The role of complement testing in dermatology

The role of complement testing in dermatology

S. Jamal and S. Jolles*

Department of Clinical Immunology, The Royal Free Hospital, London, and *National Institute for Medical Research, Division of Infection and Immunity,

Mill Hill, London, UK

Summary An up-to-date knowledge of the molecular events involved in the activation and

control of the complement cascade is essential to understand the pathogenesis of a

number of conditions presenting to dermatologists. This knowledge underpins the

pathogenesis of these conditions but allows the clinician to request the most useful tests

in terms of diagnosis and monitoring. In this review we aim to discuss complement

biology, the diseases in which complement testing is of particular relevance, the types

of laboratory tests available, their utility and interpretation. Additionally it is of critical

importance for clinicians not only to choose the most appropriate tests but also to

choose to send these to an appropriately accredited laboratory.

Introduction

Many conditions present to dermatology in which

evaluation of the complement system plays a significant

role in the diagnosis and monitoring. These include,

hypocomplementemic urticarial vasculitis syndrome

(HUVS), types 1 and 2 hereditiary angioedema (HAE),

acquired angioedema (AAE), cryoglobulinaemia, con-

nective tissue disease including systemic lupus erythe-

matosis (SLE), and complement deficiencies. The

complement system is part of the innate immune system

and consists of more than 30 heat-labile serum proteins.

These are present as inactive precursors which, once

activated, act as enzymes cleaving several molecules of

the next component in the sequence. Each precursor is

split into two or more fragments. The major fragment

usually has two biologically active sites, one binds to cell

membranes and the other enzymatically activates the

next component, while the minor fragment has inflam-

matory and chemotactic properties. The cascade is

controlled by complement inhibitors and the sponta-

neous decay of any exposed attachment sites. The main

functions of complement are to promote inflammation,

the opsonization and lysis of microorganisms, and

removal of immune complexes.

The complement cascade

There are three pathways leading to complement

activation, the classical pathway (CP), the lectin path-

way (LP) and the alternative pathway (AP). All path-

ways converge on C3, the cleavage of which results in

the formation of the C5 convertases (classical, C3b4b2a

and alternative, C3bBbP) and subsequent activation of

the final lytic pathway and membrane attack complex

(MAC) (Fig. 1).1,2

The CP is activated by immune complex formation.

The first complement component is C1 comprising of

subunits C1q, C1r, C1s. The C1q has six globular heads

which bind the Fc fragments of IgG and IgM antibodies

bound to antigen. C1s then cleaves C4 into C4a and C4b

which when surface bound acts as a binding site for C2,

which is in turn cleaved by C1s releasing C2b and in

binding C2a forms the CP C3 convertase C4b2a.

In an analogous manner to the CP the LP is activated

when lectins, synthesized mainly by the liver, bind to

carbohydrates. Mannose binding lectin (MBL), binds to

sugar residues on the microbial cell surface and

mannan associated serine proteases (MASPs), counter-

parts of C1r and C1s, cleave C4 and C2.3 From that

point C4b and C2a form the CP C3 convertase, and

activation proceeds to the terminal components.

Correspondence: S. Jolles, National Institute for Medical Research, Division

of Infection and Immunity, Mill Hill, London, UK.

E-mail: [email protected]

Accepted for publication 21 December 2004

Clinical dermatology • Review article doi: 10.1111/j.1365-2230.2005.01784.x

� 2005 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 30, 321–326 321

Unlike the CP or LP activation of the AP does not

require antibody or lectin as it continuously turns over

on a small scale termed ‘C3 tickover’. Attachment of C3

to a surface lacking complement regulators (e.g. a

bacterial or fungal cell surface) allows rapid amplifica-

tion via a feedback loop (Fig. 1) with the generation of

the AP C3 convertase (C3bBb) when factor B attaches to

target bound C3b, and is cleaved by factor D. Properdin

stabilizes the convertase. The principal C3 convertase

regulator, Factor H (a plasma protein) acts by compet-

ing with Bb preventing further activation of the

complement cascade. The remaining C3a is catabolized

to smaller products by Factor I.

The terminal pathway leads to formation of the MAC.

This begins with the enzymatic cleavage of C5 by its

convertases (classical, C3b4b2a and alternative,

C3bBbP), the remaining steps are nonenzymatic. C5b

binds C6 forming C5b6 which binds C7 to form C5b67

which is hydrophobic and inserts into cell membranes.

C8 and up to 14 C9 monomers then bind sequentially to

form a pore–the MAC leading to cell lysis.

In view of the proinflammatory and potentially

damaging nature of the complement system it is very

tightly controlled. This occurs passively in that the half-

life of the active sites on C3b and C4b are very short and

the complexes (C4b2a and C3bBb) are inherently

unstable. Active control is achieved by inhibitors and

inactivators which block initiation, prevent amplifica-

tion of the C3 and C5 convertases, and inhibit the

terminal MAC. C1-inh, a serine protease, prevents

excessive activation of the CP and LP by cleaving the

C1 complex and blocking the active sites on C1r, C1s

and MASPs.4 Convertase regulation is performed by fac-

tor I (C3b-inactivator). The inhibitors include mem-

brane bound proteins (decay accelerating factor,

complement receptor 1, membrane cofactor protein)

and plasma proteins (C4b-binding protein and factor H).

Finally CD59 on cells, protein S (vitronectin) and

Figure 1 The complement cascade. The three pathways leading to complement activation are, the classical pathway (CP), the lectin

pathway (LP) and the alternative pathway (AP) which all converge on C3, the cleavage of which results in the formation of the C5

convertases (classical, C3b4b2a and alternative, C3bBbP) and subsequent activation of the terminal pathway and membrane attack

complex resulting in cell lysis. The coloured lines show the components tested in the functional assays CH50 and AP50. Mannan binding

lectin (MBL), mannan associated serine protease (MASP), alternative pathway 50 assay (AP50), classical pathway haemolytic assay

50 (CH50), antibody (Ab), membrane attack complex (MAC).

322 � 2005 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 30, 321–326

Complement testing in dermatology • S. Jamal and S. Jolles

possibly clusterin in plasma block insertion of the MAC

into the cell membrane. Deficiency of these regulatory

proteins leads to excessive complement consumption,

inflammation, tissue destruction and depletion of C3 or

other components downstream of the missing control

protein.

Genetics

The majority of the complement components, with the

exception of properdin which has an X-linked pattern

of inheritance, are inherited in an autosomal codomi-

nant pattern (C1-inh, C2, C3, C5, C6, C7 and C9

which is more frequent in Japan). Heterozygotes

generally have levels of that component below the

mean of the normal range. C1-inh deficiency occurs

most commonly in the heterozygous state. The genet-

ics of C1, C4 and C8 are more complicated as multiple

genes are involved and all components of C1 (C1q,

C1r and C1s) are required to form a functional

complex. C4 exists in humans in two forms, C4A

which binds proteins and C4B which binds carbohy-

drate residues on cells. C4A deficiency is increased in

white patients with SLE (8–12%) vs. controls (1–3%).5

C8 is made up of three chains which are all needed for

MAC function.

Clinical conditions

HAE is, in the majority of cases, due to heterozygous

deficiency of C1-inh and is characterized by recurrent

episodes of nonpainful, nonpruritic and nonerythema-

tous subcutaneous and submucosal oedema that spon-

taneously subsides in 48–72 h. It is not accompanied by

urticarial wheals and is not responsive to antihista-

mines, steroids or b agonists.6

There are two forms of HAE, Type I which accounts

for 85% is caused by gene mutations resulting in low

levels of C1-inh, and Type II which accounts for 15% is

due to a point mutation leading to a dysfunctional C1-

inh protein present in normal to high amounts,7,8 a

third type of HAE involves C1-inh binding to albumin.

One recent study describes a further type of HAE in

which all the patients were female and had findings

consistent with HAE but normal C1-inh level and

function.9 The nature of the defect has not been

identified.

There are two types of AAE the first occurs predom-

inantly in the setting of B-cell lymphoproliferative

disorders with C1-inh depletion occurring due to the

formation of idiotype-anti-idiotype immune complexes

involving the paraprotein secreted by the tumour.10 In

the second less common form an autoantibody to C1-

inh blocks its function11 and this is most commonly

associated with lymphoproliferative disease (oligo or

monoclonal gammopathies), autoimmune haemolytic

anaemia and chronic infection.1 In both forms antigenic

C4 and functional C1-inh levels are profoundly

depressed and levels of C1q may be particularly low in

AAE differentiating this from HAE.12

Patients with HUVS have an autoantibody to the

collagen-like region of C1q which activates the CP.13–15

This is also present in about 30% of patients with SLE.

HUVS is characterized by urticaria with hypocomple-

mentaemia (low CH50, C1q, C2 and C4), skin lesions

which may persist for more than 72 h and leave

residual staining; renal involvement occurs in 40% and

obstructive lung disease has been documented.

The most common presentation of early complement

component (C1, C4 or C2) deficiency is with an

autoimmune disease and the strongest association is

with SLE. These patients also have a higher than normal

incidence of encapsulated bacterial infections. The

incidence of SLE in patients with C1q, C4 or C2

deficiency is 90%, 75% and approximately 15%,

respectively.16 Partial C4 deficiency is also associated

with SLE; 15% of patients having C4A deficiency and

patients with C4A or C4B deficiency can have border-

line normal C4 levels. SLE patients with complement

deficiencies have characteristic features with early onset

of disease, photosensitivity, less renal disease and anti-

Ro ⁄ La antinuclear antibodies in two thirds.5 Measure-

ment of C3 and C4 is useful in monitoring the disease

activity in SLE in general, but low levels should be

interpreted in the light of any component deficiency. A

two- to threefold increase in MBL deficiency has been

reported in patients with SLE17 and these patients may

have more severe infections. The clinical utility of MBL

measurement in this setting however, remains to be

established.

Patients with C3 and terminal complement compo-

nent deficiencies present with pyogenic infections

particularly with encapsulated organisms (Streptococ-

cus pneumoniae and Haemophilus influenza type b).

Deficiency of C3, the major opsonin results in severe

recurrent infections with a spectrum similar to hypo-

gammaglobulinaemia as does acquired C3 deficiency

(Factor H or I deficiency or C3 nephritic factor), it is

also associated with glomerulonephritis. Terminal

complement pathway components (C5, C6, C7, C8

and C9) and properdin result in recurrent neisserial

infections. Factor H deficiency is also associated with

glomerulonephritis and haemolytic uraemic syndrome.

Defects in the terminal pathway result in low CP



(CH50) and low AP (AP50) pathway function

(Table 1).

Leucocyte adhesion deficiency type 1 is a deficiency of

complement receptor 3, an integrin that binds the

degradation products of C3b. It is first suspected at birth

because of delayed separation of the umbilical cord and

usually results in death in childhood due to recurrent

infections and abscesses of soft tissues and mucosal

surfaces.18

Mixed cryoglobulinaemia is often caused by the

immune response to hepatitis C virus or by complexes

with rheumatoid factor. Patients with cryoglobulinae-

mia form complement fixing immune complexes which

cause inflammation in joints and nerves.19 Complement

testing reveals low C4 and normal or less commonly low

C3 (Table 1).

Acquired complement deficiency states may occur

due to the formation of C3 and C4 nephritic factors,

autoantibodies which bind to and stabilize the C3

convertases of the alternate and classical pathways

(Table 1). This results in secondary C3 deficiency and

classically presents with the triad of membranoprolifer-

ative glomerulonephritis, partial lipodystrophy and

bacterial infections20 although features such as partial

lipodystrophy may also present in isolation. Synthetic

failure of complement components may also occur in

advanced liver disease.

Laboratory testing and interpretation

Complement testing involves measurement of individual

components or measurement of function of the CP

(CH50 assay) and the AP (AP50 assay). Complement

components are measured using a nephelometer or

turbidometer and monospecific antisera or by radial

immunodiffusion (RID). The most commonly measured

components are C3 and C4 with low C4 suggesting

activation of the CP (Table 1). The specific antiserum

Table 1 Patterns of complement assay results in clinical settings. It is important to note that as many of the complement components are

acute phase reactants, decreases due to activation may be masked by increases in the synthesis rates during an inflammatory episode.

Complement component levels, e.g. C3 may also be low due to synthetic failure in particular in advanced liver disease. Transient activation

and depletion of complement components can also occur in ischaemic organ injury, trauma, burns, sepsis, viraemia and some drug

reactions. Measurement of split products can be used to determine whether activation has occurred, because their increase occurs only

when the complement enzymes are formed and active. In addition the pathway of activation can be determined; C4a and C4d are markers

of classical or lectin pathway activation, Bb is a marker of alternative pathway activation and C3a, iC3b, C3d, C5a and soluble C5b-9 can be

used to determine terminal pathway activation. These tests are however, not routinely available.

Clinical condition

Classical

pathway (CH50)

Alternative

pathway (AP50) C3 C4

C1-inh

antigenic

C1-inh

functional Process

Deficiencies

C1q, C1r ⁄ C1s, C4

or C2 deficiency*

0 Normal Normal Normal* Normal Normal Classical pathway

Factor D or properdin

deficiency

Normal 0 or very low Normal Normal Normal Normal Alternative

pathway

C3, C5, C6, C7, C8,

deficiencies

0 or very low 0 or very low Low or normal Normal Normal Normal C3 or C9

terminal

component

Consumption states

SLE, cryoglobulinaemia,

HUVS, serum sickness

Low Normal or low Normal or low Low Variable Variable Classical pathway

Sepsis, post-

streptococcal

glomerulonephritis

Normal or

slightly low

Low Low Normal Normal Normal Alternative

Pathway

Severe SLE, membrano-

proliferative GN Type 1,

major proteinuria

Low Low Low Low Variable Variable Both pathways

Failure of regulation

HAE, AAE Low Normal Normal or

slightly low

Very low Very low

to normal

Very low Classical pathway

Factor H or I deficiencies,

C3 nephritic factor

Normal to low Very low to 0 Low Normal Normal Normal Alternative pathway

*In C4 deficiency levels of C4 will be correspondingly low.



binds to the component forming an immunoprecipitate

(Ipp) suspended in aqueous solution. The nephelometer

measures the intensity of light scattered by the Ipp and

the turbidimeter the decrease in light intensity. These

values are then converted into a concentration.

The CH50 and AP50 are haemolytic assays based on

the ability of patient serum to lyse antibody-coated

sheep erythrocytes or chicken erythrocytes (which have

the unique property of activating the AP by activating

C3b),5 respectively. Briefly, in the CH50 agarose gel

containing red blood cells coated with rabbit antisheep

antibody is incubated with human serum (at 4 �C), C1binds, activating the complement cascade and MAC

which results in lysis of the erythrocytes causing a clear

zone in the plate. The antibody concentration and gel

thickness are uniform allowing quantification of the

area of lysis which is proportional to the amount of

complement components. A calibration curve is run

alongside patient sera; the diameter of the test sample is

then read from this curve and a percentage normal

complement activity is generated. The CH50 value

denotes the amount of serum required to lyse 50% of the

antibody-coated erythrocytes. In the CH100 assay the

value reported is the concentration at 100% of lysis.

The function of the lectin pathway may be measured

using an ELISA in which patients serum is plated into

wells coated with mannan.21 After MBL binds to the

mannan coated surface, the MASP enzymes cleave C4

and the resulting C4b and C4d that are deposited on the

plate can be measured using enzyme conjugated

monoclonal antibodies.

C1-inh and C3d are measured using RID where

specific antibody to it is incorporated into an agar

plate and test serum is placed into a well. An immune

complex is formed producing a precipitin ring, at

‘completion’ ) where equilibrium is achieved between

the two, the size of the ring is proportional to the

C1-inh concentration as that of the antibody is

constant.12

It is important to be aware of the external quality

assurance (National External Quality Assurance

Scheme; NEQAS) and accreditation (Clinical Pathology

Accreditation UK; CPA) of the laboratory to which

blood is sent to ensure the precision, accuracy and

reliability of the results and their interpretation. Liaison

with the Clinical Immunologist may be useful to

optimize investigations best suited for the clinical

condition and allow appropriate collection, storage

and timing of sample.

Acknowledgements

We thank Lesley MacNeill for expert assistance with

Fig. 1 and Dr Jenny Hughes for careful reading of the

manuscript.

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Key points

• Complement testing is of utility in hereditary angioede-

ma, acquired angioedema, connective tissue disease (e.g.

SLE), HUVs, mixed cryoglobulinaemia, and deficiency

states due to genetic defects and synthetic failure.

• Complement is activated by three pathways, the classical

pathway (immune complexes), the mannan binding

lectin pathway (sugars) and the alternative pathway

(bacterial cell walls).

• The main functions of complement are to promote

inflammation, the opsonization and lysis of microor-

ganisms, and removal of immune complexes.

• Complement assessment is made by both antigenic test-

ing of components and functional assays of the classical

and alternative pathways (CH50, AP50 and C1-inh).

• The laboratory should have appropriate accreditation

(Clinical Pathology Accreditation UK; CPA) and liaison

with the Clinical Immunologist may be useful in plan-

ning testing.



The role of complement testing in dermatology

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