The complement inhibitors Crry and factor H are critical for preventing autologous complement activation on renal tubular epithelial cells.

Congenital and acquired deficiencies of complement regulatory proteins are associated with pathologic complement activation in several renal diseases. To elucidate the mechanisms by which renal tubular epithelial cells (TECs) control the complement system, we examined the expression of complement regulatory proteins by the cells. We found that Crry is the only membrane-bound complement regulator expressed by murine TECs, and its expression is concentrated on the basolateral surface. Consistent with the polarized localization of Crry, less complement activation was observed when the basolateral surface of TECs was exposed to serum than when the apical surface was exposed. Furthermore, greater complement activation occurred when the basolateral surface of TECs from Crry(-/-)fB(-/-) mice was exposed to normal serum compared with TECs from wild-type mice. Complement activation on the apical and basolateral surfaces was also greater when factor H, an alternative pathway regulatory protein found in serum, was blocked from interacting with the cells. Finally, we injected Crry(-/-)fB(-/-) and Crry(+/+)fB(-/-) mice with purified factor B (an essential protein of the alternative pathway). Spontaneous complement activation was seen on the tubules of Crry(-/-)fB(-/-) mice after injection with factor B, and the mice developed acute tubular injury. These studies indicate that factor H and Crry regulate complement activation on the basolateral surface of TECs and that factor H regulates complement activation on the apical surface. However, congenital deficiency of Crry or reduced expression of the protein on the basolateral surface of injured cells permits spontaneous complement activation and tubular injury.

Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The results of immunoradiometric assays and of fluorescence-activated cell sorter analysis showed that DAF was present not only on red cells but was widely distributed on the surface membrane of platelets, neutrophils, monocytes, and B and T lymphocytes. By Western blotting, we observed small but consistent differences in the Mr of DAF from the membranes of various cell types. Quantitative studies showed that phagocytes and B lymphocytes, which presumably enter more frequently in contact with immune complexes and other potential activators of complement, had the highest DAF levels. As previously reported by others, the red cells from PNH patients were DAF deficient. When the patients' red cells were incubated in acidified serum (Ham test), only the DAF-deficient cells were lysed. In addition, we detected defects in DAF expression on platelets and all types of leukocytes. The observed patterns of DAF deficiency in these patients were consistent with the concept that the PNH cells were of monoclonal origin. In one patient, abnormal and normal cells were found only in the erythroid, myeloid, and megakaryocytic lineages. In two other patients, the lymphocytes were also DAF deficient, suggesting that a mutation occurred in a totipotent stem cell. It appears, therefore, that the lesion leading to PNH can occur at various stages in the differentiation of hematopoietic cells.

Erythrocyte membrane protein deficiencies in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by intermittent hemolytic anemia. Membrane abnormalities of blood cells from patients with PNH are the reason for the unusual sensitivity to lysis by autologous plasma complement. A patient with typical clinical disease consistent with PNH is described together with a few strategies and pitfalls for treatment. Commonly used in vitro assays are discussed that document the complement-mediated lysis of aberrant PNH erythrocytes. Membrane-associated proteins that are abnormal in PNH cells, the characteristics of these proteins, and their mechanism(s) of action are described; these include the decay accelerating factor that inhibits the C3/C5 convertases of both complement pathways on cell surfaces, the C8 binding protein that modulates a step in terminal complement lysis, and other proteins that regulate complement-mediated lysis at early or late steps of the complement cascade.

Deficiency of complement decay-accelerating factor (DAF, CD55) in non-Hodgkin's lymphoma.

We have assessed levels of surface-expressed complement regulatory proteins, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on cells from patients with hematological malignancies. Neither malignant cells nor unaffected nucleated blood cells from the patients lacked MCP. On the other hand, complete deficiency of DAF was found in 2/10 of non-Hodgkin's lymphoma (NHL), while none of the 38 patients with acute nonlymphocytic leukemia (ANLL) (14 cases), chronic myelogenous leukemia (CML) (6 cases), acute lymphocytic leukemia (ALL) (12 cases) and chronic lymphocytic leukemia (CLL) (6 cases) lacked DAF. The two patients with DAF-negative NHL had no history of paroxysmal nocturnal hemoglobinuria (PNH), and their peripheral blood cells were DAF-positive. One DAF-negative NHL exhibited T cell markers and the other those of B cell. In both cases, treatment of the DAF-negative lymphoma cells with antibody against MCP (M177) followed by Mg(2+)-EGTA-serum resulted in efficient deposition of homologous C3. These results infer that some NHL specifically lack DAF and, through treatment with M177, are targeted by homologous C3.

Robust in vitro replication of Plasmodium falciparum in glycosyl-phosphatidylinositol-anchored membrane glycoprotein-deficient red blood cells.

Red blood cells (RBCs) infected with Plasmodium falciparum are protected from complement-mediated lysis by surface membrane glycosyl-phosphatidylinositol (GPI)-anchored proteins, which include decay accelerating factor (DAF or CD55) and CD59. To determine if P. falciparum avoids or replicates less efficiently in GPI protein-deficient cells at a higher risk for complement-mediated lysis, we compared P. falciparum infectivity among control RBCs with those from subjects with paroxysmal nocturnal hemoglobinuria (PNH), a condition in which RBCs express variable levels of DAF (negative and positive) and CD59 (negative [-], intermediate [I], and high [H]). Co-cultures of 19 matched samples of control and PNH RBCs were infected with P. falciparum to directly compare parasitic invasion. Each PNH RBC sample was then assessed for P. falciparum infectivity across the spectrum of GPI protein deficiency. Identification methods included biotin-streptavidin for RBC populations, fluorescein isothiocyanate-labeled antibodies to DAF and CD59, hydroethidine for parasite DNA, and flow cytometry. The mean +/- SD parasitemias in co-cultured PNH and control RBCs were 24.7 +/- 6.9% versus 21.0 +/- 5.9% (P = 0.12). For individual PNH samples, parasitemias were significantly higher in DAF (-) cells versus DAF (+) cells (25.0 +/- 8.9% versus 19.1 +/- 8.7%; P < 0.001) and in CD59 (-) cells versus I/H cells (22.5 +/- 6.4% versus 17.6 +/- 4.2%; P < 0.0003). Across the CD59 spectrum, mean parasitemias were highest in CD59 (-) cells (24.5 +/- 6.4%), followed by CD59-H cells (19.5 +/- 5.4%), and CD59-I cells (16.4 +/- 4.8%). Expression of DAF in 12 (63%) of 19 infected PNH samples was reduced. Thus, P. falciparum does not selectively avoid RBCs with fewer GPI proteins and parasite replication in PNH cells is at least as robust as in normal RBCs.

Deficiency of phosphatidylinositol-linked membrane proteins on erythrocytes of different subpopulations in paroxysmal nocturnal hemoglobinuria.

The surface phosphatidylinositol (PI)-linked proteins on membrane of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes (PNHE) was analysed by a flowcytometer (FACS 420). It was found that the loss of acetylcholinesterase (AchE) and decay accelerating factor (DAF), two PI-linked proteins, from cell membrane of PNHE was not synchronous. The hemolysis rates of DAF (-) and AchE (-) PNHE were much higher than that of mixed population in cobra-venom factor (CoF) lysis test. Intact PNHE remaining after CoF lysis had relatively lower immunofluorescent labeling rate of AchE on membrane in comparison with normal erythrocytes. It implied that this subpopulation, in spite of being insensitive to complement lysis, was still abnormal in terms of the amount of PI-linked protein on cell membrane. When these intact PNHE remaining after CoF lysis were incubated with activated polymorphonuclear leukocytes (PMN) for three hours, immunofluorescent labeling of AchE on PNHE was prominently decreased. This indicated that reactive oxidants released from activated PMN might injure PI-linked proteins.

[Paroxysmal nocturnal hemoglobinuria (PNH) deficiency of major complement-regulatory membrane proteins on erythrocytes].

The significance of the deficiency of the major complement-regulatory membrane proteins, decay-accelerating factor (DAF) and CD59, to the lysis of paroxysmal nocturnal hemoglobinuria (PNH) red blood cells was investigated. DAF and CD59 were demonstrated to be deficient simultaneously on affected PNH red blood cells (PNH-III) by two-color FACS analysis. At least in some patients with PNH, PNH-I was also revealed to be deficient partially in DAF. Purified DAF and CD59 ameliorated the complement sensitivity of PNH red blood cells, partially and completely, respectively. Functional blocking of these molecules on nomrla human red cells by monoclonal antibodies to DAF and CD59 rendered A or AB type blood cells complement-sensitive but not O or B blood type blood cells. The differences of complement-sensitivity among blood types were revealed to reside on the step of binding of C9 to C5b-8, i. e. C9 can bind to C5b-8 more on A type blood cells than on O type blood cells. We conclude that the deficiency of DAF and CD59 play a major role for the complement sensitivity of PNH red blood cells and that other factors reported to be deficient in PNH do less than these two proteins.

The lectin-like domain of thrombomodulin ameliorates diabetic glomerulopathy via complement inhibition.

Coagulation and complement regulators belong to two interactive systems constituting emerging mechanisms of diabetic nephropathy. Thrombomodulin (TM) regulates both coagulation and complement activation, in part through discrete domains. TM's lectin like domain dampens complement activation, while its EGF-like domains independently enhance activation of the anti-coagulant and cytoprotective serine protease protein C (PC). A protective effect of activated PC in diabetic nephropathy is established. We hypothesised that TM controls diabetic nephropathy independent of PC through its lectin-like domain by regulating complement. Diabetic nephropathy was analysed in mice lacking TM's lectin-like domain (TMLeD/LeD) and controls (TMwt/wt). Albuminuria (290 µg/mg vs. 166 µg/mg, p=0.03) and other indices of experimental diabetic nephropathy were aggravated in diabetic TMLeD/LeD mice. Complement deposition (C3 and C5b-9) was markedly increased in glomeruli of diabetic TMLeD/LeD mice. Complement inhibition with enoxaparin ameliorated diabetic nephropathy in TMLeD/LeD mice (e.g. albuminuria 85 µg/mg vs. 290 µg/mg, p<0.001). In vitro TM's lectin-like domain cell-autonomously prevented glucose-induced complement activation on endothelial cells and - notably - on podocytes. Podocyte injury, which was enhanced in diabetic TMLeD/LeD mice, was reduced following complement inhibition with enoxaparin. The current study identifies a novel mechanism regulating complement activation in diabetic nephropathy. TM's lectin-like domain constrains glucose-induced complement activation on endothelial cells and podocytes and ameliorates albuminuria and glomerular damage in mice.

Inter-alpha-trypsin inhibitor attenuates complement activation and complement-induced lung injury.

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.

Additive inhibition of complement deposition by pneumolysin and PspA facilitates Streptococcus pneumoniae septicemia.

Streptococcus pneumoniae is a common cause of septicemia in the immunocompetent host. To establish infection, S. pneumoniae has to overcome host innate immune responses, one component of which is the complement system. Using isogenic bacterial mutant strains and complement-deficient immune naive mice, we show that the S. pneumoniae virulence factor pneumolysin prevents complement deposition on S. pneumoniae, mainly through effects on the classical pathway. In addition, using a double pspA-/ply- mutant strain we demonstrate that pneumolysin and the S. pneumoniae surface protein PspA act in concert to affect both classical and alternative complement pathway activity. As a result, the virulence of the pspA-/ply- strain in models of both systemic and pulmonary infection is greatly attenuated in wild-type mice but not complement deficient mice. The sensitivity of the pspA-/ply- strain to complement was exploited to demonstrate that although early innate immunity to S. pneumoniae during pulmonary infection is partially complement-dependent, the main effect of complement is to prevent spread of S. pneumoniae from the lungs to the blood. These data suggest that inhibition of complement deposition on S. pneumoniae by pneumolysin and PspA is essential for S. pneumoniae to successfully cause septicemia. Targeting mechanisms of complement inhibition could be an effective therapeutic strategy for patients with septicemia due to S. pneumoniae or other bacterial pathogens.

Structural and functional differences between decay-accelerating factor and red cell acetylcholinesterase.

The abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria, both PNH II (the moderately abnormal cells) and PNH III (the markedly abnormal cells), lack both acetylcholinesterase (AChE) activity and decay-accelerating factor (DAF) activity. Both of these activities are found on glycoprotein molecules with a molecular weight of about 70 Kd. To demonstrate that these two activities are in fact on different proteins, we have shown that binding to normal red cells of antibody to DAF does not inhibit the subsequent binding of monoclonal antibody to AChE nor AChE activity. Inhibition of DAF activity by polyclonal antibody increases the susceptibility of normal erythrocytes to lysis by complement but inhibition of AChE activity by antibody does not. The rate of decay of the C3 convertase complex of the classical pathway of complement activation was inhibited by DAF added in the fluid phase but not by AChE. When DAF was exhaustively immunoprecipitated from a solution of the erythrocyte membrane proteins, AChE remained and vice versa. These studies indicate that acetylcholinesterase and decay-accelerating factor are two different proteins, both of which are lacking on PNH II and PNH III erythrocytes.

Studies on the sensitivity to complement-mediated lysis of erythrocytes (Inab phenotype) with a deficiency of DAF (decay accelerating factor).

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.

Association of Sjögren's syndrome with hereditary angioneurotic edema: report of a case.

A case of hereditary angioneurotic edema (HANE) associated with Sjögren's syndrome is presented. One of the members of a pedigree of HANE due to deficiency of C1 inhibitor (C1INH) had a positive titer for anti-SS-A and anti-SS-B antibodies in the serum, complaining of symptom of dry eyes and dry mouth. A lip biopsy revealed lymphocytic infiltration of minor salivary glands. The patient had renal tubular acidosis (RTA). Thus the patient was diagnosed as suffering from Sjögren's syndrome with RTA.

Decreased C5b67-inhibitor activity in two families with hereditary functional deficiency of the eighth component of complement.

In addition to its role in hemolysis and host defense against Neisseria infection, the eighth component of human complement (C8) is one of several plasma proteins that are C5b67-inhibitors (C5b67-INH). The recent identification in our laboratory of two new families with hereditary deficiency of C8 provided an opportunity to study further the role of C8 as a C5b67-INH. Based on mixing and reconstitution experiments, the deficiency of C8 seemed to be due to a selective lack of the C8 beta-chain in one family and the C8 alpha-gamma subunit in the other family. Sera from individuals homozygous for the C8 abnormality were substantially deficient in C5b67-INH activity as well as totally deficient in hemolytic activity. Sera from control individuals possessed approximately 2500 C5b67-INH U/ml, whereas sera from the C8-deficient individuals had markedly depressed C5b67-INH activity, with a mean of only 428 U/ml. C5b67-INH activity was completely reconstituted in C8-deficient serum by the addition of purified human C8. We conclude that C8 constitutes the substantial majority of the C5b67-INH activity of normal human serum.