uva-dare (digital academic repository) assessment of ... · schematic overview of the complement...
TRANSCRIPT
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Assessment of complement activation in human disease
Wouters, D.
Link to publication
Citation for published version (APA):Wouters, D. (2008). Assessment of complement activation in human disease.
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
Download date: 21 Jan 2021
Assessment of complement activation (review)
47
Chapter 3
Assessment of complement activation in vivo: which assay when? (review)
Diana Wouters and C Erik Hack
Chapter 3
48
Abstract
The complement system plays a major role in host defense against infection and in inflammatory
processes. The degree of complement activation reflects the intensity of inflammation and may
be an objective index of disease activity and response to therapy. Assessment of complement
activation is largely based on measurement of residual complement factors such as C3 and C4 or
the determination of complement haemolytic function. These parameters reflect complement
activation only indirectly and are quite insensitive. A more reliable approach is to detect
activation products that are specifically produced during the activation process. Complement
activation in vivo results in the generation of activation fragments, multimolecular complexes
and the appearance of neo-epitopes on these fragments and complexes. In this review we give an
overview of the various assays that have been described to measure specific complement
activation products. Furthermore we discuss some important aspects of these activation products:
Stability in vitro, half-life in vivo, acute phase reactivity, influence of renal function and the
diagnostic value should be considered when choosing a parameter for a given condition.
Assessment of complement activation (review)
49
C3C5b-9 (MAC)
Lysis
C4bC2a
C3bBb(P)
C4 C2
C4a C2b
MBL
C1q
MASP-1
MASP-2
C1r
C1s
Immune complexesIgG, IgM
Carbohydrates
C3H2O
Lectin pathway
Classical pathway
Bacterial surfacesLPS, IgA
Alternative pathway
C3b
Opsonisation
C3a C5a
Anaphylatoxins
C4b
Ba
Terminal pathwayC3
C5b-9 (MAC)Lysis
C4bC2a
C3bBb(P)
C4 C2
C4a C2b
MBLMBL
C1qC1q
MASP-1
MASP-2
C1r
C1s
Immune complexesIgG, IgM
Carbohydrates
C3H2OC3H2O
Lectin pathway
Classical pathway
Bacterial surfacesLPS, IgA
Alternative pathway
C3b
Opsonisation
C3a C5a
Anaphylatoxins
C4b
Ba
Terminal pathway
Introduction
The complement system is part of the innate host defense system and contributes to the
inflammatory reactions against invading pathogens. Complement activation may also occur in
pathological conditions and contribute to disease symptoms. Therefore, a variety of new
complement inhibitors are presently developed for clinical application that can be used to
alleviate symptoms of complement-mediated diseases. In this review we will shortly discuss the
complement system, in particular the assays that can be used to assess activation of the system in
patients.
Complement pathways
Complement can be activated via three pathways, which all result in activation of C3, which in
its turn leads to activation of a common terminal pathway (Fig. 1). The classical pathway is
initiated by C1 binding to an appropriate activator such as immune complexes containing IgG or
IgM antibodies. After ligand binding by the C1 subcomponent C1q, the pro-enzymes C1r and
C1s, which are associated to C1q, are activated. This (auto)activation results from a fixed
position of the C1r dimer by the arms of the C1q molecule in a way that the cleavage site of one
C1r molecule completes the enzymatic pocket of the other C1r molecule via an induced-fit
mechanism, and subsequently becomes cleaved. The enzymatic pocket of the cleaved C1r
Figure 1. Schematic overview of the complement system
Chapter 3
50
molecule now becomes fixed in an active configuration and cleaves the remaining intact C1r
molecule, which also becomes activated. Active C1r then cleaves C1s, which in its turn becomes
activated and subsequently cleaves C4 as well as C2. The combined C4b and C2a fragments then
form the classical C3-convertase.
The lectin pathway differs from the classical pathway only in that mannan binding lectin (MBL)
or ficolins are the recognition molecules and MBL-associated protease (MASP)-1, 2- and 3
constitute the associated enzymes. C1q, MBL and ficolins are structurally related and are
members of the collectin family (1). Similarly, C1r, C1s and MASPs are structurally related (2,
3). Apparently, these pathways have been evolved from one archetypical pathway by gene-
duplication. The lectin pathway is activated upon MBL binding to carbohydrate-rich microbial
structures. The alternative pathway provides an antibody-independent activation pathway and
can be spontaneously initiated via a “tick-over” mechanism. This mechanism implies hydrolysis
of an internal thio-ester bond in native C3, which subsequently gets a conformation that
resembles that of C3b, and which can interact with factors B and D. This interaction generates
another C3-convertase, C3(H2O)Bb, which cleaves small amounts of fluid-phase native C3 to
give rise to low amounts of fluid phase C3b. Excessive fluid phase activation of C3 by this
mechanism is prevented by the action of factors H and I. When small amounts of C3b fix onto a
surface of for example microbes, where it is protected against the inhibitory action of factors H
and I, it can interact with factors B and D to generate another C3-convertase, C3bBb. This
activation triggered by C3b fixed on a surface where it is protected against factors I and H, is
called alternative pathway activation. Whether a surface is able to activate the alternative
pathway is determined by the carbohydrate environment of bound C3b. Negatively charged
molecules, such as sialic acid on host cells, promote binding of fH, which abrogates further
activation. In contrast, activating surfaces such as bacterial cell wall components allow binding
of fB which leads to further activation of the alternative pathway. Properdin can enhance
alternative pathway activation by stabilizing the C3bBb complex. All three pathways converge at
the level of C3, eventually leading to the formation of the terminal pathway membrane attack
complex (MAC), consisting of C5b, C6, C7, C8 and multiple molecules of C9.
Assessment of complement activation (review)
51
Effects of complement activation
Complement activation leads to direct and indirect effector mechanisms that all contribute to
protection of the host from invading organisms, often at the expense of inflammation. The MAC
complex leads to pore formation and target cell lysis. During complement activation, the
anaphylatoxins C3a and C5a are generated. In particular C5a is chemoattractive and can attract
and activate leukocytes upon binding to specific C5a receptors on these cells. C5a and C3a have
other pro-inflammatory effects as well, including enhancement of vasopermeability, smooth
muscle cell contraction, mast cell degranulation and other. Furthermore, the larger fragments of
activated C3 and C4 (C3b and C4b, respectively) act as opsonins, and facilitate phagocytosis
which is mediated by complement receptors on phagocytic cells (CR1, CR2, CR3) and
erythrocytes (CR1). In order to control excessive complement activation and to minimize
damage to host cells, there are multiple soluble and membrane bound complement regulators.
Soluble complement regulators such as C1-inhibitor (C1-inh), C4 binding protein (C4bp), factors
H and I, S-protein (vitronectin) and clusterin restrict the action of complement in the fluid phase.
Host cells are protected by membrane-bound complement regulators such as CR1, MCP (CD46),
decay accelerating factor (DAF (CD55)) and CD59.
Complement and disease
Complement plays a pivotal role in humans; on the one hand complement deficiencies lead to
increased susceptibility to autoimmune disease and infections. On the other hand, excessive
complement activation may cause host tissue damage and inflammation. Complement activation
leads to cell lysis, influx of inflammatory cells, degranulation of phagocytic cells, mast cells and
basophils, smooth muscle contraction and increase of vascular permeability. Because of these
effects, complement activation has been implicated in the pathogenesis of several diseases.
Usually, activation of complement is not the sole cause of disease, but rather exacerbates or
sustains clinical problems. Table I gives an overview of inflammatory diseases in which
complement activation is thought to be involved. The degree of complement activation reflects
the intensity of inflammation, and potentially provides an objective index of disease activity and
response to therapy. Moreover, by determining the specific pathway(s) involved, additional
information may be obtained on the pathogenic mechanisms of the disease.
The complement system may be a target for therapy. Indeed several complement inhibitors have
been or are being developed such as plasma-derived or recombinant C1-inhibitor (19), soluble
CR1 (20), anti-C5 monoclonal antibody (21) and others. Approved indications for complement
inhibitors include hereditary angio-edema (22) and paroxysmal noctural hemoglobinuria (23,
Chapter 3
52
58Complement activation products in circulationCardiopulmonary bypass
17, 18Deposits of complement in affected eyeAge related macular degeneration (AMD)
15, 16Complement activation products in senile plaquesAlzheimer’s disease (AD)
13, 14Complement activation products in CSF and areas of demyelination
Multiple Sclerosis (MS)
11, 12Deposits of complement activation products in glomeruli
Membranoproliferativeglomerulonephritis (MPGN)
9, 10Deposition of complement in infarcted areaIschaemia-reperfusion injury (myocardial infarction)
7, 8Consumption of plasma complementComplement activation products in circulation
Systemic Lupus erythematosus(SLE)
4-6Consumption of plasma complementComplement activation products in synovial fluid and plasma
Rheumatoid arthritis (RA)
RefsInvolvementDisease
58Complement activation products in circulationCardiopulmonary bypass
17, 18Deposits of complement in affected eyeAge related macular degeneration (AMD)
15, 16Complement activation products in senile plaquesAlzheimer’s disease (AD)
13, 14Complement activation products in CSF and areas of demyelination
Multiple Sclerosis (MS)
11, 12Deposits of complement activation products in glomeruli
Membranoproliferativeglomerulonephritis (MPGN)
9, 10Deposition of complement in infarcted areaIschaemia-reperfusion injury (myocardial infarction)
7, 8Consumption of plasma complementComplement activation products in circulation
Systemic Lupus erythematosus(SLE)
4-6Consumption of plasma complementComplement activation products in synovial fluid and plasma
Rheumatoid arthritis (RA)
RefsInvolvementDisease
24). In addition, complement inhibitors are evaluated in diseases such as myocardial infarction,
rheumatoid arthritis, and age related macular degeneration. In order to monitor
pharmacodynamics of complement inhibitors, convenient and reliable assays to monitor
complement activation are required.
Assays to measure complement activation in vivo
Generally, immunochemical measurement of total levels of complement components such as C3,
C4 and factor B or haemolytic assays to assess total activity levels in biological fluids are used to
evaluate complement activation. Marked reduction in serum levels of a single complement
protein points to a genetic deficiency in one of the complement components, whereas the
decrease of multiple complement components points to an activation process leading to
consumption (or decreased synthesis). To make the distinction between acquired and inherited
complement deficiency, complement activation products may be measured next to total protein
Table I. Inflammatory diseases with involvement of complement
Assessment of complement activation (review)
53
levels. Secondary complement deficiency as result of consumption is in principle accompanied
with elevated levels of activation products.
Decreased plasma levels of complement factors as an indication for activation are somewhat
insensitive, since complement levels only decrease significantly after massive activation
(presumably >10% of a given factor needs to be activated). Most complement proteins exhibit a
wide normal range and serum levels are always influenced by the balance between synthesis and
catabolism. Some complement proteins are acute phase proteins. So, an acute phase increase of
complement proteins may mask consumption during inflammatory conditions, thereby leaving
serum levels within the normal range. Altogether, a more reliable approach to assess complement
activation is detection of activation products which are specifically produced during activation,
in biological fluids. In the rest of the paper, we will review the various assays that have been
described over the last decades to measure specific complement activation products (Table II).
Analysis of complement activation products
Complement activation in vivo can be assessed by making use of the unique properties of the
complement system, such as the generation of activation fragments, multimolecular protein-
protein complexes and the appearance of neo-epitopes on these fragments and complexes.
Multimolecular protein-protein complexes may result from either complement deposition or
interaction of a complement protease with its inhibitor. Neo-epitopes on activation products are
not present on the intact proteins and may be involved in novel functional activities of the
protein obtained upon activation. The use of monoclonal antibodies against neo-epitopes
exposed on activation products in assays for activated complement components will minimize
interference of native components in the assay.
Assays for classical and lectin pathway activation
Upon activation of C1, C1-inh binds to the serine proteases C1r and C1s; thereby releasing these
components from activator-bound C1q. Early activation of the classical pathway is indicated by
the presence of stable C1-inh/C1rC1s complexes in the circulation. These complexes can be
measured either by radioimmunoassay (RIA) (25) or ELISA. In both assays, the C1-inh/C1rC1s
complexes were originally captured by antibodies against C1s and subsequently detected by C1-
inh specific antibodies. In these assays, native unbound C1s competes with C1s in the
complexes, which influences the sensitivity of the assays. To circumvent this, Fure et al
developed a modified assay in which a capturing antibody is used that recognizes a neo-epitope
on C1-inh when complexed with its substrates (26).
Chapter 3
54
Assessment of complement activation (review)
55
Ta
ble
II. O
verv
iew
of c
ompl
emen
t act
ivat
ion
para
met
ers
CP:
Cla
ssic
al p
athw
ay, L
P: L
ectin
path
way
, AP:
Alte
rnat
ive
path
way
Chapter 3
56
C1-inh/C1rC1s complexes are rapidly cleared from the circulation, which explains why
sometimes low circulating levels of these complexes are found despite evidence of complement
activation (27). Since MASP-2 is also inhibited by C1-inh, it might be valuable to develop an
ELISA to detect complexes between C1-inh and MASP-2 based on the same principle. This may
provide the first assay to specifically measure lectin pathway mediated complement activation,
since assays that can monitor activation of this pathway specifically, have not been described up
until now.
Generation of C4 cleavage products provides evidence of classical or lectin pathway mediated
complement activation. At the level of C4, several activation fragments can be measured such as
C4a, C4d and C4b/c which are formed upon enzymatic cleavage by C1s or MASP-2. C4a is a
small anaphylatoxin, which is released after proteolytic cleavage of C4. In vivo, C4a is
inactivated to C4a desArg by serum carboxypeptidase N. Quantification of the C4a desArg
fragment comprises a precipitation step prior to a double competitive inhibition RIA performed
in the supernatant (28). The reason for this precipitation step is that the antibodies used in the
assay for C4a to some extent cross-react with native C4, and hence the latter has to be removed
from samples to be tested. One disadvantage of measuring C4a is that as an anaphylatoxin, C4a
has a short half life. Moreover, C4a levels may not be appropriate to monitor renal disease, since
C4a is cleared by the kidneys and impaired clearance by the kidneys could account for high C4a
plasma concentration (29).
Using a monoclonal antibody (mAb) that reacts with a neo-epitope exposed on the activation
products C4b, C4bi and C4c (abbreviated as C4b/c) but not on native C4, C4 activation can be
quantified using a relatively simple ELISA method. This neo-epitope reflects conformational
changes resulting from disruption of an internal thio-ester bond. The neo-epitope specific mAb is
used as capturing antibody and a polyclonal rabbit anti human C4 for detection (30). However,
this ELISA, as well as other assays that detect C4 cleavage products, is influenced by in vitro
generation of the activation product. Amongst others, this is due to the fact that the thio-ester
within C4 is fragile in physiological fluids and is easily hydrolysed upon temperature changes.
Hydrolysed C4 generated in this way may lead to artificially high responses in the assay for C4
activation fragments.
In the human circulation, C4b is rapidly inactivated by factor I (using CR1, factor H or C4bp as
cofactor) resulting in the formation of C4bi. Further degradation of C4bi by factor I yields the
fragments C4c and C4d. C4d can be measured in human serum by rocket immunoelectrophoresis
(RIE) (31, 32), ELISA or nephelometry (33). The measurement of C4d is complicated by the
polymorphic nature of C4; it is well known that the C4 polymorphism lies within the C4d
Assessment of complement activation (review)
57
fragment. Using monoclonal antibodies to detect C4d, one should make sure that the antibody
reacts with all C4 allotypes. In general, measurements of C4 degradation fragments are
complicated by the allelic variation of C4 and the high frequency of heterozygous C4 deficiency.
Therefore, C4 split products given as ratio compared to levels of intact C4, may be preferred. As
C4d (and also C3d) contains the structural region that mediates binding to biological surfaces,
one can postulate that C4d levels in biological fluid insufficiently reflect total C4 activation.
However, although this cannot be denied, in general the amount of C4 fixed to an activator will
be less than 10% of the total amount of activated C4. The majority of activated C4 will be
hydrolyzed by surrounding water molecules and hence the effect of underestimation will be
limited.
Activated C4 binds covalently to surrounding molecules via its thio-ester, which becomes
exposed after proteolytic cleavage. This results in the deposition of C4 to the activator and to
proteins in the vicinity. Recently, our group described that C4 not only binds to its activator, but
also to C1q, the recognition unit of the classical pathway (34). C1q-C4 complexes can be
measured by ELISA and are specific activation products of the classical pathway. A major
advantage of measuring C1q-C4 complexes is that unlike many other complement activation
products, these complexes are very stable at different storage conditions and are not generated in
vitro, at least provided the samples contain EDTA. Complexes between activated C4 and MBL
would provide a specific assay for lectin pathway activation. MBL-C4 levels should be depicted
as ratio to total MBL, because of the large variation in MBL concentration due to its
polymorphism. However, more research is required to develop such an assay.
Elevated levels of C4d deposited on erythrocytes and thrombocytes have been described to be
highly specific for SLE and may be a useful diagnostic marker (35, 36).
During classical pathway activation, C3b has been demonstrated to bind covalently to C4b
attached to the target (37). Meri and Pangburn found that C3b also bound covalently to C4b in
the fluid phase (38). For the detection of circulating C4-C3 complexes, Zwirner described an
ELISA in which two mAbs are combined with specificities for C3b/iC3b/C3dg and C4/C4b/C4d
(39). These complexes appeared to be specific for the classical pathway. In the above mentioned
ELISA no discrepancy is made between direct C4-C3 complexes and larger immune complexes
containing both activated C4 and C3.
During classical and lectin pathway activation complexes are formed between C4b and the
inhibitor C4bp. An ELISA has been described in which these C4b-C4bp complexes are
measured (40). Since C4b bound to C4bp is easily cleaved by factor I in the serum, C4b-C4bp
complexes are rapidly lost, which may be the explanation for the limited use of this assay.
Chapter 3
58
Assays for alternative pathway activation
Alternative pathway activation can be measured by nephelometry or ELISA using monoclonal
antibodies recognizing neo-antigens on the factor B split products Ba and Bb (41, 42).
Comparable to C4a, the Ba fragment is cleared by the kidneys (43), which makes this fragment
less suitable for evaluation of complement activation in renal diseases.
During alternative pathway activation, activated C3b binds factor B which results in the cleavage
of factor B by factor D. The so formed C3bBb complex is stabilized by properdin, resulting in
the alternative pathway C3-convertase. Properdin will only bind to C3 in the presence of factor
B. Therefore, C3bBb(P) complexes can be measured in an ELISA with a properdin specific
antibody as a catching antibody and anti-C3 antibody as detecting antibody (44). Not only direct
activation of the alternative pathway by activators such as gram negative bacteria is measured in
this assay. Triggering of the amplification loop as result of initial classical or lectin pathway
activation is detected as well.
Assays for terminal pathway activation
C3 is the central protein of all three activation pathways. Thus, measurement of C3 cleavage
products provides information about overall complement activation. Various C3 activation
products can be measured in plasma (45). C3 is broken down by C3-convertases to the smaller
C3a and the larger C3b fragment. C3b is inactivated by factor I to from C3bi. Further
degradation leads to C3c and C3d fragments.
The biologically active anaphylatoxin C3a is released upon C3 activation and rapidly inactivated
to the more stable C3adesArg. Circulating C3a levels may be measured by RIA (46, 47), which
requires a pre-assay precipitation step to separate native C3 from the C3a fragment, since the
antibody used in this assay recognizes both C3 and C3a. An ELISA method developed later (48,
49) was not affected by the presence of native C3, since it makes use of a neo-epitope specific
antibody against C3a.
C3b, C3bi and C3c (C3b/c) can be measured by ELISA in which the antigen is captured by a
mAb that reacts with a neo-epitope exposed on activated C3, but not on native C3 (30, 50, 51).
The thio-ester in C3 is fragile and is easily hydrolysed upon temperature changes. Therefore, due
to improper handling of the samples, C3b/c levels may be artificially high. The C3d fragment has
a long half-life in circulation. To measure C3d in serum, the activation fragment should first be
separated from native C3 by PEG precipitation. The C3d fragment remains in supernatant, in
which it can be detected by either nephelometry or ELISA (52, 53).
Assessment of complement activation (review)
59
C5a can be used as indicator of terminal pathway activation. C5a levels can be measured by RIA
or ELISA. However, C5a levels may not be an appropriate parameter in vivo; C5a is cleared
from the circulation very rapidly by binding to high affinity receptors on neutrophils (54).
Increased concentrations of sC5b-9 (soluble terminal complement complex, consisting of S-
protein, the components C5b, C6, C7, C8 and polymerized C9), reflect complement activation
via each activation pathway. The sC5b-9 complex is only generated when the whole activation
route is completed. The sC5b-9 complexes are not detectable in normal serum or plasma, are
stable in vitro and have a relatively long half-life in vivo. S-protein levels in serum may be the
limiting factor in sC5-9 formation. The complexes can be detected by ELISA with antibodies
against the complement components C5 and C9. Particularly a monoclonal antibody against neo-
epitopes expressed on polymerized C9 has been used to further optimize assays for MAC (55,
56).
Discussion
Excessive activation of complement may harm the host by mediating inflammatory tissue
destruction and likely contributes to the pathogenesis of a number of human diseases. Detection
of complement activation is not only important for assessment of disease activity in these
diseases but may also help to monitor response to treatment. The preferred approach to evaluate
complement activation is detection of complement activation products with assays that are
specific for cleavage fragments or multimolecular complexes. Monoclonal antibodies against
neo-antigens exposed on these fragments or multimolecular complexes have been shown to be
suitable tools in these assays. Before assessing complement activation in vivo, it is important to
consider which activation parameter is most appropriate for a given clinical condition. In the
next paragraphs we will give some thoughts on aspects of activation products that may help to
select an optimal parameter for assessing complement activation in vivo.
In normal situation, most complement activation products are only present in trace amounts in
vivo, whereas they are rapidly generated in vitro. Therefore, the conditions of sample collection,
processing and storage are critical to get results that reliably reflect in vivo activation processes.
For most assays, blood should be collected in EDTA containing tubes, to prevent in vitro
activation of both the classical and alternative pathway by chelating Ca2+ and Mg2+. The plasma
should be processed soon after collection and preferably stored at -70�C until analysis.
Preferably, several aliquots of samples are stored since this may avoid repeated freezing and
thawing of samples. Repeated freezing and thawing may result in false-positive results since the
thio-ester in both C3 and C4 is fragile and gets easily hydrolyzed (57). Disruption of the thio-
Chapter 3
60
ester causes conformational changes in C4 and C3 that are similar to the changes occurring upon
activation. Nevertheless, samples are often collected and stored under suboptimal conditions,
which may lead to artificially high levels of activation products. In these situations measurement
of activation products that are stable in vitro, is preferred. C1q-C4 complexes are very stable
activation products that are not generated in vitro in the presence of EDTA (34). However, these
complexes only reflect classical pathway activation. To assess total complement activation in
samples that were not carefully processed, measurement of sC5b-9 complexes is a reliable
indicator (56).
Next to handling samples correctly, one should realize that complement activation products have
different clearance rates. This has an effect on circulating levels of these products. Table III
shows a calculation model for C3 cleavage products demonstrating to what extent serum levels
of these activation products are influenced by their different half- lives in vivo. Theoretically,
one would expect comparable molar increases of the individual C3 activation products after
induction of complement activation. However, the concentrations of these products may differ
markedly, since small fragments are in general more rapidly removed from the circulation than
larger fragments. The median normal C3 concentration in serum is 1,5 g/L, so when all C3 is
converted, the molar concentration of each C3 cleavage product would be 8283 nM. Table III
shows the molar concentration of C3a, C3b/c and C3d after acute activation of 5%, 10% or 25%
of total C3. Half-lives of these products are 7 minutes, 20 minutes and 4 hours respectively. In
case of acute conversion of 5% of total serum C3, elevated C3d levels can be measured up until
4 hours after induction of complement activation, whereas C3a and C3b/c levels have returned to
normal values already after 1 hour. Notably, the formation of C3d does not occur instantaneously
following acute activation as the breakdown of C3bi into C3c and C3d by factor I and cofactors
takes some time. At 10% C3 conversion, elevated C3b/c can be detected up to one hour after the
event. However, increased C3a levels can only be detected very shortly after the event, due to its
shorter half-life. Even at 25% C3 activation, C3a levels are again within the normal range one
hour after induction of complement activation. Thus, C3d levels stay longer increased after
induction of complement activation than levels of C3b/c and C3a values, which rapidly return
into the normal range. The comparison of activation products however can be very useful, as a
high concentration of C3d with normal C3b/c may indicate acute activation of short duration,
whereas high levels of both parameters point to strong ongoing activation. In case of mild
ongoing activation, again C3d levels may be elevated.
Assessment of complement activation (review)
61
The effect of different half-lives of C3 cleavage products on the levels that can be measured in
patients is illustrated in a study on complement activation after cardiopulmonary bypass surgery
(58). In this study, C3a and C3b/c levels were measured as parameters of complement activation.
After induction of complement activation during surgery, the molar concentration of C3b/c was
almost tenfold higher than that of C3a, whereas initially these products were equally increased.
Knowledge of the clearance rates of complement activation products is therefore important to
select the most appropriate activation products for clinical and experimental studies. In chronic
conditions, complement activation products with a long half-life are preferred like C3d or sC5b-
9. On the contrary, for acute activation processes products that are rapidly generated and cleared
are more appropriate such as C3a, C3b/c and hence elevated levels of these products point to a
recent or still ongoing activation process.
In this review we gave a short overview of the various assays that are currently available to
measure complement activation in vivo. In conclusion, for reliable assessment of complement
activation in clinical settings the following characteristics are important to consider: in vitro
stability of the activation product, half-life in vivo, acute phase reactivity, influence of renal
function, pathway specificity and diagnostic or prognostic value.
25%
10%
5%
% C3 activationat t = 0 h
321035155318112070 C3d
nd0.5322592070 C3b/c
ndndnd52070 C3a
13414621725828 C3d
ndnd13104828 C3b/c
ndndnd2828 C3a
7207311362414 C3d (nM)***
ndnd752414 C3b/c (nM)**
ndndnd1 414 C3a (nM)*
24 h4 h2 h1 h0 hParameter
25%
10%
5%
% C3 activationat t = 0 h
321035155318112070 C3d
nd0.5322592070 C3b/c
ndndnd52070 C3a
13414621725828 C3d
ndnd13104828 C3b/c
ndndnd2828 C3a
7207311362414 C3d (nM)***
ndnd752414 C3b/c (nM)**
ndndnd1 414 C3a (nM)*
24 h4 h2 h1 h0 hParameter
Table III. Calculation model for C3 cleavage products*Half-life C3a: 7 min, normal value < 6 nM; **Half-life C3b/c: 20 min, normal value < 57 nM; ***Half-life C3d: 4 hrs, normal value 41-257 nMTotal C3 concentration: 1500 μg/ml (8283 nM), nd: not detectable
Chapter 3
62
References 1. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collections and ficolins: humoral lectins of the innate
immune defense. Annu. Rev. Immunol. 21: 547-578.
2. Matsushita, M., S. Thiel, J. C. Jensenius, I. Terai, and T. Fujita. 2000. Proteolytic activities of two types of mannose-binding lectin-associated serine protease. J. Immunol. 165: 2637-2642.
3. Vorup-Jensen, T., S. V. Petersen, A. G. Hansen, K. Poulsen, W. Schwaeble, R. B. Sim, K. B. Reid, S. J. Davis, S. Thiel, and J. C. Jensenius. 2000. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2. J. Immunol. 165: 2093-2100.
4. Makinde, V. A., G. Senaldi, A. S. Jawad, H. Berry, and D. Vergani. 1989. Reflection of disease activity in rheumatoid arthritis by indices of activation of the classical complement pathway. Ann. Rheum. Dis. 48: 302-306.
5. Hietala, M. A., K. S. Nandakumar, L. Persson, S. Fahlen, R. Holmdahl, and M. Pekna. 2004. Complement activation by both classical and alternative pathways is critical for the effector phase of arthritis. Eur. J Immunol. 34: 1208-1216.
6. Petersen, N. E., J. Elmgreen, B. Teisner, and S. E. Svehag. 1988. Activation of classical pathway complement in chronic inflammation. Elevated levels of circulating C3d and C4d split products in rheumatoid arthritis and Crohn's disease. Acta Med. Scand. 223: 557-560.
7. Pickering, M. C. and M. J. Walport. 2000. Links between complement abnormalities and systemic lupus erythematosus. Rheumatology. (Oxford) 39: 133-141.
8. Manderson, A. P., M. Botto, and M. J. Walport. 2004. The role of complement in the development of
systemic lupus erythematosus. Annu. Rev. Immunol. 22: 431-456. 9. Nijmeijer, R., W. K. Lagrand, Y. T. Lubbers, C. A. Visser, C. J. Meijer, H. W. Niessen, and C. E. Hack.
2003. C-reactive protein activates complement in infarcted human myocardium. Am. J. Pathol. 163: 269-275.
10. Griselli, M., J. Herbert, W. L. Hutchinson, K. M. Taylor, M. Sohail, T. Krausz, and M. B. Pepys. 1999. C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction. J Exp. Med. 190: 1733-1740.
11. Davis, B. K. and T. Cavallo. 1976. Membranoproliferative glomerulonephritis. Localization of early
components of complement in glomerular deposits. Am J Pathol. 84: 283-298. 12. Trouw, L. A., M. A. Seelen, and M. R. Daha. 2003. Complement and renal disease. Mol. Immunol. 40: 125-
134. 13. Johns, T. G. and C. C. Bernard. 1997. Binding of complement component Clq to myelin oligodendrocyte
glycoprotein: a novel mechanism for regulating CNS inflammation. Mol. Immunol. 34: 33-38. 14. Rus, H., C. Cudrici, and F. Niculescu. 2005. C5b-9 complement complex in autoimmune demyelination and
multiple sclerosis: dual role in neuroinflammation and neuroprotection. Ann. Med. 37: 97-104.
15. Yasojima, K., C. Schwab, E. G. McGeer, and P. L. McGeer. 1999. Up-regulated production and activation of the complement system in Alzheimer's disease brain. Am J Pathol. 154: 927-936.
16. Bergamaschini, L., S. Canziani, B. Bottasso, M. Cugno, P. Braidotti, and A. Agostoni. 1999. Alzheimer's
beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner. Clin. Exp. Immunol. 115: 526-533.
17. Haines, J. L., M. A. Hauser, S. Schmidt, W. K. Scott, L. M. Olson, P. Gallins, K. L. Spencer, S. Y. Kwan, M. Noureddine, J. R. Gilbert et al. 2005. Complement factor H variant increases the risk of age-related macular degeneration. Science 308: 419-421.
Assessment of complement activation (review)
63
18. Mullins, R. F., N. Aptsiauri, and G. S. Hageman. 2001. Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis. Eye 15: 390-395.
19. Choi, G., M. R. Soeters, H. Farkas, L. Varga, K. Obtulowicz, B. Bilo, G. Porebski, C. E. Hack, R. Verdonk, J.
Nuijens et al. 2007. Recombinant human C1-inhibitor in the treatment of acute angioedema attacks. Transfusion 47: 1028-1032.
20. Weisman, H. F., T. Bartow, M. K. Leppo, H. C. J. Marsh, G. R. Carson, M. F. Concino, M. P. Boyle, K. H.
Roux, M. L. Weisfeldt, and D. T. Fearon. 1990. Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science 249: 146-151.
21. Thomas, T. C., S. A. Rollins, R. P. Rother, M. A. Giannoni, S. L. Hartman, E. A. Elliott, S. H. Nye, L. A. Matis, S. P. Squinto, and M. J. Evans. 1996. Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv. Mol. Immunol. 33: 1389-1401.
22. Carugati, A., E. Pappalardo, L. C. Zingale, and M. Cicardi. 2001. C1-inhibitor deficiency and angioedema.
Mol. Immunol. 38: 161-173. 23. Hillmen, P., N. S. Young, J. Schubert, R. A. Brodsky, G. Socie, P. Muus, A. Roth, J. Szer, M. O. Elebute, R.
Nakamura et al. 2006. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N. Engl. J Med. 355: 1233-1243.
24. Hill, A., P. Hillmen, S. J. Richards, D. Elebute, J. C. Marsh, J. Chan, C. F. Mojcik, and R. P. Rother. 2005.
Sustained response and long-term safety of eculizumab in paroxysmal nocturnal hemoglobinuria. Blood 106: 2559-2565.
25. Hack, C. E., A. J. Hannema, A. J. Eerenberg-Belmer, T. A. Out, and R. C. Aalberse. 1981. A C1-inhibitor-
complex assay (INCA): a method to detect C1 activation in vitro and in vivo. J. Immunol. 127: 1450-1453.
26. Fure, H., E. W. Nielsen, C. E. Hack, and T. E. Mollnes. 1997. A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s. Scand. J. Immunol. 46: 553-557.
27. de Smet, B. J., J. P. de Boer, J. Agterberg, G. Rigter, W. K. Bleeker, and C. E. Hack. 1993. Clearance of human native, proteinase-complexed, and proteolytically inactivated C1-inhibitor in rats. Blood 81: 56-61.
28. Gorski, J. P. 1981. Quantitation of human complement fragment C4ai in physiological fluids by competitive inhibition radioimmune assay. J. Immunol. Methods 47: 61-73.
29. Abou-Ragheb, H. H., A. J. Williams, C. B. Brown, and A. Milford-Ward. 1991. Plasma levels and mode of excretion of the anaphylatoxins C3a and C4a in renal disease. J Clin. Lab Immunol. 35: 113-119.
30. Wolbink, G. J., J. Bollen, J. W. Baars, R. J. ten Berge, A. J. Swaak, J. Paardekooper, and C. E. Hack. 1993.
Application of a monoclonal antibody against a neoepitope on activated C4 in an ELISA for the quantification of complement activation via the classical pathway. J. Immunol. Methods 163: 67-76.
31. Nitsche, J. F., E. S. Tucker, III, S. Sugimoto, J. H. Vaughan, and J. G. Curd. 1981. Rocket immunoelectrophoresis of C4 and C4d. A simple sensitive method for detecting complement activation in plasma. Am. J. Clin. Pathol. 76: 679-684.
32. Milgrom, H., J. G. Curd, R. A. Kaplan, H. J. Muller-Eberhard, and J. H. Vaughan. 1980. Activation of the fourth component of complement (C4): assessment by rocket immunoelectrophoresis and correlation with the metabolism of C4. J. Immunol. 124: 2780-2785.
33. Davies, E. T., B. A. Nasaruddin, A. Alhaq, G. Senaldi, and D. Vergani. 1988. Clinical application of new technique that measures C4d for assessment of activation of classical complement pathway. J. Clin. Pathol. 41: 143-147.
34. Wouters, D., H. D. Wiessenberg, M. Hart, P. Bruins, A. Voskuyl, M. R. Daha, and C. E. Hack. 2005. Complexes between C1q and C3 or C4: Novel and specific markers for classical complement pathway activation. J Immunol Methods 298: 35-45.
Chapter 3
64
35. Manzi, S., J. S. Navratil, M. J. Ruffing, C. C. Liu, N. Danchenko, S. E. Nilson, S. Krishnaswami, D. E. King, A. H. Kao, and J. M. Ahearn. 2004. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum 50: 3596-3604.
36. Navratil, J. S., S. Manzi, A. H. Kao, S. Krishnaswami, C. C. Liu, M. J. Ruffing, P. S. Shaw, A. C. Nilson, E. R. Dryden, J. J. Johnson et al. 2006. Platelet C4d is highly specific for systemic lupus erythematosus. Arthritis Rheum. 54: 670-674.
37. Takata, Y., T. Kinoshita, H. Kozono, J. Takeda, E. Tanaka, K. Hong, and K. Inoue. 1987. Covalent association of C3b with C4b within C5 convertase of the classical complement pathway. J. Exp. Med. 165: 1494-1507.
38. Meri, S. and M. K. Pangburn. 1990. A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b. Eur. J. Immunol. 20: 2555-2561.
39. Zwirner, J., G. Dobos, and O. Gotze. 1995. A novel ELISA for the assessment of classical pathway of complement activation in vivo by measurement of C4-C3 complexes. J Immunol Methods 186: 55-63.
40. Ito, S., T. Fujita, and N. Tamura. 1987. Determination of C4b.C4-bp complex formed by the activation of classical complement pathway using an enzyme-linked immunosorbent assay. J. Immunol. Methods 105: 145-150.
41. Senaldi, G., M. Peakman, A. Alhaq, V. A. Makinde, D. E. Tee, and D. Vergani. 1987. Activation of the alternative complement pathway: clinical application of a new technique to measure fragment Ba. J. Clin. Pathol. 40: 1235-1239.
42. Kolb, W. P., P. R. Morrow, and J. D. Tamerius. 1989. Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples. Complement Inflamm. 6: 175-204.
43. Oppermann, M., C. Kurts, R. Zierz, E. Quentin, M. H. Weber, and O. Gotze. 1991. Elevated plasma levels of the immunosuppressive complement fragment Ba in renal failure. Kidney Int. 40: 939-947.
44. Mayes, J. T., R. D. Schreiber, and N. R. Cooper. 1984. Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum. J. Clin. Invest 73: 160-170.
45. Teisner, B., I. Brandslund, N. Grunnet, L. K. Hansen, J. Thellesen, and S. E. Svehag. 1983. Acute complement activation during an anaphylactoid reaction to blood transfusion and the disappearance rate of C3c and C3d from the circulation. J Clin. Lab Immunol. 12: 63-67.
46. Hack, C. E., J. Paardekooper, A. J. Eerenberg, G. O. Navis, M. W. Nijsten, L. G. Thijs, and J. H. Nuijens.
1988. A modified competitive inhibition radioimmunoassay for the detection of C3a. Use of 125I-C3 instead of 125I-C3a. J. Immunol. Methods 108: 77-84.
47. Chenoweth, D. E., S. W. Cooper, T. E. Hugli, R. W. Stewart, E. H. Blackstone, and J. W. Kirklin. 1981. Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins. N. Engl. J Med. 304: 497-503.
48. Burger, R., G. Zilow, A. Bader, A. Friedlein, and W. Naser. 1988. The C terminus of the anaphylatoxin C3a
generated upon complement activation represents a neoantigenic determinant with diagnostic potential. J. Immunol. 141: 553-558.
49. Zilow, G., W. Naser, R. Rutz, and R. Burger. 1989. Quantitation of the anaphylatoxin C3a in the presence of C3 by a novel sandwich ELISA using monoclonal antibody to a C3a neoepitope. J. Immunol. Methods 121: 261-268.
Assessment of complement activation (review)
65
50. Hack, C. E., J. Paardekooper, R. J. Smeenk, J. Abbink, A. J. Eerenberg, and J. H. Nuijens. 1988. Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies. J. Immunol. 141: 1602-1609.
51. Hack, C. E., J. Paardekooper, and F. Van Milligen. 1990. Demonstration in human plasma of a form of C3 that has the conformation of "C3b-like C3". J Immunol 144: 4249-4255.
52. Vergani, D., L. Bevis, B. A. Nasaruddin, G. Mieli-Vergani, and D. E. Tee. 1983. Clinical application of a new nephelometric technique to measure complement activation. J. Clin. Pathol. 36: 793-797.
53. Mollnes, T. E. 1985. Quantification of the C3d split products of human complement by a sensitive enzyme-linked immunosorbent assay. Scand. J Immunol. 21: 607-613.
54. Oppermann, M. and O. Gotze. 1994. Plasma clearance of the human C5a anaphylatoxin by binding to
leucocyte C5a receptors. Immunology 82: 516-521. 55. Gawryl, M. S., M. T. Simon, J. L. Eatman, and T. F. Lint. 1986. An enzyme-linked immunoabsorbent assay
for the quantitation of the terminal complement complex from cell membranes or in activated human sera. J. Immunol. Methods 95: 217-225.
56. Mollnes, T. E., T. Lea, S. S. Froland, and M. Harboe. 1985. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand. J Immunol. 22: 197-202.
57. Mollnes, T. E., P. Garred, and G. Bergseth. 1988. Effect of time, temperature and anticoagulants on in vitro
complement activation: consequences for collection and preservation of samples to be examined for complement activation. Clin. Exp. Immunol. 73: 484-488.
58. Bruins, P., H. te Velthuis., A. P. Yazdanbakhsh, P. G. Jansen, F. W. van Hardevelt, E. M. de Beaumont, C. R.
Wildevuur, L. Eijsman, A. Trouwborst, and C. E. Hack. 1997. Activation of the complement system during and after cardiopulmonary bypass surgery: postsurgery activation involves C-reactive protein and is associated with postoperative arrhythmia. Circulation 96: 3542-3548.
59. Buysmann, S., C. E. Hack, F. N. van Diepen, J. Surachno, and I. J. ten Berge. 1997. Administration of OKT3 as a two-hour infusion attenuates first-dose side effects. Transplantation 64: 1620-1623.
60. Pfeifer, P. H., M. S. Kawahara, and T. E. Hugli. 1999. Possible mechanism for in vitro complement activation
in blood and plasma samples: futhan/EDTA controls in vitro complement activation. Clin. Chem 45: 1190-1199.
66