Primary pneumococcal peritonitis can be the first presentation of a familial complement factor I deficiency1.

Primary pneumococcal peritonitis is a rare infection that has been described in women but has not been previously linked with immunodeficiency. The complement system plays a central role in immune defence against Streptococcus pneumoniae and, in order to evade complement attack, pneumococci have evolved a large number of mechanisms that limit complement-mediated opsonization and subsequent phagocytosis. We investigated an apparently immunocompetent woman with primary pneumococcal peritonitis and identified a family with deficiency for complement factor I. Primary pneumococcal peritonitis should be considered a possible primary immunodeficiency presentation.

Further studies of the down-regulation by Factor I of the C3b feedback cycle using endotoxin as a soluble activator and red cells as a source of CR1 on sera of different complotype.

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 - the co-factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration - 22 µg/ml of extra Factor I - will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co-factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age-related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease.

Complotype affects the extent of down-regulation by Factor I of the C3b feedback cycle in vitro.

Sera from a large panel of normal subjects were typed for three common polymorphisms, one in C3 (R102G) and two in Factor H (V62I and Y402H), that influence predisposition to age-related macular degeneration and to some forms of kidney disease. Three groups of sera were tested; those that were homozygous for the three risk alleles; those that were heterozygous for all three; and those homozygous for the low-risk alleles. These groups vary in their response to the addition of exogenous Factor I when the alternative complement pathway is activated by zymosan. Both the reduction in the maximum amount of iC3b formed and the rate at which the iC3b is converted to C3dg are affected. For both reactions the at-risk complotype requires higher doses of Factor I to produce similar down-regulation. Because iC3b reacting with the complement receptor CR3 is a major mechanism by which complement activation gives rise to inflammation, the breakdown of iC3b to C3dg can be seen to have major significance for reducing complement-induced inflammation. These findings demonstrate for the first time that sera from subjects with different complement alleles behave as predicted in an in-vitro assay of the down-regulation of the alternative complement pathway by increasing the concentration of Factor I. These results support the hypothesis that exogenous Factor I may be a valuable therapeutic aid for down-regulating hyperactivity of the C3b feedback cycle, thereby providing a treatment for age-related macular degeneration and other inflammatory diseases of later life.

Specific selection of antigen-reactive lymphocytes into antigenically stimulated lymph nodes in sheep.

Sheep were primed to a variety of antigens and the efferent lymphatic from a popliteal lymph node was cannulated. The cannulated node was challenged repeatedly with PPD and all the lymph and cells removed from the animal. During this time the PBL were monitored for reactivity to all antigens (purified protein derivative of tuberculin [PPD], johnin, and keyhole limpet hemocyanin [KLH]) by the vitro transformation assay. The response of these cells to PPD was found to be gradually eliminated after repeated challenge of the cannulated node with that antigen. The response to the other antigens was unimpaired. No depletion of this response to PPD occurred in cannulated sheep when the antigen was given into a noncannulated node. In vivo delayed-type hypersensitivity skin test and helper T cell assays confirmed that there is a specific selection of antigen-reactive cells from the recirculating lymphocyte pool into antigen-stimulated lymph nodes.

Reactive lysis: the complement-mediated lysis of unsensitized cells. I. The characterization of the indicator factor and its identification as C7.

This paper describes the characteristics of the indicator factor (I) which takes part in reactive hemolysis and its identification as the seventh component of complement. I was shown to be a beta globulin with a sediment coefficient of 5.7S and a molecular weight of about 140,000. Experiments on the depletion of I activity with anti-I antiserum or with activated R euglobulin showed that I was a late acting complement component necessary for the lysis of cells after the EAC142 stage. Complement component analysis of purified I fractions excluded all known components except C7. The physicochemical characteristics of I are compatible with published data on C7. The method of quantitation described represents a convenient method of testing for C7.

Reactive lysis: the complement-mediated lysis of unsensitized cells. II. The characterization of activated reactor as C56 and the participation of C8 and C9.

It has been shown that the "activated reactor" that is produced in certain human sera by complement activation is a stable complex of the fifth and sixth component of complement (C56). On interaction with C7, the indicator factor, a complex C567 is formed which for a short time (half-life less than 1 min) has an activated binding site and can attach itself to normal red cell membranes, conferring on them the hemolytic properties of the "heat stable" complement intermediate EC 1 approximately 7, the capacity to be lysed by C8 and C9. These cells have neither antibody nor the complement components up to C3 bound on them. The binding site-activated C567c-can similarly bind to other hydrophobic surfaces, including agarose gel where it forms a "stainable line". If the complex is not bound to a surface, the binding site decays and the resulting complex will no longer give rise to lysis. However it will still inactivate C8 and C9 in solution. The sera that can generate activated reactor apparently do so because they have an excess of C5 and C6, compared to their content of C7. The phenomenon of reactive lysis thus represents complement-mediated lysis of unsensitized cells initiated at the C5 stage by a stable complex (C56) which was generated by complement activation at a distance. The immunochemistry of the phenomenon is described and some of its implications discussed.

Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59.

Schistosomiasis is a parasitic disease affecting approximately 200 million people, primarily in the third world. Schistosoma mansoni, one of the causative agents of this disease, parasitize the human mesenteric and portal blood systems while successfully evading host immune responses. During parasite penetration into the mammalian host and shortly afterwards, the larvae rapidly convert from being sensitive to being resistant to C-mediated killing. Treatment of the C-resistant parasitic forms with trypsin renders the parasite susceptible to C attack, thus indicating the presence of C inhibitory protein(s) on the parasite surface. We describe here an intrinsic schistosome C inhibitory protein (SCIP-1) that exhibits antigenic and functional similarities with the human C-inhibitor CD59. Like CD59, SCIP-1 is capable of inhibiting formation of the C membrane attack complex (MAC), probably by binding to C8 and C9 of the C terminal pathway. In addition, SCIP-1 is apparently also membrane-anchored via glycosyl phosphatidylinositol as it can be specifically released with phosphatidylinositol-specific phospholipase C. Soluble SCIP-1, partially purified from Nonidet P-40 extracts of schistosome tegument is capable of inhibiting hemolysis of sensitized sheep erythrocytes and of rabbit erythrocytes by human C. Anti-human CD59 antibodies block this activity of SCIP-1 and in addition, upon binding to intact parasites, render them vulnerable to killing by human and guinea pig C. SCIP-1 is located on the surface of C-resistant forms of the parasite, i.e., 24-h cultured mechanical schistosomula and in vivo-derived adult worms as revealed by immunofluorescence and immunogold electron microscopy studies. These results identify one of the mechanisms schistosomes use to escape immune attack.

The mechanism of action of the C3b inactivator (conglutinogen-activating factor) on its naturally occurring substrate, the major fragment of the third component of complement (C3b).

The fixation of the third component of complement (C3) results in many important biological phenomenon, among which are (a) immune adherence (1), (b) enhancement of phagocytosis (2,3), (c) the release of an anaphylatoxin which is a potent releaser of histamine (4), and (d) the feedback activation of the alternative pathway (5,6). The physiological mechanisms involving C3 fixation require the generation of a C3 convertase which may occur by two separate pathways. C3 convertase can be generated, in the form of C42, by the so-called classical pathway of activation or in the form C3b,B by the alternative or properdin pathway (7). In both cases, C3 is converted to C3b by cleavage of a small peptide, C3a. Normal human serum contains an inactivator of activated C3b. This C2b inactivator or conglutinogen-activating factor (KAF) has been shown to inhibit both immune hemolysis and the immune adherence properties of C3b and to cause cleavage of C3b in the fixed and fluid- phase stages (8-11). Although it is known that the C3b inactivator is not depleted during its reaction with C3b and that C3b treated with the C3b inactivator becomes extremely sensitive to proteolytic digestion by trypsin and "trypsin-like" enzymes (9), the exact molecular nature of the action of the C3b inactivator on C3b has not been studied. In an effort to delineate the products of this interaction, purified C3b and C3b inactivator were allowed to react for various specific lengths of time and the products of these reactions were then analyzed.

Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.

C6: synthesis by the liver in vivo.

The allotype of the sixth component of complement was determined in a patient before and after liver transplantation. The C6 phenotype changed from A before transplantation to B (the donor phenotype) within 10 days of the transplant and remained wholly of the donor phenotype at 17 wk. This demonstrates that the liver is the exclusive or predominant site of C6 synthesis in vivo in man.

Complement-mediated adipocyte lysis by nephritic factor sera.

Recent data indicate a previously unsuspected link between the complement system and adipocyte biology. Murine adipocytes produce key components of the alternative pathway of complement and are able to activate this pathway. This suggested to us an explanation for adipose tissue loss in partial lipodystrophy, a rare human condition usually associated with the immunoglobulin G(IgG) autoantibody nephritic factor (NeF) which leads to enhanced alternative pathway activation in vivo. We hypothesized that in the presence of NeF, there is dysregulated complement activation at the membrane of the adipocyte, leading to adipocyte lysis. Here we show that adipocytes explanted from rat epididymal fat pads are lysed by NeF-containing sera but not by control sera. A similar pattern is seen with IgG fractions of these sera. Adipocyte lysis in the presence of NeF is associated with the generation of fluid-phase terminal complement complexes, the level of which correlates closely with the level of lactate dehydrogenase, a marker of cell lysis. Lysis is abolished by ethylenediaminetetraacetic acid, which chelates divalent cations and prevents complement activation, and reduced by an antibody to factor D, a key component of the alternative pathway. These data provide an explanation for the previously obscure link between NeF and fat cell damage.

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin.

The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 x 10(4) C3d,g molecules per E. The finding that monocyte and neutrophil rosettes with EC3d,g were inhibited by C3d-OR, suggested that these phagocytic cells might indeed express very low numbers of CR(2), and that these CR(2) were detectable with EC3d,g and not with EC3d because C3d,g had a higher affinity for CR2 than did C3d. A fourth C3 binding site for CR(3) and conglutinin (K) was restricted to the iC3b fragment. Because of simultaneous attachment of iC3b to phagocyte CR3 and CR(3), the characteristics of iC3b binding to CR3 could only be examined with phagocytes on which the CR(1) had been blocked with anti-CR(1). Inhibition studies with EDTA and N-acetyl-D-glucosamine demonstrated a requirement for both calcium cations and carbohydrate in the binding of EC3bi to CR3 and to K. However, CR(3) differed from K in that magnesium cations were required in addition to calcium for maximum CR(3) binding activity, and NADG produced less inhibition of CR(3) activity than of K activity.

Human protectin (CD59), an 18-20-kD homologous complement restriction factor, does not restrict perforin-mediated lysis.

Human protectin (CD59) is an 18-20-kD membrane glycoprotein that restricts lysis of human erythrocytes and leukocytes by homologous complement. By directly incorporating protectin into membranes of heterologous cells we observed that protectin did not prevent perforin-mediated killing, whereas complement killing was effectively restricted. Further, no significant enhancement of cell-mediated killing or target killing by purified perforin was observed with anti-protectin antibodies. Thus, in contrast with complement lysis, restriction of lysis by protectin does not apply to cell-mediated killing.

Taking complement to the clinic--has the time finally come?

Complement has been studied for over a century and its role in promoting the effector side of antibody-mediated immune reactions and of inducing inflammation is well understood. Nevertheless, it has proved surprisingly difficult to translate this information into pharmaceutical agents that can be used to treat immunopathological and inflammatory disease. There are, however, now clear signs that this situation will change. New types of therapeutic agents to interfere with complement function are being developed and it has become apparent quite recently that some common and otherwise untreatable diseases such as age-related macular degeneration are very largely due to mutations in the complement system that leads to a hyperinflammatory state. This has stimulated a renaissance of interest in the complement system as a therapeutic target and in this short review we discuss the possible ways of taking complement to the clinic, and the indications for which this may be carried out.

Microbial immunology: a new mechanism for immune subversion.

The number of mechanisms that have evolved in microbes to subvert the immune response seems limitless. Tubercle bacilli have found a novel way to coat themselves with the C3 complement protein and invade macrophages by interactions with complement receptors.

Molecular basis of subtotal complement C6 deficiency. A carboxy-terminally truncated but functionally active C6.

Individuals with subtotal complement C6 deficiency possess a C6 molecule that is 14% shorter than normal C6 and present in low but detectable concentrations (1-2% of the normal mean). We now show that this dysmorphic C6 is bactericidally active and lacks an epitope that was mapped to the most carboxy-terminal part of C6 using C6 cDNA fragments expressed as fusion proteins in the pUEX expression system. We thus predicted that the abnormal C6 molecule might be carboxy-terminally truncated and sought a mutation in an area approximately 14% from the carboxy-terminal end of the coding sequence. By sequencing PCR-amplified products from this region, we found, in three individuals from two families, a mutation that might plausibly be responsible for the defect. All three have an abnormal 5' splice donor site of intron 15, which would probably prevent splicing. An in-frame stop codon is found 17 codons downstream from the intron boundary, which would lead to a truncated polypeptide 13.5% smaller than normal C6. This result was unexpected, as earlier studies mapped the C5b binding site, or a putative enzymatic region, to this part of C6. Interestingly, all three subjects were probably heterozygous for both subtotal C6 and complete C6 deficiency.

Restoration by purified C3b inactivator of complement-mediated function in vivo in a patient with C3b inactivator deficiency.

In a patient with lifelong increased susceptibility to infection and multiple abnormalities in complement-mediated functions, the infusion of normal plasma had been seen to produce a prolonged partial correction of serum abnormalities. It was subsequently shown that the patient was genetically deficient in the C3b inactivator and that immunochemical depletion of C3b inactivator from normal serum resulted in abnormalities similar to those found in the patient's serum, including alternative pathway C3 activation. Highly purified C3b inactivator was obtained from the euglobulin fraction of normal human serum, sterilized by filtration, and infused intravenously. Partial or complete correction of almost all the known serum abnormalities was obtained. C3b almost disappeared from the serum within 4-5 h, as did Factor C activity. Native C3, C5, and serum hemolytic activity rose to normal or near-normal levels over 4 days and were sustained for another week. Factor B, properdin, opsonic activity, and bactericidal activity reached a level at least two-five times that found before the infusion within 24 h and fell over the next 5 days. These observations prove the primary role of C3b inactivator deficiency in the patient's disease and demonstrate clearly the curcial role in vivo of C3b inactivator in modulating alternative pathway activity.

Metabolic studies of the third component of complement and the glycine-rich beta glycoprotein in patients with hypocomplementemia.

Metabolic studies using radioiodine-labeled third component of complement (C3) and the glycine-rich beta glycoprotein (GBG), a major component of the C3b-feedback pathway, were undertaken in normal subjects, in 22 patients with evidence of complement activation, and in 11 patients with various renal diseases without evidence of complement activation. In seven normal subjects GBG was found to be a rapidly metabolized protein with catabolic rates ranging from 1.7% to 2.2% of the plasma pool/h, synthesis rates from 0.14 to 0.21 mg/kg per h. and extravascular/intravascular distribution ratios from 0.81 to 1.31. In patients with reduced plasma C3, both increased C3 fractional catabolic rates and reduced C3 synthesis rates were observed, and in some patients there was evidence of increased extravascular distribution of the protein. GBG catabolism was usually increased when there was evidence of C3 activation, presumably reflecting activation of the C3b-feedback; but GBG turnover was normal or only slightly accelerated in some patients with accelerated C3 catabolism and profound hypocomplementemia, suggesting that reduced C3 synthesis had limited activation of the C3b-feedback.

Leukocytes from four patients with complete or partial Leu-CAM deficiency contain the common beta-subunit precursor and beta-subunit messenger RNA.

Deficiency of a family of three leukocyte adhesion molecules (Leu-CAM) is associated with recurrent and life-threatening bacterial infections in man. Each of the three antigens, Mo1, LFA-1, and Leu M5 has a distinct alpha subunit noncovalently associated with a common beta subunit that appears to be required for the expression of these antigens on the cell surface. To investigate the molecular basis of Leu-CAM deficiency, we studied leukocytes from four unrelated patients suffering from complete or partial Leu-CAM deficiency using immunoprecipitation of metabolically labeled proteins, RNA extraction, and Northern blot analysis. We found that B cells from all four patients synthesized a normal sized beta subunit precursor that either failed to "mature" or matured only partially to the membrane expressed form. B cells from all four patients also had a single normal sized beta subunit mRNA of approximately 3.4 kb. Leu-CAM deficiency, in these unrelated patients, is not due to the absence of the beta chain gene or to aberrant splicing of its mRNA and are consistent with a defective beta subunit gene resulting in abnormal posttranslational processing of the synthesized molecule.