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The bilateral occlusion of the renal arteries for 2h resulted in a marked decrease of serum complement activity after recirculation. The release of a proteinic factor with anticomplementary activity detectable in vitro in normal kidney supernate is considered a probable cause of the phenomenon. This factor is able to activate the alternative complement pathway in human serum.
Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.
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PURPOSE: The case of a 4-year-old boy with hemophilia B with inhibitor who developed nephrotic syndrome is described. The possible association between factor IX therapy and nephrotic syndrome in patients with hemophilia B is discussed. PATIENT AND METHODS: A chart review of a 4-year-old boy with hemophilia B and an inhibitor who developed nephrotic syndrome with transient hypocomplementemia was performed. In addition, a literature search was undertaken to determine the prevalence of this association and possible etiologic factors. RESULTS: Although the nephrotic syndrome was resistant to steroid therapy and Bebulin (Osterreichisches Institut für Haemoderivate Ges.M.B.H., Subsidiary of Immuno AG, Vienna, Austria) infusions were continued, the edema resolved and proteinuria decreased. Seven month later, proteinuria, accompanied by transient hypocomplementemia, increased again. A rise in factor IX inhibitor level was observed. The patient received no immunosuppressive therapy, and exhibited a continuous decrease in urinary protein excretion over the following months. DISCUSSION: A discussion about possible differential diagnoses and a review of the literature are presented.
OBJECTIVE: To determine if endometrial tissue specific complement (C) activation occurs in patients with endometriosis. DESIGN: Prospective. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Twenty-six women, 9 with endometriosis and 17 without endometriosis. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Uterine and ectopic endometria were evaluated by the immunofluorescence assay for the presence of activated products of the initial (C3d) and terminal (C5b-9) C pathway, C3 regulatory proteins (decay-accelerating factor and membrane cofactor protein), and terminal C regulatory proteins (clusterin and vitronectin). RESULTS: The initial (C3d) and terminal (C5b-9) C components were deposited on blood vessel walls in uterine and/or ectopic endometria of women with and without endometriosis. In the stromal compartment at both sites, C deposition was colocalized with terminal C inhibitors/cell-cell attachment factors, clusterin and vitronectin on elastic fibers. No specific staining for C proteins was detected on the glandular epithelium of uterine and ectopic endometrial tissues examined. Furthermore, intense staining of endometrial epithelium for C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein was noted on endometrial glands from women with and without endometriosis. CONCLUSIONS: Our findings demonstrate a lack of specific deposition of C in the glandular epithelial cells of endometria of women with endometriosis. The localization of C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein on glandular epithelium may suggest the involvement of intrinsic protective mechanisms on these cells from autologous C attack in vivo.
The sensitivity to lysis by complement of the erythrocytes of 56 patients with paroxysmal nocturnal haemoglobinuria (PNH) was compared to the membrane expression of decay accelerating factor (DAF, CD55), membrane inhibitor of reactive lysis (MIRL, CD59) and acetylcholinesterase (AChE). Most patients (36/50 72% in whom the analysis could be made) appeared to have erythrocytes of intermediate sensitivity to complement in the blood. These cells appeared as a discrete population of cells (PNH II cells), as a 'tail' of cells slightly less sensitive than the predominant PNH III cells (previously called PNH IIIb cells), or as a continuous spectrum of cells sensitive to complement. The PNH III cells totally lacked all three proteins (DAF, MIRL, AChE) by flow cytometric analysis whereas PNH I cells appeared to have normal or nearly normal amounts of each. The cells of intermediate sensitivity (PNH II) had coordinately decreased expression of all three proteins; the level of expression of DAF and MIRL paralleled the sensitivity of the cells to the haemolytic action of complement.
BACKGROUND: This prospective study was undertaken to assess the differences between the levels of the main plasma components of complement in allergic subjects and in those with pseudoallergic clinical manifestations. METHODS: Plasma C3 and C4 were evaluated in a total of 256 subjects examined consecutively at the allergology outpatients clinic of the Internal Medicine Division B, University of Turin. Total IgE and C1-inhibitor levels were also determined in 128 and 44 subjects respectively. RESULTS: C3 and C4 levels were not significantly different (p = 0.398 and p = 0.497) in 123 subjects with a positive and 133 with a negative prick test, nor in allergic subjects with respiratory as opposed to skin symptoms (p = 0.293 and p = 0.462), whereas the C-1 inhibitor was significantly lower (p = 0.046) in the respiratory subgroup. Total IgE was positively correlated with the C3 level (p = 0.036) in 75 allergic subjects. CONCLUSIONS: These findings suggest that plasma C3 and C4 values are not sufficient to discriminate IgE inflammation (positive prick test) and pseudoallergy (negative prick test) in the assessment of subjects with clinically suspected allergy. The positive correlation between IgE synthesis and C3 also points to an interaction between IgE synthesis and C3 regulation proteins in patients with IgE mediated diseases. Further investigation of other acute phase proteins (C reactive protein, fibrinogen) and the cytokines regulating their synthesis (IL-6) in such patients will help to clarify this correlation.
Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.
Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf(-/-)) or in the classical pathway component C4 (C4(-/-)). Clinical disease activity was markedly decreased in Bf(-/-) compared with wild-type (WT) mice (0.5 +/- 0.22 (n = 6) in Bf(-/-) vs 8.83 +/- 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4(-/-) and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf(-/-) as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf(-/-) as compared with C4(-/-) and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins.
The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself.
Nucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC-3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti-mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC-3> SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC-dependent process. Combined treatment of CA with anti-CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement-mediated lysis. Therefore, the effectiveness of antibody- and complement-based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.
Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable.
OBJECTIVE: The aim of this study was to examine the presence of the complement regulatory proteins protectin (CD59), decay accelerating factor (CD55), membrane cofactor protein (CD46) and clusterin (SP-40,40) in connection with the autoimmune exocrinopathy in Sjögren's syndrome (SS). METHODS: Labial salivary gland biopsy specimens and saliva samples were obtained from SS patients and healthy subjects. The tissue expression of these proteins was assessed by ABC immunoperoxidase techniques. Saliva was analysed by immunoblotting methods. RESULTS: Tissues from healthy subjects expressed CD59 and CD46 on the apical/luminal surfaces of acinar and ductal epithelial cells. CD59, CD55, and SP-40,40 were expressed in interstitial tissues. In SS tissues, the expression of CD55, CD59, CD46 and SP-40,40 was up-regulated, following the same patterns of tissue localization as the controls. CD46 and SP-40,40 exhibited a much higher staining intensity in SS patients compared to controls. CD55, CD59 and SP-40,40 were present at high concentrations in saliva from SS patients. CONCLUSION: The presence of complement regulators in our SS patients' saliva and the high expression of these proteins in inflamed salivary gland tissue followed the inflammatory reaction. These regulators may be involved in protecting the exocrine glands from complement mediated injury.
Controlled activation of the complement system is critical to the host-defense response of the immune system. Activated complement is responsible for the stimulation of a localized protective inflammatory response to either invading microorganisms or foreign molecules (toxins). However, the autologous activation of the complement system can have devastating consequences on many organ systems. This review discusses the various pathways involved in the activation of the complement system and the multiple levels of control established within the body to regulate activation. It also focuses on the role of complement activation in cardiovascular disease, especially myocardial ischemia and reperfusion injury as well as in cardiopulmonary bypass procedures. Lastly, this review also provides a comprehensive overview of both biologically derived proteins and chemically developed inhibitors of the complement system that range from those that are currently in the discovery stage to those that are in clinical development as novel therapeutic agents.
No episodes of clinically significant in vivo haemolysis have been reported in individuals with a novel form of decay accelerating factor (DAF) deficiency (Inab phenotype), nor do functional in vitro assays for complement-mediated haemolysis show the extreme sensitivity to lysis characteristic of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes. DAF appears to be totally deficient in the Inab erythrocytes as judged by immunochemical and functional assays. Unlike PNH, the only other described DAF deficiency (where several other phosphatidylinositol (PI)-linked membrane proteins are also absent), the only protein lacking from Inab erythrocytes appears to be DAF. The Inab phenotype seems to be an inherited specific defect in DAF whereas PNH is an acquired defect in the mechanism of insertion of PI-linked proteins into cell membranes. These findings support the view that susceptibility of PNH erythrocytes to in vivo and in vitro complement-mediated haemolysis is not due simply to DAF deficiency but to either the combined lack of several membrane proteins or to deficiency of other regulatory proteins such as the membrane attack complex inhibitor/homologous restriction factor (MIP/HRF). The findings also raise questions as to the role of erythrocyte DAF.
PURPOSE: The presence of complement activation products in the human eye during infection or inflammation has been well described. During complement activation the host must be protected from attack against self tissue; this is achieved by three membrane-bound complement regulatory proteins: membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55), and membrane attack complex inhibiting protein (CD59). This study was undertaken to analyze the expression of these proteins in the normal human eye. METHODS: Tissues were sectioned by cryostat and both polyclonal and monoclonal antibodies to MCP, DAF, and CD59 were used. Control stains were performed with nonrelevant antibodies of the same immunoglobulin subclass and normal rabbit serum as well as by omission of the primary and secondary antibodies. RESULTS: All three proteins were found to be differentially expressed in the human eye. With anti-MCP, strong staining of the corneal epithelium and weak staining of the corneal keratocytes in stroma and photoreceptor cells was observed. Staining with anti-DAF was very strong in the corneal epithelium and the ciliary body and moderate in the corneal stroma (keratocytes) and iris. In contrast, anti-CD59 stained very strongly in the corneal epithelium, corneal stroma (keratocytes), iris, choroid, and all layers of the retina, and moderately in the ciliary body. CONCLUSIONS: Identification of MCP, DAF, and CD59 in the human eye provides evidence that a regulatory system exists to protect these cells from destruction by complement-activating events. It remains to be determined if other more specialized functions exist for these proteins, especially in the case of CD59 because of its extensive expression in the retina.
Membrane attack complexes of complement occur around thyroid follicles in Graves' disease and Hashimoto's thyroiditis. The lytic potential of such complexes is controlled by membrane-bound and fluid phase regulators and we have investigated the role of these in autoimmune thyroid disease. By immunohistochemical staining, clusterin and S-protein were found in all nine thyroid specimens from patients with Graves' disease and S-protein was found in one of two Hashimoto glands. CD46, CD55 and CD59 were found on thyroid cells in all specimens. CD46 and CD55 expression occurred on thyroid cells cultured in vitro and was increased significantly by culture with interleukin-1 (IL-1) and interferon-gamma (IFN-gamma), which are known to be released by the lymphocytic infiltrate in these conditions. Blocking CD55 had a weak and inconsistent effect on complement-mediated thyroid cell killing in vitro but, in four of five experiments, blocking CD46 enhanced killing. However, the effect of blocking CD59 was greater in all cases than blocking CD46 or CD55. Expression of these fluid phase and membrane-bound proteins may be important in determining the severity of thyroid damage produced by complement fixation in Graves' disease and Hashimoto's thyroiditis.
A rapid and efficient procedure for purification from Vipera lebetina venom of a low molecular weight anticomplement protein is described. The procedure used gel filtration on Superose 12, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrated on SDS-PAGE as a single band of about 25,000 Da under nonreducing conditions and as a band of 16,000 Da under reducing conditions. Its isoelectric point was estimated to be 7.6 +/- 0.1. The isolated Vipera lebetina protein was found to decrease the hemolytic activity in human serum measured by assays for classical pathway and alternative pathway activation. The loss of the complement activity could be ascribed, at least in part, to a proteolytic cleavage of the alpha chains of C3 and C4. This protein was also found to be without action on human blood coagulation and on purified fibrinogen and Factor B.