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Biomedical subjects

D H Conrad

Publications and source records attributed to D H Conrad.

At least 91 records · Page 5Linked to original sources
PubMed1984

IgE-mediated triggering signals for mediator release from human mast cells and basophils.

Human basophils were developed in suspension culture of mononuclear cells of cord blood in the presence of conditioned medium of phytohemagglutinin-stimulated human T cells, from which interleukin 2 had been eliminated. Approximately 50-90% of cells recovered after 2-4 wk of culture were basophil granulocytes, which bear receptors with high affinity for human IgE. Sensitization of the cells with human IgE followed by challenge with anti-IgE resulted in the release of histamine and arachidonic acid (AA). In both the cultured basophils and human lung mast cells, bridging of cell-bound IgE with anti-IgE induced a transient increase in phospholipid methylation and intracellular cyclic AMP (cAMP), and these processes were followed by Ca2+ uptake and release of both histamine and AA. As demonstrated in rat mast cells, evidence was obtained that the activation of a proteolytic enzyme and phospholipid methylation induced by the bridging of IgE receptors are involved in the subsequent increase in AMP, and are an essential step for transduction of triggering signals for mediator release.

Basophils↗
PubMed1984

The murine lymphocyte receptor for IgE. I. Isolation and characterization of the murine B cell Fc epsilon receptor and comparison with Fc epsilon receptors from rat and human.

The Fc receptor for IgE (Fc epsilon R) on murine B lymphocytes was studied by using BALB/c mice infected 12 to 18 days previously with Nippostrongylus brasiliensis. B cells were enriched in the Sephadex G-10-passed lymphocytes by treating with anti-Thy-1.2 and complement (C). After stripping any cytophilic Ig with low pH, the B cells were 125I surface labeled; subsequently the membranes were solubilized with nonionic detergent, and putative Fc epsilon R components were allowed to bind to IgE-coated adsorbents. Bound radiolabel was eluted with low pH, and when examined by SDS-PAGE, was found to consist primarily of a relatively broad band centered at 49,000 m.w. (49K). Fluid-phase IgE could prevent the binding of the 49K component to the IgE solid-phase adsorbents. Rebinding studies further indicated that the 49K component exhibited a specificity for IgE, thus confirming that the 49K component was the murine B lymphocyte Fc receptor for IgE (Fc epsilon R). Some rebinding to rabbit IgG was observed, and by using 2.4G2, the monoclonal anti-Fc gamma 2b receptor (Fc gamma 2bR) antibody to isolate the IgG2b receptor, a clear distinction between the FC gamma 2bR and the 49K IgE receptor was demonstrated by SDS-PAGE analysis. Rabbit IgG was thus found to interact with both the 49K Fc epsilon R and the 59K FC gamma 2bR. The murine B lymphocyte Fc epsilon R was compared with the human B cell Fc epsilon R from the RPMI 8866 cell line and with the high affinity Fc epsilon R on rat basophilic leukemia cells by one- and two-dimensional gel analyses. The lymphocyte Fc epsilon R from mouse and human was found to be quite similar with respect to m.w. (45 to 50K) and isoelectric point (pI 4.5 to 5.0), whereas the basophil Fc epsilon R differed in both aspects.

Animals↗
PubMed1983

Use of monoclonal anti-rat IgE in a radioimmunoassay for antigen specific rat IgE.

A monoclonal mouse anti-rat IgE, ELIVB5 (B5) was used in a solid-phase radioimmunoassay (RIA) to detect antigen specific IgE in antisera from rats infected with a larval nematode. Binding of 125I-labeled B5 was specific for rat IgE and did not crossreact with human IgE. The RIA was used to demonstrate crossreactivity between antigens of closely related larval nematodes.

Animals↗
PubMed1983

Properties of two monoclonal antibodies directed against the Fc and Fab' regions of rat IgE.

Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.

Animals↗
PubMed1983

The interaction of human and rodent IgE with the human basophil IgE receptor.

The cross-reactivity of the human IgE receptor with mouse and rat IgE was studied. Using leukocytes from a patient with chronic myelogenous leukemia, in which the mononuclear fraction contained up to 75% basophils, both rat and mouse IgE were found to inhibit the binding of 125I-human IgE to the human basophilic leukemia (HBL cells). About 15-fold more rodent IgE was required for 50% inhibition of binding than unlabeled human IgE (hIgE). Dose-response studies using increasing amounts of rodent vs human 125I-IgE indicated that the HBL cells had about 8000 receptors per cell for hIgE and 5500 receptors per cell for rodent IgE. When the HBL cells were surface labeled with 125I and subsequently solubilized with non-ionic detergent, the labeled hIgE receptor could be isolated by either affinity chromatography on IgE-Sepharose (either human or rodent) or by immunoprecipitation with hIgE and anti-IgE. By SDS-PAGE on 10% gels, the receptor had a m.w. of 58,000 daltons. The solubilized receptors exhibited some rebinding to hIgE-Sepharose, and this rebinding could be inhibited by either human or rodent IgE but not by human IgG. Both the HBL cells and normal human basophils could be passively sensitized with murine IgE anti-DNP for antigen-induced histamine release. The minimum concentration of the mouse IgE antibody for sensitizing normal basophils was 20 to 200 ng/ml. Pretreatment of basophils with human IgE, but not human IgG, abrogated the capacity of the murine IgE antibody to sensitize the cells for histamine release, which indicated that the human and rodent IgE were interacting with the same receptor.

Animals↗
PubMed1983

Biochemical analysis of initial triggering events of IgE-mediated histamine release from human lung mast cells.

Mast cells were isolated from human lung tissues by counter current centrifugation elutriation, followed by flotation through Percoll gradients. Purified human mast cells released histamine upon challenge with anti-IgE. An optimal concentration of anti-IgE for maximum histamine release from human lung mast cells was comparable to that required for histamine release from normal human basophil granulocytes. Human lung mast cells could be passively sensitized with mouse monoclonal IgE antibody for antigen-induced histamine release. Bridging of cell-bound IgE molecules on human mast cells by anti-IgE or its F(ab')2 fragments induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of [3H]methyl groups into phospholipid reached a maximum at 30 sec after challenge with anti-IgE, whereas intracellular cAMP reached a maximum at 1 min. Both values declined to base line levels within 2 to 3 min. These biochemical events were followed by Ca2+ influx and histamine release. Ca2+ uptake and histamine release reached maximum at 2 to 3 min and 5 to 8 min, respectively. Neither phospholipid methylation nor initial rise in cAMP was inhibited by indomethacin, which indicates that these biochemical events are not the result of prostaglandin synthesis. However, inhibition of phospholipid methylation by inhibitors of S-adenosyl-L-methionine-mediated methylation, such as 3-deazaadenosine and S-isobutyryl 3-deazaadenosine, inhibited not only phospholipid methylation but also cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that phospholipid methylation induced by bridging of IgE receptors on human mast cells is essential for Ca2+ influx and histamine release.

Animals↗
PubMed1982

Antisera with anti-pan C3 receptor reactivity which block complement C3b, C3bi and C3d receptors.

Antisera directed at human C3 receptors were prepared by 2 different methods. In method I rabbits were immunized with EAC coated with C3 receptors of tonsil cells (AC3RS type I). In method II rabbits were immunized with insoluble aggregates of tonsil membrane constituents containing significant amounts of C3 receptors. After appropriate absorptions both types of AC3RS showed a selective reactivity with those cells known to express C3 receptors. With the EAC rosette inhibition test it is demonstrated that both types of AC3RS contain antibodies against the binding sites of C3b, C3bi and C3d receptors. The antibody titer was consistently higher in AC3RS of type II.

Animals↗
PubMed1982

The cross-reactivity of rat IgE and IgG with solubilized receptors of rat basophilic leukemia cells.

Normal rat IgG-Sepharose binds the same two receptors from solubilized rat basophilic leukemia (RBL) cells as are bound by rat IgE-Sepharose. These receptors have previously been designated R and H, and have apparent mol. wts of 45,000 and 55,000, respectively. Inhibition studies indicate that, although soluble deaggregated IgG is capable of interacting with both receptors, its affinity is higher for H than for R. Conversely, soluble IgE preferentially interacts with R, but its affinity for H is somewhat higher than that of rat IgG. Control studies show that IgG receptor interaction to be specific in that it is significantly stronger than that obtained with F(ab')2 fragments or a variety of proteins having pIs approximating that of IgG.

Animals↗
PubMed1982

Phagocytosis by rat peritoneal mast cells: independence of IgG Fc-mediated and C3-mediated signals.

Phagocytosis of sheep erythrocytes (E) bearing rabbit immunoglobulin and rat C3 by rat peritoneal mast cells was quantitated by using 51Cr-labeled E and was confirmed by electron microscopy. The relative importance of IgG Fc receptor interaction in C3-mediated phagocytosis was assessed. Removal of any traces of IgG antibody by absorption of IgM antibody with protein A-Sepharose and absorption of the other reagents with sheep E had no effect on the phagocytosis of C3b- and C3bi-coated cells. IgG antibody enhanced the phagocytosis of intermediates prepared with IgM, purified components and rat C3 (EaIgMClgp4hu oxy2hu3brat) with a graded dose response, but was only additive and not synergistic with C3. The independence of the C3- and Fc-mediated signals was confirmed by using chemically produced polymers of rabbit IgG to inhibit phagocytosis. These polymers, especially tetramers and higher aggregates, completely blocked ingestion of EAIgG, but not that of EAIgMC1423b or -3bi. When IgG was substituted for IgM in the C3b intermediate, the IgG polymers inhibited about 50% of the phagocytosis. Cumulatively, the data demonstrated that in the case of rat mast cells, the stimuli to phagocytosis induced by C3 and IgG are independent; either is sufficient by itself.

Animals↗
PubMed1982

Comparison of the receptors for IgE of various rat basophilic leukaemia cell lines. I. Receptors isolated by IgE-sepharose and IgE and anti-IgE.

REceptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of IgE-Sepharose or IgE and anti-IgE, and characterized by SDS-polyacrylamide gel electrophoresis. All cell lines were found to be associated with a receptor molecule (R) which could be isolated either with IgE-Sepharose or IgE and anti-IgE and a second receptor (H) which could only be isolated with the aid of IgE-Sepharose. The relative amounts of these two molecules, as isolated from surface iodinated cells, varied from the RBL cell line to the other and their apparent molecular weights were not identical on all cell lines. Since comparisons were made on the same gel using receptors isolated from cells labelled with different isotopes of iodine, differences in molecular weight must be considered as being intrinsic and not due to methodological variations. These results provide an explanation why differences were observed among receptors for IgE as characterized in various laboratories. In spite of the fact that the various RBL cell lines originated from the same chemically-induced tumour they have, over the years, undergone changes which are reflected in the receptors for IgE.

Animals↗
PubMed1982

Solubilization of an activity regulating C3b function from Raji cell membranes.

A fraction of isolated Raji cell membranes solubilized with 2m KBr which was capable of inhibiting C3-dependent rosettes was examined for its ability to inhibit the alternative pathway of complement. It was shown to decrease alternative pathway-dependent haemolysis of sheep erythrocytes and to accelerate decay of factor B from these cells. It had no effect on C2 decay, the classical pathway analog of factor B. Inhibitory activity solubilized from Raji cells was not removed by immunoadsorption with anti-factor H or anti-factor I, two well-characterized serum C3b-control proteins. It also differed in two functional respects from these proteins. Firstly, it failed to result in cleavage of a peptide bone in C3 which is characteristic of factor I; secondly, its inhibitory activity did not synergize with factor I in inhibiting the alternative pathway, unlike factor H. These results suggest that Raji cells contain a regulatory factor in their membranes for the alternative pathway of complement which is distinct from factors H and I.

Burkitt Lymphoma↗
PubMed1982

Comparison of the receptors for IgE of various rat basophilic leukaemia cell lines. II. Studies with different anti-receptor antisera.

Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of various anti-receptor antisera, and characterized by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Most antisera were shown to react with a receptor designated H and which, as shown previously, could be isolated by IgE-Sepharose but not by a combination of IgE and anti-IgE. Only two highly purified anti-receptor antibody preparations, which had been purified by adsorption to and elution from rat basophilic leukaemia cells, reacted primarily with a second kind of receptor molecule which had previously been designated R. Some indirect evidence was obtained which suggests that H is a highly immunogenic molecule. It was confirmed that the apparent molecular weight of H-like molecules varied on different RBL cell lines.

Animals↗
PubMed1981

Nonantibody binding of serum proteins to 5S anti-Rh fragments produced by chymotrypsin.

Chymotrypsin hydrolysis of the IgG anti-Rh antibodies Ri results in both bivalent and univalent antibody fragments. The bivalent fragments coated on Rh-positive erythrocytes are agglutinable by albumin and other serum proteins in 3% polyethylene glycol. The bivalent structure of the 5S fragment is essential for expression of this site since 5S fragments produced by trypsin and pepsin are also agglutinable, while univalent fragments produced by papain and subtilisin are not. The agglutination by albumin of the 5S fragments is not caused by residual enzyme. The reaction appears to be irreversible in that once albumin has reacted with the 5S fragment, either in the fluid phase or at the cell surface, fresh addition of albumin and PEG will not result in agglutination. The nonantibody reaction of albumin and the other serum proteins with these 5S IgG fragments is believed to be caused by hydrophobic bonding involving the intrachain disulfide in the 5S fragment and hydrophobic areas of other proteins.

Antibodies↗
PubMed1981

Cleavage of membrane bound C3bi, an intermediate of the third component of complement, to C3c and C3d-like fragments by crude leucocyte lysosomal lysates and purified leucocyte elastase.

Partial degradation of the biologically-active major fragment of the third complement component (C3b) to C3bi is catalysed by the endopeptidase C3b inactivator (I) and its co-factor, beta 1H globulin (H). Complete degradation to the fragments C3c and C3d requires an additional protease, which can be simulated in vitro by trypsin. This study was designed to identify the in vivo correlate of trypsin. Purified and 125I-labelled C3b bound to sheep erythrocytes was used as substrate. Release of label into the supernate served as an index of proteolysis. The chain structure of the peptides in the supernate or remaining bound to the erythrocytes was assessed by SDS polyacrylamide gel electrophoresis. Significant cleavage of cell-bound C3b was obtained by treatment with I, H and extracts from human peripheral blood leucocyte azurophil granules. Purified elastase also removed label in the presence of I and H. The peptide remaining on the cell had the characteristic 33K molecular weight of C3d. The activity of elastase in cleaving was blocked by alpha-1-anti-trypsin, the chloromethyl ketone, MeO-Suc-Ala-Pro-Val-Ch2Cl and by rabbit antibody to elastase. Thus, elastase purified from azurophil granules of human polymorphonuclear neutrophils (PMN) is a potent catalyst of the cleavage of C3bi to C3c- and C3d-like fragments and may contribute in vivo to the control of complement-mediated inflammation.

Animals↗
PubMed1981

Functional and partial chemical characterization of the carbohydrate moieties of the IgE receptor on rat basophilic leukemia cells and rat mast cells.

The role of the carbohydrate portion of the receptor for IgE in the interaction with IgE was investigated. Membrane carbohydrates of rat basophilic leukemia (RBL) cells and rat mast cells (RMC) were labeled by treating the cells with galactose oxidase followed by [3H]-NaBH4. IgE receptors were separated from detergent solubilized membranes and examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Pretreatment with neuraminidase markedly increased the incorporation of 3H into both the total membrane extract and into the IgE receptors. SDS-PAGE analysis demonstrated the presence of galactose in all IgE-binding components of 2 RBL cell lines and the presence of sialic acid on the major IgE-binding component. Prior saturation of the cells with IgE did not prevent the carbohydrate labeling of the receptor, although it did block the labeling of its protein part, indicating that carbohydrates are not located in the binding site. Removal of terminal sialic acid residues with neuraminidase increased the affinity of the receptor for IgE without appreciably affecting the number of receptors per cell. In order to more drastically modify the receptor carbohydrates, RBL cells were grown in the presence of Tunicamycin (TN). TN was shown to markedly inhibit the incorporation of [3H]-glucosamine into the receptor. RBL cells grown in the presence of TN expressed fewer receptors at the cell surface, as judged both by ligand binding studies and external labeling procedures. These data cumulatively suggest that the carbohydrate moieties of the receptor are not directly located in the binding site of the IgE receptor; however, the TN studies suggest that receptor carbohydrate may play a role in transport of the receptor to the plasma membrane or in its orientation thereafter.

Animals↗
PubMed1981

Characterization of the IgE receptor by tryptic mapping.

IgE receptors were labeled by lactoperoxidase-catalyzed surface radioiodination of rat basophilic leukemia (RBL) cells and rat peritoneal mast cells (RMC). After nonionic detergent solubilization and incubation with rat IgE, IgE-receptor complexes were immunoprecipitated using anti-rat IgE. The receptor for IgE was further purified by SDS-PAGE, the receptor peak, in the gel, was submitted to tryptic digestion, and the resulting peptides were analyzed by a 2-dimensional peptide mapping procedure. The peptides were then visualized by autoradiography. IgE receptors from different RBL cell lines exhibit slight differences in m.w., as judged by SDS-PAGE; however, no differences were seen in the tryptic peptide maps of receptors from the different RBL cell lines. In addition, receptors isolated from RMC also mapped identically, indicating that peptides responsible for the m.w. differences may not be labeled. The IgE-binding component of higher m.w., isolated by affinity chromatography on IgE-Sepharose, gave a distinct pattern of tryptic peptides that were different from the receptor. By using IgE-Sepharose and tryptic mapping, this 2nd and IgE-binding component was found on all RBL cell lines and on RMC. The membrane orientation of the receptor was analyzed by tryptic mapping. Tryptic maps obtained from IgE receptors labeled on intact cells (outside labeled only), membrane particles (inside and outside labeled), and in a detergent-solubilized form (all possible sites labeled) were similar, indicating that no other protein site was available to be labeled, in addition to those in the surface exposed binding site. Moreover, saturation of the receptor by IgE prevented its subsequent radioiodination, whether the receptor was labeled on intact cells, on membrane particles, or in a solubilized form, again indicating that no site other than the binding site could be labeled. Cumulatively, these data suggest that the IgE receptor on RBL cells is not a transmembrane protein.

Animals↗