The Complement System: A cascade Foreboding and unharnessed Whose waters run deep

Symposium on The Child with Recurrent Infection

The Complement System A cascade Foreboding and unharnessed Whose waters run deep.

Roger E. Spitzer, M.D.*

The state of the art concerning the complement system is tenuous. Although much progress has been made in the past 15 years, an extraordinary amount is still unknown about the basic immunochemistry of this system and, probably of more importance, its relationship to human disease. Research in this area is cumbersome with many inexact and semiquantitative methods; existing knowledge is complex and confusing, made more so by a nomenclature which preempts logic and consistency. More complement "pathways" are discovered every day, old proteins are rediscovered and renamed, and details of reaction mechanisms are escalated far out of proportion to their significance. Data on the involvement of complement in clinical disease are unwieldy, often the result of poorly controlled experiments, and of little benefit to those who care for patients. An enormous amount of information is currently available, most of which is in a very chaotic state, and of little relevance to the clinician. These considerations form the basis for the two primary objectives of this discussion: first, to provide some degree of order and understanding about the complement system; and second, to provide the reader with a format for utilizing this material in the care of children with infection. A three part discussion is therefore necessary: (1) a brief review of the basic science aspects of the complement system and its known biologic functions, (2) a review of those data concerning the role of the complement system in infectious disease, and (3) an outline for the application of these data to the care of patients with various specific infections. Two additional features of this review need to be stressed from the outset. First, this will not be an exhaustive compilation of details put forth in computer-like fashion but will be abbreviated to include only those data

* Associate Professor, Department of Pediatrics, State University of New York, Upstate Medical Center, Syracuse, New York This work was partially supported by NIH grants, AM17376 and A112721; New York State Kidney Disease Institute grant, C-71376; and a grant from the American Cancer Society, IM-l 04. Pediatric Clinics of North America-Vol. 24, No.2, May 1977

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which are important to an understanding of the role of this system in clinical disorders. Second, this reviewer feels obligated to provide a critical review which implies some degree of interpretation and bias. This would seem to be unavoidable in our present state of knowledge and these critical appraisals will be clearly indicated as opinion rather than scientific gospel as is so often the case.

IMMUNOBIOLOGY OF THE COMPLEMENT SYSTEM It is difficult to define the complement system. Many would denote complement as an auxiliary mechanism involved exclusively in the activity of antibody or solely as an effector mechanism during inflammation. It is both of these to be sure, but as will be shown, it is far more. Complement represents a biologic system involved with the entire immune process and its major role may well be to modulate or regulate a large portion of that response. The designation of complement as a biologic system reflects that broad general role. The core of this system consists of at least 12 separate and distinct serum proteins; in addition, there are a number of other serum proteins which are critical to the activation and modulation of these basic components. The 12 basic proteins and many of the auxiliary proteins function in an interrelated fashion. All of the proteins under consideration exist in plasma in an inactive or native state. When "complement" activity is initiated, these proteins interact in a sequential, orderly fashion. Cleavage of these proteins with the formation of active (and inactive) fragments followed by complex formation of one fragment with another frequently occurs. The activity of one protein is dependent on its predecessor and often determines the fate of the next component in the sequence. The term cascade has frequently been applied to these interactions and that is a useful designation. Thus the basic format of this system appears to be enzymatic cleavage of a native component, elaboration of an active fragment, complex formation, and creation of a new enzyme with separate specificities. It is through this sequential action that all of the biologic functional activity of this system is achieved. It is indeed a monumental cascade and its complexity is fearsome.

NOMENCLATURE

The basic proteins are numbered sequentially as Cl, C2, C3, etc. Unfortunately, they do not react in this same numerical sequence since they were numbered in the order of discovery. Cl is unique among these proteins in that it is a tetra-molecular complex bound together by calcium ions; these four proteins are designated Clq, Clr, CIs, and Clt.7 The auxiliary proteins are designated as factors and by letters such as factor A, factor B, etc. New proteins which are discovered are blessed with rather elaborate trivial names until their definition has been elucidated and they can be assigned a letter; initiating factor and properdin convertase are current designations. In addition, other proteins pri-

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Table 1. Proteins of the Complement System SERUM UNITS

Activation Units CIq CII CIs CIt C4 C2 IF Factor B Factor D

MOLECULAR

CON CENTRA TION

SYNONYM

WEIGHT

(/LG PER ML)

200

Cl esterase

400,000 168,000 79,000

13lE

240,000 117,000

400 30

C3NeF C3PA,GBG C3Pase, GBGase

150,000 100,000 25,000

Trace 225 Trace

190,000 70,000

20 ?

180,000 185,000 125,000 120,000 150,000 79,000

1200 75 60 60 15 Trace

Properdin Properdin convertase

Functional Units C3 C5 C6 C7 C8 C9

131C 131F

120

marily involved in regulation of this system are designated by their function rather by a number or letter such as C 1 inhibitor. Finally, as many of these proteins are cleaved into smaller fragments during reaction, the fragments have been specified by small letters following the number or capital letter f)f the parent protein as in C3a, C3b, Ba, Bb, etc. When one of these proteins assumes enzymatic activity, this active state is designated by the use of a bar over the appropriate number or letter as in Clor B to distinguish it from the native or inactive molecule. PATHWAYS OF COMPLEMENT ACTIVITY It is difficult to present this section in any simplified fashion. To provide some structure for the reader, this biologic system is arbitrarily divided into two separate and distinct units (Fig. 1). The activation units represent those proteins which act as a means of generating biologic activity. The functional unit comprises those proteins which are responsible for this biologic activity. It is to be emphasized that these divisions are arbitrary and clearly not inviolate as the reader shall subsequently see. For our purposes, however, they represent useful designations.

Activation Units CLASSICAL UNIT. The association between complement and antibody was discovered early. When IgG or IgM antibody is complexed to an appropriate antigen such as a foreign bacteria, contact with complement

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ACTIVATION ~y

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UNITS

Alternative

Classical

E.

Properdin Activator

C4

P

UNIT

Figure 1.

Pathways of complement activity.

is made rapidly and occurs through the interaction of antibody and C 1. Fixation to antibody occurs through the CIq portion of CI and enzymatic activity is generated from the CIs portion of that moleculeY Clhas specificity for C4 cleaving it into two fragments, C4a and C4b. C4b fixes to the immune complex usually at a site adjacent to the antibody-Cl site,15 ACTIVATION UNITS ,j,

C3

IMMUNE ADHERENCE } PHAGOCYTOSIS REGULATION OF B CELL FUNCTIONS BONE MARROW RE~EASE OF PMN/S RELEASE OF AG-AB COMPLEXES TRIGGERS TUMOR CELL DIVISION

~C3"

{ VIRAL NEUTRALIZATION (C4b) C2 KININ? CILIARY DYSKINESIA CHEMOTAXIS { HISTAMINE RELEASE SMOOTH MUSCLE CONTRACTION INCREASED CAPILLARY PERMEABILITY

C3b

I C5 ~C5a

CHEMOTAXI S { HISTAMINE RELEASE SMOOTH MUSCLE CONTRACTION INCREASED CAPILLARY PERMEABILITY

C5b----YEAST OPSONIZATION

,,1

C7

C5b-C6-C7 - - - CHEI10TAX I S C81 C9 C5b-C6-C7-C8-C9----CELL MEMBRANE DAMAGE Figure 2. Biologic activity of complement.

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C1 also has specificity for native C2 and cleaves that molecule into two fragments, C2a and C2b. C2a then complexes with cell-bound C4b. 64 The complex, C4b,2a, then becomes a potent enzyme whose natural substrate is native C3. The action of that enzyme is to convert native C3 into an active state and, therefore, this enzyme has been given the designation of C3 convertase. 55 It will become apparent that there are a number of C3 convertases in the various activation units but this was the first one to be described and is, therefore, referred to as the classical C3 convertase. The assembly of the C3 convertase completes this activation unit which implies a lack of biologic activity in the reactions of C1, C4, and C2. This is not entirely true as will be seen later but is true enough to be useful in organizing our thinking. ALTERNATIVE UNIT. Until 1954, the classical unit was the only known activation unit. At that time, Louis Pillemer described an alternative activation unit involving a new serum protein, properdin, and at least two other serum factors, A and B.63 In a large series of papers, Pillemer and his associates carefully detailed this reaction mechanism which bypassed antibody, C1, C4, and C2, and directly interacted with C3. To say the least, this discovery met with less than an enthusiastic reception. The waters of the cascade were unmanageable and Pillemer and his work were swept away by the torrential rush of scientific judgment. Two decades later, the "properdin system" was rediscovered. This "new" activation unit was termed the C3 activator system and required at least two distinct serum proteins designated C3 pro activator (C3PA)33 and C3 proactivator convertase (C3Pase).56 The similarity to Pillemer's system was duly noted and C3P A was subsequently shown to be identical to Pillemer's factor B while properdin was finally implicated in the early part of this unit. 34 There is little question that the formulation of this unit represents one of the most striking and important discoveries in the field of complement. Acceptance of this concept, unfortunately, occurred 20 years too late! The new activation unit was an alternative to the classical one and hence, the derivation of this now widely accepted name. The reaction mechanism of this unit is still being debated. Current concepts envision the fundamental base of this unit to be composed of a complex of C3b, a cleavage product of native C3, and factor B. The assembly of this complex is for the purpose of activating C3 and it is, therefore, another C3 convertase. The generation of C3b for the formation of this complex is critical and occurs through the action of another enzyme in this unit designated initiating factor. This material was first described in 1969 in an entirely different context and was named C3 nephritic factor (C3NeF) to denote the fact that it occurred in the serum of patients with membranoproliferative glomerulonephritis. 81 C3b may also be generated by the classical activation unit through its interaction with C3. Two vital features of the formation of the alternative C3 convertase, then, are apparent. First, any reaction which elaborates C3b can predispose to the formation of the basic structure of this activation unit. Second, the C3bfactor B complex is a self-generating enzyme by virtue of its ability to react with more native C3 and thereby elaborate more C3b. Thus the for-

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mation of this enzyme represents a very powerful amplification loop for any of the other activation units. The active part of the alternative C3 convertase resides in the factor B molecule which may exist in two different forms. It is active as an intact molecule in the C3b-factor B complex but is very unstable; it may be stabilized by the addition of activated properdin. 26•74 Of more importance, however, is that the C3b-factor B complex may interact with as yet another enzyme in this unit, factor D.27 This results in cleavage of factor B into two fragments, Ba and Bb, both with electrophoretic mobilities different from native factor B. Bb is the enzymatically-active fragment. This change in electrophoretic mobility is often used to detect the fact that factor B has reacted and the finding of Bb in the plasma of a patient is prima-facie evidence that the alternative activation unit has been invoked in vivo. Thus the complete alternative activation unit is composed of initiating factor, C3, factor B, activated P, and factor D. It is important to emphasize that the formation of the alternative C3 convertase requires the prior cleavage of C3, a process which usually involves the elaboration of biologic activity. This functional activity is minimal with initiating factor and involves only the generation of small amounts of C3b. The major cleavage of C3 occurs after the activation offactor B and that is why the reaction of initiating factor and C3 are included as part of this activation unit. In the amplification loop, however, much biologic activity may be attendant to the generation of C3b so that formation of this loop is a great exception to our arbitrary separation of activation and functional units. THE PROPERDIN ACTIVATOR UNIT. This unit involves the primary participation of properdin without the advent of factor B in the formation of a C3 convertase. Initiating factor is also part of this unit and interacts with another serum protein, properdin convertase, to convert it to its active state. 82 Activated properdin convertase then cleaves native properdin into two fragments, one with enzymatic specificity for C3 and the other with specificity for C5. Thus separate and distinct C3 and C5 convertases are generated from properdin in this activation unit. 80 The C3 convertase can also initiate the formation of the C3b-factor B dependent C3 convertase in the alternative activation unit and is yet another means of generating the amplification loop. The properdin activator unit is almost exclusively a fluid phase mechanism; in the presence of an insoluble polysaccharide, it is modulated so as to generate only small amounts of C3b which may represent an important mechanism for the in vivo regulation of the formation of the alternative activation unit at a surface site. The major function of the properdin activator unit, therefore, may well be to activate properdin, generate C3b, and control both the formation and stabilization of the more potent and important factor Bdependent C3 convertase. Clinical data, however, suggest that this unit may also function on its own in the total absence of factor B. CI BYPASS ACTIVATION UNIT. This activation unit is a combination of the classical and alternative units. It involves the activation of CI by high concentrations of IgM antibody, and the interaction of CI with factor B through an as yet unknown mechanism which may involve pro-

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perdin. 45 C4 and C2 are not involved in this unit. This, then, is a second way for the classical pathway to initiate the activity of factor B and the alternative activation unit. The exact role of this unit in various in vivo disorders is not yet clear although it has been postulated to be responsible for a small number of cases of urticaria. NONCOMPLEMENT-DEPENDENT ACTIVATION UNITS. The importance of these "units" are totally unclear. It is known that both plasmin and thrombin can directly interact with C3 and mediate its activation. 12 Further, certain bacteria can elaborate enzymes which, although most inclined to inactivate C3, may initiate a process of activation prior to cleavage and inactivation. The most interesting of these materials is that elaborated by Pseudomonas aeruginosa75 although other organisms can produce similar materials. 91 INITIATION OF THE COMPLEMENT ACTIVATION UNITS. All of the factors which govern the initiation of the various units have not been elucidated. It has already been indicated that immune complexes composed of IgG or IgM antibodies can initiate the classical unit; C-reactive protein under certain conditions also has that ability. 78 Further, it is now known that complex polysaccharides, yeast cell walls, inulin, endotoxin, IgA and IgE antibodies, and certain bacterial and viral membranes can initiate the alternative activation unit. C-reactive protein in serum can also promote this unit and therefore has a dual propensity for the initiation of complement. Similar materials can apparently initiate the properdin activator unit, and it is known that IgM antibody in large concentrations can initiate the CI bypass activation unit. It is worthwhile to stress that any reaction which generates C3b can mediate the formation of the alternative activation unit (amplification loop). The coagulation system and its multiple enzymes, as well as the kinin-forming system and the prostaglandins are also capable of interacting with or initiating the complement system. 73 The entire relationship of the complement sequence to these "inflammatory" systems is not yet clear, but it is becoming increasingly more obvious that they are all intimately intertwined. Finally, it is important to mention that venom from the Naja Naja cobra complexes with factors Band D to form a potent C3 convertase. Since the active principle in the venom is cobra C3b, this enzyme is the same as that usually seen in the alternative activation unit. 3

The Functional Unit The biologic activity which resides in the complement system is primarily derived from the activation of the terminal components, C3 to C9. Most of the information about the basic immunochemistry of these components comes from studies done with the classical C3 convertase and it is assumed that other activation units produce similar changes in C3 to C9. Activation of C3 is the pivotal step in this unit. When this occurs, C3 is cleaved into two fragments, C3a and C3b, both of which have biologic activity. C3a remains in the fluid phase while C3b fixes next to a C4b,2a site. 55 Bound C3b represents a receptor site for C5 and C4b,2a,3b then be-

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comes a C5 convertase. 76 C2a is the active portion of the C5 convertase as it was with the C3 convertase. C5, like C3, is cleaved into a smaller fragment, C5a, and a larger fragment, C5b. Both have biologic activity. Again, as with the cleavage products of C3, C5a remains in the fluid phase while C5b is bound. The deposition of C5b is necessary for the activation and deposition of C6. The enzyme C56 is unstable, but is necessary for the activation of C7. The addition of C7 and the final two components, C8 and C9, result in a large C56789 complex on the cell. 39 The assembly of the functional unit, then, is a true cascade-like process but with biologic activities attendant to the split cleavage products generated as well as to the large complexes formed on the cell surface.

BIOLOGIC FUNCTIONS OF COMPLEMENT

The precise role that the complement system plays in the biologic response of the organism is not totally or completely understood. It is only recently that we have begun to recognize that the ability of this sytem to lyse a cell reflects only a small part of its overall in vivo nmction. Moreover, it is eminently clear that in the host's resistance to infection, lysis of various organisms is of secondary importance to other biologic aspects of this system. All of the activation units began with an attack on C3. The liberation of C3a results in the chemotaxis of polymorphonuclear leukocytes as well as certain other phagocytic cells, histamine release from basophils and mast cells, smooth muscle contraction, and an increase in capillary permeability.24 It is also thought that C3a can produce ciliary dyskinesia (seen in patients with cystic fibrosis) but this feature is currently being disputed. 19 In addition, C3a has the ability to either induce the release of leukocytes from the bone marrow71 or to mediate sequestration ofleukocytes in the marginal pool. 46 It is hypothesized, therefore, that C3a is important in various types of neutrophilia and neutropenia. Certain key and critical elements of the inflammatory process are, therefore, contingent on the biologic function of C3a. The larger fragment of C3, C3b, is capable of depositing on a suitable receptor site. Two such receptor sites are known, one created by a C3 convertase and the other present as an integral part of certain cellular membranes. 42 In certain circumstances, these two different receptor sites function synchronously as in the case of C3b being deposited on a bacteria by the classical activation unit. Once this has occurred, the bound C3b then allows for the attachment of those cells which have a C3b receptor present de novo. Classically, this would be the polymorphonuclear neutrophil and such a process is necessary to initiate phagocytosis. C3b is required for internalization of an antigen but not for the process of intracellular killing. Monocytes and macrophages also have a C3b receptor, and it is presumed that it is through this site that they too initiate phagocytosis particularly in the liver and the spleen. It is of great importance that B type lymphocytes also have a cell membrane C3b receptor site. 10 These cells therefore will adhere to bound C3b; in addition, C3b from the fluid phase can deposit on these B lym-

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phocyte receptors. The attachment of C3b in this situation induces regulation of certain B cell functions such as antibody production in response to T cell-dependent antigens,25 production of lymphokines chemotactic for macrophages, and proliferation induced by B cell mitogens. 4O Some of these data have recently been challenged, particularly those dealing with antibody production, but the intriguing possibility that complement may play an important role in the early phase of the entire immune response needs to be considered. More recently, it has been noted that certain tissues, most notably the glomerulus, also have C3b receptor sites. It is postulated that these receptors function in immune clearance by the kidney.32 In addition, certain phagocytic cells and B lymphocytes also have membrane receptors for a further cleavage product of C3b, C3d. When bound C3b is cleaved, C3c is discharged into the fluid phase and C3d is left behind. 72 Since this is a reasonably rapid process in vivo, the presence of C3d receptor sites on phagocytic cells, most notably the macrophage, may be for the purpose of removal of C3d-coated cells in the liver and spleen. The function of the C3d receptor on B lymphocytes is not yet known. 70 C5 is the next reacting component in the sequence and is responsible for many activities that are similar to C3. Thus C5a can also mediate chemotaxis, histamine release, smooth muscle contraction, and capillary permeability. 77 This is clearly not a simple process of duplication of effort and one must be impressed by the specificity of the immune system. C3a and C5a apparently act at independent sites on the cell membrane producing chemotaxis and smooth muscle contraction so that tachyphylaxis for one cannot be induced by the other. 17 Further, C3a and C5a are not necessarily chemotactic for the same cell types and have certain specificities which are mediated by unknown factors. C5b can also participate in the chemotaxis of polymorphonuclear neutrophils when it is complexed with C6 and C7. 92 It has none of the other properties of C3b, however, and is probably unnecessary for phagocytosis with the one possible exception of yeast particles. 52 In that situation, C5b may be of primary importance; these data, however, have recently become suspect and the question of the role of C5b in yeast opsonization is still open. 68 The functional capabilities of the remainder of the terminal components represent relatively unexplored territory. In rabbits, but probably not in man, C6 is involved in coagulation. 36 Further, the attachment of C8 and C9 to C567 are necessary to efficiently complete the destruction of a cellular membrane leading to osmotic lysis. Our present state of knowledge does not extend beyond these limits. Several other functions of the entire unit are important, however, even though the critical steps have not yet been clearly delineated. Thus these components participate in release of immune complexes from cells, platelet release phenomena and lysis, endotoxin detoxification, viral lysis, and possibly activation of tumor cells with increased growth. The details of these activities are beyond the scope of this review. 30 Two further issues concerning the role of complement in the biology of the organism need further discussion. Since there is evidence for the

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existence of factor B in the hemolymph of the starfish 21 and it is known that C3-C9 appear earlier than C 1, C4, C2 or gamma globulin in the phylogenetic sequence,86 it is perhaps not unreasonable to postulate that the complement system initially functioned in the inflammatory process as a nonspecific host defense. As evolution proceeded, antibody and the classical activation unit were added to achieve specificity. The issue as to why these alternative units have persisted in the face of such advanced technology becomes of great interest. Tauber and associates 86 have shown that certain bacterial membranes can directly initiate the alternative activation unit. More importantly, these authors have demonstrated that certain antigenic sites on these bacterial membranes are similar to sites on several normal tissues. These data have led.to the rather exciting postulate that the alternative activation units have remained viable so as to provide a way to handle infections with these organisms without the necessity for antibody formation which right be self-destructive by virtue of cross reactivity with the normal tissues. The fact that certain types of bacteria such as staphylococci and E. coli are much more efficiently phagocytosed and killed when antibody is present and the classical C3 convertase is active, and that other types of these same bacteria are more effectively and efficiently handled by the alternative activation units, may be a manifestation of that postulate. 29 The implication, then, is that each activation unit may inherently contain some mechanism for specificity or lack of specificity which is critical to our survival. The hypothesis that the complement system functions as "grand modulator" of the immune system is worthy of further consideration. In a sense, the activity of complement regulates the activity of polymorphonuclear neutrophils and other phagocytic cells, helps to control their numbers and direction of movement, may influence coagulation and platelet release phenomena, controls the activity of antigen-antibody complexes, regulates the production of certain antibodies, may be involved in prostaglandin synthesis, and possibly controls the basic process of B cell proliferation. These diverse functions seem to indicate a regulatory rather than an effector mode of action. The fact that patients with inborn complement deficiency states have an increased incidence of collagen vascular disorders may suggest that these various rissing components are important in regulating our immune response to certain stimuli. It is perhaps naive for us to continue to classify complement activity as a mere pathway for inflammation.

MECHANISMS FOR CONTROL OF COMPLEMENT ACTIVITY

One must be concerned with the biologic constraints on this system. Although most of the active enzymes and activated cleavage products have exceedingly short half-lives, it is unlikely that control of the entire system is left to molecular chance. Thus far, multiple inhibitors and at least one accelerator have been described. In the classical activation unit, there is an inhibitor which prevents the action of C 144 and an inacti-

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vator which acts directly on C4b by cleavage of that molecule. 20 In the alternative activation unit, the most notable regulator is C3b inactivator72 which cleaves C3b into C3c and C3d. In the functional unit, an inhibitor of C5 deposition 59 and inactivators of C6 and C7 93 have been described, but their exact function and role have not been clarified. Further, serum contains an inhibitor of the C56 complex47 and two inactivators of C3a and C5a, one known as anaphylatoxin inactivator 12 and the other as chemotactic factor inactivator. 9 Most recently, an accelerator of C3b inactivator activity has been described 94 as well as a C3b stabilizer whose major function is to enhance the efficiency of C3b and prevent its destruction by C3b inactivator and accelerator. 79 The role of these materials in modulating the complement sequence is not yet clear.

SYNTHESIS, ONTOGENY, AND GENETICS

There is relatively little clear information concerning these areas. Clq, r, and s seem to have independent synthetic sites; synthesis of macromolecular Cl, however, has been found in the epithelial cells of the gastrointestinal tract.18 Macrophages apparently produce C4 and C2 while the liver synthesizes C3, C5, C6, and C9 (and possibly C2).18 Evidence for the existence of the alternative activation unit is present in the hemolymph of the starfish and other invertebrates; the classical activation unit has not been found in these species. In man, synthesis of complement can occur as early as the eighth week of gestation and precedes the appearance of immunoglobulins. Numerous genetic deficiencies exist and genetic polymorphism is present for C3 and factor B.6 Finally, synthesis of various complement components have been linked to the H-2 gene in mice and the HL-A system in man. 95 Whether this indicates a role for complement as a recognition unit is not clear.

INFECTIOUS DISEASES INVOLVING COMPLEMENT The in vitro evidence which allows that the complement system is critical to the basic process of elimination of foreign invaders is considerable. As previously detailed, this system is a dominant force in mediating inflammation, phagocytosis, and cytolysis. When the functional unit is activated, both C3a and C5a allow for histamine release from mast cells and basophils, smooth muscle contraction, an increase in capillary permeability, and leukotaxis ofneutrophils, eosinophils (C5a), and mononuclear cells. 24.77 A small molecular weight cleavage fragment (C3a?) from C3 is also responsible for bone marrow release of neutrophils. 71 Besides providing for these elements of inflammation, complement may also be involved in a direct attack on pathogenic agents by cytolysis; the entire functional unit is necessary for this process and theoretically may be activated by any of the activation units. Further, by the deposition of C3b on the surface of the offending organism, the complement system promotes phagocytosis by providing a contact point between organism and

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phagocyte and allowing for internalization. C3 activation may also be important for initiating oxidative metabolism of the polymorphonuclear neutrophil 84 and its release oflysozymal enzymes. Finally, by interaction with B type lymphocytes, C3b may also mediate the release of chemotactic factors from macrophages and further B cell proliferation and perhaps antibody formation. 25,4O Clearly, complement forms a vital link in host resistance to infection. All of these processes occur during infection with bacteria, parasites, or yeasts. With viral infestation, cytolysis and phagocytosis also occur but, in addition, complement participates in a third process, that of neutralization. 2,65 This process involves the prevention of the virion from entering a target cell or otherwise interfering with its replication. Neutralization requires either the deposition of Cl, C4, and C2 or the fixation of C3 onto the virus. Depending on the type of virus, activation and deposition of C3 can occur by the classical or the alternative activation unit. Antibody is not necessarily required and it is now known that some viruses can directly activate Cl by contact. 48 Despite these considerations, it is quite clear that many defects in complement are not associated with recurrent infections. Patients with deficiencies in an activation unit (except those who lack C3) usually do not manifest any marked increase in the incidence of infections even though there is an element of specificity to each unit and even though different units eliminate certain organisms at different rates and efficiencies. Presumably, this situation exists because these units have enough overlap in their activities to compensate for the above differences. Deficiencies in the functional unit are likewise of little importance (except for C3) since most of the functional activities necessary for host resistance are embodied in C3 activity. This feature underlines the fact that opsonization and phagocytosis are far more important than cytolysis in preventing infection. The reader must be aware that the normal physiologic process of complement activation and function can also be responsible for considerable pathology during various infectious disorders. Thus elimination of certain organisms and products of these organisms by the normal activity of complement can result in severe and dire consequences. In these instances, complement activity is initiated in an area which cannot escape the effects of inflammation (immune complexes lodging in the glomerulus) or by an abnormal initiating agent (Le., endotoxin); the resultant pathology, then, is related not to a disorder of complement per se but rather to a disorder involving complement. This is a critical distinction and will be elaborated on as we progress. It is obvious that, because of its role in host defenses, complement activity is initiated during the course of most infections. This activity is totally unrelated to the pathogenesis of the disease process. Complement is a participant, an important traveler, a conductor on the railroad; it is not, however, intimately involved in the generation of clinical symptoms. These situations, by and large, will not be included in this discussion. One must be very clear on this point for it is more than a matter of semantics. In this review, disorders involving the complement system relate only to those diseases in which complement is associated with pathogenesis. Thus numerous studies which do not fit these guidelines

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have been omitted. Deposition of various components on organisms, local accumulation of complement fragments in an area of inflammation, even systemic consumption of certain components are all data which one might expect in many infectious diseases; they cannot necessarily be misconstrued as implicating this sytem in the development of the symptoms of those diseases. These same data, however, are important in other situations in which complement is involved in pathogenesis. For our purposes, those infections which are associated with the complement system by pathogenesis have been classified into four basic groups. These depend upon which unit of complement is primarily involved and whether that unit is normal or abnormal. This system has some usefulness in grouping these disorders and allows for use of the various complement tests in diagnosis and follow-up on a somewhat organized basis.

Infections Associated with Normal Activation and Functional Units These are of several different types. Of utmost importance are those infections-bacterial, viral, parasitic, or fungal-which by their products or by virtue of the formation of immune complexes, initiate complement activity at a distant site and produce symptoms unrelated to the offending organism. Patients with gram-negative infection may go into shock, have disseminated intravascular coagulation or platelet dysfunction because of direct initiation (by endotoxin?) of their alternative activation unit. 28 Equally fulminating are the formation of circulating immune complexes following infection as with certain viral infections, such as dengue virus, 13 herpes, infectious mononucleosis,90 or with lepromatous leprosy.66 In these cases, the systemic signs of the disease are probably all due to the immune complexes. Dengue virus is particularly noteworthy for the shock associated with that disorder correlates well with the development of immune complexes and initiation of the classical activation unit. More subtle is the immune complex formation seen in the chronic infection of subacute bacterial endocarditis or that associated with infected ventricular jugular shunts. 85 Here, the complexes frequently lodge in the kidney, initiate the classical activation unit, and produce glomerulonephritis. It is noteworthy that in our experience with this disorder, serum properdin levels are reduced as well as serum levels of Cl, C4, C2, and C3. The involvement of properdin may be extensive and long lasting (as long as eight months in one of our patients). Another important example of infection associated with the normal functioning of the complement system is viral hepatitis. It is now clear that Australia antigen positivity correlates well with decreased serum levels of complement. In acute viral hepatitis, serum concentrations of C3, C4, and C6 are frequently decreased (40 to 95 per cent of patients depending on the study). It is noteworthy that the low levels of C3 only occur initially and return to normal by one week. 41.88 In addition, C3 conversion products may be found in the circulation. 87 Thus in active liver inflammation, the classical activation unit and the functional unit are initiated. In patients with this disorder who develop arthritis and other systemic

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signs, there is evidence for in vivo activation of factor B with immune complexes and cryoglobulins containing B. These data suggest the involvement of the alternative activation unit but primarily as a secondary phenomena. 89

Infection Associated with Abnormal Activation Units and a Normal Functional Unit In this category can be found patients with Clr,23 C2,22 and C4 60 deficiencies (classical activation unit). These patients may be more susceptible to certain kinds of bacterial and viral infections, but the actual incidence of these infections is not increased. The literature is exceedingly confusing, but it appears that the ultimate generation of chemotactic factors and the process of phagocytosis may be normal in these patients due to the other activation units. The rate and efficiency of these processes, however, are distinctly abnormal and, on theoretical grounds, could be responsible for organisms gaining a foothold. 31 These differences in efficiency are also related to the types of organisms being studied. 67 Further, there is substantial evidence that a cooperative effect may exist between several of the activation units. Why these features do not contribute to an actual increased incidence of infection is yet to be detennined. Using similar reasoning, it might be expected that defects in those activation units other than the classical one would also lead to an increased incidence of infections. This might be particularly true at those times when antibody and the classical unit are relatively ineffective as in the newborn period or when the offending organism is eliminated primarily by means other than antibody. Little data are available to this point. A patient with a deficiency of properdin (20 percent of normal) has been described 58 who had numerous episodes of sepsis during the first six weeks of life. As he became older, the number and severity of infections decreased and he is now perfectly well at age two despite the fact that his serum level of properdin has not increased. Patients with sickle cell disease37 have an increased incidence of infection with pneumococcus which is due to a number of factors, not the least of which is a defect in the alternative activation unit. This has previously been ascribed to an abnormality of factor B but more recently those data have been challenged and the defect ascribed to a deficiency or malfunctioning offactor D.38 Finally, newborns are known to be notoriously susceptible to infection with gram-negative organisms because of a variety of defects. One of the more important of these is the relative physiologic deficiency that exists for C3, C5,49 and factor B. 83 It has been postulated that the low serum levels of C3 and factor B may be responsible for the opsonic defects which have been noted and the increased susceptibility to infection. Data accumulated in our laboratory do not support this postulate. Despite very low levels of C3, properdin, and factor B in cord sera (115 samples), the ability of both the activation and functional units to be utilized in the sera by zymosan or cobra venom factor is normal. Further, by reactive lysis experiments, these sera are also normal implying a completely adequate functional unit for cell destruction. If there is enough of

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these materials to nonnally activate and utilize the functional unit of complement, one must wonder about the significance of the studies implicating factor B and C3 in the phagocytic and bactericidal defects. Infections Associated with Normal Activation Units but an Abnormal Functional Unit It would seem that the incidence of infection should be increased in patients with congenital defects in C5,69 C6,43 C7,14 or CB.62 In vitro abnonnalities exist in lysis of antibody-coated bacteria, and defective chemotaxis is present in the C5-deficient patients; an increased susceptibility to certain isolated bacteria in vivo has also been reported. The incidence of repeated clinical infections, however, is not very great. This observation would fit with the fact that opsonization plays a greater role in host resistance than does cytolysis. The high incidence of collagen vascular disorders in these patients as a group, however, may represent an undue susceptibility to viral infections although this has not been proved. Included in this classification are those patients with C5 dysfunction. 51 This entity is of extreme importance because, in a sense, it may well be a portent of the future. C5 in these patients is present in nonnal amounts and is even hemolytic ally active, but it will not specifically opsonize yeast particles. The patients have severe seborrhea and local and systemic infections (usually gram-negative) in a syndrome similar to that first described by Leiner. The defect has been corrected in vivo by infusions of plasma in two patients. These data are somewhat suspect because of a recently described congenitally C5-deficient patient who is not deficient in the opsonization of yeast particles. 69 A C5-like material was present in her serum, however, which may account for this finding 53 and which may be similar to that recently found in breast milk. 50 It is of considerable interest that there may be a defect associated with increased activity of the functional unit in the face of nonnal activation units. In patients with cystic fibrosis, there appears to be an excess of ciliary dyskinesia factor which is reportedly identical to C3a. 19 The evidence for this association is good, but the exact reason for the generation of C3a is not clear. Nevertheless, this material may be of some importance in the pathogenesis of the pulmonary infections in these patients.

Infections Associated with Abnormal Activation and Functional Units In this category are found the clearest examples of infection associated with complement abnonnalities. Patients with congenital C3 deficiency have decreased activity in both units. These patients have enormous problems with infection and are totally blocked in fonning the alternative activation unit and in many important biologic activities via the functional unit. At least four such patients have been described 5.8 •35,61 and all but one 61 have rather severe and life-threatening infections. Let me repeat: one does not! Understanding this patient's host defenses is obviously critical to our understanding of the role of complement in in-

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fection. It is perhaps an understatement to speculate that this patient represents an example of some additional mechanism for opsonization. One almost feels like riding through the countryside screaming "Another mechanism is coming, another mechanism is coming." Perhaps the C4 immune adherence mechanism with the recently described activation and functional units contained in C142 (without C3) has taken over.54 Perhaps the C3 bypass mechanism for C5 activation in the properdin activator unit provides compensation in this patient. Patients with severe thermal injury,l1 malnutrition,16 and perhaps newborns (though not supported by our own data as described earlier) often have defects of both units leading to increased susceptibility to infection. In patients with burns, one might think of the loss of C3 along with other plasma proteins; in patients with malnutrition, a consideration of failure of synthesis of C3 might be entertained. Evidence for both of these is present, but there is more. In burn patients, there are data to suggest the generation of an inhibitor of C3 activation. 11 In patients with malnutrition, there is evidence of increased C3 catabolism (? secondary to preexisting infection). 16 These data are not clear enough at this time to allow further interpretation. Further, not only do the hands of fate influence the level of C3, but also the offending organism may playa role in this regard. Serratia and pseudomonas organisms are able to elaborate a C3-cleaving enzyme which, at least theoretically, can protect them from attack. Thus a local C3 deficiency state can be induced which may be responsible for the increase and virulence of these organisms. Finally, there are two examples of recurrent infections related to an increased activity of both units of the complement cascade. Alper has reported one case of hypercatabolism of C3 due to some unknown C3cleaving enzyme in a patient with severe infections. 4 More importantly, a patient has been described with multiple complement abnormalities secondary to the underlying defect of an absence of C3b inactivator. 1 In this patient, the control mechanism for regulating C3b activity is missing and the alternative activation unit is in a constant state of activity. C3 is constantly utilized and the ultimate end result is hyperactivity followed by exhaustion of both the activation and functional units. This patient has severe and significant infections as expected.

Clinical Usefulness The confusion of the complementologist is of little help to the clinician when faced with a patient with recurrent infection or systemic symptoms secondary to infection. But that is to be expected with such a rapidly evolving field. The clinician is interested, perhaps mildly enthusiastic, about the basic science data but he clearly seeks some kind of perspective from which to view this work. What is sorely needed is some way to utilize these data in clinical situations. I can offer only limited direction in this regard for the bold truth is that the field of complement has not reached the stage of easy access or practical value. Before discussing what might be helpful, even on a limited basis, certain useful guidelines are worthy of attention:

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Table 2. Complement-Associated Infectious Diseases Disorders involving normal activation and functional units Active viral hepatitis (acute and chronic active) Immune complex formation in various infections Endotoxemia secondary to infection with gram-negative organisms Disorders involving abnormal activation units with a normal functional unit Congenital component deficiencies (Clr, C2, C4, P) Infections in patients with sickle cell disease and in the neonate Disorders involving normal activation units and an abnormal functional unit Congenital component deficiencies (C5, C6, C7, C8) Leiner's disease Cystic fibrosis Disorders involving abnormal activation and functional units Congenital deficiency of C3 Congenital deficiency of C3bINA Hypercatabolism of C3 Infections in patients with thermal injuries, malnutrition, and in the neonate.

1. Patients with recurrent infections may well have complement

abnormalities other than deficiencies of one component or another. These may be on a functional basis or by virtue of increased or decreased activity of one of the complement units (Table 2). 2. Standard quantitative assays for complement components are not sufficient to ascertain many of these defects and kinetic assays for complement activity and assays for complement-dependent functions need to be done. When these involve bactericidal or phagocytic studies, care must be exercised because certain defects predispose to abnormalities for one organism and not another. 3. At the present time, complement deficiencies per se probably account for only a small number of patients with recurrent infection. Patients with functional abnormalities may be of greater number, but we are just beginning to understand and appreciate this fact. Assays to identify these patients are crude and difficult to do; this area, then, must await further progress before it can be clinically useful. Now, patients with functional abnormalities are research projects by themselves; in the future, they may well form the basis for clinically relevant classification and therapy. 4. Complement assays are useful in four general areas: (a) detecting those underlying conditions which predispose to infection (Tables 3 and 4), (b) detecting certain systemic conditions resulting from infection (Tables 3 and 4), (c) predicting when infection might become a problem in certain predisposing conditions (burns, malnutrition), (d) following therapy of certain of the systemic conditions associated with infection (particularly immune complex formation, Table 4). Adequate testing for complement abnormalities in these areas include quantitation of individual proteins by assays of hemolytic activity or by radial immunodiffusion techniques, evaluation of the ability of

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Table 3. Summary of Complement Abnormalities in Infectious Diseases Predisposing to infection Congenital component deficiencies Factor B or D malfunction (sickle cell disease) Physiologic deficiency of C3, C5, P, or factor B (neonate) Component dysfunction (C5 in Leiner's disease) Excessive production of ciliary dyskinesia factor (C3a) in cystic fibrosis Hypercatabolism of C3 Congenital deficiency of C3bINA Malfunction or deficiency of C3 in patients with thermal injuries and malnutrition

Resulting from infection Initiation of the classical and/or alternative activation units and the functional unit by immune complex formation in various infections Initiation of the alternative and properdin activator activation units and the functional unit by endotoxin in infections due to gram negative organisms Initiation of the classical activation unit and functional unit in active viral hepatitis (acute and chronic active hepatitis)

Table 4. Most Common Complement Abnormalities Detected in Infectious Diseases ABNORMAL COMPLEMENT ASSAY

CONDITION

At Diagnosis

To Follow During Therapy

Predisposing to infection Congenital component deficiency

Decreased CH•• Specific component deficiency

N.A.*

Neonates

Decreased serum C3, C5, P, factor B

N.A.*

Leiner's disease

Defective yeast phagocytosis

Hypercatabolism of C3

Decreased serum C3

N.A.*

Congenital deficiency of C3bINA

Decreased serum C3bINA, C3, factor B

C3

Thermal injuries

Decreased serum C3 (early) Defective chemotaxis, phagocytosis assays

Malnutrition

Decreased serum C3

Yeast phagocytosis

Phagocytosis assays

C3

Resulting from infection Immune complex formation

Decreased serum C4, C3 properdin

EndotoxeInia

Decreased serum C3, factor B

C3

Active hepatitis

Decreased serum C4, C3

C3

* N.A. = not applicable.

C4,P

• I

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each activation unit to be initiated and mediate consumption of the functional unit, and more specific assays such as the ability to generate chemotaxis and induce opsonization. Much of this is obviously beyond the means of the usual clinical laboratory. Let me suggest a more simplified approach which, although limited, is nevertheless useful. By this approach, with three readily available and inexpensive assays, the bulk of the complement deficiencies can be detected and many of the complications attendant to infection can be ascertained. In addition, further testing can be more specifically indicated which can often be accomplished by additional commercially available assays. By this scheme and armed with the information presented in this review, many of the advantages of the more complete complement assays can be achieved. The most common complement abnormalities detected in conditions either predisposing to or resulting from infection are listed in Table 4. Also included in Table 4 are those tests which are the best ones to follow in certain conditions. It is to be emphasized that a strict reliance on Table 4 is dangerous. Many variations can occur and often more than one activation unit can be utilized. The reader is referred to the text for many of these potential variations. Despite these pitfalls, by testing for total hemolytic complement (CH 50), C3, and C4, many of these disorders can be delineated. The CH50 relies on the presence of C1-C9 and the ability of these components to lyse antibody-coated erythrocytes. The dilution of the patient's serum which is able to lyse 50 per cent of such cells is arbitrarily used as an end point (hence, the name CH50 or 50 per cent complement hemolysis). Assays for serum C3 and C4 are routinely done by radial immunodiffusion which uses precipitating antibody to measure the amount of specific protein in the serum. It is to be noted that these latter tests in no way tell us whether or not that protein is functional. All three of these tests have great variability, and the reader is cautioned about overinterpreting results. Actually, the CH50 is used for one specific purpose only so that most of the reliance is on whether C3 and C4 are normal or not. Four general situations exist: (1) When C4 is reduced, a reduction in C3 implies that the classical activation unit has been initiated and the functional unit activated. This occurs characteristically in active viral hepatitis and immune complex formation in various disorders. (2) When C3 is normal in the presence of a low C4, a congenital deficiency of C4 is suggested (also seen in hereditary angioneurotic edema, certain patients with systemic lupus erythematosus and malaria). (3) When the C4 is normal and the C3 is low, congenital deficiency of C3 or C3bINA is indicated. More importantly, these findings are characteristic of activation of the functional unit by initiation of one of the nonclassical activation units such as that which occurs in endotoxemia secondary to gram-negative sepsis. In this situation, quantitation of the serum level of factor B (commercially available as RID plates) would be necessary to differentiate congenital C3 deficiency from C3bINA deficiency, endotoxemia, or some other process initiating the alternative activation unit. It is noteworthy that both factor B and C3levels can be low when the classical activation unit is initiated and the alternative activation unit is recruited via the ampli-

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fication loop as in the arthritis secondary to viral hepatitis. (4) If both the C3 and C4 are normal, the CH50 becomes important. If the CH50 is low, the indication is of an isolated deficiency of one of the complement components and further, more specific testing is necessary. If the CH50 is normal, either the patient's complement system is normal, there is a functional deficiency somewhere in the cascade, or one of the ancillary proteins is abnormal. It is in this latter group that much misinterpretation can occur and several important disorders can be missed. It is unfortunate that no matter how extensive the testing or how astute the resident complementologist, there is no therapeutic advantage to the proper diagnosis. Perhaps that is to be expected at this time in our development, but it is a sad commentary on our priorities. Replacement therapy for Leiner's disease and C3bINA deficiency have been used to good advantage but these are the only examples of "specific treatment." Clearly, the pathway of the future is well defined. Summation It is hoped that the objectives of this review have been achieved. The extent of that achievement is, at best, debatable. To those of you who have labored to the end, the complexities will dissipate as you gain experience with this system. It will not be long before what we have discussed in this article will be merely a preamble to data which are truly of benefit in your efforts at the bedside. Evolution of a biological system proceeds stepwise and the few footprints presently available will eventually lead to a fully developed path. ACKNOWLEDGMENTS

The opportunity to write this review and much of its content reflect the hard work of my associates, most notably Ann Stitzel. As always, I am indebted to my fine technical staff, particularly Joan Urmson and Toni Putman. Mary Lou Farnett deserves a special note of gratitude for her patience and diligence in the preparation of this manuscript.

REFERENCES 1. Abramson, N., Alper, C. A., Lachman, P. J., et al.: Deficiency ofC3 inactivatorin man. J. ImmunoI., 107:19, 1971. 2. Allison, A. C.: Interactions of antibodies, complement components and various cell types in immunity against viruses and pyogenic bacteria. Transplant. Rev., 19:3, 1974. 3. Alper, C. A., and Balavitch, D. L.: Evidence that cobra venom factor (CoF) is cobra C3. J. ImmunoI., 116:1727,1976. 4. Alper, C. A., Bloch, K. J., and Rosen, F. S.: Increased susceptibility to infection in a patient with Type II essential hypercatabolism of C3. New EngI. J. Med., 288 :601, 1973. 5. Alper, C. A., Colten, H. R., Rosen, F. S., et al.: Homozygous deficiency of the third component of complement in a patient with repeated infections. Lancet, 2: 1179, 1972. 6. Alper, C. A., and Rosen, F. S.: Genetic aspects of the complement system. Adv. ImmunoI., 14 :251, 1971. 7. Assimeh, S. N., and Painter, R. H.: The identification of a previously unrecognized subcomponent of the first component of complement. J. ImmunoI., 115 :482, 1975. 8. Ballow, M., Shira, J. E., Harden, L., et al.: Complete absence of the third component of complement in man. J. Clin. Invest., 56:703, 1975.

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9. Berenberg, J. L., and Ward, P. A.: Chemotactic factor inactivator in normal human serum. J. Clin. Invest., 52:1200, 1973. 10. Bianco, C., Patrick, R., and Nussenzweig, v.: A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. Exp. Med., 132:702, 1970. 11. Bjornson, A. B., and Alexander, J. W.: Alterations of serum opsonins in patients with severe thermal injury. J. Lab. Clin. Med., 83 :372, 1974. 12. Bokisch, V. A., Muller-Eberhard, H. J., and Cochrane, C. G.: Isolation ofafragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. J. Exp. Med., 129: 1109, 1969. 13. Bokisch, V. A., Top, F. H., Russel, P. K., et al.: The potential pathogenic role of complement in dengue hemorrhagic shock syndrome. New Engl. J. Med., 289:996,1973. 14. Boyer, J. T., Gall, E. P., Norman, M. E., et al.: Hereditary deficiency of the seventh component of complement. J. Clin. Invest., 56:905, 1975. 15. Budzko, D. B., and Muller-Eberhard, H. J.: Cleavage of the fourth component of human complement by Cl esterase: Isolation and characterization of the low molecular weight product. Immunochemistry, 7:227, 1970. 16. Chandra, R. K.: Serum complement and irnmunoconglutinin in malnutrition. Arch. Dis. Child., 50:225, 1975. 17. Cochrane, C. G., and Muller-Eberhard, H. J.: The derivation of two distinct anaphylatoxin activities from the third and fifth components of hum an complement. J. Exp. Med., 127:371, 1968. 18. Colten, H. R.: Synthesis and metabolism of complement proteins. Transplant. Proc., 6 :33, 1974. 19. Conover, J. H., Conod, E. J., and Hirschhorn, K.: Studies on ciliary dyskinesia factor in cystic fibrosis. IV. Its possible identification as anaphylatoxin (C3a)-IgG complex. Life Sciences, 14:253, 1974. 20. Cooper, N. R.: Isolation and analysis of the mechanism of action of an inactivator of C4b in normal human serum. J. Exp. Med., 141 :890, 1975. 21. Day, N., Geiger, H., Finstad, J., et al.: A starfish hemolymph factor which activates vertebrate complement in the presence of cobra venom factor. J. Immunol., 109: 164, 1972. 22. Day, N. K., Geiger, H., McLean, R., et al.: C2 deficiency. Development oflupus erythema· tosus. J. Clin. Invest., 52:1601, 1973. 23. Day, N. K., Geiger, H., Stroud, R., et al.: Clr deficiency: An inborn error associated with cutaneous and renal disease. J. Clin. Invest., 51 :1102, 1972. 24. Dias da Silva, W., Eisele, J. W., and Lepow, I. H.: Complement as a mediator ofinflammation. III. Purification of the activity with anaphylatoxin properties generated by interaction of the first four components of complement and its identification as a cleavage product of C'3. J. Exp. Med., 126: 1027, 1967. 25. Dukor, P., Schumann, G., Gisler, R. H., et al.: Complement-dependent B-cell activation by cobra venom factor and other mitogens? J. Exp. Med., 139:337, 1974. 26. Fearon, D. T., and Austen, K. F.: Properdin: Binding to C3b and stabilization of the C3b-dependent C3 convertase. J. Exp. Med., 142:856, 1975. 27. Fearon, D. T., Austen, K. R., and Ruddy, S.: Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement. J. Exp. Med., 138:1305, 1973. 28. Fearon, D. T., Ruddy, S., Schur, P. H., et al.: Activation of the properdin pathway of complement in patients with gram-negative bacteremia. New Engl. J. Med., 292 :937, 1975. 29. Forsgren, A., and Quie, P. G.: Influence of the alternate complement pathway on opsonization of several bacterial species. Infection and Immunity, 10:402, 1974. 30. Frank, M. M.: Complement. Current Concepts. December, 1975, pp. 1-48. 31. Gallin, J. I., Clark, R. A., and Frank, M. M.: Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways. Clin. Immunol. Immunopathol., 3:334,1975. 32. Gelfand, M. C., Frank, M. M., and Green, I.: A receptor for the third component of complement in the human renal glomerulus. J. Exp. Med., 142: 1029, 1975. 33. GOtze, 0., and Muller-Eberhard, H. J.: The C3-activator system: An alternate pathway of complement activation. J. Exp. Med., 134:Suppl90, 1971. 34. GOtze, 0., and Muller-Eberhard, H. J.: The role of properdin in the alternate pathway of complement activation. J. Exp. Med., 139:44, 1974. 35. Grace, H. J., Brerton-Stiles, G. G., Vos, G. H., et al.: A family with partial and total deficiency of complement C3. South Afr. Med. J., 50:139, 1976. 36. Heusinkveld, R. S., Leddy, J. P., Klemperer, M. R., et al.: Hereditary deficiency of the sixth component of complement in man. II. Studies of hemostasis. J. Clin. Invest., 53:554,1974. 37. Johnston, R. B., Newman, S. L., and Struth, A. G.: An abnormality of the alternate

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pathway of complement activation in sickle-cell disease. New Engl. J. Med., 288:803, 1973. Koethe, S. M., Casper, J. T., and Rodey, G. E.: Alternative complement pathway activity in sera from patients with sickle cell disease. Clin. Exp. ImmunoI., 23 :56, 1976. Kolb, W. P., Haxby, J. A., Arroyave, C. A., et al.: Molecular analysis of the membrane attack mechanism of complement. J. Exp. Med., 135:549, 1972. Koopman, W. J., Sandberg, A. L., Wahl, S. M., et al.: Interaction of soluble C3fragments with guinea pig lymphocytes. Comparison of effects of C3a, C3b, C3c, and C3d on lymphokine production and lymphocyte proliferation. J. ImmunoI., 117:331,1976. Kosmidis, J. C., and Leader-Williams, L. K.: Complement levels in acute infectious hepatitis and serum hepatitis. Clin. Exp. Immunol., 11 :31, 1972. Lay, W. H., and Nussenzweig, V.: Receptors for complement on leukocytes. J. Exp. Med., 128:991, 1968. Leddy, J. P., Frank, M. M., Gaither, T., et al.: Hereditary deficiency of the sixth component of complement in man. I. Immunochemical, biologic and family studies. J. Clin. Invest., 53:544, 1974. Levy, L. R, and Lepow, I. H.: Assay and properties of serum inhibitor of C'l-esterase. Proc. Soc. Exp. BioI. Med., 101 :608, 1959. May, J. E., and Frank, M. M.: A new complement-mediated cytolytic mechanism-the C1-bypass activation pathway. Proc. Nat. Acad. Sci. U.S.A., 70:649, 1973. McCall, C. E., DeChatelet, L. R., Brown, D., et al.: New biological activity following intravascular activation of the complement cascade. Nature, 249:841, 1974. McLeod, B. C., Bake!..R,., and Gewurz, H.: Studies on the inhibitor ofC56-initiated lysis (reactive lysis). II. C567-INH-an inhibitor of the C567 trimolecular complex of complement. Int. Arch. Allergy Appl. ImmunoI., 47:623, 1974. Miller, B. J., Oldstone, M. B. A., and Cooper, N. C.: Complement-dependent lysis ofvesicular stomatitis virus. Fed. Proc., 35:494, 1976. Miller, M. E.: Phagocytosis in the newborn infant: Humoral and cellular factors. J. Pediat., 74:255, 1969. Miller, M. E., and Ganges, R. G.: Serum complement-like opsonic activities in human, animal, vegetable and proprietary milks. Fed. Proc., 35:359, 1976. Miller, M. E., and Nilsson, U. R: A major role of the fifth component of complement (C5) in the opsonization of yeast particles. Partial dichotomy of function and immunochemical measurement. Clin. Immunol. ImmunopathoI., 2:246,1974. Miller, M. E., and Nilsson, U. R: A familial deficiency of phagocytosis in enhancing activity of serum related to a dysfunction of the fifth component of complement (C5). New Engl. J. Med., 282:354, 1970. Miller, M. M.: Pathology of chemotaxis and random mobility. Sem. Hematol., 12:59, 1975. Moreau, S. C., and Skames, R C.: Complement-mediated bactericidal system: Evidence for a new pathway of complement action. Science, 190:278, 1975. Miiller-Eberhard, H. J., Dalmasso, A. P., and Calcott, M. A.: The reaction mechanism of ~IC-globulin (C'3) in immune hemolysis. J. Exp. Med., 123:33,1966. Miiller-Eberhard' H. J., and GOtze, 0.: C3 proactivator convertase and its mode of action. J. Exp. Med., 135:1003,1972. Naff, G. B., and Ratnoff, O.D.: The enzymatic nature ofC1r: Conversion ofCls to C1 esterase and digestion of amino acid esters by C1r. J. Exp. Med., 128:571, 1968. Neu, R L., Stockman, J. A., Spitzer, R. E., et al.: 46, xy/46, xy, 21q-Mosaicismin an infant with neutropenia and properdin deficiency. J. Med. Genet., 13:332, 1976. Okada, H., Kawachi, S., and Nishioka, K.: A new complement inhibitor in guinea pig serum. Japanese J. Exp. Med., 39:527, 1969. Osler, A. G.: Complement: Mechanisms and Functions. Foundations of Immunology Series. Osler and Weiss (eds.), Englewood Cliffs, N.J., Prentice-Hall, Inc., 1976, p. 98. Osofsky, S. G., Thompson, B. H., Lint, T. F., et al.: Homozygous C3 deficiency presenting with fever, skin rash and arthralgias and responding to whole blood transfusion. Pediat. Res., 10:391, 1976. Peterson, B. H., Graham, J. A., and Brooks, G. F.: Human deficiency of the eighth component of complement. The requirement of C8 for serum Neisseria Gonorrhoeae bactericidal activity. J. Clin. Invest., 57:283, 1976. Pillemer, L., Blum, L., and Lepow, I. H.: The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science, 120:279, 1954. Polley, M. J., and Miiller-Eberhard, H. J.: The second component of human complement: Its isolation, fragmentation by C'1 esterase, and incorporation into C'3 convertase. J. Exp. Med., 128: 533, 1968. Radwan, A. I., and Crawford, T. B.: The mechanisms of neutralization of sensitized equine arteritis virus by complement components. J. General Virology, 25:229, 1974.

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66. Rojas-Espinosa, 0., Mendez-Navarrete, I., and Estrada-Parra, S.: Presence of C1qreactive immune complexes in patients with leprosy. Clin. Exp. Immunol., 12:215, 1972. 67. Root, R K, Ellman, L., and Frank, M. M.: Bactericidal and opsonic properties ofC4 deficient guinea pig serum. J. Immunol., 109:477, 1972. 68. Rosenfeld, S. I., Baum, J., Steigbigel, R T., et al.: Hereditary deficiency of the fifth component of complement in man. II. Biological properties of C5-deficient human serum. J. Clin. Invest., 57:1635, 1976. 69. Rosenfeld, S. 1., Kelly, M. E., and Leddy, J. P.: Hereditary deficiency of the fifth component of complement in man. 1. Clinical, immunochemical, and family studies. J. Clin. Invest., 57:1626,1976. 70. Ross, G. D., Polley, M. J., Rabellino, E. M., et al.: Two different complement receptors on human lymphocytes. J. Exp. Med., 138:798, 1973. 71. Rother, K: Leucocyte mobilizing factor: A new biological activity derived from the third component of complement. Eur. J. Immunol., 2:550, 1972. 72. Ruddy, S., and Austen, K F.: C3b inactivator of man. II. Fragments produced by C3b inactivator cleavage of cell-bound or fluid phase C3b. J. Immunol., 107:742, 1971. 73. Ruddy, S., Gigli, I., and Austen, K R.: The complement system of man. New Engl. J. Med., 287:489, 1972. 74. Schreiber, RD., Medicus, R G., Gotze, 0., et al.: Properdin- and nephritic factordependent C3 convertases: Requirement of native C3 for enzyme formation and the function of bound C3b as properdin receptor. J. Exp. Med., 142: 760, 1975. 75. Schultz, D. R, and Miller, K D.: Elastase of pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors. Infection and Immunity, 10:128, 1974. 76. Shin, H. S., Pickering, J., and Mayer, M. M.: The fifth component of the guinea pig complement system. II. Mechanism of SAC14235b formation and C5 consumption by EAC1423. J. Immunol., 106:473, 1971. 77. Shin, H. S., Snyderman, R, Friedman, E., et al.: Chemotactic and anaphylatoxic fragment cleaved from the fifth component of guinea pig complement. Science, 162:361, 1968. 78. Siegel, J., Rent, R., and Gewurz, H.: Interactions of C-reactive protein with the complement system. 1. Protamine-induced consumption of complement in acute phase sera. J. Exp. Med., 140:631, 1974. 79. Spitzer, R., Stitzel, A., Florio, L., et al.: Inhibition of the alternative pathway of complement activation by a serum factor generated during transplant rejection. Immunochemistry, 13:395, 1976. 80. Spitzer, R E., Stitzel, A. E., and Urmson, J.: Interaction of properdin convertase and properdin in the alternative pathway of complement activation. Immunochemistry, 13: 15, 1976. 81. Spitzer, R E., Vallota, E. H., Forristal, J., et al.: Serum C'3lytic system in patients with glomerulonephritis. Science, 164:436, 1969. 82. Stitzel, A. E., and Spitzer, R E.: The utilization of properdin in the alternate pathway of complement activation: Isolation of properdin convertase. J. Immunol., 112:56, 1974. 83. Stossel, T. P., Alper, C. A., and Rosen, F. S.: Opsonic activity in the newborn: Role of properdin. Pediatrics, 52: 134, 1973. 84. Strauss, R G., Mauer, A. M., Asbrock, T., et al.: Stimulation of neutrophil oxidative metabolism by the alternate pathway of complement activation: A mechanism for the spontaneous NBT test. Blood, 45:843, 1975. 85. Strife, C. F., McDonald, B. M., Riley, E. J., et al.: Shunt nephritis: The nature of the serum cryoglobulins and their relation to the complement profile. J. Pediat., 88:403, 1976. 86. Tauber, J. W., Polley, M. J., and Zabriskie, J. B.: Nonspecific complement activation by streptococcal structures. II. Properdin-independent initiation of the alternate pathway. J. Exp. Med., 143:1352,1976. 87. Teisberg, P., and Gjone, E.: Circulating conversion products of C3 in liver disease. Evidence for in vivo activation of the complement system. Clin. Exp. Immunol., 14:509, 1973. 88. Thompson, R. A., Carter, R, Stokes, R P., et al.: Serum immunoglobulins, complement component levels and autoantibodies in liver disease. Clin. Exp. Immunol., 14:335, 1973. 89. Wands, J. R, Alpert, E., and Isselbacher, K J.: Arthritis associated with chronic active hepatitis: Complement activation and characterization of circulating immune complexes. Gastroenterology, 69:1286, 1975. 90. Wands, J. R, Perrotto, J. L., and Isselbacher, K J.: Circulating immune complexes and complement sequence activation in infectious mononucleosis. Am. J. Med., 60:269, 1976.

364

ROGER

E. SPITZER

91. Ward, P. A., Chapitis, J., Conroy, M. C., et al.: Generation by bacterial proteinases ofleukotactic factors from human serum, and human C3 and C5. J. Immunol., 110:1003, 1973. 92. Ward, P. A., Cochrane, C. G., and Muller-Eberhard, H. J.: The role of serum complement in chemotaxis ofleukocytes in vitro. J. Exp. Med., 122:327, 1965. 93. Wellek, B., and Opferkuch. W.: A case of deficiency of the seventh component of complement in man. Biological properties of a C7-deficient serum, and description of a C7inactivating principle. Clin. Exp. Immunol., 19:223, 1975. 94. Whaley, K., and Ruddy, S.: C3b inactivator accelerator (A-C3bINA): A serum protein of the complement system required for full expression of C3b inactivator (C3bINA) activity. Fed. Proc., 35:654, 1976. 95. Wolski, K. P., Schmid, F. R., and Mittal, K. K.: Genetic linkage between the HL-A system and a deficity of the second component (C2) of complement. Science, 188: 1020, 1975. Department of Pediatrics State University of New York Upstate Medical Center Syracuse, New York 13210