Download Biological Activities of Complement

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Biological Activities of Complement
T. BARKAS
Department of Biochemistry, University of Bath, Claverton Down,
Bath, Avon BA2 7 A Y , U.K.
Complement is a system of serum proteins whose main function is to defend the body
against infection. Although it has a reputation as a dauntingly complex system, this
results chiefly from confusion in the nomenclature, which has varied considerably with
time as more components were discovered, for example as ‘component C3’ became
subdivided into several components one of which is still called ‘C3’. Moreover, three
different nomenclatures have been used concurrently for the proteins of part of the
system. These discrepancies have been resolved and a standard nomenclature is now in
use.
Activation of the complement system
The complement system can be described in a simplified form as consisting of three
stages (Scheme 1). Probably the most important complement component is that known
as C3. This protein has a serum concentration of 1.6mglml and so represents a major
constituent of serum. The first stage of complement activation is the generation of
enzymes capable of activating component C3, the second the actual activation of
component C3, and the third comprises most of the biological activities of complement.
(i) Stage 1. Two main pathways for the activation of component C3 exist, known
as the classical and alternative pathways. The classical pathway is well-characterized
and has been extensively reviewed (Muller-Eberhard, 1968, 1969, 1975; Cooper et al.,
1971; Lachmann, 1975). The system is illustrated in Scheme 2. It is activated, in
the presence of CaZCand MgZ+, by the formation of an immune complex between
immunoglobulin M or G and antigen, here shown as an antigenic site on a cell surface.
This binding process in turn activates the complement components C1, C4, C2 and C3.
Activation of components C1 and C2 results in the generation of esterase and proteinase
activity from proenzyme forms, whereas activation of components C4 and C3 produces
fragments carrying labile binding sites, allowing the binding of complement proteins
to membranes or other proteins to occur. The reaction is a cascade-type system, in
which one active component-C1 molecule can activate many component-C3 molecules;
active component C1 can also transfer from site to site. The available data on classical
pathway components are shown in Table 1.
The alternative pathway until recently was ill-defined. However, as a result of considerable effort in the last three years, a tentative scheme for this pathway can be
proposed (Scheme 3). Recent reviews including information on this system are those by
Fearon & Austen (1976) and Fothergill &Anderson (1978). This pathway is activated by
certain immunoglobulins, such as immunoglobulin A, that do not activate the classical
pathway, and by various polysaccharides, such as bacterial endotoxin, zymosan from
yeast cell walls and suspensions of inulin. It requires the presence of Mgz+ only. The
early events in this system are still poorly understood but appear to involve a component
known as initiating factor (IF), which may be immunoglobulin-like in character
(Davis et al., 1977). This pathway is more complex because it involves component C3
at two points. First, an active complex of initiating factor, factor B and component C3
-
Generation of component-C3-activatingenzymes (bivalent cations required)
Activation of component C3
---+
Subsequent reactions
Scheme 1. Stages in the activation of complement
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c1
(Ca2' -dependent)
A
A
Scheme 2. Initial stages of the classical pathway for the activation of complement
A, Antigenic site on cell; A , antibody; -,
enzymic site;
---- +, enzymic activity.
C1, C2 etc., proenzyme forms of complement components; C1, C2a etc., active enzymes.
Table 1. Components of the classicalpathway for the activation of complement
Concn. in serum
Electrophoretic
Component
(Pgm
mobility
x Mol.wt.
Clq
180
Y2
400
Clr
101
82
168-198
Cls
110
a2
85
82
117
c2
25
81
190-220
c3
1600
c4
640
81
206
c5
80
81
190-220
C6
75
82
95
c7
55
82
110
Yl
163
C8
80
c9
230
u
79
Activating agents
Scheme 3. Alternative pathway for the activation of complement
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Table 2. Components of the alternativepathway for the activation of complement
Component
Initiating factor
Factor B
Factor D
c3
C3b
Properdin
Concn. in serum
Olglml)
Electrophoretic
mobility
-
100-200
B
B
1600
B
10-20
Y
-
-
U
U
x Mol.wt.
170
8&93
22-25
180
171
184-220
10-3 x
Mol.wt.
120-140
75-80
9
Activating
enzymes
Inactivating
enzymes
C3b
181
c3c
140
C3d
25
Scheme 4. Activation of component C3 of complement
is formed in the presence of a small-molecular-weight proteinase, factor D. This
complex, itself containing component C3, then activates further component C3 in a
similar fashion to the classical pathway, the catalytic component being factor B. If the
fragment C3b produced is deposited on a surface, in the presence of further factors B
and D, more component C3 can be activated to form clumps of bound fragment C3b.
The data available on components of the alternative pathway are shown in Table 2.
(ii) Stage 2. The actual activation of component C3 is by proteolytic cleavage.
Component C3 is a protein of two disulphide-bridged polypeptide chains (Scheme 4).
Activation consists of the removal of a small fragment, C3a, from the N-terminal
region of the a-chain. Enzymic inactivation of the fragment C3b is also by removal of
a further small fragment, C3d, from the same chain.
(iii) Stage 3. The terminal components of complement are designated components
C5, C6, C7, C8 and C9, acting consecutively.
In the case of activation by the classical pathway, the complex C,4,2,3 cleaves components C5 (a very similar molecule to component C3) into two fragments. The large
fragment C5b becomes membrane-bound, the components C6-C9 complex with fragment C5b and cytolysis can occur. In the case of the alternative pathway, the complex
containing (C3b)"B can directly activate component C5 as above. However, the stability
of the component-C5-converting enzyme is markedly enhanced by the binding of a
further component, known as properdin.
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Table 3 . Biological activities of complement
Activity
Cytolysis
Modulation of effects of immune complexes
Formation of large complexes
Binding to, and release from, cells
(a) Phagocytosis, release of lysosomal enzymes, chemotaxis
(b) Antibody production
Lymphokine production and other lymphocyte
responses
Cell-mediated antibody-dependent cytolysis
Release of complexes from lymphocytes and solubilization of precipitated complexes
(c) Release of vasoactive amines from platelets, platelet
lysis
Inactivation of bacterial lipopolysaccharide
Neutralization of viruses
Viral lysis
Components involved
Cl-C9
Clq, C3b, C4b
C3b, C4b
Alternative
pathway
c5
C1-C3
Implications in cancer
Anaphylatoxins
Kinins
C3a, C5a
C2?
Coagulation
Fibrinolysis
C6
c3, c 4
Biological activities
Complement was first described as a heat-labile factor capable of acting together
with antibody in the lysis of bacteria. Although cytolysis is probably the most widely
known aspect of complement activity, the involvement of complement components in
many different biological phenomena has been demonstrated (Table 3).
(i) Cytolysis. This very dramatic effect of complement is the only activity requiring
all the terminal components C5-C9 (see reviews by Humphrey & Dourmashkin, 1969;
Dourmashkin et al., 1972). It has been observed with erythrocytes, mammalian nucleated
cells, such as Krebs ascites cells, rat peritoneal mast cells and Chinese-hamster lung cells
(Shipley et al., 1971), platelets (Polley & Nachman, 1975), Gram-negative bacteria, the
coat of avian infectious bronchitis virus and suspensions of bacterial lipopolysaccharide.
The end result of the binding of components C5-C9 is the formation of lesions allowing the passage of ions and small molecules. Subsequent distortion then allows the efflux
of macromolecules (Green et al., 1959a,b). Characteristic ‘holes’, about lOnm in diameter, with an electron-lucent ring of 2.5 nm, can be observed by electron microscopy.
Although a correlation between the number of ‘holes’ and the calculated number of sites
of damage has been made (Borsos et a/.,1964; Humphrey & Dourmashkin, 1969),freezefracture techniques suggest that the ‘holes’ do not penetrate the full thickness of the lipid
bilayer (Iles et al., 1973; Bhakdi et al., 1974). Moreover, with liposomes, Kataoka et ( I / .
(1973) found no correlation between the appearance of ‘holes’ and the release of
contents.
The nature of the damage suffered by the membrane is not known. Complementmediated alterations of membrane proteins have been reported (Kniifermann et ul.,
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1971 ; Zimmerman & Miiller-Eberhard, 1973). However, proteolysis is unlikely to be
responsible for the formation of lesions, as these can be formed equally well on pure
lipid liposomes (Inoue & Kinsky, 1970; Kinsky, 1972). Phospholipase activity has not
been detected (Inoue & Kinsky, 1970; Lachmann eta/., 1973). Two further mechanisms
have been proposed. One is the formation of hydrophilic channels through the membrane.
Evidence for possible insertion of complement components into the membrane
has been obtained by several authors (Bhakdi et af., 1975; Hammer et a/., 1975, 1977;
Miiller-Eberhard, 1975; Kolb & Miiller-Eberhard, 1976). However, the freeze-fracture
experiments mentioned above tend to discount the formation of a completely
transmembrane channel. The second hypothesis is that the terminal components exert a
detergent-like effect on the membrane (Kinsky, 1972), causing a rearrangement of
membrane structure. Such a redisposition of membrane components is substantiated by
the freeze-fracture experiments. Moreover, a major rearrangement of membrane lipids
has been reported to occur during complement lysis (Giavedoni & Dalmasso, 1976),
and displacement of membrane phospholipid has been observed (Tnoue et al., 1977).
Changes in membrane properties have also been monitored by a decrease in
deformability correlated with binding of component C3 (Durocher et a/., 1975), nonosmotic swelling after the addition of component C9 (Valet & Opferkuch, 1975) and
increased ion-permeability of lipid bilayers after the addition of components C5-C9
(Michaels et al., 1976).
(ii) Modulation of the effects of immune complexes. The distribution of immune
complexes in the body can be markedly affected by complement. First, the size of immune
complexes can be dramatically increased by the binding of complement components,
leading to their removal by the reticuloendothelial system, or deposition in the kidney
or joints. Secondly, the localization of aggregated immunoglobulin G and, presumably,
complexes to the germinal areas of the spleen requires component C3 (Papamichail
et al., 1975). Localization of antibody-antigen complexes to splenic lymphoid follicles
may also be important in antibody production, as Klaus & Humphrey (1977) have shown
that decomplementation inhibits the formation of B-lymphocyte memory cells.
Probably the most important function of complement is the mediation of the
binding of immune complexes to various cell types. Complement receptors have been
clearly demonstrated on primate erythrocytes, on non-primate platelets and on phagocytes and lymphocytes (see Henson, 1972). Binding occurs by a stable site on fragments
C3b (Nishioka & Linscott, 1963) and C4b (Cooper, 1969). Depending on the cell type
involved, the binding of complexes can produce several effects, as follows.
( a ) Immune adherence to erythrocytes and phagocytes. Binding of complexes to
receptors on erythrocytes greatly increases phagocytosis of the complex, resulting in
more rapid engulfment of bacteria and viruses (Nelson, 1953,1956; Robineaux &Nelson,
1955). Binding of complexes directly to complement receptors on phagocytic cells also
potentiates phagocytosis (Gigli & Nelson, 1968; Wellek et al., 1975). Phagocytes are
attracted to the site of complement activation by the small fragments C3a and C5a and
the complex C5,6,7, all of which are chemotactic agents.
Binding of complement also stimulates phagocytes to release lysosomal enzymes
selectively(Go1dstein etal., 1973; Ward & Zvaifler, 1973; Schorlemmer & Allison, 1976),
and causes increased spreading of niacrophages and intense ruffling of the cell membrane
(Bianco et al., 1976).
(6) Binding to lymphocytes. B lymphocytes have receptors for fragments C3b, C3d and
C4b (reviewed by Nussenzweig, 1974). Such receptors might be of prime importance in
the production of antibody. Certain authors (Dukor & Hartmann, 1973; Dukor et al.,
1974) suggested that complement might provide the second signal required together
with antigen for the production of antibodies. However, most recent work tends to
discount this theory (Feldmann & Pepys, 1974; Pepys, 19746; Pryjma et a/., 1974;
Bitter-Suermann et a/., 1975), especially as a T-lymphocyte-independent response can
be obtained in the absence of component C3 (Pryjma & Humphrey, 1975). Antibody
responses requiring the participation of T lymphocytes may possibly require the participation of complement (Feldmann & Pepys, 1974; Pepys, 19746), perhaps in bringing
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together the two subsets of lymphocytes and the macrophages required for this response
(Pepys, 1 9 7 4 ~ ;Arnaiz-Villena & Roitt, 1975). At variance with this are reports of
T-lymphocyte-dependent responses in the absence of complement (Waldmann &
Lachmann, 1975) and normal antibody concentrations in a patient completely deficienr
in component C3 (Alper et al., 1976).
Complement has also been suggested as being responsible for the switch-over from
immunoglobulin M production to immunoglobulin G production during the course of
an immune response (Pepys, 19746; Nielsen & White, 1974) and in the induction of
immunoglobulin E production (Pepys et af., 1977).
Many other lymphocyte responses seem to be affected by complement. Cell-bound
component C4 appears to be necessary for mixed lymphocyte reactions and mitogenic
responses (Ferrone et a/., 1976). Binding to fragment C3b results in the release of
lymphokines of various types (O’Neill et al., 1975; Mackler et al., 1974; Sandberget al.,
1975) and directly stimulates DNA synthesis (Hartmann & Bokisch, 1975).
Lymphoid cells are very effective at destroying antibody-coated target cells (see
Perlmann & Holm, 1969); the efficiency in some cases can be greatly increased by added
target-cell bound complement (Perlmann et al., 1969; Lustig & Bianco, 1976; Rouse
et al., 1977). This probably results from increased binding of the complex to the effector
cell (Eden et al., 1973a; Theofilopoulos et a/., 1974; Scornik & Drewinko, 1975). Such
an effector pathway is important in that it is not inhibited by physiological concentrations of immunoglobulin G, whereas the same type of cytolysis in the absence of
complement is (Scornik, 1976; Barkas & Al-Khateeb, 1978).
Finally, complement also exerts a regulatory role in determining the length of
time complexes remain associated with lymphocytes. Low concentrations of complement
have been shown to enhance the binding of complexes to lymphocytes, whereas high
concentrations cause release. I n vivo this results in an initial rapid binding to cells,
followed by rapid release into the circulation of modified antibody-antigen complex that
is incabable of further binding to lymphocytes (Eden et al., 19736; Miller et al., 1973;
Miller & Nussenzweig, 1975~).Precipitated complexes appear to be solubilized by
a similar mechanism (Miller & Nussenzweig, 197%); this may serve as a protective
mechanism against the deposition of immune complexes (Czop & Nussenzweig, 1976).
(c) Binding to platelets. In certain species, such as the rabbit, a mechanism exists
for complement-dependent binding of complexes to platelets. This can result in either
selective release of granule contents or total lysis of the platelet. depending on whether
the complex is particulate or not (Henson & Cochrane, 1969; Henson, 1970).
Human platelets bind complexes in the absence of complement, but, in the presence of
plasma, exhibit a biphasic response, the first phase being complement-independent,
resulting in the release of 5-hydroxytryptamine (serotonin), and the second phase being
complement-dependent, resulting in the aggregation of the platelets and a further
release of granule contents. Zymosan can also produce this second release phase
(Pfueller & Liischer, 1974a,b; Zucker & Grant, 1974; Zucker e t a l . , 1974).
(iii) Inactivation o f bacterial lipopolysaccharide and neutralization of viruses. Bacterial
lipopolysaccharide is reported to be inactivated by a detergent-like activity of
component C5 (Johnson et a[., 1972).
Viruses sensitized with immunoglobulin M antibodies can be neutralized by the
sequential addition of components C1-C3 (Daniels et a[., 1969; Linscott & Levinson,
1969; Notkins, 1971; Oldstone et a / . , 1974), whereas immunoglobulin G-sensitized
viruses are less affected (Schrader & Muschel, 1975). Virally infected cells can also be
destroyed (reviewed by Porter, 1971); the antibody-directed killing of such cells by
lymphocytes is enhanced by the presence of complement (Rouse et a[., 1977).
Recent interesting developments are the reports of lysis of RNA tumour viruses by
human serum containing the complete complement system (Welsh et al., 1975). Type C
virus is reported to be lysed by serum both from primates and from non-primates (Sherwin
eta/., 1978). However, these systems do not appear to require the presence of antibody.
(iv) Implications in cancer. Some evidence exists for activation of the complement
system during tumour development. Antibody and component C3 have been shown
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to be present on tumour tissues (hie et al., 1974). The inability of the complement
system to destroy the tumour does not appear to be due to a low density of antigenic
sites (Liang & Cohen, 1975), as resistant tumour cells bind complement as efficiently as
do non-resistant strains (Yu et al., 1975; Ohanian & Borsos, 1975; Segerling et a[.,
1975~).
Correlations between inhibition of, or absence of, complement activity and tumour
development have been made. Increased concentrations of the natural inhibitor of
component C1 have been found in the serum of cancer patients (Bach-Mortensen et a/.,
1975; Spitzer et a/., 1975); the inhibitor has also been detected on the tumour cell
(Osther & Linnemann, 1973; Osther, 1974; Bach-Mortensen et al., 1975). Deficiency of
component C5 in AKR mice has been implicated in the spontaneous development of
leukaemia associated with this strain, and with impaired interferon activity (Kassel
et a/., 1973). Certain carcinogens impair the synthesis of components C4 and C2
(Colten & Borsos, 1974). Tumour cells liberate a substance capable of inactivating
chemotactic factors (Brozma & Ward, 1975).
Conversely, certain anti-tumour drugs have been found to activate the complement
system (Okuda et al., 1972),and others appear to render the tumour cell more susceptible
to the action of complement (Segerling et al., 1975a,b). The anti-tumour agent
Corynebacteriumparvum activates complement in vivo in cancer patients (Birani et al.,
1976).
(v) Anuphylatoxins and kinins. In addition to their chemotactic activity, the small
fragments C3a and C5a possess potent anaphylatoxin activity, liberating histamine
from mast cells and contracting smooth muscle. A C-terminal arginine residue is
essential for the activity (Bokisch & Miiller-Eberhard, 1970). Both the direct binding of
fragment C3a to rat mast cells (Ter Laan et al., 1974) and the direct release of histamine
by fragments C3a and C5a (Johnston et al., 1975) have been demonstrated. The amino
acid sequence of fragment C3a has been published (Hugli, 1975).
During activation of the classical pathway, a kinin is generated. This activity has
usually been ascribed to a fragment of component C2 (Klemperer et al., 1969). However,
the effect is not seen in a patient completely deficient in component C3, i.e. deficient in a
complement protein later in the chain than component C2 (Alper et al., 1976).
(vi) Coagulation and Jibrinolysis. A complement-dependent coagulation pathway
has been demonstrated (Zimmerman & Muller-Eberhard, 1971). Deficiency of
component C6 in rabbits results in impaired coagulation (Zimmerman et al., 1971),
whereas a similar defect in man does not (Leddy et al., 1973; Heusinkveld et al., 1974).
This probably results from the sensitivity of rabbit platelets to lysis by complement,
releasing platelet factor 3 and promoting coagulation, whereas human platelets are not
susceptible (Zimmerman & Kolb, 1976).
Lysis of diluted blood clots is reported to require components C4 and C3 (Taylor &
Muller-Eberhard, 1967; Schreiber & Austen, 1974).
Control
With a system potentially capable of producing such drastic effects, mention must be
made of the various safeguards built into the system to prevent the accidental triggering
of complement activation and to contain the activity spatially and temporally.
Probably the most important regulatory effect is the lability of the binding sites
generated at various steps (see Muller-Eberhard, 1969, 1975). These have half-lives of
the order of seconds or less; if binding does not occur in this space of time, the
component becomes inactivated. Certain of the intermediates also have half-lives of a
few minutes at 37°C.
Degradation by plasma proteinases and phagocytosis may also function as control
mechanisms. The rates of catabolism of complement proteins are quite high, being
0.7-2.2
of the plasma pool/h for several components.
Finally, as shown in Scheme 5, serum inhibitors exist for many of the activated
components. Inhibition of enzyme cl is stoicheiometric, whereas the other inhibitors
act enzymically.
<:
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Chemotactic factor
Anaphylatoxin
\C3a
C5a
C5,6,7
-
CI
C4b
j
C3b
C6
C7
inhibitor inhibitor i inhibitor j inhibitor inhibitor
Unstable
Unstable
(inhibitor?)
Scheme 5 . Control mechanisms in the activation of complemenl
Conclusion
As a result of considerable interest over recent years, the actual mechanisms of the
complement system have been worked out. Interest has now moved to the more
complex field of a n understanding of the biological activities of complement, resulting in
the discovery of complex interrelationships and interreactions between complement
and both the fluid-phase systems of blood, such as fibrinolysis, coagulation and kinin
generation, and cells of many types. Especially fascinating is the number of ways in which
Complement is involved in defending the body against infections, by direct lysis of antibody-coated bacteria, by enhancement of antibody-dependent cellular cytotoxicity,
by removal of complexes by immune adherence and enhanced phagocytosis, by direct
stimulation of lymphocytes and possibly by involvement in the actual production of
antibodies.
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