyme AID is also involved in class switching of the activated B cell, relating to the type of antibody the B cell will produce, and thus which type of response they will be involved in. The Ig locus contains the genetic information of all the potential subclasses, including IgD, IgM, IgG, IgE and IgA in humans and mice (42, 43). These can be further divided into subpopulations, e.g. IgG can be divided into IgG1, 2, 3 and 4 in humans and IgG1, 2a, 2b and 3 in mice. The naïve B cell expresses both IgD and IgM, making up the BCR, and changes this depending on the cytokine milieu induced by the pathogen. In simple terms, IgG subclasses are mainly related to bacterial and viral infections, IgE to the response against parasites and IgA to the protection of mucosal surfaces. IgM is the standard response from a naïve B cell and is produced without the need for class switching, thus being related to the first wave of antibody production. The role for IgD has for a long time been enigmatic, as IgD deficiency does not affect B cell development and very low serum levels of soluble IgD has been reported (44). A function for IgD earlier in the evolution has been suggested as an explanation for this (45). However, recent data has indicated a function of soluble IgD for basophil activation in the upper respiratory mucosa (46). Specialized B cell activation can also occur outside of the follicular GC reaction. In these extrafollicular foci structures activated B cells produce antibodies and can class switch, but do not undergo somatic hypermutation. This type of response is often related to TI activation, but is also evident with TD antigens and is suggested to be responsible for early class switched antibody production following B cell activation. The class switch still needs T cell interaction, which is accomplished by the migration of the B cells toward the follicular T cell zone before entering the extrafollicular foci (47). Besides antibody production the GC reaction is directly connected to generation of memory cells. These cells are long-lived antigen-specific cells that can be rapidly activated. In contrast to the naïve cells present at the first encounter with an infectious

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agent, the memory cells already have a high affinity antigen receptor and thus respond much more rapidly. The generation of high quality memory is the goal of vaccination, and can when successful even eradicate infectious diseases (48). B cell memory cells resides in several anatomical compartments including the bone marrow, the MZ and the circulation (49, 50). The persistence of the memory triggered by infections can vary dramatically, from a very short period to the generation of memory B cells with a half-life of up to 200 years (51). Several mechanisms have been proposed to account for memory B cell survival: both that the memory cells are dependent on continuous stimulation via their BCR, i.e. that antigen is retained in the body by for instance FDCs (52), and that they are independent of their BCR, by being intrinsically long-lived (53) or dependent on non-specific activation caused by other infections (54). Several non-classical mechanisms can also be involved in B cell responses; TI antigens can generate memory B cells (36, 55), GCs can be formed without T cells (56) and BAFF (B cell activating factor) overexpression leads to production of class switched (auto)antibodies independent of T cells and the GC reaction (57). The relevance for these examples is uncertain. Still they emphasize the potential complexity of immune responses, often not appreciated. 1.2.2 Antibodies (or Immunoglobulins (Igs))

After the GC process, the activated B cell develops into a plasma cell, accompanied by the downregulation of many surface markers, including the BCR. Intracellular changes related to the secretory pathway are also apparent, turning the cell into an antibody-producing factory (58, 59). The antibody molecule is built up by two identical heavy chains and two identical light chains that are held together by disulfide bonds. The structure is similar to the letter “Y”, but with great flexibility. The flexibility facilitates binding and is due to the hinge region. The structure can further be divided into a Fab (fragment, antigen binding) region, that is involved in binding the antigen, and the Fc (fragment, crystallizable) region that acts as a handle for the antibody interacting with other proteins or cells to mediate effector functions. The Fc part is only made up by the heavy chains, while the Fab part is made up by both the heavy and the light chain.

Figure 4. The IgG antibody molecule. The antibody is a glycoprotein, thus carbohydrate chains are post-translationally added to the protein structure. The subclasses differ in this regard, where for example IgE has six potential glycosylation sites on each heavy chain, while IgG has one (60). The glycosylation has implications for the antibody effector function, as the glycan alters the binding affinity of the Fc part to other proteins, like FcRs. Differences in

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the glycosylation pattern of antibodies are apparent in several disease conditions, including in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (61). Antibodies mediate their function through several different mechanisms. Alone the antibody binding can disturb interactions needed for example by a virus trying to invade a cell or neutralize a toxic product made by bacteria. By binding its target the antibody opsonizes, or flags, the antigen and thereby generate a handle that can be used to activate other mechanisms via its Fc part. These include activation of the complement system and activation of cells via binding to FcR. The antibody subclasses have different affinities for FcRs, resulting in different types of activation (62, 63). Although antibodies are mainly considered as molecules involved in activating the immune system against invading pathogens, other functions also exist. The best known example is probably the anti-inflammatory effect mediated by the IVIG (intravenous immunoglobulin) treatment. This treatment is based on the transfer of a high dose of IgG, purified from blood donors, traditionally used for patients with deficiencies in IgG, but also used for patients with autoimmune disease. Several explanations for the anti-inflammatory effect mediated by IVIG have been proposed, including blockade of FcRs and effects on the half-life of autoreactive IgG (64). Interestingly, data suggest that the effect can be attributed to the Fc part of the antibodies (65) and to their glycosylation pattern (66). 1.3 NKT CELLS

There are several smaller and/or less defined lymphocytic populations besides the conventional lymphocytes (B, T and NK cells). Of these, the small Natural Killer T cell (NKT) population has been studied in one of the publications presented in the thesis [paper I]. These unconventional T cells share characteristics of both T and NK cells including shared surface marker expression and functionality, but are very unique in that they respond to lipid and glycolipid antigens. This response is mediated by the usage of TCRs restricted for glycolipid antigens presented in CD1d, an unconventional MHC class I-like molecule. Mice only express CD1d, in contrast to humans where several CD1 molecules exist (CD1a-e). CD1d is very conserved, both structurally and functionally, between mice and humans, shown by the fact that mouse NKT cells can be activated by human CD1d and vice versa (67). CD1d is expressed on several cell populations including, as expected, antigen presenting cells, but noteworthy also on double positive thymocytes, needed for thymic NKT cell selection (68). Interestingly, the splenic MZ B cell population displays an exceptionally high CD1d expression, suggesting that they interact with NKT cells. How and when this occurs is not fully understood. The NKT cell population was first described at the end of the 1980s, more-or-less in parallel as T cells with an unusual TCR usage (69) and as T cells with reactivity against CD1 molecules (70). Mouse studies have demonstrated that the absolute major population of NKT cells are dependent on CD1d expression for its development and are therefore referred to as CD1d-restricted NKT cells. There are also non-CD1d restricted NKT cells, especially in humans, that have a variety of CD1 molecules (71). It is, however, complicated to define these populations as the co-expression of NK and T cells markers is also evident on activated non-NKT cells (72). The CD1d-restricted NKT cell population is divided into type I and type II NKT cells. The type I or invariant

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NKT (iNKT) cell population uses a limited repertoire for its TCR, namely Vα14-Jα18 and mainly Vβ2, 7 or 8 in mice, corresponding to Vα24-Jα18 and Vβ11 in humans. The population is furthermore defined by being vigorously activated by the synthetic antigen alpha-galactosylceramide (αGalCer, originally derived from a marine sponge) that, when presented in CD1d results in iNKT cell proliferation and cytokine production (73). The type II NKT cell population uses a different and, compared to iNKT cells, less limited TCR repertoire, resulting in different ligand recognition including various sulfatide isoforms (74, 75). The NKT cell population can be further divided by their surface phenotype. Especially the expression of CD4 has been addressed, but also NK1.1. These markers define functionally different subpopulations, but also different developmental stages (76, 77). Since no specific surface markers exist to define the NKT cell population the reactivity of its TCR is often used to define them. For this purpose, soluble CD1d molecule in complex with the antigen of choice is used to stain the cells. The invariant population has a very high affinity for the αGalCer:CD1d complex and is used in a panel with other markers to define them. This does not necessarily mean that all iNKT cells have the exact same reactivity. Instead the αGalCer recognition shares certain features of superantigens, although being dependent on CD1d presentation. Similar approaches with soluble CD1d have been used for several other antigens resulting in the identification of type II NKT cells with reactivity against lysophosphatidylcholine (LysoPC) (78) and sulfatides (79). One problem using this approach is that NKT cells rapidly downregulate their TCR when activated (80). NKT cells are able to influence both innate and adaptive immune responses, most easily exemplified by their potent cytokine producing ability. However, direct interaction with IgG antibodies via FcγRs has also been demonstrated (81). The role of NKT cells in autoimmune disease is debated. A protective role is attributed to them in NOD mice, which spontaneously develop autoimmune disease with similarities to autoimmune (type I) diabetes (82). The NOD mice display low levels of NKT cells controlled by several genetic loci linked to both autoimmune diabetes and SLE (83). Patients with both autoimmune diabetes and SLE, as well as with other autoimmune diseases, also have decreased levels of NKT cells in their blood (84-86). The relevance for the level of NKT cells in the blood is complicated, as it does not seem to be a good representation of numbers in different organs, as well as there is a very large inter-individual variation in humans (87). In spontaneous mouse models for SLE, opposing and often contradicting data has been presented in relation to NKT cells. A protective role is suggested in MRLlpr mice in contrast to the (NZBxNZW)F1 mice, where a pathogenic role is suggested, emphasizing the heterogeneity of the disease (88). However, old iNKT cell deficient mice on both C57BL/6 and BALB/c backgrounds develop an SLE-like disease with autoantibody production and nephritis (89). NKT cells are also implicated in the response against pathogens (90) and are potential targets for cancer therapy (91) and in vaccine development (92).

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1.4 MACROPHAGES

Phagocytosis, or the uptake of particles into specialized cellular compartments, was already described more than 100 years ago by the 1908 Nobel laureate Ilya Mechnikov (93). Several innate cell populations are involved in the phagocytic uptake of pathogens and endogenous waste, triggered by e.g. pattern recognition, complement and Fc receptors. Macrophages (Greek: makros ‘large’ and phagein ‘eat’) are together with neutrophils the most potent phagocytes in our body. Neutrophils are mainly connected to pathogen phagocytosis following the migration into inflammatory areas, while macrophages also are involved in non-pathogen-related phagocytosis. Another difference is that resident macrophages are present all over the body at strategic locations, ready to engage in uptake, although also able to migrate into tissues (94). The great heterogeneity of the macrophage population is exemplified by both osteoclasts in bones, and microglia in the central nervous system, being macrophages. Resident macrophage populations are replenished by a subpopulation of monocytes in the blood that migrates into the tissue and differentiates into macrophages (95). Monocytes can also give rise to DCs, although other DC precursor cells are described, possibly more relevant for the in vivo situation (96). Pathogens entering the body will almost immediately encounter resident macrophages (but also DCs and mast cells) that through release of soluble factors such as IL-6 and TNF-α, will activate the immune system. This initial activation is very important for the subsequent activation of the adaptive immune response. Interestingly, the activation of the adaptive cells in turn regulate macrophages, as exemplified by activated T cells producing IFN-γ, originally called macrophage activating factor (97, 98). Thus this is an example of a positive feedback loop including both innate and adaptive cells. The heterogeneity of the macrophage population is further described functionally; biochemical properties and developmental requirement have been used to divide the population into classically activated (M1) and alternatively activated (M2) macrophages. A slightly alternative division, dependent on involvement in host defense, wound healing and immune regulation, respectively, has also been proposed (99). Besides their involvement in combating infections and in the pathology related to autoimmune diseases, attention is given to macrophages in the pathogenesis of atherosclerosis. The atherosclerotic process involves macrophages in the vessel wall that in an uncontrolled fashion take up modified low density lipoprotein (LDL) and cause a local inflammatory milieu (100). Macrophages are also central to granulomas, small nodules involving the formation of multinucleated giant cells by fusion of macrophages, that are apparent in both certain infectious and non-infectious diseases (101). 1.4.1 Scavenger receptors

The scavenger receptor family is a large cluster of loosely related receptors. They are traditionally grouped together dependent on their ability to bind and mediate phagocytosis of modified LDL related to the atherosclerotic process mentioned above. As the name implies the receptor family also shares the characteristics of binding a variety of ligands of both endogenous and foreign origin, therefore sometimes being referred to as molecular flypapers (102, 103). An important involvement in the binding and phagocytosis of bacteria is described for this receptor family, a feature preserved through the evolution (104).

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At least eight classes (A-H) of the scavenger receptor family exist. In two of the papers presented in this thesis, members of the class A scavenger receptors (SR-A and/or macrophage receptor with collagenous structure (MARCO)) with great sequence homology (105) have been studied. SR-A is expressed on most macrophage populations and was determined to be responsible for macrophage adhesion to tissue culture plastic, a phenomenon traditionally used to isolate or remove macrophages from a mixed cell population (106). MARCO has a more limited expression profile, mainly occurring on splenic MZ macrophages, lymph node macrophages in the medullar cords and on peritoneal macrophages. MARCO can furthermore be potently upregulated on other cell populations, including DCs, upon activation (107-109). 1.4.2 SIGN-R1

SIGN-R1 is a mouse pattern recognition receptor belonging to the C-type lectin family. It was identified through cloning experiments looking for homologues for the human DC receptor DC-SIGN (110), a receptor that has implications for HIV infection (111). Mouse SIGN-R1 had, however, already been studied for a long time by the ERTR-9 antibody, an antibody used to define splenic MZ macrophages, for which the target was unknown (112). SIGN-R1 and MARCO expression thus partly overlap but are differently regulated; in contrast to MARCO, SIGN-R1 is downregulated upon activation (113, 114). The receptor is involved in binding different polysaccharide structures and is important for the response to certain blood-borne pathogens, at least partly by mediating a non-classical type of complement activation (115, 116). 1.5 CELL DEATH

Two main modes of cell death have been described: apoptosis and necrosis. Other modes also exist and have recently gained a lot of focus, especially autophagy, but will not be discussed herein (117). Apoptosis, or programmed cell death, is characterized by the activation of intracellular enzymes that starts degrading the cell, leading to shrinkage, bleb formation and changes in the cell membrane. The intracellular degradation prepares the cell to be removed by other cells and also to facilitate the subsequent recycling of the cellular building blocks. The appearance of phosphatidylserine (PS) on the surface (detected by Annexin V binding), activation of key enzymes (caspases) and the degradation of DNA are hallmarks of apoptosis and are used experimentally to define them. The surface appearance of PS is also involved in several of the pathways mediating phagocytic uptake of the dying cell, therefore referred to as an ‘eat-me’ signal (118). Two distinct pathways of apoptosis have been described: the intrinsic and the extrinsic. The first, also called the mitochondrial pathway, is a form that occurs when the cell itself decides that the host would benefit from its removal, e.g. due to damage to its DNA or by decreased function. The extrinsic or death-receptor pathway is, in contrast, related to that other cells engage with the targeted cell and induce the cell death. An important example of this is the Fas–FasL interaction used to remove unwanted B cells produced during the GC reaction (119). Necrosis is a type of usually accidental and non-programmed cell death related to e.g. hypoxia, extreme temperature changes or mechanical force, but also to pathogen invasion. It is characterized by swelling of the cell and disruption of the cell membrane. This fairly dramatic process leads to the release of intracellular components into the

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extracellular compartment. Since these components are usually not found outside cells this is interpreted as something that requires the attention of the immune system, discussed above as DAMPs. Necrosis is easily detected experimentally by techniques taking advantage of the disrupted cell membrane. Most often DNA-binding probes such as propidium iodine, 7AAD or DAPI are used that will only access and thus label the DNA of cells that have a compromised cell membrane. Secondary necrosis refers to the process when an apoptotic cell can no longer retain the integrity of the cell membrane, which bursts and leads to leakage of intracellular components. This process is mainly an in vitro phenomenon, as the apoptotic cells are rapidly taken care of in the in vivo situation, but can also occur in individuals with defects in removing apoptotic cells and potentially also during periods of extensive apoptosis. 1.5.1 Clearance of dying cells

A very high number of cells die in our body each day via apoptosis related to homeostatic processes. Conversely, the other modes of cell death are mainly related to trauma and infections. The generated apoptotic cells arise from several different cell populations that usually have a short life span and that are produced in high numbers. For example about 1011 neutrophils are produced each day (120). These have a lifespan of less then a day, resulting in roughly 100g of apoptotic neutrophils generated each day (based on the suggested average mass of a cell being 1ng). Furthermore, extensive homeostatic apoptosis occurs in the intestine, bone marrow and thymus. A (hypothetical) suggestion speculates that an 80 year old human would in the absence of apoptosis have a 16 km long intestine and 2000 kg of bone marrow (121). Apoptosis is also important for the development of certain organs and can thereby be detected at different set time points in the developing fetus (122). The huge numbers of generated apoptotic cells need to be removed, a process also referred to as clearance or efferocytosis. Studies in the nematode (worm) Caenorhabditis elegans (C. elegans) have been important for the understanding of how this occurs. C. elegans has a very ordered development that can easily be followed, including timed death of traceable cells. A number of genes have been identified in this model system that are involved in the engulfment of apoptotic cells (123). Although differences to the human system are obvious, several similarities exist. Especially in the functional aspect of the system; a dying cell signals what is going on and adjacent cells recognize this and rapidly engulf the dying cell via receptor-mediated mechanisms. No professional phagocytes exist in C. elegans and the dying cells are taken up by neighboring cells. This type of clearance is also evident in the human system (exemplified in (124)), but potent professional phagocytic cells also exist and are strategically located to be able to remove the dying cells. Macrophages are the prototypic population for apoptotic cell clearance, but also e.g. immature dendritic cells actively participate. The clearance is mediated both by direct interaction between receptors and altered structures on the apoptotic cell and also via soluble proteins bridging the dying cell and the phagocytic cell (125). The appearance of PS on the surface is as mentioned responsible for many of these interactions. The involvement of PS in the recognition of apoptotic cells is also seen in C. elegans (126) and thus conserved through evolution for more than 600 million years (127). Besides the direct ‘eat-me’ signals the dying cells also release soluble factors aimed to recruit phagocytic cells. Several ‘find-me’ signals are described, including

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nucleotides (128), lysophosphatidylcholine (LysoPC (129)) and sphingosine-1-phosphate (S1P (130)). 1.6 AUTOIMMUNE DISEASE

In patients with autoimmune diseases the immune system is activated against self tissues. These diseases are in general characterized by the development of high affinity autoreactive B and T cells activated against the self structures, thus related to activation of the adaptive immune system. Innate autoimmune disease is also described and is mainly related to very rare congenital defects in complement regulatory proteins and thereby improper complement activation (131). Autoimmune diseases, from now on only referring to the adaptive set of diseases, can be divided into organ-specific and systemic variants, where the former represents the major group of patients. The organ-specific diseases are characterized by autoreactive attack against autoantigens expressed in certain tissues or cells, such as the acetylcholine receptor in myasthenia gravis patients and the β-cells of the pancreas in type 1 diabetes patients. In systemic autoimmune disease multiple tissues are affected, as the name implies. A narrow definition dictates a relation to the autoreactive targeting ubiquitously occurring antigens such as DNA targeted in SLE patients. There are more than 70 chronic inflammatory diseases that are classified as having autoimmune etiology. Together these affect about 5% of the US population with a clear female bias. The complex effect on the immune system by sex hormones has been implicated in this bias, although this is far from being fully explored (132). These 70 diseases could most probably be divided into several subgroups in which the pathologies are related but the underlying mechanisms could differ. This is supported by studies of animal models where very different types of genetic alteration or other types of stimulation can cause similar pathology, something with clear implication for the treatment of affected patients. Examples from animal models: 1. Accelerated SLE-like disease is apparent in mice lacking Fas, involved in apoptosis

induction of autoreactive lymphocytes (133), and also when TLR7 (an intracellular pattern recognition receptor that can be involved in increasing autoreactive signaling from ingested endogenous chromatin) is overexpressed (134).

2. Rheumatoid arthritis-like disease develops in NOD mice with the introduction of T cell reactivity against the ubiquitous enzyme glucose-6-phosphatase isomerase (GPI) (related to the K/BxN model (135)), and also in mice immunized with cartilage-derived collagen (the collagen-induced arthritis or CIA model (136)).

These examples illustrate that similar pathologies can be caused by many different types of stimuli; supporting that certain pathologies could represent a standard response to different types of mechanisms. Thus a great potential lies in defining mechanistically different subpopulations of patients and accordingly adjusted treatment regimes. In this context a statement by Douglas Hanahan and Robert Weinberg (in relation to cancer) is of relevance: “We foresee cancer research developing into a logical science, where the complexities of the disease, described in the laboratory and clinic, will become understandable in terms of a small number of underlying principles” (137).

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1.6.1 Etiology of autoimmune diseases

The relative involvement of genetic and environmental factors differs both between and within autoimmune patient groups. Although having close relatives with autoimmune disease is a clear susceptibility factor, an unexpectedly low percentage of identical twins share their sibling’s autoimmune disease, even though they share the exact genetic code. This suggests an important role for environmental triggers in disease development, but could also involve functional (i.e. epigenetic) modifications to the genetic code explaining the disconcordance (138). However, epigenetic modifications could in turn be related to environmental triggers (139). Some human monogenetic autoimmune diseases exist, although they are very rare. Examples are defects in the Aire, Fas and Foxp3 genes (140). Autoimmune diseases generally involve defects in multiple genes, related to disease in a complex network. This complexity results in the need for enormous amounts of patients and controls to identify affected genes, with implications for what can actually be identified in genetic screens (141). Environmental factors such as infections could affect autoreactive cells in several ways, both dependent and independent of antigen specificity. Molecular mimicry, related to antigen-dependency, involves the spread of reactivity against a pathogen to self-antigens that have similar structures (142). Although several examples of this have been demonstrated, the relevance has been questioned (143). Pathogens could also push the immune system towards autoreactivity by the induction of inflammation and tissue damage, affecting the general activation of the immune system and potentially the access to autoantigens. Environmental factors also include the exposure to non-infectious agents and autoreactivity has for example been connected to exposure to mineral oils (144) and cigarette smoke (145). Research on autoimmune diseases mainly focuses on factors related to the autoreactive cells, e.g. development, regulation and effector mechanisms. However, the severity of the disease is also regulated by the targeted organ (146). This aspect suggests that genes involved in for example how the kidney responds to an autoreactive attack could dictate how severe pathology will develop. One aspect of this is that the healing of the organ is important since the autoreactive tissue-destruction could be fueled by the release of autoantigens and DAMPs. A hypothetical example is the anti-inflammatory cytokine TGF-β that, besides being related to anti-inflammatory FoxP3+ regulatory T cells, is also important for wound healing (independently of T cells) (147, 148). Thus autoimmune pathology related to TGF-β deficiency or deregulation could theoretically be related to insufficient repair of the damaged tissue. The strongest genetic factor linked to autoimmune disease is by far related to MHC variants (or haplotypes), which affects which type of peptides are presented. This underlies the importance of APC–T cell interactions and the presence of specific antigens preferentially presented in these MHC haplotypes (149).

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1.6.2 Tolerance

The term horror autotoxicus was coined more than 100 years ago by the German 1908 Nobel laureate Paul Ehrlich (sharing the prize with Ilya Mechnikov). The term refers to the body’s unwillingness to attack itself (150), but has also been used to describe autoreactivity in general. This unwillingness can be related to the more modern term tolerance. In this context tolerance is describing the process whereby the immune system tolerates the body’s own tissue antigens. Tolerance is thus broken in patients with autoimmune disease. It is mediated by self/non-self discrimination, where these different categories have unique properties that distinguish them. Tolerance was originally proposed as being related to what the body experiences early in life (tolerance-inducing) and later (immune system activating), but has since been greatly extended. The current view of self/non-self discrimination includes the involvement of specific recognition of PAMPs and DAMPs, as well as a role for the affected tissues in dictating the type of response (151). The tolerance process is commonly divided into central and peripheral processes. Central tolerance is connected to the removal of autoreactive B and T cells during development (the negative selection process). This process is rather effective but autoreactive clones can still readily be found in the naïve repertoire of healthy individuals (152). This is functionally proven in animal models for rheumatoid arthritis (the CIA model (136)), and multiple sclerosis (the experimental autoimmune encephalomyelitis (EAE) model (153)), in which disease is triggered by active immunization with autoantigens together with a strong adjuvant. Thus autoreactive clones are present in the naïve cell population and can be activated. Another important observation regarding central tolerance derives from an animal model for the autoimmune skin disease pemphigus vulgaris. In this disease pathology is related to the autoreactive attack against Desmoglein isoforms found in the skin. Interestingly, this pathology can be triggered by the transfer of B and T cells from Desmoglein 3-deficient mice to mice that express the protein, supporting the important role for central tolerance (154). In healthy individuals naïve autoreactive clones are kept under control by several peripheral tolerance processes. One mechanism in this regard relates to the need for a co-operative effort for the strong activation of B and T cells, including several cell types and soluble mediators. Thus an autoreactive B cells can not cause disease in the absence of an autoreactive T cell, which probably needs the right stimuli from a DC that in turn needs to interpret the situation as being worthy of activation. In the absence of these co-stimulatory signals the autoreactive cell is silenced or removed. This is a hypothetical example which highlights that autoreactive activation can be limited at many levels, as well as the complex etiology of this category of diseases. In this regard is it also important to distinguish between autoreactivity and autoimmune disease. Autoreactivity, probably occurring every time we experience an infection, does not necessarily need to lead to autoimmune disease development as long as the activation is limited. Peripheral tolerance is not only related to the (passive) absence of signals, but active processes are also involved; this is perhaps best exemplified by regulatory T cells that when activated dampen the immune system (155, 156).

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1.6.3 Systemic lupus erythematosus (SLE)

SLE is the prototypic systemic autoimmune disease that can affect most parts of the body. The disease affects a comparably small group of the patients with autoimmune diseases, with a prevalence of 0.04-0.2% in the US, equal to around 250 000 individuals (157). SLE is the autoimmune disease with the second highest female bias, ~90%, which is only topped by Sjögren´s syndrome (132). SLE is characterized by flares of disease followed by periods of remission. The production of autoantibodies reactive to ubiquitously expressed antigens such as DNA and chromatin-related proteins are linked to the disease pathogenesis and is used for diagnosis. The autoantibodies form immune complexes with cellular debris and end up in e.g. the kidney filtration network, capillary beds and joints. Inflammation is subsequently triggered via complement activation and by engagement of FcRs expressed on infiltrating effector cells. The direct involvement of organ-specific antibodies and autoreactive T cells in the pathology is also suggested (158, 159). Several susceptibility genes are identified; e.g. regulating the development (160) and activation (161) of autoreactive lymphocytes, as well as genes involved in the clearance of apoptotic cells (162, 163). In addition, environmental factors including exposure to sunlight and certain drugs can trigger flares (164). Several spontaneous animal models for SLE-like disease exist, including the MRLlpr and (NZBxNZW)F1 mouse models (165). 1.6.4 Cell death and SLE

As mentioned earlier, although an immense number of cells die via apoptosis each day in our body, it is very hard to find them. This is because the system removing them is rapid and efficient. However, in patients with SLE, apoptotic cells can be found in different tissues and in the circulation (163, 166). Although many factors are clearly involved in the development of SLE, several observations suggest a direct connection of the clearance defect to the development of the disease: 1. Defects in genes that are involved in apoptotic cell clearance are linked to disease

development; e.g. C1q (167), MER (168), MFG-E8 (169) and PSR (although debated (170, 171)).

2. Blockade of the clearance process leads to autoantibody production; e.g. by an antibody blocking the PS receptor TIM-4 (172).

3. Increasing the amount of apoptotic cells leads to disease manifestations and/or autoantibody production, shown by the intravenous injection of apoptotic cells from several different sources (thymocytes (173, 174), fetal liver cells (175) and splenocytes (176)), as well as by apoptosis induction in the skin by UVB irradiation (177).

Apoptosis is traditionally related to tolerance and necrosis is related to inflammation, as supported by several observations (178, 179), making the apoptosis-autoimmunity connection conceptually complicated. Nonetheless, despite the generation of an enormous amount of apoptotic cells each day we are not immunosuppressed. Additionally, repeated injection of apoptotic cells into mice triggers a transient SLE-like disease, including autoantibody production and kidney manifestation, which does not occur when injecting necrotic cells (173). Thus certain features of the apoptotic material

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can be linked to autoimmune activation. In this regard it might be relevant that intracellular enzymes activated during the apoptotic process produce modified self-antigens (180, 181). In other words, new structures (neo-epitopes) are created that could potentially bypass the effects of the central tolerance process (assuming that the neo-epitopes are not present during the negative selection process of B and T cells). Of significance, these potential autoantigens are also found grouped in cell surface structures of the apoptotic cell (182). An often described explanation for the autoreactivity linked to defects in taking care of apoptotic cells relates to the induction of secondary necrosis, sharing the modified antigens of apoptotic cells and the proinflammatory nature of necrotic cells. Although most probably true, i.e. involved in the etiology of the disease, other features of the clearance defect can be of importance. Repeated intravenous injections of apoptotic cells in mice can be used as a model for the increased levels of circulating apoptotic cells in SLE patients and trigger a transient autoantibody response, as described above. This response is evident although the cells are rapidly removed from the circulation. In line with this observation both IgM and IgG antibodies are triggered by the injection of apoptotic cells, binding directly to structures found on the apoptotic cells (183). A role for this opsonization of the apoptotic cell in relation to disease development is plausible, as the immune system will interpret the dying cell differently. The subclass of the opsonizing antibody most probably also has a role for the altered recognition (184). It is for example known that IgM binding and subsequent complement activation mediates non-inflammatory clearance of the apoptotic cell (185). Conversely, IgG opsonization could via FcγR engagement alter the involved cells and uptake pathway of the apoptotic material, potentially into a more proinflammatory compartment. An involvement of intracellular TLR7 and 9, able to bind endogenous DNA and RNA and drive autoantibody responses (134, 186), could be related to FcγR (IgG-mediated) uptake. 1.6.5 Rheumatoid Arthritis (RA)

RA is a chronic inflammatory joint disease that affects 0.5-1% of the adult population worldwide (187). It is characterized by joint infiltration by immune cells (B cells, macrophages, neutrophils and T cells), as well as the deposition of antibodies and complement. Furthermore, local activation of platelets has also recently been connected to the joint inflammation, underlining the complexity of the pathology (188). If left untreated the inflammation leads to destruction of cartilage and bone, functional disability and increased morbidity (189). Cytokine production by both infiltrating cells and by local fibroblasts has been linked to the disease development and is targeted by recently developed drugs. Anti-TNF-α antibody treatment has in particular proven successful in a substantial subpopulation of RA patients. Several other cytokines have also been implicated and suggested as therapeutic targets, including IL-1, IL-6, IL-15 and IL-18 (187). In addition, B cell depletion using targeting antibodies is beneficial, proving the involvement of B cells in RA pathology (190). Interestingly, GC-like structures containing B cells producing disease related autoantibodies, are detected in the inflamed joint (191). These types of ectopic lymphoid structures are not specific for RA and are instead reported in several chronically inflammatory conditions (192). Several different autoantibodies are detectable in RA patients. The most clinically important reactivities are the rheumatoid factor (RF, binding the Fc part of antibodies) and antibodies binding citrullinated (a post-translationally modified version of the

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amino acid arginine) proteins (193). A conceptually interesting aspect of B cells that bind antibodies (i.e. with the RF reactivity) is that antibodies that activate and are taken up by the B cells, via the RF BCR, might bind an unrelated antigen. The ‘hitch-hiking’ antigen could then activate the B cell and also be presented via MHC, thereby bypassing the need for BCR specificity of the antigen, an important aspect for the maintenance of tolerance. The RF B cells could thus be described as a B cell with no direct control of its BCR-mediated antigen uptake and might thereby become involved in driving autoreactivity against other antigens, as demonstrated in animal models (194, 195). Several animal models exist in which RA-like disease develops. These include the earlier mentioned CIA model, where joint inflammation is triggered by immunization with cartilage-derived collagen, as well as models where the administration of joint-specific antibodies causes pathology. This can be accomplished both with collagen-specific antibodies (196) or by serum transfer from the K/BxN mice that spontaneously develop joint inflammation (135).

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2 THE PRESENT INVESTIGATION

Aim The work presented in this thesis aimed to study how innate mechanisms influence autoreactive B cells and their antibodies. Specific aims: Paper I. To investigate the effect of increased levels of circulating apoptotic cells on

NKT cells and how this influences the activation of autoreactive B cells. Paper II. To study the involvement of class A scavenger receptors (MARCO and

SR-A) in the clearance of apoptotic cells and in regulation of autoreactive B cell activation.

Paper III. To study the role and effect of autoantibodies binding the scavenger receptor

MARCO Paper IV. To identify the involvement of cells and receptors in the anti-inflammatory

effect mediated by IVIG treatment.

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2.1 METHODOLOGY

Detailed descriptions of the methods used in the included papers (I-IV) are found in the respective ‘Materials and methods sections. Mice [I-IV] Knockout (CD1d, CD19, Jα18, [I], MARCO, SR-A [II-III], FcγRIIb [II and IV], MyD88 [III], CD4, JHD, RAG and SIGN-R1 [IV]), transgenic (Vα14 [I]) and CD45.1 congenic [I] mice on the C57BL/6 background were used as well as serum samples from (NZBxNZW)F1 [II] and K/BxN [IV] mice strains. Patient material [II] Serum samples from SLE patients were used with their consent. Enzyme Linked Immunosorbent Assay (ELISA) [I-III] Quantitative antibody and absorbance based method, here used to measure antibody and cytokine levels in serum samples and cell culture supernatants. Histology [I-IV] Sectioned tissue samples (spleen [I-IV], kidney [I] and joints [IV]) labeled either with fluorescent-conjugated antibodies [I-IV] or hematoxylin and eosin (H&E) [I, IV], followed by examination with fluorescence or light-based microscopy. Here used to detect: (in spleen) germinal center formation [I], the localization of labeled injected apoptotic cells [II], binding of injected labeled antibodies [III] and cellular localization and surface expression [III-IV]; (in kidney) immune complex deposition and glumeruli appearance [I]; (in joints) cellular infiltration and damage related to inflammation [IV]. TUNEL staining [III] Enzymatic method that label cells with fragmented DNA, a characteristic of apoptotic cells. Anti-nuclear antibody test (ANA) [II] Cell-based assay used together with fluorescent-conjugated antibodies and fluorescence microscopy to detect autoantibodies binding cellular components. Binding studies [II, IV] CHO and RAW cell lines transfected with MARCO, SR-A [II], SIGN-RI, hDC-SIGN and hFcγRIIb [IV] or peritoneal macrophages from WT, FcγR deficient and SIGN-RI deficient mice [IV], were used to detect binding of IVIG [IV] or apoptotic cells as well as detection of autoantibodies against MARCO and SR-A [II]. Ex vivo stimulation of splenocytes [I, III] Cells isolated from mice injected with apoptotic cells [I] or an anti-MARCO antibody [III] cultured with or with out stimulation (LPS, αGalCer and ConA). After different time points supernatants were collected and assayed for released cytokines and antibodies by ELISA [I, III] and for survival of cells as measured by FACS [I].

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Hybridoma cultures and antibody purification [III] Hybridoma cell line ED31 (anti-MARCO) was cultured and antibodies were purified from the supernatant using protein G affinity purification. Flow cytometry or FACS (fluorescence activated cell sorter) [I, III] Single cell suspensions of spleen [I, III], inguinal lymph nodes and Peyer´s patches [I] were prepared and following FcR block surface antigens were labeled with fluorescent- conjugated antibodies. Samples were analyzed using either a FACSaria or a FACScalibur using the default software or FlowJo software. Labeling of iNKT cells for FACS analysis [I] CD1d-DimerX was loaded with an excess of sonicated αGalCer and further labeled with a fluorescent antibody. This fluorescent CD1d:αGalCer complex was used together with other antibodies to specifically label iNKT. Intracellular cytokine FACS [I] Isolated splenocytes from mice injected with apoptotic cells or control were stimulated ex vivo for 5h with non-specific activators PMA and Ionomycin and Golgi stop (Brefeldin A). Following standard labeling of surface antigens were cells fixed, permeabilized and intracellular IFN-γ was labeled with fluorescent-conjugated antibodies followed by analysis using a FACSaria. In vivo B cell activation [I-III] Autoreactive response following four injections of apoptotic cells [I-III]: Weekly intravenous injection of syngenic apoptotic thymocytes followed by ELISA measurement of serum samples for autoantibody response [I-III], ex vivo stimulation of isolated splenocytes and FACS analysis of isolated spleen and lymph node cells [I, III]. T-Independent type 2 activation [I, II]: Intravenous injection of TNP-ficoll followed by ELISA measurement of serum samples for TNP-specific antibody response. T-dependent activation [I]: Intraperitoneal injection of NP-OVA in aluminum hydroxide adjuvant followed by ELISA measurement of serum samples for NP-specific antibody response and FACS analysis of germinal center formation. Adoptive cell transfer [I, IV] B cells or NKT cells purified by magnetic antibody-coated MACS beads [I] or total splenocytes [IV] were transferred intravenously to recipient mice. Mixed bone marrow chimeras [I] Irradiated mice received intravenous injection of isolated femoral bone marrow cells from WT (CD45.1 congenic) and CD1d deficient mice (CD45.2) and received antibiotics for two weeks. After seven weeks the haematopoietic cells in the mice derive from the transferred bone marrow. Mice were included in experiments and B cell activation was analyzed using FACS taking advantage of the differential CD45 allotype expression. The K/BxN model [IV] Serum transfer from the K/BxN mice to recipient mice results in rheumatoid arthritis-like joint inflammation. The inflammation was measured by scoring the swelling of paws and by histological examination of joint tissue sections. The role of different cell

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populations and receptors were here tested for their role in the anti-inflammatory effect mediated by IVIG using knockout mice strains and blocking antibodies injected intravenously. In vivo affect of anti-MARCO autoantibodies [II, III] Purified anti-MARCO antibodies were injected intravenously and the effects on the B cell response to co-injected TNP-ficoll [II] or apoptotic cells [III] were measured in serum by ELISA. Statistical analysis For comparing two unpaired samples; (non-parametric) Mann Whitney U test [I-III] and (parametric) T-test [IV]. For comparing two paired samples; (non-parametric) Wilcoxon matched pairs test [I]. For comparing multiple samples; (non-parametric) Kruskal-Wallis test with Dunns post-hoc test [I] and (parametric) ANOVA with Tukey post-hoc test [IV]. p<0.05 was considered significant using Statistica or GraphPad software. Ethical considerations These studies were conducted according to the regulations related to handling of laboratory animals and patient material and were approved by local ethical committees.

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2.2 RESULTS AND DISCUSSION

2.2.1 Invariant NKT cells limit activation of autoreactive CD1d positive B cells.

The process of removing apoptotic cells is an often overlooked process in biological research, even though a very large number of cells die in the body every day. Although apoptotic cell death is generally related to tolerance induction, increased levels of apoptotic cells are found in the circulation of SLE patients (163). This autoimmune disease is characterized by autoreactive B cells producing autoantibodies binding ubiquitously occurring antigens (e.g. DNA), subsequently becoming involved in tissue damage, e.g. to the kidneys. It is known that SLE patients have decreased levels of iNKT cells in the blood (84). Since iNKT cells recognize lipid-derivatives (presented via CD1d) and that several modifications occur in the lipid cell membrane of dying cells, we set out to investigate if iNKT cells could regulate B cell activation in response to increased levels of apoptotic cells i.e. if iNKT cells could be involved in the tolerance induction normally attributed to apoptotic cell death. B cells express high levels of CD1d, with exceptional levels on the splenic MZ B cell population. The role for this expression is, however, unclear. To address the role of iNKT cells in relation to autoreactive B cell activation, WT and two different iNKT cell-deficient mice (CD1d-/- and Jα18-/-) were injected with apoptotic cells. These mice strains differ in that CD1d-/- mice, by CD1d being a molecule essential for the development of both type 1/invariant and type 2 NKT cells, while Jα18-/- mice lack the Jα-Tcr segment only essential for the invariant (i)NKT cell population. The apoptotic cell injection-model triggers autoantibody production and subsequent kidney involvement by increasing the levels of circulating apoptotic cells, thus with similarities to SLE patients. Interestingly both iNKT cell-deficient mice displayed significantly increased levels of autoantibodies and kidney involvement compared to WT mice (Fig. 5). Old iNKT cell-deficient Jα18-/- mice (on both C57BL/6 and BALB/c background) develop a similar phenotype (89). Thus their predisposed disease can be triggered by this stimulus, suggesting the relevance for the model in relation to iNKT cell deficiency and autoreactive B cell activation.

Figure 5. Increased autoantibody response in NKT deficient mice (Jα18-/- and CD1d-/-) following four injections (day 0, 7, 14 and 21) of apoptotic cells.

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Several (potentially overlapping) mechanisms could explain the role for the iNKT cell population in this phenotype, including; A role in the development or selection of autoreactive B cells. An effect independent of interaction with B cells, for example by cytokine

production related to interacting with DCs or other CD1d expressing cells. A mechanism dependent on direct (cognate) iNKT – B cell interaction.

Performing several different types of experiments showed no obvious effect by iNKT cell-deficiency on B cell development or on general B cell activation using other standard T cell-dependent and -independent antigens. To address if a direct iNKT- B cell interaction was involved, experiments with adoptive cell transfers and mixed bone marrow chimeric mice were performed. These techniques allowed for the in vivo comparison of CD1d+/+ and CD1d-/- B cells in mice having iNKT cells, thereby directly assessing the iNKT–B cell interaction (mediated via CD1d). These experiments identified the importance of B cell expression of CD1d and thereby the involvement of a cognate interaction in the phenotype. The importance of the CD1d expression to limit autoreactive activation promoted us to investigate how CD1d expression on B cells was affected in the apoptotic cell injection-model. Interestingly, we determined that activated B cells downregulate CD1d as they enter germinal centers, and thus the cognate NKT-B cell interaction must take place before this event. This observation is also supported in the human system, where low CD1d expression in GCs has been suggested using immunohistochemistry (197). Additionally, iNKT cell-deficient mice had more GC B cells following injection of apoptotic cells, supporting the notion that the NKT cell-dependent tolerance checkpoint exist before GC entry. Increased GC entry was not a general feature of B cells in iNKT cell-deficient mice and seemed to specifically relate to autoreactive activation. Significantly, the increased autoreactive phenotype in mice lacking iNKT cells could be dampened by increasing the levels of iNKT cells in the mice by adoptive transfer. The effect of a single transfer of iNKT cells to Jα18-/- mice significantly limited both autoantibody production and levels of GC B cells in recipient mice injected with apoptotic cells (Fig. 6). This identifies a novel peripheral tolerance checkpoint for B cells relevant for autoimmune disease and a potential therapeutic target in patients with B cell-driven autoimmune disease.

Figure 6. Splenic GC B cells following four injections of apoptotic cells in WT and iNKT cell-deficient Jα18-/- mice, pretreated or not whit the transfer of iNKT.

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Two studies have indicated that iNKT cell activation can boost antigen-specific B cell activation using particles containing both B cell antigen and the potent iNKT cell activator αGalCer (80, 198). In these studies the authors present evidence for that the mechanism is related to cognate iNKT–B cell interaction following antigen-specific B cell uptake of the particles. These studies support that activated B cells interact with NKT cells and taken together these studies also illustrate that the outcome of the interaction is context-dependent, most probably related to ligand presented by CD1d. Thus, activating with αGalCer and dampening in response to apoptotic cells. The need for B cell CD1d-expression for an iNKT cell to be able to limit autoreactive B cell activation in this model indicates that the CD1d presented ligand/s could be responsible for the induction of tolerance. Whether this/these antigen/s are derived from the apoptotic cells is not known. An alternative explanation could be that B cells present endogenous tolerogenic ligands secondary to detecting apoptosis in the vicinity, or that the iNKT cell is influenced by other factors to interpret standard ligands as being tolerogenic. 2.2.2 Class A scavenger receptors regulate tolerance against apoptotic cells,

and autoantibodies against these receptors are predictive of systemic

lupus.

Several receptors are known to mediate binding and uptake/clearance of apoptotic cells. This study addressed how apoptotic cells are cleared from the circulation. To identify involved cell populations labeled apoptotic cells were injected i.v and organs were immunohistologically examined. Thirty minutes after the injection apoptotic cells were readily apparent in the splenic MZ area. Some apoptotic cells could also be found in the liver that together with the spleen are the major organs for the removal of unwanted blood-borne particles. However, the bulk of apoptotic cells under the used experimental conditions were located in the MZ. In this regard is it also probable that alveolar macrophages could engage in the apoptotic cell clearance, something we did not address. Further co-staining identified that the apoptotic cells present in the MZ were bound to the MZ macrophage population. The MZ macrophages are strategically located to scan the blood for endogenous and foreign particles. These macrophages express two members of the Class A scavenger receptor family, SR-A and MARCO, mediating phagocytosis of several endogenous and exogenous factors, including bacterial components and modified LDL (103). It was previously reported that SR-A could mediate binding to apoptotic cells (199). To address if MARCO also could be involved were binding studies with transfected cell lines performed. These studies confirmed the previous studies of SR-A and identified that MARCO could also mediate binding to apoptotic cells. As defects in clearing apoptotic cells are connected to the development of SLE, we tested if mice deficient in these receptors had an altered response to injected apoptotic cells. No obvious clearance defect was recorded in mice deficient for both these receptors (double-knockout, DKO), by examining how fast injected cells were removed from the blood. This demonstrates the redundancy in the apoptotic cell clearance system. However, DKO mice had an increased autoreactive B cell response following injection of apoptotic cells. A possible explanation for this increased response, despite

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that the blood clearance was not affected, could relate to an altered local clearance in the MZ. It is also possible that intracellular signaling in cells binding and phagocytosing the apoptotic cell is altered if they lack MARCO and SR-A expression. In addition, other phagocytic cells more likely to activate the immune system might become involved in the clearance process. The class A scavenger receptors have, like several other innate pattern recognition proteins, a collagenous domain. This group of proteins, also including e.g. C1q and surfactant proteins (SP) A and D, have collectively been called defense collagens (200, 201). Since C1q is identified as an autoantigen in SLE patients (202), we wanted to test if this could also be true for scavenger receptors. Interestingly, antibodies binding MARCO (and SR-A) were demonstrated in two mice strains that develop spontaneous lupus-like disease as well as in SLE patients. In the (NZBxNZW)F1 mice these autoantibodies were evident before onset of disease, suggesting that they could be involved in disease development. Figure 7. Anti-DNA and anti-MARCO autoantibodies in (NZBxNZW)F1 mice that spontaneously develop SLE-like disease. To functionally address the potential role of these autoantibodies an antibody binding MARCO was injected and determined to influence B cells (that do not express the receptor) to respond more strongly to co-injected TNP-ficoll (a TI-2 antigen). This supports the notion that these autoantibodies could affect B cell activation and potentially have a role in disease development. MARCO is localized close to the Bxs2 susceptibility locus that is linked to autoantibody production (anti-DNA and ANA) in BXSB mice that develop spontaneous SLE-like disease (203). A role for MARCO in apoptotic cell clearance has since our publication been supported by others, and decreased MARCO expression has been suggested to be involved in the development of lupus-like disease in the BXSB mouse strain (204). Interestingly, in that study it was suggested that MARCO-mediated uptake of apoptotic cells is independent of serum factors bridging the interaction, a finding corroborated in our study (although not included in the final paper). This suggests that MARCO binds directly to the cell membrane of the apoptotic cell, potentially to PS or oxidized phospholipids (183). Just before the publication of our study Miyake et al published data demonstrating the importance of macrophages in the MZ (MZ macrophages and the CD169 expressing metallophilic macrophages) for the tolerance-inducing effect related to the apoptotic cell. With relevance to our study, these authors determined a reduced clearance rate in the spleen of injected apoptotic cells in MZ-depleted mice, leading to the accumulation of the apoptotic cells in the MZ area (205). However, direct

30

comparison to our data is complicated as they depleted the cells using a system with controlled diphtheria toxin receptor expression and diphtheria toxin injection. Thus besides also affecting the CD169+ macrophages, this approach generates cell death in the area that could affect the later phenotype. 2.2.3 MARCO engagement triggers an inflammatory response with implication

for autoreactive B cell activation.

In paper II we speculated that the anti-MARCO autoantibodies could affect disease development by blocking the receptor and thereby its phagocytic ability, but also by triggering the release of proinflammatory soluble factors. Of relevance for this IL-12 release was detected from MARCO+ macrophages stimulated in vitro with an anti-MARCO antibody (206). In this as yet unpublished study we have further addressed the potential role of anti-MARCO autoantibodies in relation to disease with a focus on autoimmune B cell activation. The i.v injection of a monoclonal antibody binding MARCO was followed by histological examination as well as by FACS analysis, ex vivo stimulation of isolated cells and by co-injections with apoptotic cells. Since we have previously used injections of apoptotic cells to address autoimmune B cell activation we performed similar experiments with or without the simultaneous injection of an anti-MARCO antibody. Similarly to the TNP-ficoll response the response to injected apoptotic cells was increased by anti-MARCO co-injection, with a significantly increased IgG anti-DNA response at the peak of the response. However, since MARCO mediates binding to apoptotic cells it is complicated to directly address whether this increased anti-DNA response is related to the blockade of the receptor or to released proinflammatory factors. In the case of the TNP-ficoll response the direct role for blockade of MARCO is less evident, especially since depletion of the MZ macrophage population does not affect the TNP-ficoll antibody response (207) and since MARCO-deficient mice have a decreased response to injected pneumococcal polysaccharides (also a TI-2 antigen). This suggests that the increased response to TNP-ficoll in our previous study could not be solely related to the blockade of the MARCO receptor. Further evidence for that immunomodulating factors are released following MARCO engagement was detected on lymphocytes following antibody injection. Several phenotypical changes were recorded, including a rapid and dramatic decreased surface expression of CD21 on splenic and circulating B cells, a phenotype also observed in SLE patients (208). An ex vivo assay was established to address if the increased B cell activation following anti-MARCO injection could involve the influence of factors independent of blockade of the receptor. This was based on the isolation of splenocytes from anti-MARCO injected mice and subsequent stimulation in a cell culture setting. Interestingly, the data indicated that cells from anti-MARCO injected mice displayed stronger proinflammatory cytokine release and autoantibody production when stimulated ex vivo, showing that MARCO engagement could prime lymphocytes for proinflammatory activation. Several cytokines were measured in serum samples following the antibody injection in an effort to identify involved soluble mediators, including IL-6, IL-23, BAFF and TNF-α. Interestingly, a transient peak of TNF-α was recorded in the serum 1h following the injection. TNF-α is a cytokine related to the acute phase response from

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macrophages and was thus a candidate factor (209). Moreover, increased levels of this proinflammatory cytokine are typical of SLE patients (210). This induced proinflammatory milieu could potentially be involved in explaining the increased antibody response seen here and in the previous paper [II, III], although TNF-α deficient mice mount a normal response to TNP-ficoll (211). It is nonetheless likely that several factors are released locally following the engagement of MARCO that could be difficult to measure systemically. Targeting cells with antibodies are used in the clinic to deplete cells. A concern related to the described phenotypes is that the MZ macrophages are possibly depleted by the anti-MARCO antibody injection. This cell death could then potentially trigger an inflammatory response and also affect the uptake of injected antigens. However, no evidence for cell death of the MARCO+ macrophage, or any other cell population was determined using several different techniques. Rapid disappearance of CD21 (and CD62L) as well as increased binding of Annexin V, all of which we see in our model, has been connected to signaling via the purinergic P2X7 receptor (212, 213). This receptor binds extracellular ATP and is a member of a large family of ligand-gated ion channels (214). Since extracellular ATP levels are normally kept at a minimum, signaling via the receptor is interpreted as a pro-inflammatory trigger. The receptor is expressed on hematopoetic cells and is interesting in relation to autoimmune disease as mice deficient for the receptor are protected from joint inflammation in a model of arthritis (215). Furthermore, the P2x7

gene is located close to the human SLE susceptibility locus SLEB4, and it is known that polymorphisms exist that increase the signaling from the receptor and thereby increase the proinflammatory effect of ATP (216). As we have demonstrated that MARCO binds apoptotic cells, a process often related to tolerance induction, proinflammatory signaling by an anti-MARCO antibody presented herein is somewhat unexpected. However, MARCO also binds bacterial components such as LPS. Thus it is evident that the outcome of the binding by this kind of broadly binding receptors is related to the combined signal mediated by all involved interactions. We conclude that injected antibodies binding MARCO can directly cause several effects that mimic what occurs in SLE patients, including the release of pro-inflammatory cytokines and an increased activation of autoreactive B cells.

Figure 8. Suggested model were (A) engagement of the MARCO receptor on positive cells lead to (B) the release of factors that (C) affect cells in a gradient-like way, i.e. more effect on cells located proximal to the MARCO+ cell compared to more distally located cells. The effects described includes downregulation of CD21, increased binding of Annexin V and increased pro-inflammatory cytokine production from stimulated splenic cells as well as increased autoantibody production ex vivo (with LPS stimulation) and in vivo (with injections of apoptotic cells).

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2.2.4 Identification of a receptor required for the anti-inflammatory activity of

IVIG.

In this study we investigated requirements for the anti-inflammatory affect of IVIG (high dose intravenous immunoglobulins) in the K/BxN serum transfer model. The K/BxN model induce a joint inflammation mainly mediated by the transfer of antibodies that, by engaging activating FcγRs on joint-infiltrating cells, cause tissue pathology (217). Studies in this model system have shown a protective roll (limitation of joint inflammation) for the IVIG treatment involving induction of the inhibitory FcγRIIb on infiltrating effector macrophages (218). This effect is not directly mediated by an IVIG interaction with the effector macrophages; instead it involves a central regulatory cell interacting with the IVIG preparation. Furthermore, only a fraction of the IVIG preparation mediates this effect, directly related to the glycosylation pattern of the involved IgG molecules. Specifically, the active fraction has a terminal α2,6 sialylation of the N-linked glycan attached at asparagine (Asn) 297 located in the Fc domain of the molecule (64, 219). A proposed model explaining this suggests that these IgG species are formed during steady-state conditions and are thereby involved in maintaining an anti-inflammatory state. During activation of the immune system this changes and antibodies lacking the terminal α2,6 sialylation are instead produced, allowing for a stronger response (66). Using several approaches to identify cells and receptors mediating the IVIG effect in the K/BxN model, an important role for the SIGN-R1 receptor expressed on MZ macrophages and for the spleen was identified. Furthermore, SIGN-R1 was found to directly bind to the active form (α2,6 sialylated) of IVIG. A transfer experiment with splenocytes from IVIG injected mice also supported the proposed model of a central (SIGN-R1+) regulatory cell population affecting the peripheral diseases-causing effector population.

Figure 9. Showing that SIGN-R1 deficient mice, in contrast to WT (C57BL/6) mice, are not protected from K/BxN-mediated joint inflammation by the active α2,6 Fc IVIG preparation. [Copyright (2008) National Academy of Sciences, U.S.A.]

The human homologue for the identified IVIG receptor is DC-SIGN, which was further demonstrated to bind the active α2,6 Fc IVIG preparation. DC-SIGN is not specifically expressed on splenic macrophages, but is instead a DC marker. Together, the data thus suggests that the anti-inflammatory effect by IVIG involves DCs and DC-SIGN in the human setting. This is interesting as the human immunodeficiency virus (HIV) interacts with DC-SIGN (220). Thus the virus might use an inherent anti-inflammatory mechanism operational in the host, normally involved in fine-tuning of the immune system, to escape recognition.

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2.3 FINAL REFLECTIONS AND FUTURE PERSPECTIVES

Three major findings are presented in this thesis: That NKT cells are ‘guardians of the autoreactive GC’, i.e. limit the GC entry of

certain autoreactive B cells. That MARCO is an autoantigen with a potential role in disease development. The complex role of the splenic MZ macrophage that combine its strategic location

with the ability to both activate and dampen the immune system, as well as being involved in the removal of apoptotic cells.

The experiments in paper I identified iNKT cells as promising therapeutic targets in SLE patients. The observation that increasing the iNKT cell numbers in deficient mice could significantly dampen the autoreactive response following injections of apoptotic cells is central. Several approaches could be used to expand the iNKT population in patients, including the injection of αGalCer or αGalCer-loaded DC (91). However, this will result in potent cytokine production and potential exacerbation of the disease (221). A better approach would be to identify tolerance-inducing iNKT cell ligands presented when apoptotic cells are injected and use these in therapy. The anti-MARCO autoantibodies (found in paper II and tested in paper III) are mechanistically interesting in regard to disease development. However, they could also potentially be used as a diagnostic tool. These autoantibodies arise before disease onset in the (NZBxNZW)F1 mice strain and thus could be diagnostically important. Since scavenger receptors have been implicated in the development of atherosclerosis (222), the anti-MARCO autoantibodies are also potentially interesting in relation to the increased incidence of atherosclerosis in SLE patients (223). We are currently addressing this in a relevant SLE cohort. The multiple roles for the MZ macrophage population (described in papers II-IV) are fascinating. However, their localization to the MZ indicates that the population will continuously encounter numerous different types of endogenous and sometimes foreign particles and molecules in the blood. It is therefore not unexpected that the population also can respond in numerous ways. These MZ macrophage studies describe in vivo examples for that the outcome of an interaction is decided by the combination of all triggered signals. MARCO engagement alone, by an antibody, is a proinflammatory trigger, perhaps related to the response seen when the receptor engages blood-borne bacteria. In contrast, MARCO engagement by apoptotic cells is not a proinflammatory trigger, presumably related to the simultaneously engagement of other receptors recognizing the apoptotic cell or other anti-inflammatory factors (e.g. certain cytokines). Furthermore, the SIGN-R1 interaction with IVIG most probably need co-receptors for its anti-inflammatory effect since two different antibodies binding the receptor were not able to induce the response (instead they blocked it). This could of course be dependent on how the antibody binds, i.e. if it has an agonistic or antagonistic function. A potential relevant observation for the MZ macrophage data is that heterogeneity exists in the population, whereby not all MARCO+ macrophages are SIGN-R1+, but all SIGN-R1+ macrophages are double-positive for MARCO. One explanation for this could be that SIGN-R1 is downregulated upon activation, shown by both activating the macrophage with LPS (113) and by injection of the SIGN-R1 specific antibody 22D1 (114). However, op/op mice that lack macrophage colony stimulating factor (M-CSF)

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and that thus have several macrophage defects, have MARCO+ MZ macrophages that lack SIGN-R1 expression (224). This suggests that MARCO+ and MARCO+ SIGN-R1+ cells could represent two distinct MZ macrophage subpopulations, something that could have implications for the responses presented herein. A lot of focus has been given in the thesis to the injection of apoptotic cells and the subsequent production of autoantibodies. Several aspects of this model have been discussed earlier, including a potential initial role for opsonizing autoantibodies in helping clearance of the apoptotic cell. The antigen specificity in the initial response could be questioned as polyclonal activators such as LPS trigger autoantibody production (exemplified in paper I, figure 1I). Instead, non-specific cell death-related mechanisms could be involved in the initial activation, e.g. related to the release of DAMPs (225). Thus these autoantibodies could be associated with the acute phase response in which numerous soluble factors are released in response to inflammatory stimuli. However, later responses in the model generate GC formation, especially in the absence of iNKT cells, and thus an antigen-specific response is triggered, producing class-switched autoantibodies binding e.g. DNA. An assumption that can be made is that IgG opsonization of apoptotic cells (evident following injection of apoptotic cells) shows that the antigen has activated a B cell, and thereby influences how other cells interpret the antigen. In individuals with predisposition for disease an IgG binding the apoptotic cell could be involved in breaking tolerance to the autoantigens present in the opsonized dying cell. This could especially be relevant to individuals with defects in FcγRIIb, associated to SLE development (226, 227), as the balancing inhibitory signaling mediated by the receptor will be absent from interactions with the IgG Fc part. The nature of the B cell–antibody relationship is in this regard also of importance as antibody involvement is not regulated after being produced. This is in sharp contrast to T cells, where effector functions are directly regulated by antigen-specific activation. Thus the initial (helpful) autoantibody response (an ‘autoreactive reflex’) could in the chronic situation be involved in bypassing regulatory mechanisms and thereby be involved in the development of disease in susceptible individuals. The opsonization could for example alter the uptake pattern of the apoptotic cell and lead both to uptake by cells and into compartments more linked to immune activation. This ‘opsonization hypothesis’ could compliment the role for secondary necrosis often used to explain the connection between apoptotic cells and SLE disease. The hypothesis is currently being tested experimentally. With the herein presented work new insights into the somewhat conflicting view on apoptotic cells as inducers of tolerance but also, during certain conditions, as activators of the immune system has been presented. The focus has been on how autoreactive B cells are activated. However, the question has implications for not only autoimmune disease, but also cancer, infection and transplantation immunology where a shift in the balance between tolerance and activation could be part of both the problem and the solution.

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3 POPULÄRVETENSKAPLIG SAMMANFATTNING

Immunförsvaret är det system i kroppen som försvarar oss mot patogener (grekiska för ”sjukdomsalstrare”), alltså vanligtvis virus och bakterier som kan orsaka förkylningar och urinvägsinfektioner men också betydligt mer allvarliga sjukdomar som HIV. Även parasiter och vissa typer av svampar kan invadera/infektera oss och skapa sjukdom och klassificeras därmed också som patogener. Immunförsvaret är uppbyggt av ett komplext nätverk av celler och lösliga molekyler som har uppgiften att skydda oss. Vanligtvis delas immunförsvaret upp i en enklare och en mer avancerad del. Den enklare delen kallas ofta för medfödd, eller ”innate” som det står i avhandlingens titel. De mer avancerade delarna kallas adaptiva. De enklare delarna är trots denna uppdelning inte per definition mindre viktiga för oss än de adaptiva delarna. Ett exempel på en enkel men viktig funktion är vår hud som mer eller mindre passivt fungerar som en skyddande barriär gentemot skadliga organismer utanför vår kropp. Även aktiva mekanismer, som till exempel känner igen och kan identifiera specifika strukturer på patogener och därmed aktivera immunförsvaret, ingår i den medfödda delen av vårt immunförsvar. Flera delar av detta mer primitiva immunförsvar har bevarats genom evolutionen och finns därmed ofta även hos de mest primitiva organismerna som funnits på jorden i 1000-tals miljoner år. Den adaptiva delen av immunförsvaret dök upp betydligt senare i evolutionen. Den är uppbyggd runt två cellpopulationer som kallas B- och T-celler. De adaptiva B- och T-cellerna skiljer sig från de evulotionellt mer primitiva delarna av immunförsvaret genom att de adaptiva cellerna kan komma ihåg vad vi varit infekterade med, med andra ord generera ett minne. Detta minne är vad man försöker åstadkomma vid vaccinationer, alltså då en ofarlig preparation av patogenen injiceras i en person för att lära personens immunförsvar hur patogenen ser ut. Det genererade minnet gör att kroppen kan svara snabbare och bättre vid en framtida infektion med den aktuella patogenen. Denna avhandling fokuserar framför allt på hur B-celler aktiveras. Denna aktivering följs av att B-cellerna skickar ut målsökande molekyler, s.k. antikroppar. Den ovan nämnda minnesprocessen är inte specifikt studerad i avhandlingsarbetet. Istället har reglering av B-cells aktivering studerats i relation till det medfödda immunförsvaret, alltså hur det medfödda immunförsvaret kan påverka hur B-celler aktiveras. För förståelsen av hur B-celler fungerar är det viktigt att känna till att vid en infektion, förorsakad av t.ex. en bakterie, produceras en stor mängd olika antikroppar som skyddar oss genom att binda (eller målsöka) olika delar av bakterien. Vårt immunförsvar skyddar inte bara utan kan tyvärr också skada oss genom att bli felaktigt aktiverat gentemot oss själva. Immunförsvaret misslyckas alltså i dessa fall med att avgöra vad som är kroppseget (oss själva=ofarligt) och det som är främmande och därmed potentiellt farligt. Den kategori av sjukdomar som kan bli ett resultat av denna missbedömning kallas autoimmuna. Såsom namnet antyder angriper immunförsvaret alltså kroppen vid dessa sjukdomar (auto=själv). Upp till 5 % av befolkningen är drabbade av dessa autoimmuna sjukdomar vilka bland annat inkluderar ledgångsreumatism, multipel skleros (MS), typ 1 diabetes (också kallad barndiabetes), Sjögrens syndrom samt, med relevans för denna avhandling, systemisk lupus erythematosus (SLE). Hos SLE-patienter bildas, förutom skyddande antikroppar som

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bildas i samband med infektioner, också sjukdomsskapande antikroppar som binder och skadar kroppsegna strukturer. Dessa kallas autoantikroppar. Orsakerna till varför vissa människor får autoimmuna sjukdomar är komplexa och varierar både mellan och inom de olika sjukdomarna. Både arv, alltså faktorer relaterade till gener/DNA, samt miljöfaktorer är inblandade i förklaringen. Den första delen av denna avhandling behandlar framför allt hur celler som dör i vår kropp är kopplade till autoimmuna sjukdomar, och SLE i synnerhet, med särskilt fokus på aktivering av de tidigare nämnda B-cellerna. Det finns någonstans i närheten av 10 000 miljarder (1013) celler i vår kropp, alltså ungefär 1000 gånger fler än det finns människor på jorden. För att alla dessa celler ska fungera korrekt måste de bytas ut med jämna mellanrum. Hur ofta varierar väldigt mycket beroende på celltyp. Den ständigt pågående processen med att byta ut celler genererar därmed hela tiden en stor mängd döda celler, i detta fall karaktäriserade som apoptotiska. De apoptotiska cellerna städas bort direkt av specialiserade celler och den döda cellens byggstenar kan därigenom återanvändas i ett effektivt kretslopp. Kanske så många som 0,1-1 % av våra celler dör/byts ut varje dag. Många patienter med SLE har problem med att ”städa bort” celler som dör och ökade nivåer av de döda cellerna kan därmed hittas i till exempel patienternas blod. Celler som tillhör immunförsvaret är viktiga för att ta om hand de apoptotiska cellerna och har alltså inte bara funktioner i förhållande till virus och bakterier. För att förhindra att immuncellerna som städar bort de apoptotiska cellerna felaktigt aktiveras har de apoptotiska cellerna en inneboende dämpande effekt på immunförsvaret. Det är med andra ord paradoxalt att SLE-patienter, med ett överaktivt immunförsvar, kan ha förhöjda nivåer av apoptotiska celler, vilket tvärt om borde dämpa deras immunförsvar. I de tre första artiklarna i denna avhandling har den paradoxala kopplingen mellan apoptotiska celler och SLE-lik sjukdom studerats. Detta har gjorts genom att öka mängden apoptotiska celler i cirkulationen på möss genom intravenösa injektioner. Detta leder till en autoimmun aktivering av bland annat B-celler som producerar sjukdomsskapande autoantikroppar. I delarbete I studerade vi hur en cellpopulation som heter NKT-celler* interagerar med de potentiellt sjukdomsskapande B-cellerna. Vi kunde se att möss som saknar NKT-celler fick ett mycket starkare sjukdomsskapande svar mot de injicerade apoptotiska cellerna. Dessutom såg vi att detta svar kunde hindras om mössen behandlades så nivåerna av NKT-celler ökades. Det är sedan tidigare känt att SLE-patienter har låga nivåer av NKT-celler och vår studie identifierar att detta kan vara direkt inblandat i deras sjukdomsutveckling. Dessutom visar resultaten att NKT-cellerna kan vara ett lovande behandlingsmål i patienterna. En behandling baserad på dessa resultat skulle följaktligen kunna baseras på att antingen öka nivåerna av NKT-celler i patienterna eller på att påverka de NKT-celler patienterna fortfarande har så att de stoppar sjukdomsskapande B-celler. * En anekdot i sammanhanget är att den kemiska struktur, kallad αGalCer, som ofta används för att studera NKT-celler isolerades från det marina svampdjuret Agelas mauritianus av en forskningsavdelning på det japanska ölbryggeriet Kirin.

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Anledningen till att SLE-patienter har ökade nivåer av apoptotiska celler i blodet kan som sagt oftast spåras till att dessa individer har defekter i systemet som ska se till att de döda cellerna städas bort. I delarbete II och III studerades två medlemmar i en familj av så kallade scavenger-receptorer** som tillhör det medfödda immunförsvaret. Scavenger är engelska för renhållning och denna familj av receptorer är inblandade i många olika städuppgifter. De två studerade receptorerna heter SR-A och MARCO och sitter på cellytan av framförallt en cellpopulation som heter makrofager. Makrofager tillhör det medfödda immunförsvaret och kategoriseras ofta som renhållningsarbetare i kroppen. I delarbete II identifierades att MARCO-receptorn kan binda till apoptotiska celler och att specifika MARCO-uttryckande celler i mjälten är inblandade i att städa bort döda celler från blodet. Dessutom såg vi att MARCO-receptorn (felaktigt) kan angripas av autoantikroppar i både SLE-patienter och i möss som utvecklar SLE-lik sjukdom. I delarbete III utökades studien av vad ett autoimmunt angrepp mot MARCO-receptorn kan leda till hos de SLE-patienter där autoantikropparna hittades. I studien fann vi att dessa autoantikroppar kan skapa flera av de aktiverande effekter som är karaktäriserande för SLE-patienter. De identifierade autoantikropparna skulle med andra ord kunna vara inblandade i sjukdomsutvecklingen och skulle också kunna spela en viktig roll i den kliniska diagnostiken av SLE-patienter. I delarbete IV studerades en behandlingsmetod som heter IVIG. Denna behandling består av att en stor mängd antikroppar, isolerade från tusentals blodgivare, ges till patienten. Traditionellt har IVIG-behandlingen används för patienter som saknar antikroppar. Det har dock tidigare upptäckts att denna behandling också kan vara till hjälp för patienter med vissa autoimmuna sjukdomar. IVIG används därför ibland vid behandling av dessa patienter. Mekanismen för hur IVIG fungerar i patienterna med autoimmuna sjukdomarna är däremot inte helt känd. I denna studie identifierade vi att de MARCO-uttryckande makrofagerna, som vi studerade i delarbete II och III, fungerar som en centralt reglerande cell och förmedlar de dämpande signalerna vid IVIG-behandling. Vidare såg vi att detta var beroende av en receptor som heter SIGN-R1, uttryck på den aktuella makrofagens cellyta. IVIG-behandlingen fungerar således i denna sjukdomsmodell genom att SIGN-R1 binder till den aktiva komponenten i IVIG. Sammantaget identifierar dessa studier flera mekanismer, tillhörande det medfödda immunförsvaret, som reglerar hur autoreaktiva B-celler kan bli inblandade i autoimmuna sjukdomar. Dessa mekanismer kan därmed vara intressanta mål för behandling och diagnostik av patienter med sjukdomarna. Till exempel skulle NKT-cellerna kunna påverkas så att de stoppar sjukdomsskapande B-celler i SLE-patienter. IVIG-behandlingen skulle också kunna effektiviseras genom att mer specifikt binda till de celler som skapar de dämpande signalerna som är kärnan i behandlingen. Dessutom skulle autoantikroppar mot MARCO-receptorn kunna mätas i blodet och därigenom användas för att bättre diagnostisera SLE-patienter. ** Celler använder receptorer för att upptäcka och påverka vad som händer runt dem. Receptorer är väldigt små i förhållande till cellerna och 1000-tals receptorer kan finnas både på cellens yta och inuti den.

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4 ACKNOWLEDGEMENTS

I’m grateful to many people for contributing in different ways to this thesis: My supervisor Mikael Karlsson (the hat maker!) for accepting me as a student and teaching me about lab work, immunology, American punk rock and what to do in a sake bar. My co-supervisor Ola Winqvist for showing the business side of science and for your positive attitude. Annika Scheynius for your involvement in my PhD education and for your commitment in solid scientific work. Karl Tryggvason for letting me use the tools your group has generated to study the MARCO receptor. Timo Pikkarainen for skillful help with laboratory work and for your general knowledge in molecular biology and the scavenger receptor family and also the hard(est) working Juha Ojala for practical help. Sanna Cardell and Linda Löfbom for important contributions to the NKT cell manuscript and interesting discussions. Shozo Izui for providing important mice samples. Jeff Ravetch for allowing me to spend time in your lab and for your inspiring way of addressing scientific questions. Several people made my stay at Rockefeller University very enjoyable: Rob Anthony for doing science and kicking ass, for great guitar moves and endless laughs. Kate Jeffrey for shaping up Rob (in a good way), for your skilful help with the NKT cell manuscript and enjoyable company. Patrick Smith for always helping out (including explaining difficult words). Vanessa Bryant and Reuben Richards for your spectacular dinner parties and welcoming way. Tamer Shabaneh, Rene Ott and Brad Rosenberg for adding flavor to my stay. Also former members of the Ravetch lab that I’ve met at conferences including Tracy “good times” McGaha, Falk Nimmerjahn and Pierre Bruhns. The late Ann Kari Lefvert for accepting me as a summer student at CMM in 2004. Maria Kakoulidou who supervised me during that period, showing me real lab work for the very first time and Priya Sakthivel for finishing the project I was involved in. Elisabet Svenungsson for providing SLE patient material and statistical analysis for the follow up study regarding the anti-MARCO autoantibodies. Gunilla Karlsson Hedestam for inspiration and interesting discussions. Birgitta Heyman and Christian Rutemark for an interesting collaboration. Hans Grönlund for your knowledge about what to do with proteins. Lisa Westerberg for “interesting” discussions regarding B cell development and for showing us around in Boston. Susanna Brauner, Robert Saxelin and Ame Beyeen for continuous discussions around science stuff and other things. Bob Harris for dissecting the language in this thesis. Patrik Magnusson, I’m sure you one day will catch a sea cucumber, don’t give up...

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Le groupe: Sara and Emilie for teaming up on the NKT cell paper and for valuable help writing applications, papers and this thesis as well as for all the good times in the lab and elsewhere. Emma for your friendship and humor. Yunying for your observations that often makes me laugh and for your cooking advice. Evelina, Kajsa and Mattias for support and for adding to the “Le Groupe” feeling. All other people at L2:04 including: Anne and Gerd for administrative help. Catharina for being one of the most important people in the lab, organizing the chaos. Agneta for lending cell culture medium and for stories from California and Uppsala. Malin for illustrations. Peter for knowing things I don’t. Present and former members of the room where we try to write: Theresa, Mats, Sara “sitt inte i drag” Johansson, Lotta, Ricardo, Tiiu “tipex” Saarne, Patricia “Patsy McNeal” Torregrosa Paredes, Uffe, Mattias and Marissa. My mother Marie for being the only one in the family who could help me with my science homework and for inspiring my career choice. My father Hans for being the kindest person I know. My sister Erika for showing that nothing is impossible. My grand father (and former chemist) Georg for your genuine interest in my work. Also my wonderful relatives and in-law family for your support. Örjan for being the band leader of my musical life as well as a dear friend. Dan Anders for the big smile, spirited heart and the trips to Berlin. Krister “K” Jönsson for carrying the K with honors. Daniel H for questioning the idea of “truth”, held so dearly by the natural science society. Daniel W for the sweet sailing trips, although I haven’t seen that seal yet. Johan for your humor and for the good times in Leiden and elsewhere. Max for doing inspiring things. Finally to Eva, the most valuable player. I heard a child arguing that when you find a girlfriend you should not tell her where you live or work. I screwed up… Thanks for making it all worthwhile.

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5 REFERENCES

1. Zandman-Goddard, G., and Y. Shoenfeld. 2005. Infections and SLE. Autoimmunity

38:473-485. 2. Janeway, C.A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu Rev

Immunol 20:197-216. 3. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu Rev Immunol

21:335-376. 4. Sokol, C.L., G.M. Barton, A.G. Farr, and R. Medzhitov. 2008. A mechanism for the

initiation of allergen-induced T helper type 2 responses. Nat Immunol 9:310-318. 5. Iwasaki, A., and R. Medzhitov. Regulation of adaptive immunity by the innate immune

system. Science 327:291-295. 6. Foell, D., H. Wittkowski, and J. Roth. 2007. Mechanisms of disease: a 'DAMP' view of

inflammatory arthritis. Nat Clin Pract Rheumatol 3:382-390. 7. Greaves, D.R., and S. Gordon. 2009. The macrophage scavenger receptor at 30 years of

age: current knowledge and future challenges. J Lipid Res 50 Suppl:S282-286. 8. Agrawal, A., P.P. Singh, B. Bottazzi, C. Garlanda, and A. Mantovani. 2009. Pattern

recognition by pentraxins. Adv Exp Med Biol 653:98-116. 9. Ogden, C.A., and K.B. Elkon. 2006. Role of complement and other innate immune

mechanisms in the removal of apoptotic cells. Curr Dir Autoimmun 9:120-142. 10. Lai, A.Y., and M. Kondo. 2008. T and B lymphocyte differentiation from hematopoietic

stem cell. Semin Immunol 20:207-212. 11. Matthews, A.G., and M.A. Oettinger. 2009. RAG: a recombinase diversified. Nat

Immunol 10:817-821. 12. Rajewsky, K. 1996. Clonal selection and learning in the antibody system. Nature

381:751-758. 13. Bach, F.H. 1976. Genetics of transplantation: the major histocompatibility complex.

Annu Rev Genet 10:319-339. 14. Bergtold, A., D.D. Desai, A. Gavhane, and R. Clynes. 2005. Cell surface recycling of

internalized antigen permits dendritic cell priming of B cells. Immunity 23:503-514. 15. Batista, F.D., and N.E. Harwood. 2009. The who, how and where of antigen

presentation to B cells. Nat Rev Immunol 9:15-27. 16. Cyster, J.G. 2003. Lymphoid organ development and cell migration. Immunol Rev

195:5-14. 17. Alvarez, D., E.H. Vollmann, and U.H. von Andrian. 2008. Mechanisms and

consequences of dendritic cell migration. Immunity 29:325-342. 18. Martin, G.S., D.M. Mannino, S. Eaton, and M. Moss. 2003. The epidemiology of sepsis

in the United States from 1979 through 2000. N Engl J Med 348:1546-1554. 19. Morris, D.H., and F.D. Bullock. 1919. The Importance of the Spleen in Resistance to

Infection. Ann Surg 70:513-521. 20. Altamura, M., L. Caradonna, L. Amati, N.M. Pellegrino, G. Urgesi, and S. Miniello.

2001. Splenectomy and sepsis: the role of the spleen in the immune-mediated bacterial clearance. Immunopharmacol Immunotoxicol 23:153-161.

21. Robinette, C.D., and J.F. Fraumeni, Jr. 1977. Splenectomy and subsequent mortality in veterans of the 1939-45 war. Lancet 2:127-129.

22. Brendolan, A., M.M. Rosado, R. Carsetti, L. Selleri, and T.N. Dear. 2007. Development and function of the mammalian spleen. Bioessays 29:166-177.

23. Kraal, G., and R. Mebius. 2006. New insights into the cell biology of the marginal zone of the spleen. Int Rev Cytol 250:175-215.

24. Cesta, M.F. 2006. Normal structure, function, and histology of the spleen. Toxicol Pathol 34:455-465.

25. Loder, F., B. Mutschler, R.J. Ray, C.J. Paige, P. Sideras, R. Torres, M.C. Lamers, and R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med 190:75-89.

41

26. Huston, J.M., M. Ochani, M. Rosas-Ballina, H. Liao, K. Ochani, V.A. Pavlov, M. Gallowitsch-Puerta, M. Ashok, C.J. Czura, B. Foxwell, K.J. Tracey, and L. Ulloa. 2006. Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med 203:1623-1628.

27. Cinamon, G., M.A. Zachariah, O.M. Lam, F.W. Foss, Jr., and J.G. Cyster. 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat Immunol 9:54-62.

28. LeBien, T.W., and T.F. Tedder. 2008. B lymphocytes: how they develop and function. Blood 112:1570-1580.

29. Zandvoort, A., and W. Timens. 2002. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general first-line defense against blood-borne antigens. Clin Exp Immunol 130:4-11.

30. Fagarasan, S., and T. Honjo. 2000. T-Independent immune response: new aspects of B cell biology. Science 290:89-92.

31. Lopes-Carvalho, T., and J.F. Kearney. 2005. Marginal zone B cell physiology and disease. Curr Dir Autoimmun 8:91-123.

32. Rowley, B., L. Tang, S. Shinton, K. Hayakawa, and R.R. Hardy. 2007. Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors. J Autoimmun 29:236-245.

33. Mongini, P.K., K.E. Stein, and W.E. Paul. 1981. T cell regulation of IgG subclass antibody production in response to T-independent antigens. J Exp Med 153:1-12.

34. Dalakas, M.C. 2008. B cells as therapeutic targets in autoimmune neurological disorders. Nat Clin Pract Neurol 4:557-567.

35. Lanzavecchia, A. 1990. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu Rev Immunol 8:773-793.

36. Foote, J.B., and J.F. Kearney. 2009. Generation of B cell memory to the bacterial polysaccharide alpha-1,3 dextran. J Immunol 183:6359-6368.

37. Klein Klouwenberg, P., and L. Bont. 2008. Neonatal and infantile immune responses to encapsulated bacteria and conjugate vaccines. Clin Dev Immunol 2008:628963.

38. Dempsey, P.W., M.E. Allison, S. Akkaraju, C.C. Goodnow, and D.T. Fearon. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271:348-350.

39. Klaus, G.G., J.H. Humphrey, A. Kunkl, and D.W. Dongworth. 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol Rev 53:3-28.

40. Teng, G., and F.N. Papavasiliou. 2007. Immunoglobulin somatic hypermutation. Annu Rev Genet 41:107-120.

41. Schwickert, T.A., R.L. Lindquist, G. Shakhar, G. Livshits, D. Skokos, M.H. Kosco-Vilbois, M.L. Dustin, and M.C. Nussenzweig. 2007. In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446:83-87.

42. Reinhardt, R.L., H.E. Liang, and R.M. Locksley. 2009. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat Immunol 10:385-393.

43. Stavnezer, J., J.E. Guikema, and C.E. Schrader. 2008. Mechanism and regulation of class switch recombination. Annu Rev Immunol 26:261-292.

44. Roes, J., and K. Rajewsky. 1991. Cell autonomous expression of IgD is not essential for the maturation of conventional B cells. Int Immunol 3:1367-1371.

45. Wilson, M., E. Bengten, N.W. Miller, L.W. Clem, L. Du Pasquier, and G.W. Warr. 1997. A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc Natl Acad Sci U S A 94:4593-4597.

46. Chen, K., W. Xu, M. Wilson, B. He, N.W. Miller, E. Bengten, E.S. Edholm, P.A. Santini, P. Rath, A. Chiu, M. Cattalini, J. Litzman, B.B. J, B. Huang, A. Meini, K. Riesbeck, C. Cunningham-Rundles, A. Plebani, and A. Cerutti. 2009. Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils. Nat Immunol 10:889-898.

47. MacLennan, I.C., K.M. Toellner, A.F. Cunningham, K. Serre, D.M. Sze, E. Zuniga, M.C. Cook, and C.G. Vinuesa. 2003. Extrafollicular antibody responses. Immunol Rev 194:8-18.

48. Breman, J.G., and I. Arita. 1980. The confirmation and maintenance of smallpox eradication. N Engl J Med 303:1263-1273.

42

49. Lanzavecchia, A., and F. Sallusto. 2009. Human B cell memory. Curr Opin Immunol 21:298-304.

50. Paramithiotis, E., and M.D. Cooper. 1997. Memory B lymphocytes migrate to bone marrow in humans. Proc Natl Acad Sci U S A 94:208-212.

51. Amanna, I.J., N.E. Carlson, and M.K. Slifka. 2007. Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 357:1903-1915.

52. Dogan, I., B. Bertocci, V. Vilmont, F. Delbos, J. Megret, S. Storck, C.A. Reynaud, and J.C. Weill. 2009. Multiple layers of B cell memory with different effector functions. Nat Immunol 10:1292-1299.

53. Maruyama, M., K.P. Lam, and K. Rajewsky. 2000. Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407:636-642.

54. Bernasconi, N.L., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298:2199-2202.

55. Obukhanych, T.V., and M.C. Nussenzweig. 2006. T-independent type II immune responses generate memory B cells. J Exp Med 203:305-310.

56. de Vinuesa, C.G., M.C. Cook, J. Ball, M. Drew, Y. Sunners, M. Cascalho, M. Wabl, G.G. Klaus, and I.C. MacLennan. 2000. Germinal centers without T cells. J Exp Med 191:485-494.

57. Groom, J.R., C.A. Fletcher, S.N. Walters, S.T. Grey, S.V. Watt, M.J. Sweet, M.J. Smyth, C.R. Mackay, and F. Mackay. 2007. BAFF and MyD88 signals promote a lupuslike disease independent of T cells. J Exp Med 204:1959-1971.

58. van Anken, E., E.P. Romijn, C. Maggioni, A. Mezghrani, R. Sitia, I. Braakman, and A.J. Heck. 2003. Sequential waves of functionally related proteins are expressed when B cells prepare for antibody secretion. Immunity 18:243-253.

59. Ma, Y., and L.M. Hendershot. 2003. The stressful road to antibody secretion. Nat Immunol 4:310-311.

60. Gould, H.J., and B.J. Sutton. 2008. IgE in allergy and asthma today. Nat Rev Immunol 8:205-217.

61. Arnold, J.N., M.R. Wormald, R.B. Sim, P.M. Rudd, and R.A. Dwek. 2007. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25:21-50.

62. Nimmerjahn, F., and J.V. Ravetch. 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310:1510-1512.

63. Baudino, L., S. Azeredo da Silveira, M. Nakata, and S. Izui. 2006. Molecular and cellular basis for pathogenicity of autoantibodies: lessons from murine monoclonal autoantibodies. Springer Semin Immunopathol 28:175-184.

64. Nimmerjahn, F., and J.V. Ravetch. 2008. Anti-inflammatory actions of intravenous immunoglobulin. Annu Rev Immunol 26:513-533.

65. Debre, M., M.C. Bonnet, W.H. Fridman, E. Carosella, N. Philippe, P. Reinert, E. Vilmer, C. Kaplan, J.L. Teillaud, and C. Griscelli. 1993. Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura. Lancet 342:945-949.

66. Kaneko, Y., F. Nimmerjahn, and J.V. Ravetch. 2006. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313:670-673.

67. Brossay, L., and M. Kronenberg. 1999. Highly conserved antigen-presenting function of CD1d molecules. Immunogenetics 50:146-151.

68. Coles, M.C., and D.H. Raulet. 2000. NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J Immunol 164:2412-2418.

69. Fowlkes, B.J., A.M. Kruisbeek, H. Ton-That, M.A. Weston, J.E. Coligan, R.H. Schwartz, and D.M. Pardoll. 1987. A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family. Nature 329:251-254.

70. Porcelli, S., M.B. Brenner, J.L. Greenstein, S.P. Balk, C. Terhorst, and P.A. Bleicher. 1989. Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes. Nature 341:447-450.

71. Godfrey, D.I., H.R. MacDonald, M. Kronenberg, M.J. Smyth, and L. Van Kaer. 2004. NKT cells: what's in a name? Nat Rev Immunol 4:231-237.

43

72. Assarsson, E., T. Kambayashi, J.K. Sandberg, S. Hong, M. Taniguchi, L. Van Kaer, H.G. Ljunggren, and B.J. Chambers. 2000. CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo. J Immunol 165:3673-3679.

73. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M. Taniguchi. 1997. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science 278:1626-1629.

74. Cardell, S., S. Tangri, S. Chan, M. Kronenberg, C. Benoist, and D. Mathis. 1995. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J Exp Med 182:993-1004.

75. Blomqvist, M., S. Rhost, S. Teneberg, L. Lofbom, T. Osterbye, M. Brigl, J.E. Mansson, and S.L. Cardell. 2009. Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells. Eur J Immunol 39:1726-1735.

76. McNab, F.W., D.G. Pellicci, K. Field, G. Besra, M.J. Smyth, D.I. Godfrey, and S.P. Berzins. 2007. Peripheral NK1.1 NKT cells are mature and functionally distinct from their thymic counterparts. J Immunol 179:6630-6637.

77. Coquet, J.M., S. Chakravarti, K. Kyparissoudis, F.W. McNab, L.A. Pitt, B.S. McKenzie, S.P. Berzins, M.J. Smyth, and D.I. Godfrey. 2008. Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population. Proc Natl Acad Sci U S A 105:11287-11292.

78. Chang, D.H., H. Deng, P. Matthews, J. Krasovsky, G. Ragupathi, R. Spisek, A. Mazumder, D.H. Vesole, S. Jagannath, and M.V. Dhodapkar. 2008. Inflammation-associated lysophospholipids as ligands for CD1d-restricted T cells in human cancer. Blood 112:1308-1316.

79. Jahng, A., I. Maricic, C. Aguilera, S. Cardell, R.C. Halder, and V. Kumar. 2004. Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide. J Exp Med 199:947-957.

80. Berzins, S.P., M.J. Smyth, and D.I. Godfrey. 2005. Working with NKT cells--pitfalls and practicalities. Curr Opin Immunol 17:448-454.

81. Kim, H.Y., S. Kim, and D.H. Chung. 2006. FcgammaRIII engagement provides activating signals to NKT cells in antibody-induced joint inflammation. J Clin Invest 116:2484-2492.

82. Cardell, S.L. 2006. The natural killer T lymphocyte: a player in the complex regulation of autoimmune diabetes in non-obese diabetic mice. Clin Exp Immunol 143:194-202.

83. Esteban, L.M., T. Tsoutsman, M.A. Jordan, D. Roach, L.D. Poulton, A. Brooks, O.V. Naidenko, S. Sidobre, D.I. Godfrey, and A.G. Baxter. 2003. Genetic control of NKT cell numbers maps to major diabetes and lupus loci. J Immunol 171:2873-2878.

84. Wither, J., Y.C. Cai, S. Lim, T. McKenzie, N. Roslin, J.O. Claudio, G.S. Cooper, T.J. Hudson, A.D. Paterson, C.M. Greenwood, D. Gladman, J. Pope, C.A. Pineau, C.D. Smith, J.G. Hanly, C. Peschken, G. Boire, and P.R. Fortin. 2008. Reduced proportions of natural killer T cells are present in the relatives of lupus patients and are associated with autoimmunity. Arthritis Res Ther 10:R108.

85. Wilson, S.B., S.C. Kent, K.T. Patton, T. Orban, R.A. Jackson, M. Exley, S. Porcelli, D.A. Schatz, M.A. Atkinson, S.P. Balk, J.L. Strominger, and D.A. Hafler. 1998. Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature 391:177-181.

86. van der Vliet, H.J., B.M. von Blomberg, N. Nishi, M. Reijm, A.E. Voskuyl, A.A. van Bodegraven, C.H. Polman, T. Rustemeyer, P. Lips, A.J. van den Eertwegh, G. Giaccone, R.J. Scheper, and H.M. Pinedo. 2001. Circulating V(alpha24+) Vbeta11+ NKT cell numbers are decreased in a wide variety of diseases that are characterized by autoreactive tissue damage. Clin Immunol 100:144-148.

87. Berzins, S.P., K. Kyparissoudis, D.G. Pellicci, K.J. Hammond, S. Sidobre, A. Baxter, M.J. Smyth, M. Kronenberg, and D.I. Godfrey. 2004. Systemic NKT cell deficiency in NOD mice is not detected in peripheral blood: implications for human studies. Immunol Cell Biol 82:247-252.

88. Godfrey, D.I., and M. Kronenberg. 2004. Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest 114:1379-1388.

89. Sireci, G., D. Russo, F. Dieli, S.A. Porcelli, M. Taniguchi, M.P. La Manna, D. Di Liberto, F. Scarpa, and A. Salerno. 2007. Immunoregulatory role of Jalpha281 T cells in aged mice developing lupus-like nephritis. Eur J Immunol 37:425-433.

44

90. Tupin, E., Y. Kinjo, and M. Kronenberg. 2007. The unique role of natural killer T cells in the response to microorganisms. Nat Rev Microbiol 5:405-417.

91. Chang, D.H., K. Osman, J. Connolly, A. Kukreja, J. Krasovsky, M. Pack, A. Hutchinson, M. Geller, N. Liu, R. Annable, J. Shay, K. Kirchhoff, N. Nishi, Y. Ando, K. Hayashi, H. Hassoun, R.M. Steinman, and M.V. Dhodapkar. 2005. Sustained expansion of NKT cells and antigen-specific T cells after injection of alpha-galactosyl-ceramide loaded mature dendritic cells in cancer patients. J Exp Med 201:1503-1517.

92. Barral, P., J. Eckl-Dorna, N.E. Harwood, C. De Santo, M. Salio, P. Illarionov, G.S. Besra, V. Cerundolo, and F.D. Batista. 2008. B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo. Proc Natl Acad Sci U S A 105:8345-8350.

93. Rabinovitch, M. 1995. Professional and non-professional phagocytes: an introduction. Trends Cell Biol 5:85-87.

94. Wermeling, F., M.C. Karlsson, and T.L. McGaha. 2009. An anatomical view on macrophages in tolerance. Autoimmun Rev 9:49-52.

95. Gordon, S., and P.R. Taylor. 2005. Monocyte and macrophage heterogeneity. Nat Rev Immunol 5:953-964.

96. Liu, K., and M.C. Nussenzweig. Origin and development of dendritic cells. Immunol Rev 234:45-54.

97. Nathan, C.F., H.W. Murray, M.E. Wiebe, and B.Y. Rubin. 1983. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 158:670-689.

98. Billiau, A., and P. Matthys. 2009. Interferon-gamma: a historical perspective. Cytokine Growth Factor Rev 20:97-113.

99. Mosser, D.M., and J.P. Edwards. 2008. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958-969.

100. Hansson, G.K. 2005. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352:1685-1695.

101. Helming, L., and S. Gordon. 2007. The molecular basis of macrophage fusion. Immunobiology 212:785-793.

102. Krieger, M. 1992. Molecular flypaper and atherosclerosis: structure of the macrophage scavenger receptor. Trends Biochem Sci 17:141-146.

103. Peiser, L., S. Mukhopadhyay, and S. Gordon. 2002. Scavenger receptors in innate immunity. Curr Opin Immunol 14:123-128.

104. Ramet, M., A. Pearson, P. Manfruelli, X. Li, H. Koziel, V. Gobel, E. Chung, M. Krieger, and R.A. Ezekowitz. 2001. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15:1027-1038.

105. Bowdish, D.M., and S. Gordon. 2009. Conserved domains of the class A scavenger receptors: evolution and function. Immunol Rev 227:19-31.

106. Fraser, I., D. Hughes, and S. Gordon. 1993. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 364:343-346.

107. Chen, Y., F. Wermeling, J. Sundqvist, A.B. Jonsson, K. Tryggvason, T. Pikkarainen, and M.C. Karlsson. A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses. Eur J Immunol

108. Elomaa, O., M. Kangas, C. Sahlberg, J. Tuukkanen, R. Sormunen, A. Liakka, I. Thesleff, G. Kraal, and K. Tryggvason. 1995. Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell 80:603-609.

109. Granucci, F., F. Petralia, M. Urbano, S. Citterio, F. Di Tota, L. Santambrogio, and P. Ricciardi-Castagnoli. 2003. The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia. Blood 102:2940-2947.

110. Park, C.G., K. Takahara, E. Umemoto, Y. Yashima, K. Matsubara, Y. Matsuda, B.E. Clausen, K. Inaba, and R.M. Steinman. 2001. Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN. Int Immunol 13:1283-1290.

111. Wu, L., and V.N. KewalRamani. 2006. Dendritic-cell interactions with HIV: infection and viral dissemination. Nat Rev Immunol 6:859-868.

112. Dijkstra, C.D., E. Van Vliet, E.A. Dopp, A.A. van der Lelij, and G. Kraal. 1985. Marginal zone macrophages identified by a monoclonal antibody: characterization of immuno- and enzyme-histochemical properties and functional capacities. Immunology 55:23-30.

45

113. Groeneveld, P.H., T. Erich, and G. Kraal. 1986. The differential effects of bacterial lipopolysaccharide (LPS) on splenic non-lymphoid cells demonstrated by monoclonal antibodies. Immunology 58:285-290.

114. Kang, Y.S., J.Y. Kim, S.A. Bruening, M. Pack, A. Charalambous, A. Pritsker, T.M. Moran, J.M. Loeffler, R.M. Steinman, and C.G. Park. 2004. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen. Proc Natl Acad Sci U S A 101:215-220.

115. Kang, Y.S., S. Yamazaki, T. Iyoda, M. Pack, S.A. Bruening, J.Y. Kim, K. Takahara, K. Inaba, R.M. Steinman, and C.G. Park. 2003. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran. Int Immunol 15:177-186.

116. Kang, Y.S., Y. Do, H.K. Lee, S.H. Park, C. Cheong, R.M. Lynch, J.M. Loeffler, R.M. Steinman, and C.G. Park. 2006. A dominant complement fixation pathway for pneumococcal polysaccharides initiated by SIGN-R1 interacting with C1q. Cell 125:47-58.

117. Hotchkiss, R.S., A. Strasser, J.E. McDunn, and P.E. Swanson. 2009. Cell death. N Engl J Med 361:1570-1583.

118. Fadok, V.A., D.R. Voelker, P.A. Campbell, J.J. Cohen, D.L. Bratton, and P.M. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207-2216.

119. Choi, Y.S. 1997. Differentiation and apoptosis of human germinal center B-lymphocytes. Immunol Res 16:161-174.

120. Dancey, J.T., K.A. Deubelbeiss, L.A. Harker, and C.A. Finch. 1976. Neutrophil kinetics in man. J Clin Invest 58:705-715.

121. Melino, G. 2001. The Sirens' song. Nature 412:23. 122. Zakeri, Z., D. Quaglino, and H.S. Ahuja. 1994. Apoptotic cell death in the mouse limb

and its suppression in the hammertoe mutant. Dev Biol 165:294-297. 123. Reddien, P.W., and H.R. Horvitz. 2004. The engulfment process of programmed cell

death in caenorhabditis elegans. Annu Rev Cell Dev Biol 20:193-221. 124. Walsh, G.M., D.W. Sexton, M.G. Blaylock, and C.M. Convery. 1999. Resting and

cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils. Blood 94:2827-2835.

125. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2:965-975.

126. Wang, X., Y.C. Wu, V.A. Fadok, M.C. Lee, K. Gengyo-Ando, L.C. Cheng, D. Ledwich, P.K. Hsu, J.Y. Chen, B.K. Chou, P. Henson, S. Mitani, and D. Xue. 2003. Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12. Science 302:1563-1566.

127. Blaxter, M. 1998. Caenorhabditis elegans is a nematode. Science 282:2041-2046. 128. Elliott, M.R., F.B. Chekeni, P.C. Trampont, E.R. Lazarowski, A. Kadl, S.F. Walk, D.

Park, R.I. Woodson, M. Ostankovich, P. Sharma, J.J. Lysiak, T.K. Harden, N. Leitinger, and K.S. Ravichandran. 2009. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461:282-286.

129. Lauber, K., E. Bohn, S.M. Krober, Y.J. Xiao, S.G. Blumenthal, R.K. Lindemann, P. Marini, C. Wiedig, A. Zobywalski, S. Baksh, Y. Xu, I.B. Autenrieth, K. Schulze-Osthoff, C. Belka, G. Stuhler, and S. Wesselborg. 2003. Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell 113:717-730.

130. Gude, D.R., S.E. Alvarez, S.W. Paugh, P. Mitra, J. Yu, R. Griffiths, S.E. Barbour, S. Milstien, and S. Spiegel. 2008. Apoptosis induces expression of sphingosine kinase 1 to release sphingosine-1-phosphate as a "come-and-get-me" signal. Faseb J 22:2629-2638.

131. Meri, S. 2007. Loss of self-control in the complement system and innate autoreactivity. Ann N Y Acad Sci 1109:93-105.

132. Whitacre, C.C. 2001. Sex differences in autoimmune disease. Nat Immunol 2:777-780. 133. Watanabe-Fukunaga, R., C.I. Brannan, N.G. Copeland, N.A. Jenkins, and S. Nagata.

1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314-317.

134. Pisitkun, P., J.A. Deane, M.J. Difilippantonio, T. Tarasenko, A.B. Satterthwaite, and S. Bolland. 2006. Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication. Science 312:1669-1672.

46

135. 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.

136. Holmdahl, R., M.E. Andersson, T.J. Goldschmidt, L. Jansson, M. Karlsson, V. Malmstrom, and J. Mo. 1989. Collagen induced arthritis as an experimental model for rheumatoid arthritis. Immunogenetics, pathogenesis and autoimmunity. Apmis 97:575-584.

137. Hanahan, D., and R.A. Weinberg. 2000. The hallmarks of cancer. Cell 100:57-70. 138. Javierre, B.M., A.F. Fernandez, J. Richter, F. Al-Shahrour, J.I. Martin-Subero, J.

Rodriguez-Ubreva, M. Berdasco, M.F. Fraga, T.P. O'Hanlon, L.G. Rider, F.V. Jacinto, F.J. Lopez-Longo, J. Dopazo, M. Forn, M.A. Peinado, L. Carreno, A.H. Sawalha, J.B. Harley, R. Siebert, M. Esteller, F.W. Miller, and E. Ballestar. Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res 20:170-179.

139. Martino, D.J., and S.L. Prescott. Silent mysteries: epigenetic paradigms could hold the key to conquering the epidemic of allergy and immune disease. Allergy 65:7-15.

140. Ulmanen, I., M. Halonen, T. Ilmarinen, and L. Peltonen. 2005. Monogenic autoimmune diseases - lessons of self-tolerance. Curr Opin Immunol 17:609-615.

141. Weiss, K.M., and J.D. Terwilliger. 2000. How many diseases does it take to map a gene with SNPs? Nat Genet 26:151-157.

142. Gross, D.M., T. Forsthuber, M. Tary-Lehmann, C. Etling, K. Ito, Z.A. Nagy, J.A. Field, A.C. Steere, and B.T. Huber. 1998. Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 281:703-706.

143. Benoist, C., and D. Mathis. 2001. Autoimmunity provoked by infection: how good is the case for T cell epitope mimicry? Nat Immunol 2:797-801.

144. 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.

145. Costenbader, K.H., and E.W. Karlson. 2006. Cigarette smoking and autoimmune disease: what can we learn from epidemiology? Lupus 15:737-745.

146. Liu, K., Q.Z. Li, A.M. Delgado-Vega, A.K. Abelson, E. Sanchez, J.A. Kelly, L. Li, Y. Liu, J. Zhou, M. Yan, Q. Ye, S. Liu, C. Xie, X.J. Zhou, S.A. Chung, B. Pons-Estel, T. Witte, E. de Ramon, S.C. Bae, N. Barizzone, G.D. Sebastiani, J.T. Merrill, P.K. Gregersen, G.G. Gilkeson, R.P. Kimberly, T.J. Vyse, I. Kim, S. D'Alfonso, J. Martin, J.B. Harley, L.A. Criswell, E.K. Wakeland, M.E. Alarcon-Riquelme, and C. Mohan. 2009. Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans. J Clin Invest 119:911-923.

147. Crowe, M.J., T. Doetschman, and D.G. Greenhalgh. 2000. Delayed wound healing in immunodeficient TGF-beta 1 knockout mice. J Invest Dermatol 115:3-11.

148. Gorelik, L., and R.A. Flavell. 2002. Transforming growth factor-beta in T-cell biology. Nat Rev Immunol 2:46-53.

149. Fernando, M.M., C.R. Stevens, E.C. Walsh, P.L. De Jager, P. Goyette, R.M. Plenge, T.J. Vyse, and J.D. Rioux. 2008. Defining the role of the MHC in autoimmunity: a review and pooled analysis. PLoS Genet 4:e1000024.

150. Silverstein, A.M. 2001. Autoimmunity versus horror autotoxicus: the struggle for recognition. Nat Immunol 2:279-281.

151. Matzinger, P. 2002. The danger model: a renewed sense of self. Science 296:301-305. 152. Wardemann, H., S. Yurasov, A. Schaefer, J.W. Young, E. Meffre, and M.C.

Nussenzweig. 2003. Predominant autoantibody production by early human B cell precursors. Science 301:1374-1377.

153. Morgan, I.M. 1947. Allergic Encephalomyelitis in Monkeys in Response to Injection of Normal Monkey Nervous Tissue. J Exp Med 85:131-140.

154. Aoki-Ota, M., K. Tsunoda, T. Ota, T. Iwasaki, S. Koyasu, M. Amagai, and T. Nishikawa. 2004. A mouse model of pemphigus vulgaris by adoptive transfer of naive splenocytes from desmoglein 3 knockout mice. Br J Dermatol 151:346-354.

155. Mueller, D.L. Mechanisms maintaining peripheral tolerance. Nat Immunol 11:21-27. 156. Wing, K., and S. Sakaguchi. Regulatory T cells exert checks and balances on self

tolerance and autoimmunity. Nat Immunol 11:7-13. 157. Rahman, A., and D.A. Isenberg. 2008. Systemic lupus erythematosus. N Engl J Med

358:929-939.

47

158. Bagavant, H., and S.M. Fu. 2005. New insights from murine lupus: disassociation of autoimmunity and end organ damage and the role of T cells. Curr Opin Rheumatol 17:523-528.

159. Chan, O.T., L.G. Hannum, A.M. Haberman, M.P. Madaio, and M.J. Shlomchik. 1999. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J Exp Med 189:1639-1648.

160. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, and M.C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med 201:703-711.

161. Blank, M.C., R.N. Stefanescu, E. Masuda, F. Marti, P.D. King, P.B. Redecha, R.J. Wurzburger, M.G. Peterson, S. Tanaka, and L. Pricop. 2005. Decreased transcription of the human FCGR2B gene mediated by the -343 G/C promoter polymorphism and association with systemic lupus erythematosus. Hum Genet 117:220-227.

162. Kim, S.J., D. Gershov, X. Ma, N. Brot, and K.B. Elkon. 2003. Opsonization of apoptotic cells and its effect on macrophage and T cell immune responses. Ann N Y Acad Sci 987:68-78.

163. Gaipl, U.S., A. Kuhn, A. Sheriff, L.E. Munoz, S. Franz, R.E. Voll, J.R. Kalden, and M. Herrmann. 2006. Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun 9:173-187.

164. Kanta, H., and C. Mohan. 2009. Three checkpoints in lupus development: central tolerance in adaptive immunity, peripheral amplification by innate immunity and end-organ inflammation. Genes Immun 10:390-396.

165. Taneja, V., and C.S. David. 2001. Lessons from animal models for human autoimmune diseases. Nat Immunol 2:781-784.

166. Ren, Y., J. Tang, M.Y. Mok, A.W. Chan, A. Wu, and C.S. Lau. 2003. Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis Rheum 48:2888-2897.

167. Botto, M., C. Dell'Agnola, A.E. Bygrave, E.M. Thompson, H.T. Cook, F. Petry, M. Loos, P.P. Pandolfi, and M.J. Walport. 1998. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 19:56-59.

168. Scott, R.S., E.J. McMahon, S.M. Pop, E.A. Reap, R. Caricchio, P.L. Cohen, H.S. Earp, and G.K. Matsushima. 2001. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411:207-211.

169. Hanayama, R., M. Tanaka, K. Miyasaka, K. Aozasa, M. Koike, Y. Uchiyama, and S. Nagata. 2004. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 304:1147-1150.

170. Li, M.O., M.R. Sarkisian, W.Z. Mehal, P. Rakic, and R.A. Flavell. 2003. Phosphatidylserine receptor is required for clearance of apoptotic cells. Science 302:1560-1563.

171. Bratton, D.L., and P.M. Henson. 2008. Apoptotic cell recognition: will the real phosphatidylserine receptor(s) please stand up? Curr Biol 18:R76-79.

172. Miyanishi, M., K. Tada, M. Koike, Y. Uchiyama, T. Kitamura, and S. Nagata. 2007. Identification of Tim4 as a phosphatidylserine receptor. Nature 450:435-439.

173. Mevorach, D., J.L. Zhou, X. Song, and K.B. Elkon. 1998. Systemic exposure to irradiated apoptotic cells induces autoantibody production. J Exp Med 188:387-392.

174. Mukundan, L., J.I. Odegaard, C.R. Morel, J.E. Heredia, J.W. Mwangi, R.R. Ricardo-Gonzalez, Y.P. Goh, A.R. Eagle, S.E. Dunn, J.U. Awakuni, K.D. Nguyen, L. Steinman, S.A. Michie, and A. Chawla. 2009. PPAR-delta senses and orchestrates clearance of apoptotic cells to promote tolerance. Nat Med 15:1266-1272.

175. Enzler, T., G. Bonizzi, G.J. Silverman, D.C. Otero, G.F. Widhopf, A. Anzelon-Mills, R.C. Rickert, and M. Karin. 2006. Alternative and classical NF-kappa B signaling retain autoreactive B cells in the splenic marginal zone and result in lupus-like disease. Immunity 25:403-415.

176. Grader-Beck, T., L. Casciola-Rosen, T.J. Lang, R. Puliaev, A. Rosen, and C.S. Via. 2007. Apoptotic splenocytes drive the autoimmune response to poly(ADP-ribose) polymerase 1 in a murine model of lupus. J Immunol 178:95-102.

177. O'Brien, B.A., X. Geng, C.H. Orteu, Y. Huang, M. Ghoreishi, Y. Zhang, J.A. Bush, G. Li, D.T. Finegood, and J.P. Dutz. 2006. A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse. J Autoimmun 26:104-115.

48

178. Gallucci, S., M. Lolkema, and P. Matzinger. 1999. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 5:1249-1255.

179. Huynh, M.L., V.A. Fadok, and P.M. Henson. 2002. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation. J Clin Invest 109:41-50.

180. Utz, P.J., T.J. Gensler, and P. Anderson. 2000. Death, autoantigen modifications, and tolerance. Arthritis Res 2:101-114.

181. Casciola-Rosen, L., F. Andrade, D. Ulanet, W.B. Wong, and A. Rosen. 1999. Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity. J Exp Med 190:815-826.

182. Casciola-Rosen, L.A., G. Anhalt, and A. Rosen. 1994. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 179:1317-1330.

183. Chang, M.K., C.J. Binder, Y.I. Miller, G. Subbanagounder, G.J. Silverman, J.A. Berliner, and J.L. Witztum. 2004. Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory. J Exp Med 200:1359-1370.

184. Getahun, A., and B. Heyman. 2006. How antibodies act as natural adjuvants. Immunol Lett 104:38-45.

185. Kim, S.J., D. Gershov, X. Ma, N. Brot, and K.B. Elkon. 2002. I-PLA(2) activation during apoptosis promotes the exposure of membrane lysophosphatidylcholine leading to binding by natural immunoglobulin M antibodies and complement activation. J Exp Med 196:655-665.

186. Nickerson, K.M., S.R. Christensen, J. Shupe, M. Kashgarian, D. Kim, K. Elkon, and M.J. Shlomchik. TLR9 regulates TLR7- and MyD88-dependent autoantibody production and disease in a murine model of lupus. J Immunol 184:1840-1848.

187. Firestein, G.S. 2003. Evolving concepts of rheumatoid arthritis. Nature 423:356-361. 188. Boilard, E., P.A. Nigrovic, K. Larabee, G.F. Watts, J.S. Coblyn, M.E. Weinblatt, E.M.

Massarotti, E. Remold-O'Donnell, R.W. Farndale, J. Ware, and D.M. Lee. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production. Science 327:580-583.

189. Gravallese, E.M. 2002. Bone destruction in arthritis. Ann Rheum Dis 61 Suppl 2:ii84-86. 190. Keystone, E., P. Emery, C.G. Peterfy, P.P. Tak, S. Cohen, M.C. Genovese, M. Dougados,

G.R. Burmester, M. Greenwald, T.K. Kvien, S. Williams, D. Hagerty, M.W. Cravets, and T. Shaw. 2009. Rituximab inhibits structural joint damage in patients with rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitor therapies. Ann Rheum Dis 68:216-221.

191. Weyand, C.M., and J.J. Goronzy. 2003. Ectopic germinal center formation in rheumatoid synovitis. Ann N Y Acad Sci 987:140-149.

192. Hjelmstrom, P. 2001. Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines. J Leukoc Biol 69:331-339.

193. Uysal, H., K.S. Nandakumar, C. Kessel, S. Haag, S. Carlsen, H. Burkhardt, and R. Holmdahl. Antibodies to citrullinated proteins: molecular interactions and arthritogenicity. Immunol Rev 233:9-33.

194. Leadbetter, E.A., I.R. Rifkin, A.M. Hohlbaum, B.C. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416:603-607.

195. Rifkin, I.R., E.A. Leadbetter, B.C. Beaudette, C. Kiani, M. Monestier, M.J. Shlomchik, and A. Marshak-Rothstein. 2000. Immune complexes present in the sera of autoimmune mice activate rheumatoid factor B cells. J Immunol 165:1626-1633.

196. Holmdahl, R., K. Rubin, L. Klareskog, E. Larsson, and H. Wigzell. 1986. Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies. Arthritis Rheum 29:400-410.

197. Exley, M., J. Garcia, S.B. Wilson, F. Spada, D. Gerdes, S.M. Tahir, K.T. Patton, R.S. Blumberg, S. Porcelli, A. Chott, and S.P. Balk. 2000. CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes. Immunology 100:37-47.

49

198. Leadbetter, E.A., M. Brigl, P. Illarionov, N. Cohen, M.C. Luteran, S. Pillai, G.S. Besra, and M.B. Brenner. 2008. NK T cells provide lipid antigen-specific cognate help for B cells. Proc Natl Acad Sci U S A 105:8339-8344.

199. Platt, N., H. Suzuki, Y. Kurihara, T. Kodama, and S. Gordon. 1996. Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro. Proc Natl Acad Sci U S A 93:12456-12460.

200. Sigal, L.H. 2003. Molecular biology and immunology for clinicians 25: defense collagens. J Clin Rheumatol 9:267-272.

201. Brodsky, B., and N.K. Shah. 1995. Protein motifs. 8. The triple-helix motif in proteins. Faseb J 9:1537-1546.

202. Uwatoko, S., and M. Mannik. 1988. Low-molecular weight C1q-binding immunoglobulin G in patients with systemic lupus erythematosus consists of autoantibodies to the collagen-like region of C1q. J Clin Invest 82:816-824.

203. Hogarth, M.B., J.H. Slingsby, P.J. Allen, E.M. Thompson, P. Chandler, K.A. Davies, E. Simpson, B.J. Morley, and M.J. Walport. 1998. Multiple lupus susceptibility loci map to chromosome 1 in BXSB mice. J Immunol 161:2753-2761.

204. Rogers, N.J., M.J. Lees, L. Gabriel, E. Maniati, S.J. Rose, P.K. Potter, and B.J. Morley. 2009. A defect in Marco expression contributes to systemic lupus erythematosus development via failure to clear apoptotic cells. J Immunol 182:1982-1990.

205. Miyake, Y., K. Asano, H. Kaise, M. Uemura, M. Nakayama, and M. Tanaka. 2007. Critical role of macrophages in the marginal zone in the suppression of immune responses to apoptotic cell-associated antigens. J Clin Invest 117:2268-2278.

206. Jozefowski, S., M. Arredouani, T. Sulahian, and L. Kobzik. 2005. Disparate regulation and function of the class A scavenger receptors SR-AI/II and MARCO. J Immunol 175:8032-8041.

207. Kraal, G., H. Ter Hart, C. Meelhuizen, G. Venneker, and E. Claassen. 1989. Marginal zone macrophages and their role in the immune response against T-independent type 2 antigens: modulation of the cells with specific antibody. Eur J Immunol 19:675-680.

208. Wilson, J.G., W.D. Ratnoff, P.H. Schur, and D.T. Fearon. 1986. Decreased expression of the C3b/C4b receptor (CR1) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus erythematosus. Arthritis Rheum 29:739-747.

209. Beutler, B., and A. Cerami. 1986. Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature 320:584-588.

210. Svenungsson, E., G.Z. Fei, K. Jensen-Urstad, U. de Faire, A. Hamsten, and J. Frostegard. 2003. TNF-alpha: a link between hypertriglyceridaemia and inflammation in SLE patients with cardiovascular disease. Lupus 12:454-461.

211. Marino, M.W., A. Dunn, D. Grail, M. Inglese, Y. Noguchi, E. Richards, A. Jungbluth, H. Wada, M. Moore, B. Williamson, S. Basu, and L.J. Old. 1997. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci U S A 94:8093-8098.

212. Elliott, J.I., A. Surprenant, F.M. Marelli-Berg, J.C. Cooper, R.L. Cassady-Cain, C. Wooding, K. Linton, D.R. Alexander, and C.F. Higgins. 2005. Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes. Nat Cell Biol 7:808-816.

213. Sengstake, S., E.M. Boneberg, and H. Illges. 2006. CD21 and CD62L shedding are both inducible via P2X7Rs. Int Immunol 18:1171-1178.

214. Chen, L., and C.F. Brosnan. 2006. Regulation of immune response by P2X7 receptor. Crit Rev Immunol 26:499-513.

215. Labasi, J.M., N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M.M. Payette, W. Brissette, J.R. Wicks, L. Audoly, and C.A. Gabel. 2002. Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response. J Immunol 168:6436-6445.

216. Elliott, J.I., J.H. McVey, and C.F. Higgins. 2005. The P2X7 receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products. Arthritis Res Ther 7:R468-475.

217. Ji, H., K. Ohmura, U. Mahmood, D.M. Lee, F.M. Hofhuis, S.A. Boackle, K. Takahashi, V.M. Holers, M. Walport, C. Gerard, A. Ezekowitz, M.C. Carroll, M. Brenner, R. Weissleder, J.S. Verbeek, V. Duchatelle, C. Degott, C. Benoist, and D. Mathis. 2002. Arthritis critically dependent on innate immune system players. Immunity 16:157-168.

50

218. Bruhns, P., A. Samuelsson, J.W. Pollard, and J.V. Ravetch. 2003. Colony-stimulating factor-1-dependent macrophages are responsible for IVIG protection in antibody-induced autoimmune disease. Immunity 18:573-581.

219. Anthony, R.M., F. Nimmerjahn, D.J. Ashline, V.N. Reinhold, J.C. Paulson, and J.V. Ravetch. 2008. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320:373-376.

220. Steinman, R.M., A. Granelli-Piperno, M. Pope, C. Trumpfheller, R. Ignatius, G. Arrode, P. Racz, and K. Tenner-Racz. 2003. The interaction of immunodeficiency viruses with dendritic cells. Curr Top Microbiol Immunol 276:1-30.

221. Zeng, D., Y. Liu, S. Sidobre, M. Kronenberg, and S. Strober. 2003. Activation of natural killer T cells in NZB/W mice induces Th1-type immune responses exacerbating lupus. J Clin Invest 112:1211-1222.

222. Lundberg, A.M., and G.K. Hansson. Innate immune signals in atherosclerosis. Clin Immunol 134:5-24.

223. Gustafsson, J., I. Gunnarsson, O. Borjesson, S. Pettersson, S. Moller, G.Z. Fei, K. Elvin, J.F. Simard, L.O. Hansson, I.E. Lundberg, A. Larsson, and E. Svenungsson. 2009. Predictors of the first cardiovascular event in patients with systemic lupus erythematosus - a prospective cohort study. Arthritis Res Ther 11:R186.

224. Ito, S., M. Naito, Y. Kobayashi, H. Takatsuka, S. Jiang, H. Usuda, H. Umezu, G. Hasegawa, M. Arakawa, L.D. Shultz, O. Elomaa, and K. Tryggvason. 1999. Roles of a macrophage receptor with collagenous structure (MARCO) in host defense and heterogeneity of splenic marginal zone macrophages. Arch Histol Cytol 62:83-95.

225. Green, D.R., T. Ferguson, L. Zitvogel, and G. Kroemer. 2009. Immunogenic and tolerogenic cell death. Nat Rev Immunol 9:353-363.

226. Tsuchiya, N., Z. Honda, and K. Tokunaga. 2006. Role of B cell inhibitory receptor polymorphisms in systemic lupus erythematosus: a negative times a negative makes a positive. J Hum Genet 51:741-750.

227. Bolland, S., Y.S. Yim, K. Tus, E.K. Wakeland, and J.V. Ravetch. 2002. Genetic modifiers of systemic lupus erythematosus in FcgammaRIIB(-/-) mice. J Exp Med 195:1167-1174.