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Complement research: the impact of molecular genetics
immunology Today,voL 7, No. 2, 1986 ii
The structure and function of the complement system, its genetics, its role in the physiological or pathological immune response and in host defence are now being analysed with the powerful techniques of molecular biology. New developments in complement research were discussed at a recent workshop representing the major sdentific meeting in this area of immunology*. Since the last complement workshop in Mainz, Germany, two years ago, a substantial number of cDNA and genomic DNA clones encoding most complement components and related proteins have been isolated. These cloned complement genes have facilitated the structural comparison of functionally related and of polymorphic proteins. They have allowed examination of the molecular basis of genetic defects and of the regulation of complement synthesis, and have permitted the chromosomal localization of complement genes.
Polymorphismsand homologiesbetween complementproteins The components C4, C2 and factor B are of special interest because they are coded by genes of the major histocompatibility complex (MHC). Comparison of the complete primary structure of murine C4 and the related, haemolytically inactive sexlimited protein (Sip) showed not only the expected marked homology indicating gene duplication but also a deletion in Sip close to the corresponding cleavage site for Cls in C4 (M. Nonaka, Kanazawa; R. Ogata, La Jolla). The absence of haemolytic activity in Sip might be due to this deletion. Despite the close homology there is a selective regulation of transcription for these two, closely linked proteins, apparently determined by the flanking sequences (E. Georgatosou, Paris). Similarly, two closely linked human C4 isotypes, C4A and C4B, differ only in six amino acids despite their marked difference in haemolytic activity. Both loci are highly polymorphic. Null alleles, total deficiencies and duplications were identified at the protein level. At the genomic level most of the null alleles
news and tCeatures Complementresearch:the impact of molecular genetics from ReinhardBurger appeared normal; only a few resuited from gene deletions (M. Caroll, Boston). Differences in four amino acids in the C4d moiety of C4B were identified as the basis for the expression of Chido antigen determinants (C.Y. Yu, Oxford). Several groups reported progress in the structural analysis of the regulatory proteins at the cDNA level. The deduced amino acid sequence of C4b-binding protein (C4BP) revealed homology regions of about 60 amino acids with factor H, C2 and factor B (its Ba-fragment) (L. Chung, Oxford; T. Kristensen, La Jolla). All these proteins have the ability to interact with C3b or C4b. The homology region in this family of proteins (and also in the complement receptor CR1, see below) might, therefore, function as C3b/ C4b binding site. C2a and Bb bear regions homologous with catalytic domains of other serine proteases (D. Bentley, Oxford). Human C1 inhibitor shows homology with the protease inhibitors antithrombin III and C~l-antitrypsin (A. Davis, Boston). The availability of appropriately characterized cDNA clones facilitates a more detailed analysis of complement polymorphisms with a much higher resolution than previously. D. Bentley (Oxford) identified a restriction fragment length polymorphism (RFLP) of C2 and demonstrated three C2 variants, indistinguishable by typing at the protein level, in individuals bearing a common C2 allele.
Regulation of complementsynthesisby lymphokines
An interesting connection between mediators involved in cellular immune response and the complement system was described by the group of H. Colten (Boston). These studies might provide a model system for other immunologically relevant proteins and are concerned with the regulatory mechanisms for synthesis of the MHC class III products C2 and factor B. The genes for *TheXlthInternationalComplementWorkshopwas C2 and B are separated by only 0.6 heldin Miami,Forida,USA,3-5 November1985. kb. The lymphokine interferon ~/
(IFN--y) is a well-known modulator of MHC class II antigens (la-antigen) expression. S. Cote (Boston) demonstrated that IFN-~ induces an increase in synthesis of the MHC class Ill products C2 and B by human monocytes. Synthesis of both proteins is influenced at the pretranslational level, but the two closely linked proteins were affected independently as shown by different kinetics of transcription. Interleukin 1 (IL-1) influences the synthesis of factor B but not of C2 by hepatocytes. The basis for this selective action on the adjacent genes was studied by D. Perlmutter (Boston) by using murine L cells transfected with a cosmid bearing both the human C2 and factor B genes and expressing both proteins constitutively. IL-1 induced an increase of factor B expression but not C2, again by a pretranslational mechanism. The IL-1 regulation might affect the short segment between the two genes and one should be able to identify structural basis for this selectivity. IL-1 induced an increase in the density of the complement receptors CR1 and CR3 on human neutrophils (I.M. Berger, Cleveland).
Complement receptors A cDNA clone for CR1 (the C3b/ C4b receptor) was described by W. Wong (Boston). RFLP was demonstrated for the polymorphic variants of this protein permitting a distinction of the structural allotypes F and S. In addition, an independent RFLP of CR1 correlated with the genetically controlled number of CR1 per erythrocyte indicating that the quantitative CR1 expression is directly associated with the CR1 gene (J. Wilson, Boston). The CR1 cDNA clone contained repeating units related to the proteins H and C4BP (L. Klickstein, Boston). This corresponds again with the functional relation of these proteins, which share the ability to bind to C3b (see above). The proteins CR1, H and C4BP represent a linkage group located on the human chromosome 1. Several abstracts were directed at functional analysis of complement
~) 1986, Elsevier Science Publishers B.V, Amsterdam
0167 - 4919/86/$02.00
27
-news and eatures receptors at the protein level. The importance of rapid phosphorylation processes in CR1 function was reported by P. Changelian (Boston) and S. Wright (New York). Phorbol myristate acetate (PMA) induces phagocytosis by polymorphonuclear neutrophils. Incubation of neutrophils with PMA resulted in activation of protein kinase C and subsequent phosphorylation of CR1. This reaction exhibited a marked specificity in that it occurred only in phagocytic cells but not, e.g., in B cells or erythrocytes. This covalent modification of CR1 therefore seems to correlate with the acquired, increased phagocytic activity of the cells. In contrast, by appropriate stimulation of B-cell lines, phosphorylation of CR2 was induced arguing for a role of this receptor in B-cell function (see below). C. Mold (La Jolla) used the isolated CR2 protein (C3d receptor)incorporated into liposomes for a direct confirmation that the CR2 molecule in fact has also the capacity for the binding of Epstein-Barr virus. This dual receptor function apparently is mediated by distinct molecular sites because soluble C3d does not interfere with virus binding.
28
Complement and immune responses A new area of research was established at the Basel Institute for Immunology. F. Melchers (Basel) described the finding that cross-linked human C3d is one of the essential growth factors in the cell cycle of preactivated murine B cells. C3d regulates B-cell growth by binding to the C3d receptor on the lymphocyte surface. Cross-linked C3d gives a positive signal to the B cell for the entry from the G1 phase into the S phase. C3d replaces the macrophage:derived growth factors ('efactors') required for passing this 'restriction point' in the cell cycle of activated B cells. In contrast, soluble C3d has an inhibitory effect. This report clearly revived a quite controversial field; namely the role of complement in the induction of the antibody response. It produces a welcome and potent stimulus for discussions and additional work e.g., analysis of synthetic C3d peptides in this system (J. Lambris, La Jolla). F. Praz (Paris) reported that the Ba frag -0 ment of factor B also exhibits a marked mitogenic effect on preactivated murine B cells.
Independent pieces of evidence fit into Melcher's hypothesis, in particular the finding that - despite some exceptions (E. Klein, Stockholm)Epstein-Barr virus infection enhances or induces expression of C3dreceptor (CR2) in the membrane of previously virus-negative Burkitt's lymphoma lines (J. Cohen, Paris). One can speculate about a role of this host cell (not virus) encoded protein and its ligand C3d in proliferation of Fpstein-Barr virus transformed B cells. Similar supportive evidence was provided by M. Barel (Paris) showing that binding of antibody to CR2 induces enhanced B-cell proliferation. A role of C3 in the induction of the antibody response was also suggested by E. Boettger (Mainz) using a different approach. In the absence of C2 or C4 the activation of C3 via the classical pathway is prevented. C2deficient or C4-deficient guinea pigs exhibited a grossly impaired antibody formation to the antigen bacteriophage CX 174 especially at low antigen concentrations. The residual ability to mount a humoral immune response of these deficient animals remained unaffected after artificial depletion of C3 with cobra venom factor (CVF). In contrast, in normal guinea pigs the antibody formation was suppressed by decomplementation, again dependent on the antigen dose. Therefore, due to the absence of either C3 or of C3-cleaving enzymes there appears to be an insufficient ability to use C3 during the induction phase of an immune response resulting in an impaired antibody response. These data argue against a generalized immunosuppressive function of the complement fragments which are generated in excess during the CVF-mediated decom plementation. U. Hadding (Mainz) suggested a direct role of C3 in MHC-controlled immune phenomena. After treatment of human monocytes with C3b the expression of la antigens was reduced and, concomitantly, the capacity to function as antigenpresenting cells in an in-vitro system for T-cell proliferation was inhibited. The physiological role of C3 in the immune response has been analysed by numerous groups for more than a decade. Considering the recent progress with the improved, less heterogenous, and well defined assay systems, a more detailed outline of the various biological func-
Immunology Today, vol. 7, No. 2, 1986
tions of this versatile molecule and their reaction sequence appears to be in sight. A recently described guinea pig strain with an inherited C3-deficiency might prove helpful for further analysis. These animals have a C3 s-chain rendered functionally inactive by an insertion in the C3d-region (H. Auerbach, Boston).
Membrane associated regulatory proteins The decay accelerating factor (DAF), described years ago, has experienced a renaissance as shown by a substantial number of contributions to this topic. This membraneassociated protein accelerates the physiological decay of C3 convertases of both the classical and alternative pathways and prevents their assembly. It apparently has the capacity for binding to the catalytic site of these enzymes (K. WadeyPangburn, Tyler). M. Medof (New York) reported the presence of DAF on a wide variety of epithelial cells and exocrine glandular cells in many different tissues (e.g. cornea and bladder). In addition, DAF was found in various secretions like saliva and urine. The widespread distribution of DAF, especially on surfaces in intimate contact with the complement and other protein systems, might reflect its important- perhaps not yet known - biological function. No general agreement was reached in discussions on the role of DAF and acetylcholinesterase abnormalities in the increased susceptibility for lysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. A new leucocyte membrane glycoprotein (gp 45-70) was shown by T. Seya (St. Louis) to possess a highly efficient cofactor activity (50 times superior to H) for the initial inactivation of C3b, and also of C4b. It was renamed, therefore, membrane cofactor protein and may be a major regulatory protein. A regulatory role was discussed for two newly described erythrocyte proteins which interfered with the insertion of C5b9 into the membrane and with channel formation (G. H~nsch, Heidelberg; L. Zalman, La Jolla). Susceptibility of tumor cellsto lysis Antibody-sensitized melanoma cells differ in their susceptibility to lysis by complement. Susceptible cells bound C3b to their surface and retained it (M. Panneerselvam, Wash-
immunology Today, vol. 7, No. 2, 1986
ington). Resistant cells initially bound even three times higher amounts of C3b but exhibited a rapid C3b inactivation by cleavage to C3d. Pretreatment of the resistant cells with adriamycin induced susceptibility and inhibited the degradation of C3b. immobilized adriamycin could replace the soluble drug with an even higher efficiency and did not have the cytotoxic side effects. Apparently, a surface-mediated mechanism is involved in this phenomenon. The molecular basis is still obscure; one might consider some drug effects on toxic oxygen derivatives or interaction with regulatory proteins.
The C1 component cDNA clones were identified for the C1 subunits and should facilitate the structural analysis of this highly complex molecule. Defective transcripts, possibly caused by a mutation, for the B-chain of Clq have been demonstrated in one Clq-deficient patient (R. McAdam, Oxford). The French groups (hosts of the next International Complement Workshop in Chamonix, September 1987) have succeeded in localizing the peptide bonds which are cleaved during Clr activation. They identified twin domains as the core of C1 r involved in catalytic function and presented a sterical model for interaction of the different C1 subunits in order to explain the intramolecular activation events (G. Arlaud, Grenoble). B.Ziccardi (La Jolla) postulated a mechanism of feedback inhibition. He demonstrated that nascent C3b and C4b generated in the course of complement activation bind to the Fc portions of antibodies in immune complexes, thereby preventing further C1 activation.
RecombinantCSa Three similar contributions described the production of C5a by expression of appropriate gene segments in E. coil (A. Franke, Groton; G. Carter, Abbot Park; G. Fey, La Jolla). The product exhibited most of the biological functions of normal C5a. Site-specific mutagenesis in the
news and features C5a should permit the sites involved in receptor be identified; modulation ing events for cellular seems feasible.
molecular binding to of triggerfunctions
The membrane attack complex C5b-9 The exact reaction sequenceinvolved in lysis of cells by the membrane attack complex remains controversial and some questions are unresolved. New elements emerged e.g. through biophysical approaches, molecular genetics, or through the similarities between complement lesions and cytolytic granules in cytotoxic lymphocytes. An impressive example of the effectiveness of the combined application of molecular genetics and immunological techniques was presented by K. Stanley (Heidelberg). Based on the amino acid sequence derived from a cDNA clone, he pro posed a topographical map of C9 and a model for insertion of C9 into the membrane. According to his computer-aided model, C9 does not contain linear hydrophobic sequence for interaction with membrane lipids. In contrast, the secondary structure is characterized by amphi pathic elements permitting insertion into the membrane. Monoclonal antibodies reactive with defined C9 segments (obtained by cloning in an expression vector) permitted localization of individual determinants. A. Esser (Gainesville) emphasized the importance of a 'non-ordered' lipid bilayer induced by C5b-8 as prerequisite for association of C9 with the membrane. This conclusion was based on experiments with membrane-restricted photolabelling probes. The concept that poly-C9 is the actual lytic agent was challenged by S. Bhakdi (Giessen). The degree of C9-polymerization and thereby the C9 content differs in C5b-C9 complexes and is responsible for the observed heterogenous pore size of the complement ring lesions. He described a method for producing defined, homogenous complexes containing - at least statistically - a single C9 in association with C5b-8 which, nevertheless, are haemolytically active. Nucleated cells seem to
escape complement lysis either by calcium-dependent internalization (D. Carney, Baltimore) or by concentrating most of the C5b-C9 complexes in minor areas of the cell surface membrane which are shed in the form of vesicles (B. Morgan, Gainesville). The molecular mechanisms and site of lethal damage of bacteria by complement remain controversial and are not fully understood. K. Joiner (Bethesda) proposed that the presence of sufficient, multimeric C9 within the C5b-9 complex is required to cause efficient damage, whereas J. Dankert (Gainesville) found no need for poly-C9 formation. PoiyC9 may provide a trigger signal for an energy-dependent bacterial process which finally affects the inner membrane and causes release of cytoplasmic constituents. A bridge between cell-mediated lytic events and complement lesions was established by the demonstration of shared domains in the corresponding proteins as detected by cross-reactivity of monoclonal antibodies (E. Podack, Valhalla; J. Tschopp, Epalinges). The family of related proteins includes C9 and per'forin from T-cell cytolytic granules, C8 e-chain, C7, C6, and the functionally unrelated receptor for lowdensity lipoprotein. At present, the emphasis in complement research seems to be on the interaction of regulatory compo nents (particularly of the membrane bound proteins) with their ligands; the identification of functional domains, preferentially defined by peptides; the modulation of the immune response by complement; the effect on cellular functions; and the structural analysis of related proteins belonging to multigene families. The rapid development in the last two years and the increased spreading of molecular genetics promises much information for the next workshop in 1987. The abstracts of the Xlth International Complement Workshop were published as a special issue of the journal Comple ment, 2, 3-92 (1985) Reinhard Burger is at the Institute for Immunology, University of Heidelberg, 6900 Heidelberg, FRG.
29
The structure and function of the complement system, its genetics, its role in the physiological or pathological immune response and in host defence are now being analysed with the powerful techniques of molecular biology. New developments in complement research were discussed at a recent workshop representing the major sdentific meeting in this area of immunology*. Since the last complement workshop in Mainz, Germany, two years ago, a substantial number of cDNA and genomic DNA clones encoding most complement components and related proteins have been isolated. These cloned complement genes have facilitated the structural comparison of functionally related and of polymorphic proteins. They have allowed examination of the molecular basis of genetic defects and of the regulation of complement synthesis, and have permitted the chromosomal localization of complement genes.
Polymorphismsand homologiesbetween complementproteins The components C4, C2 and factor B are of special interest because they are coded by genes of the major histocompatibility complex (MHC). Comparison of the complete primary structure of murine C4 and the related, haemolytically inactive sexlimited protein (Sip) showed not only the expected marked homology indicating gene duplication but also a deletion in Sip close to the corresponding cleavage site for Cls in C4 (M. Nonaka, Kanazawa; R. Ogata, La Jolla). The absence of haemolytic activity in Sip might be due to this deletion. Despite the close homology there is a selective regulation of transcription for these two, closely linked proteins, apparently determined by the flanking sequences (E. Georgatosou, Paris). Similarly, two closely linked human C4 isotypes, C4A and C4B, differ only in six amino acids despite their marked difference in haemolytic activity. Both loci are highly polymorphic. Null alleles, total deficiencies and duplications were identified at the protein level. At the genomic level most of the null alleles
news and tCeatures Complementresearch:the impact of molecular genetics from ReinhardBurger appeared normal; only a few resuited from gene deletions (M. Caroll, Boston). Differences in four amino acids in the C4d moiety of C4B were identified as the basis for the expression of Chido antigen determinants (C.Y. Yu, Oxford). Several groups reported progress in the structural analysis of the regulatory proteins at the cDNA level. The deduced amino acid sequence of C4b-binding protein (C4BP) revealed homology regions of about 60 amino acids with factor H, C2 and factor B (its Ba-fragment) (L. Chung, Oxford; T. Kristensen, La Jolla). All these proteins have the ability to interact with C3b or C4b. The homology region in this family of proteins (and also in the complement receptor CR1, see below) might, therefore, function as C3b/ C4b binding site. C2a and Bb bear regions homologous with catalytic domains of other serine proteases (D. Bentley, Oxford). Human C1 inhibitor shows homology with the protease inhibitors antithrombin III and C~l-antitrypsin (A. Davis, Boston). The availability of appropriately characterized cDNA clones facilitates a more detailed analysis of complement polymorphisms with a much higher resolution than previously. D. Bentley (Oxford) identified a restriction fragment length polymorphism (RFLP) of C2 and demonstrated three C2 variants, indistinguishable by typing at the protein level, in individuals bearing a common C2 allele.
Regulation of complementsynthesisby lymphokines
An interesting connection between mediators involved in cellular immune response and the complement system was described by the group of H. Colten (Boston). These studies might provide a model system for other immunologically relevant proteins and are concerned with the regulatory mechanisms for synthesis of the MHC class III products C2 and factor B. The genes for *TheXlthInternationalComplementWorkshopwas C2 and B are separated by only 0.6 heldin Miami,Forida,USA,3-5 November1985. kb. The lymphokine interferon ~/
(IFN--y) is a well-known modulator of MHC class II antigens (la-antigen) expression. S. Cote (Boston) demonstrated that IFN-~ induces an increase in synthesis of the MHC class Ill products C2 and B by human monocytes. Synthesis of both proteins is influenced at the pretranslational level, but the two closely linked proteins were affected independently as shown by different kinetics of transcription. Interleukin 1 (IL-1) influences the synthesis of factor B but not of C2 by hepatocytes. The basis for this selective action on the adjacent genes was studied by D. Perlmutter (Boston) by using murine L cells transfected with a cosmid bearing both the human C2 and factor B genes and expressing both proteins constitutively. IL-1 induced an increase of factor B expression but not C2, again by a pretranslational mechanism. The IL-1 regulation might affect the short segment between the two genes and one should be able to identify structural basis for this selectivity. IL-1 induced an increase in the density of the complement receptors CR1 and CR3 on human neutrophils (I.M. Berger, Cleveland).
Complement receptors A cDNA clone for CR1 (the C3b/ C4b receptor) was described by W. Wong (Boston). RFLP was demonstrated for the polymorphic variants of this protein permitting a distinction of the structural allotypes F and S. In addition, an independent RFLP of CR1 correlated with the genetically controlled number of CR1 per erythrocyte indicating that the quantitative CR1 expression is directly associated with the CR1 gene (J. Wilson, Boston). The CR1 cDNA clone contained repeating units related to the proteins H and C4BP (L. Klickstein, Boston). This corresponds again with the functional relation of these proteins, which share the ability to bind to C3b (see above). The proteins CR1, H and C4BP represent a linkage group located on the human chromosome 1. Several abstracts were directed at functional analysis of complement
~) 1986, Elsevier Science Publishers B.V, Amsterdam
0167 - 4919/86/$02.00
27
-news and eatures receptors at the protein level. The importance of rapid phosphorylation processes in CR1 function was reported by P. Changelian (Boston) and S. Wright (New York). Phorbol myristate acetate (PMA) induces phagocytosis by polymorphonuclear neutrophils. Incubation of neutrophils with PMA resulted in activation of protein kinase C and subsequent phosphorylation of CR1. This reaction exhibited a marked specificity in that it occurred only in phagocytic cells but not, e.g., in B cells or erythrocytes. This covalent modification of CR1 therefore seems to correlate with the acquired, increased phagocytic activity of the cells. In contrast, by appropriate stimulation of B-cell lines, phosphorylation of CR2 was induced arguing for a role of this receptor in B-cell function (see below). C. Mold (La Jolla) used the isolated CR2 protein (C3d receptor)incorporated into liposomes for a direct confirmation that the CR2 molecule in fact has also the capacity for the binding of Epstein-Barr virus. This dual receptor function apparently is mediated by distinct molecular sites because soluble C3d does not interfere with virus binding.
28
Complement and immune responses A new area of research was established at the Basel Institute for Immunology. F. Melchers (Basel) described the finding that cross-linked human C3d is one of the essential growth factors in the cell cycle of preactivated murine B cells. C3d regulates B-cell growth by binding to the C3d receptor on the lymphocyte surface. Cross-linked C3d gives a positive signal to the B cell for the entry from the G1 phase into the S phase. C3d replaces the macrophage:derived growth factors ('efactors') required for passing this 'restriction point' in the cell cycle of activated B cells. In contrast, soluble C3d has an inhibitory effect. This report clearly revived a quite controversial field; namely the role of complement in the induction of the antibody response. It produces a welcome and potent stimulus for discussions and additional work e.g., analysis of synthetic C3d peptides in this system (J. Lambris, La Jolla). F. Praz (Paris) reported that the Ba frag -0 ment of factor B also exhibits a marked mitogenic effect on preactivated murine B cells.
Independent pieces of evidence fit into Melcher's hypothesis, in particular the finding that - despite some exceptions (E. Klein, Stockholm)Epstein-Barr virus infection enhances or induces expression of C3dreceptor (CR2) in the membrane of previously virus-negative Burkitt's lymphoma lines (J. Cohen, Paris). One can speculate about a role of this host cell (not virus) encoded protein and its ligand C3d in proliferation of Fpstein-Barr virus transformed B cells. Similar supportive evidence was provided by M. Barel (Paris) showing that binding of antibody to CR2 induces enhanced B-cell proliferation. A role of C3 in the induction of the antibody response was also suggested by E. Boettger (Mainz) using a different approach. In the absence of C2 or C4 the activation of C3 via the classical pathway is prevented. C2deficient or C4-deficient guinea pigs exhibited a grossly impaired antibody formation to the antigen bacteriophage CX 174 especially at low antigen concentrations. The residual ability to mount a humoral immune response of these deficient animals remained unaffected after artificial depletion of C3 with cobra venom factor (CVF). In contrast, in normal guinea pigs the antibody formation was suppressed by decomplementation, again dependent on the antigen dose. Therefore, due to the absence of either C3 or of C3-cleaving enzymes there appears to be an insufficient ability to use C3 during the induction phase of an immune response resulting in an impaired antibody response. These data argue against a generalized immunosuppressive function of the complement fragments which are generated in excess during the CVF-mediated decom plementation. U. Hadding (Mainz) suggested a direct role of C3 in MHC-controlled immune phenomena. After treatment of human monocytes with C3b the expression of la antigens was reduced and, concomitantly, the capacity to function as antigenpresenting cells in an in-vitro system for T-cell proliferation was inhibited. The physiological role of C3 in the immune response has been analysed by numerous groups for more than a decade. Considering the recent progress with the improved, less heterogenous, and well defined assay systems, a more detailed outline of the various biological func-
Immunology Today, vol. 7, No. 2, 1986
tions of this versatile molecule and their reaction sequence appears to be in sight. A recently described guinea pig strain with an inherited C3-deficiency might prove helpful for further analysis. These animals have a C3 s-chain rendered functionally inactive by an insertion in the C3d-region (H. Auerbach, Boston).
Membrane associated regulatory proteins The decay accelerating factor (DAF), described years ago, has experienced a renaissance as shown by a substantial number of contributions to this topic. This membraneassociated protein accelerates the physiological decay of C3 convertases of both the classical and alternative pathways and prevents their assembly. It apparently has the capacity for binding to the catalytic site of these enzymes (K. WadeyPangburn, Tyler). M. Medof (New York) reported the presence of DAF on a wide variety of epithelial cells and exocrine glandular cells in many different tissues (e.g. cornea and bladder). In addition, DAF was found in various secretions like saliva and urine. The widespread distribution of DAF, especially on surfaces in intimate contact with the complement and other protein systems, might reflect its important- perhaps not yet known - biological function. No general agreement was reached in discussions on the role of DAF and acetylcholinesterase abnormalities in the increased susceptibility for lysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. A new leucocyte membrane glycoprotein (gp 45-70) was shown by T. Seya (St. Louis) to possess a highly efficient cofactor activity (50 times superior to H) for the initial inactivation of C3b, and also of C4b. It was renamed, therefore, membrane cofactor protein and may be a major regulatory protein. A regulatory role was discussed for two newly described erythrocyte proteins which interfered with the insertion of C5b9 into the membrane and with channel formation (G. H~nsch, Heidelberg; L. Zalman, La Jolla). Susceptibility of tumor cellsto lysis Antibody-sensitized melanoma cells differ in their susceptibility to lysis by complement. Susceptible cells bound C3b to their surface and retained it (M. Panneerselvam, Wash-
immunology Today, vol. 7, No. 2, 1986
ington). Resistant cells initially bound even three times higher amounts of C3b but exhibited a rapid C3b inactivation by cleavage to C3d. Pretreatment of the resistant cells with adriamycin induced susceptibility and inhibited the degradation of C3b. immobilized adriamycin could replace the soluble drug with an even higher efficiency and did not have the cytotoxic side effects. Apparently, a surface-mediated mechanism is involved in this phenomenon. The molecular basis is still obscure; one might consider some drug effects on toxic oxygen derivatives or interaction with regulatory proteins.
The C1 component cDNA clones were identified for the C1 subunits and should facilitate the structural analysis of this highly complex molecule. Defective transcripts, possibly caused by a mutation, for the B-chain of Clq have been demonstrated in one Clq-deficient patient (R. McAdam, Oxford). The French groups (hosts of the next International Complement Workshop in Chamonix, September 1987) have succeeded in localizing the peptide bonds which are cleaved during Clr activation. They identified twin domains as the core of C1 r involved in catalytic function and presented a sterical model for interaction of the different C1 subunits in order to explain the intramolecular activation events (G. Arlaud, Grenoble). B.Ziccardi (La Jolla) postulated a mechanism of feedback inhibition. He demonstrated that nascent C3b and C4b generated in the course of complement activation bind to the Fc portions of antibodies in immune complexes, thereby preventing further C1 activation.
RecombinantCSa Three similar contributions described the production of C5a by expression of appropriate gene segments in E. coil (A. Franke, Groton; G. Carter, Abbot Park; G. Fey, La Jolla). The product exhibited most of the biological functions of normal C5a. Site-specific mutagenesis in the
news and features C5a should permit the sites involved in receptor be identified; modulation ing events for cellular seems feasible.
molecular binding to of triggerfunctions
The membrane attack complex C5b-9 The exact reaction sequenceinvolved in lysis of cells by the membrane attack complex remains controversial and some questions are unresolved. New elements emerged e.g. through biophysical approaches, molecular genetics, or through the similarities between complement lesions and cytolytic granules in cytotoxic lymphocytes. An impressive example of the effectiveness of the combined application of molecular genetics and immunological techniques was presented by K. Stanley (Heidelberg). Based on the amino acid sequence derived from a cDNA clone, he pro posed a topographical map of C9 and a model for insertion of C9 into the membrane. According to his computer-aided model, C9 does not contain linear hydrophobic sequence for interaction with membrane lipids. In contrast, the secondary structure is characterized by amphi pathic elements permitting insertion into the membrane. Monoclonal antibodies reactive with defined C9 segments (obtained by cloning in an expression vector) permitted localization of individual determinants. A. Esser (Gainesville) emphasized the importance of a 'non-ordered' lipid bilayer induced by C5b-8 as prerequisite for association of C9 with the membrane. This conclusion was based on experiments with membrane-restricted photolabelling probes. The concept that poly-C9 is the actual lytic agent was challenged by S. Bhakdi (Giessen). The degree of C9-polymerization and thereby the C9 content differs in C5b-C9 complexes and is responsible for the observed heterogenous pore size of the complement ring lesions. He described a method for producing defined, homogenous complexes containing - at least statistically - a single C9 in association with C5b-8 which, nevertheless, are haemolytically active. Nucleated cells seem to
escape complement lysis either by calcium-dependent internalization (D. Carney, Baltimore) or by concentrating most of the C5b-C9 complexes in minor areas of the cell surface membrane which are shed in the form of vesicles (B. Morgan, Gainesville). The molecular mechanisms and site of lethal damage of bacteria by complement remain controversial and are not fully understood. K. Joiner (Bethesda) proposed that the presence of sufficient, multimeric C9 within the C5b-9 complex is required to cause efficient damage, whereas J. Dankert (Gainesville) found no need for poly-C9 formation. PoiyC9 may provide a trigger signal for an energy-dependent bacterial process which finally affects the inner membrane and causes release of cytoplasmic constituents. A bridge between cell-mediated lytic events and complement lesions was established by the demonstration of shared domains in the corresponding proteins as detected by cross-reactivity of monoclonal antibodies (E. Podack, Valhalla; J. Tschopp, Epalinges). The family of related proteins includes C9 and per'forin from T-cell cytolytic granules, C8 e-chain, C7, C6, and the functionally unrelated receptor for lowdensity lipoprotein. At present, the emphasis in complement research seems to be on the interaction of regulatory compo nents (particularly of the membrane bound proteins) with their ligands; the identification of functional domains, preferentially defined by peptides; the modulation of the immune response by complement; the effect on cellular functions; and the structural analysis of related proteins belonging to multigene families. The rapid development in the last two years and the increased spreading of molecular genetics promises much information for the next workshop in 1987. The abstracts of the Xlth International Complement Workshop were published as a special issue of the journal Comple ment, 2, 3-92 (1985) Reinhard Burger is at the Institute for Immunology, University of Heidelberg, 6900 Heidelberg, FRG.
29