Correlation of C3b-receptor activity and diphenylhexatriene polarization in a murine macrophage cell line.
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Isolation of an iC3b forming enzyme from peritoneal polymorphonuclear leukocytes of guinea pigs.
We have investigated fluid phase cleavage of C3b by peritoneal polymorphonuclear leukocytes of guinea pigs and found that polymorphonuclear leukocytes expressed an iC3b forming enzyme as well as C3b receptor with maturation in peritoneal cavity. The iC3b forming enzyme was found to be distinct from C3bINA, a physiological iC3b forming enzyme in plasma, since the activity was inhibited by monoiodoacetic acid and did not require a cofactor plasma protein, beta 1H, for the cleavage of C3b into iC3b. The iC3b forming enzyme is gradually released upon incubation of PMN at 37 degrees C. The molecular weight of the iC3b forming enzyme was estimated to be 48,000 from gel filtration on Sephadex G-200.
Animals↗
Stimulation and priming of human neutrophils by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor: qualitative and quantitative differences.
Granulocyte colony-stimulating factor(G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increased neutrophil C3bi-receptor expression and adherence and rapidly (less than 10 min) primed neutrophils to enhanced O2- release and membrane depolarization stimulated by chemotactic peptide. Direct triggering of O2- release in suspended neutrophils was also provoked by GM-CSF but not by G-CSF. GM-CSF-induced O2- release was inhibited by cyclic AMP agonists and cytochalasin B. The biological activity was greater in non-glycosylated GM-CSF than in glycosylated GM-CSF, whereas it was identical in glycosylated and non-glycosylated G-CSFs. Direct stimulation and priming by GM-CSF were consistently greater than those by G-CSF and the combined addition of the optimal concentrations of G-CSF and GM-CSF resulted in the effects of GM-CSF alone. These findings indicate that the effects of G-CSF and GM-CSF on neutrophil functions are qualitatively and quantitatively different from each other.
Alprostadil↗
Biochemical basis of HLA-DR and CR3 modulation on human peripheral blood monocytes by lipopolysaccharide.
The biochemical events leading to enhanced membrane expression of HLA-DR and CR3 by human peripheral blood monocytes (MO) following exposure to bacterial lipopolysaccharide (LPS) were examined. In a previous study we demonstrated that an increase in intracellular calcium was necessary, but not sufficient, for MO to increase membrane expression of both antigens within 1 hr of addition of LPS. The present study was initiated to examine the other biochemical requirements which lead to the MO response to LPS. Enhanced expression of both antigens following addition of LPS was dependent on microfilament function, but independent of microtubule function and of protein synthesis. Inhibition of formation of cyclooxygenase or lipoxygenase metabolites of arachidonic acid had no effect on HLA-DR or CR3 modulation by LPS. A role for phosphatidylinositol metabolism was suggested by the inhibition of the MO response to LPS by dibutyryl cAMP and theophylline and by the enhanced expression of both antigens following addition of phorbol diesters. However, H-7, a putative inhibitor of protein kinase C, did not alter the MO response to LPS or phorbol diesters. These results suggest that LPS enhances expression of HLA-DR and CR3 by inducing redistribution of these antigens from an intracellular pool. The data also support a role for the generation of hydrolysis products of phosphatidylinositol, leading to calcium redistribution and activation of protein kinase C or other kinases, in the MO response to LPS.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Enhancement of the function of neutrophil type-1 and type-3 complement receptors by human leukocyte inhibitory factor (LIF).
The lymphokine leukocyte inhibitory factor (LIF) has previously been documented to enhance several neutrophil (PMN) functions, including stimulated chemotaxis and superoxide generation, phagocytosis and adherence of opsonized targets, and antibody-dependent cellular cytotoxicity. The present studies were designed to investigate the effects of LIF on PMN function mediated by the complement components C3b and C3bi. LIF induced a dose-dependent increase in superoxide production generated by opsonized zymosan (up to 97.1 +/- 31.4% at 16 U LIF/ml; P less than 0.01). While neither control nor LIF-treated PMN were capable of inducing phagocytosis of either C3b- or C3bi-opsonized sheep erythrocytes (E) directly, exposure to LIF caused a significant (P less than 0.05) increase in their adherence to E (137.4 and 59.4%, respectively). Specificity for complement receptor function was confirmed by the ability of anti-CR1 antibody to block adherence of LIF-treated PMN to EAC3b (77.0% inhibition) and anti-CR3 antibody to block adherence to EAC3bi (70.2% inhibition). Increased C3b and C3bi function may have been due, at least in part, to increased expression of their respective surface membrane receptors. Thus, using indirect immunofluorescence, LIF induced a 38.2% increase in fluorescence of the anti-CR1 antibody and a 96.1% increase in anti-CR3 binding. These studies describe an additional mechanism through which LIF may have an important pro-inflammatory role in vivo.
Antibodies, Monoclonal↗
Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments.
Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.
Burkitt Lymphoma↗
Solubilization of immune precipitates by complement in the absence of properdin or factor D.
Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.
Antibodies↗
Expression of complement-regulatory proteins in normal and UW-preserved human liver.
BACKGROUND/AIMS: Somatic cells are protected against complement-mediated injury by specialized membrane proteins, known as complement-regulatory proteins (CRP). The knowledge of the pattern of CRP expression in the liver is important to evaluate the role of complement-mediated injury in graft rejection. METHODS: We determined the distribution of four main CRP: membrane cofactor protein (MCP), decay accelerating factor (DAF), protectin, and complement receptor 1 (CR1) in 30 histologically normal livers, 13 samples from University of Wisconsin cold-storage solution (UW)-preserved tissue and 17 postoperative biopsies of UW-preserved allografts. RESULTS: In normal liver, hepatocytes expressed only MCP. Bile duct cells were reactive for MCP and protectin. Sinusoidal endothelial cells expressed MCP and protectin but displayed no or faint expression of DAF. Endothelial cells of portal vessels and centrilobular veins expressed high levels of DAF, MCP, and protectin. No expression of CR1 was observed. No change in CRP expression was usually detected after UW preservation, except for protectin, induced on hepatocytes in 9 samples of UW-preserved liver tissue and in 9 allografts. CONCLUSIONS: Hepatocytes and sinusoidal endothelial cells, which have a defective expression of CRP, might be at risk for complement-mediated injury. However, this risk is not aggravated after UW preservation.
Adenosine↗
A microtiter plate enzyme-linked immunosorbent assay for measuring C3b receptors on human erythrocytes.
An enzyme-linked immunosorbent assay was developed for quantitation of complement C3b receptors (C3bR) on human erythrocytes fixed in monolayer to microtiter plates. The disadvantages of macro test tube systems (large consumption of sample material and reagents, tedious washing procedures, cell loss and hemolysis) were avoided, and the fixed cells could be stored. In return a modest reduction in antigenicity induced by glutaraldehyde was inevitable. The calibration curve expressed a detection limit of about 10% and discrimination between levels of C3bR in the interval of about 10-120% of a standard erythrocyte with a high uptake of anti-C3b-receptor antibodies. The determinations of C3bR in normals as well as in patients varied within these limits, and showed a preponderance of low levels in the patients. The between-day coefficient of variation was 10.3%; the within-assay interplate and intraplate determinations respectively gave coefficients of variation of 9.7% and 6.2%. The method is suitable for further development and use for investigation of erythrocyte-bound immune complexes.
Antibodies, Monoclonal↗
Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography.
The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.
Amino Acid Sequence↗
Studies on the binding of C3b-coated microspheres to human neutrophils.
A method is described for the quantitation of C3b receptors on human neutrophils using a mixture of C3b-coated fluorescent and C3b-coated non-fluorescent microspheres. The method measures the "sterically available' C3b receptors on the cells, for example, the receptors available to opsonized bacteria. The use of mixtures of fluorescent and non-fluorescent microspheres resulted in lowered fluorescence intensities of the microsphere-coated neutrophils that were well within the fluorescence limitations of fluorescence activated cell analyzers or sorters used in the assay procedure. These mixtures also allowed the distribution of the C3b-coated microspheres around the neutrophils to be easily visualized in the fluorescence microscope. The binding of the C3b-coated microspheres to the neutrophils was shown to be receptor mediated by typical saturable binding kinetics, by complete inhibition by fluid phase C3b, but not by other proteins and by nearly complete inhibition by anti-C3b receptor antibody. Several parameters that could affect the binding of C3b-coated microspheres to neutrophils were studied; these included time and temperature of incubation of the microspheres with the cells, the diameter of the microspheres, the C3b content of the C3b-coated microspheres, the presence of metal ions, azide, EDTA, protein (BSA, IgG), soybean trypsin inhibitor in the buffers, and the method of isolation of the neutrophils. The C3b-coated microspheres were evenly distributed around the neutrophils in almost all of the cases; however, the neutrophils used in these studies were not activated and were not phagocytosing. The method is extremely reproducible and sensitive in detecting small changes in number of C3b receptors on cells.
Complement C3b↗
Enumeration of CR1 complement receptors on erythrocytes using a new method for detecting low density cell surface antigens by flow cytometry.
A sensitive enhancing system was developed for detecting low density cell surface antigen by flow cytometry. The system termed the 'super avidin-biotin system' (SABS) uses biotinylated antibody, phycoerythrin-streptavidin (StreptA-PE), biotinylated goat anti-streptavidin antibody, and StreptA-PE. CR1 complement receptor antigenic sites were quantified on erythrocytes from healthy individuals and patients with antibodies against human immunodeficiency virus (HIV) using SABS and a conventional radioimmunoassay (RIA) with monoclonal anti-CR1 antibody. As little as 50 sites/cell were detected using either SABS or RIA. Accurate quantification of CR1 antigenic sites was achieved within the range of 100-1300 sites/cell. Similar results were obtained using either of the two methods. Intra-assay and day-to-day reproducibilities using SABS were 2% and 12% respectively, comparable to those of conventional RIA measurements. In addition to enumeration of CR1 on erythrocytes for clinical purposes, the use of SABS may probably be extended to a wide number of situations where a sensitive detection of low density cell surface antigen is needed.
Acquired Immunodeficiency Syndrome↗
Receptors for C3b on the neutrophil surface associate with the cytoskeleton-containing cell residue subsequent to cell lysis with non-ionic detergent. Cytoskeletal-CR1 interactions.
Radioiodinated Fab' and F(ab')2 fragments were prepared from 57F monoclonal antibody which is specific for the C3b receptor (CR1) on human cells. We find that both Fab' and F(ab')2 fragments bind to CR1 on human neutrophils and then dissociate, at 0 degree C, with half-lives of 172 and 35 min, respectively. In addition to binding to cell surface CR1, both antibody fragments bind specifically to the isolated non-ionic detergent-insoluble cellular residue (NDIR) which remains after cell lysis and solubilization in Triton X-100. We also find that Fab' and F(ab')2 antibody fragments remain associated with the NDIR after the radiolabeled antibody fragments are allowed to bind to cell surface CR1, the unbound fragments removed, and the cells subsequently lysed in non-ionic detergent. Because the cytoskeletal matrix (together with the cell nucleus) makes up a substantial portion of the NDIR, these results suggest that some fraction of cell surface CR1 may be associated in vivo with the cytoskeletal matrix. However, post-lysis association of cell surface-CR1-antibody fragment complexes to the NDIR occurs. Examination of the binding kinetics of this interaction reveals that less than 10% of cell surface CR1 exists bound to the NDIR prior to cell lysis. While the nature of post-cell lysis association of CR1 with the NDIR is unknown, several modulating effects are noted. For example, binding of bivalent cross-linking agents to CR1 on cells followed by a 37 degrees C incubation is known to induce internalization of cell surface CR1. We find that binding of F(ab')2 antibody fragments under these conditions causes a 50% decrease in association of this 57F fragment to the NDIR. Pretreatment of the cells at 37 degrees C with the chemotactic peptide N-formyl-methionyl-leucylphenylalanine similarly caused a 50% decrease of (Fab'-labeled) CR1 association with the NDIR cell fraction. These results support the hypothesis that chemotactic peptide serves to enhance CR1-mediated adherence to C3b-bearing targets by increasing the density of CR1 on the cell surface rather than by inducing cytoskeletal-dependent, detergent-stable, CR1 redistribution on the cell surface.
Antibodies, Monoclonal↗
Demonstration of human erythrocyte C3b receptors (CR1) by haemadsorption and indirect haemagglutination techniques.
A closed chamber haemadsorption (HA) technique with tissue sections and an indirect haemagglutination (IHA) technique with monoclonal anti-C3b receptor (CR1) antibody were used to measure CR1 on human erythrocytes (E) (ECR1). In the HA technique the indicator cells were sheep E coated with rabbit IgM antibodies (A) and various amounts of human C3b (EAC). The ECR1 activity was classified according to the degree of haemadsorption of EAC. High ECR1 activity was found in 24% of healthy blood donors, intermediate activity in 62%, low activity in 12% and no activity in 2% of the blood donors. The results obtained using the IHA technique showed a good correlation with the results obtained using the HA technique. Both techniques are sensitive and the results are highly reproducible. The techniques are easy to perform and do not require expensive reagents or equipment. The HA and IHA techniques therefore offer an alternative to other techniques for measuring ECR1 activity.
Adolescent↗
Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion.
A highly reproducible two-color fluorescence cytometric method is described for determining the amount of receptor-mediated and of non-specific phagocytosis by human neutrophils of polystyrene microspheres that have been covalently coated with C3b, iC3b, IgG, mixtures of these, BSA or human F(ab')2. The method includes a correction for externally bound particles and thus measures net phagocytosis. The method involves the incubation of neutrophils with coated green fluorescent microspheres in buffer alone or with cytochalasin D to inhibit phagocytosis followed by staining the externally bound microspheres with red fluorescent antibodies to the immobilized ligand, and determining the green and red fluorescence in a dual laser fluorescence activated flow cytometer. The red to green fluorescence ratio of the mixtures containing cytochalasin D allows for a correction of the green fluorescence due to externally bound microspheres in the mixtures not containing cytochalasin D. The corrected green intensities thus represent net phagocytosis. The specificity of receptor-mediated phagocytosis was confirmed by inhibition with fluid-phase C3b or iC3b or monoclonal antibodies to the receptors. The method can be applied to the determination of both adherent and suspension phagocytosis and can be used as a general model of the phagocytosis of bacteria by neutrophils.
Buffers↗
Human non-transformed monocyte-derived macrophage cell lines.
We have demonstrated that human blood monocyte-derived macrophages can be passaged from primary cultures and replicate. Passaged cells have typical macrophage characteristics: they are non-specific esterase positive, phagocytic, and respond to 3 days of treatment with interferon-gamma with enhanced production of superoxide on stimulation with PMA. The passaged cells express Fc, CR1, CR3 and FMLP receptors. Both primary and passaged cultures constitutively produce CSF-1 after 3 weeks in culture. Cultures studied between 7 and 16 weeks in culture produce 3712 +/- 478 U of CSF-1 per 10(6) cells. Randomly selected lines were examined to look for cell proliferation by looking at numbers of cells over time and by labelling cells with tritiated thymidine to determine the number of cells synthesizing DNA. In addition, the cells can be frozen at the time of isolation and stored for at least 1 year, and then thawed and shown to retain functional activity. Human monocyte-derived macrophages can be cultured as finite cell lines.
Carboxylesterase↗
Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion.
The density of CR1 (the C3b receptor, CD35) on erythrocytes from normal individuals is determined by a codominant bi-allelic system associated with a single base mutation within an intron of the CR1 structural gene, leading to an additional polymorphic HindIII endonuclease site. The CR1 genotype is determined by HindIII digestion of genomic DNA and Southern blotting. We have developed a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of interest followed by HindIII endonuclease digestion and agarose gel electrophoresis which permits a more rapid and reliable determination of the CR1 genotype. The method is suitable for large scale clinical studies in diseases with altered expression of CR1 on erythrocytes.
Base Sequence↗
A flow cytometric assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes.
A flow cytometric assay (FCA) was developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It was possible to measure these parameters accurately with intra- and interassay coefficients of variation of 2.0% and 6.5% respectively. The method was able to discriminate between low and high levels of erythrocyte CR1, C3d and C4d. Comparison with a previously described RIA method gave excellent correlation coefficients with r2 values of 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay was used to measure CR1, C3d and C4d on the erythrocytes of 98 healthy individuals and the 95% upper limits for C3d and C4d were established. There was a wide distribution of CR1 levels amongst these individuals but their C3d and C4d levels were low and often not above background. The possible application of this method in clinical medicine is discussed.
Antigens, CD↗