List of Papers

This thesis is based on the following Papers, which are referred to in the text by their Roman numerals.

I Mathsson L*, Åhlin E*, Sjöwall C, Skogh T, Rönnelid.

Cytokine induction by circulating immune complexes and signs of in-vivo complement activation in systemic lupus erythemato-sus are associated with the occurrence of anti-Sjögren's syn-drome A antibodies. Clin Exp Immunol. 2007 Mar;147(3):513-20.

II Åhlin E, Mathsson L, Eloranta MJ, Jonsdottir T, Gunnarsson I, Rönnblom L, Rönnelid J. Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE Manuscript

III ElShafie AI*, Åhlin E*, Mathsson L, Elghazali G, Rönnelid J. Circulating immune complexes (IC) and IC-induced levels of GM-CSF are increased in Sudanese patients with acute visceral Leishmania donovani infection undergoing sodium stiboglu-conate treatment. Implications for disease pathogenesis J Immunol. 2007 Apr 15;178(8):5383-9.

IV Åhlin E, Elshafie AI, Nur A. M., Hassan El Safi S, Rönnelid J. Occurrence of rheumatoid arthritis-associated autoantibodies in Sudanese patients with Leishmania donovani infection Manuscript

*Authors contributed equally

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11 Immunity: A brief overview ..................................................................... 12 Hypersensitivity reactions ........................................................................ 13 Autoimmunity .......................................................................................... 14 Autoantibodies ......................................................................................... 16 Systemic Lupus Erythematosus ................................................................ 17 Leishmania donovani infection ................................................................ 21 Immune complexes in health and disease ................................................ 23

Complement activation ........................................................................ 24 Cellular receptors ................................................................................. 25 Cytokine induction............................................................................... 27

Aims .............................................................................................................. 30 Paper I ...................................................................................................... 30 Paper II ..................................................................................................... 30 Paper III .................................................................................................... 30 Paper IV ................................................................................................... 30

Methods ........................................................................................................ 31 Patients ..................................................................................................... 31 Immune complex purification and measurement ..................................... 32

PEG precipitation of IC ....................................................................... 32 C1q-binding assay for isolation of circulating IC ................................ 33 Measurement of levels of circulating IC.............................................. 33

Preparation of mononuclear cells and culture conditions ......................... 33 Complement activation as a marker of disease activity ........................... 33 Cytokine enzyme-linked immunosorbent assays (ELISA) ...................... 34 Immunoglobulin G ELISA ....................................................................... 35 Functional blocking of Fc-receptors ......................................................... 35 Detection of autoantibodies ...................................................................... 35

Immunofluorescence and immunodiffusion techniques for ANA detection ............................................................................................... 35 Line blot assay for anti-nuclear antibodies .......................................... 36 Measurement of anti-CRP ................................................................... 36 Measurment of anti-CCP ..................................................................... 36 Measurement of RF ............................................................................. 37

Statistics ................................................................................................... 37

Results and discussion .................................................................................. 38 Paper I ...................................................................................................... 38 Paper II ..................................................................................................... 40 Paper III .................................................................................................... 42 Paper IV ................................................................................................... 43

General conclusions and future perspectives ................................................ 45

Populärvetenskaplig sammanfattning ........................................................... 48 Bakgrund .................................................................................................. 48 Målet för avhandlingen ............................................................................ 49 Delstudier ................................................................................................. 49

Arbete I och II ...................................................................................... 49 Arbete III ............................................................................................. 50

Slutsatser av delarbetena .......................................................................... 51

Acknowledgements ....................................................................................... 52

References ..................................................................................................... 54

Abbreviations

Ab Antibody

ACPA Antibodies against citrullinated proteins and/or peptides

ACR American College of Rheumatology

ADCC Antibody dependent cell mediated cytotoxicity

ANA Anti-nuclear antibody

ANOVA Analysis of variance

APC Antigen presenting cell

C Complement component

CCP Cyclic citrullinated peptides

CD Crohn's disease

CeD Celiac disease

COI Cut-off index

CR Complement receptor

CRP C-reactive protein

DC Dentritic cell

DID Double immunodiffusion

dsDNA Double stranded DNA

DTH Delayed type hypersensitivity

EBV Eppstein-Barr virus

ELISA Enzyme-linked immunosorbent assay

Fab Fragment, antigen binding

FcR Fragment, crystallisable receptor

GM-CSF Granulocyte macrophage colony-stimulating factor

HCV Hepatitis C virus

HEPES hydroxyethyl-piperazineethanesulfonic acid

HLA Human leukocyte antigen

HSM Hepatosplenomegaly

IC Immune complex(es)

IFN Interferon

Ig Immunoglobulin

IL Interleukin

LPS Lipopolysaccharide

mAb Monoclonal antibody

MHC Major histocompatibility complex

MMF Mycophenolate mofetil

MS Multiple sclerosis

NIBSC National Institute for Biological Standards and Controls

NK Natural killer cell

OD Optical density

PBMC Peripheral blood mononuclear cell

PCNA Proliferating cell nuclear antigen

PDC Plasmacytoid dentritic cell

PEG Polyethylene glycol

PEST Penicillin-streptomycin

PKDL Post kala-azar dermal leishmaniasis

PM Polymyositis

RA Rheumatoid arthritis

RF Rheumatoid factor

RNP Ribonuclear protein

SLE Systemic lupus erythematosus

SLEDAI Systemic lupus erythematosus disease activity index

Sm Smith antigen

SSA Sjögren's syndrome A antigen

SSB Sjögren's syndrome B antigen

T1D Type 1 diabetes

Th T helper cell

TLR Toll-like receptor

TNF Tumour necrosis factor

UV Ultraviolet

VL Visceral leishmaniasis

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Introduction

The common issue in my thesis work has been to investigate functional mechanisms underlying immune complex (IC)-driven inflammation in rheumatic diseases and tropical infections. I will present and discuss data on the contribution of specific autoantibodies and classical complement activa-tion on the capacity of IC from SLE patients to induce cytokines. I will also present results on autoantibody formation in association with IC in the para-sitic infection caused by the Leishmania donovani parasite. How disease activity and treatment influence the magnitude of IC-induced cytokines in L. donovani infection will also be discussed. Knowledge about these mecha-nisms is of importance for understanding of the pathological significance of autoantibodies in IC formation and pathology in SLE and in parasite-induced inflammation.

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Immunity: A brief overview In mammals there are two main types of immune defence, innate and adap-tive. Innate immunity is the first line of defence and is not specific to any certain pathogen but constitutes a protection including anatomical, physio-logical and phagocytic barriers. In addition, chemical mediators such as the complement cascade lyses microorganisms and/or facilitate phagocytosis as a part of the non-specific host defence. Most components of innate immunity are present before an infectious agent is introduced. If microbes nonetheless bypass the anatomical and physiological barriers protecting the body, phago-cytes and natural killer (NK)-cells detect the immediate presence and nature of the infection. In response to recognition of certain highly conserved mo-lecular patterns expressed by different groups of microbes, i.e. extracellu-larly expressed oligosaccharides on bacteria and parasites or intracellular molecules such as double stranded RNA present in replicating viruses, these cells and the complement cascade become activated to eliminate or neutral-ise the organism. If the same pathogen is encountered a second time the in-nate response will react in a similar manner as with the first encounter.

Adaptive immunity, involving T- and B-lymphocytes, mobilises when there is an antigenic challenge and can be summarised in the terms of: speci-ficity, diversity, memory, and the ability to discriminate between self/non-self antigens. Antigen exposure results in differentiation of B-cells into memory cells and antibody-secreting plasma cells. Due to affinity matura-tion, the process by which B-cells produce antibodies with increased affinity for antigen during the course of an immune response, later exposure to the same antigen results in a response that occurs faster and with higher specific-ity than during the first exposure and is often more effective in neutralising and clearing the pathogen.

When tissues are damaged by for example physical trauma or an invading microorganism the innate immune system becomes activated. As described above, certain molecular components of microorganisms trigger these cells to immediately respond to the intruder. In addition, a variety of chemical mediators are released which enhance the immune response by increasing the influx of phagocytes to the site and by activating complement. Some mediators are released from damaged cells, and others are generated by sev-eral plasma enzyme systems. Among these chemical mediators are proteins called acute-phase proteins, which dramatically increase in concentration in response to tissue-damaging infections. C-reactive protein (CRP) is one of the major acute phase proteins and is released by the liver. CRP has many biological functions, including enhancement of complement activation and induction of cytokines. This sequence of events is referred to as the inflam-matory response.

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Activation of the adaptive immune system is also capable of inducing in-flammation through several mechanisms, e.g. activation of T-cells or by antibody binding to a pathogen. The innate and the adaptive systems do not operate independently in the inflammatory response; instead they interact and function cooperatively. Activation of an innate response results in sig-nalling via cell-to-cell interactions and by release of signalling molecules such as cytokines and chemokines that stimulate and direct subsequent adap-tive responses. Likewise, the adaptive system produces signals and compo-nents that increase the effectiveness of innate responses; upon activation B-cells release antibodies (as part of the humoral immune response) directed against pathogens and by binding to their targets facilitates complement-mediated killing. Conversely, T-cells release cytokines to direct and regulate both innate and adaptive immune responses but might also function as effec-tor cells by the release of cytotoxic substances and expression of death re-ceptor ligands. The inflammatory response is thus important for the organi-sation of a specific immune response to the invasion, but is also important in the repair and regeneration of tissue once the inflammatory response has subsided.

During an inflammatory process both inflammatory and anti-inflammatory cytokines are secreted. To maintain control over the inflammation a local balance between inflammatory and anti-inflammatory factors must be main-tained. If the inflammatory response is insufficient the life of the organism is in danger, while an excessive response might lead to systemic inflammation.

Hypersensitivity reactions Immune activation occasionally results in reactions that are adverse and may be both damaging and cause discomfort, and that in some cases are also fatal for the host. These kinds of reactions may develop in the course of either humoral or cell-mediated responses. In 1963 Gell and Coombs classified the undesirable reactions produced by a normal immune system into four types of hypersensitivities [1]. Mutual to all four types is the prerequisite of the host to have previously encountered the antigen giving rise to the reaction.

Type I is mediated by IgE antibodies that induce the release of histamine and other biologically active molecules in response to antigens. Significant IgE responses are normally only mounted as defence to parasites, but in some individuals abnormalities result in stimulation of IgE production in response to non-parasitic antigens. These antigens are referred to as allergens and the IgE mediated effector functions result in the manifestations related to many of the common allergies, including hay fever, asthma and food aller-gies.

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Type II and III reactions stem from antibodies binding to the patient’s own tissues and can activate both complement and mediate cell destruction by antibody-dependent cell-mediated mechanisms. The antigens recognised can be either naturally occurring autoantigens or they can be extrinsic, such as antigens arising from an infection with a pathogen. If the antibody target is on the surface of a cell or tissue the reaction is regarded as type II hypersen-sitivity. A typical example is autoimmune hemolytic anemia where antibod-ies against the body’s own reticulocytes results in haemolysis through com-plement activation and/or cell mediated killing. Antibody-antigen interac-tions occurring in the circulation may result in formation of IC that when deposited give rise to a type III reaction at the site. Typical manifestations include vasculitis, glomerular nephritis and systemic inflammation.

Type IV reactions are mediated by sensitised T cells which when the anti-gen is recognised release cytokines that activate phagocytic and cytotoxic cells resulting in direct cellular damage. A typical example of type IV hyper-sensitivity is graft rejection.

Autoimmunity The ability to sense the difference between self-antigens (components of the own body, such as nucleic acids and proteins) and non-self structures (for-eign antigens) is central to the development of a healthy immune response. Cells that recognise foreign antigens are retained while cells directed against self-structures are killed or inactivated. As the immune system has great capacity to protect the body from a vast amount of intruders it is dependent on meticulous mechanisms to prevent its destructive abilities from attacking self. In certain individuals or due to certain circumstances this highly regu-lated immune system loses the ability to discriminate between self and non-self, resulting in hyperactive responses against the body’s own cells and tissues. Such autoimmune reactions result in inflammation and tissue dam-age and can lead to many forms of autoimmune diseases.

Autoimmune diseases affects 3-5% of the general population [2, 3]. The clinical disease manifestations are very diverse, involving many different organ systems and can be classified into organ-specific or systemic diseases. In organ-specific diseases the immune reactivity is directed against a certain antigen that is only present in that organ, such as thyroperoxidase and thy-roglobulin in cells of the thyroid gland in autoimmune thyroditis. In auto-immune diseases affecting multiple organs, no particular cell type seems to be targeted. Autoantigenic targets may instead be found in all cell types (e.g. chromatin and centromeres). Common to all autoimmune diseases is the damage to tissues and organs as a result of an immune response mounted against self-antigens.

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The exact causes of autoimmune diseases are largely unknown, but are thought to be dependent on multiple genetic alterations. The properties of the host’s genes affect susceptibility to autoimmune disease at several levels [4]:

Alterations in genes that affect the overall immunoreactivity may predispose an individual to many different types of autoimmune diseases.

The activity of other genes can result in an effect of the alterations in the overall immunoreactivity to be concentrated to a certain an-tigen or tissue.

Genes that act by modulating the target tissue also contribute to susceptibility.

Many of the genetic risk loci that have been associated with autoimmune syndromes overlap in several diseases [5]. Lymphoid tyrosine phosphatase (Lyp) is a powerful inhibitor of T cell activation and is encoded by the PTPN22 gene. Polymorphisms in this gene have been associated with a number of autoimmune diseases such as, SLE, type 1 diabetes (T1D), RA, and Crohn’s disease (CD) [6-8]. STAT4 is a transcription factor involved in cytokine signalling that is important in transducing signals in T cells and monocytes. Alterations in genes encoding STAT4 have been suggested to be involved in the development of SLE, RA, T1D, and in Sjögren’s syndrome [9-11]. In addition to the genes depicted in Figure 1, genes within the MHC region including HLA class I and II is associated with many autoimmune diseases. The class I and II genes encode membrane glycoproteins that pre-sent antigens for recognition by T lymphocytes.

Susceptibility is also heavily influenced by environmental factors that can affect immunoreactivities at all levels (Figure 1). Infections have been im-plicated as potential triggers of autoimmune diseases [12], either through molecular mimicry, causing immunoreactivity against both foreign and self-antigens [13, 14], or indirectly by influencing cytokine production. Infec-tions with Epstein-Barr virus (EBV), one of the most common viruses in humans, has been suggested to have a role in the pathogenesis of multiple autoimmune diseases including SLE, RA, and primary Sjögren’s syndrome [15]. Tobacco smoke has been reported to interact strongly with genetic fac-tors creating a combined risk for the development of RA [16]. Smoking has also been associated with SLE [17]. It is known that smoking can modify immune responses in several ways, including induction of inflammation, immune suppression, altered cytokine profiles, apoptosis, and damage to DNA resulting in formation of anti-DNA antibodies [18].

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Figure 1. Autoimmune diseases are associated with a number of genetic and envi-ronmental factors that also overlap in several diseases, suggesting that common mechanisms may lead to autoimmunity. These factors affect susceptibility at several levels involved in immunoreactivity. Figure modified from [5] and [19]. Data on genome wide associations reviewed in [5]. (CeD: Celiac disease, CD: Crohn’s dis-ease, T1D: Type 1 diabetes, MS: Multiple Sclerosis, SLE: Systemic Lupus Erythe-matosus, RA: Rheumatoid Arthritis)

Autoantibodies Autoantibodies are immunoglobulins (Ig) that bind antigens originating from the same organism. Many (but not all) autoimmune diseases are character-ised by the presence of circulating or tissue-deposited autoantibodies. In some autoimmune diseases autoantibodies even precede diagnosis [19, 20], provide high specificity for a certain disease and may thus be suitable as biomarkers, aiding both in diagnosing the patient and enabling therapeutic intervention very early in the disease process [21]. Pathogenicity of autoan-tibodies results from autoantibodies binding directly to their target organs or by autoantibodies reacting with free molecules forming pathogenic IC (these mechanisms will be discussed in more detail later in this thesis).

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In systemic autoimmune diseases such as SLE, autoantibodies probably arise from disturbances in homeostatic processes related to clearance of dying cells, antigen-receptor signalling or effector- cell functions, whereas in or-gan- specific diseases autoantibody production might be stimulated by local-ised inflammation. Cross-reactivity between microbial targets and self-antigens (molecular mimicry) is also a possibility [22].

Antibodies reactive to self-structure also occur in the healthy individual. These naturally occurring autoantibodies bind both exogenous antigens on various microbes, as well as self-antigens [23], and are referred to as natural antibodies. Due to their polyreactivity to different microbial components they provide an addition to the first-line of defence against infections [24-26]. The properties of these autoantibodies differ from autoantibodies asso-ciated with pathological processes in their ability to bind their target anti-gens. In contrast to pathological autoantibodies which are usually of high affinity, the polyreactive natural antibodies bind at low affinity [23]. In addi-tion, autoreactive cells also take part during the early phases of any normal immune response but are retained from causing prolonged inflammation in the absence of co-stimulatory signals or cytokines needed for evoking an immune response [27].

Systemic Lupus Erythematosus Systemic lupus erythematosus is an autoimmune disease associated clini-cally with symptoms involving several organ systems, including skin rashes, arthritis, pleuritis, carditis, glomerulonephritis and neurological manifesta-tions. The disease is heterogeneous, with various combinations of four of the eleven clinical criteria (summarised in Table 1) required for case classifica-tion [28, 29], although these criteria are mainly used for grouping patients for research purposes. Instead, SLE may be suspected in the clinic if there are signs of systemic inflammation involving at least two organ systems with signs of autoimmune serology, including the occurrence of antinuclear anti-bodies [30] that are typical for SLE and occur in the majority of patients.

The risk for SLE development between monozygotic twins is 20–40% as compared to dizygotic twins and other full siblings with a 2–5% risk [31, 32]. Prevalence is higher in females with a ratio of nearly 10:1 with an in-crease to almost 20:1 among adult women before menopause. The total prevalence in Sweden is around 60/100 000 [33]. Disease exacerbations are often treated with glucocorticoids while disease-modifying drugs such as anti-malarials (hydroxychloroquine), azathiprine, mycophenolate mofetil (MMF), cyclosporine A, and methotrexate are used for maintenance treat-ment [34, 35]. Renal disease is the most severe complication and is the most critical predictor of mortality in SLE [36]. These patients are usually treated with cyclophophamide or MMF [35, 37]. However, in patients with severe

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glomerulonephritis that fail first-line cyclophosphamide therapy, B-cell de-pletion by treatment with anti-CD20 antibodies is an alternative therapeutic intervention [38]. New approaches for treatments that are still in the clinical trial state include biological agents aiming at targeting T- and B -cell interac-tions, cytokines, and B-cell activation.

Immunopathological features including systemic inflammation and vascu-litis, including deposition of IC, are the basics of the pathology underlying SLE. The cooperation between genes controlling immune response, inflam-mation, regulation of transport and clearance of IC, and apoptosis, are im-portant factors in disease development. Environmental factors such as UV-light, infections, hormones and chemical substances also influence the risk of developing SLE. But even though much is known about both the genetics and environmental factors, it is difficult to explain the exact reason for why a certain individual develops SLE, possibly because of a complex interplay between genes and environment causing the disease.

Table 1. The revised ACR classification of SLE is based on 11 criteria. For identifi-cation of patients in clinical studies, a person should be regarded as having SLE if any four or more of the eleven criteria are present, serially or simultaneously, during any interval of observation [28, 29].

Criterion Definition

1. Malar rash Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds

2. Discoid rash Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions

3. Photosensitivity Skin rash as a result of unusual reaction to sunlight, by patient his-tory or physician observation

4. Oral ulcers Oral or nasopharyngeal ulceration, usually painless, observed by a physician

5. Arthritis Non-erosive arthritis involving 2 or more peripheral joints, charac-terized by tenderness, swelling, or effusion

6. Serositis Pleuritis or pericariditis

7. Renal disorder Persistent proteinurea or cellular casts

8. Neurologic disorder Seizures or Psychosis

9. Hematologic disorder Hemolytic anemia or leukopenia or lymphopenia or thrombocyto-penia

10. Immunologic disorder Positive LE cell preparation or abnormal anti-DNA titer or anti-SM presence or Positive finding of antiphospholipid abs on: abnormal serum levels of anticardiolipid abs or false positive test for syphilis.

11. Antinuclear antibodies An abnormal titer of antinuclear antibody by IF or an equivalent assay

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Autoantibodies in SLE The presence of autoantibodies is a hallmark of SLE and is demonstrated many years before the diagnosis of SLE when patients do not show any symptoms. The appearance of autoantibodies also seems to follow a predict-able course, with a gradual increase of specific autoantibodies before disease onset [19].

Autoantibodies directed against nuclear structures such as double- or sin-gle-stranded DNA, nucleoproteins, histones, and nuclear RNA occur in the majority of patients with untreated SLE. Table 2 briefly describes autoanti-bodies occurring in SLE and in other autoimmune diseases.

Table 2. Brief description of SLE-associated autoantibodies (reviewed in [39]).

Autoantibody Antigen Clinical significance Anti-RNP/Sm Group of 9-70kDa ribonuclear- pro-

teins involved in pre-mRNA splicing. Anti-SM: high specificity to dissemi-nated SLE but occurs only in few patients. Anti-RNP: in SLE, MCTD, and other rheumatologic diseases.

Anti-SSA (Ro52 and/or Ro60)

Ribonuclear protein composed of five RNA molecules and a 60kDa protein. The 52kDa protein is also / (discussed to be) associated with the SSA com-plex.

Found in up to 90 % of Sjögren’s, patient. In SLE 50%, neonatal SLE; 100%. Can cause heart block in neona-tal SLE. Isolated Ro52 is not regarded as SSA positivity.

Anti-SSB RNA associated phosphoprotein Found in Sjögren’s (40-80%), (often both SS-A/B). 10-20% in SLE

Anti-PCNA Cofactor of DNA polymerase. Expres-sion is cell-cycle- dependent

Specific (questioned) for SLE but low prevalence

Anti-dsDNA Antibodies against native dsDNA, bind to the backbone of the double helix.

Specific for SLE, besides anti-Sm accounted as serological marker.

Anti-nucleosomes

Functional subunits of chromosomes consisting of histones and dsDNA

Found in both Scleroderma and SLE, using newer tests more specific to SLE.

Anti-histones DNA associated proteins stabilising the double helix

Found in 50-80% of SLE patients (particularly in drug induced) and RA (15-50%)

Anti-ribosomal P-proteins

Subunits of the ribosomes Specific for SLE but found in only 10% of patients

Anti-double stranded DNA and anti-Sm antibodies show high specificity for patients with SLE. The Sm antigen is a small ribonuclear protein bound to a set of core proteins and other proteins associated with the RNA molecule. Anti-Sm antibody titres are usually constant in the patient while anti-DNA antibody titres vary over time and with disease activity [40]. Anti-SSA is traditionally not considered a disease activity marker but a marker of the diagnoses primary Sjögren's syndrome and SLE, in the latter case associated with photosensitivity, interstitial lung disease and homozygous deficiency of complement components C2 or C4. Anti-SSA is also regarded as a marker of

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sub acute cutaneous lupus erythematosus [41], but do not seem to exhibit variations in relation to disease activity [42].

A prominent increase in apoptosis and impaired ability to clear nuclear debris from the circulation is a characteristic finding in SLE probably con-tributing to the broad spectrum of autoantibody formation against these structures. Apoptosis, or programmed cell death, is a highly regulated proc-ess essential for normal function in all multicellular organisms. Under nor-mal circumstances apoptotic cells are rapidly removed through phagocytosis by cells of the innate immune system. Apoptotic death is also suggested to provide antigens that induce specific immunological unresponsiveness to subsequent challenging doses of the antigen and thereby retaining the im-mune system to respond in an unfavourable manner to self [43]. In the later stages of apoptosis nuclear proteins sequester in blebs on the surface of dy-ing cells. During this process potential immunogenic material become avail-able for phagocytosis by antigen presenting cells. Multiple mechanisms are involved in the interactions between the phagocytic and the dying cell, in-cluding: signal transduction that regulate inflammation, receptors on phago-cytic cells, and plasma derived molecules that bind to the apoptotic cell sur-faces. Experimental studies suggest that abnormalities in this process are an important source of autoantigens in SLE [44] and induction of systemic autoimmunity [45].

In SLE patients neutrophils and macrophages have been reported to have a higher rate of spontaneous apoptosis and diminished response to soluble factors, such as IFNγ or extracellular matrix-bound proteins that also precede apoptosis [46]. In addition, the clearance of apoptotic cells is impaired and non-engulfed material tends to accumulate in lymph nodes [47]. Further evidence for the association between defective clearance and the occurrence of SLE include the associations described between genetic alterations affect-ing certain components of the classical complement pathway which are of importance in clearance of cellular debris, as reviewed by Manderson et al [48].

Collectively, the increased exposure of nuclear antigens and a defective clearance is probably of importance for the formation of autoantibodies to these structures in SLE. Hypothetically, autoantigens present in apoptotic blebs are made available to antigen presenting cells presenting these autoan-tigens to T-lymphocytes. This will in turn activate B cells to produce anti-bodies to these autoantigens.

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Leishmania donovani infection Leishmaniasis is a disease caused by protozoan parasites belonging to the genus Leishmania. Several species within the genus cause infections in hu-mans including e.g. L. donovani, L. mexicana, L. major, and L. braziliensis [49]. The different species are morphologically similar but can be distin-guished by DNA analysis or monoclonal antibody reactivities. Human infec-tions may be asymptomatic (i.e. subclinical), cause cutaneous leishmaniasis characterised by skin sores which erupt weeks to months after infection, or may lead to a severe visceral disease in which internal organs are parasitized [50]. The disease is transmitted by parasites residing in sandflies (Figure 2, following page) and is responsible for an estimated half a million deaths each year, making it the second largest parasitic killer after malaria [51]. Visceral Leishmaniasis (VL), also called Kala-azar, is the most severe form of leishmaniasis and is caused by parasites of the L. donovani family, includ-ing L. infantum in Africa, Asia, and Europe and L. chagasi in South Africa [52]. Diagnosis is usually made by aspiration of bone marrow, spleen, or by lymph node biopsy, whereby amastigotes can be detected. Clinical findings are characterised by fever, weakness, weight loss and massive enlargement of the spleen. Although not diagnostic, very high concentrations of IgG are commonly found and might be indicative of infection.

The treatment is usually sodium stibogluconate, a pentavalent antimony compound [53]. The antifungal drug Amphotericin B is used in VL patients not responding to antimony treatment [54]. If left untreated the disease will most certainly result in death of the infected patient. However, with proper therapy the mortality rate is markedly reduced.

Post-Kala-azar dermal leishmaniasis (PKDL) is a complication of VL characterised by severe rashes in mostly young patients who have recovered from VL following antimony treatment but PKDL occurs also during treat-ment. No firm evidence exists that predicts which VL patients will develop PKDL, but an interesting finding is the high expression of IL10 in VL pa-tients subsequently developing PKDL following Leishmania therapy [55].

The susceptibility to VL is different between tribes in the Sudan (personal communication with Amir Elshafie) and development of PKDL is highly divergent in India compared to the Sudan. In Sudan 50-60% of VL patients develop PKDL whereas in India only 5-10% develop the disease [56]. This might be due to differences in the host immune responses against the para-site itself, to the response to treatment, or both. Polymorphisms in genes controlling innate and adaptive immunity have been suggested as possible candidates [57].

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Figure 2. When the sandfly takes a blood meal from an infected host it ingests macrophages containing amastigotes (the form the parasite takes in the vertebrate host). After dissolution of the macrophages, the free amastigotes differentiate into promastigotes in the gut of the sandfly where they multiply and then migrate to the pharynx. During the next bite the parasites are transmitted. The cycle in the sandfly takes approximately 10 days. After an infected sandfly bites a human, the promas-tigotes are engulfed by macrophages, where they transform into amastigotes. The infected cells die and release progeny amastigotes that rapidly invade new macro-phages where they multiply and subsequently migrate to internal organs. The life cycle of the parasite is completed when the fly again ingests macrophages contain-ing the amastigotes. Figure from Wikimedia commons, printed with permission from Wikimedia commons.

Autoantibodies in L. donovani infection Antibodies against self-structures are also demonstrated in infectious dis-eases, the most commonly occurring being rheumatoid factors (RF). These are autoantibodies that are reactive with the Fc-portion of IgG. The classic RF is an IgM antibody with reactivity against IgG-Fc but IgA and IgG RF can also be found. In patients with RA, RFs are detected in sera of a majority of patients [58] but are also demonstrated in other rheumatic diseases as well as in infectious and inflammatory diseases [59, 60].

Antibodies against citrullinated proteins and/or peptides (ACPA) have come forward as a more specific serological marker for RA, with higher diagnostic specificity making it superior to RF in the laboratory diagnostics and is now included in the recently published RA criteria [61]. The event of citrullination of proteins and peptides occurs naturally during inflammation and is a post-translational modification of arginine through deamination [62]. The presence of antibodies against cyclic citrullinated peptides (CCP) has also been demonstrated in a number of infectious diseases, e.g. Pulmo-

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nary Tuberculosis, Hepatitis C viral (HCV) infection, Type 1 Autoimmune Hepatitis and in Leishmania infection [63-68]. Importantly, anti-CCP posi-tivity evident in non-RA disease sera seems to not always be citrulline-dependent as demonstrated in autoimmune hepatitis [66]. Additionally, in HCV-related infections a number of other autoantibodies with other speci-ficities are detectable [69].

There are clinical case reports on autoreactivity in Leishmania infections, including signs of vasculitis with the presence of RF and antinuclear anti-bodies [70]. Polyclonal B-cell activation demonstrated in Leishmania-infected patients is suggested to be associated with the presence of autoanti-bodies against smooth muscles, DNA, and antibodies against various haptens [71]. As a consequence, a strong inflammatory reaction giving rise to a broad production of natural autoantibodies that target known autoantigens might possibly account for at least some autoantibody positivity observed in Leishmaniasis. But although it seems that some infections might yield falsely positive autoantibody tests, there is a possibility that in isolated cases infections could be associated with the appearance of autoantibodies through the mechanisms of molecular mimicry [13, 14]. An appealing hypothesis of a microbial source of autoantigen is immunity to the bacteria Porphyromo-nas gingivalis associated with peridontitis. It has been shown that P. gin-givalis (in contrast to other bacteria commonly found in the oral cavity) is able to citrullinate proteins, suggesting that P. gingivalis-mediated citrullina-tion provides a molecular mechanism for generating antigens that could give rise to autoantibodies to these structures [72, 73].

Immune complexes in health and disease Immune complexes consist of antibodies associated with their corresponding antigens. In the healthy individual IC facilitates removal of foreign antigens from the circulation as a part of the normal immune response, e.g. in con-junction to vaccination and infections [74]. Antibodies forming IC and fac-tors of the complement system that opsonise the material facilitates binding via complement- and IC- interacting receptors and thereby facilitating clear-ance by cells of the reticuloendothelial system [75, 76]. Immune complexes are normally transported along with the erythrocytes to the mononuclear phagocytic system in liver and spleen, where they are cleared from the circu-lation. The complement factor C3b facilitates binding of IC to complement receptors (CR1) on erythrocytes and binding of opsonised IC to erythrocytes also limits direct interaction between IC and circulating leukocytes. As a result of IC formation many immune modulating effects are elicited, exerted through complement activation, Fc-receptor mediated phagocytosis and cy-tokine production.

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In situations with saturating amounts of IC, when erythrocytes are unable to eliminate all antigen antibody complexes, i.e. in SLE and certain infections, cross-linking of Fc- and/or complement-receptors on mononuclear phago-cytes may lead to their activation, with induction of effector functions such as cytokine secretion and phagocytosis to such extent that tissue damage occur. In autoimmune diseases, autoantigen- and autoantibody-containing IC are demonstrated whereas in infectious diseases IC often contains antigens from pathogens and the corresponding antibodies. The magnitude of the reactions depends both on the quantity and size of IC as well as their distri-bution within the body. Antibodies binding a planted antigen forming IC in situ results in a localised reaction whereas IC formed in the circulation result in a reaction developing wherever the complexes are deposited. In particular, IC deposition occurs at sites were filtration of plasma occurs, i.e. kidney, blood vessels, and in the synovium of the joints [77]. In infectious diseases complexes of antibodies and viral-, bacterial-, and parasitic- antigens induce a variety of reactions such as skin rashes, arthritic symptoms and glomeru-lonephritis [77], these symptoms being commonly evident in autoimmune diseases that stem from reactions mediated through IC formed by autoanti-bodies reacting with their corresponding self-structures.

Complement activation The complement system has been known for a long time to be activated in exacerbations of SLE, particularly reflecting nephritic activity. Activation of the classical complement pathway is also demonstrated in Leishmania dono-vani infection [68, 69]. The early steps in activation of the complement sys-tem occur via three distinct pathways, known as the classical, alternative and lectin pathways. The final steps leading to a membrane attack are the same for all three pathways.

Immune complexes activate the complement system through the classical pathway. Activation is initiated by immune complex formation or through binding of antibodies to antigens on a suitable target such as a bacterial cell. The IgM pentamer and the IgG subclasses IgG1, IgG2, and IgG3 are able to activate the classical pathway. When an antigen-IgM complex is formed a conformational change in the Fc portion of the IgM molecule exposes a binding site for the initial classical component C1, consisting of C1q that binds first, and two other molecules held together in a complex. Each C1 molecule must bind by its C1q globular head to at least two Fc sites to obtain a stable C1-antibody interaction. To achieve this, the pentameric form of IgM assumes a configuration exposing at least three binding sites for C1q when an antigen is bound to the IgM molecule. Conversely, an IgG molecule contains only a single C1q binding site in the Fc domain. As a consequence, C1q binding is only achieved when two IgG molecules are within 30-40 nm

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of each other, either as part of an IC (type III reaction) or individually bound to their target tissue (type II reaction).

During the complement cascade that culminates in formation of the mem-brane attack complex, various peptides are generated along the way. Smaller fragments resulting from complement cleavage, C3a, C4a, and C5a, are col-lectively termed anaphylatoxins. These fragments bind to receptors on mast cells and basophils and induce degranulation, with release of pharmacologi-cally active mediators that in turn induce increased vascular permeability and smooth-muscle contraction. With large amounts of immune complexes present this can lead to a tissue-damaging type III reaction.

Patients suffering from hypocomplementemia, with deficiencies in early complement components C1, C2, and C4, are predisposed for the develop-ment of immune complex-mediated diseases. This demonstrates the impor-tance of the early steps in complement activation where C3b is generated. As a consequence, low function of the classical complement pathway reduces the ability of opsonising IC which in turn leads to reduced ability to clear IC from the circulation and subsequently to higher levels of IC. Another form of complement deficiency associated with SLE that also leads to elevated IC levels are the reduced numbers of complement receptors on erythrocytes and macrophages [40, 78], possibly either as a result of excessive burden or by intrinsic factors.

Cellular receptors

Fc-receptors During microbial infections or autoimmune diseases activated B cells pro-duce large amounts of antibodies. Innate immune effector cells abundantly express cell surface receptors (FcRs) specific for the Fc-part of antibodies. Antibody-FcR interactions result in signalling capable of recruiting innate immune effector cells and thus link the specificity of the humoral immune system to the powerful effector functions provided by cells of the innate immune system [79, 80].

The Fc receptor-family consists of different subgroups based on the iso-type of the interacting antibody. The Fc-receptors for IgG (FcγR) comprise the largest family and mediate many biological functions such as phagocyto-sis, antibody-dependent cell-mediated cytotoxicity, induction of inflamma-tory cascades and modulation of immune responses [81-84]. FcγRs are pre-sent on most cell types of the innate and adaptive immune systems, including B cells, T cells, NK cells, macrophages, mast cells, dendritic cells and neu-trophils [85]. Cells having cytotoxic potential expressing these receptors bind to the Fc-part of a particular antibody isotype and thus to the target cells to which the antibody has earlier bound and subsequently mediate cell lysis.

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IC-FcR interactions occurring on macrophages on the other hand lead to production of factors capable to recruit more phagocytic cells to the site.

Fc-receptors for IgG (FcγRI, FcγRII, and FcγRIII) are highly expressed on the human mononuclear phagocytes, granulocytes and monocytes. FcγRI is a high affinity receptor that preferentially binds monomeric IgG and is unique among these receptors in its inducibilty by IFNγ [86]. FcγRII is a low affinity receptor for complexed or polymeric IgG and FcγRIII is a medium-affinity receptor for complexed IgG. Both FcγRII and RIII have sub-types on mononuclear phagocytes: FcγRIIa and RIIIa are stimulatory receptors, while FcγRIIb exhibits an inhibitory function [87]. FcγRIIIb is expressed on neu-trophils and promotes phagocytosis.

Activating and inhibitory receptors are co-expressed and the outcome of the IC/antibody-FcR interaction is a response that under normal conditions is highly balanced to maintain homeostasis [88]. If not, accumulation of IC and phagocytic cells will cause a prolonged inflammatory response damaging the local tissue. The regulation of immune responses by IgG is an area of grow-ing interest, and will hopefully provide mechanisms for how specific anti-bodies formed during infections or autoimmune diseases differently regulate immune responses in the host.

Toll-like receptors The toll-like receptors (TLRs) are a family of receptors recognising struc-tures such as LPS, glycolipids, peptidoglycans, lipopeptides, and nucleic acids, that are shared by large groups of microorganisms and are thus impor-tant in defence against viral, bacterial and parasitic infections [89, 90]. TLRs are located on either the plasma membrane or intracellularly in endosomal compartments in dendritic cells (DCs) and NK cells. T and B lymphocytes also express TLRs. Signals transduced through TLRs cause transcriptional activation that leads to synthesis and secretion of pro-inflammatory cyto-kines such as IFNα, IL12 and TNFα. TLR-1, -2, -4 -5, and -6 are all cell surface receptors for mainly bacterial structures while TLR3 is expressed intracellularly and mediates cytokine production induced by double-stranded RNA, whereas single-stranded RNA is recognised by TLR-7 and -8. Un-methylated bacterial DNA triggers effector functions through TLR9.

Many antigens that are incorporated in IC activate intracellular TLRs and require binding to antibodies in order to reach the endosomal compartment. Recognition starts by interaction between the Fc-part of an IC-bound anti-body with FcγRIIa. This interaction then mediates endocytosis followed by co-localisation of FcγRIIa and TLR7 or TLR9 in the endosome with subse-quent cellular activation [91].

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Cytokine induction Cytokines are involved in a broad array of activities that affect the immune system in numerous ways, including innate and adaptive immunity, inflam-mation and haematopoiesis. The cytokines are often arranged into functional groups depending on their targets and effects, although many play roles in more than one functional category. A general classification is Th1 or Th2 cytokines. Th1 associated cytokines are generally regarded as being pro-inflammatory and enhance cellular responses while Th2 cytokines are anti-inflammatory and mediate antibody production. Th17 cells are considered distinct from Th1 and Th2 cells and are important in defence against certain microbes (e.g. Candida and Staphylococcus). In pathological conditions Th2 cytokines promote B-cell hyperactivity that can cause type II and -III reac-tions, while over-production of Th1 and Th17 cytokines mediate T cell hy-peractivity [92]. In addition, Th17 cells are thought to be particularly impor-tant in development of autoimmunity [93, 94].

A number of cell types respond to IC by cytokine production, including monocytes [95-97], macrophages [98], monocytoid dendritic cells [99] and plasmacytoid dendritic cells (PDCs) [100]. In both SLE and in Leishmania infection the balance between pro- and anti-inflammatory cytokines influ-ences the clinical manifestations.

Cytokines in SLE The cytokines IL4, IL6, IL10, and IL13 stimulate plasma cells to produce antibodies. In SLE increased levels of IL10 [101, 102] and IL6 [103, 104] have been demonstrated and both these cytokines have been shown to be involved in the production of autoantibodies against double-stranded DNA in SLE [105]. There are also Papers reporting a correlation between IL10 and SLE disease activity, either measured as serum levels [106] or as ratios versus other cytokines, for example IFNγ or IL12 [107, 108]. In addition, immune complexes from SLE patients induce IL10 from healthy peripheral blood mononuclear cells (PBMC) through FcγRII-dependent mechanisms [109].

Interleukin-12 is an important cytokine in the physiology of the develop-ment of Th1 cells, being responsible for many cell-mediated functions as well as a strong promoter of the synthesis of IFNγ, which in turn also pro-motes a Th1 responses [110]. Furthermore, IL12 is able to change an estab-lished predominant humoral response to involve more cell-mediated func-tions. Deficient IL12 production is apparent in SLE patients and is thought to be associated with other abnormalities in cytokine production, primarily with an increase in IL10 production [111]. Interleukin-10 promotes Th2 cells by down-regulating the secretion of IL12, which in turn results in a humoral immune response stimulating B cells to produce more antibodies.

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Interferon-α is a cytokine normally produced by PDCs in response to viral DNA or RNA through TLRs and that induces an antiviral state in most cell types by increasing MHC class I expression and activation of NK-cells. Interferon-α is also known to promote survival and differentiation of acti-vated lymphocytes [112, 113], and this could induce loss of tolerance due to activation of autoreactive T- and B-lymphocytes. In SLE serum, levels of IFNα are often elevated [114] and its immune-modulatory effects are of im-portance for SLE pathogenesis as levels correlate both to disease activity and severity [115, 116]. Expression of FcγRIIa has been observed on IFNα-producing PDCs that have been demonstrated to produce IFNα following TLR ligation by SLE IC-containing endogenous DNA and RNA. These IC are endocytosed after FcγRIIa interactions [100, 117, 118]. This suggests a mechanism of immune cell activation and a link between IC and SLE pa-thology in which the involvement of both the autoantigen and autoantibody component of SLE-IC are critical.

The importance of IC in SLE pathology is also further demonstrated by defective Fc-receptor mediated clearance of IC in lupus patients [119, 120] and there are also reports describing a decreased expression of FcγRII on monocytes of SLE patients [121]. In addition, polymorphisms of FcγRIIa are associated with SLE [122].

Cytokines in Leishmania infection Unfavourable clinical outcomes of Leishmania infection have traditionally been associated with either Th2 dominance or a defective Th1 response. However, more recent studies instead argue that other immunosuppressive or immune-evading mechanisms contribute to pathogenesis, as reviewed in [123]. Reports have implicated TLR2 in recognition of immune responses to the surface molecule lipophosphoglycan on Leishmania parasites [124] al-though the L. donovani parasite in some way evades the induction of in-flammation normally induced by TLR ligation. The strategies adopted by the parasite to silence TLR2-stimulated pro-inflammatory responses are not well understood.

Numerous studies in mice have demonstrated that the production of IL12 by APCs and IFNγ by T cells are required for control of parasite reproduc-tion [125-128]. Furthermore, the balance between IL10 and IL12 and/or IFNγ is of importance for acquired resistance to the parasite [129, 130]. Se-cretion of IL10 as a result of ligation of FcγRs by IgG bound to Leishmania amastigotes plays a crucial role during development of infection by inhibit-ing macrophage activation and thereby contributing to parasite growth [131]. In gene deleted mice lacking FcγRs, enhanced control of Leishmania infec-tion is demonstrated by a reduction in numbers of cells producing the anti-inflammatory cytokines IL4 and IL10, although the ability of Leishmania to parasitize cells is maintained [132].

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Levels of circulating IC are increased in chronic leishmaniasis [133] and immune suppression is often evident in parallel to maximal IC load and can be measured as depression of humoral immunity and delayed type hypersen-sitivity [134] as well as by depressed mitogen responses [135]. The immune suppression might depend on IC-induced production of IL10 [136]. As there are high loads of circulating parasite antigens during acute infections, and a concomitant increase in IC levels, the immunosuppressive activities induced by IC might thus promote parasite replication and disease progression.

The hematopoietic growth factor GM-CSF has many stimulatory effects on monocytes/macrophages that are helpful during intracellular infections, including enhancement of phagocytic and metabolic functions and release of other pro-inflammatory cytokines [137]. In a small placebo controlled study Leishmania chagasi-infected patients treated with GM-CSF had a signifi-cantly reduced number of secondary infections, along with increased neutro-phil counts [138]. In experimental studies addition of GM-CSF to human monocytes in vitro increased their leishmanicidal effects [139] and in BALB/c mice production of GM-CSF is increased during infection [140], arguing for potentially disease-limiting effects of Leishmania infection-induced GM-CSF.

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Aims

Paper I Paper I was a follow-up on the Paper by Rönnelid et al [109] in which IL10 induction by IC from SLE patients was demonstrated to be dependent on FcγRII. The aim of Paper I was to investigate the influence of complement activation and patient autoantibody profile on the properties of IC from SLE patients to induce cytokine production. The second aim was to examine the relationship between IC-induced levels of IL10 and IL12.

Paper II The findings in Paper I indirectly implicated anti-SSA in immune complex formation, but did not formally prove that anti-SSA antibodies participate in the formation of SLE IC to a greater degree than other ANA-associated autoantibodies. In Paper II the aim was to analyse the enrichment in IC of a larger panel of SLE-associated autoantibodies. Secondly we wanted to inves-tigate whether certain autoantibodies accumulate in circulating IC during SLE flares.

Paper III To explore IC-induced cytokine production in Sudanese patients with Vis-ceral Leishmaniasis, the most severe form of Leishmania donovani infection. In addition, I wanted to study the possible effect of antimony treatment and disease activity on IC formation.

Paper IV To investigate the presence of the autoantibodies anti-CCP and RF in Suda-nese patients infected with the Leishmania donovani parasite and to see if there was an association between IC levels and the occurrence of autoanti-bodies.

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Methods

Patients In Paper I, anonymised serum samples were obtained from patients with a clinical diagnosis of SLE and who had previously been investigated concern-ing autoantibody profile, classical complement function and levels of C3 and C3d as measures of disease activity.

For Paper II sera were collected from SLE patients chosen to represent di-vergent autoantibody profiles. Nine of the patients were sampled during both inactive and active disease. Four pairs were obtained from patients with SLE nephritis among whom the inactive sample were defined by a 50% reduction in disease activity as determined by SLEDAI (SLE disease activity index) [141, 142]. Five of the serum sample pairs were obtained from patients with SLE who had previously been investigated concerning classical complement function and levels of C3 and C3d as measures of disease activity. Among the latter, the more inactive disease state was defined by sera having 50% decrease in the C3d/C3 ratio and 50% increased classical complement func-tion, or 100% amelioration of any of the two activity measures.

The Leishmania-infected patients in Papers III and IV were obtained from Tabarakalla rural hospital in Gadarif state, along the lower Atbara River in Gallabat Province, eastern Sudan. The area is located 70 km southeast from the Gadarif town (map shown in Figure 3, following page) and is endemic for L. donovani. The samples were collected by my colleague Amir Elshafie who performed a detailed clinical history of all patients including tribe, resi-dence, occupation, marital status, medical treatment, abdominal pain, vomit-ing, nausea, previous history of bleeding tendency, urinary tract infection and insect bites, and family history of VL, hypertension or diabetes mellitus. A general clinical examination was then conducted with particular reference to simultaneous enlargement of liver and spleen (hepatosplenomegaly, HSM),

enlargement of lymph nodes and recurrent fever for more than one month. Swollen/enlarged lymph nodes were classified as being ‘localised’ if only found at one site and ‘generalised’ if found at two or more sites. The oral and nasal mucous membranes were clinically examined for evidence of mucosal leishmaniasis. Thick and thin blood films for detection of Plasmodium para-sites were examined from all individuals who either had fever, looked ill, or

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had splenomegaly, and those with positive blood films for malaria were ex-cluded. An inguinal lymph node aspiration was performed on those clinically

suspected of having VL (i.e. all individuals with fever for more than two months, left upper quadrant pain, lymphadenopathy, splenomegaly, or wast-ing). Those with a negative result underwent bone marrow aspiration from the superior posterior iliac crest. The smears were fixed with methanol, stained with Giemsa, and examined using an oil-immersion lens. As there are no laboratory tests for diagnosing PKDL those patients were diagnosed on clinical grounds, on the appearance and distribution of the rash after anti-mony treatment in previously diagnosed VL patients.

Figure 3. The Leishmania-infected patients in Papers III and IV were sampled by my colleague Amir Elshafie at Tabarakalla rural hospital in Gadarif state. The area is situated along the lower Atbara River in Gallabat Province, eastern Sudan. The area is located 70 km southeast from the Gadarif town.

Immune complex purification and measurement PEG precipitation of IC Precipitations of IC from sera were performed in accordance to an earlier described method [19]. In brief, serum samples were mixed with ice-cold 5% (w/v) polyethylene glycol (PEG-) 6000 and incubated overnight at 4oC. The following day serum samples were diluted three times with 2.5% PEG 6000 in phenol red-RPMI. The diluted sera were then added on top of 2.5% PEG 6000 supplemented with 5% (w/v) human serum albumin and centrifuged. In this combined procedure the precipitates are purified and free antibodies and antigens are washed away. After centrifugation the precipitates were diluted to the initial serum volume and immediately used in cell culture experiments.

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C1q-binding assay for isolation of circulating IC Immune complexes were isolated from sera using C1q coated plates (Bin-dazyme C1q binding kit; Binding Site, Birmingham, UK). Serum samples were diluted 1:10 in the sample buffer provided by the manufacturer before addition to C1q coated plates for one hour at room temperature (RT). Plates were then washed three times before addition of elution buffer (25% metha-nol pH 11.2) for two minutes under mild shaking. The elution condition used has been shown to effectively break IC formed with low-molecular weight organic molecules without causing loss of antibody recovery or activity [143]. Eluted samples were instantly brought to neutral pH by dilution in the sample buffer used for autoantibody analysis. Samples were then immedi-ately analysed for autoantibody content.

Measurement of levels of circulating IC Levels of circulating IC were measured by a solid-phase C1q assay (Bin-dazyme C1q binding kit; Binding Site, Birmingham, UK) according to the instructions from the manufacturer. The range of the assay was 1.23-100 Eq/mL.

Preparation of mononuclear cells and culture conditions Buffy coats were obtained from healthy blood donors and separated over a Ficoll-Hypaque gradient. After two washes in PBS the cells were suspended in RPMI-1640 supplemented with 1% glutamine, 1% HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 1% PEST (Penicillin-Streptomycin). Cells were cultured in a serum-free system using 4% Ultroser G as supplement. Ultroser G is according to the manufacturer free from cy-tokines and complement factors. Cell suspensions were diluted to 1*106 peripheral blood mononuclear cells (PBMC)/mL with this complete medium for cell culture experiments.

The responses to IC are known to vary among individuals, with some do-nors yielding low responses. Therefore cells from two different PBMC do-nors were always investigated in parallel and thereafter data from the re-sponder yielding the strongest responses was used for statistical analyses.

Complement activation as a marker of disease activity In Papers I and II, complement activation was used as a marker for active SLE. Functional activity of the classical pathway was measured according to the protocol by Nilsson and Nilsson [144] in which patient and control sera

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are diluted 1:5 and mixed with a 40% solution of sheep erythrocytes coated with rabbit IgM. The reaction is stopped by the addition of 0.01M EDTA and samples are centrifuged. Each supernatant is then added to 96-well plates and the absorbance is measured. The function of the classical pathway is then defined by the ratio of hemolysis (measured as absorbance at 540nm) in the patient samples and in the control serum pool. Levels of C3 were measured using rate nephelometry (Immage; Beckman Coulter, Stockholm, Sweden) according to the manufacturer's instructions. C3d levels were also measured using rate nephelometry and an unconjugated polyclonal rabbit anti-C3d antibody (A0063; Dako Sweden AB, Stockholm, Sweden). Prior to C3d measurements the sera were mixed with 20% PEG 6000 and incubated for 90 min at 4°C before centrifugation at 1920g for 30 min at 4°C.

If disease activity is to be assessed in a patient with unknown diagnosis but where SLE can be suspected, complement levels provide a rather poor measure, as complement protein levels are likely to vary among different individuals. However, when assessing samples from the same individual taken at different time points, changes in complement activity can reflect changes in disease activity. Assessment of split products such as C3d associ-ated with activation of complement is also more accurate than assessments of total C4 and C3 levels. It has also been reported that C3d, and particularly the C3d/C3 ratio, provide sensitive markers for disease activity in SLE [145]. In our studies patients with decreased function of the classical com-plement pathway and/or raised C3d/C3 ratios were regarded as having active disease.

Cytokine enzyme-linked immunosorbent assays (ELISA) PBMC cultures were incubated for 20 hours after the newly precipitated IC were added. Supernatants were subsequently analysed using ELISA for the measurement of IL10, IL6, IL12p40 IL1β, IL1ra, TNFα, TNF-Rp75 and GM-CSF. All analyses had been established in the laboratory for the meas-urement of IC-induced cytokine responses in vitro and are described in detail elsewhere [95-97, 99, 109, 146]. Mouse monoclonal antibodies were used for capture. For detection, biotinylated polyclonal goat antibodies were used for TNFα, IL1β, and IL1ra. For TNF-Rp75, IL6, GM-CSF and IL12p40, biotinylated monoclonal mouse antibodies were employed. Measurements of IL10 with ordinary IL10 antibodies had shown some IL10 contents in PEG precipitates. As this might be an artefact caused by rheumatoid factor-like antibodies in the precipitates, we instead used F(ab´)2-fragments of antibod-

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ies for detection and capture of IL10 in studies I and III. None of the other cytokines were detected in IC preparations (unpublished data).

Immunoglobulin G ELISA Total levels of IgG were determined using an in house sandwich-ELISA for immunoglobulin detection. Fab´2 fragments of an Fcγ-chain specific rabbit anti-human IgG (309-006-008, Jackson Immune research Lab. Inc., West Grove, USA) antibody were used for coating of ELISA plates (Nunc Max-isorb, Roskilde, Danmark). For detection alkaline phosphatase (ALP)-conjugated goat Fab´2 antibody (109-056-098, Jackson Immune research Lab. Inc.) against human γ-chain were used. Samples were diluted in PBS-Tween 20 before being analysed and a serum with known immunoglobulin concentration was used as a standard. All analysed samples were at dilutions within the linear range of the standard curve. This ELISA has been used in earlier studies of PEG precipitated IC in samples from serum and synovial fluids from RA patients [95].

Functional blocking of Fc-receptors For blocking experiments anti-FcγRII mAb (IV.3 (Fab); Medarex, Princeton N.J, USA) or anti-FcγRIII (3G8 (F(ab')2); Medarex) were added to the cell cultures and left to stand at 4°C for 30 min before addition of dissolved PEG precipitates. The antibody concentration used was 1.5µg/ml as preliminary experiments had demonstrated an equivalent blocking effect using either 1.5 or 4 µg/ml. The IV.3 antibody has earlier been shown to react specifically with FcγRIIa [147, 148].

Detection of autoantibodies Immunofluorescence and immunodiffusion techniques for ANA detection In Paper I ANA and anti-dsDNA antibodies were analysed utilising im-munofluorescence (IF) microscopy on HEp-2 cells and Crithidia luciliae, respectively (both from Immunoconcepts, Sacramento, CA, USA). Antibod-ies against SSA, SSB, U1-snRNP and Sm were analysed by double radial immunodiffusion (DID) for 48 h (Immunoconcepts). These analyses were performed at the routine clinical immunology lab at Uppsala University hos-pital.

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Line blot assay for anti-nuclear antibodies A commercial line blot assay (Euroline ANA profile 3; Euroimmune, Lübeck, GER) containing the SLE- associated autoantigens: RNP/Sm, Sm, SSA/Ro60, SSA/Ro52, SSB, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosomes, histones, and ribosomal P antigen was used to meas-ure autoantibody content in serum and in parallel IC. Samples were analysed according to the instructions from the manufacturer. In brief, diluted (1:100) samples were incubated together with strips coated with parallel lines of purified antigens. After three successive washes, bound antibodies were detected in a second incubation step using enzyme-labelled anti-human IgG. The washing step was repeated before staining was performed for up to 30 strips in parallel using automated equipment (EuroBlotMaster, Euroimmun). Paired samples obtained at different occasions and PEG precipitates or C1q isolated IC obtained from such paired sera were always investigated in paral-lel. The intensity of the reaction for each positive band was determined with densitometry using the EuroLineScan software (Euroimmun). The recom-mended cut-off (10 densitometry units) was used to define which autoanti-body specificities were present in sera, PEG precipitates and IC isolated by C1q binding.

To compare levels of specific autoantibodies in the patient’s sera and in PEG-precipitates autoantibody levels were normalised against the total level of IgG in each sample. Autoantibody enrichment was defined by the ratio between the normalised autoantibody levels in PEG-precipitate and sera.

Measurement of anti-CRP Autoantibodies to CRP were measured as described previously [149]. In brief, ELISA plates were coated with native human CRP (Sigma, St Louis, MO, USA). Patients sera diluted in PBS was then added and incubated for 60min. An ALP-conjugated rabbit anti-human IgG, specific for γ-chains was used for detection. Results were expressed as the OD at 405nm after subtrac-tion of background OD obtained on uncoated plates. A reference sample from an SLE patient at flare was always included. The cut-off value for posi-tive samples was calculated from the 95th percentile obtained in a control cohort. These experiments were performed by Christopher Sjövall, Linköping University.

Measurment of anti-CCP Anti-CCP was measured using the Immunoscan RA Mark 2 assay (Euro-Diagnostica, Malmö, Sweden). Anti-CCP positivity was determined in ac-cordance with manufacturer’s instructions with 25 U/ml used as a cut-off. The assay does not yield quantitative levels below the company-defined cut-

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off. For that reason we extended the standard curve to obtain values also below 25 U/ml (extended range 3.126-1600 U/mL).

A control ELISA plate with cyclic peptides containing arginine instead of citrulline in the relevant peptide positions was generously provided by Jör-gen Wieslander at Euro-Diagnostica and was used to evaluate citrulline- specific reactivity. The cut-off value for the arginine control was determined arbitrarily by the absorbance corresponding to 25 U/mL in the standard curve for the citrulline (CCP) variant. Results were then calculated as cut-off index (COI): observed arginine OD450 / citrulline cut-off OD450 as according to Vannini et al [66]. To test if IC present in the investigated samples influ-enced anti-CCP reactivity we adsorbed C1q binding IC from sera and evalu-ated CCP reactivity afterwards. Sera were diluted 1:50 and incubated for 2 hours on C1q coated plates (Bindazyme C1q binding kit; Binding Site, Bir-mingham, UK). The samples were directly after incubation transferred to the CCP plate and analysed for anti-CCP according to the manufacturer of the anti-CCP test kit.

Measurement of RF RF was measured by nephelometry (Immage, Beckman Coulter), and ex-pressed in international units/mL (IU/mL) with values >20 IU/mL regarded as positive. The analysis was standardised using the NIBSC 64/002 reference serum. The nephelometer do not express quantitative data below 20 IU/mL, and RF negative samples were given the value 0 IU/mL when comparing different groups.

Statistics The Mann-Whitney U-test was used for comparisons between groups in unpaired design, whereas the Wilcoxon signed-rank test was employed for paired comparisons in Papers I, II, III and IV. For correlations both Pear-son’s (Paper IV) and the Spearman's rank correlation test was used (Paper I, II, and III). Differences between proportions were analysed using both the chi2-test and Fisher’s exact test for smaller groups. Two-way analysis of variance (ANOVA) was used to investigate the combined effects of dual variables in Papers I and III. P-values below 0.05 were considered signifi-cant.

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Results and discussion

Paper I In Paper I it was demonstrated that increased complement activation, both measured as decreased function of the classical complement pathway and as raised C3d/C3 ratios, correlated with increased IC-induced IL10 production. In addition, decreased levels of C3 were associated with increased IL12p40 production. When samples were divided by the presence of anti-SSA and anti-SSB, it was revealed that PEG precipitates from patients positive for both antibodies induced higher levels of IL10 and IL6 as compared to PEG-precipitates from sera lacking those antibodies. The autoantibody effect was specific for anti-SSA and anti-SSB, and could not be shown for the other SLE-associated autoantibodies anti-dsDNA, anti-U1-snRNP or anti-CRP.

Precipitation using PEG to obtain IC is particularly suitable for the rela-tively small blood volumes used in these investigations. The quick procedure also facilitates experiments whereby cells from the same donor are stimu-lated with relatively large numbers of precipitated IC-samples in parallel. Successive centrifugations to remove free antibodies and antigens could probably result in dissociation of small or loosely bound IC. Even though the combined washing/purification step removes most of un-specifically precipi-tated molecules there is a risk of carrying over of other high molecular weight proteins in parallel to IC when treating serum samples with PEG [150, 151 ]. Nonetheless, the correlation between levels of C1q-binding cir-culating IC and levels of cytokines induced by PEG precipitates, and the fact that the levels of serum PEG precipitate-induced cytokines can be specifi-cally reduced by pre-treatment of the responder PBMC with blocking anti-bodies against FcγRIIa [95], both argue for IC-mediated effects.

It would not have been feasible to perform cell culture experiments to analyse the impact of antibody status and complement function in a larger number of samples than was analysed concerning IC-induced cytokine pro-duction. We therefore instead examined levels of circulating IC measured by C1q binding. Both classical complement function and the occurrence of anti-SSA influenced circulating IC levels, with a strong interaction in ANOVA analysis between decreased complement function and the occurrence of anti-SSA antibodies. In our experiment this interaction was only evident as an effect of anti-SSA in serum samples with complement activation and not in samples with normal complement profiles. No corresponding association

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with complement and IC levels was recorded for anti-dsDNA, the combina-tion of anti-SSA and anti-SSB, anti-U1-snRNP or for anti-CRP antibodies. None of the anti-SSA positive patients had any known homozygous defi-ciency for C2 or C4 as this could have caused a bias in the analysis. In the case of complement deficiencies complement function values in the analysis are zero or close to zero but no such values were observed among the inves-tigated patients.

When SLE samples were analysed regarding antibodies against CRP 40% (50/125) had increased levels as compared to healthy controls. The elevated levels were associated with decreased C3 levels and increased C3d/C3 ratios suggesting a relation to complement activation. It has earlier been reported that that anti-CRP antibodies are associated with SLE disease activity [152] and thus the associations demonstrated here confirm these previous findings. The prevalence of anti-CRP antibodies in this study also concords with pre-vious observations [152, 153]. Anti-CRP antibody levels did not differ be-tween SLE sera with and without ANA-associated autoantibodies, elevated levels of CRP or IC levels. Immune complexes from SLE patients have been demonstrated to contain CRP [154]. Consequently, a positive anti-CRP anti-body test could be explained by the presence of circulating IC. Nonetheless, in this study we demonstrated that PEG-precipitated and re-solubilised IC from SLE sera did not induce falsely positive anti-CRP tests in our analysis.

Others have demonstrated the interplay between in vitro production of IL10 and IL12 by PBMC from SLE patients to correlate inversely [111, 155-157], but the converse is also reported [158]. In this study we determined either positive or no correlation between IC-induced levels of IL10 and IL12p40. Our data imply that the earlier described inverse correlation be-tween IL10 and IL12 in SLE does not depend on circulating IC. One must also take into account not only the degree of complement activation in vivo during creation of IC, but also to consider the availability of an intact com-plement system during cell culture studies. This concept has been studied earlier in our research group [96, 146]. In our present experimental set-up we circumvent this by using a serum-free cell culture system.

Immune complex-mediated activation of the classical complement path-way is a hallmark of flare and ongoing organ damage in SLE and studies support that anti-dsDNA antibodies are involved in this process [159]. Ac-cording to the literature it seems that levels of other SLE-related autoanti-bodies, such as anti-SSA/SSB, do not exhibit variations in relation to disease activity [42]. Our results imply that in active SLE, anti-SSA and anti-SSB antibodies forming IC with antigens released from apoptotic cells result in complement activation and leukocyte production of cytokines. Conversely, in quiescent disease autoantigens are not released, leaving the circulating autoantibodies uncomplexed.

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To summarise; anti-SSA and anti-SSB antibodies are regarded as disease markers, but have not previously been associated with disease activity in SLE. The result of the present study indicates that these antibodies might be of importance in formation of circulating IC, with my hypothesis that anti-SSA and possibly anti-SSB antibodies participate directly in the inflamma-tory process in SLE by enhancing IC formation and subsequent cytokine production.

Paper II Findings in Paper I implicated anti-SSA in immune complex formation, but the formal proof that anti-SSA participates in the formation of circulating IC in SLE to a greater degree than other ANA-associated autoantibodies was lacking. I therefore investigated to what extent different autoantibodies ac-cumulated in SLE IC, and whether such accumulation was dependent on SLE disease activity.

To analyse to what extent the different autoantibody specificities accumu-lated in circulating IC, the levels in serum and PEG-precipitates were nor-malised against total IgG in each sample. Enrichment was then defined as the ratio between the normalised autoantibody levels in PEG-precipitate and in sera. All investigated autoantibody specificities except anti-dsDNA were enriched in circulating IC as compared with parallel SLE sera. The RNA- associated autoantibodies, and in particular autoantibodies against RNP/Sm and SSA/Ro52, exhibited the highest degree of enrichment in SLE PEG pre-cipitates. To further confirm our data I also analysed the autoantibody speci-ficities in IC bound to solid phase C1q. Immune complexes bound to C1q only contained autoantibodies directed to RNP/Sm, Sm, SSA 60kDa, Ro52, SSB, and nucleosomes; except the latter these are the same antibodies that had the highest levels of enrichment in IC isolated by PEG precipitation. The defined types of autoantibody accumulating in IC were thus in concordance using both methods.

The first aim of this study was to characterise the relative enrichment in IC of various SLE-associated autoantibodies. Patient selection was thus in the first place based on the diversified presence of autoantibodies. The sec-ond priority was to find paired sera from active and inactive SLE respec-tively, representing these various autoantibodies. Paired samples were col-lected from two different cohorts, one from the Department of Rheumatol-ogy at Uppsala University Hospital and the other from Karolinska University Hospital at Solna, Stockholm. Definition of active or inactive disease was based on markedly different degree of complement activation in the Uppsala patients and on SLEDAI in the Stockholm patients. Samples from active and inactive disease differed concerning levels of circulating C1q-binding IC, but no difference was noted in anti-dsDNA levels. This can perhaps explain

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the fact that I determined no differences in autoantibody enrichment between active and inactive SLE as I had hypothesised that at least anti-SSA autoan-tibodies would show a more pronounced enrichment in IC during times of SLE flares. The acquisition of these paired samples representing different autoantibody profile was difficult, however.

Immune complex-mediated inflammation is typical for SLE flares and ongoing organ damage and particularly with kidney involvement. Many studies support that anti-dsDNA antibodies are involved in SLE nephritis [159]. Three main hypotheses exist concerning the deposition of anti-dsDNA in kidneys: as preformed circulating IC, or as monomeric antibodies reacting either with planted nuclear antigens or cross reacting with kidney autoanti-gens [160]. In general, the two latter hypotheses concerning deposition of non-complexed autoantibodies are brought up [161]. No such theories have been published concerning other SLE-associated autoantibodies. The pres-ence of autoantibodies with other specificities in addition to anti-dsDNA, including antibodies to Sm, SSA, SSB, the collagen-like region of C1q, and chromatin have been demonstrated in glomeruli from SLE patients with kid-ney involvement [162]. The same study also reported that the enrichment of antibodies to Sm, SSA, and SSB in kidney extracts exceeded that of antibod-ies to dsDNA. In our present study of circulating IC, the same RNA-associated specificities (RNP/Sm, Sm, SSA/Ro60, SSA/Ro52, and SSB) were more enriched than the anti-dsDNA antibodies, possibly reflecting that different mechanisms are involved in the localisation in SLE kidneys of autoantibodies with different specificities.

Levels of anti-dsDNA in IC have also been shown to decrease very early during SLE flares, possibly due to tissue deposition of anti-dsDNA antibod-ies [163]. In addition, positively charged antibodies [164], especially anti-dsDNA antibodies [165] bind more strongly to glomerular basement mem-brane compared to non-cationic antibodies in SLE. There are also several studies demonstrating that anti-dsDNA antibodies become more cationic during flares [166-168]. As a consequence, anti-dsDNA may bind their tar-get forming IC in situ in the kidney and do not form IC in the circulation to the same extent as the RNA-associated autoantibodies. Tron et al. have re-ported that DNA-anti-DNA complexes only constitute a minor part of circu-lating IC detected by PEG-assay [169], suggesting either that other antigen-antibody systems constitute the major IC components in SLE sera, or that the assay utilised did not detect IC-bound anti-DNA. These data are therefore in agreement with our finding of low enrichment of anti-dsDNA and other autoantibodies against DNA-associated autoantigens, as compared to autoan-tibodies against RNA-associated nuclear proteins.

Hypothetically, the pathogenicity of the RNA-associated autoantibodies forming IC in the circulation could result from a type III reaction with com-plement activation and ensuing inflammation due to leukocyte infiltration at

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the site of IC deposition. Anti-dsDNA, conversely, might cause tissue dam-age as a consequence of a type II reaction occurring at the antigenic target organ of the autoantibody.

Paper III In Paper III we observed that IC precipitated from Leishmania infected pa-tients induced significantly higher levels of GM-CSF and IL10, as well as of IL6 and IL1 receptor antagonist as compared with precipitates from matched healthy Sudanese controls. The induction of IL6, IL10 and GM-CSF was most prominent. The induction of GM-CSF by PEG-precipitated IC was especially prominent in acute VL patients receiving sodium stibogluconate treatment and a parallel association with the level of circulating IC in sera measured by C1q binding was demonstrated.

When performing ANOVA on the data, elevated levels of C1q binding IC and GM-CSF were associated both with acute VL and with ongoing sodium stibogluconate therapy. Levels of both IC and GM-CSF displayed a strong interaction with the two variables (therapy and disease activity) in the analy-sis. This infers that active disease and ongoing therapy in synergy predispose to elevated levels of IC in sera and induction of GM-CSF by precipitated IC.

Numerous studies have described increased levels of IC containing parasite antigens during Leishmania infection [55, 170-176]. The cytokine GM-CSF induced by Leishmania antigens [177] might be either beneficial by activat-ing macrophages to become leishmanicidal [178, 179], or detrimental by stimulating a Th1-driven DTH response and possibly also facilitating Leishmania-associated kidney engagement [180-182]. Earlier longitudinal studies on IC levels in treated VL patients have shown decreasing IC levels after institution of sodium stibogluconate therapy and one earlier study [171] compared levels of IC before and 1.5 months after start of therapy, whereas our patients were sampled during the very first days after treatment start. This concords with our study in which patients that were currently not under treatment but have had treatment earlier (more than 1.5 months ago) dis-played lower IC levels. Presumably, shortly after antimony therapy there are most likely a massive number of parasites being destroyed, releasing large amounts of antigen into the circulation and increasing the load of IC.

Patients with active VL most probably have higher parasite burdens than do VL patients with subacute disease. This could in turn account for the dif-ferences observed in IC levels and PEG IC-induced levels of GM-CSF be-tween patients with acute and subacute VL. The synergistic effect between levels of C1q-binding IC and IC-induced cytokine responses on the one hand and disease activity and ongoing therapy on the other might also reflect this. The increase in IC levels and IC-induced GM-CSF very early after sodium

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stibogluconate treatment indicates the importance of longitudinal studies including the very first days after treatment. Level of IC responses the very first days after treatment could possibly correlate to clinical symptoms such as skin rash and signs of kidney involvement.

Paper IV In this study we have demonstrated that Sudanese patients infected with the Leishmania donovani parasite exhibit serological similarities with RA pa-tients from Sudan and elsewhere.

Among VL and PKDL patients the majority (86% and 69% respectively) were RF positive. Among healthy Sudanese controls 11% had RF levels above the cut-off and in a cohort of healthy Swedish controls 2% were re-corded. The difference in RF levels between the healthy control groups was far from being statistically significant (p=0.87). When we assessed the levels of RF between the other groups we determined that the VL group had higher RF levels as compared with PKDL patients, but there was no difference be-tween the Sudanese RA and VL patients. However, RA patients had higher levels as compared to PKDL patients. Each of three disease groups had sig-nificantly higher RF levels than the healthy Sudanese controls.

Levels of C1q binding IC were determined in all groups demonstrating that the VL patients had the highest levels as compared to both PKDL and RA patients. There was no difference between PKDL and RA patients al-though both groups were higher than both Sudanese and Swedish healthy controls. There was no difference between the two healthy control groups.

Among the VL patients, 12% were found to be anti-CCP positive. Anti-CCP reactivity was also detected in 4 % of the PKDL patients. In the Suda-nese healthy control group one anti-CCP positive was identified and in the Swedish control group 3/100 were positive. The levels of anti-CCP among VL patients correlated well to the levels of C1q binding IC. In the anti-CCP positive RA group no such association was evident. To rule out the possibil-ity that anti-CCP positivity was due to cross-reactions with IC, or that the CCP-reactivity might be bound to IC, I adsorbed C1q-binding IC from sera and evaluated CCP reactivity afterwards. This procedure did not diminish the anti-CCP reactivity in either the VL group or among anti-CCP positive RA patients.

The citrulline specificity among anti-CCP positive patients was analysed using a control plate containing non-citrullinated cyclic peptides as target antigen. Among the anti-CCP positive samples in the VL group there was no difference in reactivity against CCP and the non-citrullinated control pep-tide. The same pattern was determined for the two anti-CCP positive PKDL patients. This non-citrulline specific reactivity was in strict contrast to the anti-CCP positive Sudanese RA patients in which anti-CCP reactivity was

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specific for CCP and with very low reactivity against the arginine-containing control peptides.

An interesting observation is that two groups of infections in which a non-citrulline-specific and non-arthritis-associated ACPA response has been reported represent microbes localising intracellularly in tissue macrophages. Both Leishmania parasites [68] as well as tuberculosis [63, 67] represent intracellular infections in tissue macrophages. The findings of ACPA re-sponses in hepatitis C [64, 65, 183] where the infection primarily resides in the hepatocytes might not seem in accordance with such a hypothesis. It has been suggested, however, that macrophages are also infected by hepatitis C virus, as reviewed in [184]. Furthermore, autoimmune hepatitis type 1 in which arthritis-independent ACPA reactivity also has been reported [66] is associated with macrophage activation both in the early [185] as well as in later fibrotic stages [186].

The anti-CCP reactivity among RA patients is most often either totally negative or at very high levels. In the present study the mean anti-CCP reac-tivity among positive VL patients was 49 U/ml, representing 1.96 times the cut-off value. This is in contrast to Swedish anti-CCP positive RA patients exhibiting a mean level of 1128 U/ml, 45.1 times the cut-off using the same commercial test [187]. Together with our finding that CCP reactivity in VL patients was not restricted to citrulline; this argues that the anti-CCP reactiv-ity in VL is an effect of the extensive inflammation and immune activation more than a sign of shared pathogenic characteristics with anti-CCP positive arthritis. The lower levels of the anti-CCP positive samples among VL pa-tients could also be due to naturally occurring autoantibodies, which bind with lower affinity [23] and are possibly expanded through polyclonal B-cell activation in these severely ill patients [71]. The strong association between C1q-binding IC levels and anti-CCP among VL patients might also mirror the suggested relation to the strong inflammatory response. Intriguingly, in a study of hepatitis patients [183] the two patients exhibiting (non-specific) ACPA reactivity were the ones having IC in the form of cryoglobulins.

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General conclusions and future perspectives

In both SLE and in infections with L. donovani large amounts of potentially immunogenic material are present in the circulation and are believed to have impact on disease pathogenesis. In SLE autoantigens from dying cells are abnormally exposed to the immune system as a consequence of deficiencies in regulation of apoptosis and removal of apoptotic material, whereas in Leishmaniasis, large amount of parasitic antigens are possibly released shortly after antimony therapy. Formation of IC is a natural consequence in the process of removal of these antigens, and by interactions with the players of the innate immune system, IC facilitate their removal. However, when this system becomes overloaded or imbalanced, systemic inflammation or autoimmunity may be induced by the overproduction of IC.

As I have demonstrated in SLE, certain autoantibodies against nuclear struc-tures are particularly prone to form these potentially pathogenic IC, espe-cially during active disease as the levels of IC were highest among patients that were anti-SSA positive and displayed increased complement activation. Additionally, I demonstrate that IC from patients having anti-SSA and -SSB autoantibodies induce higher amounts of IL10 and IL6 in vitro. Both IL10 and IL6 induce B-cell survival and plasma-cell differentiation and conse-quently antibody production. Persistent IC-induced production of these cyto-kines might thus contribute to increased autoantibody production, further IC production, and thereby contribute to a vicious cycle in SLE maintaining systemic inflammation, as described in (Figure 4).

In Paper II, I demonstrate that autoantibodies directed against RNA-associated antigens are present to a larger extent in circulating IC in SLE than are anti-dsDNA antibodies. This finding supports the association of higher levels of IC in sera among patients having the RNA-related autoanti-body anti-SSA. I also tested the hypothesis that RNA-related autoantibodies are more prone to form IC during active disease, but I could not determine such an association, possibly due to patient selection. My opinion is that this concept warrants further investigations before conclusions are drawn.

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Figure 4. My data support that in SLE, RNA associated autoantibodies in particular form IC in the circulation whereas anti-dsDNA bind its target in-situ, causing local-ised inflammation. In both SLE and L. donovani infection large amounts of antigens (nuclear and parasitic, respectively) are released which result in extensive IC forma-tion. I have demonstrated that circulating IC from both diseases are capable of in-ducing IL6 and IL10. Persistent production of these cytokines contributes to in-creased autoantibody production, further IC formation, and thereby contributes to a vicious cycle maintaining systemic inflammation. In L. donovani GM-CSF was also induced by IC, a cytokine that has been associated with kidney engagement [180].

Secretion of IL10 as a result of ligation of FcγRs by IgG bound to Leishma-nia amastigotes plays a crucial role during development of the immune re-sponse towards the parasite by inhibiting macrophage activation and thereby contributing to parasite growth [131]. In addition, recent studies argue that immunosuppressive or immune-evading mechanisms contribute to Leishma-nia pathogenesis and that IL10 might have a central role [123]. I have dem-onstrated increased IL10 and IL6 production in association with IC in VL patients. Overproduction of IL10 with polyclonal B-cell activation and hy-pergammaglobulinemia fits with systemic inflammation in VL, and IC-induced IL10 could thus also contribute to the strong inflammatory response. Elevated levels of IL10 might possibly also lead to depressed ability to mount a sufficient immune response to the parasite. In our studies immune complexes from VL patients also induced GM-CSF. This cytokine could possibly be either beneficial by activating macrophages to become leishmanicidal [178, 179] or detrimental for the patient by stimulating a Th1-driven DTH and possibly also facilitating Leishmania-associated kidney engagement [180-182].

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To conclude, IC have central roles in the pathogenesis of both SLE and Leishmania infection through induction of cytokines that are important in disease progression. Blockade of receptors involved in IC-induced cytokine production or suppression of effector cells activated by IC might be ways to reduce pathogenic cytokine production in these diseases.

My impression is that research regarding the properties of IC has tradi-tionally mostly concerned the classically described functions including anti-gen neutralisation, complement activation or IC-mediated phagocytosis, whereas how the antibody characteristics contribute to the cytokine-inducing properties of IC are not that well covered in the literature and thus warrants further studies. The results from my studies in SLE indicates that IC contain-ing specific autoantibodies are involved directly in the inflammatory process in SLE by forming IC in the circulation, whereas other specificities deposit as individual antibodies forming IC in situ. To confirm this hypothesis fur-ther studies are needed. An interesting continuation and extension of these data would be to examine whether the mechanisms and properties for IC to induce cytokines that we have studied in experimental systems in solution also applies to tissue-bound IC in SLE. Knowledge of these processes is of importance in the development of new therapies and may provide increased ability to foresee symptoms that are related to IC formation.

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Populärvetenskaplig sammanfattning

Bakgrund

När kroppens vävnader skadas av till exempel mekanisk skada eller infek-tion aktiveras dess försvarsmekanismer. Genom att känna igen strukturer som är gemensamma för många olika mikroorganismer kan kroppens för-svarsceller omedelbart agera mot inkräktare. I blodet finns dessutom en mängd olika proteiner som aktiveras av främmande ämnen och av skador på vävnaden. Flera av dessa ingår i komplementsystemet som kan borra hål i den främmande mikroorganismens cellmembran genom att bilda ett protein-komplex. Dessa proteiner verkar även aktiverande på försvarscellerna. Andra proteinsystem hjälper till att återställa vävnaden efter skador genom att reglera bl.a. blodkärlstillväxt och koagulation. Den process som kroppen sätter igång för att försvara sig och reparera de skador som den utsatts för kallas inflammation.

För att reglera försvarsprocessen utsöndrar kroppen små signalmolekyler, så kallade cytokiner. De kan verka både förstärkande och hämmande på det kroppsegna försvaret. Cytokinerna signalerar också till de celler som tillver-kar antikroppar. Antikroppar är mycket specifikt riktade mot sitt mål och hjälper på så vis till att vägleda förvsvaret, men är även viktiga för att rensa bort främmande ämnen från kroppen. Antikroppar som bundit sitt mål kan även de aktivera komplementsystemet.

För att behålla kontroll över inflammationen måste balans upprätthållas mellan alla dessa faktorer. Om det inflammatoriska svaret är otillräckligt kan infektionen bli livshotande, medan ett överdrivet försvar kan leda till så star-ka reaktioner att omfattande skador uppstår på den egna vävnaden. Det in-flammatoriska svaret är alltså viktigt för organisationen av det specifika im-munsvaret mot infektionen, men också för reparation och återuppbyggnad av vävnad efter att den primära orsaken till inflammationen försvunnit.

Immunförsvarets förmåga att skydda kroppen mot en stor mängd olika inkräktare är beroende av precisa mekanismer för att förebygga att dess de-struktiva förmåga inte attackerar den egna vävnaden. Hos vissa personer eller vid särskilda omständigheter kan det starkt reglerade immunförsvaret förlora förmågan att skilja mellan kroppsegna och främmande strukturer, vilket resulterar i reaktioner riktade mot kroppens egna celler och vävnader. Sådana reaktioner orsakar så kallade autoimmuna sjukdomar där inflamma-tionen kan leda till svåra vävnadsskador.

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Immunkomplex består av antikroppar som funnit sitt mål och på så vis bildat en sammansatt struktur, ett komplex. I en frisk människa svarar immunkom-plexen som en del i det normala immunförsvaret för att rensa ut potentiellt skadligt material från cirkulationen. Vid vissa sjukdomstillstånd kan dock situationer uppstå där mekanismerna för att rensa ut immunkomplex inte räcker till; överskottet av immunkomplex kan då leda till reaktioner som är skadliga för individen. Den autoimmuna sjukdomen systemisk lupus erythematosus (SLE) är ett typexempel på en sjukdom där immunkomplex-ens skadliga effekter är centrala. Andra situationer där dessa mekanismer kan bli överbelastade är vid svåra och långdragna infektioner.

Vid autoimmuna sjukdomar bildas dessa komplex av antikroppar riktade mot kroppsegna strukturer (sk autoantikroppar) medan immunkomplex vid infektionssjukdomar ofta innehåller antikroppar riktade mot material från den infekterande organismen. Omfattningen av reaktionerna beror både på mängden och storleken av komplexen samt deras fördelning i kroppen. An-tikroppar som binder sitt mål på en vävnadsyta resulterar i en lokal reaktion medan komplex som bildas i blodomloppet resulterar i att en reaktion ut-vecklas varhelst komplexen fastnar. Detta sker i synnerhet på platser där filtrering av blodplasman sker, d.v.s. i njurarna, i blodkärlens väggar och i lederna. Typiska symptom är hudutslag, ledinflammation och njursvikt.

Målet för avhandlingen Det övergripande målet med min avhandling har varit att studera faktorer som bidrar till immunkomplexassocierade sjukdomstillstånd vid SLE och infektioner orsakade av den tropiska parasiten Leishmania donovani. Jag har fokuserat på betydelsen av autoantikroppar och på immunkomplex-stimulerad cytokinproduktion.

Delstudier Arbete I och II Syftet med delarbete I var att undersöka vilken inverkan sjukdomsaktivitet och förekomst av speciella autoantikroppar hos SLE-patienter hade på mängden immunkomplex, och dessa immunkomplex förmåga att stimulera produktionen av cytokiner. Vi fann att immunkomplex från patienter med förhöjd komplementaktivering (ett tecken på aktiv sjukdom) inducerade högre nivåer av cytokinerna IL6 och IL10. De patienter som dessutom hade en särskild typ av autoantikroppar, anti-SSA och anti-SSB, inducerade allra mest cytokiner. Biokemiska mätningar av nivåerna av immunkomplex i blo-det avslöjade en synergistisk effekt mellan autoantikroppar och komple-

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mentaktivering, det vill säga att effekten av anti-SSA på immunkomplexni-våer förstärktes då patienten också hade ökad komplementaktivering. Dessa fynd tyder på att anti-SSA antikroppar är inblandade i bildningen av SLE-immunkomplex, men formella bevis för att de förekommer i immunkomplex i större utsträckning än andra autoantikroppar saknades.

I delarbete II isolerade jag därför cirkulerande immunkomplex som jag sedan analyserade för förekomst av ett flertal olika autoantikroppar som är associerade med autoimmuna sjukdomar. Jag fann att samtliga undersökta typer av autoantikroppar utom en ansamlades i cirkulerande immunkomplex jämfört med de som fanns i fri form i blodcirkulationen. Autoantikroppar associerade med RNA i cellkärnan (anti-SSA och anti-SSB tillhör denna grupp), var dock betydligt mer anrikade i SLE-immunkomplex än antikrop-par riktade mot DNA.

Slutsatserna i delstudie I och II stöder båda teorin att autoantikroppar mot RNA-associerade strukturer är mer benägna att bilda cirkulerande immun-komplex vid SLE, i motsats till antikroppar mot DNA-associerade antigen, vilka inte i samma utsträckning bildar immunkomplex i cirkulationen.

Arbete III I delstudie III undersökte jag immunkomplexinducerad cytokinproduktion hos sudanesiska patienter med visceral leishmaniasis (VL), orsakad av Leishmania donovani-parasiten. Jag studerade hur behandling och sjuk-domsaktivitet inverkade på bildningen av immunkomplex och effekten av dessa immunkomplex i provrörsförsök. Jag konstaterade att immunkomplex från Leishmania-infekterade patienter inducerade högre nivåer av cytokiner-na GM-CSF och IL10, samt av IL6, än immunkomplex från friska personer. Induktionen av GM-CSF var särskilt kraftig hos akut sjuka VL-patienter som nyligen fått den i Sudan vanliga behandlingen med antimonsalt. Det samma gällde för nivån av immunkomplex i cirkulationen hos dessa patienter.

Arbete IV Immunkomplex och förekomst av autoantikroppen reumatoid faktor (RF) är båda vanligt förekommande både vid reumatiska sjukdomar och vid infek-tionssjukdomar. Produktion av RF är mycket vanligt vid Leishmania-infektion där immunkomplexnivåerna också är förhöjda. Autoantikroppar mot citrullinerade proteiner (anti-CCP) är en mycket specifik markör för reumatoid artrit (RA), men har även påvisats vid ett antal infektionssjukdo-mar. I delarbete IV undersökte jag förekomsten av anti-CCP, RF, och nivå-erna av immunkomplex i sudanesiska patienter infekterade med Leishmania donovani-parasiten. Jag fann att Leishmania-infekterade patienter ofta var RF-positiva, hade förhöjda immunkomplexnivåer och att ett stort antal hade antikroppar mot CCP. Men tvärtemot vad vi fann hos Sudanesiska RA pati-

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enter så var CCP-reaktiviteten inte begränsad till CCP utan reagerade lika bra mot en kontrollpeptid. Det fanns en stark statistisk koppling mellan mängden immunkomplex och mängden anti-CCP-antikroppar i cirkulationen hos Leishmania-patienterna, men jag kunde utesluta att immunkomplexen var orsaken till de uppmätta höga anti-CCP-nivåerna. Förekomsten av CCP-antikroppar hos de parasitinfekterade patienterna har troligvis inte att göra med likheter med RA avseende sjukdomsförloppet, utan är antagligen ett resultat av den kraftiga inflammation som kännetecknar VL-patienter. Våra resultat understryker vikten av att undersöka citrullinspecificiteten avseende anti-CCP-reaktivitet i nya icke artritassocierade patientgrupper.

Slutsatser av delarbetena Sammanfattningsvis har jag funnit att olika autoantikroppstyper på olika sätt bidrar till immunkomplexassocierad inflammation via potentiellt olika me-kanismer vid SLE och VL. Vid både SLE och Leishmania donovani-infektion bildas stora mängder immunkomplex. Jag har visat att immunkom-plex i blodcirkulation från båda sjukdomarna kan stimulera produktion av cytokinerna IL6 och IL10. Den kontinuerliga produktionen av dessa cytoki-ner bidrar till ökad antikroppsproduktion, ytterligare immunkomplexbild-ning, och kan därmed skapa en ond cirkel som upprätthåller inflammationen. Kunskap om dessa mekanismer leder till ökad förståelse av de bakomliggan-de orsakerna till många symptom vid reumatisk sjukdom, men även vid in-flammation orsakad av parasiter. Vetskap om dessa förlopp är viktiga vid utvecklandet av nya behandlingsformer och kan ge ökade möjligheter att förutse symptom som är relaterade till immunkomplexförekomst. Blockering av de cellytereceptorer vars stimulering orsakar immunkomplexinducerad cytokinproduktion eller hämning av de celler som aktiveras av immunkom-plexen kan vara sätt att minska den skadliga cytokinproduktionen vid dessa sjukdomar.

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Acknowledgements

This work was carried out at the Unit of Clinical Immunology, Department of Oncology, Radiology and Immunology, Faculty of Medicine, Uppsala University. This investigation was supported by grants from The Swedish Research Council, ALF grants from Uppsala University Hospital, the Swed-ish League against Rheumatism, the King Gustav Vth 80-years foundation, the Groschinsky foundation, the Swedish Fund for Research without Animal Experiments, the Sund foundation for Rheumatological Research, the Brunnberg foundation and the Rudberg foundation.

In addition, I would like to express my sincere gratitude to a number of per-sons who have made this thesis possible:

Johan Rönnelid, my main supervisor, for being a very good supervisor and friend, and for sharing your knowledge about both technical and clinical aspects of research. Thank you also for being a good travel companion, es-pecially on our trip to NY.

Amir Elshafie, for good collaborations and for your always positive attitude.

Linda Mathsson, for introducing me to the lab and for being a good lab-companion.

My co-supervisors, Karlis Pauksens and Bo Nilsson for scientific input.

Bob Harris, for linguistic advice.

All other co-authors for nice collaborations.

The patients that have provided samples. Without you there would have cer-tainly not been any thesis and I hope that it will contribute to the understand-ing of your diseases.

Past and present members of our group and at the Unit of Clinical Immunol-ogy.

The personnel at the routine lab for letting me use all necessary equipment.

53

The administrative staff for all the help with practical issues.

Stort tack till släkt och vänner för allt annat kul utanför arbetstiden, så som träningsläger, matupplevelser, resor, osv.

Tack också till grabbarna från tiden vid SLU och senare som verkligen för-gyllde studietiden.

Pappa, Mamma, Ida, Oskar, Hanna, Håkan, Arvid, Eskil och Elsa. Och Eng-lunds: Lennart, Sinikka, Lina, Martin, Nora, Vilgot, Otto, Joel & Kristin. Tack för all uppmuntran och stöd!

Viktigast av alla är Hille, Sixten, och bullen i ugnen. Ni är bäst! –Sixten, när snön försvinner ska vi cykla trixigastigen. Med trampor.

54

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