THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 67: 346-353. 1976 Vol. 67 , No.3 Copyright © 1976 by The Williams & Wilkins Co. Printed in U.S.A.

IMMUNOCHEMISTRY AND IMMUNOBIOLOGY OF THE

IRMA GIGLI, M.D. Departments of Dermatology and Medicine, Irvington House Institute, New York University School of Medicine, New York , New York, U.S.A.

The complement system, a complex mixture of centrations in blood and in their physicochemical at least 15 sequentially activated plasma proteins, characteristics (Tab.). constitutes one of the primary effector systems of immunologically induced inflammatory reactions. The First Component (Cl) The proteins are present in plasma in nonactivated form. Activation is by a series of specific proteo­ The first component of the classic pathway is present in its inactivated form as a calcium­ lytic reactions, which can be initiated by dependent macromolecular complex of three sub­ antigen-antibody complexes, fungal and bacterial units, C1q, Clr, and CIs [4]. There have been substances, and certain cellular and humoral pro­ reports of another subcomponent necessary for full teolytic enzymes. C1 hemolytic activity [5]; however, C1 has been The molecular events in the activation of the totally reconstituted from highly purified prepara­ complement system can be divided between those tions of C lq and the proenzyme forms of C lr and involved in the classic activation mechanism (C1 , CIs at concentrations comparable to those of the C4, C2, and (:3) and those which participate in the subunits in the euglobulin fraction of serum con­ alternative or pathway (IF, P , B, D). taining Cl [6]. Clq is a glycoprotein with an The latter pathway functions independently of C1, unusual amino acid composition, containing C4, and C2 but requires C3. The final step in the hydroxylysine, hydroxyproline, and glycine [7]. sequence, assembly of C5- C9, can be accomplished This composition is similar to that of basal mem­ by both pathways. In addition, there are at least brane proteins [8]. The hydroxylysine residues three normal plasma proteins which exert a regula­ appear to have glucosylgalactosyl residues coupled tory effect on the complement reaction sequence to them, another feature of basal membrane pro­ [1 ]. teins. The hemolytic activity of C1q is lost rapidly The products and byproducts of complement on digestion with collagenase [9] or heating [10], activation have distinct biologic significance. They and hence a collagen-like sequence, which is es­ mediate structural and functional alterations of sential for its hemolytic activity, must be a struc­ cellular membranes, and activation of specific tural feature of the molecule. Binding of C1q to IgG cellular functions. is less sensitive to collagenase or heating, suggest­ PROTEINS OF THE CLASSIC PATHWAY AND THEIR ing that the interaction between IgG and Clq may ACTIVATION be through a noncollagen-like portion of the mole­ The classic pathway is composed of 11 distinct cule. Digestion with pepsin fragments the noncol­ proteins [2 ,3] which differ markedly in their con- lagen portion, with subsequent loss of binding [11]. The molecular weight (MW) of C lq is agreed to Dr. Gigli was a fellow of the John Simon Guggenheim be close to 400,000 daltons [9,12,13]; however, Foundation and the recipient of a Research Career there is some controversy as to the subunit struc­ Development Award (AM 46409) from the National ture [9 ,14]. Recent work has proposed that C1q is Institutes of Health. composed of nine non-covalently linked subunits of Reprint requests to: Dr. I. Gigli, Department of Der­ matology, New York University Medical Center, 550 42,000 to 47,000 MW, consisting of six dimers of First Avenue, New York, New York 10016. two nonidentical chains (A-B) and three dimers Abbreviations: of two identical chains (C-C) [11] with MWs of DFP: diisopropyl phosphofluoridate 24,000, 22,500, and 21,000, respectively [14]. Based NF: nephritic factor P: properdin on chemical data, a model for C1q has been - IF: initiating factor proposed in which the collagen-like regions in each The components onne classic complement system are A, B, and C chain are combined to form a triple designated numerically, Cl, C2, C3, C4, C5 , C6, C7 , C8, helical structure of the same type as is found in C9; the three subcomponents of Cl are referred to as Clq, collagen. Each major triple helix is joined to Clr, and CIs; and the components of the alternative pathway are designated by capital letters, IF, P , B, D. another via the C-chain interchain disulfide bond Physiologic fragments of components resulting from to give a pair of 67,000 MW units [15]. The cleavage by enzymes which are indigenous to the comple­ suggested structure of C1q fits with the biologic ment system are distinguished by small arabic letters, e.g. , and . Enzyme activity is indicated by features such as the presence of six binding sites for placing a bar above the designation of the component in immunoglobulins. which the activity resides. C1r, first described by Lepow et al [16], was 346 Sept.1976 THE COMPLEMENT SYSTEM 347

TABLE. Physicochemical characteristics of proteins of the complement system Approxi- Electro- mate NameD Molecular serum weight phoret ic Fragments mobility concen- tration (Jl g/ml)

Classic activation: C1q 400,000 1'2 190 C1r 168,000 (3 CIs 86,000 ex 120 C4 «(31E) 206,000 (3 1 430 , C4b C2 117,000 (3 2 30 C2a, C2b C3 «(31C) 180,000 (31 1600 C3a, C3b Alternative pathway activation: Properdin 186,000 1' 2 15 Initiating factor 150,000 1' 1 (nephritic factor analog, IF) 25 ,000 ex2 Trace (C3PAase, GBGase) Factor B 93,000 (3 2 225 ex Fragment, GAG, (C3PA, GBG) 1'Fragment, GGG Terminal sequence: C5 «(31F) 185,000 (31 85 C5a,C5b C6 95,000 (32 75 C7 110,000 (3 2 60 C8 163,000 1'1 80 C9 79,000 ex 230

a Synonyms are given in parentheses found to activate CIs and to hydrolyze some CIs consists of a single polypetide chain of 83 ,000 synthetic ester substrates; therefore it was thought MW. CIS activated by Clr has a similar MW, but to be C1f [17]. More recently, both a precursor and gives two peptide chains of 56 ,000 and 27,000 MW an active form of C1r have been isolated in highly under dissociating conditions [23,24]. DFP inhibits purified forms. C1r has a MW of 188,000 and an 8 both the esterase and hemolytic activity of ClS. val ue of 7.5 which fell to 68 upon activation by Using 32P-labeled DFP, it was shown that a single trypsin [18]; this C1f was no longer able to form a molecule is bound per molecule of CIS. The labeled complex with C1q and CIs in Ca2+ . In contrast, DFP was found to be present in the 27 ,000-MW spontaneously activated Clf has a MW of 168,000 fragment [25], which must therefore contain the and retained the power to recombine with C1q and enzymatic active site. A study of the sequence of CIS [19] . Under dissociating conditions C1r is a this peptide disclosed a similarity to the sequence single polypeptide chain of approximately 83, 000 found in the active site of other serine esterases MW; but activated C If consists of two polypeptide such as trypsin, chymotrypsin, thrombin, and chains of MWs 56,000 and 27 ,000 [20]. Activation plasmin [11]. must therefore involve cleavage of at least one The Second Component (C2) polypeptide bond in the proenzyme. The active site of C1r is located in the 27,000 MW fragment, C2 is a trace protein in blood. It has a MW of as evidenced by its binding of [32P ]diisopropyl 117,000 and an 8 value of 4.5. CIS is known to split phosphofluoridate (DFP) [20]. DFP treatment in­ C2 into two fragments, C2a and C2b; however, hibits CIs activation by C1r [21]. these products have not been either isolated or CIS has been identified as the component analyzed. carrying the major esterase activity of C1 [16 J. The Third and Fifth Components (C3 and C5) Purified CIS will hydrolyze C4 and C2 and a variety of synthetic peptide esters, such as the C3 and C5 have similar MWs (180,000) and both N-acetyl-L-arginine ethyl ester, and the p-toluene­ contain two covalently linked chains of 110,000 and sulfonyl-L-arginine methyl esters of both aromatic 70,000 MW [26,27]. C3 and C5 are substrates of basic amino acids [22]. two different enzymes formed consecutively during CIS and CIs have both been purified. Although complement activation. A small fragment of the CIs has been reported to activate spontaneously, 110,OOO-MW chain is split in each case to give highly purified stable CIs has been obtained [6]. fragments C3a and C5a, leaving the major frag- 348 GIGLI Vol. 67, No.3 ments C3b and C5b with a transient capacity to bind to membranes. In addition, C3b has a binding region for properdin (P) [28]; and C5b for C6, C7, and C8 [29].

The Fourth Component (C4) C4 is one of the two natural substrates of CIS, with MW 206,000. Both a two [30] and a three [31] covalently linked chain structure have been postu­ lated for C4. Mild reduction of C4, followed by gel filtration, has recently confirmed the three-chain structure [32]. The isolated polypeptide chains have MWs of 100,000, 76,000, and 33,000, respec­ tively. C IS splits C4 into at least two portions, C4b and C4a [33, 34]. C4a, a 6,000-MW fragment, is derived from the 100,000-MW chain in a limited proteo­ lytic reaction [31] which is thought to involve a FIG. 1. Activation of the classic pathway of the com­ single peptide cleavage. plement system. its biologic role may be visualized as twofold: first, The Sixth to Ninth Components (C6-C9) it may be an essential component in C1r activation C6, C7, C8, and C9 have been less well charac­ by forming a stable complex of C1q-C1r; secondly, terized. They react reversibly with C5, forming a it may restrict CIs activation and, in this manner, product with a sedimentation velocity of approxi­ act as a controlling mechanism in the activation of mately 15S, which is slower than that obtained the classic component system. with the activated molecules, 26S [29]. There are several reports of nonspecific activa­ tion of C1 by membranes of different origins [39]. The Classic Activation Mechanism In addition, plasmin and trypsin [40] are assumed C1 activation occurs specifically by binding of to be responsible for the nonspecific activation of the C1q subunit to the Fc portion of aggregated Cl. immunoglobulin [35] . However, under certain con­ It has long been appreciated that C4 precedes C2 ditions, the proresses of binding and activation can in the immune hemolytic sequence, even though be separated [36]. The specificity of C1 binding both are natural substrates of CIs [41]. The reason and activation is high; full activation follows for this order became aEparent with the finding binding of C1 to aggregates of the IgM and IgG that the activity of C1 upon C2 is markedly immunoglobulins (human IgG 1, IgG2, and IgG3) enhanced by the prior interaction of CI, either free but little, if any, follows binding to monomers. in the fluid phase or cell bound (EACi), with C4. It N ei ther IgA, IgD [37], nor IgE [38] binds or was postulated that the ci-C4 interaction alters activates Cl. the configuration of the CI molecule, exposing the The very unusual hexamer structure of C1q, enzymatic site responsible for C2 utilization [42]. correlated with its ability to combine with four to Studies on the species specificity of the compo­ six IgG molecules [7], suggests that the binding nents involved and some physicochemical proper­ sites of C1q may be located in the peripheral ties support this view. CI of human origin inacti­ globular portions of the molecule [ll] (Fig. 1). vates guinea-pig C4 far less than it inactivates Although there is no evidence of the development human C4. Similarly, guinea-pig C4 fails to en­ of enzymatic activity in C1q on binding, it results hance human C2 utilization and, indeed, causes in C1r activation. It is possible that this reaction inhibition. C1 heated at 56°C for 30 min loses its may proceed through a conformational change in hemolytic activity but retains its capacity to bind the globular portion of the C1q molecule, which is to antibody-coated erythrocytes and to hydrolyze transmitted through the relatively rigid collage­ C4 but not C2. This suggests that the binding of CI nous portion to the central area to which C1r may to sensitized erythrocytes is relatively heat stable, be bound. Activation of C1r leads to activation of as is the esterase activity, but that the cLi site the proenzyme CIs [24]. which facilitates hydrolysis of C2 is heat labile In the intact C1 molecule, the conditions for CIs [42]. The fact that heat-treated ci' can still be activation are unusual in that the enzyme (CIr) bound by sensitized erythrocytes, a property asso­ and the substrate (CIs) are believed to be present ciated with the subunit C1q, and that it retains its in an equimolar ratio. However, using isolated C1r esterase activity, due to the ClS subunit, suggests and CIs in the absence of Ca 2+, it has been possible that the heat-labile C4 binding site will be in Clf. to demonstrate that 1 mole of Clf can activate In contrast to these data, a C4 binding site on CIs several moles of CIs. Addition of Ca2+ restricts distinct from the enzymatic site has been postu­ activation to an equimolar ratio [21]. Since Ca His lated [43]. an intrinsic component of the intact C1 molecule, The action of the C 1 enzyme on C4 leads to Sept. 1976 THE COMPLEMENT SYSTEM 349 cleavage of C4 and physical uptake of the major Properdin (P) fragment, C4b, onto membranes [44]. The C14 Activated properdin CP} is a highly basic globu­ complex generated acts on C2, allowing the major lin [52] with MW 184,000, consisting of four fragment of C2 (C2a) to fuse with C4b, resulting in non-covalently bound subunits of similar MW the formation of a protein-protein complex C142, (46,000-53,000). The subunits can recombine to the classic C3 convertase, with new enzymatic form 88,000-MW dimers which partially retain the activity [45]. This enzym~is unstable at 37°C, antigenic and biologic properties of the native reverting to the original C 14 state [46]; the addi­ molecule [53]. Properdin exists in serum in precur­ tion of fresh C2 restores its C3-converting activity. sor form and activation proceeds without appreci­ C3 convertase acts on C3, inducing the cleavage of able .changes in its molecular size. the C3 molecule into two fragments, C3a and C3b. During this step, the major fragment, C3b, either Factor B associates with its activating enzyme, generating C1423, or appears in the fluid phase as an inactive This (3 globulin (C3 pro activator, C3PA; glycine­ product, C3i. The lesser fragment, C3a, is released rich (3 globulin, GBG) [54,55] is the precursor of a into the fluid phase. The complex formed, C1423, C3 cleaving enzyme. It consists of a single peptide acquires a new enzymatic activity (C5 convertase) chain with a MW of 93,000; upon activation, it is which has as substrate C5 [47,48 J. Although fluid­ split into two antigenic ally distinct fragments, one phase C3 convertase is capable of initiating these behaving as a 63,000-MW 'Y-globulin and the oth~r reactions, a cell-bound position for C3 convertase, as a 30,000-MW a-globulin. The I' fragment (B) as on EAC142, favors the deposition of the cleavage was originally designated C3 activator (C3A) and products C3b and C5b on the cell surface and the when isolated, retains some of its ability to cleave assembly of C5-C9 on the membrane in a cytolyti­ C3 and hydrolyze N-a-acetyl-glycyl-L-lysine-meth­ cally active form. yl ester [56] . PROTEINS OF THE ALTERNATIVE PATHWAY AND Factor D (C3 Proactivator Convertase, C3PAase) THEIR ACTIVATION This is a trace protein of serum which has been In 1954, Pille mer and his associates [49] de­ recently isolated in its active CD) [57] and precur­ scribed a mode of complement utilization initiated sor (D) forms [58]. Although isolated factor D is by a naturally occurring lipopolysaccharide, zymo­ readily activated by trypsin [59], its physiologic san, by which the C3 contained in serum was mechanism of activation has not yet been eluci­ inactivated to a much greater extent than were C1, dated. Factor :is, but not D, is susceptible to C4, and C2; the mechanism was independent of inactivation by DFP. It has been functionally specific antibodies and involved a distinct serum identified with C3PAase [60]. protein, properdin. The action of properdin 2 +, B), required Mg a heat-labile factor (Factor a C3 (Factor A) hydrazine-sensitive factor (Factor A), and a factor present in the pH 5.5 euglobulin fraction of serum. The hydrazine-sensitive factor of the properdin This pattern of complement utilization differs system, factor A, has been shown to be identical to markedly from that observed with the classic C3 C3 [60,61]. Restoration of factor A activity in convertase, and it is attributed to activation of C3 serum rendered deficient by treatment with and the terminal complement sequence via the hydrazine can be demonstrated upon addition of properdin system, which includes both an activa­ purified C3. Treatment of purified C3 with hydra­ tion pathway and an amplification loop. At least zine abolished its C3 hemolytic activity and factor five distinct proteins have been recognized as part A function. of the system (Tab.). Activation Mechanism Initiating Factor (IF) Although antigen-antibody complexes prepared Although originally properdin was described as from guinea-pig 1'1 antibodies [62], aggregated the IF of the alternative pathway, recent data F(ab')2 fragments [63], and aggregated human IgA indicate that this protein does not establish the and IgE myeloma proteins [38] are capable of initial interaction with the activators of the alter­ activating the alternative pathway, direct nonim­ native pathway. A trace protein in normal human munologic activation by endotoxin or a variety of serum, identical to a factor described in patients bacterial lypopolysaccharides also occurs [64]. with hypocomplementemic glomerulonephritis, Activation of the alternative pathway by zymo­ nephritic factor (NF) [50], appears to be an essen­ san appears to require IF early in the sequence tial early-acting component of the system [51]. (Fig. 2). Serum depleted of IF by immunoadsorp­ This normal serum protein, called the initiating tion does not support C3 consumption upon incu­ factor, was found to be a heat-stable 6-7S pseudo­ bation with inulin or zymosan in the presence of globulin with the electrophoretic mobility of a normal levels of P, B, and C3. Addition of purified 'Yl-globulin. Antiserum to NF detects IF; thus, IF IF results in the consumption of C3. Using fluores­ and NF may represent identical serum proteins ceinated antisera to C3 and P, it has been shown [51 ]. that C3 is deposited if zymosan is incubated in 350 GIGLI Vol. 67, No.3 altered IgA. F(ab')2 ' polysaccharides THE TERMINAL ATTACK SEQUENCE (). IIF Attack of C5 by either the classic C5 convertase "'- ~ IF C~~ ~I (C1423) or the alternative pathway convertase Mg l C3.B. D Mg" (C3PB) initiates a process which results in the C31FB C3 1 assembly of the multimolecular complex C5-C9 (Fig. 3). C5b, the larger of the two fragments I i _ +t- C 5, 6, 7,8, 9 produced by C5 cleavage, interacts with C6 to form ~ 5-C3 B,o Mg > 5-C3B ~5-C5-9 C3 a Cg6 complex [74J. Reaction of membrane-bound 1'B"'" rP Cg6 with C7 leads to the formation of a stable C567 C 3 . P -----..5 - C3 p'!<==/=;;:;.> 5 - C3 P B co_mplex on the cell surface [75]. In theJ1uid phase, C56 reacts with C7 to form nascent C567 which is B,D Mg* ~6C3 _ C5 C 3 transiently capable of binding directly to cell 5-C 3 surfaces which have not reacted with antibody or 5-C5 the earlier components of complement [76,77]. The 5 = Site on a membrane yc 6.7.8,9 trimolecular complex C567, either bound to a S-C5-9 membrane or in free solution, interacts with C8 in FIG. 2. Activation of the alternative pathway of the a purely absorptive process without any further complement system. enzymatic activity. C8 in the C5-C8 complex binds C9, completing the formation of the molecular serum depleted of C4 and P, but not if the serum is assembly responsible for membrane damage [29]. free of B, :5, or IF. P is detected on zymosan only The binding capacity of nascent C5-C9 decays when the conditions allow C3 deposition [51] . rapidly with the accumulation of biologically inac­ From these experiments it can be concluded that tive products in the fluid phase [78,79]. IF, in cooperation with Band D, form a C3 Experimental evidence suggests that C8 is the convertase, generatin[ C3b to which P is capable of molecule which executes the cytolytic function of binding. Binding of P to C3b [65], but not of P the terminal complement sequence. Whereas the [51 ,66], has bee~ demonstrated using purified P membrane of a target cell carrying C567 remains and ~ and EAC43. Activ~tion of P is initiated by intact, it becomes altered upon absorption of C8. EAC43 and requires B, D, and Mg2 +. Since P is In the absence of C9, highly purified C8 causes the bound to preformed EAC43B in the presence of cells to undergo'low-grade lysis [80]. The role of C9 EDTA (ethylenediamine tetraacetate), EAC43B may be visualized as the enhancement of C8 must be the P-activating principle [67]. Binding of function. P stabilizes the enzymatic site by prolonging the CONTROL MECHANISMS decay rate of the C3]3 enzyme [65,66J. Large amounts of P and B with C3 in the absence The complement reaction sequence is subject to of is form a C3 convertase in which cleavage ______... 0 0 o enzyme-substrate interaction between D and B 000 .: { 8 7 5 0 0 0 and becomes instrumental in activating a system ~ ~ 0 Alter~atlve 5 Membrane which acts on C3. C3B, whether present in the Palhway Convertase ® drf6 damage fluid phase or bound to a membrane as EAC3B is (C38. C3P8) ~ 7 5 - ' ® unstable at 37 ac because of the B decay [28]. . (j) Assembly of C3B on the surface of cells induces ly­ FIG. 3. Terminal sequence of complement activation. sis when the components C3-C9 anre added [72,73]. Attack mechanism. Sept. 1976 THE COMPLEMENT SYSTEM 351 enzymatic site of CIs [83] to form an inactive lymphoblastoid cells [106,107]. It has postulated complex. The C3b inactivator (C3bINA) [84,85] that C3b binding triggers B-cell activation either splits cell-bound or fluid-phase C3b into two frag­ directly or indirectly by facilitating cell coopera­ ments, C3c and C3d. As a result, the hemolytic tion [108]. activity of C423 and the C3b-dependent activation of factor B are destroyed [60,86], serving to control REFERENCES the amplification loop in which these factors par­ 1. Gigli I: Transplant Proc 6:9, 1974 ticipate. 2. Nelson RA, Jensen J , Gigli I. Tamura R: Immuno­ chemistry 3: 111 , 1966 In addition, the biologic functions mediated by 3. Ruddy S, Gigli I, Austen KF: N Engl J Med 287:489, C3b, and phagocytosis [87], are 1972 suppressed. Both C3a and C5a are controlled by 4. 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Nicol PAE, Lachmann PJ: Immunology 24:259, invariably fails to reveal by direct immunofluorescent 1973 techniques the deposition of IgA complement compo­ 73. Fearon DT, Austen KF, Ruddy S: J Exp Med nents as well as properdin and properdin Factor B. 138: 1305, 1973 74. Shin HS, Snyderman R, Friedman E, Mellors A, Positive fluorescence is found in periblister areas and Mayer MM: Science 162:361, 1968 normal skin. 75. Mayer MM: Proc Nat! Acad Sci USA 69:2954,1972 Lazarus: What is the mechanism of membrane lysis 76. Thompson RA, Lachmann PJ: J Exp Med 131:629, by complement? 1970 Gigli: C8 in combination with C9 may play an 77 . Goldman IN, Ruddy S, Austen KF: J Immunol important role in the production of membrane damage 109:353, 1972 seen in complement lysis. 78. Koethe SM, Austen KF, Gigli 1: J Immunol110:390, Harber: The size of the hole in colloid osmotic 1973 79 . Kolb WP, Muller-Eberhard HJ: J Exp Med 138:438, hemolysis has been demonstrated to be less than lOA. 1973 The complement hole is 3 to 10 times larger in electron 80. Stolfi R: J Immunol 110:46, 1968 micrographs. Colloid osmotic hemolysis is compatible 81. Lepow IH, Leon MA: Immunology 5:222, 1962 with damage to the ATP "pump" and rapid K+ loss. This 82. Gigli I, Ruddy S, Austen KF: J Immunol 100:1154, has not been shown to be applicable in complement lysis. 1968 Cohen: In immunologically related inflammatory 83. Rosen FS, Alper CA, Pensky J, Klemperer MR, processes there is a propensity of specific-type circulating Donalson VH: J Clin Invest 50:2143, 1971 leukocytes and cellular counterparts within tissues to 84. Tamura N, Nelson RA: J Immunol 99:582, 1967 85 . Ruddy S, Austen KF: J Immunol 107:742, 1971 localize, accumulate, and/or interact in specific organs or 86. Alper CA, Rosen FS, Lachmann PJ: Proc Nat! tissues and to segregate with other cell types. This often Acad Sci USA 69:2910, 1972 occurs without apparent relationship to exact areas of 87. Gigli I, Nelson RA: Exp Cell Res 51:45, 1968 immune complex deposition or inactivation of putative Sept. 1976 THE COMPLEMENT SYSTEM 353 responsible chemical mediators. On the supposition that lymphocytes had Factor B detectable on their surface. complement fragment cellular . attachments could be Austen: A chemotactic factor must work in concert relevant, can you tell us for which cell types there is with a permeability factor which creates a marginating definite evidence for receptors and for which products of cell pool which then responds to the specificity of the the complement cascade? chemotactic factor. Gigli: Complement receptors have been found in a Krueger: Do complement component fragments, variety of lymphoid cells. The receptor for Clq does not namely, C5a and C3a, interact with specific receptors on seem to play a significant role. B-Iymphocytes have any cell surfaces? Can these factors be identified by receptors for native C4 and C3 and for their major imm unofl uorescence? fragments C4b, C3b, C4d, and C3d. Bokisch recently Gigli: With fluorescein-conjugated antisera, C3a and reported that 40 to 50% of human peripheral blood C5a have been demonstrated to bind to mast cells.