Control of the function of substrate-bound C4b-C3b by the complement receptor Cr1.

The complement fragments C3b and C4b are the main ligands for the membrane receptor CR1. We showed elsewhere that CR1 functions as an essential cofactor for the factor I-mediated enzymatic breakdown of membrane-bound C3b (*C3b) into C3c and * C3dg . One of the main findings of the present paper is that CR1 also promotes the degradation of bound C4b (*C4b) into C4c and *C4d. On a weight basis, the cofactor activity of CR1 in the cleavage of *C4b present on the cell intermediate EAC14 is 10(3)-fold greater than that of the serum cofactor C4-binding protein ( C4bp ). An additional finding is that the effect of CR1 on either *C3b or *C4b is modulated by the presence of the other ligand in its vicinity; that is, *C4b degradation by CR1 plus I is enhanced by neighboring *C3b and vice versa. For example, upon uptake of optimal amounts of *C3b onto EAC142 and the assembly of the C3-convertase EAC1423 , the activity of CR1 in generating C4c is enhanced 5-10 times further. Conversely, when the number of *C3b molecules on EAC1423 is relatively small (or when EAC1423 has been converted by I plus H into EAC1423i ), the presence of neighboring *C4b enhances the conversion of *C3b (or *iC3b) into C3c plus * C3dg . The enhancing effect of *C3b on the cleavage of *C4b by I is observed only if the cofactor of this reaction is CR1. Indeed, the activity of I or I plus C4bp on *C4b is significantly inhibited when *C3b is fixed and the main product of the reaction is * iC4b . Taken together, these findings suggest that degradation of *C4b will be more effective when enough C3b molecules are fixed nearby, thus facilitating the interaction of *C4b*3b clusters with CR1-bearing cells, and that under physiological conditions, *C4b activity can be efficiently controlled by CR1.

Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.

Decay-accelerating factor (DAF), extracted from the stroma of human erythrocytes, was purified to homogeneity and incorporated into the membrane of sheep red cell complement intermediates, where its functional properties were analyzed. Incorporation of DAF into the cell membranes was temperature dependent, took place on pronase- or trypsin-treated erythrocytes, and did not depend on prior deposition of antibody, C1 or C4. Serum lipoproteins (high and low density) effectively inhibited DAF incorporation, but had no effect on the activity of DAF after its association with the cell membrane. The incorporated DAF could not be removed from the red cell surface by repeated washings in the presence of high salt concentration but was solubilized when the stroma were extracted with 0.1% Nonidet P-40. The presence of DAF in the membrane of EA did not affect the deposition of C1 and C4, but as few as 10(2) DAF molecules per cell profoundly inhibited the assembly of C3 and C5 convertases of both the classical and alternative pathways. The DAF inhibitory effect on EAC14 or EAC43 was not overcome by supplying an excess of C2 or factor B, but the alternative pathway C3 convertase could be assembled in the presence of Ni++, or nonphysiological concentrations of Mg++, which enhances the binding affinity of factor B for C3b. The DAF effect on EAC14 or EAC143 was entirely reversed by treating the cells with specific anti-DAF antibodies, showing that DAF did not alter the structure of C4b or C3b. Taken together, the experimental evidence suggests that DAF interacts directly with membrane-bound C3b or C4b and prevents subsequent uptake of C2 and factor B. DAF can function only within the cell membrane. Indeed, the decay dissociation of the C4b2a enzyme on DAF-containing sheep intermediates was not changed by varying the cell concentration. DAF-treated EA had no influence on the decay of nontreated EAC142 present in the same mixture. Moreover, the inhibitory activity of intact human erythrocytes on C4b2a was not blocked by antibodies to DAF, but was abolished by antibodies to the C3b/C4b receptor (CR1). When incorporated into the membrane of rabbit erythrocytes, human DAF inhibited their lysis by human complement. In conclusion, on the basis of these and previous results, it appears that DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane-bound C4b interacts endogenously with complement receptor CR1 of human red cells.

Activation of the classical complement pathway on the membrane of autologous cells results in the deposition of C4b on their surface and in the assembly of the C3 convertase C4b2a, one of the amplifying enzymes of the cascade. Here we study the sequence of events leading to irreversible inactivation of the potentially harmful C4b bound to human red cells. We show that deposited C4b interacts endogenously with complement receptor type 1 (CR1) present on the membrane of the same red cell. Complexes containing CR1 and C4b are found in extracts of membranes of C4b-bearing red cells after treatment of the intact cells with a bifunctional crosslinking reagent. The amount of complexed CR1 increases with the number of deposited C4b molecules. Only small amounts of free CR1 are observed on red cells bearing as few as 1,900 molecules of C4b, suggesting that the binding avidity between C4b and endogenous CR1 is high. In agreement with this observation, we find that the deposited C4b inhibits the exogenous cofactor activity of the red cell CR1 for the factor I-mediated cleavage of target-bound clustered C3b. The C4b bound to the human red cells is cleaved by the serum enzyme C3b/C4b inactivator (factor I) and a large fragment (C4c) is released in the incubation medium. The cleavage is totally inhibited by mAbs against CR1, showing that the complement receptor is an essential cofactor for the activity of I. When the number of bound C4b per red cell is relatively small (less than 1,000 molecules) the substrate for the enzymatic activity of factor I is mostly or exclusively the C4b bound endogenously to CR1. Indeed, the kinetics or the extent of cleavage of C4b are not affected by greatly augmenting the concentration of exogenous CR1 or of C4b-bearing red cells in the incubation mixture, thereby increasing the frequency of collisions between CR1 on the surface of one cell with C4b deposited on the membrane of a different cell. On the basis of the present and prior observations, we speculate that both DAF and CR1 act endogenously to inactivate the function of autologous red cell-bound C4b and prevent the progression of the cascade. DAF binding prevents the formation of the C3 convertase, C4b2a. The cleavage and irreversible inactivation of C4b only occurs after the concerted activities of endogenous CR1 and serum factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The results of immunoradiometric assays and of fluorescence-activated cell sorter analysis showed that DAF was present not only on red cells but was widely distributed on the surface membrane of platelets, neutrophils, monocytes, and B and T lymphocytes. By Western blotting, we observed small but consistent differences in the Mr of DAF from the membranes of various cell types. Quantitative studies showed that phagocytes and B lymphocytes, which presumably enter more frequently in contact with immune complexes and other potential activators of complement, had the highest DAF levels. As previously reported by others, the red cells from PNH patients were DAF deficient. When the patients' red cells were incubated in acidified serum (Ham test), only the DAF-deficient cells were lysed. In addition, we detected defects in DAF expression on platelets and all types of leukocytes. The observed patterns of DAF deficiency in these patients were consistent with the concept that the PNH cells were of monoclonal origin. In one patient, abnormal and normal cells were found only in the erythroid, myeloid, and megakaryocytic lineages. In two other patients, the lymphocytes were also DAF deficient, suggesting that a mutation occurred in a totipotent stem cell. It appears, therefore, that the lesion leading to PNH can occur at various stages in the differentiation of hematopoietic cells.

Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids.

Decay-accelerating factor (DAF) is a 70 kD membrane regulatory protein that prevents the activation of autologous complement on cell surfaces. Using immunohistochemical methods and a radioimmunometric assay based on mAbs to DAF, we found large amounts of membrane-associated DAF antigen on the epithelial surface of cornea, conjunctiva, oral and gastrointestinal mucosa, exocrine glands, renal tubules, ureter and bladder, cervical and uterine mucosa, and pleural, pericardial and synovial serosa. Additionally, we detected soluble DAF antigen in plasma, tears, saliva, and urine, as well as in synovial and cerebrospinal fluids. While plasma, tear, and saliva DAF are larger than erythrocyte (Ehu) membrane DAF by Western blot analysis, urine DAF is slightly smaller (67,000) in Mr. Unlike purified Ehu DAF, however, urine DAF is unable to incorporate into the membrane of red cells. Although its inhibitory activity on the complement enzyme C3-convertase is lower than that of Ehu DAF, it is comparable to that of serum C4 binding protein (C4bp). Biosynthetic studies using cultured foreskin epithelium and Hela cells disclosed DAF levels (approximately 2 X 10(5) molecules/cell) exceeding those on blood cells. In addition, these studies revealed the synthesis of two DAF species, one with apparent Mr corresponding to that of epithelial cell membrane DAF and the other to urine DAF, suggesting that the urine DAF variant arises from adjacent epithelium. The function of DAF in body fluids is unknown, but the observation that urine DAF has C4bp-(or factor H-)like activity shows that it could inhibit the fluid phase activation of the cascade.

Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N-acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N-acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl-phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.

The gene encoding decay-accelerating factor (DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.

Delay-accelerating factor (DAF) protects host cells from complement-mediated damage by regulating the activation of C3 convertases on host cell surfaces. Using a panel of hamster-human somatic cell hybrids, the DAF gene was mapped to human chromosome 1. In situ hybridization studies using human metaphase cells further localized the gene to bands 1q31-41, with the largest cluster of grains at 1q32. This establishes the close linkage of the DAF gene to genes for four other proteins (C3b/C4b receptor or complement receptor 1, C3d receptor or complement receptor 2, factor H, and C4-binding protein) that share 60-amino-acid homologous repeats as well as complement-regulatory or -receptor activity, thereby enlarging the complement-regulatory gene family on the long arm of human chromosome 1.

Decay accelerating factor is essential for successful corneal engraftment.

In contrast to immune restrictions that pertain for solid organ transplants, the tolerogenic milieu of the eye permits successful corneal transplantation without systemic immunosuppression, even across a fully MHC disparate barrier. Here we show that recipient and donor expression of decay accelerating factor (DAF or CD55), a cell surface C3/C5 convertase regulator recently shown to modulate T-cell responses, is essential to sustain successful corneal engraftment. Whereas wild-type (WT) corneas transplanted into multiple minor histocompatibility antigen (mH), or HY disparate WT recipients were accepted, DAF's absence on either the donor cornea or in the recipient bed induced rapid rejection. Donor or recipient DAF deficiency led to expansion of donor-reactive IFN-gamma producing CD4(+) and CD8(+) T cells, as well as inhibited antigen-induced IL-10 and TGF-beta, together demonstrating that DAF deficiency precludes immune tolerance. In addition to demonstrating a requisite role for DAF in conferring ocular immune privilege, these results raise the possibility that augmenting DAF levels on donor corneal endothelium and/or the recipient bed could have therapeutic value for transplants that clinically are at high risk for rejection.

Primed CD8(+) T-cell responses to allogeneic endothelial cells are controlled by local complement activation.

CD8 T cells primed by transplantation recognize allogeneic class I MHC molecules expressed on graft vascular endothelium and contribute to allograft injury. We previously showed that immune cell-derived complement activation fragments are integral to T cell activation/expansion. Herein we tested the impact of local complement production/activation on T cell/endothelial cell (EC) interactions. We found that proinflammatory cytokines upregulated alternative pathway complement production by ECs, yielding C5a. We further found that ECs deficient in the cell surface C3/C5 convertase regulator decay accelerating factor (DAF, CD55) induced greater CD8 T-cell proliferation and more IFNgamma(+) and perforin(+) effector cells than wild-type (WT) ECs. Allogeneic C3(-/-) EC induced little or no CD8 responses. Abrogation of responses following C5a receptor (C5aR) blockade, or augmentation following addition of recombinant C5a demonstrated that the effects were mediated through T-cell-expressed-C5aR interactions. Analyses of in vivo CD8 cell responses to transplanted heart grafts deficient in EC DAF showed similar augmentation. The findings reveal that EC-derived complement triggers secondary CD8 T-cell differentiation and expansion and argue that targeting complement and/or C5aR could limit T-cell-mediated graft injury.

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.

Activation of the alternative complement pathway by exposure of phosphatidylethanolamine and phosphatidylserine on erythrocytes from sickle cell disease patients.

Deoxygenation of erythrocytes from sickle cell anemia (SCA) patients alters membrane phospholipid distribution with increased exposure of phosphatidylethanolamine (PE) and phosphatidylserine (PS) on the outer leaflet. This study investigated whether altered membrane phospholipid exposure on sickle erythrocytes results in complement activation. In vitro deoxygenation of sickle but not normal erythrocytes resulted in complement activation measured by C3 binding. Additional evidence indicated that this activation was the result of the alterations in membrane phospholipids. First, complement was activated by normal erythrocytes after incubation with sodium tetrathionate, which produces similar phospholipid changes. Second, antibody was not required for complement activation by sickle or tetrathionate-treated erythrocytes. Third, the membrane regulatory proteins, decay-accelerating factor (CD55) and the C3b/C4b receptor (CD35), were normal on sickle and tetrathionate-treated erythrocytes. Finally, insertion of PE or PS into normal erythrocytes induced alternative pathway activation. SCA patients in crisis exhibited increased plasma factor Bb levels compared with baseline, and erythrocytes isolated from hospitalized SCA patients had increased levels of bound C3, indicating that alternative pathway activation occurs in vivo. Activation of complement may be a contributing factor in sickle crisis episodes, shortening the life span of erythrocytes and decreasing host defense against infections.

Synthesis of aberrant decay-accelerating factor proteins by affected paroxysmal nocturnal hemoglobinuria leukocytes.

Paroxysmal nocturnal hemoglobinuria (PNH) leukocytes fail to express decay-accelerating factor (DAF) but contain DAF mRNA transcripts resembling those in normal cells. To further investigate the nature of the DAF defect in affected cells, patients' polymorphonuclear and mononuclear leukocytes (PMN and MNC) were biosynthetically labeled and newly synthesized DAF proteins examined. Analyses of greater than 98% surface DAF-negative PMN and MNC from a patient with PNH III erythrocytes showed precursor DAF protein approximately 3 kD smaller in each cell type than in normal cells. The proportion of precursor to mature (O-glycosylated) DAF protein was increased and soluble DAF protein was detected in the medium. Studies of 70-80% surface DAF-negative PMN and MNC from four patients with type II erythrocytes showed mixtures of the 3 kD smaller and normal DAF precursors. Partitioning with Triton X-114 detergent and biosynthetic labeling with the anchor precursor [3H]ethanolamine indicated that the abnormal peptides lacked glycosyl-inositolphospholipid membrane-anchoring structures. Thus, in PNH cells nascent DAF polypeptides are synthesized. Some of the abnormal pro-DAF molecules are processed in the Golgi and some are released extracellularly.

Decay-accelerating factor protects human tumor cells from complement-mediated cytotoxicity in vitro.

The disialoganglioside GD2 is expressed on a wide spectrum of human tumor types, including neuroblastomas and melanomas. Upon binding of 3F8, a murine monoclonal antibody (MAb) specific for GD2, neuroblastomas and some melanomas are sensitive to killing by human complement, whereas some melanomas are not. To investigate the mechanism underlying these differences in complement mediated cytotoxicity, complement-insensitive melanoma cell lines were compared with respect to expression of the decay-accelerating factor (DAF), a membrane regulatory protein that protects blood cells from autologous complement attack. While DAF was undetectable among neuroblastomas, it was present in complement-insensitive melanomas. When the function of DAF was blocked by anti-DAF MAb, C3 uptake and complement-mediated lysis of the insensitive melanoma lines were markedly enhanced. F(ab')2 fragments were as effective in enhancing lysis as intact anti-DAF MAb. The DAF-negative and DAF-positive melanoma cell lines were comparably resistant to passive lysis by cobra venom factor-treated serum. The data suggest that in some tumors, DAF activity accounts for their resistance to complement-mediated killing. The ability to render these cells complement-sensitive by blocking DAF function may have implications for immunotherapy.

Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes exhibit abnormalities in decay accelerating factor (DAF), acetylcholinesterase, and resistance to autologous C5b-9 attack. To investigate the nature of the lesion underlying PNH cells, we examined the relationship of these abnormalities to one another. Analyses of DAF in acetylcholinesterase-negative erythrocytes revealed that these two abnormalities involve functionally independent molecules, coincide precisely in the same cell populations, and are similarly expressed in PNH II and more complement-sensitive PNH III erythrocytes. The DAF and acetylcholinesterase deficiencies contrast with the C3b/C4b receptor (CR1) deficit, which is less profound and similarly distributed in complement-insensitive cell populations. Hemolytic studies showed that defective resistance to autologous C5b-9 attack is mediated by another mechanism. Whereas reconstitution of PNH II erythrocytes with DAF completely corrected their complement sensitivity, DAF reconstitution of PNH III erythrocytes restored their ability to circumvent C3b uptake but had no effect on their heightened susceptibility to reactive lysis. Assays of complement-insensitive (PNH I) erythrocytes surviving after reactive lysis disclosed partial DAF and acetylcholinesterase deficits. These findings indicate that the PNH lesion involves multiple membrane components and that PNH I erythrocytes are also abnormal.

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Glycophosphatidylinositol-anchored protein deficiency as a marker of mutator phenotypes in cancer.

Phosphatidylinositol glycan-A (PIGA) is a gene that encodes an element required for the first step in glycosylphosphatidylinositol (GPI) anchor assembly. Because PIGA is X-located, a single mutation is sufficient to abolish cell surface GPI-anchored protein expression. In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator (Mut) phenotypes in cancer. We examined eight Mut colon cancer lines and four non-Mut colon cancers as controls. In every case, flow cytometric analyses of cells sorted for low fluorescence after staining for GPI-linked CD59 and CD55 revealed negative peaks in the Mut lines but not in the controls. Single cell cloning of purged and sorted GPI-anchor- HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations. Pretreatment of the Mut lines with anti-CD55 or anti-CD59 antibodies and complement or with the GPI-anchor-reactive bacterial toxin aerolysin enriched for the GPI-anchor- populations. Expansion of purged GPI-anchor+ cells in the Mut lines and analyses using aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10(-5) to 10(-4), values similar to the mutation frequencies of the hprt gene. This novel approach allows for the detection of as yet undescribed repair or replication defects and in addition to its considerably greater ease of use than existing techniques and in principle would not require the production of cell lines.

Coexistence of autologous antibodies and decay-accelerating factor, an inhibitor of complement, on human renal tumor cells.

Autologous immunoglobulin was detected on the cell surface of tumor cells freshly isolated from cancerous kidneys of patients with renal cell carcinoma by flow cytometry after staining with murine anti-human IgG monoclonal antibodies. Cells isolated in parallel from macroscopically normal regions of the tumorous kidneys were not specifically stained with the anti-human IgG reagents. In further studies, tumor cells were stained with an antibody to decay-accelerating factor (DAF), a known inhibitor of complement. Flow cytometry of these cells revealed that nearly all tumor cells expressed DAF, and that the intensity of staining with the anti-DAF monoclonal antibody correlated with the staining of cells with anti-IgG. The results suggest that tumor cells coated with autologous antibody may be resistant to complement-mediated cytotoxicity in vivo through the expression of high levels of DAF.

Cloning and characterization of the mouse PIG-A gene.

Currently there is no experimental animal model for studying paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic anemia linked to mutations of the PIG-A gene. In this study, we cloned and characterized the mouse PIG-A gene. Sequencing of mouse PIG-A cDNA showed that it encodes a 485 amino acid-long protein. Northern hybridizations identified a major mRNA transcript of 3.6 kb and PCR amplifications identified four smaller alternative splice products. Exon:intron junctional analyses of the mouse PIG-A genome showed 6 exons (1(> or = 60 bp), 2(780 bp), 3(133 bp) 4(133 bp), 5(207 bp), and 6(2276 bp)), the latter 5 of which encompass the coding region. Chromosomal mapping using C57BL/6J x M. Spretus backcross DNA localized the mouse PIG-A gene near the telomeric end of the mouse X chromosome. The isolation of the mouse PIG-A gene opens the possibility for the development of a mouse model of PNH.

Expression of the DAF (CD55) and CD59 antigens during normal hematopoietic cell differentiation.

Expression of decay-accelerating factor (DAF or CD55) and of CD59 during hematopoietic cell development in normal bone marrow and on peripheral blood leukocytes were characterized by three-color immunofluorescence experiments. With this technique cell subsets were identified by forward light scatter, orthogonal light scatter, and two cell-surface antigens. For each cell lineage, specific combinations of two monoclonal antibodies labeled with different fluorochromes were used. DAF or CD59 were then quantitated on the defined cell subsets from the fluorescence signal of the respective antibody conjugated with a third fluorochrome. Early uncommitted hematopoietic progenitor cells (CD34+, CD38-) all expressed both proteins homogeneously. Initial commitment to the erythroid (CD71+, CD45dim), myeloid (CD33+), or B lymphocyte (CD10+) lineages was not associated with changes in DAF or CD59 levels. With erythroid development, i.e., after loss of CD45 and decrease of CD71, expression of both proteins decreased. With myeloid maturation, expression of CD59 remained constant and expression of DAF varied. During neutrophil maturation, DAF decreased initially and then reemerged on maturing neutrophils concurrently with the appearance of CD16 (Fc gamma RIII), whereas during monocyte maturation, DAF increased concurrently with up-regulation of CD14. With B cell development, expression of DAF increased concurrently with down-regulation of CD10 and up-regulation of CD20, whereas expression of CD59 diminished slightly late in B cell maturation. Analysis of peripheral blood elements showed that monocytes, neutrophils, and B lymphocytes expressed both proteins homogeneously, but that in contrast to other cell subsets, which all expressed CD59, only a subset of (CD3+) T lymphocytes and (CD16+) Natural killer cells expressed DAF. The absence of DAF was not related to CD4 or CD8 expression or to the presence of activation markers (CD25+, CD38+), memory cell markers (CD58+, CD45RO+), or virgin T cell markers (CD45RA+), but was correlated with expression of CD11b (CR3) and CD11c (gp150/95). Although CD21+ (CR2) and CD35+ (CR1) cells all expressed DAF, CD11a (LFA-1) levels correlated inversely with those of DAF. Although the presence of CD55 and CD59 on early progenitor cells and throughout hematopoietic cell development is consistent with the requirements for both proteins in protection of host cells from complement-mediated injury, the physiological relevance of the unique patterns of variation for each cell lineage is unclear. Nevertheless, the availability of a detailed DAF and CD59 expression map in normal marrow will facilitate analyses of alterations during hematopoietic development that may occur in hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH).