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.     

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59

CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required.     

Species-specific restriction of complement by HRF20 (CD59) generated by cDNA transfection.

The 20-kDa homologous restriction factor (HRF20, CD59) is a phosphatidyl inositol-anchored membrane glycoprotein that inhibits the formation of human complement membrane attack complexes. The cDNA of HRF20 was transfected into Chinese hamster ovary (CHO) cells resulting in expression of human HRF20 protein on the cell surface anchored via glycosylphosphatidyl inositol. The transfected CHO cells were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rat and guinea pig complement. Therefore, species specificity between HRF20 and complement is maintained in HRF20 generated on the CHO cells following transfection with HRF20 cDNA.     

Enhanced expression of decay-accelerating factor and CD59/homologous restriction factor 20 in intestinal metaplasia, gastric adenomas and intestinal-type gastric carcinomas but not in diffuse-type carcinomas

AIMS: Variable expression of the complement regulatory proteins, decay-accelerating factor, CD59/homologous restriction factor 20 (HRF20) and membrane cofactor protein has been shown in human gastrointestinal malignancies, but their expression in gastric cancer has not been fully described. Thus, we immunohistochemically defined the distribution of these proteins in human normal gastric mucosa, intestinal metaplasia, adenomas and gastric cancers. METHODS AND RESULTS: Gastric tissues were obtained by endoscopic biopsy or surgical resection and stained with mouse monoclonal antibodies to decay-accelerating factor, CD59/HRF20, and membrane cofactor protein. In the normal gastric mucosa, membrane cofactor protein was diffusely stained on the basolateral surface of epithelial cells, whereas the expression of decay-accelerating factor and CD59/HRF20 was inconspicuous. In intestinal metaplasia, adenoma and intestinal-type gastric carcinoma cells, decay-accelerating factor and HRF20 were intensely stained on the apical surface; membrane cofactor protein retained its location on the basolateral surface. In diffuse-type gastric carcinomas, the expression of decay-accelerating factor, CD59/HRF20 was lost, but membrane cofactor protein was present on the tumour cell surface. CONCLUSIONS: These findings suggest that membrane cofactor protein plays a primary role in the regulation of complement activation in normal and neoplastic gastric cells and that the expression pattern of the complement regulatory proteins is closely related to gastric carcinoma development.     

Expression of the complement regulatory proteins CD21, CD55 and CD59 on Burkitt lymphoma lines: their role in sensitivity to human serum-mediated lysis.

On a panel of nine human B cell lines we showed that the expression of the complement regulatory factors complement receptor type 2 (CR2; CD21), decay-accelerating factor, (DAF; CD55) and homologous restriction factor (HRF20, CD59) is not correlated. All lines expressed DAF, six lines carried detectable amounts of CR2 and three carried HRF20. Upon incubation in human serum, under conditions which allowed the activation of complement through the alternative pathway, the CR2-carrying lines bound C3 fragments and two of them (Ramos and one of its two sublines) were damaged. These two lines had the lowest DAF expression, less than 50% of the cells reacted with the IA10 monoclonal antibody. By modulating the expression of the complement regulatory molecules, the lytic sensitivity of the B cell lines could be altered. Blockade of DAF on the HRF20-, CR2+ lines with the specific monoclonal antibodies increased their sensitivity to lysis by human serum. With the DAF- and HRF20+ cells significant lytic effect was obtained only when they were pretreated with both of the specific antibodies. Interferon-gamma or tumor necrosis factor-alpha treatment elevated the amount of CR2 on the low-CR2 expressor line (Ramos/HR1K) which thereafter bound higher amounts of C3 fragments and was lysed when incubated in human serum. This line had relatively low DAF level and lacked HRF20. The cytokine treatment did not alter the expression of these molecules. The CR2+ Ramos and the CR2- Rael cells were treated with 5-azacytidine which induced HRF20 and increased DAF expression. In parallel with this change Ramos cells became resistant to C-mediated lysis. The experiments with the panel of human B cell lines showed thus that cytolysis through activation of complement in homologous serum can be regulated at several steps by cell surface molecules. While expression of CR2 was required for C3 fixation, DAF and HRF20 inhibited lysis. By independent modulation of the quantities of these molecules, cells acquired or lost their sensitivity.     

Rapid transformant selection by human complement using HRF20 (CD59) cDNA as a selection marker

The 20 kDa homologous restriction factor (HRF20, CD59) prevents formation of membrane attack complexes of human complement. Transfection of the cDNA of HRF20 to heterologous cells, which are sensitive to human complement, thereby renders these cells resistant to human complement attack. This property can then be used as a selection marker of transfection. In addition, the procedure is short and can be performed within 8 h. Since only 1 h is required for incubation with complement, the damage to the transfected cells is limited compared with damage resulting from selection for drug resistance which requires 10 days or more of incubation in the presence of harmful agents.     

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.     

IgM natural antibody against an asialo-oligosaccharide, gangliotetraose (Gg4), sensitizes HIV-I infected cells for cytolysis by homologous complement

Although cells are usually resistant to homologous complement,the IgM natural antibody against gangliotetraose (Gg4), an asialo-oligosaccharideof GM1, was found to cause cytolysis of HIV-1 infected cellsby homologous complement. Due to its size, IgM might enablethe initiation of the complement cascade away from membranecomplement inhibitors such as decay accelerating factor andmembrane cofactor protein. Furthermore, HRF20 (CD59), whichrestricts formation of membrane attack complexes (MAC) of complementon homologous cell membranes, was significantly decreased onHIV-infected cells and therefore formation of MAC on cell membraneswould be facilitated. IgM antibodies reactive with HIV-infectedcells could result in the elimination of infected cells viacomplement-mediated cytolysis in HIV-infected patients, sinceall tested sera from HIV-positive hemophilia patients who havesurvived for >12 years contained IgM antibody activity againstHIV-infected MOLT4 cells in a preliminary experiment. Therefore,administration of IgM antibodies reactive with HIV-infectedcells may be effective in the treatment of HIV carriers.     

Complement Regulatory Proteins on the Sperm Surface: Relevance to Sperm Motility

PROBLEM: To determine whether complement regulatory proteins are present on human spermatozoa and whether antibodies to these proteins adversely affect sperm motility. METHOD OF STUDY: Human sperm membrane proteins were solubilized and subjected to Polyacrylamide gel electrophoresis followed by Western blot analysis against antibodies to complement component 1 inhibitor (C1-INH), decay-activating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and homologous restriction factor (HRF; CD59). Spermatozoa, obtained by a swim-up technique, were incubated in medium (control 1) and medium supplemented with antibodies to human albumin (control 2) and antibodies to these complement regulatory proteins. We used a computerized sperm motion analysis to determine the effect of these antibodies on sperm motion characteristics. RESULTS: Complement regulatory proteins such as C1-INH, CD55, CD46, and CD59 were found in the sperm extracts as shown by Western blot analysis. The heat-treated (56°C, 30 min) IgG fraction of antibodies to these proteins significantly reduced sperm motility in general and other motion parameters. Addition of complement did not affect these results except in the antibodies to CD46 in which the reducing action was further amplified. CONCLUSIONS: Our data suggest that C1-INH, CD55, CD46, and CD59 are present on the sperm surface. These proteins may have biological functions, such as affecting sperm motility, besides the complement regulatory functions. In infertile men and women with antibodies that recognize one or more of these complement regulatory proteins, there may be problems related to poor sperm motility and survival in the reproductive tracts.     

Thyroid follicular cell function after non-lethal complement membrane attack

Terminal complement complexes have been identified around thyroid follicles in Graves' disease and Hashimoto's thyroiditis, and the concentrations of such complexes are increased in the sera of these patients, suggesting a role for complement activation and membrane attack complexes (MAC) in autoimmune thyroiditis. This has been investigated further using cultured human and rat thyroid cells. Thyrocytes were resistant to lysis by homologous complement, in contrast to the effects of heterologous (rabbit) complement. The formation of non-lethal amounts of MAC, using reactive lysis or classical pathway activation, significantly reduced cAMP production by these cells in response to thyroid-stimulating hormone (TSH) (P less than 0.01); similar effects were seen with thyroid-stimulating antibodies. Thyroid cells were able to recover rapidly from complement attack after washing and incubation for 30 min. Non-lethal MAC formation also resulted in reactive oxygen metabolite production, detected by luminol-dependent chemiluminescence in three out of five thyroid cell preparations tested. Ionomycin, but not TSH, also stimulated reactive oxygen metabolite production. These results suggest that repeated or continuous sub-lethal complement attack on thyroid cells may exacerbate hypothyroidism in Hashimoto's thyroiditis, or partially counter the effects of thyroid-stimulating antibodies in Graves' disease. Furthermore, the production of reactive oxygen metabolites in these circumstances could increase the intra-thyroidal inflammatory response; oxygen radical scavenging by anti-thyroid drugs (which are concentrated by thyrocytes) may account in part for the amelioration of thyroiditis observed with such treatment.     

Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro,and an insight into mechanism(s) of resistance

Human lung cancer expresses cell membrane complement inhibitory proteins (CIP). We investigated whether human lung cancer cell lines also express cell-membrane CIP molecules and whether the biology of CIP molecules in these cell lines differs from that of CIP in normal human respiratory epithelium in culture. The cell lines ChaGo K-1 and NCI-H596 were compared with normal human nasal epithelium in primary cultures in respect to the level of cell membrane CIP expression of membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59, in respect to the level of cell resistance to complement-mediated lysis, and in respect to the contribution of cell membrane CIP to cell resistance against complement-mediated lysis. We found, using flow cytometry, that both human lung cancer cell lines expressed MCP, DAF and CD59, as did normal nasal epithelial cells. However, normal cells showed a large subpopulation of low DAF-expressing cells (60% of all cells) and a smaller subpopulation of high DAF-expressing cells (40%), while the lung cancer cell lines showed only one cell population, of high DAF expression. In addition, both lung cancer cell lines expressed higher MCP levels, and NCI-H596 cells showed higher levels of CD59. Cell resistance to complement-mediated lysis of both lung cancer cell lines was much higher than that of normal cells. Fifty percent normal human serum, under the same concentrations of complement activators, induced lysis of less than a mean of 10% of lung cancer cells, while lysing up to a mean of 50% of nasal epithelial cells. Lung cancer cell resistance to complement was due to its ability to prevent significant activation of complement upon its cell membrane, as manifested by a failure of complement activators to increase cell membrane deposition of C3-related fragments. The exact mechanism for this resistance remains obscure. Unexpectedly, neutralizing antibodies, anti-MCP and anti-DAF were entirely ineffective and anti-CD59 was only slightly effective (18% mean cell lysis) in increasing the susceptibility of the lung cancer cell lines to complement, while the same antibodies were very effective in facilitating complement-mediated lysis of the normal nasal epithelial cells (50% mean cell lysis with CD59 MoAb). On the other hand, detachment of DAF and CD59 by phosphatidylinositol-specific phospholipase C (PIPLC) from the lung cancer cell lines abrogated their resistance to lysis. We suggest that the biology of cell membrane CIP molecules in human lung cancer cell lines is different from that of CIP in normal respiratory epithelial cells. Human lung cancer cell lines are able to prevent significant complement activation upon its cell membrane and are therefore especially resistant to complement-mediated lysis. Complement resistance may serve this common and highly lethal human cancer as an escape mechanism from the body's immunosurveillance and prevent effective immunotherapy with tumour-specific MoAbs.     

The Influence of Tumour Necrosis Factor-γ, Interleukin-1β and Interferon-γ on the Expression and Function of the Complement Regulatory Protein CD59 on the Human Colonic Adenocarcinoma Cell Line HT29

CD59 is a 18–25kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up–regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose–dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-γ and interleukin-1β. A dose–dependent increase in the level of CD59 expression was also seen using low concentrations of interferon-γ (IFN-γ), while CD59 expression on cells cultured with high IFN-γ concentrations was comparable to non–stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non–stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape.     

Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers.

Human cells are relatively resistant to lysis by the homologous complement system. Here we describe the mechanism of action of a recently discovered and widely distributed 18,000-20,000 molecular weight (MW) membrane glycoprotein (CD59), which appears to act as a major protective element against complement-mediated lysis (hence called protectin). When incorporated into heterologous erythrocyte membranes, protectin efficiently prevented cell lysis by human serum. Neutralization with antibody of the naturally occurring protectin on human erythrocytes or on nucleated K562 cells increased their susceptibility to lysis by homologous complement. During complement activation, protectin became incorporated into the membrane attack complex (MAC). By interacting with newly exposed regions in the C5b-8 complex and in aggregating C9 it limited the number of C9 molecules associating with the C5b-8 complex to a C8:C9 ratio of 1:1.5 instead of a normal average of 1:3.5. The results demonstrate directly that protectin is a powerful inhibitor of complement cytolysis and acts by inhibiting the C5b-8 catalysed insertion of C9 into the lipid bilayer.     

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.     

Relative roles of decay-accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement-mediated lysis.

Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane-bound C regulatory proteins, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 70-kDa molecule under nonreducing conditions in 2% deoxycholate extracts of HUVEC, MCP as a 63-kDa protein and CD59 as a 20-kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol-phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor. The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab')2 fragments of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab')2 anti-CD59 and to a lesser, but significant, extent in the presence of F(ab')2 anti-DAF. F(ab')2 anti-MCP did not significantly alter the susceptibility of HUVEC to complement-mediated lysis.     

Decreased expression of 20-kD homologous restriction factor (HRF20, CD59) on T lymphocytes in Epstein–Barr virus (EBV)-induced infectious mononucleosis

HRF20 (CD59) is one of the membrane-associated complement regulatory proteins. The characteristic function of CD59 is to prevent membrane attack complex (MAC) formation on the cell surface and to protect the cell from complement-mediated cell lysis. We examined the expression of CD59 antigen on T cell subpopulations in patients with acute infectious mononucleosis (IM) and analysed the relationship between the amount of CD59 expression and activation-induced cell death of mature T cells with apoptosis. Decreased expression of CD59 on CD8⁺ T cells, especially on CD45RO⁺ and HLA-DR⁺ activated T cells, was marked in acute IM patients. In contrast, activated CD4⁺ T cells from IM patients expressed as much CD59 antigen as CD4⁺ T cells from healthy volunteers. After incubation-induced cell death, viable CD8⁺ T cells showed normal amounts of CD59 antigen on their surface. CD59^dim CD8⁺ T cells were more susceptible to apoptosis than CD59^bright CD8⁺ T cells. These findings suggest that decreased expression of CD59 on CD8⁺ T cells may discriminate the susceptibility of activated CD8⁺ T cells to activation-induced cell death in IM.     

Membrane attack complex of complement and 20 kDa homologous restriction factor (CD59) in myocardial infarction

In order to investigate the mechanism of deposition of the complement membrane attack complex (MAC) in cardiomyocytes in areas of human myocardial infarction, the 20 kDA homologous restriction factor of complement (HRF20; CD59) and complement components (C1q, C3d and MAC) were analysed immunohistochemically using specific antibodies. Myocardial tissues obtained at autopsy from nine patients who died of acute myocardial infarction were fixed in acetone and embedded in paraffin. The ages of the infarcts ranged from about 3.5 h to 12 days. In cases of myocardial infarction of 20 h or less, MAC deposition was shown in the infarcted cardiomyocytes without loss of HRF20. Where the duration was 4 days or more, the cardiomyocytes with MAC deposition in the infarcted areas also showed complete loss of HRF20. Outside the infarcts, HRF20 in the cardiomyocytes was well preserved without MAC deposition. The present study suggests that the initial MAC deposition in dead cardiomyocytes can occur as a result of degradation of plasma-membrane by a mechanism independent of complement-mediated injury to the membrane. Loss of HRF20 from dead cardiomyocytes may not be the initial cause of MAC deposition, but may accelerate the deposition process of MAC in later stages of infarction.     

CD59 expressed by human endothelial cells functions as a protective molecule against complement-mediated lysis.

CD59 is a 18-20-kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single 20-kDa molecule in 2% deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti-CD59 enhanced markedly the susceptibility of HUVEC to C-mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C-mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.     

The control of homologous lysis.

Complement activation unleashes powerful effector mechanisms against which host cells are protected by homologous restriction factors. These factors are glycolipid-anchored membrane proteins that either induce C3 convertase dissociation (for example decay-accelerating factor) or prevent the full development of the membrane attack complex (for example homologous restriction factor and CD59). In this article Peter Lachmann explores the biology and biochemistry of these important and intriguing molecules.