complement and autoimmunity

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DIAGNOSIS OF AUTOIMMUNITY Complement and autoimmunity Eleonora Ballanti Carlo Perricone Elisabetta Greco Marta Ballanti Gioia Di Muzio Maria Sole Chimenti Roberto Perricone Published online: 25 April 2013 Ó Springer Science+Business Media New York 2013 Abstract The complement system is a component of the innate immune system. Its main function was initially believed to be limited to the recognition and elimination of pathogens through direct killing or stimulation of phagocytosis. However, in recent years, the immunoregulatory functions of the complement system were demonstrated and it was determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate and adaptive responses. When the delicate mechanisms that regulate this sophisticated enzymatic system are unbalanced, the complement system may cause damage, mediating tissue inflammation. Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjo ¨gren’s syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis. Complement deficiencies have been associated with an increased risk to develop autoimmune dis- orders. Because of its functions, the complement system is an attractive therapeutic target for a wide range of diseases. Up to date, several compounds interfering with the complement cascade have been studied in experimental models for autoimmune diseases. The main therapeutic strategies are inhibition of complement activation components, inhibition of complement receptors, and inhibition of membrane attack complex. At present, none of the available agents was proven to be both safe and effective for treatment of autoimmune diseases in humans. Nonetheless, data from preclinical studies and initial clinical trials suggest that the modulation of the complement system could constitute a viable strategy for the treatment of autoimmune conditions in the decades to come. Keywords Complement system Á Complement deficiency Á Autoimmune disease Á Anti-complement therapy Introduction The complement system is a component of the innate immune system, which consists of physical, cellular, and chemical elements. The immune system has evolved to protect the human body against pathogens or other dan- gerous elements, and its responses can be divided into innate and adaptive immune responses. Traditionally, the main function of the complement system was believed to be limited to the recognition and elimination of pathogens through direct killing and/or stimulation of phagocytosis (innate responses) [1, 2]. In recent years, the immunoreg- ulatory functions of the complement system were demon- strated and it was determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate and adaptive responses [3]. The contribution of complement to the development of humoral immunity has been confirmed through a series of elegant studies, and a body of data has accumulated demonstrating E. Ballanti Á E. Greco Á M. Ballanti Á G. Di Muzio Á M. S. Chimenti Á R. Perricone (&) Rheumatology, Allergology and Clinical Immunology, Department of Internal Medicine, Unit of Rheumatology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy e-mail: [email protected] C. Perricone Reumatologia, Dipartimento di Medicina Interna e Specialita ` Mediche, Sapienza Universita ` di Roma, Rome, Italy Roberto Perricone 123 Immunol Res (2013) 56:477–491 DOI 10.1007/s12026-013-8422-y

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DIAGNOSIS OF AUTOIMMUNITY

Complement and autoimmunity

Eleonora Ballanti • Carlo Perricone • Elisabetta Greco •

Marta Ballanti • Gioia Di Muzio • Maria Sole Chimenti •

Roberto Perricone

Published online: 25 April 2013

� Springer Science+Business Media New York 2013

Abstract The complement system is a component of the innate immune system. Its main function was initially believed

to be limited to the recognition and elimination of pathogens through direct killing or stimulation of phagocytosis.

However, in recent years, the immunoregulatory functions of the complement system were demonstrated and it was

determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate

and adaptive responses. When the delicate mechanisms that regulate this sophisticated enzymatic system are unbalanced,

the complement system may cause damage, mediating tissue inflammation. Dysregulation of the complement system has

been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus

erythematosus, vasculitides, Sjogren’s syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and

rheumatoid arthritis. Complement deficiencies have been associated with an increased risk to develop autoimmune dis-

orders. Because of its functions, the complement system is an attractive therapeutic target for a wide range of diseases. Up

to date, several compounds interfering with the complement cascade have been studied in experimental models for

autoimmune diseases. The main therapeutic strategies are inhibition of complement activation components, inhibition of

complement receptors, and inhibition of membrane attack complex. At present, none of the available agents was proven to

be both safe and effective for treatment of autoimmune diseases in humans. Nonetheless, data from preclinical studies and

initial clinical trials suggest that the modulation of the complement system could constitute a viable strategy for the

treatment of autoimmune conditions in the decades to come.

Keywords Complement system � Complement deficiency � Autoimmune disease � Anti-complement therapy

Introduction

The complement system is a component of the innate

immune system, which consists of physical, cellular, and

chemical elements. The immune system has evolved to

protect the human body against pathogens or other dan-

gerous elements, and its responses can be divided into

innate and adaptive immune responses. Traditionally, the

main function of the complement system was believed to

be limited to the recognition and elimination of pathogens

through direct killing and/or stimulation of phagocytosis

(innate responses) [1, 2]. In recent years, the immunoreg-

ulatory functions of the complement system were demon-

strated and it was determined that the complement proteins

play an important role in modulating adaptive immunity

and in bridging innate and adaptive responses [3]. The

contribution of complement to the development of humoral

immunity has been confirmed through a series of elegant

studies, and a body of data has accumulated demonstrating

E. Ballanti � E. Greco � M. Ballanti � G. Di Muzio �M. S. Chimenti � R. Perricone (&)

Rheumatology, Allergology and Clinical Immunology,

Department of Internal Medicine, Unit of Rheumatology,

University of Rome Tor Vergata, Via Montpellier 1,

00133 Rome, Italy

e-mail: [email protected]

C. Perricone

Reumatologia, Dipartimento di Medicina Interna e Specialita

Mediche, Sapienza Universita di Roma, Rome, Italy

Roberto Perricone

123

Immunol Res (2013) 56:477–491

DOI 10.1007/s12026-013-8422-y

that the activation of the complement system is also critical

to the development of T cell immunity [4].

The complement system comprises more than 30 plasma

and membrane-bound proteins [3, 5]. The activation of

these proteins occurs through three possible pathways: the

classical, the alternative, and the lectin pathways. All three

pathways are activated according to a cascade system, with

activation of one factor leading to the activation of the

next.

Pathways of complement system activation

The pathways of complement system activation constitute

enzyme cascades, analogous to the coagulation, fibrinoly-

sis, and kinin pathways. Several complement proteins are

cleaved during activation of the system, and the fragments

are generally designated with suffixes ‘‘a’’ and ‘‘b.’’ All

three pathways lead to the cleavage of C3 and finally

converge at the activation of C5 (Fig. 1). The activation of

the classical pathway is dependent on immunoglobulins

IgM or IgG, present in immune complexes (IC), and

binding to the C1 complex [6]. The binding of C1q to

antibody leads to activation of C4 and C2 inducing the

formation of the C4bC2a complex, also known as classical

pathway’s C3 convertase [7]. If the activation occurs

through the alternative pathway, factor D, a serine protease,

cleaves factor B, which is complexed with spontaneously

hydrolyzed iC3b, leading to formation of Ba and Bb. Bb

and C3b generate the C3 convertase of the alternative

pathway, C3bBb. Properdin increases the stability of this

enzyme [8]. The lectin pathway becomes activated, when

either mannose-binding lectin (MBL) or ficolins recognize

carbohydrate patterns on microbes and activate C2 and C4

through MBL-associated serine proteases (MASP), with

the formation of the same C3 convertase as the classical

pathway, C4bC2a [7]. Likewise, the classical and lectin

pathways generate the same C5 convertase (C3bC4bC2a),

whereas the alternative pathway generates a different C5

convertase (C3bBbC3b). The activation of C5 produces

C5a (a potent chemoattractant) and C5b which forms the

membrane attack complex (MAC) C5b-9, able to cause cell

lysis. In addition to cell lysis, effects of complement acti-

vation include activation of granulocytes and endothelia by

sublytic quantities of MAC; deposition of C3 fragments

(e.g., C3b, iC3b) on membranes and/or particles leading to

opsonization and enhanced phagocytosis; immune complex

clearance; clearance of apoptotic bodies; alterations in

immune cell signal transduction, adhesion activation and

cytokine production; anaphylatoxin-mediated effects [4].

Regulation of the complement system

To avoid excessive complement activation and thus protect

the host, the complement system is tightly controlled by

proteins present in the fluid phase and on cell membranes

[9]. Interestingly, evidence shows that many pathogenic

Fig. 1 Activation of the complement system occurs through three

possible pathways: the classical, the alternative, and the lectin

pathway (A). All the pathways lead to the cleavage of C3 and finally

converge at the activation of C5, with possible formation of the

membrane attack complex (A). To avoid excess complement

activation, the complement system is tightly controlled by several

regulatory proteins that act at different points of complement cascade

(A). When the mechanisms that regulate this delicate balance do not

work correctly, complement (that usually mediates beneficial func-

tions) may cause damage by induction and augmentation of inflam-

mation (B). Its intervention has been implicated in the pathogenesis of

several autoimmune diseases (C)

478 Diagnosis of Autoimmunity (2013) 56:477–491

123

microorganisms interact with these complement regulators

to elude the immune system [10].

Several proteins regulating the complement system

decrease the activity of C3 convertases in the classical and

alternative pathways, enzymes that catalyze key reactions

during the activation of the complement system. Moreover,

important control proteins are involved in the regulation of

C1 activity and of the MAC [9]. When the mechanisms that

regulate this delicate balance do not work properly or there

is an over-activation of the complement system exceeding

the capacity of the regulatory mechanisms, the complement

system may cause damage, by induction and amplification

of inflammation [3] (Fig. 1).

The importance of the regulatory mechanisms is evident

in hereditary angioedema (HAE). In this disease, the defi-

ciency of C1 inhibitor (C1INH), a multi-functional serine

protease inhibitor, causes inappropriate activation of the

complement system as well as of the other plasma enzy-

matic systems (contact system, coagulation system) that

results in recurrent episodes of angioedema involving the

skin and the mucosa [11].

Paroxysmal nocturnal hemoglobinuria is another

example of the consequences of the failure to regulate the

complement system. In such disease, a somatic mutation in

the phosphatidylinositol glycan class A gene causes a

deficiency of a protein required for the synthesis of gly-

cosylphosphatidylinositol, the lipid that anchors several

proteins to cell membranes [12]. As a result, two molecules

anchored by glycosylphosphatidylinositol, decay-acceler-

ating factor or CD55 (which regulates the formation of C3

convertase) and CD59 (which inhibits the formation of the

membrane attack complex), are lacking on cell membrane,

thus increasing susceptibility of erythrocytes to comple-

ment, leading to intravascular hemolysis [12].

Complement system as mediator of tissue damage

Several studies demonstrated that the complement system

is involved in inflammatory tissue damage. The activation

of the complement system in the tissues occurs through IC,

which trigger the classical complement pathway [5].

Moreover, in ischemic tissues, phospholipids and mito-

chondrial proteins, normally sequestrated in the cells, are

exposed and are able to activate the complement system

either directly by binding C1q or MBL or indirectly by

binding natural antibodies or C-reactive protein (CRP) [5].

CRP can activate the classical pathway by binding C1q.

Furthermore, necrotic cells and tissues lack the regulatory

molecules that normally prevent the binding of comple-

ment proteins [5].

Inappropriate activation of the complement system

causes the release of several pro-inflammatory mediators,

such as anaphylatoxins C3a and C5a that, in turn, are able

to stimulate the synthesis of other pro-inflammatory

mediators [13]. C5a is also a potent chemoattractant for

neutrophils, monocytes, and eosinophils [13]. In addition,

the MAC can contribute to inflammation and tissue dam-

age. Formation of the terminal complement complex C5b-9

can lead to cell death by necrosis or apoptosis [13].

The contribution of the complement system to tissue

damage is exemplified by necrosis following ischemia. In

fact, the activation of the complement system has been

demonstrated in areas of myocardial infarction and cerebral

stroke [5, 14].

Testing the complement system

Assays to test the complement levels in serum are one of

the standard measures used in the clinical management of

patients with autoimmune diseases. The complement sys-

tem is prone to in vitro artificial activation. Therefore, the

conditions of sample collection and storage are critical.

With some exceptions, samples for complement diagnosis

should be collected in ethylenediaminetetraacetic acid

(EDTA). Plasma should be separated soon after collection

and transferred immediately to the laboratory on ice or

frozen at -20 �C or preferably at -70 �C [15]. In the

majority of laboratories, measures are restricted to immu-

nochemical determinations of C3 and C4, and sometimes

C1q. The concentration of these proteins is usually asses-

sed by nephelometry or radial immunodiffusion by using

polyclonal antibodies, but more sensitive assays (sensitive

enzyme-linked immunosorbent assays—ELISA—or Wes-

tern blot) are available too.

A smaller number of laboratories routinely provide a

functional measurement of the activity of the whole com-

plement pathway from classical pathway activation

through to formation of the membrane attack complex,

such as the CH50 (complement hemolysis 50 %) test.

Although many variants have been described, hemolytic

assays are still based on protocols first described by Mayer

[16]. The test is very useful as it provides a profile of the

whole complement pathway. Tests for complexes or split

products formed during complement activation allow to

discriminate specific activation of the system from non-

specific [17]. Early tests to detect the cleavage of com-

plement proteins included counterimmunoelectrophoresis

or crossed immunoelectrophoresis. In the last decades,

ELISA tests using monoclonal antibodies have been

developed to specifically detect complement components,

such as alternative pathway proteins (native factor B, factor

D, and factor H, factor B split products Ba and Bb, C3-

fragments C3b/iC3b/C3dg) and anaphylatoxins (C3a and

C5a) in human blood plasma [18, 19]. Assays to measure

Diagnosis of Autoimmunity (2013) 56:477–491 479

123

these molecules are not widely available and require spe-

cial sample handling to a degree that makes routine clinical

use difficult. Complement components may represent a

target of autoantibody responses. Autoantibodies may

occur against individual components, convertase com-

plexes, complement regulating proteins, and complement

receptors. More clinically relevant autoantibodies are C3

nephritic factor, anti-C1q, and anti-C1INH that can be

detected through ELISA [20, 21].

In recent years, promising techniques to assess com-

plement activation within particular tissues have been

introduced. Non-invasive detection of complement activa-

tion through magnetic resonance imaging (MRI)-based

method has been proposed [22]. These results may open up

new avenues to develop tools for the monitoring of disease

progression in complement involving diseases.

Complement system and autoimmunity

The relation between the complement system and auto-

immunity is apparently paradoxical. If, on the one hand,

the complement system is activated and contributes to

tissue damage in autoimmune diseases, on the other hand,

deficiency of the complement proteins leads to autoim-

munity and it is challenging reconciling these two aspects.

The complement system is involved in the pathogenesis

and contributes to the clinical manifestations of several

autoimmune diseases (Fig. 1), such as systemic lupus

erythematosus (SLE), anti-glomerular basement membrane

disease, vasculitides, Sjogren’s syndrome (SS), anti-

phospholipid antibody syndrome (APS), systemic sclerosis

(SSc), dermatomyositis (DM), and rheumatoid arthritis

(RA) [23].

Paradoxically, complement system deficiencies are also

associated with autoimmune diseases. The prevalence of

complement system’s deficiencies in the population is

difficult to assess. Most heterozygotic complement-defi-

cient individuals are clinically normal. Generally, only C1-

INH (autosomal dominant), properdin (X-linked recessive),

and homozygotic complement-deficient individuals present

with clinical diseases. Deficiencies of components of the

complement system may result in a wide variety of clinical

presentations, including recurrent bacterial infections,

HAE, rheumatic disorders, leukocyte adhesion deficiency,

and hemolytic uremic syndrome [24].

Autoimmune disorders, mainly SLE, can be the com-

mon clinical manifestation of genetically determined dis-

orders of the complement system, and they are most

frequently observed in patients with deficiencies of the

early components of the classical pathway. Whereas over

30 % of patients with C2 deficiency and nearly 80 % of

patients with either C3 or C4 deficiency present

autoimmune manifestations, less than 10 % of the patients

with deficiencies of C5–9 develop similar clinical evi-

dences. The most common presentation is SLE, but discoid

lupus, dermatomyositis, scleroderma, and glomerulone-

phritis were also reported [25].

Several theories have been proposed to explain the

association between deficiencies of components of the

complement system and autoimmune diseases. The

majorities of these focus on the inadequate clearance of IC

in presence of reduced levels of complement system’s

components [26]. An excess of IC may deposit in the tis-

sues, resulting in inflammatory damage and release of

autoantigens that trigger an autoimmune response. Alter-

natively, the presence of high concentration of apoptotic

cells (AC) due to poor clearance may be sufficient to elicit

an autoimmune response [27].

In particular, among complement proteins, a primary

role in clearance activity is attributed to the first component

of the complement C1q. This can directly and indirectly

opsonize apoptotic cells for phagocytosis by macrophages.

It can directly bind AC and mediate AC phagocytosis

through CD91 or LRP-1 (low-density lipoprotein receptor–

related protein 1) on macrophages. It can also bind indi-

rectly, through CRP and IgM, to cause complement acti-

vation and C3 deposition [28, 29].

A third hypothesis suggests that the complement system

favors the development of tolerance against self [30].

The complement system is needed for the elimination of

self-reactive lymphocytes during maturation of the immune

system. Hence, deficiencies of the complement system

result in lack of normal B cell tolerance and production of

autoantibodies [31, 32].

Moreover, recent data seem to demonstrate that com-

plement system’s proteins, in particular C1q, act as a strong

signal for dendritic cells (DCs) [33]. Mature DCs are the

most powerful antigen-presenting cells (APCs) able to

induce and direct the differentiation of naive T cells.

Depending on the maturation signal and the cytokine

environment, DCs may stimulate CD4? helper, CD8?

cytotoxic or regulatory T cells, inducing tolerance. C1q

plays an important role in the initiation of DCs maturation

and induction of cytokine production, suggesting that, in

the absence of this complement system’s protein, DC

functions might be impaired. Therefore, C1q-containing IC

and C1q-opsonized apoptotic cells and pathogens most

probably play a role in both the induction and regulation of

immunity and autoimmunity [33]. Benoit et al. have

recently identified specific molecular pathways induced by

C1q. They demonstrated that C1q bounds to autologous

apoptotic lymphocytes modulating the expression of genes

associated with JAK/STAT signaling, chemotaxis, immu-

noregulation, and NLRP3 inflammasome activation in

LPS-stimulated human monocyte-derived macrophages.

480 Diagnosis of Autoimmunity (2013) 56:477–491

123

These results underline the role of C1q in suppressing

macrophage induced inflammation as well as providing

potential therapeutic targets to control macrophage polar-

ization and thus inflammation and autoimmune reactions

[34]. The protective role of the complement system from

autoimmune conditions is also expressed by the role of

complement receptors 1 (CR1) and 2 (CR2), the defect of

which has been associated with increased susceptibility to

autoimmune diseases [26]. These receptors, found on

erythrocytes, hematopoietic cells, follicular dendritic cells,

and B cells, are capable of binding C3 activation frag-

ments. CR2 binds C3d and iC3b. It has been demonstrated

that CR2 is able to amplify antigen-induced B cell acti-

vation through surface IgM (sIgM), to rescue peripheral B

cells from sIgM-mediated apoptosis, to promote antigen

processing and presentation of C3d-bound targets, to

modulate the expression of costimulatory molecules, to

stabilize the B cell receptor in lipid rafts, and to target IC to

germinal centers in secondary lymphoid organs [26]. Many

of these functions may occur via interactions of CR2 with

CD19 and CD81 on the B cell surface, where these

receptors form a multi-molecular signal transduction

complex.

Whereas the role as an activating receptor of CR2 is

better defined, the exact function of CR1 on B cells is yet to

be determined [35]. CR1 binds the C3b and with lower

affinity, iC3b and C4b, and it is capable of transmitting

both activating and inhibitory signals to human B cells.

Expression of CR2 and CR1 on human B cells has been

studied in some human autoimmune diseases. It was

observed that patients with SLE had abnormalities in the

expression of both CR2 and CR1 on B lymphocytes.

Peripheral B cells showed a marked decrease in both CR2

and CRl density when compared to control subjects [27,

36]. RA patients also had fewer CR1- and CR2-positive B

cells and decreased receptor expression compared to

healthy subjects [37, 38].

Complement system and autoimmune diseases

Systemic lupus erythematosus

SLE is the prototype of a multi-organ autoimmune disease.

As found in several other autoimmune disorders, SLE path-

ogenesis is multifactorial lying on genetic and environmental

factors and on abnormalities of both the innate and the

adaptive immune system [39]. The role of the complement

system in the pathogenesis of SLE is apparently paradoxical.

On the one hand, active SLE is accompanied by the activation

of the complement pathway, and there is evidence that the

complement system contributes to inflammatory tissue

damage. On the other hand, patients with hereditary

deficiencies of complement proteins of the classical pathway

are at increased risk for SLE, suggesting the protective role of

complement system’s proteins [5]. Hereditary homozygous

deficiencies of C1q, C1r and C1s (whose defect is usually

inherited together), and C4 are each strongly associated with

susceptibility to SLE, with frequencies of 93, 57, and 75 %,

respectively [5]. In contrast, 10 % of the subjects with C2

deficiency, the most frequent hereditary deficiency of the

complement system’s classical pathway, develop SLE [27].

The hypothesis invoked to explain the involvement of the

complement system in the pathogenesis of SLE is known as

the ‘‘waste-disposal’’ hypothesis [40]. According to this

hypothesis, complement system’s proteins play a role in

clearing IC and apoptotic cells from both tissues and circu-

lation. Failing this activity of the complement system, waste

material—consisting of partially degraded components of

the cytoplasm and nucleus—could accumulate and elicit an

autoimmune response. The ‘‘waste-disposal’’ hypothesis

envisages the following steps. The first step is the failure to

clear autoantigens [5]. The second step is the uptake of

autoantigen by immature dendritic cells in the presence of

inflammatory cytokines, which causes these cells to mature

into APCs, allowing the presentation of autoantigens to T

cells [5]. The third step is the provision of help by T cells to

autoreactive B cells, which have taken up autoantigen by

means of their immunoglobulin receptors. Such B cells

mature into plasma cells that secrete autoantibodies [2].

SLE-like disease associated with complement system’s

deficiencies clinically differs from SLE disease without

complement system’s deficiencies. In patients with SLE-

like disease and complement system’s deficiency, the onset

is at a younger age; renal, pulmonary, and pericardial

involvement are less conspicuous, but annular photosensi-

tive skin rashes are more prominent. Fulminant glomeru-

lonephritis, progressive nephritis, and renal failure are also

far less common in patients with SLE-like disease and

complement system’s deficiency. Antinuclear antibodies,

anti-DNA antibodies are usually relatively low or absent in

patients with SLE-like disease, whereas anti-Ro (SSA)

antibodies are often elevated [24].

The severity of SLE-like disease varies according to the

position of the missing component within the classical

pathway of activation (C1 [ C4 [ C2) [41]. Inherited

deficiencies of C1q and C4 are invariably associated with

the development of a severe SLE-like disease early in life,

while C2 deficiency is only weakly associated with a

milder form of SLE [41].

In the case of C3 deficiency, the clinical picture is rather

different [5]. It is characterized by recurrent pyogenic

infections, membranoproliferative glomerulonephritis,

rashes and it is rarely associated with SLE.

The initial cause of complement activation in SLE is

thought to be the formation of high levels of IC that

Diagnosis of Autoimmunity (2013) 56:477–491 481

123

activate complement via the classical pathway [40]. Key

mediators of tissue damage induced by the complement

system are the anaphylatoxins (C3a and C5a) and MAC.

Histologic data indicate that the activation of the comple-

ment system substantially contributes to tissues damages in

patients with SLE. Deposits of C3, C4, and associated

complement fragments are easily detected in biopsies of

inflamed tissues from patients with SLE [42]. In these

patients, the MAC is localized in the basement membrane

zone of cutaneous lesions and, compared with clinically

normal tissues, more prominent MAC deposits have been

observed in inflamed tissues [43].

Immunofluorescence studies in SLE patients have

shown deposits of immunoglobulins and complement in

renal glomeruli and vessels in spleen, heart, skin, and liver

[44, 45]. There is evidence that in SLE nephritis, the

activation of the complement system is triggered by the

deposition of IC in the glomerulus, either through the blood

flow or by in situ formation [44, 45].

In patients with active SLE, low complement concentra-

tions and activation of the complement system are charac-

teristic findings and are routinely evaluated for diagnostic

and disease monitoring purposes [17]. They can be consid-

ered conventional biomarkers for disease activity [46].

In vivo complement activation can be demonstrated by

tests for complexes or split products formed during the

activation of the complement system. Several studies have

shown that such measurements are more sensitive than

regular measurement of CH50 or complement native

components, such as C1q, C4, and C3, to verify comple-

ment activation and disease activity [17].

In addition to the previously mentioned reduced expres-

sion of complement receptors on B cells in SLE patients [47,

48], these are also reduced on erythrocytes. On red blood

cells, CR1 acts as a binding molecule for particles carrying

C3b clusters (for example parasites and bacteria) further

transporting them to the fixed mononuclear phagocytic

system in the liver, spleen, and bone marrow [47]. There is

evidence that reduction in CR1 levels on erythrocytes cor-

relates with disease activity in SLE [48].

Approximately a third of patients with SLE show ele-

vated levels of anti-C1q antibodies. Experimental models

seem to demonstrate that they are directed against a neo-

epitope of C1q not expressed in the intact C1q complex

[47]. These autoantibodies are associated with severe dis-

ease, with occurrence of glomerulonephritis and with

intense activation of classical pathway [47].

Rheumatoid arthritis

To date, the pathogenesis of RA is not fully understood but

there is increasing evidence of the role played by compo-

nents of the complement system’s cascade [49]. Animal

models suggest that the immunization with type II collagen

(CII), the major constituent protein of cartilage in diar-

throdial joints, induces autoimmune arthritis resembling

RA (collagen-induced arthritis or CIA) [50–53]. C3 or

factor B deficient mice immunized with bovine CII showed

reduction or complete remission of clinical or histologic

signs of arthritis [54]. Moreover, the systemic administra-

tion of anti-C5 monoclonal antibody (mAb) prevented the

onset of arthritis and improved established disease [55].

While increased complement system activation is plau-

sibly related to the onset and/or augmentation of inflam-

mation in RA, complement system’s deficiencies may also

induce RA. In particular, C1q and C2 deficiency are cor-

related with the development of RA [56, 57].

The plasma levels of most of the complement system’s

components are maintained by hepatic synthesis. We

evaluated plasma levels of complement system’s compo-

nents in 114 patients with active RA. Mean C3 and C4

plasma levels observed in patients with active RA were

significantly higher than in controls. This finding is con-

sistent with the presence of an underlining inflammatory

process [58] and confirms that complement system’s pro-

teins may act as acute phase proteins, with increased

hepatocyte synthesis occurring in response to inflammatory

cytokines [59–62].

However, it has to be noted that the synthesis of com-

plement system’s components also occurs in extrahepatic

sites and in chronically inflamed tissues such as the rheu-

matoid joint [63–66]. Synovial membrane cells which seem

to be responsible for the synthesis of complement system’s

components include lining cells, fibroblasts, mononuclear

phagocytes, and endothelial cells [67].

Evidence of complement system activation in the

synovial fluid is abundant. Levels of complement proteins

are generally depressed in the synovial fluid of patients

with RA, reflecting consumption of complement. More-

over, elevated levels of several complement cleavage

products, such as C3a, C3c, C5a, sC5b-9, Bb, C1-C1INH

complexes, have been observed in the synovial fluid [68–

71]. Some studies demonstrated low levels of MAC

inhibitors in synovial fluid and tissue of RA patients. This

observation might justify lytic or sublytic attacks on local

cells [70, 72].

Several mechanisms of complement system activation

have been proposed in RA patients. RA patients have

increased levels of circulating IC [73, 74], also including

rheumatoid factors (RFs), and they can activate the clas-

sical pathway of the complement system [75–78].

Although the activation of the classical pathway is pre-

dominant [79], the alternative pathway is also activated in

RA synovium as demonstrated by decreased levels of

factor B and properdin and increased levels of Ba in the

synovial fluid [80]. Increased complement activation via

482 Diagnosis of Autoimmunity (2013) 56:477–491

123

the lectin pathway could also play a role in RA. Changes in

IgG glycosylation cause an increase in binding of MBL

resulting in increased complement activation [81].

Trouw et al. [82], from in vitro observations, suggested

that anti-cyclic citrullinated peptide/protein antibodies

(anti-CCP) from RA patients may activate complement via

both the classical and the alternative pathways. Another

potential trigger for complement system activation in RA

patients could be CRP, since this acute phase protein can

activate the complement system both in vitro and in vivo

[83–87]. Furthermore, it appears that molecules present in

or released from cartilage, such as fibromodulin (FM), may

have a role in activating the complement system by inter-

acting with the globular head of C1q [88]. Several studies

failed to demonstrate a correlation between local and sys-

temic activation of the complement system in RA and

reported higher levels of complement cleavage products in

synovial fluid than in plasma [68, 89, 90]. These findings

are consistent with a prevalent local activation of the

complement system’s cascade. It has to be noted the pos-

sible association between the activation of the complement

system and disease activity. Some authors reported a cor-

relation between levels of complement system’s cleavage

products and disease activity in RA patients [68, 91].

Other inflammatory arthritides

There is limited availability of data in literature about role

of complement system’s proteins in other inflammatory

arthritides.

Studies conducted in psoriatic patients have shown

increased plasma concentrations of iC3b, C4d, and Bb

fragments, especially in patients with erythrodermic pus-

tular psoriasis and psoriatic arthritis (PsA) [92]. Partsch

et al. [93] have reported relatively low levels of the C3c

cleavage product in synovial fluid from PsA patients,

similar to that observed in patients with osteoarthritis.

A possible implication of complement activation in the

pathogenesis of PsA is supported by the finding of impaired

expression of complement regulators in these patients.

Triolo et al. [94] reported low expression of erythrocyte

membrane-anchored CD59, an important membrane

inhibitor of MAC. In keeping with this observation,

increased SC5b-9 plasma levels were detected in patients

with active disease and an inverse correlation was also

found between SC5b-9 plasma levels and CD59 expression

levels [94]. Rivas et al. found a statistically significant

decrease in CR1 density on erythrocyte membranes in PsA

patients with polyarthritis compared to controls. An inverse

correlation between CRl concentration and the articular

index of PsA patients has been reported, and this finding is

suggestive of a correlation between CRl levels and disease

severity in PsA patients [95].

Our group has recently conducted a study evaluating the

complement system in PsA treated with anti-tumor necro-

sis factor agents that demonstrated that moderate to severe

PsA disease is associated with higher C3 and C4 plasma

levels with respect to healthy subjects [96]. This finding is

compatible with the involvement of complement system’s

proteins during the acute phase response [97].

Systemic sclerosis

SSc is a chronic multi-system autoimmune disease that is

clinically highly heterogeneous. The two widely recog-

nized subsets of SSc are limited cutaneous and diffuse

cutaneous SSc. The disease affects the skin and multiple

internal organs leading, eventually, to fibrosis.

Although many studies have been conducted to inves-

tigate its pathogenesis, this is not fully understood and,

although the role of the complement system has been

investigated, no definitive conclusions can be drawn.

Reduced complement levels in SSc patients were first

observed in 1967 [98]. Senaldi et al. subsequently dem-

onstrated higher level of complement fragments in patients

with SSc, in particular those with diffuse cutaneous SSc,

with respect to controls. They postulated a direct correla-

tion between the complement system activation and clini-

cal severity [99]. In the light of these findings, low C3

levels were identified as candidate parameter for inclusion

in the American College of Rheumatology (ACR) classi-

fication criteria for SSc [100]. In 2001, hypocomplemen-

temia was included among the parameters used to assess

the disease activity score by the European Scleroderma

Study Group [101].

More recently Hudson et al. studied a cohort of 321

patients with SSc. It was observed that there was no dif-

ference in the clinical presentation of the disease between

patients with normal or low complement levels [102].

However, hypocomplementemia was associated with fea-

tures of overlap disease. In particular, inflammatory myo-

sitis and vasculitis were more frequent in patients with low

levels of complement system’s proteins compared to

patients with normal values [102]. Further studies are

needed to assess the role of the complement systems in the

pathogenesis of SSc.

Antiphospholipid antibodies syndrome

APS is characterized by thrombosis, recurrent miscarriage,

and pregnancy complications in the presence of anti-

phospholipid (aPL) antibodies. The pathogenic mecha-

nisms underlying aPL antibodies induced thrombosis are

not completely understood. It is well established that

activated complement fragments themselves have the

capacity to bind and activate inflammatory and endothelial

Diagnosis of Autoimmunity (2013) 56:477–491 483

123

cells as well as to induce a prothrombotic phenotype, either

through the MAC directly or through C5a receptor (CD88)-

mediated effects [103, 104].

To better understand these mechanisms, several murine

models have been developed. A study focused on APS

induced by passive transfer of human aPL-IgG antibodies has

demonstrated that complement blockade at the point of C3

activation prevents fetal loss and growth retardation [105].

Girardi et al. reported that, in a murine model, the

activation of the complement system’s cascade by the IC is

required for APS development. The model provided con-

vincing evidence that the activation of both classical and

alternative pathways is needed to generate sufficient C5

cleavage to cause fetal loss [106].

Recent experiments in mice have underscored the

importance of fetal regulation mechanisms to modulate

potentially damaging maternal immune responses derived

from the activation of the complement system. These

studies have focused on complement receptor 1–related

gene/protein y (Crry), a membrane-bound intrinsic com-

plement regulatory protein that blocks C3 and C4 activa-

tion on self-membranes in mice [107]. Adequate

complement system inhibition is essential for a normal

pregnancy, and this is demonstrated by the finding that

Crry deficiency in utero progressively leads to embryonic

death [108].

Moreover, studies performed in rats showed that CD59,

an inhibitor of C5b-9 assembly and insertion, protects

against thrombotic microangiopathy, demonstrating that

C5b-9 plays a critical role in the pathogenesis of throm-

bosis [109].

Interestingly, Pierangeli et al. found that mice deficient in

complement components C3 and C5 were resistant to

thrombosis and endothelial cell activation induced by aPL

antibodies. Furthermore, inhibition of C5 activation using

anti-C5 mAb prevented thrombophilia induced by aPL

antibodies. They showed that the activation of the comple-

ment system mediates two important effects of aPL anti-

bodies: induction of thrombosis and endothelial activation

[110]. Holers et al. [105] found that a C3 convertase inhibitor

can prevent fetal loss and growth restriction, and that mice

deficient in complement C3 are resistant to fetal injury

induced by aPL antibodies. Finally, Salmon and Girardi

explained pregnancy loss during APS as an inflammatory

disorder. They found that aPL antibodies targeted to decidual

tissues interfere with pregnancy by engaging the classical

pathway of complement system activation, followed by

amplification through the alternative pathway. Considering

the evidence that heparin inhibits the activation of the

complement system in vitro, it was postulated that heparin

may prevents pregnancy loss by inhibiting complement

activation on trophoblasts [111]. A recent study on humans

demonstrated that the activation of the complement system

also contributes to the development of APS in humans. It was

found that serum complement levels were significantly lower

in patients with primary APS than in patients with APS

secondary to non-SLE connective tissue diseases. Patients

with APS had significantly higher levels of C3a or C4a than

healthy controls, suggesting complement consumption

[112]. These studies highlight the importance of developing

and testing targeted complement inhibitory therapy for

patients with APS.

Inflammatory myopathies

Inflammatory myopathies can be differentiated into three

groups: dermatomyositis (DM), polymyositis, and inclu-

sion body myositis. There is increasing evidence that

complement-mediated microangiopathy plays a pathogenic

role in DM [113].

The primary antigenic target in DM is the endothelium

of the endomysial capillaries. Putative antibodies directed

against endothelial cells activate C3 [114]. In its turn,

activated C3 leads to formation of C3b and C4b fragments

and MAC, which can be detected early in the course of the

disease both in serum and in the capillaries before

inflammatory or structural changes are observed in the

muscular tissue [114]. Capillary complement deposits

induce swollen endothelial cells, vacuolization, capillary

necrosis, perivascular inflammation, ischemia, and

destruction of muscle fibers [114]. The characteristic

perifascicular atrophy reflects endofascicular hypoperfu-

sion, more prominent distally. In the later stage of the

diseases, a striking reduction in the number of capillaries

per muscle fiber associated with compensatory dilatation of

the lumen of the remaining capillaries is observed. Cyto-

kines and chemokines related to the activation of the

complement system are released. Cytokines and chemo-

kines upregulate vascular cell adhesion molecule (VCAM-

1) and intercellular adhesion molecule (ICAM-1) on the

endothelial cells and facilitate the egress of activated T

cells to the perimysial and endomysial spaces [114].

Muscle biopsy in DM reveals the deposition of the MAC

on endothelial cells [115]. Moreover, the deposition of

MAC was found in a high proportion of biopsies from skin

lesions of DM patients and was absent in unaffected skin,

suggesting that the complement system is also involved in

the pathogenesis of skin lesions [116]. The immunopath-

ologic mechanism associated with polymyositis and

inclusion body myositis is driven by CD8-positive cells and

to a lesser extent by the complement system.

Sjogren’s syndrome

SS is a chronic autoimmune disorder of the exocrine glands

with associated lymphocytic infiltrates of the affected

484 Diagnosis of Autoimmunity (2013) 56:477–491

123

glands. Dryness of the mouth and eyes results from the

involvement of the salivary and lacrimal glands [117].

SS occurs in a primary form not associated with other

diseases and in a secondary form that complicates other

rheumatic conditions.

In patients with primary SS, there is growing interest in

determining the negative prognostic significance of low

complement levels. The prevalence of hypocomplemente-

mia in SS patients has been evaluated in several studies.

Skopouli et al. [118] detected low C3 levels in 4 (2 %) and

low C4 levels in 44 (17 %) of 261 Greek patients, while Io-

annidis et al. [119] found low C3 in 17 (3 %) and low C4 in 122

(20 %) of 601 Greek patients in a multicenter study. Recently,

Theander et al. [120] reported low C3 levels in 98 (25 %) and

low C4 levels in 105 (27 %) of 386 Swedish patients. Ramos-

Casals et al. [121] observed low C3 values in 42 (12 %), low

C4 values in 39 (12 %), and low CH50 in 51 (15 %) of 336

Spanish patients. In the latter study, patients with SS and HCV

infection presented a higher frequency of hypocomplemente-

mia with respect to patients with primary SS, 76 and 24 %,

respectively. The proportion was even higher (78 %) in patient

with SS and HCV-related cryoglobulinemia.

Low complement levels seem to be associated with

systemic SS features, including extraglandular features

(fever, articular and cutaneous involvement, vasculitis and

peripheral neuropathy) and immunological markers (cryo-

globulinemia, RF) [121]. Hypocomplementemia is also a

marker of unfavorable outcomes in primary SS because it

is associated with lymphoma development and death. In

particular, low C4 levels showed the closest statistical

association with mortality and lymphoproliferation [119–

121]. These results support the inclusion of complement

level determination as a predictor of SS outcome both at

diagnosis and routinely during the clinical follow-up.

Vasculitides

Vasculitides are defined by the presence of leukocytes in

the vessel wall with reactive damage. Depending on the

specific vasculitic disorder, affected vessels vary in size,

type, and location. Many vasculitic disorders are caused by

IC, and the activation of the complement system is

involved in their pathogenesis. In particular, complement

involvement was demonstrated in small vessel vasculitides,

as described in the next paragraphs, but complement-

dependent cytotoxicity against endothelial cells has been

implicated in large vessel vasculitides too, such as Taka-

yasu’s arteritis [122].

ANCA-associated vasculitides

The anti-neutrophil cytoplasmic autoantibody (ANCA)-

associated vasculitides (AAVs) include Wegener’s

granulomatosis, microscopic polyangiitis, Churg–Strauss

syndrome, ‘‘renal-limited’’ vasculitis and certain drug-

induced vasculitis syndromes [123]. These small vessel

vasculitides are characterized by necrotizing inflammation

of the vessel wall, particularly of small arteries, arterioles,

capillaries, and venules, in conjunction with the presence

of ANCAs. In AAV, ANCAs are directed against pro-

teinase 3 (PR3) or to myeloperoxidase (MPO).

In AAVs, the adaptive immune response, expressed by

the ANCAs, interacts with innate immunity, in particular

with neutrophils and the complement system. Together

they target the endothelium, resulting in necrotizing vas-

culitis [124].

In vitro data demonstrate that in AAVs, the complement

system constitutes an amplification loop for ANCA-

induced neutrophil activation. Schreiber et al. [125]

showed that supernatants from ANCA-activated neutro-

phils activate the complement system via the alternative

pathway, resulting in the production, among others, of C5a.

C5a was able to prime neutrophils for ANCA-induced

activation, and blocking of the C5a receptor on neutrophils

abrogated this process.

Murine models have shown that complement depletion

prevented MPO-ANCA glomerulonephritis and mice defi-

cient in C5 or complement factor B did not develop pauci-

immune necrotizing crescentic glomerulonephritis

(NCGN) [126]. In agreement with these experimental data,

the complement components MAC, C3d, and factor B

could be detected in diseased glomeruli of patients with

AAVs. The alternative pathway component factor B

colocalized with MAC, but the classical pathway compo-

nent C4d could not be detected [127]. According to these

findings, compounds interfering with the complement

cascade should be explored as therapeutic options for

ANCA-associated vasculitides.

Cryoglobulinemia

Cryoglobulins (CG) are an abnormal group of serum pro-

teins that share the common property of reversible pre-

cipitation at low temperatures. It is widely accepted that the

majority of CG are either intact monoclonal immuno-

globulins or IC in which one component, usually IgM,

exhibits antibody activity to IgG. The latter are known as

mixed CG [128].

Monoclonal CG are usually associated with hemato-

logical disorders, whereas mixed CG are found in many

infectious and systemic disorders. Essential mixed cryo-

globulinemia shows a striking association with hepatitis C

virus infection ([90 %). It is a systemic vasculitis (leuk-

ocytoclastic vasculitis) affecting cutaneous vessels and

multiple visceral organs [129]. The classical pathway of the

complement system is usually activated in both essential

Diagnosis of Autoimmunity (2013) 56:477–491 485

123

and secondary cryoglobulinemia. Decreased C4 and C2

levels are observed together with slightly altered C3 levels.

Late complement components are also insignificantly

affected, although modest elevations have been reported.

Diminished serum levels of complement components may

reflect ongoing consumption by CG-containing IC [130,

131].

Henoch-Schonlein purpura/IgA nephropathy

Henoch-Schonlein purpura nephritis (HSPN) and IgA

nephropathy (IgAN) are currently considered related dis-

eases. Both diseases display similar histologic features and

IgA abnormalities. The common clinical pattern of IgAN is

an indolent progressive disease with slowly increasing

proteinuria and loss of the renal function associated with

episodes of macroscopic hematuria in half of the patients.

In the majority of patients, HSPN is characterized by acute

onset followed by full recovery [132]. The activation of the

complement pathway is likely to be involved in the path-

ophysiology of glomerular lesions. Glomerular deposition

of MBL, L-ficolin, MASP, C4d are reported in the vast

majority of patients with HSPN and IgAN. These findings,

together with the absence C1q, support the predominant

activation of the lectin pathways of the complement system

as pathophysiological mechanism [133]. The deposition of

complement fragments, derived from the activation of the

lectin pathway, has been shown to be associated with a

higher degree of proteinuria and hematuria as well as with

more severe histologic lesions in both HSPN and IgAN

patients [134, 135]. These findings emphasize the need for

further studies to assess the potential significance of the

measurement of blood and urinary complement splits

products and MAC to evaluate the disease activity.

Potential therapies targeting complement cascade

in autoimmune disease

The complement system is increasingly recognized to have

a causal link with tissue damage during ischemic, inflam-

matory, and autoimmune diseases. This makes the com-

plement system an attractive target for the treatment of a

wide range of diseases, such as connective tissue diseases,

glomerulonephritis, myocarditis, multiple sclerosis, type I

diabetes mellitus, asthma, myocardial infarction, paroxys-

mal nocturnal hemoglobinuria, vasculitis, and many others

[14, 15, 136–138].

An issue that deserves further consideration is the side

effects potentially associated with the modulation of

complement system’s activity in the long term. Prolonged

systemic suppression may, for instance, increase the sus-

ceptibility to bacterial infections [56].

Several compounds interfering with complement system

cascade have been studied in experimental models for

autoimmune diseases. The main therapeutic strategies are

inhibition of complement activation components, inhibition

of complement receptors, and inhibition of MAC [136].

Different molecules believed to have complement modu-

lation properties have been studied in several animal

models for arthritis. These include soluble CR1 [139] (that

suppresses complement system activation at the main

gathering point C3), C3a [140], and C5a [141] receptor

antagonists (that can be used to control the anaphylatoxins

C3a and C5a), recombinant CD59 [142] (that inhibits the

formation of MAC). Although PMX53, a C5a mimetic

compound that binds the C5a receptor, has shown

encouraging results in rats, where a significant improve-

ment of arthritis was observed with no side effect [141], its

use in RA patients did not fill the expectation [143].

The involvement of the complement system in the

pathogenesis of SLE was proven by the findings that an

inhibitory anti-C5 mAb blocks the development of glo-

merulonephritis in the (NZB 9 NZW)F1 animal model for

SLE [144], that fB_/_ MRL/lpr mice are protected from

renal disease [145], and that mice in which C3 activation

was blocked using transgenic expression of soluble Crry

[146] or recombinant Crry (a Fc fusion protein designated

Crry-Ig) [147] did not develop glomerulonephritis. Two

complement inhibitors, soluble complement receptor 1

(TP10), and a monoclonal anti-C5 antibody (Eculizumab),

have been shown to inhibit complement safely and promise

to be used therapeutically in lupus nephritis [148].

Additional animal models have assessed the comple-

ment system as potential therapeutic target for autoimmune

diseases, such as APS, PM/DM, autoimmune myocarditis,

multiple sclerosis, type 1 diabetes mellitus, asthma [136].

However, these did not translate into viable treatment

options for autoimmune diseases in humans.

Only two complement modulators have been approved

for use in human. Eculizumab binds to the complement

protein C5 inhibiting its cleavage and is indicated for the

treatment for paroxysmal nocturnal hemoglobinuria [149].

Plasma-derived C1 esterase inhibitor is indicated for the

treatment for hereditary angioedema [150].

An additional strategy to be considered is the replace-

ment of missing complement system’s proteins, to reverse

the effects of complement deficiencies. However, this

approach presents several potential drawbacks. Indeed,

purified or engineered complement proteins are not avail-

able for treatment purposes, and whole plasma preparations

would have to be used, with obvious complications of

plasma treatment. Secondly, the replacement of a missing

complement protein may be followed by complement

activation and tissue damage. Finally, exposure to a protein

that is genetically deficient may cause development of

486 Diagnosis of Autoimmunity (2013) 56:477–491

123

antibodies. At present, only anecdotal observations have

been reported [47]. The modulation of the complement

system is one of the benefits associated with the use of high

dose intravenous immunoglobulins (IVIg) in autoimmune

conditions. The complement systems modulating effect

exhibited by IVIg can be explained by several mechanisms.

First, activated C3 and C4 may bind to immunoglobulin

molecules, which then serve as scavengers, hence avoiding

in situ deposition of these fragments [151]. Furthermore,

C1q may bind to immunoglobulin leading to a deviation of

C1 binding from its target to the IVIg [151]. Third, IVIg

may enhance the inactivation of C3 in complex with

immunoglobulins and thus down-regulate the C3 conver-

tase activity [150]. Finally, IVIg are able to cause a mild

and controlled activation of the complement system. This

is not harmful per se, and it may reduce a pathological

activation observed in the pathogenesis of autoimmune

disease [151]. A broadly applicable anti-C therapeutic

agent, useful in acute and chronic conditions, should be

inexpensive, highly specific, either have a very long plasma

half-life or be active orally and be able to block the path-

ological activation of the complement system while caus-

ing minimal disruption of the systemic complement

function [152]. None of the currently available agents

satisfy these requirements, but data derived from preclini-

cal studies and initial clinical trials suggest that comple-

ment modulation could become an important therapeutic

strategy in autoimmune conditions in the next decades.

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