Molecular aspects of complement activation

0098-2997/81 / 040209-65532.50/0 Copyright©1981 Pergamon Press Ltd

Molec. Aspects Med. Vol. 04, pp. 209 to 273, 1981 Printed in Great Britain. All rights reserved

MOLECULAR

A S P E C T S OF C O M P L E M E N T

K. Whaley

ACTIVATION

and A. Ferguson

University of Glasgow Department of Pathology,

Western Infirmary, Glasgow

CONTENTS INTRODUCTION

211

COMPLEMENT COMPONENTS The Classical Pathway

212 212 212 215 215 216 216 217 218 218 219 219 223 224 224 224 225 226 226 227 227 227 229 229 230 230 230 232 232

Clq C1r Cls The C1 macromolecule C4 C2 Activation of the classical pathway

Antibody-dependent activation Terminal Components

C3 C5 C6 and C7 C8 C9 The C5b-9 membrane attack complex The Alternative Pathway

Introduction Proteins of the alternative pathway

C36 Factor B Factor ~ Properdin Cobra venom factor (CoVF) C3 nephritic factor (C3NeF) Assembly of the alternatice pathway C3 and C5 convertases Analogies between the classical and alternative pathways Activation of the alternative pathway

MODULATION OF COMPLEMENT ACTIVATION

233 233 235 235 236

CT-inhibitor C4-binding protein C3b inactivato-r ~IH globulin 209

K. Whaley and A. Ferguson

210

S protein Anaphylotoxin inactivator Initiation of the alternative pathway activation

238 239 240

BIOLOGICAL ACTIVITIES OF C O M P L E M E N T Cytolysis Anaphylotoxins and chemotaxis Modulation of immune-complex mediated effects Micro-organisms and complement opsonisation Antiviral activity of complement

241 241 243 24~ 244 244

BIOSYNTHESIS

244 245 245 245 245 246 246 246 247 247 247 247 247 247 248 248

OF COMPLEMENT

PROTEINS

ci c4 c2 c3 c5

C6 C7 and C8 C9 Alternative pathway components Control proteins

Cl-inhibitor C3b-inactivator and ~2H globulin Ontogeny of complement Genetically-determined polymorphism of complement components Acquired abnormalities of the complement system in human disease

CONNECTIVE

TISSUE

DISEASES

Systemic lupus erythematosus (SLE) Rheumatoid arthritis Infectious diseases Renal disease Complement and skin disease Genetically-determined deficiencies of complement components Complement deficiencies associated with recurrent infection Complement deficiency and connective tissue diseases

RE FE RENCE S

?:4g 248 249 250 251 251 252 252 254

255

I NTRODUCT

I ON

The complement system is a group of self-assembling plasma proteins which constitutes the principle mediator of antigen-antibody reactions. In 1884 Grohmann I showed that blood plasma was capable of killing bacteria, and Buchner 2'3 later showed that fresh serum was bactericidal. Buchner termed this activity alexin and noted that is was sensitive to heat (55°C for 30 minutes) and dialysis against distilled water at 0°C for 18 to 36 hours. From these experiments Buchner concluded that the lytic activity of fresh serum was due to serum enzymes, and made the important suggestion that serum-mediated bacteriolysis could be used to study the interaction of proteins. Bordet 4'5 proved that at least two factors in immune serum were required for bacteriolysis. A heat-stable factor, increased by immunisation and reacting specifically with the immunising organism, is now known to be antibody. The second factor Bordet termed alexin was shown to be heat-labile, and was present in both immune and non-immune animals. The heat-stable factor was incapable of killing bacteria in the absence of alexin. Alexin was also capable of lysing antibodycoated erythrocytes 5. During the early years of the twentieth century a number of workers showed that alexin, or complement, was not a single serum constituent, and four factors were identified on the basis of euglobulin precipitation, heatsensitivity, treatment with zymosan or cobra venom and ammonia-sensitivity. The euglobulin contains CI, or midpiece, while the pseudoglobulin was termed endpiece. Heat-inactivated serum was devoid of CI and C2, zymosan and cobra venom-treated serum was deficient in C3, and ammonia destroyed C4 (reviewed by Rapp and Borsos~). These studies showed the sequential nature of complement activation, and the existence of cellular intermediate products in immune haemolysis. C3 activity was considered to be due to a single component, and it was not until 1958 that Rapp ? showed that a mathematical analysis of the haemolytic reaction of C3 was consistent with it being more than one factor. It is now known that C3 activity determined by these old techniques is a complex of six factors 8. With the advent of improved methods of plasma protein fractionation the complement system has been shown to consist of at least eighteen proteins. Their physicochemical nature, molecular interactions, and biological activities are becoming better understood. The purpose of this paper is to describe the components of the complement system, their molecular interactions during activation and the biological activities generated during activation. The mechanisms controlling complement

211

212

K. Whaley and A. Ferguson

activation, the cells which synthesise individual components, the factors controlling synthesis and the genetically determined deficiencies of these components will be discussed.

COMPLEMENT

COMPONENTS

At least 18 plasma proteins constitute the complement system. For the purposes of presentation they are divided into four functionally separate groups: two groups, the classical and alternative pathway proteins, are concerned with the generation of enzymes with cleave C3 and C5, one group forms a multimolecular protein complex which is responsible for complement-mediated cytolysis, and the fourth group constitutes a group of proteins which modulate complement activation. The components of the classical pathway and the membrane attack complex are numbered numerically, CI, C2, C3, C4, C5, C6, C7, C8 and C9; the components of the alternative pathway are given alphabetical symbols, factor B (B), factor D (D) and properdin (P). Finally the control proteins are named on the basis of their activities.

The C l a s s i c a l

Pathway

The classical pathway consists of five distinct proteins, C|q, C1r, C1s and C4 and C2 (Table I). The first three proteins interact to form the multimolecular complex CI 9 held together by calcium ions I°. The biochemistry of these proteins will be described and their molecular interactions discussed later.

Clq C1q, the recognition molecule of the classical pathway, is a globulin which has a molecular weight of 400,000. It interacts with CX2 domain of IgG II and probably with C~4 domain of IgM 12, antibody present in antigen-antibody complexes. This rather unusual protein consists of collagen-like triple helix distributed amongst six identical subunits (Fig. I). Each subunit contains three different polypeptide chains (termed A, B and C) 13, all of which have molecular weights of approximately 23,000. The A and B chains are disulphide-linked and the C chains are present as disulphide-linked dimers. Amino acid sequencing of these three chains has shown that they are very similar, each having about 80 residues of typical collagen-like sequence beginning close to the N terminus 14 All three chains contain the repeating triplet GIy-X-Y, where Y is often hydroxyproline or hydroxylysine 14. This similarity to collagen is increased by the presence of glucosylgalactosyl disaccharide substitutions on the hydroxylysine residues 15'16 The C terminal regions are very similar and these form a globular unit at the end of each triple helix section. The N terminal parts of the six triple helices are linked by non-colavent bonds to form a larger collagen-like fibril. Recent amino acid sequencing data have shown breaks in the GIy-X-Y sequence; in the A chain, threonine is inserted between positions 38 and 39, and alanine is substituted for glycine at position 36 in the C chain. These substitutions generate a distortion in the helix, consistent with the divergence of the six subunits. The structure of C1q is shown in Figure I, the structure agreeing with the reported electron microscopic appearances of the molecule 17 The globular protein structures at the carboxyterminus "recognise" the changes in the IgG or IgM molecules in antigen-antibody complexes. The collagen-like helix binds the C|r and C1s subcomponents 18

Electrophoretic mobility

y

~

B

~

~

Molecular weight

400,000

90,000

90,000

204,000

100,000

Component

C1q

C1r

C1s

C4

C2

20

430

80

100

250

I

3

1

1

18 (6x 3)

Polypeptide chain structure

+

+

?

?

-

Genetic polymorphism

Classical Pathway Proteins

Serum concentration (Mg/ml)

TABLE I

C2a C2b

C4a C4b

H & L chains

H & L chains

-

Cleavage products

0

Anaphylotoxic Part of C3 and C5 convertases opsonin Part of C3 and C5 convertases ?

0

Cleaves C4 and C2

~O

_,~+. 0..~

>

o

B

B

B B

o

Cleaves C1s

Binds with immune complexes

Biological activity

B

o

5"

~D

~r

214

K. Whaley and A. Ferguson STRUCTURE OF HUMAN CIq 6A+ 6 B+6C Chains (each approx. 23000 Mol.wt.)

18 chains

Reduction ~or Oxidation C

BN c

C C C

9 Subunits ( 6A- B dimers + 3 C-C dimers)

C I

I

,

II

N-Termingl non collagen-like regions of 2-8 resid ues

il

,

C I

3

Collagen-like regions Non collagen-like of 78 residues regions of 103-108 residues

~; ...~

j

Globular C-Terminal regions

,•]3

Structural units

3

Helicalends of structural units form fibril to yield intact molecule of 410,000M.W. 6",.,, ~ ~., "', 7.0 nm"~-- L, 11.5nm xl.hnm~,

"""

~I

'.'.L~/6."~

Regionof the molecule thought to be left intact after collager~se - digestion ie, the globular peripheral portions. Intact

"['~1[

Region of molec ule thought to be left molecule !ntoct after pepsin digestion at pH4.Z, 11-2nm Nkd~,,ji ie, the six connecting strands plus , L/L,.'I,~L f i bril-like end-piece ' VI,'I)II

4-5nm 1

The proposed p e p t i d e s t r u c t u r e of Clq. The dimensions given are averages of those of Shelton and coworkers (?roc. Nat. Acad. Sci. USA, 69, 65-68, 1972), as e s t i m a t e d from e l e c t r o n microscopy s t u d i e s . Proposed t r i p l e h e l i x s e c t i o n , t h a t i s , c o l l a g e n - l i k e regions of tbe molecule. The following comparisons can be made. Length of collagenlike fibre + fibril endpiece = 11.5+ 11.2 n m = 22.7 nm. Length of triple helix proposed from sequence studies = 8 . 0 x 0 . 2 9 = 23.2 nm. (Reprinted by permission of the authors, Porter, R.R. and Reid, K.B.M., from Nature, 275, 699-704, 1978. Copyright © 1978. Macmillan Journals Ltd.) Fig.

The Biochemistry of Inflammation: Complement Activation

215

C1r

C1r is a single polypeptide chain zymogen having a molecular weight of 90,000 (Fig. 2). It is very similar to C1s, but exists as a non-covalent dimer, and has a different substrate specificity (C]s being its only natural substrate). The spectrum of synthetic esters hydrolysed by C1r differs from that of CTs 19. Further more the affinity labelling reagent mCP (PBA)-F which labels C~s does not label C~r 19. C~r, the active enzyme consists of two disulphide-bridged polypeptide chains, having molecular weights of 56,000 and 27.000. The active site of C]r has been shown to be present on the smaller chain, as this binds 32P-labelled DFP and C~ inhibitor 2°'21. N terminal analysis suggests that the smaller chain is probably derived from the C-terminal of precursor C 1 r 22 . The amino acid sequence of the smaller chain is similar to that of CIs 22 and other serum proteases 23 Active site ¢,

.

.

.

.

J C l r Dimer Zymogen

Fig. 2

.

.

7

"

t'__'

mill

L chain

C l r Dimer

Diagrammatic model for actication of Cir. H chain = heavy chain, L chain = light chain, .... non-covalent bonds. Modified from Ref. 166.

Cls

The inhibition of the enzymatic activity of functionally purified CI by DFP suggested that C1s was a serine protease 2~. It is now known that C1s circulates as a zymogen consisting of a single polypeptide chain of 90,000 molecular weight (Fig. 3) 2 S ' 2 6 . Following activation it splits into two disulphide bridged chains of 56,000 and 27,000, the smaller chain binding 32P-DFP and CT-inhibitor, and therefore carrying the active site of the enzyme 25'27-29 The N terminal amino acid of precursor C1s is at the C terminus of the smaller chain, and sequence studies have revealed that the N-terminal sequence and the sequence around the active site of the enzyme are similar to other serine proteases 22'27

L cha.in CIs Zymogen

Fig. 3

Cls

~'Active s i t e

Diagrammatic model for activation of C1s molecule. H chain = heavy chain, L chain = light chain. Modified from Ref. 166.

216

K. Whaley and A. Ferguson

In addition to enzymatically activating C4 and C2, C1s shows a plasmin-like speci-ficity in the spectrum of synthetic esters, it is capable of hydrolysing 22 Although C1s exists as a monomer, in the presence of Ca ++ and C1r, a tetramer is formed consisting of 2 moles Clr and 2 moles C1s 21'22'30 These conditions are those found naturally in serum.

The C1 macromolecule The assembly of a CI complex from the individual constituents Clq, C1r and C1s produces a molecule with a smaller sedimentation coefficient than the native CI complex isolated from serum 9'31 To date no other protein constituent of the CI complex has been isolated. It is doubtful that other protein constituents exist as the functional activity of other properties of the CI molecule are restored by the combination of C1q with the precursor forms of C1r and C1s 32 The molar ratios of the CI subcomponents in the CI complex in serum appears to be one mole C1q to two moles each of C1r and C1s, although for maximum haemolytic activity one mole of C1q must bind with 5 moles each of C1r and C1s 33. The mode of activation of precursor C1r within the CI macromolecule is unknown. Ziccardi and Cooper 34 suggested that C1r was conformationally altered following the binding of CI to immune complexes by the C1q subunit. As a result an enzymatic site on precursor C1r was exposed, which autocatalysed the molecule. More recent work has suggested that C1s is essential for this initial conformational change to occur 33 CTs does not catalyse the activation of C1r but the presence of precursor C1s, CTs or CTs inactivated by DFP is essential for C1r activation.

C4 Human C4 is a pseudoglobulin having a molecular weight of 204,00035'36 The molecule consists of three disulphide-linked polypeptide chains, a, B and X, having molecular weights of 93,000, 78,000 and 33,000, respectively (Fig. 4). C4a -

I

I.

-

C4b

Eli

--

I

o

I / /

g

;

s

//

+

C4bp

/ /

/

/

,¢ C4C

....

Fig. 4

~)~

]s

C4,"

s

s

,~:

~TK

Diagrammatic model f o r enzymatic c l e a v a g e of C4 o c c u r r i n g d u r i n g complement a c t i v a t i o n

The Biochemistry of Inflammation: Complement Activation

217

Carbohydrate residues are present on all three chains. The functional activity of C4 is destroyed by treatment with hydrazine, armnonia, potassium thiocyanate or dialdehyde dextran37'3S.lt is now known that nucleophilic amines bind to the labile binding site of C4 and prevent its binding to cell membranes s6~'365 Upon activation by C~ or C~s, the ~ chain on C4 is cleaved, to release C4a fragment of 6,000 molecular weight from the amino terminal end, and C4b 3s C4a and C4b remain associated at pH 7.4, and can only be dissociated on lowering the pH to 4.5. C4a migrates anodally in polyacrylamide gel electrophoresis, whereas C4b is more cathodal. Amino acid analysis shows that C4a possesses a relatively higher content of basic amino acids (cystine, proline, glycine) than C4, and a lower content of histidine, threonine, valine and leucine 39. As a result of the cleavage of the chain, a labile binding site is exposed on C4b, which can then bind to membrane receptors or antibodies ~°-42 Because the labile binding site is available for a period of 50 milliseconds, approximately only one in 300 C4b molecules binds to antibody or cell membranes, the remainder becoming phase C4b and probably playing little role in further complement activation 43. Degradation of C4b on cell surfaces or in the fluid-phase involves the actions of an enzyme, C3b inactivator, and a high molecular weight cofactor which has been termed C4-binding protein (C4bp). Although neither of these proteins on its own produces proteolysis of C4b, when incubated together with C4b the ~i chain is cleaved in two places yielding three fragments (~2, ~3, ~4) with molecular weights of 47,000, 25,000 and 17,000 daltons respectively 44. The B and y chains are not degraded in this process (Fig. 4). The ~3 and ~4 fragments are linked by disulphide bridges to the B chains, and together with the y chain form the degradation product C4c. The ~2 fragment is not covalently linked to the rest of the molecule and forms the C4d fragment, which migrates as an ~ globulin on electrophoresis 4~. As a result of the actions of C3blNA and C4bp, C4b loses its ability to interact with C2 to form a C~-2, the classical pathway C3 convertase, and its ability to participate in the immune adherence phenomenon by its interaction with C4b receptors. C2

The second component of human complement is a 100,000 dalton molecular weight 8 globulin (Fig. 5) 4s'46. On isoelectric focussing the protein resolves into at f-

I

SH

ci;

I

- Active

1

Site [ C2a

;R SH

C2b ~4b

C2

- binding site

C2 Rapid Decay Activated

Active Site

C21) Oxidised C2 Activated C2 Stable disulphide brid~e

Fig. 5

C4b bh)din~ site

Diagranraatic model f o r a c t i v a t i o n of human C2 and o x i d i s e d human C2 . . . . . = n o n - c o v a l e n t bonds.

218

K. Whaley and A. Ferguson

least three major components having pI values between 6.2 and 6.547 . C2 consists of a single polypeptide chains, which is cleaved by C~ or C~s. The resultant cleavage products C2a and C2b have molecular weights of 70,000 and 30,000 daltons, respectively 4s'46 C2a migrates more anodally and C2b more cathodally than native C2 ~8. C4 and C2 have a relatively high affinity for each other, reversibly forming a complex in free solution ~9. Activated C2 apparently binds to C4b by the C2b fragment. Decay of C~-~ occurs because C2a, the peptide carrying the enzymatic site of C42, rapidly decays from the complex 45. Effective molecule titration of C2 has shown that its haemolytic efficiency is extremely low 5°, only 5% of molecules becoming cell-bound. Treatment of C2 with iodine results in a 10-20 fold increase in heamolytic activity, associated with increased stability of the component in its active form, and a decreased rate of its dissociation from 4-251. This action results from the oxidation of two free sulphydryl (SH) groups to form an intramolecular disulphide bond. Reduction of these free SH groups by p-hydroxymercuribenzoate or p-chloromercuribenzoate partially inactivates C2, emphasising the importance of these groups for haemolytic activity s2. C2 is a rather unusual complement component with the catalytic site being present on the larger rather than the smaller fragment.

Activation

of the classical

pathway

Antibody-dependent activation When antibody interacts with antigen, it acquires the ability to bind C1q. IgA, IgD, IgE and IgG4 do not activate the classical pathway whereas IgGl, IgG2 and IgG3 and IgM doS3. Of the IgG subclasses, IgG3 is the most potent activator, followed by IgGl and IgG2 in descending order of activity 5~. For activation of CI, C1q must bind the heavy chain of two antibody molecules. Thus a single molecule of IgM can activate CI. To ensure that two molecules of IgG were sufficiently approximated (on the surface of an erythrocyte) for activation of CI to occur, 800 IgG molecules are required 55's6 Since antibody molecules and C1q are normally present together in the plasma in the absence of overt complement activation, it is probable that conformational changes occur in the antibody molecule which trigger activation. Conformational changes in the antibody molecule, the proximity of several antibody molecules close together or both these factors may be important 57'58 The Cy2 domain of IgG has been shown to possess the binding site for C1q. Trypsin digestion of acid-treated Fc fragments have full C1-binding activity sg. As the C~2 domain prepared by this process were single, one domain only is required to bind C1q, although there are two in the intact IgG molecule. Reduction of disulphide bonds which permits CT2 domain separation but does not impair the ability to bind C1q 6° The C1q binding area on IgM is thought to be the C~4 domain 12. Interestingly, the amino acid sequences of the C1q-binding domains of different immunoglobulins do not show any obvious correlation between structure and function sS. Isolated Fc fragments or subfragments of IgG46°, IgA 61 and IgM 62 have been shown to bind CI even when their parent molecules failed to demonstrate this function. It is therefore possible that all immunoglobulins possess the appropriate structure for CI binding, but this is often concealed in the intact molecule. A corollary would be that when antigen reacts with antibody, conformational changes occur, perhaps in the quaternary structure of the domains ~3, and that non-complement-fixing antibodies have lost the ability to undergo this conformational change which exposes the C1-binding site. Using circular dichroism and polarisation of fluorescence conformational changes have been shown to occur in the Fc region when antigen binds to the Fab region of the antibody molecule 64-67 Unfortunately identical changes have been shown to occur when the monovalent loop-region of lysozyme was used as the antigen and when multivalent antigens were used. As monovalent antigens do not cause complement binding, the significance of these changes is unclear 67 The disulphide bridges of the immunoglobulin hinge region are important for transferring conformational change from Fab to Fc regions. Complement fixing ability of immunoglobulins

The Biochemistry of Inflammation: Complement Activation

219

diminishes as the interchain disulphide bridges are reduced ~ . Chemical modification of tryptophan 69 and tyrosine 7° residues have supported the idea that the complement-binding site is situated within the Cy2 domain. Certain synthetic peptides containing the critical tryptophanyl tyrosine are capable of complement activation 7°. These data suggest that the complement binding site is situated around the tryptophan residue in position 277. The degree of polymerisation of antibody required for the binding of C1q has been extensively studied. Monovalent haptens reacted with antibody do not usually activate complement, although it has been suggested that rabbit antibody to nonadecalysyl-C dinitrophenyl-lysine possesses this activity 71. The use of bifunctional haptens has shown that at least four immunoglobulin molecules must be linked together to give significant complement activation s5'72 Digestion of C1q by collagenase permits the isolation of the globular subunits which bind to antigen-antibody complexes 73. The presence of six globular subunits per C1q molecule suggests that one molecule of C1q can bind six molecules of immunoglobulin. Ultracentrifuge strudies have shown that this figure may be as high as 12 or 1874 . The avidity with which CI binds to antigen-antibody complexes is not related to the degree of complement activation. Extremely good binding may sometimes fail to cause activation, and poor binding frequently causes marked activation 75. w

Activation of complement is probably equally efficient whether the antigen is a soluble protein precipitated by antibody or is an antibody-coated erythrocyte 7~. Activation of the C1-complex and the formation of the classical pathway C3 convertase, C42, have been described in detail earlier (Fig. 6). The interaction of C-reactive protein with C-polysaccharide or choline phosphatides results in the consumption of complement 77'78 Activation may also be achieved by the interaction of polyanions with polycations, such as heparin and protamine 79. The significance of these findings is obscure, but perhaps such modes of activation could be important during infections or inflammation. Plasmin will activate precursor C1r only when it is present in the CI complex. As a result C~ is generated and complement consumption occurs 8° This is thought to be one of the major reasons for the complement activation which occurs in patients with C~-inhibitor deficiency.

Terminal Components (Table 2) C3 C3 is the bulk protein of the complement system, its serum concentration ranging from 768 ~g/ml to 1700 ~g/ml. It is a euglobulin, having a molecular weight of 190,000 daltons, consisting of two polypeptide chains of 110,000 daltons (~ chain) and 75,000 daltons (~ chain), respectively (Fig. 7) sl-s3 Both disulphide bridges and non-covalent forces hold the chains together. Generation of C3 converting enzymes during complement activation causes cleavage of the ~ chain of C3. The point of cleavage is an arginyl serine bond in position 77, and the fragment cleaved is termed C3a, which consists of the N-terminal 1-77 amino acids 83-8~ The larger fragment, C3b, consists of the residual ~ chain and the intact B chain. Under physiological conditions C3a remains bound to C3b by non-covalent forces, but the two fragments can be dissociated from each other under acid conditions 85, Following cleavage of C3 by C3 convertases an unstable binding

Fig. 6

V

@+ @@ @+

Suggested assembly of the early components of the classical pathway of complement on antibody bound to all surfaces. The heads of the C1q molecule bind to the CH2 domain of the antibody and the C1r2-Ca2-C1s complex to the collagenous tails of C1q through Cir. The activated CTs formed splits C4 and C2. Most C~ and C~ is inactivated in the serum but some C4 binds either to the Fab part of the antibody or to the cell surface. C2 binds to adjacent C4 molecules and forms C3 convertase, but is inactivated before reading more distant C~ molecules which are ineffective in the haemolytic sequence. (Reprinted by permission of the authors, Porter, R.R. and Reid, K.B.M., from Nature, 275, 699-704, 1978. Copyright © 1978. Macmillan Journals Ltd.)

ASSEMBLY OF EARLY COMPONENTS OF COMPLEMENT ON CELL BOUND ANTIBODY

@

@

i

CELL SURFACE

o

cD

m

>

Q_

E

o

Electrophoretic mobility

B

B

B $ y B

Molecular weight

190,000

185,000

128,000 121,000 153,000 79,000

Component

C3

C5

C6 C7 C8 C9

60 60 80 50

75

1300

Serum concentration (~g/ml)

TABLE 2

I I 3 I

2

2

+ + + -

+

Polypeptide Genetic chain polystructure morphism

C5a C5b

C3c C3d C3e

C3a C3b

Cleavage products

Terminal Sequence Proteins

)

0..~

_~.

o

> ) Part of membrane attack ) complex

)

(D

3

"O

3

o

..

3 3

o

3

o

Chemotactic anaphylotoxin Part of membrane attack complex

Anaphylotoxin I) Part of alternative pathway C3 and C5 convertases 2) Part of classical pathway C5 convertase 3) Opsonins 4) Solubilisation of complexes ? ? Mobilisation of PMN from bone marrow

Biological activity

o

K. Whaley and A. Ferguson

PPP

C3a ~

a

I

IIOK

I

I

Eli ....

I

TM

C3b

......

,

I

I

I

C3blNA

C3b|

02

~.

o'

C3d

C3e

l

D

.....

V-w

C3c

I!•

II ,

I ~

~

9K 60K

o3

Fig. 7

401( 2'7K 23K 10K

b

Diagrammatic model depicting enzymatic cleavage products of the C3 molecule occurring during complement activation.

site is generated, which is capable of binding with cell membranes. This binding site has a short half-life of approximately 50 milliseconds, after which the binding site decays, presumably due to molecular rearranegement into a more stable configuration. This decayed form is termed C3i, and contains both C3a and C3b, linked non-covalently 85. The rapidity of the molecular rearrangement of C3b results in the binding of only 10% of C3b molecules. The labile binding site of C3 is blocked by the actions of nitrogen and oxygen nucleophiles366'36?.Current data suggest the presence of an internal thioester bond in native C3, which is required for haemolytic activity 366 The site of membrane binding of C3b differs from that of C4b; using ferritinlabelled anti-C3 and anti-C4, it has been chown that C3 and C4 are distributed differently s6. C3 occupies sites on the cell membrane, from which C4 was completely absent. C4 molecules were approximately 2,000-30,000 A apart, where C3 molecules were much closer together, their average distance apart being 50-300 %. Assuming that cell-bound C4 is active in a C3 convertase, then C3b must bind to areas remote from the site of cleavage. C3b, the major cleavage product of C3, has a molecular weight of 171,000 daltons, and consists of the intact ~ chain of C3 and the residual ~ chain (101,000 daltons), from which C3a has been cleaved. Apart from its ability to bind to cell membranes by its labile binding site, C3b can bind to C3b receptors by means of a separate and stable binding site. C3b receptors are present on the membranes of polymorphonuclear leukocytes, mononuclear phagocytes, B lymphocytes 87-89, human erythrocytes 9°, and the epithelial cells of Bowman's capsule 91, Langerhan's cells 92 and hepatocytes 93 The C3b receptor from human erythrocytes has been isolated: it has a single polypeptide chain having a molecular weight of 205,000 daltons 94 There appear to be 900 C3b receptor sites/human erythrocytes, 60,000 sites/polymorphonuclear leukocyte, 61,500/monocyte and 36,300/B lymphocyte 9s. The role of the C3b receptor in opsonisation and phagocytosis will be discussed later. C3b molecules which bind close to the C3 convertase (of either the classical or alternative pathways) alters the specificity of the enzymes to that of a C5 convertase 96'97 the enzymatic site being on C2a for the classical pathway enzyme, and on Bb (see below) for the alternative pathway convertase. C3b acts to bind C5 and thereby approximate it to the enzymatic site for cleavage 96'9s C3b activity is modulated by the presence of two control proteins, C3b Jnactivator (C3blNA) and BIH globulin. Although absolute proof is lacking, it is thought that C3blNA is an enzyme whereas ~IH binds stoichiometrically to C3b 99-I°I When C3blNA and BIH are incubated separately with C3b no cleavage occurs, but when both are

The Biochemistry of Inflammation: Complement Activation

223

incubated simultaneously the ~ chain is cleaved (Fig. 7), leaving a molecule consisting of three polypeptide chains (two ~ chain fragments and the intact S chain, all held together by disulphide bonds. The ~ chain fragments have molecular weights of 60,000 (C3b ~-60) and 40,000 (C3b ~-40) daltons I°2'I°3. For the formation of C3c and C3d a further proteolytic reaction occurs. Serine proteases such as trypsin and plasmin produce cleavages of the ~ chain to release C3d, a 23,000 dalton fragment and C3e, a fragment of 10,000 daltons. C3c consists of the intact chain and two ~ chain fragments of 40,000 daltons and 27,000 daltons (Fig. 7). Treatment of C3b with C3bINA and $IH results in loss of factor B 99'I°3 and C5 I°3'I°~ binding ability, and inability to bind to C3b receptors I°3

C5 C5 is a similar protein to C3, being a daltons (Fig. 8) I°5, and consisting of bridges and non-covalent bonds I°5'I°6 115,000 daltons and the ~ chain 75,000 90% of its activity being destroyed by

D-globulin with a molecular weight of 190,000 two polypeptide chains linked by sulphide The ~ chain has a molecular weight of daltons. C5 is thermolabile, approximately incubation at 56°C for 10 minutes.

CSx

I

oi ,,,,

CSb

GI ~P l .a e.m. l.. .

i i ~

. . . . .

"

I

pl,ls ma trypsin

r ~C5d?

~

. . . . .

- - ~ - - --

' ~

C5c?

p

-

~ ~

~

P

i

I

'

I I

I

I

enzymes

orl • 21 • 2lI • 2111 • 3

~4[ ~ - - -

~'

,

~4II ~5

= 12K =25K = 15.7K = 10.7K = 14.7K : 9K = ILK = 30K

i I L

Fig. 8

.

.

.

.

.

.

.

.

.

Diagrammatic model depicting enzymatic cleavage products of C5 occurring during complement activation. Modified from Ref. 166.

Cleavage of C5 by C5 convertase or trypsin leads to the formation of C5a and C5b. CSa is a 12,000 molecular weight peptide derived from the chain of C5, and has anaphylotoxic and chemotactic properties. C5a has been purified and sequenced. It consists of the N-terminal 1-74 amino acids of the ~ chain of C51°7. The 74 amino acids account for a molecular weight of 8,200 and a single complex oligosaccharide unit attached to the asparagine residue at position 64 accounts for the remainder of the molecular weight. Numerous similarities between the primary structure of C5a and C3a point to their common genetic ancestry I°7. The anaphylotoxic activity is completely, and the chemotactic activity partly lost following removal of the C-terminal arginine residue by the plasma enzyme carboxypeptidase B (also known as anaphytoxin inactivator) i°s'l°9 C5a can also be released from C5 by a neutrophil neutral protease 11°-112, and other neutrophil enzymes degrade C5a. Trypsin, as might be anticipated from its actions on C3, will also cleave the ~ chain of C5 to form C5a and C5b I13. Indeed, trypsin will produce several secondary cleavages in the ~ chain of C5b, and some of these

224

K. Whaley and A. Ferguson

cleavage peptides appear to have biological activity il3 C5b consists of two polypeptide chains linked by disulphide bridges and noncovalent bonds: the ~ chain is 103,000 daltons and the B chain is 75,000 daltons. Following cleavage of the ~ chain of C5 by C5 convertases, trypsin or neutrophil neutral proteases, a labile binding site is exposed, and for a short period (approximately 50 milliseconds) C5b can bind to cell membranes. Loss of the labile binding site presumably follows a conformational change in the molecule to form a more stable fluid-phase structure. Less than 4% of cleaved C5 molecules become membrane bound, and approximately I in 7 of these molecules are haemolytically active I14. The functional activity of ce11-bound C5b decays, but this decay is not due to dissociation of the C5b molecule from the cell membrane ii~ Decay of C5b is prevented by the subsequent interaction of C6 and C7, hence the EACI-7 intermediate is stable and will lyse on exposure to C8 and C9. The labile binding site on C5b is stabilised by binding with C6 in the fluid phase. The C5b6 complex can travel considerable distances and bind to cell membranes in the presence of C7. The subsequent interaction of C8 and C9 may lead to cytolysis of cells most sensitised by antibody. This phenomenon has been termed reactive lysis iiS'ii6.

C6 a ~ d C? C6 and C7 are similar molecules, each being composed of single polypeptide chains, with molecular weights of 128,000 and 121,000 respectively liT. Both are B-globulins and contain some degree of ~-helial structure. C5, C6 and C7 enter into a reversible association in free solution ii8 C6 binds to C5b, and only when C5b6 has been formed will C7 bind to form C5b67, which is an equimolar trimolecular complex iig. During the assembly of this complex on cell membranes and perhaps in the fluid phase neoantigens are expressed, suggesting that conformational changes in the constituent molecules have occurred i2° The C5b67 was at one time thought to be chemotactic, but there is some doubt as to the validity of this assertion.

C8 C8 is a 7-globulin with a molecular weight of 150,000 daltons. It consists of three polypeptide chains, ~ 77,000, B 63,000 and y 14,000 daltons, respectively. The ~ and y chains are held together by disulphide bridges, whereas the B chain is linked only by non-covalent forces i2i. Labelling of the native mo]ecule showed that the tyrosine residues on the ~ chain could not be labelled in the native molecule, whereas following denaturation labelling was readily achieved 12i It is probable that the ~ chain is located in the interior of the native molecule, and has a hydrophobic character which contributes to the ability of C8 to initiate membrane damage. The binding site of C8 on the C5b67 complex involves all three molecules as antisera to C5, C6 or C7 will inhibit C8 uptake 122. Binding of C8 to C5b67 initiates the cytolytic reaction; lysis, however, is a slow process, and is speeded up by the incorporation of C9 into the complex i23

C9 C9 is the terminal component of the complement system. It is a B-globulin with a molecular weight of 79,000 daltons, consisting of a single polypeptide chain i24 C9 is resistant to mercapto-ethanol (10 -2 M/L), or p-chloromercuribenzoate (10 -~ M/L). It is sensitive to heat; 56°C for 5 minutes destroys 50% of its activity. Oxidation with potassium metaperiodate (10 -2 M/L) causes complete loss of activity,

The Biochemistry of Inflammation: Complement Activation

225

suggesting that the carbohydrate moiety is functionally important 124. C9 is consumed in the reaction with C 5 b - ~ 125. The dose response curves of the lyric reaction show that C9 induces lysis of EACI-8 by a single membrane lesion caused by a single effective molecule 126. Using radiolabelled C9 it is now known that six C9 molecules are incorporated into every lytic C5b-9 complex 125. The lytic activity of C9 on EACI-8 cells can be substituted for by the chelating agents O-phenanthroline of bipyridine 124'127 The lyric activity of phenanthroline on EACI-8 was dependent on the input of C8, was temperature-dependent and inhibited by bivalent iron. In these respects its actions are identical with C9, and it is therefore possible that C9 acts by the withdrawal or transfer of ferrous iron from a critical site on the cell membrane, perhaps from cell-bound C8.

The C5b-9 membrane attack complex C5, C6, C7, C8 and C9 in their native forms exhibit affinity for each other in free solution and undergo reversible protein-protein interactions. C5 associates with C6 and C7, C8 associates with C5, C6 and C7, and C9 associates with C5678 and with C8128. When all five components are ultracentrifuged together a 22.5S complex consisting of C56789 can be detected in addition to free components 128. Following cleavage of C5, by C5 convertase or trypsin, C6 binds more avidly to C5b, presumably because C5b has undergone a conformational change to permit adsorptive binding. C7 then binds to C~b-6 to form the equimolar trimolecular C5b67 complex. The three constituent proteins are distributed in a triangular fashion, one protein being at the corner of each triangle. The molecular weight of the C5b--7 complex is approximately 365,000 daltons. The triagnular arrangement of C5b67 is essential for C8 binding: C8 is thought to bind the central part of the triangle, its binding site involving all three proteins, as antibodies to C5, C6 and C7 will inhibit its binding 122. Uptake of C8 by C5b67 increases linearly with input until saturation occurs, at one molecule of C8 for each C5b67 complex 122 Thus C5b-8 is a tetramolecular complex, probably in a tetrhedral conformation. C8 and C9 tend to self-associate in free solution, sedimenting as a I0.2S complex 128. C9 cannot bind to C5b67 but once C8 has entered the complex binding rapidly occurs. The binding site for C9 is on the C8 molecule as antibody to C8, but not antibodies C5, C6 or C7, which will inhibit C9 uptake 122. Using radiolabelled C9 it has been shown that the C5b-9 complex has a molecular weight of 995,000 daltons 129. The only enzymatic step in the formation of the membrane attack complex is cleavage of C5 to form C5b. The remaining steps are all non-enzymatic reactions, possibly involving molecular rearrangements of the individual constituent molecules. The C5b-9 complex in a dimeric form is inserted into the lipid bilayer of the cell membrane, probably because it is hydrophobic 13° The ability to bind lipid increases as the complex assembles. None of the constituent molecules in isolation nor C5b6 will bind lipid, whereas 399 moles of phospholipid are bound per mole of C5b67, and 410 moles of phospholipid are bound per mole of C5b-8, and 1443 moles of phospholipid are bound per mole of C5b-9133 The C5b-9 complex is capable of binding to cell membranes for less than 0.1 seconds, following which time it loses its labile binding site. This could be due to molecular rearrangement of the molecule, although in serum two proteins, the S protein and VLDL, will bind to the labile binding site and prevent insertion of the complex into other cell membranes 132'133 The S protein was first identified as an unknown protein constituent of the C5b-9 complex isolated from serum following complement activation 129. S protein and VLDL are therefore examples of control proteins of the complement system.

226 The

K. Whaley and A. Ferguson Alternative Pathway

In troduction In 1954 it was reported that zymosan, a complex polysaccharide derived from baker's yeast, interacted with the late acting components (designated as C3, but including C5, C6, C7, C8 and C9) while sparing the early components (CI, C4, C2) 134 It was shown that zymosan interacted with a plasma protein, properdin (Latin pro perdere, to prepare to destroy), which bound to the zymosan particles and could be eluted using high ionic strength buffer 134 Properdin-depleted serum (RP), prepared by incubation with zymosan at 17°C, was unable to kill certain strains of gram-negative bacteria 13s, neutralize certain viruses 13~'136'137 or to lyse erythrocytes from patients with paroxysmal nocturnal haemoglobinuria 13~ Activity was restored by the addition of properdin eluted from the zymosan particles. Further work showed that a number of cofactors were required for the expression of properdin activity. These factors included a hydrazine-sensitive factor, termed factor A 13s, and a heatlabile factor, termed factor B 139 Thus the properdin system was thought to be an alternative pathway for the activation of the membrane attack sequence of complement sparing CI, C4 and C2. The interactions of the constituents of this pathway interacted with a wide variety of complex polysaccharides such as those present in zymosan, endotoxin, inulin or dextran, and formed a natural and non-specific defence mechanism in normal serum. The concept of the properdin system was disputed by Nelson 14°, who suggested that properdln was a natural IgM antibody, and that the hydrazine-sensitive and heatsensitive co-factors were C4 and C2, respectively. It was suggested that IgM antibodies fixed relatively small quantities of CI, C4 and C2, but much larger amounts of the terminal components. In the absence of present day protein fractionation techniques the controversy was insoluble and work on the properdin system virtually ceased. However, in 1968 properdin was isolated in pure form and was shown to be a gamma globulin, distinct from all known immunoglobulin classes 141 Other workers showed that endotoxin added to guinea pig serum activated the terminal sequence, yet sparing CI, C4 and C2142'I~3 Antibody to C2 which prevented the lysis of antibody coated erythrocytes in guinea pig serum was unable to block the consumption of the terminal components by lipopolysaccharide 144. C4 deficient guinea pig serum was shown to sustain lipopolysaccharide-induced consumption of the terminal components 145, and C2-deficient human serum shown to contain a heatlabile factor necessary for opsonisation 146. In addition to complex polysaccharides, it was shown that certain immunoglobulins or immunoglobulin fragments could consume the terminal components yet sparing CI, C4 and C2. For instance Schur and Becker 147 showed that antigen-aggregated rabbit F(ab)~ fragments had this property, whereas the Fc fragment was required for activation of CI. Guinea pig Yl antibodies and the human myeloma proteins IgG~, IgAl and IgA2 cannot activate CI, but Sandberg and her colleagues 148-152 showed that they consumed the terminal components and that the F(ab)~ fragment was required for this process. The final confirmation that an alternative pathway for complement activation was present stemmed from observations made at the turn of the century, in which it was shown that snake venoms administered intravenously resulted in destruction of erythrocytes and leukocytes, loss of serum bactericidal properties and death 153'Is4 In 1965 Klein and Wellensiek 155 showed that cobra venom consumed C3 and C5, and shortly afterwards a number of workers 156'158 isolated the active component. This component is a 7S B2-glycoprotein of 140,000 daltons molecular weight and has been termed cobra venom factor. Analysis of the reaction of cobra venom factor with serum revealed that a heat labile factor was required to generate C3 cleaving activity. This protein was

The Biochemistry of Inflammation: Complement Activation

227

called C3 proactivator Is9 and was later shown to be identical with Pillemer's factor B and glycine-rich B-glycoprotein 16°. A second factor required for the formation of cobra venom factor C3 convertase was later identified as the serine protease factor ~161,162. Only when cobra venom factor, factor B and factor D were mixed together was a stable C3 convertase activity generated 163. During this process factor B was cleaved into two fragments of ~ and X electrophoretic mobility 16°.

Proteins of the alternative pathway (Table 3, Fig. 9) Surface Discrimination

Heparin

c~

Sialic Acid

i

C 3bBbP

~:

C3bBb P

pL~smin

C 3 a/

/B c36B~++ ~C3b

/

C3b

C3b~A

Fig. 9

Molecular interactions of the alternative pathway. The ability of surface constituents to dictate turnover is represented. Reducing surface sialic acid restricts BIH activity and therefore turnover is enhanced. Increasing surface heparin restores BIH activity and turnover is restricted.

C3b

The major cleavage product of C3 is an integral part of the C3 and C5 convertases generated during alternative pathway activation.

Factor B

This protein is identical with glycine-rich B-glycoprotein 16° and C3 proactivator 159. It is a pseudoglobulin with a molecular weight of 93,000 daltons, a sedimentation coefficient of 6.2 Svedberg units and migrates as a B-globulin on im~nunoelectrophoresis 164'165 It consists of a single polypeptide chain containing 10.6% carbohydrate by weight (5.4% hexose, 4.2% acetyl hexosamine, 0.9% acetyl neuraminic acid and 0.1% fucose) 166 Factor B is inactivated by heating at 52°C for 20 minutes and by treatment with 1.0 M potassium thiocyanate. During activation of the alternative pathway in whole serum, or during the incubation of purified factor B with C3b and D or trypsin (5% w/v), factor B is cleaved into two peptides, one migrating as an a globulin (Ba), the other as a X globulin (Bb). The molecular weights of these peptides are 30,000 daltons and 63,000 daltons, respectively 164'167 Isolated Bb retains some C3 cleaving activity and therefore carries the active site of the alternative pathway C3 convertase 159

y

~

25,000

22,000

B

93,000

C3b

P

B

181,000

Component

Electrophoretic mobility

Molecular weight

30

2

150

?

4

I

I

2

?

?

+

See C3

Genetic polymorphism

-

-

Ba Bb

C3bi C3c C3d C3e

Cleavage products

Pathway Proteins

Polypeptide chain structure

Alternative

Serum concentration (pg/ml)

TABLE 3

activity

Stabilises C3--3-b-B-~

Cleaves B

? Chemotactic Part of C3 and C5 convertases Activates macrophages

? ligand for CR3 receptor ? ? Mobilises PMN from bone m a r r o w

Biological

E o

m

>

O_

E -<

00

The Biochemistry of Inflammation: Complement Activation

229

Bb activity is resistant to treatment with 10 -3 M DFP 168, showing that it is not a serine esterase. Bb will hydrolyse N-a-acetyl glycyl L-lysine methyl ester I~9. Bb activity decays from the alternative pathway C3 and C5 convertases, and can be regenerated by the addition of fresh B in the presence of ~170, It is not known whether Bb or Ba binds to C3b, but by analogy with the classical pathway C3 convertase, it is possible that the enzyme of the alternative pathway is C3bBaBb, from which Bb, which carries the enzymatic site, decays.

Factor Factor D is a single polypeptide chain protein with a molecular weight of 24,000 daltons 162'163 and a sedimentation coefficient of 3.0 Svedberg units. It migrates as an a globulin in fresh serum, but as a B globulin following its purification 171 This change in electrophoretic mobility is thought to be due to a serum cofactor, a pseudoglobulin of 50,000-80,000 daltons molecular weight which is present in factor B-depleted serum 171. The biological significance of this finding is difficult to envisage. D is a serine esterase, its activity being inhibited by DFP (10 -3 M/L) 16s There is some controversy as to whether it exists in plasma as a zymogen, as an early report 168 has not been confirmed by a subsequent study 172. If a zymogen form of factor D does exist, its mode of activation is unknown. Factor D catalyses the formation of the C3 and C5 convertases of the alternative pathway. This action is mediated by its ability to enzymatically cleave factor B into the fragments Ba and Bb, once it has bound to C3b 172. It cannot cleave factor B in isolation. As trypsin can substitute for factor D activity 173, and form Ba and Bb even in the absence of C3b, the requirement for B binding to C3b cannot be to expose the enzymatically susceptible peptide bond. It is therefore probable that during binding to C3b, factor B is induced to fit into the substrate binding site of factor D. This binding site is considered to be cryptic as factor D does not react with known plasma enzyme inhibitors, nor with factor B, its natural substrate, unless C3b acts as a cofactor 172 Although trypsin will substitute for factor D activity, no naturally occurring plasma enzymes possess this activity. Thus the requirement of factor D for the activation of the alternative pathway is absolute. Factor D has no esterolytic activity towards a number of synthetic esters and no proteolytic activity towards the B chain of insulin 172 The extended NH2-terminal amino acids of factor D shows strong homologies with other serine proteases, especially trypsin_and kallikrein. There is no increased homology with the first 20 residues of C1r and C~s 174.

Properdin Properdin is a highly asymmetric non-immunoglobulin X globulin which has a molecular weight of 186,000 daltons, and a sedimentation coefficient of only 5.2 Svedberg units. It consists of four identical subunits of 46,000 daltons which are linked by non-covalent forces 175. Properdin contains 9.8% carbohydrate by weight, consisting of hexose, hexosamine, fucose and sialic acid 166. Properdin is cleaved by trypsin into two fragments, the larger, PL (150,000 daltons) and the smaller PS (68,000 daltons). The molecular weight of these fragments corresponds to their sedimentation coefficients, which suggests that the asymmetry of the intact properdin molecule occurs as a result of a folding which yields two stable positions linked by a region susceptible to proteolysis 17~. It is also possible that properdin is amorphous and following proteolysis the fragments adopt a stable globular conformation 176. SDS polyacrylamide gel electrophoresis shows that PL and PS each consist of four identical subunits with molecular weights of 68,000 and 13,000

230

K. Whaley and A. Ferguson

respectively, suggesting that the subunits of properdin were cleaved in identical regions 17~ Properdin binds to C3b and stabilises the alternative pathway C3 and C5 convertases by retarding the decay of Bb in a dose-dependent fashion 178. Properdin exists as a precursor in which form it can be isolated from plasma. Precursor properdin lacks the ability to induce C3 turnover in properdin-depleted serum unless an activating agent such as zymosan is present, whereas activated properdin causes C3 turnover when added to properdin-depleted serum 178. The mechanism whereby precursor properdin is converted to its activated form has been the subject of some controversy. Initially it was suggested that properdin underwent a proteolytic cleavage reaction during which a fragment (or fragments) of 20,000 daltons were removed and resulted in a conversion of the electrophoretic mobility from B to y179 More recent investigations showed that precursor properdin could be converted to activated properdin by binding to EC3b, and by freeze thawing, and the reverse process can be promoted by the incubation of activated properdin in high concentrations (0.8 M) guanidine hydrochloride 17s These findings suggest that the change from native to the inactivated form of properdin is purely conformational. This conclusion has been supported by the findings that the molecular weight and electrophoretic mobilities of native and activated properdin were identical, as were the NH2 and COOH terminal amino acid analysis 17s. The circular dichroism spectra of native and activated properdin differ significantly. The spectra are typical of proteins which show no a-helix or B-structure and are thought to be present in a random coil formation 17s

Cobra venom factor (CoVF) CoVF is a BI glycoprotein with a molecular weight of 150,000. It can be isolated from the venom of the Asiatic hooded cobra NajQ naja, or the Egyptian cobra Naja naje. Upon addition to normal serum it causes intense activation of the alternative pathway 18° The protein complexes with factor B and in the presence of forms a stable C3 and C5 convertase 181. CoVF is cobra C3b which is resistant to proteolysis by human C3b inactivator Is2 and CoVF-dependent convertases are also resistant to the destabilising action of BIH 183

C3 nephritic factor (C3NeF) Nephritic factor occurs in the sera of some patients who have mesangiocapillary glomerulonephritis. These patients tend to be persistently hypocomplementaemic, with low serum levels of C3, but usually with normal levels of CI, C4 and C2 Is~ The sera of these patients contain IgG which when added to normal serum in the presence of Mg-EGTA will cause activation of the alternative pathway with C3 consumption185o Nephritic factor has been shown to be an antibody to the unstable C3-convertase (C3bBb) of the alternative pathway 186-188 Usually both C3b and Bb contribute to the antigenic determinant, and thus nephritic factor physically binds to C3b and Bb, thereby retarding the natural decay of the enzyme 189 The nephritic factor-stabilised convertase is resistant to the decay accelerating activity of the control protein BIH 19° Nephritic factor is thus an immunoconglutinin (i.e. an antibody to an activated complement component).

A s s e m b l y of the a l t e r n a t i v e

p a t h w a y C3 a n d C5 c o n v e r t a s e s

The reaction sequence involved in the generation of the alternative pathway C3 and C5 convertases depends primarily upon the interaction of C3b with factor B. This interaction requires the presence of magnesium ions. The reaction produced by C3bB is thought to have slight C3 cleaving activity 191, although the possibility that trace contamination of factor B with factor D has not been excluded satisfactorily. Factor D then cleaves factor B to form the C3 cleaving enzyme C~SB-~,

The Biochemistry of I n f l a m m a t i o n : C o m p l e m e n t A c t i v a t i o n

231

which i s termed the u n s t a b l e a l t e r n a t i v e pathway C3 e o n v e r t a s e as Bb r a p i d l y decays from t h e complex 191, the r a t e of decay b e i n g t i m e and t e m p e r a t u r e d e p e n d e n t . P r o p e r d i n b i n d s t o C3b, and as a r e s u l t o f t h i s b i n d i n g r e t a r d s the decay o f Bb 177" Thus C3bBbP i s termed t h e p r o p e r d i n - s t a b i l i s e d a l t e r n a t i v e pathway C3 c o n v e r t a s e . The degree o f s t a b i l i s a t i o n of C3bBb i s dependent upon the i n p u t of p r o p e r d i n : optimal stabilisation b e i n g a c h i e v e d w i t h a p r o p e r d i n c o n c e n t r a t i o n of 250 n g / m l . This c o n c e n t r a t i o n p r o l o n g s the h a l f - l i f e of the c o n v e r t a s e from 2-4 m i n u t e s a t 30°C t o o v e r 30 m i n u t e s 177. P r o p e r d i n n o t o n l y b i n d s t o C3b, b u t can t r a n s f e r from one C3b molecule to another 177 Thus maximum stabilisation can be achieved with fewer molecules of properdin than there are molecules of C3bBb. In the classical pathway C3b is essential for the formation of the C5 convertase; presumably it acts as a Inhibition of C5 and B acUvi~v by complement componems 1oo

1oo

B

•

C3b~A

75

25

0 [llpUl r~l II)I)II)I)TI(,III (J]= ,nil}

Fig.

10

(a)

I n h i b i t i o n of c l a s s i c a l pathway C5 e o n v e r t a s e a c t i v i t y by C3blNA ( e - e ) , B ( o - o ) , p r o p e r d i n ( ~ - a ) and ~IH (A - A ) . EAC14°xy23b were p r e p a r e d w i t h 1.5 h a e m o l y t i e C3b s i t e s in the presence of 50 effective molecules of purified C5 (5 ~g/ml). 100 ~I EACI4°xy23b (I x 10S/ml DGVB ++) were incubated with 100 ~I C3blNA, B, P or BIH at 37°C for 15'. C5, C6, C7, C8 and C9 (50 em/ml) were then added, followed by a further incubation of 60' at 37°C. The ordinate shows % inhibition of lysis. The results show that B. P and BIH inhibit C5 cleavage as a result of their binding to C3b.

(b)

Inhibition of formation of alternative pathway C3 convertase by C3blNA (e-e), BIH ( A - A ) , and C5 (o-o). 100 ~i EAC43b (I x 10S/ml DGVB ++) bearing 1.8 haemolytic C3b sites when exposed to excess (5 ~g/ml) B and D (10 units/ml) were incubated with 100 ~I DGVB ++ containing C3blNA, BIH or C5 at 37°C for 15' 100 ~i B and D were added, followed by a further 30' incubation at 30 ° to allow f o r ~ t i o n of EAC43bBb. Addition of C3-C9 using rat serum diluted 1/15 in 0.4 M EDTA/GVB resulted in lysis. The percentage of inhibition of lysis is shown on the ordinate. The results show that by binding to C3b, BIH and C5 inhibit B binding. Properdin enhances lysis by retarding the decay of Bb.

232

K. Whaley and A. Ferguson

template for C5 ensuring the correct presentation for cleavage by C2a. C3b plays a similar role in the alternative pathway. C3b therefore plays two distinct roles: the first is to serve as a cofactor for factor B prior to cleavage of factor B by factor D, i.e. in the formation of the C3 convertase, and, secondly, it plays a separate role in the formation of the C5 convertase 97. Although factor B and C5 bind to C3b, binding of factor B inhibits C5 binding and conversely C5 binding inhibits factor B binding (Fig. 10). Thus there is steric inhibition between these two molecules, and for the formation of a C5 convertase an extra C3b molecule is required. Following C5 cleavage the assembly of the membrane attack complex is identical to that of the classical pathway.

Analogies between the classical and alternative pathways Both C~ and factor D are serine esterase enzymes inhibited by DFP. Both are destroyed by heating to 56°C for 20 minutes. The differences between them are that CT is a trimolecular complex with a molecular weight of approximately 700,000 daltons, whereas D, a_single protein, is the smallest known complement component of 24,000 daltons. CI contains a recognition molecule C1q which is the trigger for classical pathway activation. Recognition and activation of the alternative pathway depends upon the interaction of C3b with various surface structures (see below). C4b in the classical pathway and C3b in the alternative pathway bind to C2 and factor B, respectively, to form C3 convertases. Both C4b and C3b are ammonia and hydrazine-sensitive, but heat stable factors. Both C4b and C3b are enzymatically inactivated by C3b inactivator. C2 and factor B have many properties in common. They are pseudoglobulins with electrophoretic mobility, consisting of single polypeptide chains of similar molecular weights. Both are cleaved by serine esterases into large and small fragments, their active sites being on the larger fragments (C2a and Bb respectively) which is unusual for complement proteins. C42 and C3bBb are both subject to decay and can be reconstituted by the addition of fresh C2 or factor B respectively. Finally in man, the genes controlling the electrophoretic polymorphism of C2 and factor B are located on the short arm of the sixth chromosome, in the region of the major histocompatibility complex 192 Indeed it has been suggested that factor B and C2 represent the products of gene reduplication 192

Activation of the alternative pathway The question of how alternative pathway activation is initiated has remained a central issue over the past decade. Searches for a factor, or factors, which act as recognition units led to the idea that initating factor, a normal plasma protein, was responsible for this process 193. A number of observations suggest that an initiating factor is not required for this process. The most important observation is that C3b, the major cleavage product of C3, is a constituent of the alternative pathway C3 and C5 convertases. Thus C3b, formed by classical or alternative pathway activation or by the action of enzymes such as plasmin, can trigger the alternative pathway in a positive feedback fashion. Therefore C3b, once formed, should perpetually stimulate the alternative pathway until C3 and/or factor B are completely consumed. This does not occur because in plasma there exist two proteins, C3b inactivator (C3bINA)~and BIH globulin, which act in concert to limit alternative pathway activation. If these two control proteins can effectively control altern*C3bINA and BIH are now called factors I and H respectively.

The Biochemistry of Inflammation: Complement Activation

233

ative pathway activation two questions are raised: I)

Why does recruitment of the positive feedback loop occur in the presence of control proteins?

2)

How do substances which initiate alternative pathway activation circumvent these controls?

In order to answer these two questions the actions of the control proteins must first be discussed.

MODULATION OF COMPLEMENT ACTIVATION The requirement for mechanisms to control complement activation is obvious when one considers the variety of biologically active products generated by the system (see below). The high catabolic rates for the complement proteins suggests that the system is undergoing continuous activation which serves to re-emphasise the need for adequate control. During the early stages of complement activation cleavage of C4, C3 and C5 exposes their labile binding sites, which for less than 100 milliseconds can bind to cell membranes or other structures. Following this period, conformational changes occur which ensure that their binding activities are lost 194. As a consequential extreme lability of these binding sites, the functional efficiency of C4, C3 and C5 is low, so cleavage of large numbers of molecules is required to ensure continuation of the system. The natural decay of the C3 and C5 convertase offers a further level of control. C2a decays from C4b2a and C4b--~a3b19S, Bb decays from C3bBb and C3b--~ 177, and C5b activity decays on cell membranes. Regeneration of these enzymes occurs if the native molecules are reintroduced and the components required for their activation are present. A third level of control is provided by the presence of certain plasma proteins which modulate the activities of certain complement components (Table 4).

C~-inhibitor C~-inhibitor is an a2 neuraminoglycoprotein consisting of a single polypeptide chain having a molecular weight of 105,000 daltons and a sedimentation coefficient of 4 Svedberg units. Purified preparations of C~-inhibitor show a minor band with a molecular weight of 96,000 daltons 28. This band has the same immunological and functional properties of the major band, but its significance has not been explained It contains 35% carbohydrate by weight, of which 12% is hexose and 17% N-acetyl neuraminic acid 176 The molecule is destroyed by treatment with heat (56°C for 30 minutes), acid i
C3b inactivator

Anaphylotoxin inactivator

300,000

88,000

90,000

C4 binding protein

S protein

540,000 590,080

CIinhibitor

150,000

105,000 90,000

Component

BIH

Molecular weight

~

a

$

S

B

~

Electrophoretic mobility

?

?

300

50

?

180

Serum concentration (Mg/ml)

8

I

I

2

8

I

?

?

?

?

+

Genetic polymorphism

-

-

Cleavage products

Control Proteins

Polypeptide chain structure

TABLE 4

activities

insertion Removes C terminal arginine from c3a and C5a

Binds to C5b-9 to prevent into membrane

I) Binds to C3b to accelerate delay of C3bBb and C3bBbp 2) With C3b inactivator responsible for cleavage of C3b to C3bc

Degrades C4b and C3b, in concert with C4 binding protein and BIH

2) With C3b inactivator responsible for degrading C4b to C4c and C4d

c4-~

I) Binds to C4b to accelerate decay of

Inhibits certain serine proteases including CTr and CTs

Biological

>

m

:T

~O

The Biochemistry of Inflammation: Complement Activation

235

and one C~-inhibitor molecule was associated with one CTs molecule. The molecular weight of the CTr, C~s, C~-inhibitor complex was 382,000 daltons, indicating that it is composed of one C~r, one C~s and two C~-inhibitor molecules. Therefore, from each C~ complex, C~-inhibitor must release two CTr, C~s CT-inhibitor complexes. Deficiency of C~-inhibitor results in the clinical picture of hereditary angiooedema T M in which the uncontrolled activity of C~ increases C4, C2202 and C3 turnover 203'204 with the generation of C2-kinin, a vasoactive peptide released from C2 by the action of plasmin ~°5

C4-binding protein C4-binding protein is a glycoprotein consisting of a number, probably eight, of identical subunits of 70,000 daltons molecular weight linked by disulphide bridges. On SDS polyacrylamide gel electrophoresis run under non-reducing conditions, purified C4 binding protein migrates as two distinct bands with molecular weights of 540,000 and 590,000 daltons. Both high molecular weight and low molecular weight C4 binding protein are immunologically and functionally identical 2 ~ . The protein has a sedimentation coefficient of 10.7 Svedberg units, and migrates as a 8globulin on immunoelectrophoresis in the absence of divalent cations. In the presence of calcium C4 binding protein migrates as a gamma globulin. C4 binding protein binds to C4b, in a cation-independent reaction, which is not inhibited by DFP. Complexes of C4b and C4 binding protein have sedimentation coefficients between 15 and 17 Svedberg units. One molecule of C4 binding protein appears to be saturated by 4 or 5 molecules of C4b 2°6. This finding, if confirmed, suggests that the subunits of C4 binding protein, although having the same molecular weight, perhaps are functionally and structurally different. Although the reaction between C4b and C4 binding protein is cation-independent, stable complexes between theqe molecules do not occur in EDTA-plasma 2°6 C4 binding protein acts as an essential cofactor for C3b inactivator in the enzymatic degradation of C4b, as has been described in the section dealing with C4 ~4'2°? Shirishai and Stroud 208 isolated an I0S globulin which acted as a cofactor for C3b inactivator in the proteolysis of C4b: it is now apparent that the I0S globulin and C4 binding protein are the same protein. Following the formation of the classical pathway C3 convertase, C4b2a, C4 binding protein will bind with C4b and accelerate the decay of the enzyme, probably by displacing C2a from the complex 2°9 No data are yet available as to the effects of C4 binding protein on the rate of decay of the classical pathway c5 convertase.

C3b inactivator C3b inactivator is a glycoprotein with a molecular weight of 93,000 daltons, consisting of two polypeptide chains linked by disulphide bridges and non-covalent forces. The two polypeptide chains have molecular weights of 55,000 and 42,000 daltons. C3b inactivator is highly heterogeneous in terms of its surface change, as seen by alkaline polyacrylamide gel electrophoresis and isoelectric focussing 21° The carbohydrate content of C3b inactivator is 10.7% by weight (excluding sialic acid) consisting of 7.5% hexose and 3.2% glucosamine. Most of the carbohydrate is on the smaller polypeptide chain 211. C3b inactivator was originally called conglutinogen activating factor (KAF) as it interacted with hemolytic intermediates coated with C3b, rendering them agglutinable by a ruminant plasma protein, conglutinin 212.

236

K. Whaley and A. Ferguson

Although it is widely assumed that C3b inactivator is an enzyme, and BIH is its cofactor, there is no definite evidence that exposure of fluid-phase C3b to C3b inactivator in the absence of BIH results in its proteolysis. However, purified C3b inactivator will interact with cell bound C3b in the absence of ~IH to block the participation of C3b in the classical pathway C5 convertase 99, and the alternative pathway C3 and C5 convertases 97'99'213 Immune adherence, the binding of C3b-coated particles with C3b receptors, is also inhibited 99. As C3b inactivator does not bind tightly to C3b it is possible that C3b inactivator produced a limited cleavage cell-bound C3b. Complete degradation of fluid-phase C3b to C3 and C3d requires C3b inactivator, BIH globulin (see below) and another protease such as trypsin or plasmin 211 Hereditary deficiency of C3b inactivator 214'els or it immunochemical removal from serum 216 results in uncontrolled alternative pathway turnover. This occurs because C3b formed during normal low-grade turnover of the complement system is not degraded, and so interacts with factors B and D, and properdin to form C3bBbP, which amplifies C3 cleavage.

BIH globulin BIH globulin is a glycoprotein consisting of a single polypeptide chain having a molecular weight of 150,000 daltons, on SDS polyacrylamide gel electrophoresis and by equilibrium sedimentation in the analytical ultracentrifuge 99. Its elution pattern on gel filtration chromatography corresponds to a molecular weight of 300,000 daltons, suggesting that the molecule is asymmetric. This asymmetry must be rigid rather than subject to random coil formation as the sedimentation coefficients determined by analytical ultracentrifugation were the same at two different concentrations of BIH. The sedimentation coefficients determined in the analytical ultracentrifuge and in sucrose density gradients were 5.6 and 6.4 respectively. The carbohydrate content of $IH is approximately 10% and includes hexose 4.3% by weight and sialic acid 4.3% by weight 217. Digestion of BIH with neuraminidase, mannosidase or fucosidase or ~-galactosidase does not reduce its functional activity (Fig. 11) 217. Indeed the activity of sialic acid-depleted ~IH is enhanced by approximately 40% 368 . Digestion of BIH with trypsin (1% w/w) results in a rapid cleavage (within minutes) of the molecule, giving a major peptide which migrates more anodally (Fig. 12). After 10 minutes a further cleavage reaction is obvious, and the two products become even more anodal. ~IH binds to C3b stoichiometrically 99-I°I, and as a result plays an important role in the control of the alternative pathway at three levels. Following its binding to C3b, it inhibits the C5 convertase activity of EAC4-~-b I°4, presumably because it sterically hinders C5 binding to C3b. However, our recent studies 217 have shown that factor B and properdin also inhibit the C5 convertase activity of EAC423b. Indeed, the inhibition produced by properdin and factor B exceeds that of BIH (Fig. 10). Once ~IH has bound to C3b, factor B cannot bind to C3b and therefore the formation of the alternative pathway C3 and C5 convertases (C3bBb and C3bBbP) is inhibited 99. The activity is directed to inhibiting factor B binding as BIH does not block properdin uptake as shown by failure of BIH (and also C5 and B) to block properdin-mediated agglutination of EAC43b 217. BIH has been shown to inhibit the uptake of radiolabelled factor B by membrane-bound C3b I°°'t°I C5 also inhibits factor B uptake by EAC43b, although it is only about half as effective as BIH (Fig. 10). Thus the role of ~IH in modulating C3b activity is directed to the alternative pathway rather than the classical pathway. Following the formation of C3bBb or C3bBbP, C3b inactivator can no longer enzymatically attack C3b; the susceptible peptide bound on C3b is concealed by Bb. BIH can still bind to C3b and in so doing, displaced Bb from the complex and thereby renders C3b susceptible to the action of C3b inactivator 99'19°. Once ~IH has bound to C3b the rate and extent of the inactivation of C3b by C3b inactivator is potentiated 99. BIH does not

The Biochemistry of Inflammation: Complement Activation

Fig.

11

237

Effect of digestion of ~IH by carbohydrates on its electrophoretic mobility. Top well NHS, top trough anti-NHS. Anode to the right. Well 2 -- native ~IH; wells 3-6 -- BIH digested with fucosidase (3), mannosidase (4), neuraminidase (5) and $-galactosidase (6). Troughs contain anti BIH. The electrophoretic mobility of BIH is only altered by neuraminidase digestion. The removal of negatively charged sialic acid residues renders BIH more cathodal in its migration.

potentiate the inactivation of C4b by C3b inactivator 99. The extent of BIH-mediated potentiation of the inactivation of C3b by C3b inactivator is limited by the amount of C3b inactivator available. A plateau of C3b inactivation is reached, the level of which depends upon C3b inactivator input (Fig. 13). Indeed, we now measure C3b inactivator functional activity in serum by adding excess BIH (400 ng/ml) to serum dilutions, and examine their ability to limit the formation of EAC34b--~ 21s. In a previous study, before the role of ~IH was recognised, we found that when C3b inactivator functional activity in serum was plotted against C3b inactivator protein concentration, a good correlation was achieved only when C3b inactivator activity was expressed logarithmically 213 When excess BIH is added to the assay, there is a direct linear correlation. ~IH is also required for the complete proteolytic degradation of C3b by C3b inactivator. The final enzymatic step is mediated by enzymes such as plasmin 211

238

K. Whaley and A. Ferguson

Fig.

12

Immunoelectrophoresis of BIH before (well 2) and after (wells 4-7) digestion with trypsin (1% w/w) at 37°C. Well 3 -- $IH incubated with trypsin + soybean trypsin inhibitor. Wells 4-7 represent the results of digestions lasting, I, 3, 5 and 10 minutes. A single cleavage reaction occurs within one minute, resulting in the formation of a single arc which is more anodal than native ~IH (wells 4-6). After 10 minutes digestion a further cleavage reaction results in the formation of two arcs, one similar in position to the arc in wells 4-6, and the second migrating even more anodally. Top well NHS, top trough anti-NHS. Remaining troughs anti BIH. Anode is to the right.

Hereditary deficiency of $IH has not yet been reported, but the immunochemical depletion of ~IH from serum results in the uncontrolled turnover of the alternative pathway which can be abrogated by the addition of pure BIH but not C3b inactivator 218.

protein the S protein has a molecular weight of 88,000 daltons based on SDS polyacrylamide gel electrophoresis. It is a glycoprotein as the band stains with periodic acid Schiff stains 133. This protein has been identified as an extra protein band in

The Biochemistry of Inflammation: Complement Activation

239

HH Potentiation of C3b Inactivation by C3blNA

3.0

. . . . - - @ C3bINA + ~IH 2.0

Z' 1.0

-x P o t e n t i a t i o n Effect

//" ×

, , ~

62.5 125

250

500

$1H

, 10000

Input of ~IH (ng / m l )

Fig.

13

BIH-mediated potentiation of the inactivation of C3b by C3b inactivator (C3blNA). EAC43b cells bearing limited C3b were incubated (37°C for 30 minutes) in the absence of C3blNA or in the presence of sufficient C3blNA to produce 0.2Z' units of inhibition (Z' = -In [% lysis in test sample / % lysis in control]). BIH (concentrations shown on abscissa) was added to all but the control tubes. Following washing the cells were exposed to excess B and D to allow formation of a C3 convertase, and then for lysis to occur C3-C9 (EDTA-treated rat serum) was added. BIH alone (o-o) has a slight inhibitory activity, whereas in the presence of C3blNA ( e - e ) a marked potentiation of C3b inactivation occurred. When the effects of BIH alone and C3blNA alone were subtracted from the effect of both proteins together, then the degree of potentiation is observed (x-x). This shows a plateau effect of B|H concentrations over 250 ng/ml. Thus ~IH potentiation is limited by the available C3blNA.

SDS polyacrylamide gel electrophoresis of the C5b-9 complex purified from serum in which complement activation has occurred 129. It cannot bind to C5b or C5b6, but can bind when C7 is incorporated into the complex 133 The C5b-9 complex is capable of being integrated into cell membranes, resulting in lysis. There is probably continuous low grade formation of C5b and therefore the C5b-9 complex would be spontaneously formed. The S protein competes for the membrane binding site of the C5b6-7 complex and blocks its cytolytic potential 133 The S protein prevents dimerization of the C5b-9 complex, the dimer being the membranolytic form of the complex 13° Very low density lipoproteins (VLDL) act in an identical fashion to the S protein to inhibit the insertion of the C5b6-7 complex into cell membranes 132.

Anaphylotoxin inactivator Anaphylotoxin inactivator, also known as carboxypeptidase B, is a plasma protein with a molecular weight of 310,000 daltons, consisting of 8 subunits each of 36,000 daltons 219. The sedimentation coefficient of the protein is 9.5 Svedberg

240

K. Whaley and A. Ferguson

units, and it migrates as an ~ globulin on immunoelectrophoresis 219. The action of the protein is to remove the COOH terminal arginine residue from C3a and from C5a, thereby removing their anaphylotoxic activities. The chemotactic activity of C5a is, however, not destroyed by this treatment, although C5a desarg requires a serum cofactor for its chemotactic activity 22° Anaphylotoxin inactivator hydrolyses hippuryl-L-arginine and hippuryl-L-lysine and its actlons are inhibited by EDTA, EACA, phenanthrollne • • • 219 . • " and arglnlne

I n i t i a t i o n of the a l t e r n a t i v e p a t h w a y a c t i v a t i o n Our c u r r e n t ideas on how i n i t i a t i o n of the a l t e r n a t i v e pathway a c t i v a t i o n occurs are derived from two basic o b s e r v a t i o n s : ].

P u r i f i e d C3, f a c t o r s B and D and properdin in f r e e s o l u t i o n form a low e f f i c i e n c y C3 cleaving enzyme. WhenC3h i s formed as a r e s u l t of C3 cleavage by t h i s enzyme, C3BbP, a higher e f f i c i e n c y enzyme C3bBbP i s formed221

2.

Hereditary d e f i c i e n c y of C3b i n a c t i v a t o r214'214, or i t s immunochemical removal from serum216, r e s u l t s in uncontrolled turnover of the a l t e r n a t i v e pathway. These o b s e r v a t i o n s show t h a t C3b i s being formed continuously and C3b i n a c t i vator i s required to control i t s a c t i v i t y , and so l i m i t the a l t e r n a t i v e p a t h way "tickover".

Thus, in the absence of control proteins, unrestricted formation of C3bBbP occurs. It is therefore obvious that in order to initiate alternative pathway activation in the presence of control proteins, circumvention of these regulatory activities must occur. When cobra venom factor is introduced into serum, alternative pathway activation occurs. Cobra venom factor is cobra C3b which is resistant to human C3b inactivator 182, and the convertase it forms with factors B and D is resistant to BIH decay-dissocation Is3 Nephritic factor, an antibody to C3bBb 18~-188, holds the two proteins together because they both contribute to the antigenic determinant. Therefore, nephritic factor stabilises the C3 convertase and in this form it resists $1H-mediated decaydissociation 19° When complex polysaccharides of the surface of yeasts such as zymosan, or gramnegative bacteria are introduced into serum there is marked alternative pathway activation. This phenomenon has been explained by Fearon and Austen 222'223, who have shown that during the normal low-grade fluid phase turnover of C3, some C3b binds to the surface of these particles, and in some way their surfaces offer a microenvironment which is protected from the activities of the control proteins. The observation that these particles were relatively deficient in sialic acid, whereas sheep erythrocyte membranes were relatively rich in sialic acid 224 offered an interesting approach to this problem. Sheep erythrocytes are not activators of the alternative pathway, but following neuraminidase treatment they acquired this ability 22S'226 Furthermore, inbred strains of mice have erythrocytes of varying sialic acid content, and the ability of these erythrocytes to "activate" the alternative pathway was inversely proportional to their sialic acid content 227 Mating a strain with erythrocytes with a high sialic acid content, with a strain having erythrocytes with a low sialic acid content, and studying the FI and F2 hybrid progeny, and their backcrosses with the parent strains, it became obvious that the sialic acid content of the erythrocytes and their ability to activate the alternative pathway were transmitted in a simple Mendelian fashion, which could not be segregated 227. The data suggested that both features are controlled by a single autosomal locus. The association constant for the binding of BIH with C3b is

The Biochemistry of Inflammation: Complement Activation

241

decreased on surfaces having a low sialic acid content, thereby favouring the formation of C3-b-B-b228. Conversely, if one couples heparin to zymosan, its ability to activate the alternative pathway decreases as the number of bound heparin molecules increases 229. This has been shown to be due to a progressive increase in the number of C3b molecules which are susceptible to the modulatory influences of SIH and C3b inactivator 229. There are therefore two surface molecules which restrict alternative pathway turnover, sialic acid and N-sulphated mucopolysaccharide, both of which facilitate the inactivation of particle-bound C3b by the regulatory proteins. The ability of bacteria and yeasts to activate the alternative pathway is, of course, a basic host defence mechanism, which requires a very primitive recognition system. Assembly of C3bBbP on their surfaces permits further C3 and C5 cleavage and ensures their being coated with C3b which facilitates their removal through interaction with macrophage C3b receptors, or lysis by the C5b-9 complex. Increasing magnesium concentration of serum causes alternative pathway turnover by increasing the association of factor B for C3b, while not altering the association of BIH for C3b 22s It is therefore reasonable to assume that agents which initiate alternative pathway activation, simply amplify pre-existing lowgrade turnover of the system. Methylamine-treated C3 acts as C3b in its ability to form C3-~-b 369. It has been postulated that the initial C3 convertase of the alternative pathway may be formed from the native C3 in the absence of proteolysis by nucleophiles including water ~69 These nucleophiles react with a thioester group on the ~ chain of C3369. In diseases associated with classical pathway activation such as systemic lupus erythematosus or rheumatoid arthritis, alternative pathway activation occurs because C3b production outstrips the ina~tivatin~ capacity of C3b inactivator and BIH.

BIOLOGICAL ACTIVITIES OF COMPLEMENT Cytolysis The cytolytic consequences of complement activation first drew attention to the existence of the complement system, as it was noted that a heat-labile serum component was required in addition to antibody for the lysis of bacteria. It is now known that activation of the complement system can cause lysis of nucleated and nonnucleated mammalian cells, bacteria, platelets, mycoplasmae and viruses. The observations that complement can induce lysis of liposomes 23° shows that proteolysis is unlikely to cause the lytic event, and the failure to detect phospholipase activity suggests that lipolysis is not a factor 231'232 The most probable explanation for cytolysis is that the C5b-9 complex is inserted into the cell membrane to form a hydrophilic channel which permits the passage of water and electrolytes, eventually leading to osmotic lysis 232 Electron microscopic studies of complement-lysed cell membranes reveals characteristic "holes" about I0 nm diameter with a 2.5 nm electron-lucent ring (Fig. 14) 23~ The characteristic lesions do not appear until the complete C5b-9 membrane attack complex has been assembled on the cell membrane 23S'236 However, at the C5b67 stage 'folacious' particles can be seen projecting from the cell membrane235; and following the addition of C8 these particles enlarge and develop a variable number of arms. At the C5b-9 stage the typical hollow cylinders appeared, projecting from the cell membrane and partly penetrating it 23s As C5b-9 complex isolated from lysed cell membranes is identical with the complement lesion in the cell membranes, and is capable of being re-incorporated into artificial lipid vesicles 237, it is fa{rly safe to conclude that the C5b-9 complex is inserted into cell membranes to become the "complement lesion". Recent studies have shown that the membrane attack (C5b-9) complex isolated from lysed cell membranes is in dimeric form, and it is this dimer which has the electron microscopic appearances of the complement lesion 13°

K. Whaley and A. Ferguson

242

Fig.

14

Erythrocyte ghost treated with C5b-9. A number of typical complement lesions may be seen on the surface, and also projecting from the membrane (x 180,0OO). (From Dourmashkin, R.R. (1978), I~lu~o~ogy, 35, 205-212. Courtesy of the author and editor).

Although integration of the C5b-9 complex explains the release of membrane lipid 232'238, intercalar particle aggregation 239, rearrangement of membrane lipids 23s, and non-osmotic membrane swelling 24°, the exact mechanism by which lysis is produced is not clear. The centre of the cylinder takes up the silico-tungstate stain and therefore could represent at transmembrane channel. The evidence that the C5b-9 complex penetrates the full thickness of lipid vesicles is conflicting, although it has been shown to increase the ion permeability of lipid bilayers 241 It is possible that the transmembrane channel does not pass through the centre of the C5b-9 complex, but rather the lipophilic C5b-9 complex grossly distorts the lipid bilayers, and a channel forms immediately adjacent to the complex. Once the C5b-9 complex has been assembled on the cell membrane there are at least three distinct steps required for lysis. A temperature-dependent step is associated with the insertion of the complex into the cell membrane. Prior to this step the complex is trypsin-sensitive, whereas after insertion it is trypsin-resistant. The trypsin resistance is not due to the burying part of the complex within the cell membrane, but rather it appears to be a property of the C5b-9 dimer, which because of its covalent and non-covalent interactions becomes resistant to trypsin and chymotrypsin 13°. Following the insertion of the complex into the membrane two further steps can be distinguished, one which is blocked by high molarity EDTA 2~2

243

The Biochemistry of Inflammation: Complement Activation Only when these three steps have been completed are the cell contents released: this release being blocked in the presence of 5% albumin 242.

Anaphylotoxins and chemotaxis Cleavage of C3 or C5 by their respective convertases, or proteolytic enzymes, yields C3a and C5a respectively. Both C3a and C5a possess anaphylotoxin activity, the potency of C5a being approximately ten-fold that of C3a 243. Anaphylotoxins bind to receptors on the membranes of mast cells and basophils with resulting degranulation. These granules contains vasoactive amines such as histamine, and their release is associated with increased vascular permeability. Smooth muscle contraction is induced by anaphylotoxins, acting on specific receptors on smooth muscle cells, and not because of their histamine-releasing activity. C5a, but not C3a, is chemotactic for polymorphonuclear leukocytes and macrophages 244. The chemotactic activity is preserved following degradation of C5a by anaphylotoxin inactivator, although in this case C5a des Arg requires a serum cofactor for its activity 22° The trimolecular complex C5b67 has been said to possess chemotactic properties 24S but evidence that the complex possessed chemotactic properties after steps have been taken to deliverately eliminate C5a contamination has not been obtained. The in vitro assembly of the alternative pathway C3 convertase C3bBb is associated with the generation of chemotactic activity 246, perhaps because the Ba fragment is release: Ba, but not Bb or native B, is chemotactic for polymorphonuclear leukocytes 247. The Bb fragment has been shown to interact with macrophage membranes, increasing the ruffling of the membrane and causing cytoplasmic spreading 24s.

Modulation of immune-complex mediated effects The interaction of immune complexes with the proteins of the complement completely alters their biological activities.

system

Although in vivo experiments have shown that the recognition and gross removal of complexes from the circulation by the cells of the mononuclear phagocyte system is a complement-independent event 249, in vitro studies have shown that the attachment of immune complexes to macrophages is greatly enhanced once they have been incubated with fresh serum 2s° This incubation allows them to become coated with C3b and permits them to interact with the C3b receptor. Degradation of the complexes is increased, not because internalisation or intracellular degradation is enhanced, but rather because the attachment of complexes to the macrophages is enhanced 2s° The discrepancy between these in vivo and in vitro observations probably results from differences in the size of complexes used. The in vivo studies used small soluble complexes which were probably incapable of activating the complement system efficiently, whereas the in vitro studies employed large complement-activating complexes. When immune complexes are injected intravenously into normal mice, some bind to the cellular constituents of the blood and some stay in the plasma. Within a short period of time the cell-bound complexes become dissociated from the cell membranes and circulate in the plasma from which they are removed 2sl This complex releasing activity, which may play a role in protecting the body from the harmful effects of immune complexes, requires an intact alternative pathway 252. Insoluble antigenantibody complexes are solubilised by their interaction with the complement system. This process requires an intact alternative pathway but proceeds more efficiently in the presence of an intact classical pathway 253-25s The solubilisation process is not enzymatic but depends upon the intercalation of C3b, and perhaps C4b into the antigen-antibody lattice 2s2'2S3 As a result of this intercalation process,

244

K. Whaley and A. Ferguson

the size of the complexes is reduced, and the injurious effects of insoluble complexes may be abrogated.

immune

The presence of C3b or C4b on immune complexes permits their interaction with their specific receptors on the surfaces of certain cells such as human erythrocytes, mononuclear phagocytes, polymorphonuclear leukocytes, and B-lymphocytes. This interaction is termed immune adherence 9°. The biological consequences of this phenomenon vary depending upon the type of cell involved; for instance, phagocytic cells show increased phagocytosis 2s6 and increased intracellular killing of bacteria 257 The role of C3b receptors on lymphocytes continues to be controversial, but perhaps they may be important in antibody production. It has been suggested that complement together with antigen provides a second signal for antibody production 2s8, but this is probably incorrect, especially as T-lymphocyte-independent antibody responses can be obtained in the absence of c3esg. T-lymphocyte-dependent antibody responses require C32s9, perhaps because C3b could assemble macrophages and the two lymphocyte subsets required for this response. In contrast normal antibody responses occurred in a patient with genetically determined complete deficiency of C3 e6° The formation of B memory cells is impaired on decomplemented rabbits 261, probably because C3 is required for the transport of aggregated IgG and immune complexes into germinal centres 2~2 Rabbit, but not human, platelets have C3b receptors, and C3b-coated complexes stimulate selective secretion of granule contents, or total lysis of the platelets, depending upon the size of the complex. Neutrophil lysosomal contents are also secreted following the binding of C3b-coated particles to C3b receptors 2~3 Human platelets bind immune complexes by Fc receptors. When zymosan is incubated with human plasma it acquires the ability to stimulate the platelet secretory response. The reaction requires an intact alternative pathway and fibrinogen, but is independent of the classical pathway components 26~

Micro-organisms and complement opsonisation C3b or C4b on bacterial surfaces facilitates their attachment to macrophages. C3b can coat gram-negative bacteria by the alternative pathway as described earlier. In immune individuals opsonisation may be achieved by IgG antibody, C4b, C3b, or all three.

Antiviral a c t i v i t y of complement Neutralisation of herpes-type viruses by antibody is enhanced by C1, C4 and C22~s RNA tumour viruses possess a C]q receptor, which binds C1q, with activation of CI. Virolysis occurs in human but not guinea pig serum 266 Lysis of antibody-coated measles virus infected cells occurs by the alternative pathway 267. The reaction depends upon the intact (Fab')-2 fragment. Antibodydependent virolysis by leukocytes is enhanced by the addition of complement 268

BIOSYNTHESIS OF C O M P L E M E N T

PROTEINS

Four cell types have been shown to be capable of synthesising complement components, hepatocytes, mononuclear phagocytes, fibroblasts and the epithelial cells of the gastrointestinal and genitourinary tracts 269.

The Biochemistry of Inflammation: Complement Activation

245

C7

Haemolytically active macromolecular CI has been shown to be synthesised by the columnar epithelial cells of the human and guinea pig intestine 270-272 , peritoneal macrophages 273 in short term culture by normal colon. Large quantities of macromolecular CI and C1s were synthesised by normal human colon, adenocarcinoma of the colon and the transitional epithelial cells of the bladder, urethra and renal pelvis in long term primary cultures 274'27s, and smaller quantities by monocytes and fibroblasts 27s'276 Although macromolecular CI is synthesised by guinea pig and human peritoneal macrophages, the rates of production of CI and the C1q subcomponent suggest that the CI subcomponents are synthesised independently, perhaps by different cells 277 The biological importance of these observations is obscure, but clinical studies have shown that the serum concentrations of the CI subcomponent~ vary independently of each other. Secretion of C1q by macrophages is impaired by the addition of 2,2'-dipyridyl to the culture medium 277 This is thought to be due to inhibition of post-translational hydroxylation of proline y lysine residues, thereby preventing the formation of the triple helical structures of the collagen-like regions of C1q. Hydroxylated C1q may not be secreted, or may be rapidly degraded, intra- or extracellularly. There is evidence from SDS-PAGE studies that C1q synthesised fn vitro by epithelial cells, fibroblasts, consists of 180,000 dalton molecular weight subunits 27S'276 which are larger than the subunits of serum C1q. These could be C1q dimers, precursor or pro C1q molecules.

C4 The macrophage appears to be the principle site of C4 biosynthesis. This conclusion is principally based upon the incorporation of radio-labelled amino acids into C4 protein by macrophages in culture 278-2s° and the accumulation of functionally active C4 in macrophage culture supernatants 28°-2s2 Human monocytes in culture synthesise functionally inactive C4283'284 The explanation for the failure of monocytes to produce a functionally active molecule is because C4 is synthesised as the single polypeptide chain proc-C4285 Thus monocytes lack the enzyme to convert pro-C4 to functionally active C4. Functionally active C4 has been shown to be synthesised by established cell lines from normal rat liver or hepatomas 286. At least one of these lines has many characteristics of hepatic parenchymal cells. Cell-free synthesis studies have shown that C4 in the guinea pig, mouse and rabbit is synthesised as a single chain precursor molecule (pro-C4) which requires posttranslational proteolysis for conversion to functionally active three polypeptide chain C4287'288

C2 C2 is synthesised in functionally active form by monocytes and other cells of the mononuclear phagocyte series 283'284'289 Although cell lines derived from chemically-induced guinea pig hepatomas produce C2, it is probable that the Kupffer cell is the primary site of hepatic C2 synthesis 269.

C3 The liver appears to be the primary site of C3 synthesis in the human.

Using

246

K. Whaley and A. Ferguson

prolonged agarose electrophoresis, Alper and his colleagues 29° showed that the phenotypic C3 variant of a liver transplant recipient changed from the rare C3FS to C3SS, the phenotype of the donor. Human foetal liver and postnatal liver synthesise functionally active C3291 , and immunofluorescence studies showed hepatocytes staining for C3292 Cultures of pieces of rheumatoid synovial membrane synthesise functionally active C3293 , although synovial fluid macrophages and macrophages derived from the surface of the rheumatoid synovial membrane synthesise non-functioning C3294 . These findings suggest that other cells in the synovial membrane synthesise the functionally active molecules. Human monocytes and macrophages also synthesise functionally inactive C3283'284 The C3 synthesised by monocytes in culture consists of a two-polypeptide chain molecule indistinguishable from the functionally active C3 molecules found in plasma 295, and there is no obvious explanation for its lack of activity. Studies of C3 synthesis in cell-free systems show that it is synthesised as a single polypeptide chain pro C3 molecule which undergoes a limited post-translational proteolytic cleavage to convert it to the active form 296'297 Minute quantities of single chain pro C3 and C4 are found in human plasma 289'259, suggesting that in the human also C3 and C4 are synthesised as single chain precursor molecules.

C5

C5-deficient mice can be reconstituted by bone marrow transplants taken from normal mice 3°°. Spleen cells from the recipients synthesise C5, 3-4 weeks post-transplantation. In normal mice it has been shown that only the adherent cells of the spleen are capable of C5 synthesis 3°I Experiments on human tissues have shown that functionally active C5 is synthesised by lung, liver, spleen and foetal intestine 3°2. Incorporation of radiolabelled amino acids into C5 protein occurs in cultures of human thymus, placenta, peritoneal cells, and bone marrow 3°2 It is likely that a specific cell type present in all these tissues is responsible for C5 synthesis, but to date the cell type has not been identified although it is probably the mononuclear phagocyte. Human monocytes in culture synthesise C5, but the molecule is haemolytically inactive 284, probably because it is degraded following secretion 2~s Like C3 and C4, C5 is synthesised as a single polypeptide chain precursor which is converted to functionally active C5 by post-translational proteolysis 3°3'3°4 Functionally active C5 is synthesised by several rat hepatoma cell lines 286 and Kupffer cell-depleted primary rat hepatocyte cultures 269. However, the contribution of the hepatocytes to C5 synthesis remains an enigma, as mouse and guinea pig liver apparently do not synthesise this molecule.

C6 In vitro cultures

of rabbit liver slices synthesise C63°5. More recently, isolated rabbit liver organ cultures were shown to incorporate 14C-labelled amino acids into C63°6 The conclusion that the liver is the site of C6 production has been confirmed by the observation that the C6 phenotype present in the serum of a patient following liver transplantation was that of the donor and not the recipient 3°7 Furthermore, human monocytes in culture do not synthesise C6 when measured by functional assay 283

C7 and C8

The biosynthesis of C7 has not yet been studied, and data on C8 biosynthesis are derived from studies of tissues taken from foetal pigs 3°8. Between 17 and 112 days

The Biochemistry of Inflammation: Complement Activation

247

of intrauterine life C8 synthesis was detected in the spleen, liver, lung, intestine and kidney, but not in the lymph nodes, thymus or bone marrow. The cell of origin was not determined.

C9 The l~ng-term rat hepatoma cell line (MHICI) synthesise C9, in addition to albumin, C3, C1-inhibitor and C53°9. Primary rat liver cell cultures enriched in parenchymal cells have been shown to synthesise functionally active C9269 .

Alternative pathway components Factor B, D and properdin are synthesised by huamn monocytes and macrophages derived from rheumatoid arthritis synovial fluids 28~'294 Guinea pig peritoneal macrophages have also been shown to synthesise factor B, D and properdin 31°. All three alternative pathway components are synthesised in functionally active form.

Control proteins Cl-inhibitor Using immunofluorescence, 5-I0% of hepatocytes stain for C~-inhibitor, whereas Kupffer cells were negativee92o Hepatic tissue in short term cultures has been shown to synthesise C1-inhibitor, which can be detected by both functional and immunochemical assay systems T M

C3b inactivator and ~IH globulin Human monocytes and macrophages derived from the inflamed synovial joints of patients with rheumatoid arthritis have been shown to synthesise C3b inactivator and BIH globulin in their functionally active forms 2s~'29~

The biosynthesis

of C4 binding protein has not yet been studied.

Ontogeny of complement Using immunofluorescent, immunochemical and functional assays, the time of onset of synthesis of complement components has been studied in the foetus. These investigations have been assisted by investigations of materno-foetal differences in the electrophoretic polymorphic variations of individual components, and measuring the concentration of complement components in the cord blood of foetuses born to mothers with genetically-determined complement deficiencies. The results of these studies have shown that complement components are synthesised early in foetal life and that complement components do not cross the placenta. Serum concentrations of C3, C4, C5, properdin and factor B in the newborn are approximately one-half to two-thirds of the maternal serum level 31~ Normal adult values are achieved between the ages of one and three years. C1-inhibitor synthesis of ted 291'302 Functionally

is synthesised by foetal liver by 4 weeks of age 312, and by 8 weeks C2 and C4 by liver macrophages, and C3 by foetal liver can be detecC5 synthesis is not detected until 14 weeks of gestation 3°2'312'313 active CI was synthesised by the colon from a foetus of 19 weeks

248

K. Whaley and A. Ferguson

gestation 272 In the pig, C1q is synthesised in the intestine after 48 days gestation and after 66 days synthesis was detected in lymph nodes and spleen 314 C8 synthesis in foetal pigs was shown to occur in kidney, liver, spleen and lung by 48 days of gestation 3°8.

Genetically-determined

p o l y m o r p h i s m of c o m p l e m e n t c o m p o n e n t s

Many of the complement proteins studied to date exhibit genetically-determined electrophoretic polymorphism (Table 5). The polymorphic variability of complement components is determined by autosomal co-dominant genes 166. The polymorphic variants of C2, C4 and factor B are controlled by genes on the short arm of chromosome 6, amid the genes controlling the major histocompatibility complex 192. C4, C2, C3, C6 and factor B exhibit genetically-determined polymorphism. Polymorphism of other complement components has not yet been shown to have a genetic basis 166 The functional activity of particular polymorphic variants and their possible disease associations are currently under investigation.

Acquired abnormalities

of t h e c o m p l e m e n t s y s t e m i n h u m a n

disease

Over the past two decades it has become increasingly apparent that complement activation occurs in a number of disease states, and that the products of complement activation may play a pathogenetic role. The conclusions are based upon: (I) the finding of hypocomplementaemia in patients, the degree of which parallels disease activity; (2) the deposition of complement components at the site of tissue injury; (3) hypersynthesis and hypercatabolism of complement components in disease states; (4) the observations that the lesions of complement-mediated tissue injury in experimental animals resemble the lesions in human diseases.

CONNECTIVE

Systemic lupus

erythematosus

T I S SUE D I S E A S E S

(SLE)

The d e t e c t i o n of c i r c u l a t i n g immune complexes a l s ' 3 1 6 , reduced serum l e v e l s of the c l a s s i c a l pathway components C1, C4 and C2317 , t h e g l o m e r u l a r d e p o s i t i o n of immune complexes and c l a s s i c a l pathway components a l a ' 3 1 9 and the h y p e r c a t a b o l i s m of C432° show t h a t a c t i v a t i o n of the c l a s s i c a l pathway o c c u r s in SLE. In the routine laboratory abnormalities of the classical pathway are more frequently observed than abnormalities of the alternative pathway: generally speaking the serum levels of immune complexes rise during exacerbations and levels of complement proteins drop. Examination of serum samples taken serially over long periods of time shows that the measurements which correlate best with disease activity are immune complexes and C4, with the former providing a somewhat more sensitive index than the latter 316. Concentrations of CI subcomponents change with disease activity but rarely fall into the subnormal range, and C3 may be normal in patients who have severe disease and pronounced complement activation, especially in the absence of renal disease 317 Serum concentrations of the regulatory protein C~-inhibitor are usually elevated; its detection in the glomeruli by immunofluorescence 3zI suggests that it is hypercatabolised. Although complement activation in SLE is primarily by the classical pathway,

The Biochemistry of Inflammation: Complement Activation

249

significant alternative pathway activation occurs, as shown by reduced serum levels of factor B and properdin 322'232, the presence of factor B cleavage products 32~ glomerular localisation of factor B and properdin 319'321, and increased catabolism of factor B 3 2 0 ' 3 2 5 and properdin 326 Although the mean serum levels of factor B and properdin are frequently reduced in patients who have severe disease 323, mild cases of SLE rarely have low levels of either protein, despite markedly reduced C4 levels. Despite the normal levels of the alternative pathway proteins, however, increased turnover of factor B can be shown by the presence of increased levels of its cleavage product Ba. Similarly increased C3 turnover in SLE is shown by elevated levels of the cleavage product, C3d, despite normal concentrations of C3321 . The increased turnover of the alternative pathway in SLE is probably secondary to the intense activation of the classical pathway, as nephritic factor-like substances cannot be detected in SLE sera. If, as seems likely, the increased alternative pathway turnover seen in SLE is secondary to the generation of C3b, by the classical pathway, then the serum concentrations of the control proteins C3b inactivator and BIH ought to be of central importance in controlling the extent of turnover. Reduced serum concentrations of C3blNA and BIH were found during exacerbations of SLE, especially when C3 levels were reduced. The reductions in levels of 61H were more pronounced than those of C3blNA 323. However, the serum concentrations of C3b inactivator and ~IH were unrelated to either the catabolic or synthetic rates of factor B or to the intravascular/extravascular distribution of the protein 3eI. This finding argues against the absolute levels of these control proteins and does not dictate the extent of alternative turnover in SLE, but there is considerable evidence for increased utilisation of BIH in this disease. Whenever C3 is detected in the glomeruli of renal biopsies from these patients, BIH is always present 327 and their distribution patterns are identical. Likewise, ~IH may be found along with C3 in skin biopsies from these patients 32s The evidence supporting hypercatabolism of C3blNA in SLE is less conclusive. Occasionally this enzyme may be detected in the glomeruli during immunofluorescence examination 327 but its pattern of distribution is focal and segmental, which is difficult to explain. The infrequent detection of C3blNA in SLE renal biopsies is almost certainly due to the weak binding of C3blNA to C3b and it is impossible to detect consumption of the protein during complement activatinn ~n vitpo 329.

Rheumatoid

arthritis

In contrast to SLE, in which complement activation is seen in the blood, in RA serum complement levels are usually elevated except in patients with vasculitis 33° However, when the synovial fluid is examined, evidence of complement activation is obvious. High concentrations of immune complexes 316'331 and reduced concentrations of CI and its subcomponents C4 and C2332'333 the presence of C4 cleavage products 3a4 all show classical pathway activation occurs in synovial fluid. Although CT-INH levels are occasionally reduced in RA synovial fluid, the mean levels do not differ from those found in DJD ~21 Reduced concentrations of B and p335,336 and the presence of factor B cleavage products 321'337'338 show that alternative pathway activation occurs. As we have been unable to detect nephritic factor-like substances in RA synovial fluid, it is probable that activation of the alternative pathway in RA results from C3 turnover by the classical pathway convertase. Although the serum concentrations of C3bINA and BIH are high in RA, their synovial fluid concnetrations are sometimes extremely low, but their mean levels do not differ from those found in DJD. As in SLE, both in RA serum and synovial fluid the concentrations of C3blNA and ~IH correlate well with levels of C3, B and p213,339, but again no correlation could be found between the concentrations of C3blNA or BIH and turnover of C3 or factor B.

250

K. Whaley and A. Ferguson

This is unexpected as absolute deficiency of C3bINA 214'215 and immunochemical depletion of either C3bINA 216 or BIH 21s results in uncontrolled alternative pathway turnover. However, to reduce turnover of the alternative pathway to its normal level in these depleted sera, one requires to add only between 25 and 50% of their original concentrations. Thus under normal conditions there is a great deal of "spare" control protein available, which may account for the lack of correlation between B turnover and control protein concentration. It is also possible that B turnover may be produced by tissue proteases rather than the more specific mechanisms involved in complement activation. Such a mechanism has been proposed by Goldstein and Weissmann 34°

Infectious

diseases

The role of complement in infectious diseases was stemmed from the original observation of Bordet 4'S, who showed that serum-mediated lysis of Vibrio cholerae required heat stable (antibody) and heat labile (complement) components. The complement system acts in at least three ways to combat infection: coating of organisms with C3b enhances adherence to C3b receptors, and therefore enhances phagocytosis and promotes the intracellular killing of bacteria. Formation of C5a attracts polymorphonuclear leukocytes and monocytes into the area, and the C5b-9 complex results in lysis of certain bacteria. Deficiency of C3 results in recurrent severe bacterial infections, similar to the picture of hypogammaglobulinaemia. There appears to be an undue frequency of infections caused by pneumococci. Patients with deficiencies of C5, C6, C7 and C8 present with recurrent bacteraemia, either with the gonococcus or meningococcus (Table 5). Deficiencies of CT subcomponents C4 and C2 are not associated with recurrent infections, probably because the alternative pathway is intact. Treponema pallidium, Neisseriae, leptosperae, pseudonomas, Yibrio cholera and gram-negative enterobacteria are lysed by antibody and complement ~ l . Many organisms, especially mycobacteriae and gram-positive organisms, are not lysed by complement. In these cases it is thought that the antigen in the cell wall is situated too far from the plasma membrane for complement activation to exert its bacteriolytic activity. During infections with these organisms opsonisation must be important. In most bacterial infections serum complement levels are elevated 342, but some patients at the onset of gram-negative endotoxaemia show evidence of reduced serum levels of the alternative pathway components, factor B and properdin. These patients later develop shock, whereas those who have normal levels of alternative pathway components survive 3~3 The patients who develop shock also have reduced serum levels of the control proteins C3b inactivator and BIH 34~. In contrast, patients who have established gram-negative endotoxic shock have activation of both classical and alternative pathways, as shown by reduced serum levels of C4, C3, factor B and properdin 34s Increased turnover of both pathways was seen during periods of shock, and in episodes of endotoxaemia which ended fatally ~4S The prognostic importance of the serum levels of complement components and their cleavage products is currently under evaluation. Whether complement activation plays a pathogenetic role in shock or whether the increased activation is simply a host response to the increased demand remains to be answered, as the management of endotoxic shock depends upon this knowledge. For instance, if complement activation were pathogenetic, then plasmaphoresis with removal of phlogistic molecules would be indicated. Conversely, if complement activation was beneficial, then the administration of fresh frozen plasma would be therapeutically advantageous.

The Biochemistry of Inflammation: Complement Activation Renal

251

disease

A number of experimental forms of glomerulonephritis in laboratory animals have been shown to be complement-dependent. Although in human glomerulonephritis complement is thought to be of major pathogenetic importance, the evidence is usually indirect, being based on the findings of deposition of glomerular complement components and reduced serum concentrations of total haemolytic complement or individual components. Occasional studies showing the presence of cleavage product of C3 and factor B in the plasma, or reduced plasma half-lives of radiolabelled purified components support this conclusion. In investigating the role of complement activation in glomerulonephritis, it is difficult to establish whether activation is occurring in the circulation, within the kidney, or in both. For instance, in membranous glomerulonephritis serum complement component concentrations are normal, whereas glomerular deposition of complement components is invariably present 346. In this situation complement activation could be occurring exclusively within the glomerulus, although increased synthesis of components could mask systemic hypercatabolism. In contrast in the subendothelial type of membranoproliferative glomerulonephritis 3~6 and systemic lupus erythematosus 321'323 reduced serum concentrations of complement components and their localisation within the glomeruli suggest that complement activation is occurring within the cirulcation, and that glomerular deposition could be secondary to this. Alternatively, intraglomerular complement activation could be occurring independently of activation within the circulation. In dense-deposit deisease, very little C3 is seen in the glomeruli, and as systemic complement activation is pronounced, it is probable that glomerular deposition of C3 occurs secondarily to systemic activation. No evidence exists, however, to substantiate these possibilities in the human, although in experimental serum sickness and tissue sections, evidence for the intraglomerular activation of complement has been obtained 347 In membranous glomerulonephritis, glomerular deposition of C1q, C1s, C4, C3 and C5 pathway with little properdin shows that the classical pathway activation is predominant 346 In contrast, in SLE and type I membrano-proliferative glomerulonephritis, marked activation of both classical and alternative pathways occurs, as shown by reduced serum levels and the glomerular deposition of components 321'323' 32~,346 Type II membrano-proliferative glomerulonephritis is unusual as there is little or no deposition of immunoglobulin, but traces of C3 are seen in the glomeruli. However, there is marked activation of the alternative pathway, as shown by reduced serum levels of C3, factor B and properdin, but normal, or only slightly reduced, concentrations of CI, C4 and C2 Is4'34G This profile of alternative pathway activation is explained on the basis that in the serum of these individuals is a substance termed C3 nephritic factor (C3NeF). C3NeF is an immunoconglutinin which is an antibody directed against a determinant (or determinants) of C3bBb 186-Is8 When C3NeF binds to C3bBb it stabilises the enzyme and prevents BIH from dissociating factor B from the complex 19°. The nephritic factor stabilised in the fluid phase convertase can only cleave C3, which explains why C5 levels are usually normal in this disease 184'346 Acute post-strptococcal glomerulonephritis is associated with marked reductions in serum C3 levels, but with normal or only mildly depressed concentrations of CI, C4 and C2348. Properdin levels are markedly reduced, but factor B levels are usually only slight subnormal. The profile is that of alternative pathway activation, which often returns to normal prior to the resolution of the renal lesion. Interestingly, acute post-streptococcal glomerulonephritis is the only disease in which factor B is regularly detected in the glomerulus 3u9.

Complement

and

skin

disease

The skin lesions of systemic and discoid lupus erythematosus

show deposition of

252

K. Whaley and A. Ferguson

antigen-antibody complexes and classical and alternative pathway components along the dermo-epidermal junction. Similar findings occur in pemphigus, a disease in which bullae occur in the skin. The fluid from these bullae shows reduced levels of complement components, suggesting that local complement activation is occurring 35°. This local activation may be related to the presence of a high molecular weight (>19S), anticomplementary factor in the blister fluid, or to the deposition of circulating immune complexes which some of these patients have in their sera. In bullous pemphigoid, the presence of subepidermal bullae is associated with the presence of a circulating antibody to the basement membrane zone in 80% of patients. Deposition of C1q, C4, C3 and C5 in the basement membrane zone is frequent, whereas factor B is rarely detected T M It is thought that the binding of the basement membrane zone antibody in the tissues activates the classical pathway to produce the clinical features of the disease. Dermatitis herpetiformis is a skin disease characterised by the presence of vesicles and bullae within the epidermis, associated with intense pruritus. IgA is deposited in normal and lesional skin the dermal papillae below the basement membrane zone. C3 is detected in the same region, but classical pathway components are usually absent. The alternative pathway proteins properdin and factor B are sometimes present, which suggests that deposition of IgA containing immune complexes may selectively activate the alternative pathway T M Cutaneous necrotising vasculitis usually involves the lower limbs, presenting as palpable purpura. The disease is thought to be due to the deposition of antigenantibody complexes in the walls of the small blood vessels, leading to complement activation and intense polymorphonuclear leukcocyte infiltration. Immunofluorescence studies showing the presence of immunoglobulins, C1q and C3 in the early lesions, reduced serum levels of C1q, C4, C2 and C3, and the detection of circulating immune complexes, support this notion.

Genetically-determined

d e f i c i e n c i e s of c o m p l e m e n t c o m p o n e n t s

The documentation of genetically determined deficiencies of complement components has greatly enhanced our knowledge of the biological significance of the complement system in terms of resistance to bacterial infection. Deficiencies of all the classical pathway terminal components have been described (Table 5), whereas complete deficiencies of alternative pathway proteins are so far unrecognised. Deficiencies of all the complement components are inherited in autosomal recessive or codominant fashion, the parents being heterozygous for the deficiency and having approximately 50% of the normal level of the component. Deficiency of C2 occurs most commonly, and it has been estimated that the gene frequency is about one per cent. Deficiency states are detected by noticing the absence of haemolytic eomplement activity in the patients' sera. The only exception to this is homozygous C9 deficiency, in which patients have reduced serum haemolytic activity. This occurs 35 because C5b-8 can produce lysis, although less efficiently than when C9 is present

Complement d e f i c i e n c i e s a s s o c i a t e d

with recurrent

infection

Homozygous deficiency of C1q, C3 and C5, C6, C7 and C8 are associated with recurrent bacterial infections. Deficiency of C1q was associated with recurrent skin lesions and chronic infections 353, whereas the inheritance of an antigenically and functionally deficient form of C1qwas associated with an immune complex glomerulonephritis 354 C3 deficiency was associated with severe recurrent infections, including septicaemia,

Absent

Absent

Absent

Absent

Absent

Absent

Absent

Absent

Both

Absent

Both

Absent

C1q

C1r

C1s

C4

C2

C3

C5

C6

C7

C8

C9

CT-INH

C3blNA

Levels 20% of normal

Both

Component

Anaphylotoxin inactivator

Protein absent or non f u n c t i o n a l

TABLE 5

?

Autosomal recessive

Autosomal dominant

It

IV

11

IS

"

"

"

"

"

"

Autosomal recessive

C1q deficiency unknown C1q dysfunctional protein Codominant

Mode of transmission

Reduced levels in other family members

Yes

Heterozygotes get disease

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

?

No

No

No

No

No

No

No

No

Yes

Yes

No

No

No

Yes Yes

No

HLA linkage

No

Heterozygotes detected

SLE

SLE

Recurrent urticaria and angioedema

Recurrent pyogenic infections

Hereditary angioedema, SLE

Normal

SLE, xeroderma pigmentosa, recurrent Neisserial bacteraemia

Raynaud's phenomenon, ankylosing spondylitis, recurrent Neisserial bacteraemia

Recurrent neisserial bacteraemia, Raynaud's phenomenon

Recurrent Neisserial bacteraemia, SLE

Recurrent pyogenic infections

SLE, juvenile rheumatoid polymyositis

SEE

Glomerulonephritis,

Glomerulonephritis,

Recurrent infection, glomeruonephritis Recurrent infection, glomerulonephritis

Disease Associations

Genetically Determined Deficiencies of Complement Components

O1

~O

< m o

>

3 o

3

C~ o

~

3

3

O

3 ~

W ~" o

254

K. Whaley and A. Ferguson

and deficiencies of C5, C6, C7 and C8 are associated with recurrent Neisserial bacteraemias, either with the meningococcus or the gonococcus 355'35~ Serum from C3-deficient patients fails to generate complement-mediated opsonic activity and c5-mediated chemotactic activity. Elimination of Neisseriae requires lysis by serum, and deficiencies of C5, C6, C7 and C8 predispose to recurrent bacteraemias by this group of organisms. Patients with C2 deficiency normally do not have an increased risk of infection. However, two patients with C2 deficiency associated with low factor B levels had recurrent septicaemias 357. Presumbably patients with C2 deficiency opsonise bacteria via an intact alternative pathway, and when factor B levels are reduced this ability is compromised sufficiently to produce clinical symptoms.

Complement deficiency and connective tissue diseases Approximately 40% of the reported patients with homozygous C2 deficiency have SLE and 20% have various other connective tissue diseases. There is also an increased frequency of heterozygous C2 deficiency in SLE and adult and juvenile rhematoid arthritis 3S8 From Table 5 is can be seen that connective tissue diseases have been reported in other complement deficiency states with the exception of C3, C6 or C3b inactivator deficiency. Although there must be an association between connective tissue diseases and complement deficiency states, the precise relationship cannot yet be stated with confidence, as errors in sampling must arise from the screening of patients with connective tissue diseases for complement deficiency. The association of complement deficiency with connective tissue disease in which complement activation is reputed to play a pathogenetic role is curious; one would have expected complement deficiency to exert a protective effect. Three possible explanations can be proposed. Firstly, CI, C4 and C2 increase the efficiency of neutralisation of certain viruses 265 and oncornaviruses activate the classical pathway in the absence of antibody, with resultant viral lysis 26~. Clearly, therefore, complement deficiency could permit certain viruses to replicate, and perhaps produce disease. Against this argument is the observation that only a few viruses show enhanced neutralization and that patients with complement deficiency do not show an undue frequency of viral infections. A second mechanism, whereby complement deficiency could lead to disease, would be by failure of opsonisation of immune complexes. Thus complexes may circulate and fail to be removed by normal host defence mechanisms and be deposited in the tissues with consequent inflammation. The third explanation stems from the findings that the genes controlling C2, C4 and factor B polymorphism, and C2 and C4 deficiency (i.e. structural genes) are located on the sixth chromosome in the region of the major histocompatibility complex. The association of complement deficiency with connective tissue disease may occur because certain cell membrane antigens coded for by genes within the HLA complex may act as receptors for infectious organisms, or be similar in structure to antigens present in foreign organisms, thereby provoking cross-tolerance. There are also an increasing number of reports of complement proteins in certain lymphocyte membranes. C4 inhibits the mixed lymphocyte reaction in the human 42 Perlmann and coworkers 359 have shown that C8 in lymphocyte membranes is required for cytotoxic reactions. Factor B is present in lymphocyte membranes and can form a C3 convertase with cobra venom factor 3~°, and Budzco and coworkers 3~I have shown that that certain malignant lymphoma cell lines carry a preformed alternative pathway C3 convertase. Recently the cross-linking of C5 in lymphocyte membranes by anti-C5

The Biochemistry of Inflammation: Complement Activation

255

antibodies has been shown to increase DNA synthesis 362 Thus the complement system may not only be a plasma protein system, but also a system of membrane proteins, perhaps involved in cell co-operation. This proposal is supported by the observations of Dierich and Landen 363, who were able to form bridges between cells bearing different complement components which interact with each other.

ACKNOWLEDGEMENT This work was supported by grants HERT 532 and 564 from the Scottish Hospital Endowment Research Trust.

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