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A Nicholson-Weller

Publications and source records attributed to A Nicholson-Weller.

At least 55 records · Page 3Linked to original sources
PubMed1989

Ca2+-activated K+ efflux limits complement-mediated lysis of human erythrocytes.

The lytic effect of complement on human erythrocytes has been reported by others to increase when Na+ is substituted for K+ in the external medium. In this paper we have investigated the hypothesis that net loss of K+ through a K+ transport pathway protects erythrocytes from complement-induced colloidosmotic swelling and lysis. Antibody-sensitized human erythrocytes containing different intracellular cation concentrations (nystatin treatment) were exposed to low concentrations of guinea pig serum in media of different cation composition; complement lysis was assessed by the release of hemoglobin and the volume of the surviving cells estimated by their density distribution profiles. Complement-dependent swelling and lysis of erythrocytes (a) were limited by the presence of an outwardly directed K+ electrochemical gradient and (b) were enhanced by carbocyanine, a specific inhibitor of the Ca2+-activated K+ transport pathway, and by absence of Ca2+ in the external medium. We propose that during complement activation a rising cytosolic calcium triggers the Ca2+-activated K+ permeability pathway, the Gardos effect, produces a net K+, Cl- and water loss, and thus limits the colloidosmotic swelling and lysis of erythrocytes.

Animals↗
PubMed1989

Monocyte antigen CD14 is a phospholipid anchored membrane protein.

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.

Amino Acid Sequence↗
PubMed1989

Cytoplasmic lipid bodies of human neutrophilic leukocytes.

The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids.

Arachidonic Acid↗
PubMed1988

Complement induces a transient increase in membrane permeability in unlysed erythrocytes.

The effects of low concentrations of human serum on antibody-sensitized sheep erythrocytes (EA) were studied. We report that exposure to low concentrations of serum induced a large but transient increase in the membrane permeability of those EA that do not lyse. This change in the permeability of the erythrocyte membrane resulted in net uptake of Na+ and decrease in cell K+, without affecting the total internal cation content. Although exposure to serum also allowed for net uptake of larger molecules like L-glucose, it did not lead to cell swelling. Experiments with sera genetically deficient in one of the terminal complement components showed that C8, but not C9, was required to produce the observed change in membrane permeability. Therefore, we propose that the C5b-8 complex can mediate the transient increase in permeability observed in unlysed erythrocytes during complement activation by whole serum.

Animals↗
PubMed1988

Release of C8 binding protein (C8bp) from the cell membrane by phosphatidylinositol-specific phospholipase C.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol-containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Bacterial Proteins↗
PubMed1987

Enhancement of complement-mediated lysis of dithiothreitol-treated erythrocytes involves increased C9 insertion and polymerization.

Treatment of human erythrocytes with dithiothreitol (DTT) increases the sensitivity of normal cells to complement (C)-mediated lysis. We have investigated the mechanism through which DTT increases cell susceptibility to complement by comparing the interactions of complement proteins with DTT-treated erythrocytes and with normal cells. In addition, we have studied the effect of DTT on the physical state of the erythrocyte membrane. Results indicated that the DTT primarily affects the interactions of the late components of complement with the cell membrane. In particular, the insertion efficiency of C9 and its ability to form tubular poly-C9 are enhanced on DTT-treated cells. Electron spin resonance (ESR) spectroscopic analyses of the treated and untreated membranes showed essentially no correlation between bulk membrane fluidity and the DTT-induced change in lytic susceptibility, suggesting no gross disruption of the membrane lipid structure by DTT. In view of the fact that DTT-treated erythrocytes have been proposed as a possible model for the abnormally complement-sensitive erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) which are deficient in a 75,000 mol. wt membrane protein called decay accelerating factor (DAF), we explored the possibility that DAF might be affected by DTT. Studies with anti-DAF F(ab')2 antibodies indicated that DAF activity is protected from DTT-treatment. These results are reinforced by the observation that DTT-treatment of DAF-deficient Type III PNH-E also led to enhanced lysis of PNH-E, implying that DTT affects membrane structures other than DAF. Thus, we conclude: (1) that DTT increases the lytic susceptibility of human erythrocytes to late components of human complement by modifying membrane structures to facilitate C9 insertion and polymerization, and (2) that DTT-treated erythrocytes are not a suitable model for PNH erythrocytes.

Biopolymers↗
PubMed1987

Decay accelerating factor (DAF) peptide sequences share homology with a consensus sequence found in the superfamily of structurally related complement proteins and other proteins including haptoglobin, factor XIII, beta 2-glycoprotein I, and the IL-2 receptor.

Amino acid sequence data derived from tryptic peptides of the decay accelerating factor indicate that this complement regulatory protein contains a sequence with homology to the superfamily of structurally related complement proteins, including the C4 binding protein, factor H, complement receptor type 1, complement receptor type 2, Ba, C1r, and to their non-complement relatives, including beta 2-glycoprotein I, factor XIIIb, the alpha 1 chain of haptoglobin, and the interleukin 2 receptor. Identifying DAF as a member of the superfamily of structurally related complement proteins provides evidence that DAF may contain a functionally important C4b and C3b binding domain.

Amino Acid Sequence↗
PubMed1986

Natural killer cells are deficient in the surface expression of the complement regulatory protein, decay accelerating factor (DAF).

Decay-accelerating factor (DAF) is a 75,000 m.w. membrane protein that inhibits autologous complement C3 activation at the cell surface. One-color direct immunofluorescence with anti-DAF antibody and cytofluorographic analysis indicates that normal human monocytes and granulocytes are uniform in expression of DAF, whereas 23% of peripheral blood lymphocytes are DAF deficient. A two-color indirect immunofluorescence method, used to define the phenotype(s) of the DAF-deficient lymphocytes, was less efficient in DAF detection and led to overestimation of the fraction of deficient cells. Nonetheless, the difference between DAF expression by natural killer cells, identified by the CD16 and HNK-1 antigens, was marked. DAF deficiency was intermediate in cells expressing the CD2, CD3, CD4, or CD8 markers. On the basis of the phenotypic definition of natural killer cells and their contribution to the lymphocyte population, it is concluded that a uniform deficiency of DAF on natural killer cells accounts for about one-half of the DAF-deficient lymphocytes in peripheral blood of normal donors. The finding of a complete DAF deficiency in the lymphocytes from a patient with a lymphoproliferative disorder with the predominant proliferation of CD2+, CD3+, CD8+, HNK-1+ large granular lymphocytes gives additional support for the association of DAF-deficiency with natural killer cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.

Animals↗
PubMed1985

Deficiency of the complement regulatory protein, "decay-accelerating factor," on membranes of granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria are deficient in decay-accelerating factor, a membrane protein that inhibits the complement C3 convertases. We studied the expression of this protein on leukocytes and platelets from four patients with paroxysmal nocturnal hemoglobinuria, using cytofluorographic analysis and antibody to decay-accelerating factor. The granulocytes and monocytes had a bimodal distribution of fluorescence, indicating antigen-deficient and antigen-positive subpopulations of cells. In contrast, granulocytes and monocytes from normal donors and patients with other diseases had no antigen-deficient cells. Platelets from the four patients with paroxysmal nocturnal hemoglobinuria had less fluorescence than normal platelets. Furthermore, surface-radiolabeled granulocytes and platelets from one of the four patients, which were maximally deficient in decay-accelerating factor, also lacked antigen that was immunoprecipitable by specific antibody to this protein. Thus, paroxysmal nocturnal hemoglobinuria is a clonal disorder characterized by deficient membrane expression of decay-accelerating factor on granulocytes, monocytes, and platelets, as well as on erythrocytes.

Adult↗
PubMed1985

Enhanced complement-mediated lysis of type III paroxysmal nocturnal hemoglobinuria erythrocytes involves increased C9 binding and polymerization.

The interaction of terminal complement proteins (C5-C9) with normal erythrocytes and type III paroxysmal nocturnal hemoglobinuria erythrocytes (PNH-E) has been compared in terms of binding of the C5-9 complex, C9 polymerization, and C9 insertion into membranes. Complement components C5, C7, and C8 bind equally well to both types of erythrocytes, whereas the binding of C9 to PNH-E is 5-6 times greater than that to normal erythrocytes. The kinetics of C9 binding was compared with the kinetics of lysis for both types of cells under conditions leading to 100% lysis. There was a noticeable lag time between C9 binding and lysis of normal erythrocytes, but the lysis of PNH-E proceeded without a lag and the kinetics of lysis more closely paralleled C9 binding. The efficiency of C9 insertion was similar for both types of cells, but C9 polymerization was significantly enhanced on PNH-E. These data indicate that the enhanced susceptibility of type III PNH-E toward lysis by C5-9 can be correlated with abnormally high C9 binding and increased formation of poly(C9).

Carrier Proteins↗
PubMed1985

Normal function of CR1 on affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which affected erythrocytes (E) are abnormally sensitive to lysis by autologous complement. Affected E from patients with PNH (PNH-E) are deficient in an E membrane regulatory protein of complement, decay-accelerating factor (DAF). Because a functional defect in a second membrane regulatory protein of complement, CR1 (C3b receptor), has also been hypothesized, severely affected PNH-E (type III PNH-E) were tested for abnormalities in CR1 by four methods. E from two patients with 100% type III PNH-E had 3201 and 6783 sites per cell for binding of 125I-labeled rabbit polyclonal F(ab')2 anti-CR1. These values fall within the normal range of CR1 antigenic sites per cell (1267 to 7915, mean = 5,014 +/- 155 SEM) established by assaying the E from 113 healthy donors. The Ka of CR1 on type III PNH-E for 125I-labeled C3b dimer was 2.06 X 10(7) M-1, and the Ka values for the binding of the same ligand to the E from two healthy individuals were 2.45 X 10(7) M-1 and 1.58 X 10(7) M-1. In an assay designed to measure the capacity of human E (Eh) to accelerate the decay of the classical C3 convertase deposited on 1 X 10(7) bystander sheep E (EAC1gp,4bh,2agp), the half-life (t 1/2) of this convertase was diminished from 18.1 min (range 15.2 to 22.9) to 8.1 min (range 7.4 to 8.5) by the addition of 1 X 10(7) normal Eh, to 6.2 min by 100% type III PNH-E, and to 7.5 min by Eh pretreated with an IgG fraction of human antiserum directed against the D antigen of the Rh system. In contrast, Eh (t 1/2 = 7.4) pretreated with a saturating dose of F(ab')2 anti-CR1, and CR1-deficient Eh (less than 10 CR1 molecules/E) from a patient with systemic lupus erythematosus, showed a loss of convertase decay-accelerating capacity to t 1/2 = 11.6 and t 1/2 = 12.4, respectively. Type III PNH-E pretreated with anti-CR1 demonstrated a total loss of their decay-accelerating capacity (t 1/2 = 19.9). In an assay of I cofactor activity, soluble C3b was rapidly converted to iC3b by purified I plus Eh or type III PNH-E, whereas CR1-deficient Eh exhibited less than 5% the I cofactor activity of normal Eh.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-Antibody Complex↗
PubMed1985

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.

Blood Platelets↗
PubMed1984

Potentiation of virulence by group B streptococcal polysaccharides.

The ability of group B streptococcal (GBS) capsular polysaccharides to potentiate virulence was examined. Incubation of soluble type Ia or III polysaccharide in hypogammaglobulinemic human serum inhibited classical complement-dependent opsonophagocytic killing of type Ia strain 515. When functional complement components were measured, C1 activity increased in sera with added polysaccharide whereas C4 activity decreased 80%-90%. Incubation of purified C1 with type Ia polysaccharide inhibited lysis of EAC4 cells in a C1 transfer assay. In a mouse lethality model, tail-vein injection of 50 micrograms of type Ia or III polysaccharide decreased the 50% lethal dose (LD50) from 3.2 X 10(6) to 2.2 X 10(5). Total hemolytic complement levels in mice immediately after polysaccharide injection were increased over levels in control mice, but this difference was not seen 30 min later. The LD50 in mice depleted of C3 (with cobra venom factor) was 6 X 10(5) and simultaneous injection of polysaccharide did not further lower the LD50.

Animals↗
PubMed1983

Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired defect of bone marrow stem cells in which the affected clones produce erythrocytes (also granulocytes and platelets) with membranes that are abnormally sensitive to complement-mediated lysis. Abnormal erythrocytes (E) from patients with PNH (PNH-E) are 3-5 times more sensitive (type II PNH-E) or 15-25 times more sensitive (type III PNH-E) to lysis in vitro by human complement than normal E from unaffected individuals and the functionally normal E that arise from unaffected clones and the functionally normal E that arise from unaffected clones in PNH patients (type I PNH-E). After complement activation by either the classical or alternative pathway, abnormal amounts of C3b are deposited on the membranes of PNH-E compared with normal E, suggesting that the PNH-E membrane cannot regulate the events responsible for C3b deposition. Two proteins that decrease the stability of the classical and alternative pathway C3 convertases on target cells have been isolated from normal human E stroma: the 70,000 Mr decay accelerating factor of stroma (DAF) and the 250,000 Mr C3b receptor (C3bR). Specific immune precipitates of solubilized membranes from 125I-surface-labeled normal E demonstrate both proteins. In contrast, specific immune precipitates of PNH-E from three patients show C3bR but are deficient in DAF; type II PNH-E are relatively deficient and type III PNH-E are totally deficient in DAF. Antibody that neutralizes the activity of isolated DAF is adsorbed by intact normal E under conditions in which it is weakly adsorbed by type II PNH-E and not adsorbed by type III PNH-E. The deficiency of DAF antigen in PNH-E, as assessed by lack of immunoprecipitation and antibody adsorption, could explain the abnormal sensitivity of PNH-E to complement-mediated lysis and suggests that DAF may protect the membranes of normal E from damage resulting from autologous complement activation.

Antigen-Antibody Complex↗
PubMed1982

Antibody-independent activation of C1 by type Ia group B streptococci.

A prototype fresh clinical isolate of type Ia group B streptococci (strain 515) can be opsonized by serum containing low levels of antibody. This opsonizing process can also occur in hypogammaglobulinemic serum, confirming its antibody independence, but it does require Ca++ ions and the second component of complement (C2). When formalin-fixed organisms are reacted with whole serum, C4 and C3 are cleaved. These data are strong evidence that this organism can directly activate C1 in the absence of antibody and that this activation leads to the formation of a classical C3 convertase (C4b2a), which cleaves C3. The observation that opsonization can occur in hypogammaglobulinemic serum suggests that complement alone, presumably the fixation of C3b, is sufficient for phagocytes to ingest this pathogen.

Complement Activation↗