Quantification of the CD55 and CD59, Membrane Inhibitors of Complement on HIV-1 Particles as a Function of Complement-Mediated Virolysis

Previous studies have demonstrated that the murine monoclonal antibody (MoAb) NM-01 activates the human complement classical pathway resulting in lysis of human immunodeficiency virus (HIV). The present study was performed to determine the availability of the V3-loop of gp120 relative to the complement regulatory proteins, CD55 (DAF) and CD59 (HRF20) molecules on HIV. The results demonstrate that CD55 and CD59 exist on HIV virions, along with gp120 molecules. These findings suggest that activation of human complement on free viral particles is induced by MoAb NM-01 and that this occurs regardless of the presence of CD55 and CD59 molecules. The destruction of viral particles was demonstrated by a decrease in infectivity. The involvement of human complement in this process was confirmed with an immunoelectron microscopy technique by the presence of a human C9 to prove membrane attack complex (MAC). The results indicate that NM-01 can induce complement activation because of the ratios of CD55 and CD59 to gp120 molecules on HIV virions. The availability of the gp120 V3 domain on the virion is sufficient for binding of NM-01 and thereby the formation of MAC that results in virolysis.     

Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.     

Is the membrane attack complex of complement an enzyme?

Summary Recent studies on the functional activities of the membrane attack complex of complement, C5b-9, are reviewed. A new speculative hypothesis has been advanced to account for the ability of complement to mediate lysis of various targets. This hypothesis has three major elements: 1) that the membrane attack complex is an enzyme; 2) that the substrate for this putative enzyme is a membrane constituent; 3) that the substrate specificity of the putative enzyme is dependent on the species source of individual complement components within the C5b-9 complex.Abbreviations E = sheep red cells - A = rabbit IgM anti-Forssman antibody - Hu or hu = human - GP or gp = guinea pig     

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.     

Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9.

The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9-inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species.     

Membrane vesiculation protects erythrocytes from destruction by complement

Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Virion-Associated Complement Regulator CD55 Is More Potent than CD46 in Mediating Resistance of Mumps Virus and Vesicular Stomatitis Virus to Neutralization

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.     

Changes in plasma membrane phospholipids inhibit antibody-mediated lysis

A variety of mechanisms have been proposed to explain how tumors evade immune destruction. This work has identified one such mechanism that determines susceptibility to immune lysis; membrane phospholipid composition altered susceptibility to antibody plus complement (Ab+C)-mediated lysis. Effects on antibody plus complement-mediated lysis were correlated with levels of major histocompatibility complex (MHC) molecules but not inherent resistance to complement damage. This cellular mechanism could be a means by which tumor cells escape immune detection and destruction.     

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.     

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.     

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.     

The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.

A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.     

Aurin Tricarboxylic Acid Protects against Red Blood Cell Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinemia

Objectives

Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage.

Design and Methods

We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera.

Results

We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes.

Conclusions

We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.     

Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin-ation, we have shown that isolated myelin activates serum complement in the absence of myelin-specific antibody and that membrane attack complex of complement is the required factor in antibody-mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin-associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b-9 neoantigen. The subunit structure of C5b-9 was determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late-acting complement components, C5-C9, was assembled in the target myelin membranes.     

Complement channels in membranes: Inhibition with a monoclonal antibody to a neoantigen of polymerized C9

The channels produced by complement in red cell membranes are heterogeneous, with diameters of ～0.5 to ～12.0 nm. We investigated the relationship of the components of the membrane attack complex, C5b through C9, to the functional transmembrane channels >3 nm in diameter. Radiolabelled macromolecules were incorporated into resealed red cell membrane ghosts which were then treated with complement. A monoclonal antibody to a neoantigen in polymerized C9 inhibited macromolecule diffusion through the complement channels. There was also inhibition with polyclonal antisera to C9 but not with antisera to any of the other components of the membrane attack complex. The results demonstrate a functional correlation of the larger complement lesions with the previously described poly C9 tubular structures.     

Elimination of terminal complement complexes in the plasma membrane of nucleated cells: influence of extracellular Ca2+ and association with cellular Ca2+

Nucleated cells, unlike erythrocytes, are able to survive limited complement attack by eliminating potentially cytolytic complement channels from the plasma membrane (PM) by processes that involve, plasma membrane (PM) by processes that involve, but may not be limited to, endocytosis. The observation that C5b-9 channels, as well as C5b-8 and C5b-7 intermediates, are rapidly eliminated from the cell surface of nucleated cells has prompted us to examine whether terminal complement complexes stimulate membrane events that lead to accelerated elimination of these complexes. We have suggested previously that ion flux through terminal complement complexes might influence the rate of elimination on the basis of our finding that terminal complement complexes with larger functional channel sizes are more rapidly eliminated. In this study, we examined the role of Ca2+ on the elimination rate of terminal complement complexes in the PM of Ehrlich cells, because changes in Ca2+ flux across the PM are known to influence many metabolic activities including endocytosis. To determine the elimination rate for terminal complement complexes by functional analysis, cells bearing C5b-7 or C5b-8 complexes with or without a sublytic dose of C9 were incubated at 37 degrees C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. The initial elimination rates for the terminal complement complexes were compared in the presence of 0.015, 0.15, and 1.5 mM CaCl2 in the medium. Sufficient lowering of the extracellular Ca2+ concentration, (Ca2+)o, resulted in prolonging the elimination of each of the terminal complement complexes to a different extent. The effect of (Ca2+)o on the elimination rate was most pronounced for C5b-8 in the presence of a sublytic number of C5b-9, with less of an effect on C5b-8 alone, and the least effect with C5b-7. The elimination rates for terminal complement complexes were also determined by measuring the persistence of C5b antigen on the cell surface at 37 degrees C in the presence of various (Ca2+)o by using fluorescence-activated cell sorter analysis and were comparable with that obtained by functional analysis. Examination of the effect of terminal complement complexes on the cellular Ca2+ concentration, (Ca2+)i, revealed that these complexes increased the (Ca2+)i in proportion with the known functional pore size of the terminal complement complex in the PM. In addition, Quin 2, which can buffer internal Ca2+ transients, was found to increase the susceptibility of Ehrlich cells to lysis by C5b-9, further suggesting a relationship between the (Ca2+)i and the elimination process.(ABSTRACT TRUNCATED AT 400 WORDS)     

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.