The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells.

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.

GPI-anchored complement regulatory proteins in seminal plasma. An analysis of their physical condition and the mechanisms of their binding to exogenous cells.

We analyzed and compared the properties of three glycosylphosphatidylinositol (GPI)-anchored proteins. CD59, CD55 (both C regulators), and CDw52, and of the transmembrane C regulator CD46 in seminal plasma (SP). We demonstrated previously that anchor-intact SP CD59 is present on the membranes of vesicles (prostasomes) and that cells acquire this protein during incubation with SP. We now report that this acquisition is due partly to adherence of prostasomes to cells and partly to a second mechanism which may involve micellar intermediates. Using fluorescent labeling, ultracentrifugation, and density gradient centrifugation, virtually all CD46 was present on prostasomes whereas CD59, CD55, AND CDw52 were also detected in a form which remained in the 200,000 g supernatant and equilibrated at higher density than prostasomes in gradients. All three GPI-linked proteins eluted at high molecular mass during size exclusion chromatography of this nonprostasome fraction. As documented by videomicroscopy and biochemical analysis, cells acquired new copies of the GPI-linked proteins during incubation with the nonprostasome fraction as well as with prostasomes. These data demonstrate the presence in SP of a stable population of membrane-free, GPI-linked proteins available for transfer to cells. Binding of these proteins to spermatozoa and pathogens in SP may confer new properties on their membranes including increased resistance to C attack. Finally, our data raise the possibility that lipid-associated GPI-linked proteins may be suitable for therapeutic applications.

Control of the complement system.

The complement system has developed a remarkably simple but elegant manner of regulating itself. It has faced and successfully dealt with how to facilitate activation on a microbe while preventing the same on host tissue. It solved this problem primarily by creating a series of secreted and membrane-regulatory proteins that prevent two highly undesirable events: activation in the fluid phase (no target) and on host tissue (inappropriate target). Also, if not checked, even on an appropriate target, the system would go to exhaustion and have nothing left for the next microbe. Therefore, the complement enzymes have an intrinsic instability and the fluid-phase control proteins play a major role in limiting activation in time. The symmetry of the regulatory process between fluid phase and membrane inhibitors at the C4/C3 step of amplification and convertase formation as well as at the MAC steps are particularly striking features of the self/nonself discrimination system. The use of glycolipid anchored proteins on membranes to decay enzymes and block membrane insertion events is unlikely to be by chance. Finally, it is economical for the cofactor regulatory activity to produce derivatives of C3b that now specifically engage additional receptors. Likewise, C1-Inh leads to C1q remaining on the immune complex to interact with the C1q receptor. Thus the complement system is designed to allow rapid, efficient, unimpeded activation on an appropriate foreign target while regulatory proteins intervene to prevent three undesirable consequences of complement activation: excessive activation on a single target, fluid phase activation, and activation on self.

Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1.

This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms.

Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis.

We demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 micrograms/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC. This phenomenon could be demonstrated not only if cells were incubated with purified SP CD59 but also if unfractionated SP were used. Further, CD59 in unfractionated SP bound to washed spermatozoa, increasing their membrane content of the protein. The mechanism by which this protein retains its GPI anchor while apparently present in the fluid phase is of interest and was further investigated. Using the techniques of high-speed centrifugation, fast performance liquid chromatography fractionation, and electron microscopy, we found that all detectable SP CD59 was associated with vesicular extracellular organelles. These organelles, named "prostasomes," were previously known to be present in SP and to interact with spermatozoa, although their function was uncertain. Interaction of heterologous E with prostasomes rendered the cells more resistant to lysis by human MACs. We propose that these organelles represent a pool of CD59 from which protein lost from spermatozoa, perhaps as a result of low level complement attack or of normal membrane turnover, can be replenished.

Complement in human reproduction: activation and control.

The behaviour of the complement system during human reproduction is now the focus of much scientific attention. The presence of antisperm antibodies in the reproductive tracts of some infertile individuals, and of complement in cervical and ovarian follicular fluid, suggests that complement-mediated damage of spermatozoa is involved in some cases of infertility. Further, deposition of maternal IgG and of complement in the extrafetal tissues indicates that complement activation occurs within the fetoplacental unit. Recently, three complement-regulatory proteins--decay-accelerating factor, membrane cofactor protein and CD59--have been detected on spermatozoa and in the extrafetal tissues. It is likely that these inhibitors are essential for normal reproductive function. This article reviews current understanding of the interaction of the complement system with cells and tissues involved in reproduction, with emphasis on the nature and function of the controlling proteins.

Characterization of the membrane attack complex inhibitory protein CD59 antigen on human amniotic cells and in amniotic fluid.

A functional complement system and the potential for its activation are present in human amniotic fluid. We have recently demonstrated that CD59 antigen is present and functionally active on human amniotic epithelial cells (HAEC). We have now further examined the role of this protein on HAEC and have also demonstrated its presence in amniotic fluid (AF). CD59 Ag on HAEC is similar in size to the erythrocyte protein and is anchored via glycosyl phosphatidylinositol. The AF protein retained the capacity to incorporate into target cells and protect against lysis by complement. These data suggest that HAEC secrete into AF a form of CD59 Ag which retains inhibitory activity and which may be important in protection of the foetus from maternal complement in utero.

Membrane attack complex (MAC)-mediated damage to spermatozoa: protection of the cells by the presence on their membranes of MAC inhibitory proteins.

Although antibody and complement are known to cause immobilization and killing of spermatozoa in vitro the components of the complement system mediating these effects remain undefined. Here we have examined the effects of the membrane attack complex (MAC) on spermatozoa and demonstrate that spermatotoxic effects are dependent on assembly of the complete MAC. We subsequently examined the presence and functional significance of the complement regulatory proteins decay accelerating factor (DAF), MAC-inhibiting protein (MIP) and CD59 antigen on spermatozoa. Both DAF and CD59 antigen were present on the membranes of these cells. Neutralization of CD59 antigen with specific antibodies increased the susceptibility of the cells to MAC-mediated damage, suggesting a role for this molecule in the protection of spermatozoa from complement-mediated damage in the female reproductive tract.

The complement-inhibiting protein, protectin (CD59 antigen), is present and functionally active on glomerular epithelial cells.

Protectin (CD59 antigen) is a 20-kD phosphatidyl-inositol-linked membrane protein that inhibits formation of the membrane attack complex (MAC) of complement on homologous cells. Although the antigen has been identified in a number of human tissues, until recently a functional role had been demonstrated only in circulating cells. Using immunofluorescence techniques we have shown the presence of protectin on human glomerular epithelial cells (GEC) in culture and on GEC, tubular epithelial cells and endothelial cells in frozen sections of normal human renal cortex. In addition, we present evidence that this protein functions in protection of GEC from homologous complement: cultured cells incubated with the Fab2 fragment of a monoclonal anti-protein antibody were markedly more susceptible to killing by homologous serum than were cells in the absence of Fab2 anti-protectin. These findings suggest that this protein may be important in the maintenance of glomerular integrity in vivo, and may be of relevance in certain renal diseases.

Protection of human amniotic epithelial cells (HAEC) from complement-mediated lysis: expression on the cells of three complement inhibitory membrane proteins.

Cultured human amniotic epithelial cells (HAEC) were found by immunofluorescence microscopy to express three complement inhibitory membrane proteins, CD59 antigen, decay-accelerating factor (DAF) and membrane attack complex (MAC) inhibitory protein (MIP), on their surfaces. The effects of incubation with Fab2 fragments of monoclonal antibodies (mAb) raised against these proteins on susceptibility of sensitized cells to lysis by homologous complement was examined. Percentage cell lysis was markedly increased in the presence of Fab2 anti-CD59 and to a lesser, but significant, extent in the presence of Fab2 anti-DAF. Fab2 anti-MIP did not alter the sensitivity of the cells to lysis by complement.