Indian Journal of Experimental Biology
Vol. 53, November 2015, pp. 701-713
Complement and membrane-bound complement regulatory proteins as biomarkers
and therapeutic targets for autoimmune inflammatory disorders, RA and SLE
Nibhriti Das*
Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110 029, India
Received 26 March 2015; Revised 10 June 2015
Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system
consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get
activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical,
alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement
receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive
immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant
uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement
regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important
information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the
clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that
affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement
back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and
therapeutic targets for autoimmune diseases with focus on SLE and RA.
Keywords: Autoimmune diseases, Complement receptor type 1 (CR1), Decay accelerating factor (DAF), Inflammation,
Mannose binding lectin (MBL) pathway, Membrane cofactor protein (MCP), Membrane attack complex (MAC),
Polymannans, Rheumatoid Arthritis (RA), Systemic lupus erythematosus (SLE).
Complement was identified more than 100 years ago
as a result of its ‘complementary’ bactericidal activity
and its role in phagocytosis of cellular debris1. This
pro-inflammatory system consists of about 40 proteins
in the form of inactive enzyme precursors (zymogens),
soluble or cell-bound regulatory proteins and receptors
specific for effecter peptides of the system2. These
effector peptides are generated by activation of the
zymogens in the system by three different pathways
triggered by different molecular patterns like antigenantibody complexes, altered self antigens and pathogen
associated molecules3. Active peptides and a membrane
attack complex (MAC) are generated which effect a
——————
*Correspondence:
E-mail: [email protected]
Present add: Department of Biochemistry, Nayati Health Care and
Research Pvt. Ltd., Gurgaon-122 002, Haryana, India.
Abbreviations: C, complement; CIA, collagen-induced arthritis;
CR1, complement receptor type 1; DAF, decay accelerating
factor, MAC, membrane attack complex; MASPs, MBL associated
proteins; MBL, mannose binding lectin; MCP, membrane cofactor
protein; RA, rheumatoid arthritis; RCA, regulators of complement
activation; SLE, Systemic lupus erythematosus; TCC, terminal
complement complexes.
wide range of functions including injurious effects to
self. Complement regulatory proteins keep complement
activation under control and prevent complement
mediated injury to self in normal health3.
Several studies including ours have suggested that
the levels of active complement peptides and
expression of complement regulatory proteins get
altered in autoimmune diseases and many other
disease conditions3,4. This has formed the basis of
exploring complement and complement regulatory
proteins as biomarkers and therapeutic targets. While
each of the complement components hold promise as
biomarkers, research has geared up to explore the
suitability of complement regulatory proteins especially,
those encoded by regulators of complement activation
(RCA) and membrane-bound complement regulatory
proteins as biomarkers3.
Strategies in the development of complement-based
therapeutics range from use of anti-complement
antibodies,
enhancing
complement
mediated
phagocytosis of undesired elements and pathogens,
inhibiting complement mediated entry of pathogens
to infect the cells, inhibitory structural analogues,
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INDIAN J EXP BIOL, NOVEMBER 2015
increasing complement mediated regulation
autoinjury, use of DNA mimetics, etc5,6.
of
Mechanisms of complement activation
Activation of the complement pathways occurs
in a sequential manner by proteolytic cleavages
of zymogens followed by their association7.
Complement activation can take place by three
different pathways, depending on the molecular
triggers. These are; Antibody-dependent classical
pathway8 antibody-independent alternative pathway9
and mannose binding lectin (MBL) triggered
pathway10. Overview of the complement activation
pathways are depicted in Fig. 1.
All the pathways converge at C3-convertase,
resulting in the formation of pro-inflammatory
anaphylatoxins (C3a and C5a), chemotaxins (C5a),
opsonins (C3b, C4b, iC3b, C3d, C3dg) and a terminal
lytic complex C5-C9(n), known as membrane attack
complex (MAC)11,12.
The classical pathway
The classical pathway is initiated by antigenantibody complexes containing complement fixing
IgG or IgM antibodies and by other molecules
generated as a result of an inflammatory reaction such
as C-reactive protein or serum amyloid protein8,10,13.
This pathway can also be activated in an antibodyindependent manner by viruses and gram-negative
bacteria1, and its components are named as C1-C9.
Activation of the classical pathway is initiated by
binding of C1q, a part of the first component of
complement (C1), to the Fc portions of immunoglobulins that further activates the C1r and C1s
esterases, the subcomponents of C1. C1s esterase
cleaves C4 and C2 to form bimolecular complex
C4b2a which has C3 convertase enzymatic activity.
The C3 convertase cleaves C3 and generates a small
fragment C3a and a large fragment C3b. Attachment
of C3b to C4b2a complex forms the C4b2a3b
complex, which has C5 convertase activity10.
The alternative pathway
The alternative pathway is triggered by
carbohydrates, lipids and proteins found on foreign
and non-self surfaces9,14. It provides a rapid defense
against certain pathogens14. C3 is constantly
hydrolyzed at a low level (“tick over”) to form C3b,
which binds to targets such as bacteria. Factor B is
then recruited to the bound C3b followed by Factor
D that cleaves Factor B to form the C3 convertase
C3bBb, which is stabilized by the presence of
plasma properdin15. The spontaneously generated
C3 convertase C3bBb cleaves C3 to produce more
C3b autocatalytically and form C5 convertase
C3bBbC3b. Thus, alternative pathway is devoid
of C1, C2 and C4 but has Factor B, D and also P as
additional components.
The mannan binding lectin (MBL) pathway
The MBL pathway is activated when either
mannose binding lectin or Ficolin bind to
carbohydrate moieties on surfaces of pathogens
Fig. 1—Pathways of complement activation. [The classical, MBL and alternative pathways converge into a final common pathway
when C3 convertase (C3 con) cleaves C3 into C3a and C3b. Ab (antibody); Ag (antigen); MAC (membrane attack complex); MASP
(MBL-associated serine protease); MBL (mannose-binding lectin)]
DAS N: COMPLEMENT SYSTEM AS BIOMARKERS AND THERAPEUTIC TARGETS FOR RA & SLE
including yeast, bacteria, parasites and viruses11.
It has all the components of the classical pathway
except C1 which is replaced by MBL or Ficolin.
Both, MBL and Ficolin circulate in the serum as
complexes with MBL associated proteins (MASPs).
Binding to pathogens induces conformational
changes resulting in auto-activation of MASP2
which cleaves C4 and C2 to form C3 convertase
C4bC2a16. The rest of the cascade is identical to that
of the classical pathway.
Membrane attack complex (MAC)
Amplification of either of the pathways leads to
generation of C5b which then binds sequentially to
C6-C9 components to form membrane attack
complex (MAC). Binding of C6 to C5b induces a
conformational change in C6 making it capable of
binding C7. The C5b-7 complex is hydrophobic and
capable of binding to lipid membranes. C8 in turn
may bind either to a cell-bound or a soluble C5b-7
complex. The C5b678 complex creates a small
pore- (10 Å dia) that can lead to the lysis of
red blood cells but not of nucleated cells. In the
final step, 10-17 molecules of C9 bind and
polymerize to form a single C5b678 complex.
During polymerization, the C9 molecules undergo
transition, so as to insert into the membrane 17. The
intermediate complexes C5b-7, C5b-8 and C5b-9
formed during the pore assembly are referred to as
terminal complement complexes (TCCs), while
C5b-9, the final complex and the most effective at
inducing cell death, is referred to as the membrane
attack complex (MAC) 17. The completed MAC
has a tubular form and functional pore size of
70-100 Å. Through the central channel of MAC,
ions and small molecules can freely diffuse. The
cell is unable to maintain its osmotic stability and is
killed by an influx of water and loss of electrolytes18.
When the number of channels assembled on the
surface of nucleated cells is limited, sublytic MAC
can activate target cells (neutrophils, endothelial
and epithelial cells) to secrete inflammatory
mediators, such as reactive oxygen metabolites,
prostaglandins and thromboxanes. Sublytic C5b-9
protects cells from apoptotic cell death through
post-translational regulation of Bad which inhibits
the mitochondrial pathway of apoptosis. Also,
C5b-9 inhibits caspase-8 activation; downregulates
FasL expression, Bid cleavage and induces
significant increase in FLIP cleavage 19.
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Peptides generated during complement activation
and functions of the complement system
Complement activation via all the three pathways
leads to generation of small anaphylatoxins C3a, C4a
and C5a. These anaphylotoxins target a broad spectrum
of immune and non-immune cells. Also they act as
chemo-attractants of the leukocytes. C5a is the most
potent anaphylotoxin and chemotaxin. These peptides
trigger inflammatory pathways by interacting with their
receptors on the granulocytes and other cells. C3b and
C4b are opsonins that facilitate phagocytosis of
pathogens, other danger signals and clear immune
complexes. The terminal complement complex/(MAC)
pokes holes on the cell membranes and lyse the cells18,20-26.
In addition, Complement proteins play an important role
in modulating adaptive immunity and in bridging innate
and adaptive responses27. Complement regulates CD4+ and
CD8+ T cell activation and effecter functions28. C3d or
C3dg coated immune complexes bind to CR2-CD19CD81 co-receptor and decrease the threshold required
for B cell activation29. In addition, CR2 enhances B -cell
memory and CR1 regulates B cell responses. CD46 has
been found to regulate the proliferation and effector
functions of CD4+ and CD8+ T cells30. C3a and C5a via
their receptors C3aR and C5aR on dendritic cells, T cells
and macrophages also influence various adaptive
immune responses (Table 1)31-34.
Complement regulatory proteins—Complement
activation is under stringent control by a large number
of complement regulatory proteins (Table 2).
Table 1—Complement Peptides and their functions Complement
peptide Functions Complement Receptor
C3a,C5a
Anaphylaxis and
chemotaxis,provoke
inflammation,regulation
of adaptive immunity.
C3b,C3bi,C4b
Opsonization,
facilitation of
phagocytosis,
ADCC,clearance of
immunecomplexes,
regulation of adaptive
immunity,facilitate entry
of certain opsonized
pathogens like M. tb.
Induction of B-cell
responses.
Poke holes into the
bacterial cell membranes
and lysis of the cell,
Induction of apoptosis.
C3d
C5b6789(n),
MAC
C3a,C5a receptors
on granulocytes,
dendritic cells,
T cells and
macrophages.
CR1,CR3,CR4.
CR3 and CR4 are
integrins.
CR2 on the
B-lymphocytes
Plasma membrane
lipid bi layer.
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INDIAN J EXP BIOL, NOVEMBER 2015
Table2—complement regulatory proteins
Regulator/
soluble/membranebound/Pathway
C1 inhibitor
(C1Inh)/soluble/
Classical Pathway
C4 BP
Mechanism of Action
Inhibits Serine protease,
dissociating C1r2s2 from C1q.
Binds with C4b and inhibits the
formation of C3 convertase.
Factor
Binds with C3b; cofactor for
H(FH)/soluble/Alternative cleavage of C3b by factor I.Inhibits
Pathway.
formation of C3 convertase.
Fluid phase inhibitor.
Factor-I /soluble
Serine protease: cleaves C4b or C3b
using C4bBP, CR1, factor H, DAF,
or MCP as cofactor
Serum proteases/soluble
Blocks fluid-phase MAC
S protein/
Binds soluble C5b67 and prevents
Vitronectin/soluble
its insertion into cell membrane
Anaphylatoxin
Inactivates anaphylatoxin activity
inactivator/soluble
of C3a,C4a,andC5a by
carboxypeptidase N removal of
C-terminal Arg
Membrane-cofactor
Block formation of C3 convertase
protein (MCP)/
by binding C4b or C3b; cofactor
membrane-bound/
for factor I-catalyzed cleavage
Classical, alternative and of C4b or C3b C3bBb
lectin pathway
Decay-accelerating
Accelerates dissociation of C4b2a
factor (DAF or CD55)/
and C3bBb(classical and alternative
membrane-bound/
C3 convertases)
Classical, alternative and
lectin pathway
Membrane inhibitor of
Bind to C5b678 on autologous cells,
reactive lysis
blocking binding of C9
(Protectin/HRF20/ MIRL
or CD59)/ membranebound
The regulation is achieved either by intervening the
cascade amplification by binding proteins (C4bp,
C3bp), inhibiting the activation (C1 inhibitor),
inhibiting the formation of the end MAC (CD59,
s-protein, clusterin), accelerating the decay of the
active fragments (DAF, CR1) and acting as a cofactor
(CR1, MCP) for the ultimate destruction of active
peptides by factor I, a serine protease. Factor I is the
master regulatory protein as it degrades the active
complement fragments in no time with the help of
the co-factors and decay accelerating proteins35.
Most stringent regulation is at the level of C3 or C4
by a group of complement regulatory proteins
encoded by a single gene cluster called regulators of
complement activation (RCA) gene family36.
Complement as biomarker—With regard to human
disease, the complement system has been shown to
play a role in the pathogenesis of various immune-
mediated disorders, including dermatologic, renal,
neurologic, and rheumatic diseases37.
Complement and complement regulatory proteins
as biomarkers for RA and SLE
Exaggerated complement activation, deficiency of
complement components or modulation of complement
regulatory proteins had been observed in autoimmune
disorders. While active complement peptides
and MAC may cause significant cellular injury in
autoimmune disorders, complement regulatory
proteins protect the host against complement mediated
autoinjury38.
Rheumatoid arthritis (RA)
There is increasing evidence that the complement
system could play an important role in the
pathogenesis of RA. Complement activation could be
induced by immune complexes consisting of type II
collagen (CII) and CII autoantibodies39 or by IgG and
rheumatoid factor in vitro40. Similar mechanisms
are activated by increased release of histamine in
response to C5a by synovial mast cells, which have
been shown to bear significant amounts of C5aR in
RA compared with OA39,40. Assembly of TCC on cell
membranes results in the formation of, which can lead
to lytic attacks on synovial cells and chondrocytes,
facilitated by low levels of the membrane-associated
inhibitor of the MAC protectin (CD59) on synovial
cells MAC41,42. Since synovial cells as well as other
nucleated cells are rather resistant to lytic attacks,
the primary effect of the MAC in RA appears to be
sublytic attacks on the cells. It results in a release
of reactive oxygen metabolites, prostaglandin E2,
leukotriene B4, and interleukin-6 from synovial cells
which may lead to enhanced DNA damage in
rheumatoid synovium and increased collagenase
specific mRNA expression by synovial fibroblasts43.
These effects are supported by low concentrations
of the fluid-phase MAC inhibitors clusterin and
vitronectin in synovial fluid. Of interest, the “hot
zone,” in which complement activation results in
actual damage, appears to be the microvasculature, a
fact that is illustrated by the intensive amounts of
C5b-9 close to activated lymphocytes in this area43.
Certain complement proteins may also contribute to
joint destruction. C1s, which is increased by TNF in
chondrocytes, is involved in cartilage degradation44.
More important, complement, in particular C1s in
conjunction with matrix metalloproteinase 9, has a
physiologic role in remodeling of cartilage in the
DAS N: COMPLEMENT SYSTEM AS BIOMARKERS AND THERAPEUTIC TARGETS FOR RA & SLE
growth plate and in fracture healing44,45. Findings in
studies using animal models of RA also support
the hypothesis that complement is required for
the development of arthritis. In DBA/1LacJ mice with
collagen-induced arthritis (CIA), systemic administration
of a specific anti-C5 monoclonal antibody inhibits
terminal complement activation in vivo and prevents
the subsequent on set of arthritis in immunized mice,
and ameliorates established disease46. Wang and
colleagues47 also concluded that activated TCC plays
an important role in the induction as well as the
progression and manifestation of disease. Moreover,
C5-deficient mice do not develop CIA48.
Many studies have shown high levels of
complement components and active complement
metabolites C2, C3, C3a, C5a, and TCC C5b–9 in
serum, SF, and ST. Furthermore, positive correlations
have been shown between SF complement activation
(e.g., levels of plasma C3dg, a C3 activation
metabolite) and local as well as general disease
activity in RA and between C2 and C3 expression in
RA synovium and inflammation49-53.
Systemic lupus erythematosus (SLE)
Measuring serum complement levels had been
considered the "gold standard" for monitoring disease
activity in SLE patients. Six decades ago, Vaughan et al.
revealed the association between decreased complement
and active SLE54. On usefulness of these assays in
monitoring SLE, some studies have concluded that
measuring C proteins are valuable for predicting the
disease course while other studies have deduced that
measuring C proteins are of minimal value55,56.
Conventionally, complement activation is assessed by
functional assays quantifying hemolytic activity
(CH100 or CH50 which measure total complement
activity) or by static measures of serum complement
components such as C1q, C2, and C4 (classical
pathway activation), factor B (alternative pathway
activation), or C3 (representing the common terminal
pathway)55,56. Low concentrations of complement
components due to increased catabolism are found in
a majority of patients with active and severe SLE55,56.
The sensitivities and specificities for the tested
components were 70 and 70%, respectively, for
CH50, 64 and 91% for C3 and 64 and 65% for C4. In
further analysis by recursive partitioning on the same
data set, the combined sensitivity and specificity
forCH50, C3 and C4 were 74 and 88%57. In a more
recent study, low levels of C1q were found to have a
high specificity for SLE (96%) but the sensitivity was
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low (20%)58. However, according to some studies
measurement of complement proteins in the plasma
doesn’t provide a reliable indication of the disease
activity59. This is because apart from the disease
process, level of C also varies due to difference in the
rates of synthesis among patients. In an effort to
improve the value of complement measurements in
the assessment of disease activity, attempt has been
made to measure the levels of serum markers of
complement activation, such as anaphylatoxin
levels60, C1r-C1s-C1 inhibitor complexes55, C4d
levels61, iC3b or C3dg levels62 and MAC/TCC levels63
to replace the static measurement of complement
proteins. Such measurements are more sensitive than
regular measurement of CH50 or complement
components such as C1q, C4 and C364-66. However,
none of these assays has been widely adopted in
clinical practice. Each of these complement activation
products is highly unstable in vivo and, therefore,
difficult to measure with any useful reliability outside
a research setting making them unsuitable for clinical
diagnosis. Moreover, antibodies to complement
protein, especially C1q, can cause rapid complement
activation, which does not reflect disease activity66.
Some reports state that markers of complement
activation in tissues correlate better with the presence
of inflammation, e.g., the presence of the membrane
attack complex is more prominent in inflamed tissues
like kidney67,68 or skin69 compared with clinically
normal tissues from patients with SLE.
Proliferative glomerulonephritis (WHO class III and IV)
and that C1q concentration decreased prior to clinical
manifestations of flares of the disease70. Low C1q levels
have also been shown to predict the histopathological
outcome of lupus nephritis71. C3d measurement in urine
has also been suggested to be of value for evaluation of
active SLE glomerulonephritis71. In sequentially
followed patients, evidence of activation of the early
part of the classical pathway measured by C1INH2C1r-C1s complexes is detected prior to C3 activation64.
For a better predictive value, sequential measurements
of C are required for monitoring disease activity.
Since complement activation occurs in several
co-morbidities of SLE, especially infections, the
specificity of C level as a disease marker is low, even
though the assays well discriminate active disease
from the remission state. Furthermore, artefacts
caused by complement activation in vitro during
coagulation and other handling procedures add to
the problem.
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INDIAN J EXP BIOL, NOVEMBER 2015
Membrane- bound complement regulatory proteins
as biomarkers
Studies with gene knockout mice have suggested that
membrane bound complement regulatory proteins may
critically determine the sensitivity of host tissues to
complement injury in autoimmune and inflammatory
disorders71. The upregulation of many of the membrane
bound molecules have been reported in inflammatory
tissues and organs affected by autoimmune diseases.
In vitro studies have also revealed that the expression
of the CRPs can be modulated by ICs, several
cytokines like TNF-alpha, IL-1beta, TGF-beta, and
IFN-gamma, etc.72. The membrane-bound complement
regulatory proteins decay-accelerating factor (DAF,
CD55), membrane cofactor protein (MCP, CD46),
complement receptor-1 and CD59 provide protection
from autologous complement-mediated injury. DAF
and MCP act at the level of the C3 convertase. In
contrast, CD59 inhibits the terminal pathway of
complement activation, preventing the incorporation of
C9 into the MAC72.
The most studied complement regulatory protein
is CR1. Role of other CRPs like C1 inhibitor, DAF,
MCP, CD59 and others are beginning to emerge.
Following is a brief account of the studies those
suggest potential of CR1, DAF, MCP and CD59 as
the biomarkers for RA and SLE72.
Complement receptor type 1 (CR1, CD35)
Rheumatoid arthritis (RA)—It had been observed
that CR1 expression goes down on erythrocytes and
leukocytes73,74. The reduced level of CR1 on these
cells is speculated as a key mechanism contributing to
immune complex overload and exaggerated
complement activation in RA. Existing reports have
indicated that CR1 has a negative regulatory role in
the activation of human B and T lymphocytes. The
underlying mechanism is not clear. Several studies
have documented modulation of CR1 levels in RA.
Lowered expression of E-CR1 has been reported in a
number of studies. However, studies on leukocytes
have reported increased expression of CR174,75.
In view of the exaggerated complement activation
in RA and significance of complement receptor 1
(CR1/CD35) as a complement regulatory protein
(CRP), this author observed lower levels of leucocytecomplement receptor 1 (L-CR1) transcript in 45 RA
patients compared to 66 controls, and their
correlations with the levels of CIC, C3d and DAS28
at different time-points in RA patients suggested CR1
as a potential disease marker for RA76.
Systemic lupus erythematosus (SLE)—Walport and
colleagues77,78 observed low CR1 and deposition of
C4 and C3 fragments on erythrocytes in active SLE.
A later study79 demonstrated that the combined
detection of high levels of erythrocyte-bound C4d and
low levels of CR1 on erythrocytes had high sensitivity
(72%) and specificity (79%) for SLE. E-CR1 got
much attention due to the fact that E-CR1 plays an
important role in the clearance of immune complexes,
overload of which is a major cause of the pathological
manifestations in SLE80,81. In addition to E-CR1,
lower levels of CR1 on leukocytes and glomerular
podocytes were also reported in SLE82. Other
Studies83,84 have documented a decline in B cell and
neutrophil CR1, which correlated positively with the
decline in E-CR1 in SLE. Another study85 showed that
recombinant sCR1 could restore the immune complex
binding ability of CR1-deficient erythrocytes. These
findings strongly suggested a key role of deficient
CR1 expression in the pathogenesis of SLE.
Further studies from our lab showed a drastic
decline in the levels of u-CR1 in SLE86 which
correlated with expression of glomerular CR1.
Therefore, u-CR1 was suggested as a potential marker
to diagnose glomerular involvement in SLE. Earlier,
we observed E-CR1 to be a prognostic marker for
glomerulonephritis and rheumatoid arthritis87. Later
we found marked decline in the levels of CR1
transcript and protein and negative correlations
between the levels of leucocyte CR1 transcript and
protein with the disease activity and severity of
SLE88. This suggested L-CR1 transcript as a disease
activity marker for SLE. Further studies are needed in
this direction to be more conclusive about the
usefulness of leucocyte CR1 as a bio marker for
SLE88. Membrane cofactor protein (MCP, CD46)
Rheumatoid arthritis (RA)—In RA, in a retrospective
and follow-up study, we found a marked decline of
neutrophil and PBMC MCP transcript in patients with
rheumatoid arthritis which correlated negatively with
the disease activity89. Studies elucidating the role of
MCP in SLE modulation are limited. There is a report
of elevated serum levels of soluble MCP (sMCP) in
patients with SLE90. Also, the level was significantly
higher in case of patients with active SLE compared
to those with inactive SLE and the level declined with
cortisol treatment. Longitudinal analysis of soluble
sMCP showed that sMCP levels decreased in parallel
with the levels of anti-dsDNA and correlated with
reduced CH50 levels90. However, the source as well
as the function of this serum sMCP is not clear. An
DAS N: COMPLEMENT SYSTEM AS BIOMARKERS AND THERAPEUTIC TARGETS FOR RA & SLE
immunohistochemical study of the expression of
MCP in the kidney tissues of patients with renal
diseases, including lupus nephritis has shown
significantly increased intensity of MCP in the
affected glomeruli when compared to normal91. Since
there are a massive deposition of C3 and immunoglobulin in the glomeruli of lupus nephritis patient92,
the C3 regulatory protein MCP might have a
protective role in the glomerular tissue injury and the
increased MCP could be a result of up-regulated
synthesis of MCP in the glomerular cells.
Systemic lupus erythematosus (SLE)—In another
study, we have suggested that the expression of
leukocyte MCP at the mRNA level is closely related
to disease activity in SLE. Further, we speculated a
protective role of MCP in response to increased
disease burden. Moreover, the follow-up study
suggested MCP as a potential disease marker93.
Decay accelerating factor (DAF, CD55)
Not much is known about the role of DAF in the
disease process of SLE; however, evidence strongly
indicates its possible role in autoimmune disease
modulation. A study of the autoimmune disease in
DAF knockout MRL (DAFKO MRL/lprIgh) mice has
shown more aggressive disease development in such
mice characterized by accelerated and aggravated
dermatitis and lymphadenopathy as well as increased
antichromatin autoantibody production94. Injection of
a subnephritogenic dose of rabbit anti-mouse
glomerular basement membrane serum has induced
glomerular disease in DAF knockout mice but not in
wild-type controls, with DAF knockout mice
displaying an increased glomerular volume95.
Enhanced expression of DAF has been documented in
glomerular cells in the presence of glomerular injury96
indicating an enhanced protective role of this CRP to
complement attack. On neutralizing DAF with
antibody, the glomerular epithelial cells have been
demonstrated to be more susceptible to complement
mediated cytotoxicity97, which elucidates the
functional importance of DAF on glomerular cells.
However, there is a contradictory report stating
a slight but statistically significant decrease of
DAF level on RBCs of SLE patients with
diffuse proliferative glomerulonephritis, which was
dependent on the stage of the disease activity98.
Another study reported a deficiency of red cell bound
DAF in SLE patients with autoimmune hemolytic
anemia99, indicating the modulatory role played by
DAF in the different clinical manifestations of SLE.
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A study from our lab on the level of DAF in
patients with Rheumatoid arthritis reported
significantly reduced expression of this protein on the
erythrocytes and a significant inverse relationship
between DAF expression and in vitro complement
activation100. We observed increased erythrocyte DAF
in SLE patients and an enhanced expression of the
same in the glomeruli of SLE patients. Also, we found
a significant decline in the levels of leukocyte DAF in
RA89, but increase in SLE with significant
correlations with the disease activity101.
CD59 (Protectin)
Rheumatoid arthritis (RA)—Expression of CD59
is significantly decreased on the synovial lining,
stromal cells and EC102. Moreover, injection into the
rat knee joint of an anti-rat CD59 mAb induces a
spontaneous complement-dependent arthritis and
CD59-deficient mice are prone to enhanced antigeninduced arthritis103.
Systemic lupus erythematosus (SLE)—Several
studies have demonstrated increased serum levels of
MAC C5b-9 in active SLE patients indicating the
potential role of C-mediated tissue injury in the
disease process17,19,44,49,50,53. Since CD59 is an
important surface molecule protecting the autologous
cells from MAC mediated lysis, it possibly plays an
important role in the modulation of complement
mediated injury in SLE. Tamai et al.104 first reported
an up-regulation of CD59 in the glomerular cells of
lupus nephritis patients, which was followed, by a few
other reports of increased CD59 in the glomeruli of
SLE patients. Suppression of CD59 by monoclonal
antibody has shown a dose dependent increase MAC
deposition and exacerbated tubulo-interstitial injury in
rats further elucidating the role of CD59 in the
maintenance of normal integrity of kidney105,106.
Cultured glomerular epithelial, endothelial and
mesangial cells have been shown to exhibit increased
susceptibility to C -mediated lysis in the presence
of neutralizing antibodies in vitro107,108. A study on
CD59a gene knockout mice, has demonstrated that
mice lacking the mCd59a gene are more susceptible to
accelerated nephrotoxic nephritis than matched
controls109. These mice develop greater glomerular
cellularity early in the disease process and more
severe glomerular thrombosis and proteinuria at later
time points. The excess C9 deposition reflects the
presence of a greater quantity of MAC and most likely
represents the mechanism whereby the absence of
CD59a causes greater tissue injury. Most of the
708
INDIAN J EXP BIOL, NOVEMBER 2015
studies on the role of CD59 in autoimmunity deal
with the expression of CD59 in the kidney of patients
with renal diseases.
An increased expression of CD59 on the RBCs of
SLE patients with diffuse proliferative glomerulonephritis was also observed in our lab110. There is a
contradictory report showing decreased level of CD59 on
the red cells of SLE patients with autoimmune hemolytic
anemia. Thus, the expression of CD59 on erythrocytes
may vary with the varied manifestations of SLE. A
follow-up study carried by us showed increased levels
of CD59 transcript during the active stage of disease
that declined on remission and reverted to the first day
high levels during flares. Preliminary studies in our lab
found a decreased expression of CD59 in leucocytes of
the patients with RA at the transcript and protein level.
Detailed studies are warranted.
Therapeutic implications
In view of the diversity of the clinical disorders
involving abnormal complement activity or
regulation, which include both acute and chronic
diseases and affect a wide range of organs, diverse yet
specifically tailored therapeutic approaches may be
needed to shift complement back into balance.
A large number of complement therapeutics on the
market or in clinical trials. Many more are on
development111. They hold promise in the treatment
of organ transplantation, ischemia-reperfusion injury,
coronary artery disease, myocardial infarction, stroke,
infection, cancer, immunosuppression, paroxysmal
nocturnal hematuria, glomerulonephritis, rheumatoid
arthritis, and acute respiratory distress syndrome, and
have also been used in the coating of extracorporeal
circuits in cardiopulmonary bypass and dialysis112.
Strategies in the development of complementbased therapeutics range from use of anti-complement
antibodies,
enhancing
complement
mediated
phagocytosis of undesired elements and pathogens,
inhibiting complement mediated entry of pathogens to
infect the cells, inhibitory structural analogues,
increasing complement mediated regulation of
autoinjury, use of DNA mimetics etc3.
The endogenous soluble complement-inhibitors,
antibodies or low molecular weight antagonists that
either block key proteins of the cascade reaction or
neutralize the action of the complement-derived
anaphylatoxins have successfully been tested in
various animal models over the past years. Promising
results consequently led to first clinical trials5.
A strategy can be inhibiting initial component of
one or more of the activation pathways like blocking
the activation of factor D (Alternative pathway), C1q
(Classical pathway) or MBL, (Lectin pathway)113.
Most of the approaches to inhibit complement have
focused either on blocking at the level of C3 that
implies a general and broad inhibition of the system,
or on selective blocking of C5 activation with
subsequent inhibition of C5a and C5b-9 (TCC)
formation114. Another strategy may be neutralizing
C3a, C5a or using their receptor antagonists115.
Compstatin, a 13-residue cyclic peptide was
isolated using combinatorial peptide libraries to
identify C3 binding peptides, later characterized in
detail and named compstatin116. This peptide blocks
cleavage of C3 and is highly specific for binding
to C3. No interaction with other cascade proteins has
been demonstrated. It was found to reduce complement
and granulocyte activation in an in vitro model of
extracorporeal circulation117,118 and to protect against
hyperacute xenograft rejection ex vivo119. Compstatin
works only in primates, and therefore, animal studies
are limited. Soulika et al. demonstrated inhibition of
heparin-protamine-induced complement activation in
a primate in vivo model120.
Monoclonal antibodies that specifically inhibit
terminal complement activation while preserving the
critical functions of the early complement cascade
have now been developed. These antibodies target the
C5 complement protein, blocking its cleavage and the
subsequent generation of potent proinflammatory
molecules121. Two different anti-C5 single chain Fv
(ScFv), one that inhibits both release of C5a and
assembly of the TCC (TS-A 12/22) and another that
selectively blocks formation of the TCC (TS-A 8),
could represent potential therapeutic agents to be
used in patients with RA121.
Early complement proteins are critical in the
clearance of immune complexes and apoptotic bodies,
and their absence predisposes individuals to SLE.
Alternatively, TCC activation is associated with
exacerbations of disease and damage to tissues and
organs, particularly in lupus nephritis122.
Case-controlled and follow-up studies carried out
in our laboratory suggest deficient leukocyte DAF,
MCP, CR1, and CD59 expression in RA and deficient
E-CR1,G-CR1 and L-CR1 expression SLE3,73,76,80,86,89.
Replenishing these proteins along with administration
of anti-C5 may be an effective therapeutic strategy for
these diseases. Anti-C5 therapeutics have recently
DAS N: COMPLEMENT SYSTEM AS BIOMARKERS AND THERAPEUTIC TARGETS FOR RA & SLE
been investigated both in an animal model of SLE
and in humans122. Recombinant soluble membranebound complement regulatory proteins are promising
therapeutic targets for complement mediated
inflammatory disorders. Strategy is to target these
soluble proteins to the cell membrane or to the
specific site to enhance their endogenous activity as
complement inhibitors123-125.
Two complement inhibitors, soluble complement
receptor 1 (TP10)125 and a monoclonal anti-C5 antibody
(Eculizumab) have been shown to inhibit complement
safely and now are being investigated in a variety of
clinical conditions126. Eculizumab has shown to reduce
hemolysis and has been approved by the FDA in
paroxysmal nocturnal hemoglobinuria127. Although still
no clinical trial has been performed in SLE, they hold
promise to be used therapeutically in SLE125.
Conclusion
With increasing understanding of the role of
complement and complement regulatory proteins in
inflammatory and autoimmune disorders and
literature culminating on RA and SLE in this context,
it is clear that many of the cascade proteins especially
C1q, C3, C3a, C4, C5, C5a along with TCC hold
promise as biomarkers. Among complement regulators,
expression levels of membrane-bound complement
regulatory proteins especially CR1 and MCP may
help in assessing the disease prognosis.
Several small molecules recognized as complement
inhibitors, antibodies against initial, central and terminal
complement components and receptor antagonists
along with various formulations of recombinant
soluble CR1 and soluble forms of other membranebound complement regulatory proteins are under trial
as therapeutics .Prevention of TCC and anaphylatoxin
formation and blockade of C3a/C5a receptor rather
than blanket immune-suppression may be an effective
therapeutic strategy against RA and SLE.
Acknowledgements
Author acknowledges the Council of Scientific and
Industrial Research (CSIR), New Delhi and Department
of Science and Technology (DST), Govt. of India,
New Delhi for funding their work on this topic.
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