Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro,and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage, DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-α, IL-1β, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.     

Effect of IL-1beta,TNF-alpha and IL-4 on complement regulatory protein mRNA expression in human articular chondrocytes

Most of the cells possess complement regulatory proteins (CRPs) that protect them against complement-mediated damage. In our previous work we revealed that human articular chondrocytes express CPRs. Moreover, increase of CRPs expression after treatment of chondrocytes with proinflammatory cytokines IL-1beta and TNF-alpha has been demonstrated by ELISA technique. Chondroprotective cytokine IL-4 stimulated expression of CD46 only. In this work RT-PCR technique was used to evaluate the expression of mRNA of cell surface CPRs in cultured isolated articular human chondrocytes after treatment with IL-1beta, TNF-alpha and IL-4. Chondrocytes stimulated with IL-1beta and TNF-alpha showed augmented levels of CD46, CD55 and CD59 mRNA. Treatment with IL-4, however, increased only the level of CD46 mRNA. These results confirm and extend our previous observations. CD35 mRNA was not found. Expression of complement regulatory proteins on chondrocytes and its upregulation by cytokines stimulating matrix degradation could be important for the protection of these cells against complement-mediated lysis, which might be caused by immunocomplexes deposited in articular cartilage in inflammatory joint diseases.     

Expression of the complement regulatory proteins decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 in the normal human uterine cervix and in premalignant and malignant cervical disease.

The membrane-bound complement regulators decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), and CD59 are broadly expressed proteins that act together to protect host tissues from autologous complement. Comparison of expression profiles of these proteins between normal and pathological tissues could reveal a mechanism by which tumor cells evade complement-mediated killing. Expression of the regulators was therefore examined in the normal human uterine cervix, in cervical intraepithelial neoplasia (CIN; n = 23), and in cervical squamous carcinomas (n = 6). DAF and MCP were reciprocally expressed in normal ectocervical epithelium. MCP was confined predominantly to the basal and parabasal layers with more extensive expression in metaplastic squamous epithelium. An apparent expansion in MCP expression was observed in more severe premalignant lesions whereas cervical carcinoma were uniformly MCP positive. By contrast, DAF expression appeared unaltered in premalignant lesions and variable in carcinomas. However, increased DAF was observed in stromal cells directly adjacent to infiltrating tumor cells. A low molecular weight DAF product was detected in tumors, and preliminary evidence suggests this may be derived from stromal cells. Overall, changes in expression of C3 convertase regulators in both the stromal and epithelial compartments may be important for evasion of immune surveillance in cervical cancer.     

Tissue distribution of the rat analogue of decay-accelerating factor

In humans, decay-accelerating factor (DAF) is a widely distributed, cell-bound inhibitor of the complement activation enzymes and plays a key role in regulating complement activation, preventing the generation of anaphylotoxins and opsonins, and protecting against complement-mediated lysis. Rodent analogues of DAF have recently been identified, providing a new avenue for the analysis of function. Rat DAF was cloned in our laboratory. Here we describe the generation of monoclonal antibodies (mAbs) against rat DAF, using transfected cells as immunogen, and their use in the analysis of the distribution of DAF in the rat by flow cytometry, Western blot analysis and immunohistochemistry. One of the mAbs was found to block the complement inhibitory function of rat DAF, offering the prospect of neutralization of DAF function in vivo. The antibodies have also been used for purification of DAF from rat erythrocytes by affinity chromatography. Rat DAF purified in this manner was similar in molecular mass to human DAF. The purified protein incorporated into lipid membranes, confirming the presence of a glycolipid anchor, and incorporated protein strongly inhibited the rat C3 convertase. Rat DAF was strongly expressed on endothelia throughout the animal and was also present in most tissues and organs. DAF expression was weak or absent in the brain and on circulating and spleen-resident T cells. Strong DAF expression observed in the kidney was restricted to the glomerulus and Bowman's capsule. DAF expression in the testis was found only in association with the later stages of spermatogenesis.     

Expression of decay-accelerating factor is reduced on hyperplastic synovial lining cells in rheumatoid synovitis.

Decay-accelerating factor (DAF), a membrane inhibitor of homologous complement activation, is present in synovial cells lining joint space and detected in synovial fluid. DAF is considered to protect synovial membrane from complement-mediated injury associated with articular inflammation. We studied the immunohistopathological features of DAF molecules in synovial membrane of rheumatoid synovitis using a DAF-specific monoclonal antibody, 1C6. Reacting molecules with the 1C6 antibodies in synovial tissue extracts formed a 70-kD band in Western blot analysis. DAF was strongly detected on the flat synovial lining cells, but weakly on the hyperplastic and multi-layered lining cells in rheumatoid synovitis. The latter cells reacted with anti-Leu-M3 antibodies specific for a cell surface marker of activated macrophages, sometimes accompanied by C3 and IgM deposition on the superficial synovial membrane. These results suggest that active rheumatoid synovitis characteristically with hyperplastic synovial lining cells is out of control by DAF, thereby permitting further complement-mediated injury.     

Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.     

Role of apoptosis signal-regulating kinase 1 in complement-mediated glomerular epithelial cell injury

In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.     

Decay-accelerating factor in the cardiomyocytes of normal individuals and patients with myocardial infarction

Summary The presence of decay-accelerating factor (DAF) was clearly demonstrated on the surface of normal cardiomyocytes. In patients who had died of myocardial infarction (MI) cardiomyocytes displayed different appearances: outside the ischaemically damaged region the myocytes showed no significant variations in DAF expression when compared with controls without MI. Within myocardial zones damaged by ischaemia, however, apparently normal myocytes showed large gaps in surface staining of DAF or formed clusters which were entirely devoid of reactivity with anti-DAF antibodies. The number of DAF-deficient myocytes increased with the extent of necrosis and also with the number of days between onset of MI and death. Even though injury to myocytes is to a large extent related to anoxia and to the presence of free oxygen radicals, the complement system also appears to be involved; DAF may have protective functions against complement-mediated injury. We speculate that phospholipase may be involved in the removal of DAF from the cardiomyocyte surface.This work was supported in part by grant no. 3.157.88 from the Swiss National Foundation for Scientific Research and a contribution from Sandoz Ltd. Pharma Division, Basel     

Role of early- or late-phase activation of p38 mitogen-activated protein kinase induced by tumour necrosis factor-alpha or 2,4-dinitrochlorobenzene during maturation of murine dendritic cells

Dendritic cells (DCs) are maturated by a variety of stimuli. However, the precise mechanisms underlying the maturation of DCs are not fully understood. In the present study, we analysed the effects of tumour necrosis factor-alpha (TNF-alpha) and 2,4-dinitrochlorobenzene (DNCB) on phenotypic maturation and p38 mitogen activated protein kinase (MAPK) activity, using a murine DC line. TNF-alpha markedly increased the surface expression of major histocompatibility complex (MHC) and costimulatory molecules, CD86 and CD80, on DCs. DNCB more markedly enhanced the surface expression of costimulatory molecules, but showed less stimulatory capability on MHC molecules, compared with TNF-alpha. Simultaneous treatment of DCs with TNF-alpha and DNCB showed additive enhancement of costimulatory molecule expression. TNF-alpha activated p38 MAPK in DCs only at an early time-point (15 min). In contrast, DNCB activated p38 MAPK at later time-points (3-6 hr). SB203580, a specific inhibitor of p38 MAPK, partially or markedly inhibited the phenotypic changes of DCs induced by TNF-alpha or DNCB, respectively. In addition, N-acetyl-l-cysteine, a reducing supplier, completely inhibited the DNCB-induced expression of MHC and costimulatory molecules, but not those induced by TNF-alpha. These findings demonstrate that TNF-alpha and DNCB activate the p38 MAPK pathway at an early and a late phase, respectively, and thereby induce DC maturation through different signal pathways.     

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.     

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.     

Signal transduction via a protein associated with a glycosylphosphatidylinositol-anchored protein, decay-accelerating factor (DAF/CD55)

Decay-accelerating factor (DAF/CD55) is a glycosylphosphatidylinositol- anchored protein which is known to have signal transducing capacity and to be associated with several proteins. To determine the signal transducer in the DAF-forming complex, we purified DAF-associated proteins from Raji B cells using an anti-DAF mAb (1C6)-bound affinity column and established five mAb against them. Among these, mAb 2E12- G7(IgM/kappa) reacted with a variety of intact cells, including peripheral blood mononuclear cells (PBMC), as well as cells from T and B cell lines, as shown by cytofluorimetric analyses. The Mr of 2E12-G7 antigen was estimated to be 43 kDa by surface biotinylation and immunoblotting analysis. This antigen was demonstrated in 1C6 immunoprecipitates, but not in anti-CD59 (another GPI-anchored complement regulatory factor)-immunoprecipitates. Sequential treatment with 1C6 F(ab')2 and then with anti-mouse Ig F(ab')2 stimulated PBMC to induce tyrosine phosphorylation on proteins of 45, 72, 78 and approximately 100 kDa. Also, mAb cross-linked to 2E12-G7 stimulated PBMC to induce tyrosine phosphorylation on proteins of 72, 78 and approximately 100 kDa. Furthermore, when 2E12-G7 and 1C6 immunoprecipitates were incubated with [gamma-32P]ATP, the main constituents detected in both were phosphorylated proteins of 26, 32 and 62 kDa. Thus, DAF-associated 2E12-G7 antigen transduces a signal, similar to the DAF molecule.      

Complement regulatory proteins and autoimmunity

To discriminate self from non-self is an essential issue in the immune system. Autologous cells are protected against complement-mediated cell injury by the self-recognition mechanism using complement regulatory proteins composed of complement receptor type 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55) and homologous restriction factor (protectin, CD59). Recently, the up-regulation of these molecules has been widely shown in inflammatory tissues and organs affected by autoimmune diseases, and in vitro assays have revealed that immune complexes or several cytokines, including interferongamma, tumor necrosis factor alpha, interleukin 1beta and transforming growth factor beta, can up-regulate these molecules. In contrast, it has been found that expression of these complement regulatory proteins is markedly decreased on autologous cells undergoing apoptosis. These findings suggest that complement regulatory proteins have dual roles at inflammatory sites: enhancement of cellular resistance to complement attack and acceleration of the clearance of cells injurious to the organism due to complement-mediated mechanisms. To assist the former function, a therapeutic approach using recombinant soluble complement regulatory proteins may provide a promising strategy for the treatment of autoimmune diseases.     

C5b-8 Step Lysis of Swine Endothelial Cells by Human Complement and Functional Feature of Transfected CD59

The authors established several swine endothelial cell (SEC) lines expressing human CD59 by transfection of cDNA, and assessed the function of the transfectant molecules in comparison with those of membrane cofactor protein (MCP) and decay-accelerating factor (DAF) in an in vitro hyperacute rejection model of swine to human discordant xenograft. At the usual expression rate, DAF and MCP protected SEC from human complement mediated cell lysis, but CD59 did not block human complement attack on SEC. However, CD59 protects SEC from cell lysis when sufficiently expressed as in human umbilical vein (HUVEC). The authors examined why CD59 needed so many molecules to protect human complement-mediated SEC lysis and found that SEC underwent lysis by human C5b-8. The degree of C5b-8 step lysis of SEC was approximately 70% of the total activity (C5b-9). Additionally, CD59 protected human complement activation less efficiently at the C5b-8 step than at the C9-step. Therefore, to overcome human complement mediated SEC lysis, C8 activity must be inhibited by dense expression of CD59.     

Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin).

Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the surrounding extracellular matrix.