Complement decay accelerating factor (DAF)/CD55 in cancer

The complement system is a powerful innate mechanism involved in protection of the host against pathogens. It also has a role in the clearance of apoptotic cells and has been implicated in a range of pathologies including autoimmunity and graft rejection. The control of complement is mediated through the complement regulatory proteins (CRPs). These are present on most cells and protect normal cells from complement-mediated attack during innate activation. However, in a range of pathologies and cancer, these molecules are up or down regulated, sometimes secreted and even lost. We will review the expression of CRPs in cancer, focussing on CD55 and highlight other roles of the CRPs and their involvement in leukocyte function. We will also provide some data providing a potential mechanism by which soluble CD55 can inhibit T-cell function and discuss some of the implications of this data.This article is a symposium paper from the “Robert Baldwin Symposium: 50 years of Cancer Immunotherapy”, held in Nottingham, Great Britain, on 30 June 2005.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

Effect of periodontopathogen lipopolysaccharides and proinflammatory cytokines on CD46, CD55, and CD59 gene/protein expression by oral epithelial cells

Membrane-anchored complement regulatory proteins (CRPs), including CD46, CD55, and CD59, protect host cells from complement attack. In the present study, we investigated whether periodontopathogen lipopolysaccharide and proinflammatory cytokines modulate CRP gene/protein expression in human oral epithelial cells. The lipopolysaccharide of Treponema denticola and Tannerella forsythia were the most potent for increasing the gene expression of CD55 and CD59, and to a lesser extent CD46, after a 48-h stimulation. An lipopolysaccharide-induced upregulation of epithelial cell-surface CRP was also demonstrated. The stimulation of epithelial cells with lipopolysaccharide was associated with interleukin-6 (IL-6) and IL-8 secretion. Although these two cytokines had no effect on CD46 and CD55 gene expression in epithelial cells, IL-1β and tumor necrosis factor-α induced a significant upregulation. The cell-surface expression of CRP was also increased by the stimulation of epithelial cells with cytokines. The CD46, CD55, and CD59 gene/protein expression was upregulated by periodontopathogen lipopolysaccharide and proinflammatory cytokines. It can be hypothesized that, when faced with bacterial challenges and inflammatory conditions associated with active periodontal sites, oral epithelial cells may respond by increasing CRP gene/protein expression to avoid cell lysis by the complement system, which is activated during periodontitis.     

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.     

Immunolocalization of calcineurin and FKBP12, the FK506-binding protein, in Hassall's corpuscles of human thymus and epidermis

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays an important role in various physiological functions including T cell activation. This enzyme is a target molecule for the immunosuppressants, cyclosporin A and FK506. In the present study, we investigated immunohistochemical localization of calcineurin and FKBP12, an FK506-binding protein, in human thymus and epidermis. The catalytic subunit (calcineurin A) of calcineurin was abundantly expressed in Hassall's corpuscles which were localized in the thymic medulla and represented the terminal stages of thymic medullary epithelium. The regulatory subunit (calcineurin B) of calcineurin was also expressed in high amounts in Hassall's corpuscles. In the epidermis, which shows similarities to Hassall's corpuscles, both subunits were also abundantly expressed, and their expression increased with the differentiation of keratinocytes. FKBP12 was observed to be expressed abundantly, both in Hassall's corpuscles and the entire epidermis. These findings suggest that the differentiated forms of the two cell types, which are the thymic medullary epithelial cell and the keratinocyte, are the target for pharmacological actions of FK506.     

Formation of Hassall's corpuscles in vitro by the thymic epithelial cell line IT-26 R 21 of the rat

Summary In the monolayer of an established epithelial cell line from the rat thymus, IT-26R21, characteristic cell aggregates quite similar to Hassall's corpuscles were formed. These aggregates were examined by light and electron microscopy, and immunohistochemically. Their interpretation as Hassall's corpuscles is based on the following observations: (1) The aggregates are formed in the monolayer of cells that greatly resemble medullary epithelial cells of the thymus. (2) They consist of flattened epithelial cells in a concentric pattern with one or more degenerating cells in the center. (3) Loss of microvilli suggests that these cells are keratinizing. (4) The aggregates show strongly positive reactions in immunofluorescent staining with antikeratin and antiprekeratin.When Hassall's corpuscles increase in size, cellular proliferation is somewhat suppressed. Both in vivo and in vitro, they may be interpreted as an expression of a changing growth pattern in confined spaces and thus seem to have little immunological function.     

Destruction of Hassall's corpuscles by macrophages in the sheep thymus

Summary The dissolution of Hassall's corpuscles by macrophages has been demonstrated in the sheep thymus. The findings indicate that enlarged Hassall's corpuscles are rapidly broken down by macrophages at the end of gestation or immediately after birth and replaced by newly formed corpuscles, and that these cyclic changes in Hassall's corpuscles persist, under normal physiological conditions, throughout life.     

Epithelial cell heterogeneity in mammalian thymus: Monoclonal antibody to high molecular weight keratins exclusively binds to Hassall''s corpuscles

Summary Hassall's corpuscles represent a subset of medullary thymic epithelial cells whose origin and function within the thymus still remain largely unknown. The present study shows that Hassall's corpuscles can be defined by their intracellular content in specific keratin subunits. Two monoclonal anti-keratin antibodies were used: KL1, directed to high molecular weight keratins, and KL4, specific for high and medium molecular weight polypeptides.In vivo, KL1 exclusively binds to Hassall's corpuscles of five mammalian species including mouse, rat, guinea-pig, rabbit and pig. Thus KL1 appears as an exclusive marker of Hassall's corpuscles in a large number of mammals.In vitro, thymic epithelial cells gave rise in certain species to Hassall's corpuscles. In contrast to itsin vivo reactivity, KL1 never labelled Hassall's corpuscles developedin vitro. These data strongly support the following conclusions: (1) Hassall's corpuscles derive from medullary epithelial cells; (2) they represent advanced stages of thymic epithelial maturation; (3) thymic epithelial cell differentiation is impairedin vitro. Furthermore, this study provides additional evidence that thymic epithelium heterogeneity reflects different stages in epithelial maturation.     

Critical protection from renal ischemia reperfusion injury by CD55 and CD59

Renal ischemia-reperfusion injury (IRI) is a feature of ischemic acute renal failure and it impacts both short- and long-term graft survival after kidney transplantation. Complement activation has been implicated in renal IRI, but its mechanism of action is uncertain and the determinants of complement activation during IRI remain poorly understood. We engineered mice deficient in two membrane complement regulatory proteins, CD55 and CD59, and used them to investigate the role of these endogenous complement inhibitors in renal IRI. CD55-deficient (CD55(-/-)), but not CD59-deficient (CD59(-/-)), mice exhibited increased renal IRI as indicated by significantly elevated blood urea nitrogen levels, histological scores, and neutrophil infiltration. Remarkably, although CD59 deficiency alone was inconsequential, CD55/CD59 double deficiency greatly exacerbated IRI. Severe IRI in CD55(-/-)CD59(-/-) mice was accompanied by endothelial deposition of C3 and the membrane attack complex (MAC) and medullary capillary thrombosis. Complement depletion in CD55(-/-)CD59(-/-) mice with cobra venom factor prevented these effects. Thus, CD55 and CD59 act synergistically to inhibit complement-mediated renal IRI, and abrogation of their function leads to MAC-induced microvascular injury and dysfunction that may exacerbate the initial ischemic assault. Our findings suggest a rationale for anti-complement therapies aimed at preventing microvascular injury during ischemia reperfusion, and the CD55(-/-)CD59(-/-) mouse provides a useful animal model in this regard.     

Mouse CD4CD25 T regulatory cells are protected from autologous complement mediated injury by Crry and CD59

Self cells depend on surface complement regulators to protect them from autologous complement mediated attack. CD4⁺CD25⁺foxp3⁺ T regulatory (Treg) cells are critical in maintaining immune homeostasis, however, which complement regulators are expressed on them and how they are protected from autologous complement attack remains unknown. We report here that mouse Treg cells express virtually no DAF or CR1. Instead, all of them express Crry and approximately half of them express CD59. Both Crry^−/− and CD59^−/− Treg cells exhibit greater complement mediated injury than WT Treg cells. These results clarify the status of cell surface complement regulators on mouse Treg cells and indicate that both Crry and CD59 are required to protect Treg cells from autologous complement mediated injury. Additionally, these data also argue that different from previous assumption, at least in mice, CD4⁺CD25⁺foxp3⁺ Treg cells are not homogenous and could be further divided into subgroups based on CD59 expression.     

Costimulation via CD55 on human CD4+ T cells mediated by CD97

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4(+) T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55(-/-) mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4(+) T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.     

Specific Acquisition of Functional CD59 but Not CD46 or CD55 by Hepatitis C Virus

Viruses of different families encode for regulators of the complement system (RCAs) or acquire such RCAs from the host to get protection against complement-mediated lysis (CML). As hepatitis C virus (HCV) shares no genetic similarity to any known RCA and is detectable at high titers in sera of infected individuals, we investigated whether HCV has adapted host-derived RCAs to resist CML. Here we report that HCV selectively incorporates CD59 while neither CD55, nor CD46 are associated with the virus. The presence of CD59 was shown by capture assays using patient- and cell culture-derived HCV isolates. Association of CD59 with HCV was further confirmed by Western blot analysis using purified viral supernatants from infected Huh 7.5 cells. HCV captured by antibodies specific for CD59 remained infectious for Huh 7.5 cells. In addition, blocking of CD59 in the presence of active complement reduced the titer of HCV most likely due to CML. HCV produced in CD59 knock-down cells were more significantly susceptible to CML compared to wild type virus, but neither replication, assembly nor infectivity of the virus seemed to be impaired in the absence of CD59. In summary our data indicate that HCV incorporates selectively CD59 in its envelope to gain resistance to CML in serum of infected individuals.     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four- to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.     

Myoepithelial cells in human thymus: staining, polarization and fluorescence microscopic studies.

Myoid cells in human thymus were studied around the turn of the century, and alterations in patients with cardiovascular disease were reported. It was therefore deemed of interest to reinvestigate these long forgotten cells. The configurational staining, polarization and fluorescence microscopic properties of smooth myofibrils in thymic epithelial cells were identical with those of classical myoepithelial cells, smooth muscle, and A bands of striated muscle. Cross-striated myoid cells could not be found in thymus of children. Myoepithelial cells formed a layer at the surface of thymic lobules; others were scattered throughout the cortex and medulla. In addition, the medulla contained seemingly hypertrophic myoepithelial cells. Hassall's corpuscles consisted of layers of myoepithelial cells. Hammar (1905) regarded epithelial cells with smooth myofibrils in human thymus as equivalents of the cross-striated myoid cells in lower vertebrates. The myoepithelial cells observed in this study are apparently identical with the smooth myoid cells of early anatomists; the hypertrophic myoepithelial cells correspond to the unicellular Hassall's corpuscles. The functions of these cells are not yet clear; the wide variations from case to case in the same age group indicate that the myoepithelial cells are affected by a variety of diseases.     

Identification of the individual residues that determine human CD59 species selective activity

Formation of the cytolytic membrane attack complex of complement on host cells is inhibited by the membrane-bound glycoprotein, CD59. The inhibitory activity of CD59 is species restricted, and human CD59 is not effective against rat complement. Previous functional analysis of chimeric human/rat CD59 proteins indicated that the residues responsible for the species selective function of human CD59 map to a region contained between positions 40 and 66 in the primary structure. By comparative analysis of rat and human CD59 models and by mutational analysis of candidate residues, we now identify the individual residues within the 40-66 region that confer species selective function on human CD59. All nonconserved residues within the 40-66 sequence were substituted from human to rat residues in a series of chimeric human/rat CD59 mutant proteins. Functional analysis revealed that the individual human to rat residue substitutions F47A, T51L, R55E, and K65Q each produced a mutant human CD59 protein with enhanced rat complement inhibitory activity with the single F47A substitution having the most significant effect. Interestingly, the side chains of the residues at positions 47, 51, and 55 are all located on the short single helix (residues 47-55) of CD59 and form an exposed continuous strip parallel to the helix axis. A single human CD59 mutant protein containing rat residue substitutions at all three helix residues produced a protein with species selective activity comparable to that of rat CD59. We further found that synthetic peptides spanning the human CD59 helix sequence were able to inhibit the binding of human CD59 to human C8, but had little effect on the binding of rat CD59 to rat C8.     

Fbw7/hCDC4 dimerization regulates its substrate interactions

Background

Membrane complement regulatory proteins (mCRPs) inhibit complement-mediated killing of human cells by human complement, a property that confers protection from complement to malignant breast cancer cells and that thwarts some immunotherapies. Metabolic mechanisms may come into play in protecting cancer cells from the complement system subsequent to relatively low levels of complement deposition.

Results

In differentiating these mechanisms, two types of human breast cancer cell lines, MCF7 (adenocarcinoma) and Bcap37 (medullary carcinoma) were cell-cycle synchronized using glutamine-deprivation followed by restoration. These cells were examined for the expression of two mCRPs (CD59 and CD55), and for subsequent susceptibility to antibody-mediated complement-induced membrane damage. After glutamine restoration, MCF7 and Bcap37 cells were synchronized into the G2/M phase and an average increased expression of CD59 and CD55 occurred with a corresponding resistance to complement-mediated damage. Blocking CD59 inhibitory function with monoclonal antibody revealed that CD59 played a key role in protecting unsynchronized Bcap37 and MCF7 cancer cells from the complement membrane attack complex. Interestingly, glutamine-deprivation did not significantly affect the expression of proteins e.g., the surface level of CD59 or CD55, but did increase the susceptibility to complement-mediated killing. One possible explanation is that glutamine-deprivation may have slowed the turnover rate of mCRPs, preventing the cells from replacing pre-existing mCRPs, as they became neutralized by covalent C4b and C3b depositions.

Conclusion

Taken together the findings are consistent with the conclusion that future immunotherapies should aim to achieve a highly specific and profound activation and deposition of complement as well as to disrupt the synthesis and expression of CD59 and CD55 by the cancer cells.     

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells

Complement is the canonical innate immune system involved in host defense and tissue repair with the clearance of cell debris. In contrast to the robust armory mounted against microbial nonself-pathogens, complement is selectively activated on altered self (i.e. apoptotic and necrotic cells) to instruct the safe demise by poorly characterized mechanisms. Our data shed new light on the role of complement C1q in sensing nucleic acids (NA) rapidly exposed on apoptotic Jurkat T cell membranes and in driving C3 opsonization but without the lytic membrane attack complex. DNA/RNase-treated apoptotic cells failed to activate complement. We found that several other apoptotic cell models, including senescent keratinocytes, ionophore-treated sperm cells, and CMK-derived platelets, stained for cleaved caspase 3 were rapidly losing the key complement regulator CD46. CD46 from nuclear and membrane stores was found to cluster into blebs and shed into microparticles together with NA, phosphatidylserine, C1q, and factor H. Classical and alternative pathways of complement were involved in the recognition of H2O2-treated necrotic cells. Membrane attack complex was detected on necrotic cells possibly as a result of CD46 and CD59 shedding into soluble forms. Our data highlight a novel and universal paradigm whereby the complement innate immune system is using two synergistic strategies with the recognition of altered self-NA and missing self-CD46 signals to instruct and tailor the efficient removal of apoptotic and necrotic cells in immunoprivileged sites.     

Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis

NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.     

Expression of the complement regulatory protein CD59 on human spermatozoa: Characterization and role in gametic interaction

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identifield on the human sperm surface by means of H19, an IgG₁ anti-protectin mouse monoclonal antibody. Using Indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome-reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol-specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement-mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test. © 1994 Wiley-Liss, Inc.