Download Functional Role of Immune Complexes in Rheumatic

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 erythematosus are associated with the occurrence of anti-Sjögren's syndrome 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 stibogluconate 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
ACPA
Antibody
Antibodies against citrullinated proteins and/or peptides
ACR
ADCC
ANA
ANOVA
APC
C
American College of Rheumatology
Antibody dependent cell mediated cytotoxicity
Anti-nuclear antibody
Analysis of variance
Antigen presenting cell
Complement component
CCP
Cyclic citrullinated peptides
CD
CeD
COI
CR
CRP
DC
DID
dsDNA
DTH
EBV
ELISA
Fab
FcR
GM-CSF
HCV
HEPES
HLA
Crohn's disease
Celiac disease
Cut-off index
Complement receptor
C-reactive protein
Dentritic cell
Double immunodiffusion
Double stranded DNA
Delayed type hypersensitivity
Eppstein-Barr virus
Enzyme-linked immunosorbent assay
Fragment, antigen binding
Fragment, crystallisable receptor
Granulocyte macrophage colony-stimulating factor
Hepatitis C virus
hydroxyethyl-piperazineethanesulfonic acid
Human leukocyte antigen
HSM
IC
IFN
Ig
IL
Hepatosplenomegaly
Immune complex(es)
Interferon
Immunoglobulin
Interleukin
LPS
mAb
MHC
MMF
MS
Lipopolysaccharide
Monoclonal antibody
Major histocompatibility complex
Mycophenolate mofetil
Multiple sclerosis
NIBSC
NK
OD
PBMC
PCNA
PDC
PEG
PEST
PKDL
National Institute for Biological Standards and Controls
Natural killer cell
Optical density
Peripheral blood mononuclear cell
Proliferating cell nuclear antigen
Plasmacytoid dentritic cell
Polyethylene glycol
Penicillin-streptomycin
Post kala-azar dermal leishmaniasis
PM
RA
RF
RNP
SLE
SLEDAI
Sm
SSA
SSB
T1D
Th
TLR
TNF
UV
VL
Polymyositis
Rheumatoid arthritis
Rheumatoid factor
Ribonuclear protein
Systemic lupus erythematosus
Systemic lupus erythematosus disease activity index
Smith antigen
Sjögren's syndrome A antigen
Sjögren's syndrome B antigen
Type 1 diabetes
T helper cell
Toll-like receptor
Tumour necrosis factor
Ultraviolet
Visceral leishmaniasis
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 activation 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 parasitic 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 mechanisms is of importance for understanding of the pathological significance of
autoantibodies in IC formation and pathology in SLE and in parasite-induced
inflammation.
11
Immunity: A brief overview
In mammals there are two main types of immune defence, innate and adaptive. Innate immunity is the first line of defence and is not specific to any
certain pathogen but constitutes a protection including anatomical, physiological 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, phagocytes and natural killer (NK)-cells detect the immediate presence and nature
of the infection. In response to recognition of certain highly conserved molecular patterns expressed by different groups of microbes, i.e. extracellularly 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 neutralise the organism. If the same pathogen is encountered a second time the innate 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: specificity, diversity, memory, and the ability to discriminate between self/nonself antigens. Antigen exposure results in differentiation of B-cells into
memory cells and antibody-secreting plasma cells. Due to affinity maturation, 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 specificity 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 several 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 inflammatory response.
12
Activation of the adaptive immune system is also capable of inducing inflammation 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 signalling via cell-to-cell interactions and by release of signalling molecules
such as cytokines and chemokines that stimulate and direct subsequent adaptive responses. Likewise, the adaptive system produces signals and components that increase the effectiveness of innate responses; upon activation Bcells release antibodies (as part of the humoral immune response) directed
against pathogens and by binding to their targets facilitates complementmediated killing. Conversely, T-cells release cytokines to direct and regulate
both innate and adaptive immune responses but might also function as effector cells by the release of cytotoxic substances and expression of death receptor ligands. The inflammatory response is thus important for the organisation 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 maintained. 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 allergies.
13
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 hypersensitivity. A typical example is autoimmune hemolytic anemia where antibodies against the body’s own reticulocytes results in haemolysis through complement activation and/or cell mediated killing. Antibody-antigen interactions 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 antigen is recognised release cytokines that activate phagocytic and cytotoxic
cells resulting in direct cellular damage. A typical example of type IV hypersensitivity 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 (foreign 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 regulated immune system loses the ability to discriminate between self and nonself, resulting in hyperactive responses against the body’s own cells and
tissues. Such autoimmune reactions result in inflammation and tissue damage 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 thyroglobulin in cells of the thyroid gland in autoimmune thyroditis. In autoimmune 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.
14
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 antigen 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 present 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 implicated as potential triggers of autoimmune diseases [12], either through
molecular mimicry, causing immunoreactivity against both foreign and selfantigens [13, 14], or indirectly by influencing cytokine production. Infections 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 factors 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].
15
Figure 1. Autoimmune diseases are associated with a number of genetic and environmental 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 disease, T1D: Type 1 diabetes, MS: Multiple Sclerosis, SLE: Systemic Lupus Erythematosus, RA: Rheumatoid Arthritis)
Autoantibodies
Autoantibodies are immunoglobulins (Ig) that bind antigens originating from
the same organism. Many (but not all) autoimmune diseases are characterised 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 autoantibodies 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).
16
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 organ- specific diseases autoantibody production might be stimulated by localised inflammation. Cross-reactivity between microbial targets and selfantigens (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 [2426]. The properties of these autoantibodies differ from autoantibodies associated with pathological processes in their ability to bind their target antigens. In contrast to pathological autoantibodies which are usually of high
affinity, the polyreactive natural antibodies bind at low affinity [23]. In addition, 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 clinically with symptoms involving several organ systems, including skin rashes,
arthritis, pleuritis, carditis, glomerulonephritis and neurological manifestations. The disease is heterogeneous, with various combinations of four of the
eleven clinical criteria (summarised in Table 1) required for case classification [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 antibodies [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 increase 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 treatment [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
17
glomerulonephritis that fail first-line cyclophosphamide therapy, B-cell depletion 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 interactions, cytokines, and B-cell activation.
Immunopathological features including systemic inflammation and vasculitis, including deposition of IC, are the basics of the pathology underlying
SLE. The cooperation between genes controlling immune response, inflammation, regulation of transport and clearance of IC, and apoptosis, are important factors in disease development. Environmental factors such as UVlight, 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 identification 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
6. Serositis
Fixed erythema, flat or raised, over the malar eminences, tending to
spare the nasolabial folds
Erythematous raised patches with adherent keratotic scaling and
follicular plugging; atrophic scarring may occur in older lesions
Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation
Oral or nasopharyngeal ulceration, usually painless, observed by a
physician
Non-erosive arthritis involving 2 or more peripheral joints, characterized by tenderness, swelling, or effusion
Pleuritis or pericariditis
7. Renal disorder
Persistent proteinurea or cellular casts
8. Neurologic disorder
Seizures or Psychosis
2. Discoid rash
3. Photosensitivity
4. Oral ulcers
5. Arthritis
9. Hematologic disorder
Hemolytic anemia or leukopenia or lymphopenia or thrombocytopenia
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
18
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 predictable course, with a gradual increase of specific autoantibodies before disease
onset [19].
Autoantibodies directed against nuclear structures such as double- or single-stranded DNA, nucleoproteins, histones, and nuclear RNA occur in the
majority of patients with untreated SLE. Table 2 briefly describes autoantibodies occurring in SLE and in other autoimmune diseases.
Table 2. Brief description of SLE-associated autoantibodies (reviewed in [39]).
Autoantibody Antigen
Anti-RNP/Sm
Clinical significance
Group of 9-70kDa ribonuclear- proteins involved in pre-mRNA splicing.
Anti-SM: high specificity to disseminated SLE but occurs only in few
patients. Anti-RNP: in SLE, MCTD,
and other rheumatologic diseases.
Anti-SSA (Ro52 Ribonuclear protein composed of five Found in up to 90 % of Sjögren’s,
and/or Ro60)
RNA molecules and a 60kDa protein. patient. In SLE 50%, neonatal SLE;
The 52kDa protein is also / (discussed 100%. Can cause heart block in neonatal SLE. Isolated Ro52 is not regarded
to be) associated with the SSA complex.
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- Specific (questioned) for SLE but low
prevalence
sion is cell-cycle- dependent
Anti-dsDNA
Antibodies against native dsDNA,
Specific for SLE, besides anti-Sm
bind to the backbone of the double
accounted as serological marker.
helix.
AntiFunctional subunits of chromosomes
Found in both Scleroderma and SLE,
nucleosomes
consisting of histones and dsDNA
using newer tests more specific to
SLE.
Anti-histones
DNA associated proteins stabilising
Found in 50-80% of SLE patients
the double helix
(particularly in drug induced) and RA
(15-50%)
Specific for SLE but found in only
Anti-ribosomal Subunits of the ribosomes
10% of patients
P-proteins
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
19
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 contributing to the broad spectrum of autoantibody formation against these
structures. Apoptosis, or programmed cell death, is a highly regulated process essential for normal function in all multicellular organisms. Under normal 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 immune 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 dying cells. During this process potential immunogenic material become available for phagocytosis by antigen presenting cells. Multiple mechanisms are
involved in the interactions between the phagocytic and the dying cell, including: signal transduction that regulate inflammation, receptors on phagocytic cells, and plasma derived molecules that bind to the apoptotic cell surfaces. 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 affecting 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 autoantigens to T-lymphocytes. This will in turn activate B cells to produce antibodies to these autoantigens.
20
Leishmania donovani infection
Leishmaniasis is a disease caused by protozoan parasites belonging to the
genus Leishmania. Several species within the genus cause infections in humans including e.g. L. donovani, L. mexicana, L. major, and L. braziliensis
[49]. The different species are morphologically similar but can be distinguished by DNA analysis or monoclonal antibody reactivities. Human infections 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, including 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 treatment. No firm evidence exists that predicts which VL patients will develop
PKDL, but an interesting finding is the high expression of IL10 in VL patients 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 parasite itself, to the response to treatment, or both. Polymorphisms in genes
controlling innate and adaptive immunity have been suggested as possible
candidates [57].
21
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 promastigotes are engulfed by macrophages, where they transform into amastigotes. The
infected cells die and release progeny amastigotes that rapidly invade new macrophages where they multiply and subsequently migrate to internal organs. The life
cycle of the parasite is completed when the fly again ingests macrophages containing 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 diseases, 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. Pulmo22
nary Tuberculosis, Hepatitis C viral (HCV) infection, Type 1 Autoimmune
Hepatitis and in Leishmania infection [63-68]. Importantly, anti-CCP positivity evident in non-RA disease sera seems to not always be citrullinedependent as demonstrated in autoimmune hepatitis [66]. Additionally, in
HCV-related infections a number of other autoantibodies with other specificities are detectable [69].
There are clinical case reports on autoreactivity in Leishmania infections,
including signs of vasculitis with the presence of RF and antinuclear antibodies [70]. Polyclonal B-cell activation demonstrated in Leishmaniainfected patients is suggested to be associated with the presence of autoantibodies 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 Porphyromonas gingivalis associated with peridontitis. It has been shown that P. gingivalis (in contrast to other bacteria commonly found in the oral cavity) is
able to citrullinate proteins, suggesting that P. gingivalis-mediated citrullination 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 conjunction to vaccination and infections [74]. Antibodies forming IC and factors of the complement system that opsonise the material facilitates binding
via complement- and IC- interacting receptors and thereby facilitating clearance 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 circulation. 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 cytokine production.
23
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 phagocytes 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 distribution 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 glomerulonephritis [77], these symptoms being commonly evident in autoimmune
diseases that stem from reactions mediated through IC formed by autoantibodies 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 donovani infection [68, 69]. The early steps in activation of the complement system 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
24
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 membrane attack complex, various peptides are generated along the way. Smaller
fragments resulting from complement cleavage, C3a, C4a, and C5a, are collectively termed anaphylatoxins. These fragments bind to receptors on mast
cells and basophils and induce degranulation, with release of pharmacologically 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 development of immune complex-mediated diseases. This demonstrates the importance 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 produce 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 isotype of the interacting antibody. The Fc-receptors for IgG (FcγR) comprise
the largest family and mediate many biological functions such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, induction of inflammatory cascades and modulation of immune responses [81-84]. FcγRs are present on most cell types of the innate and adaptive immune systems, including
B cells, T cells, NK cells, macrophages, mast cells, dendritic cells and neutrophils [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.
25
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 mediumaffinity 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 neutrophils 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 growing interest, and will hopefully provide mechanisms for how specific antibodies 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 structures such as LPS, glycolipids, peptidoglycans, lipopeptides, and nucleic
acids, that are shared by large groups of microorganisms and are thus important 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 cytokines 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. Unmethylated 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 antibody with FcγRIIa. This interaction then mediates endocytosis followed by
co-localisation of FcγRIIa and TLR7 or TLR9 in the endosome with subsequent cellular activation [91].
26
Cytokine induction
Cytokines are involved in a broad array of activities that affect the immune
system in numerous ways, including innate and adaptive immunity, inflammation 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 proinflammatory and enhance cellular responses while Th2 cytokines are antiinflammatory 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 reactions, while over-production of Th1 and Th17 cytokines mediate T cell hyperactivity [92]. In addition, Th17 cells are thought to be particularly important 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 influences 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 development 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 promotes a Th1 responses [110]. Furthermore, IL12 is able to change an established predominant humoral response to involve more cell-mediated functions. 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.
27
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 activated 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 importance for SLE pathogenesis as levels correlate both to disease activity and
severity [115, 116]. Expression of FcγRIIa has been observed on IFNαproducing 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 pathology 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] although the L. donovani parasite in some way evades the induction of inflammation 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 reproduction [125-128]. Furthermore, the balance between IL10 and IL12 and/or
IFNγ is of importance for acquired resistance to the parasite [129, 130]. Secretion of IL10 as a result of ligation of FcγRs by IgG bound to Leishmania
amastigotes plays a crucial role during development of infection by inhibiting macrophage activation and thereby contributing to parasite growth [131].
In gene deleted mice lacking FcγRs, enhanced control of Leishmania infection is demonstrated by a reduction in numbers of cells producing the antiinflammatory cytokines IL4 and IL10, although the ability of Leishmania to
parasitize cells is maintained [132].
28
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 hypersensitivity [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 significantly reduced number of secondary infections, along with increased neutrophil 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 infectioninduced GM-CSF.
29
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 investigate whether certain autoantibodies accumulate in circulating IC during
SLE flares.
Paper III
To explore IC-induced cytokine production in Sudanese patients with Visceral 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 Sudanese patients infected with the Leishmania donovani parasite and to see if
there was an association between IC levels and the occurrence of autoantibodies.
30
Methods
Patients
In Paper I, anonymised serum samples were obtained from patients with a
clinical diagnosis of SLE and who had previously been investigated concerning 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 divergent 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 function, 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, residence, occupation, marital status, medical treatment, abdominal pain, vomiting, 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 parasites were examined from all individuals who either had fever, looked ill, or
31
had splenomegaly, and those with positive blood films for malaria were excluded. 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 wasting). 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 antimony 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.
32
C1q-binding assay for isolation of circulating IC
Immune complexes were isolated from sera using C1q coated plates (Bindazyme 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% methanol 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 immediately analysed for autoantibody content.
Measurement of levels of circulating IC
Levels of circulating IC were measured by a solid-phase C1q assay (Bindazyme 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-(2hydroxyethyl)-1-piperazineethanesulfonic acid), and 1% PEST (PenicillinStreptomycin). Cells were cultured in a serum-free system using 4% Ultroser
G as supplement. Ultroser G is according to the manufacturer free from cytokines 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 donors yielding low responses. Therefore cells from two different PBMC donors were always investigated in parallel and thereafter data from the responder 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
33
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 associated 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 complement 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 measurement 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-
34
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 Maxisorb, 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 PBSTween 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 immunofluorescence (IF) microscopy on HEp-2 cells and Crithidia luciliae,
respectively (both from Immunoconcepts, Sacramento, CA, USA). Antibodies 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 hospital.
35
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 measure 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 parallel. The intensity of the reaction for each positive band was determined with
densitometry using the EuroLineScan software (Euroimmun). The recommended cut-off (10 densitometry units) was used to define which autoantibody 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 subtraction of background OD obtained on uncoated plates. A reference sample
from an SLE patient at flare was always included. The cut-off value for positive 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 (EuroDiagnostica, Malmö, Sweden). Anti-CCP positivity was determined in accordance with manufacturer’s instructions with 25 U/ml used as a cut-off.
The assay does not yield quantitative levels below the company-defined cut36
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 Jörgen Wieslander at Euro-Diagnostica and was used to evaluate citrullinespecific 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 influenced anti-CCP reactivity we adsorbed C1q binding IC from sera and evaluated CCP reactivity afterwards. Sera were diluted 1:50 and incubated for 2
hours on C1q coated plates (Bindazyme C1q binding kit; Binding Site, Birmingham, 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 expressed 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 Pearson’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 significant.
37
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 PEGprecipitates 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 relatively small blood volumes used in these investigations. The quick procedure
also facilitates experiments whereby cells from the same donor are stimulated 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 precipitated 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 circulating IC and levels of cytokines induced by PEG precipitates, and the fact
that the levels of serum PEG precipitate-induced cytokines can be specifically reduced by pre-treatment of the responder PBMC with blocking antibodies 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 production. We therefore instead examined levels of circulating IC measured by
C1q binding. Both classical complement function and the occurrence of antiSSA influenced circulating IC levels, with a strong interaction in ANOVA
analysis between decreased complement function and the occurrence of antiSSA 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
38
with complement and IC levels was recorded for anti-dsDNA, the combination 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 deficiency 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 investigated 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 previous observations [152, 153]. Anti-CRP antibody levels did not differ between 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 antibody 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, 155157], 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 between 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 complement 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 pathway is a hallmark of flare and ongoing organ damage in SLE and studies
support that anti-dsDNA antibodies are involved in this process [159]. According to the literature it seems that levels of other SLE-related autoantibodies, 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.
39
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 antiSSA and possibly anti-SSB antibodies participate directly in the inflammatory 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 accumulated in SLE IC, and whether such accumulation was dependent on
SLE disease activity.
To analyse to what extent the different autoantibody specificities accumulated in circulating IC, the levels in serum and PEG-precipitates were normalised 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 RNAassociated autoantibodies, and in particular autoantibodies against RNP/Sm
and SSA/Ro52, exhibited the highest degree of enrichment in SLE PEG precipitates. To further confirm our data I also analysed the autoantibody specificities 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 second priority was to find paired sera from active and inactive SLE respectively, representing these various autoantibodies. Paired samples were collected from two different cohorts, one from the Department of Rheumatology 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
40
the fact that I determined no differences in autoantibody enrichment between
active and inactive SLE as I had hypothesised that at least anti-SSA autoantibodies 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 autoantigens [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 presence 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 kidney involvement [162]. The same study also reported that the enrichment of
antibodies to Sm, SSA, and SSB in kidney extracts exceeded that of antibodies to dsDNA. In our present study of circulating IC, the same RNAassociated 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 antibodies [163]. In addition, positively charged antibodies [164], especially antidsDNA antibodies [165] bind more strongly to glomerular basement membrane 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 target 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 reported that DNA-anti-DNA complexes only constitute a minor part of circulating IC detected by PEG-assay [169], suggesting either that other antigenantibody 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 autoantibodies 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 complement activation and ensuing inflammation due to leukocyte infiltration at
41
the site of IC deposition. Anti-dsDNA, conversely, might cause tissue damage 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 patients 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 analysis. 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 activating 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) displayed 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 differences observed in IC levels and PEG IC-induced levels of GM-CSF between 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
42
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 patients 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 recorded. 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 between the Sudanese RA and VL patients. However, RA patients had higher
levels as compared to PKDL patients. Each of three disease groups had significantly 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 although 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. AntiCCP reactivity was also detected in 4 % of the PKDL patients. In the Sudanese 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 possibility 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 peptide. 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
43
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 responses 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 reactivity 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 reactivity 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 patients 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.
44
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 structures are particularly prone to form these potentially pathogenic IC, especially 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 consequently antibody production. Persistent IC-induced production of these cytokines 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 RNAassociated 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 autoantibody 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.
45
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 localised inflammation. In both SLE and L. donovani infection large amounts of antigens
(nuclear and parasitic, respectively) are released which result in extensive IC formation. I have demonstrated that circulating IC from both diseases are capable of inducing IL6 and IL10. Persistent production of these cytokines contributes to increased 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 Leishmania amastigotes plays a crucial role during development of the immune response 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 Leishmania pathogenesis and that IL10 might have a central role [123]. I have demonstrated increased IL10 and IL6 production in association with IC in VL
patients. Overproduction of IL10 with polyclonal B-cell activation and hypergammaglobulinemia fits with systemic inflammation in VL, and ICinduced 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 Th1driven DTH and possibly also facilitating Leishmania-associated kidney
engagement [180-182].
46
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 traditionally mostly concerned the classically described functions including antigen 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 containing 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 further 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.
47
Populärvetenskaplig sammanfattning
Bakgrund
När kroppens vävnader skadas av till exempel mekanisk skada eller infektion aktiveras dess försvarsmekanismer. Genom att känna igen strukturer
som är gemensamma för många olika mikroorganismer kan kroppens försvarsceller 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 proteinkomplex. 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 tillverkar 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 starka reaktioner att omfattande skador uppstår på den egna vävnaden. Det inflammatoriska svaret är alltså viktigt för organisationen av det specifika immunsvaret 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 destruktiva 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 inflammationen kan leda till svåra vävnadsskador.
48
Immunkomplex består av antikroppar som funnit sitt mål och på så vis bildat
en sammansatt struktur, ett komplex. I en frisk människa svarar immunkomplexen 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 immunkomplexens 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. Antikroppar som binder sitt mål på en vävnadsyta resulterar i en lokal reaktion
medan komplex som bildas i blodomloppet resulterar i att en reaktion utvecklas 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 immunkomplexstimulerad 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 blodet avslöjade en synergistisk effekt mellan autoantikroppar och komple49
mentaktivering, det vill säga att effekten av anti-SSA på immunkomplexnivåer förstärktes då patienten också hade ökad komplementaktivering. Dessa
fynd tyder på att anti-SSA antikroppar är inblandade i bildningen av SLEimmunkomplex, 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 antikroppar riktade mot DNA.
Slutsatserna i delstudie I och II stöder båda teorin att autoantikroppar mot
RNA-associerade strukturer är mer benägna att bilda cirkulerande immunkomplex 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 sjukdomsaktivitet inverkade på bildningen av immunkomplex och effekten av
dessa immunkomplex i provrörsförsök. Jag konstaterade att immunkomplex
från Leishmania-infekterade patienter inducerade högre nivåer av cytokinerna 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 infektionssjukdomar. Produktion av RF är mycket vanligt vid Leishmaniainfektion 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 infektionssjukdomar. I delarbete IV undersökte jag förekomsten av anti-CCP, RF, och nivåerna 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 pati50
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 CCPantikroppar 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 mekanismer vid SLE och VL. Vid både SLE och Leishmania donovaniinfektion bildas stora mängder immunkomplex. Jag har visat att immunkomplex i blodcirkulation från båda sjukdomarna kan stimulera produktion av
cytokinerna IL6 och IL10. Den kontinuerliga produktionen av dessa cytokiner bidrar till ökad antikroppsproduktion, ytterligare immunkomplexbildning, och kan därmed skapa en ond cirkel som upprätthåller inflammationen.
Kunskap om dessa mekanismer leder till ökad förståelse av de bakomliggande orsakerna till många symptom vid reumatisk sjukdom, men även vid inflammation 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 immunkomplexen kan vara sätt att minska den skadliga cytokinproduktionen vid dessa
sjukdomar.
51
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 Swedish 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 persons 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, especially 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 labcompanion.
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 certainly not been any thesis and I hope that it will contribute to the understanding of your diseases.
Past and present members of our group and at the Unit of Clinical Immunology.
The personnel at the routine lab for letting me use all necessary equipment.
52
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 förgyllde studietiden.
Pappa, Mamma, Ida, Oskar, Hanna, Håkan, Arvid, Eskil och Elsa. Och Englunds: 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.
53
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