Surface Decay-accelerating Factor Research Articles

Synergistic Therapeutic Vascular Cytoprotection against Complement-Mediated Injury Induced via a PKCα-, AMPK-, and CREB-Dependent Pathway

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

Enhanced oncolysis mediated by Coxsackievirus A21 in combination with doxorubicin hydrochloride

Virotherapy is an emerging strategy for the treatment of cancer that utilizes both replication-competent and genetically modified viruses to selectively kill tumor cells. We have previously shown that Coxsackievirus A21 (CVA21), a common-cold producing enterovirus, is an effective oncolytic agent against human melanoma, prostate, and breast cancer xenografts in vivo. CVA21 specifically targets and lytically infects susceptible cells expressing the CVA21 cellular receptors, intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF). Herein, the efficacy of CVA21 administered in combination with doxorubicin hydrochloride as a new therapeutic regimen for cancer was investigated. Flow cytometric analysis demonstrated that the human breast, colorectal, and pancreatic cancer cell lines examined expressed moderate levels of surface ICAM-1 and DAF, whilst a normal breast cell line expressed only minimal levels. When CVA21 was combined with doxorubicin hydrochloride, synergistically enhanced cell death was observed when CVA21 was administered both simultaneously or 24h prior to doxorubicin hydrochloride exposure. Doxorubicin hydrochloride had no effect on CVA21 replication. Through the use of an orthotopic (MDA-MB-231-luc) xenograft SCID mouse model of human breast cancer we showed that a single intravenous injection of CVA21 in combination with an intraperitoneal injection of doxorubicin hydrochloride resulted in significantly greater tumor reduction compared to either agent alone. Overall, these findings highlight the exciting potential of CVA21, administered in combination with doxorubicin hydrochloride, as a new therapeutic regimen for cancer.

Increased morbidity and mortality in murine cytomegalovirus-infected mice following allogeneic bone marrow transplant is associated with reduced surface decay accelerating factor expression

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo-BMT). The manifestations of CMV infection can range from neurological and haematological abnormalities to diminished graft survival and, in extreme cases, death. Many clinical studies have shown a direct correlation between cytomegalovirus infection and increased morbidity and mortality post allo-BMT, yet the exact mechanism is not well understood. Although driven primarily by T cell responses, the role of complement activation in acute and chronic graft-versus-host disease (GVHD) has also become more evident in recent years. The present studies were performed to examine the effects of murine cytomegalovirus (MCMV) infection on decay accelerating factor (DAF) and MCMVs role in exacerbating morbidity and mortality post-allo-BMT. Mice infected previously with a sublethal dose of MCMV (1 × 10⁵ plaque-forming units) have reduced expression of DAF on lung tissues and lymphocytes following allo-BMT. More importantly, mortality rates post-allo-BMT in recipient DAF knock-out mice receiving wild-type bone marrow are increased, similar to wild-type MCMV-infected recipient mice. Similarly, DAF knock-out mice showed greater intracellular interferon (IFN)-γ production by lung CD8 T cells, and infection with MCMV further exacerbated both intracellular IFN-γ production by CD8 T cells and mortality rates post-allo-BMT. Together, these data support the hypothesis that MCMV infection augments morbidity and mortality post-allo-BMT by reducing surface DAF expression.

Potent Oncolytic activity of human enteroviruses against human prostate cancer

Oncolytic virotherapy offers a unique treatment modality for prostate cancer, especially stages that are resistant to current therapies, with the additional benefit of preferentially targeting tumor cells amongst an environment of healthy tissue. Herein, the low pathogenic enteroviruses; Coxsackievirus A21 (CVA21), as well as a bio-selected variant of Coxsackievirus A21 (CVA21-DAFv) and Echovirus 1 (EV1) are evaluated as novel oncolytic agents against human prostate cancer. The surface expression of viral receptors required for enterovirus cell attachment/entry, including intercellular adhesion molecule-1 (ICAM-1), decay-accelerating factor (DAF) and integrin alpha(2)beta(1) on a number of human prostate cancer lines was assessed by flow cytometry. Susceptibility to viral oncolysis was determined via in vitro cell lysis assays performed on cell monolayers cultured in micro titer plates. The in vivo oncolytic efficacy of the enteroviruses was assessed using xenograft models in immune compromised SCID-mice following systemic challenge. The majority of prostate cancer lines tested expressed surface ICAM-1 and/or DAF, or alpha(2)beta(1), facilitating significant degrees of oncolysis following in vitro viral challenge. Systemic delivery of each of the three viruses induced reduction of xenograft tumor burdens in vivo, and a therapeutic dose-response was demonstrated for escalating doses of EV1 in the LNCaP animal model. Enteroviruses CVA21, CVA21-DAFv, and EV1 are potentially potent oncolytic agents against human prostate cancer.

Systemic targeting of metastatic human breast tumor xenografts by Coxsackievirus A21

Breast cancer is the most commonly diagnosed malignancy in women worldwide. Metastatic development is associated with poor prognosis and current therapies provide only limited success. Virotherapy is an emerging strategy for the treatment of cancer that utilizes both replication-competent and genetically modified viruses to selectively kill tumor cells. We have previously shown that Coxsackievirus A21 (CVA21), a wild-type common-cold producing enterovirus, is an effective oncolytic agent against human melanoma xenografts in vivo. CVA21 specifically targets and lytically infects susceptible cells expressing the CVA21 cellular receptors, intercellular adhesion molecule-1 (ICAM-1) and/or decay-accelerating factor (DAF). Herein, the efficacy of CVA21 as a therapeutic agent against human breast cancer was investigated both in vitro and in vivo. Flow cytometric analysis revealed that the human breast cancer cell lines examined expressed significantly elevated levels of surface ICAM-1 and DAF compared to normal breast cell lines, and that all cancerous lines were more susceptible to lytic infection by CVA21 than the normal cells. Through the use of subcutaneous (T47D cells) and orthotopic (MDA-MB-231-luc cells) xenograft SCID mouse models it was demonstrated that a single intravenous injection of CVA21 produced significant regression of pre-established tumors in vivo, as well as targeting and elimination of metastases in the orthotopic model. Taken together, these findings highlight the exciting potential of CVA21 as a therapeutic agent against both primary and metastatic human breast cancer.

Interaction with Coxsackievirus and Adenovirus Receptor, but Not with Decay-Accelerating Factor (DAF), Induces A-Particle Formation in a DAF-Binding Coxsackievirus B3 Isolate

Although many coxsackie B viruses interact with decay accelerating factor (DAF), attachment to DAF by itself is not sufficient to initiate infection. We examined the early events in infection that follow virus interaction with DAF, and with the coxsackievirus and adenovirus receptor (CAR). Interaction with soluble CAR in a cell-free system, or with CAR on the surfaces of transfected cells, induced the formation of A particles; interaction with soluble or cell surface DAF did not. The results suggest that CAR, but not DAF, is capable of initiating the conformational changes in the viral capsid that lead to release of viral nucleic acid.

Increased serum concentrations and surface expression on peripheral white blood cells of decay-accelerating factor (cd55) in patients with active ulcerative colitis

Inflammatory stimuli induce expression and release of decay-accelerating factor (DAF), a complement-regulatory protein present on peripheral-blood cells. Therefore, in ulcerative colitis (UC), an inflammatory colonic disease in which activated leukocytes are involved, DAF may be released from leukocytes into the circulation. In this study we compared serum DAF concentrations and surface DAF expression on peripheral-blood cells in patients with UC with disease activity. Peripheral-blood samples were obtained from 60 patients with UC (30 with active and 30 with inactive disease) and 19 healthy volunteers. Serum DAF concentrations were determined by means of immunoassay, and surface DAF expression on blood cells was examined with the use of flow cytometry. Serum DAF concentrations in patients with active disease (mean 48.6 ng/mL) were significantly higher than those in patients whose disease was in remission (33.3 ng/mL; P = .0003) and those in healthy controls (32.3 ng/mL; P = .0007). Surface DAF expression on neutrophils, CD14+ monocytes, and subsets of lymphocytes in patients with active UC was significantly increased compared with that in patients with UC in remission and in healthy controls. The increased serum DAF concentrations and surface DAF expression on leukocyte fractions in patients with active disease fell to significantly lower levels when the disease had gone into remission after medical therapy. Serum DAF concentrations are increased in UC patients in relation to disease activity. The likely source of increased DAF concentrations is peripheral-blood leukocytes that have been activated as part of the UC disease process.

In Vivo and in Vitro Evidence Suggest That HB-EGF Regulates Endometrial Expression of Human Decay-Accelerating Factor

Human endometrium expresses the critical complement component C3 in a cyclic fashion, with the highest expression in the secretory phase. As activated complement can kill cells, self or foreign, the secretory endometrial epithelium protects itself by concomitant expression of complement-protective proteins. The objectives of our present study were to describe the spatial and temporal regulation of the complement-protective protein decay-accelerating factor (DAF) in human endometrium and to identify local regulators of its expression. To describe the cyclic regulation of DAF, immunohistochemistry was performed using the IH4 monoclonal antibody on secretory phase endometrial biopsies taken from normal fertile volunteers in LH-timed cycles (n = 114). DAF expression in human endometrium was predominantly localized to the apical membrane of glandular and luminal epithelium. DAF expression, as assessed by histological scoring analysis, was minimal in the proliferative and early secretory phases and increased markedly on approximately day LH +7 (lumen) and LH +8 (glands). Maximal expression was seen in both glands and lumen by LH +8, and this persisted into menses. Using the RL95-2 endometrial epithelial cancer cell line as a model system, we next examined the cellular regulation of DAF. Treatment with E2 and progesterone, alone or in combination, had little effect on DAF expression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment increased cell surface and total DAF protein, increasing the signal by 260% on flow cytometry and by 200% on Western blot analysis. Stimulation of DAF protein expression was dose dependent, with maximal expression seen at 1 ng/ml. The stimulatory effects of HB-EGF were also observed at the mRNA level. EGF had effects similar to those of HB-EGF on DAF mRNA and protein expression, suggesting that the HB-EGF effect was mediated at least in part by the Her1 EGF receptor subunit. These studies suggest that DAF expression in the midsecretory phase is stimulated by HB-EGF or other members of the EGF family and may function to protect the epithelial integrity of human endometrium in the face of increased complement expression.

In vivo and in vitro evidence suggest that HB-EGF regulates endometrial expression of human decay-accelerating factor.

Human endometrium expresses the critical complement component C3 in a cyclic fashion, with the highest expression in the secretory phase. As activated complement can kill cells, self or foreign, the secretory endometrial epithelium protects itself by concomitant expression of complement-protective proteins. The objectives of our present study were to describe the spatial and temporal regulation of the complement-protective protein decay-accelerating factor (DAF) in human endometrium and to identify local regulators of its expression. To describe the cyclic regulation of DAF, immunohistochemistry was performed using the IH4 monoclonal antibody on secretory phase endometrial biopsies taken from normal fertile volunteers in LH-timed cycles (n = 114). DAF expression in human endometrium was predominantly localized to the apical membrane of glandular and luminal epithelium. DAF expression, as assessed by histological scoring analysis, was minimal in the proliferative and early secretory phases and increased markedly on approximately day LH +7 (lumen) and LH +8 (glands). Maximal expression was seen in both glands and lumen by LH +8, and this persisted into menses. Using the RL95-2 endometrial epithelial cancer cell line as a model system, we next examined the cellular regulation of DAF. Treatment with E2 and progesterone, alone or in combination, had little effect on DAF expression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment increased cell surface and total DAF protein, increasing the signal by 260% on flow cytometry and by 200% on Western blot analysis. Stimulation of DAF protein expression was dose dependent, with maximal expression seen at 1 ng/ml. The stimulatory effects of HB-EGF were also observed at the mRNA level. EGF had effects similar to those of HB-EGF on DAF mRNA and protein expression, suggesting that the HB-EGF effect was mediated at least in part by the Her1 EGF receptor subunit. These studies suggest that DAF expression in the midsecretory phase is stimulated by HB-EGF or other members of the EGF family and may function to protect the epithelial integrity of human endometrium in the face of increased complement expression.

Phospholipid-anchored and transmembrane versions of either decay-accelerating factor or membrane cofactor protein show equal efficiency in protection from complement-mediated cell damage.

Decay-accelerating factor (DAF) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that protects cells from complement-mediated damage by regulation of the C3 convertase. To investigate the role of the GPI anchor in the function of DAF, the cDNA encoding human DAF was expressed by transfection in Chinese hamster ovary (CHO) cells. Testing of these DAF transfectants in an antibody plus human complement-mediated cytotoxicity assay demonstrated that DAF protects these cells from cytotoxicity, and that the level of protection increases with expression of surface DAF. A cDNA construct encoding a transmembrane version of DAF (DAF-TM) protects CHO transfectants from cytotoxicity with equal efficiency to DAF. This DAF-TM construct used the TM and cytoplasmic domains of membrane cofactor protein (MCP); an alternate TM version of DAF constructed with the TM and cytoplasmic domains of HLA-B44 showed equivalent protection. The protection from cytotoxicity involved a decrease in the deposition of C3 on the cell, consistent with the effect of DAF on the C3 convertase. A second pair of anchor variants, MCP and a GPI-anchored construct, MCP-PI, were also equivalent in their complement protection. The equivalent function of GPI-anchored and TM versions of a protein was not expected based on the hypothesized increased lateral mobility of GPI-anchored proteins, which should confer a functional advantage in contacting ligand, in this case, C3b or C4b, on the cell surface. These data suggest either that GPI-anchored and TM versions of a protein have equal lateral mobility in the membrane, or else that increased lateral mobility is not advantageous to DAF or MCP in carrying out their complement inhibitory roles. Furthermore, DAF and MCP demonstrated approximately equal protection of cells from complement-mediated cytotoxicity, suggesting that DAF and MCP provide overlapping levels of protection to cells against damage mediated by the complement system.

An internally positioned signal can direct attachment of a glycophospholipid membrane anchor.

All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.

Phorbol esters increase synthesis of decay-accelerating factor, a phosphatidylinositol-anchored surface protein, in human endothelial cells.

A number of cell-surface proteins are anchored by a phosphatidylinositol (PI)-glycan moiety. These proteins can be released by PI-specific phospholipases C (PI-PLC). Decay-accelerating factor (DAF) is such a cell-surface protein that protects cells from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have studied the regulation of DAF synthesis in human umbilical vein endothelial cells (HUVEC), a cell that has the highest level of surface DAF among those human cells that have been studied. HUVEC DAF was measured by immunoradiometric assay of detergent extracts and of cell supernatants after treatment of cells with a bacterial (Bacillus thuringiensis) PI-PLC. Eighty percent of the HUVEC DAF (4 to 8 x 10(5) molecules/cell) was released by exogenously added PI-PLC, indicating that it is predominantly PI-anchored. The level of PI-PLC-sensitive HUVEC DAF was increased three- to fourfold by overnight treatment of cultures with the protein kinase C activators, PMA (1 to 10 nM), phorbol-12,13-dibutyrate (10 to 100 nM), and teleocidin A (1 to 10 nM) under conditions where cell number, protein, and lactate dehydrogenase remain unchanged. This DAF synthesis was blocked by the protein kinase C inhibitor K-252a in a dose-dependent manner (ED50 = 0.06 microM). The biologically inactive phorbols, 4-alpha-phorbol-12 myristate-13-acetate (1 microM) and 4-alpha-phorbol-12, 13-didecanoate (1 microM) did not increase DAF levels. The newly expressed DAF in PMA-stimulated cells was still largely PI-anchored. In contrast, another PI-anchored protein, alkaline phosphatase, was not altered by PMA treatment, demonstrating that the PMA effect is not uniform among all surface proteins. The increased expression of DAF only was evident 8 h after PMA addition and was blocked by the RNA and protein synthesis inhibitors, actinomycin D and cycloheximide, indicating that both transcription and translation are required for DAF synthesis induced by phorbol esters. It is concluded that protein kinase C activators cause selective induction of endothelial cell DAF and that DAF synthesis involves protein kinase C activation.

Endocytosis and shedding of the decay accelerating factor on human polymorphonuclear cells.

The decay-accelerating factor (DAF) is a cell membrane glycoprotein that functions in the control of C activation. We studied the modulation of membrane DAF on polymorphonuclear cells (PMN) by using anti-DAF antibodies. Fluorescence-activated cell sorter analysis showed that DAF expression was reduced by 43 +/- 7% on resting or stimulated cells that were held at 37 degrees C for 30 min when compared with those kept on ice. Most of this reduction occurred within the first 15 min, and was followed by a gradual further decrease in surface DAF. PMN that were held at 37 degrees C for varying periods of time before DAF measurement had a gradual decrease suggestive of release of DAF from the PMN membrane or endocytosis. To examine the latter, PMN were reacted with anti-DAF at 0 degree C, followed by 125I-Fab'2 secondary antibodies at either 0 degree C or 37 degrees C, and subsequently treated with pronase. Thirty +/- 11% of the 125I remained bound to cells kept at 37 degrees C compared to 2% in those held at 0 degrees C. Internalization was further confirmed by electron microscopy. In PMN that were not exposed to pronase, 26 +/- 2% of the surface-associated 125I was released at 37 degrees C compared with 7% at 0 degrees C. Immunoprecipitation and SDS-PAGE of surface-labeled PMN showed that the temperature-dependent released DAF had a lower m.w. than membrane DAF. Immunofluorescent studies revealed that 37 degrees C mediated the redistribution of DAF from a homogeneous pattern into caps. These results show that under the conditions studied DAF is partially internalized and partially released from the PMN membrane to the fluid phase; the latter may contribute to the presence of DAF in body fluids.

Association of cytoskeletal re-organization with capping of the complement decay-accelerating factor on T lymphocytes.

Recent studies have identified cell-associated proteins that are membrane anchored by glycosyl-inositol-phospholipid structures but the biologic implications of this mode of membrane attachment are incompletely understood. Among proteins anchored in this way is the decay-accelerating factor (DAF), a complement (C) regulatory factor that functions on blood cell surfaces to prevent autologous C attack. As one approach to investigate the functional consequences of glycosyl-inositol-phospholipid-anchoring of DAF in T lymphocytes, the effects of crosslinking surface DAF molecules were compared to those of crosslinking conventionally by anchored cluster of differentiation (CD) proteins. Upon incubation with anti-DAF mAb and anti-murine IgG, DAF re-distributed to a pole of the cell with a t1/2 at 37 degrees C of 4.4 min as compared to t1/2 of 3.5 to 7 min for CD3, CD4, and CD8. Re-distribution of DAF occurred independently of CD2, CD3, CD4, or CD8. Anti-DAF immunoprecipitates of membrane extracts of cells chemically cross-linked with dithiobis(succinimidylpropionate) contained only monomeric DAF. Immunofluorescent staining demonstrated clustered actin, tubulin, and vimentin beneath the capped DAF protein. Pre-treatment of cells with colchicine or 8-azidoadenosine 3',5'-cyclic phosphate, but not lumicolchicine, resulted in reduction of the t1/2 for DAF to 1 to 2.6 min. Conversely, treatment of cells with cytochalasins B or D completely blocked DAF capping. The results indicate that, upon cross-linking, glycosyl-inositol-phospholipid-anchored DAF molecules undergo capping similar to conventionally anchored CD molecules and that DAF capping is associated with cytoskeletal reorganization.

Increased expression of complement decay-accelerating factor during activation of human neutrophils.

Decay-accelerating factor (DAF) is a membrane protein that protects blood cells from damage by autologous complement. Using monoclonal antibodies in both direct-binding studies and flow cytometry, we found that resting neutrophils (polymorphonuclear leukocytes [PMN]) expressed 10(4) DAF molecules on their surface, and that surface DAF expression more than doubled when the cells were activated. Upregulation of surface DAF occurred within minutes, paralleled the upregulation of complement receptor types 1 and 3 (CR1 and CR3), and was not dependent on new protein synthesis. It was unaffected by EDTA but was inhibited by 10 microM trifluoperazine, suggesting involvement of intracellular Ca2+ and calmodulin or protein kinase C. Upon activation, the affected PMN lacking surface DAF from patients with paroxysmal nocturnal hemoglobulinuria failed to increase DAF expression. In contrast, these cells increased CR1 and CR3 expression normally, suggesting that DAF deficiency in affected cells involves abnormal synthesis or packaging of DAF for intracellular storage. Translocation of DAF to the cell surface induced by chemoattractants may be important in allowing PMN to survive and function at inflammatory sites where there is rapid complement turnover.

Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein.

Decay-accelerating factor (DAF) is a 70,000 Mr membrane protein that inhibits amplification of the complement cascade on the cell surface, and protects cells from damage. Purified DAF can be reincorporated into the membrane of red cells and is functional. DAF is deficient in paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by increased sensitivity of erythrocytes to complement lysis. We show here that DAF is part of a newly described family of membrane proteins anchored to the lipid bilayer by means of phosphatidylinositol (PI). Treatment with PI-specific phospholipase C (PIPLC) releases 70-80, 60, and 10% of cell surface DAF from mononuclear cells, neutrophils, and erythrocytes, respectively. The PIPLC-released DAF (DAF-S) is slightly smaller (67,000 Mr) than the membrane form. DAF and DAF-S cannot be distinguished antigenically. Furthermore, DAF-S has lost its ability to significantly inhibit the C3-convertase, as well as its ability to incorporate into cell membranes. Since DAF can only inhibit C3-convertase endogenously, i.e., within the membrane of the same cell, it is likely that the loss of activity of DAF-S is causally related to its inability to reincorporate in the lipid bilayer. As shown by others, the complement-sensitive red cells from PNH patients lack acetylcholinesterase, which is also anchored to the membrane by PI (9). Thus it is possible that the molecular defect in PNH lies in the biosynthetic pathways leading to the attachment of PI to the polypeptide chains, in the transport of these proteins to the surface, or in their release by the action of endogenous phospholipases. From a practical standpoint the specific release of DAF by PIPLC could facilitate killing of tumor cells by amplifying the effects of the complement cascade on the surface of antibody-sensitized cells.

Surface Membrane Expression by Human Blood Leukocytes and Platelets of Decay-Accelerating Factor, a Regulatory Protein of the Complement System

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol-reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.

Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol- reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.