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D H Conrad

Publications and source records attributed to D H Conrad.

At least 37 records · Page 2Linked to original sources
PubMed1997

Induction of B cell apoptosis by co-cross-linking CD23 and sIg involves aberrant regulation of c-myc and is inhibited by bcl-2.

A novel system to study the effects of co-cross-linking CD23/FceRII and sIg on murine B lymphocytes utilizes a highly multivalent form of anti-Ig prepared by covalently linking anti-Ig antibodies to a DNP-dextran backbone. CD23-sIg co-cross-linking is accomplished by the addition of DNP-specific monoclonal IgE. Previous studies demonstrated that co-cross-linking CD23 and sIg significantly inhibited mouse B cell proliferation, especially at high doses of the multivalent anti-Ig. Interestingly, examination of early activation signals reveals no difference in B cells subjected to co-cross-linking conditions as compared to B cells activated with anti-Ig alone. Total cellular protein tyrosine phosphorylation levels are unchanged by co-cross-linking. Analysis of B cell mRNA reveals that co-cross-linking the receptors does not alter the expression levels of ornithine decarboxylase 8 h after stimulation as compared to the controls. In contrast, levels of the proto-oncogene c-myc were significantly elevated 1 h after inducing B cell activation under co-cross-linking conditions. However, it remains unclear whether this aberrant c-myc regulation plays any role in inducing apoptosis. In addition, on day 3 after stimulation, the co-cross-linking of CD23 and sIg resulted in the formation of apoptotic B cells, determined by both photomicroscopy of the B cell cultures and FACS analysis of B cell nuclei. B cells obtained from bcl-2 transgenic mice proliferated as well as controls, and failed to undergo apoptosis when CD23 and sIg were co-cross-linked on their surface. These studies indicate that co-cross-linking of CD23 with B cell sIg inhibits B cell proliferation by a mechanism that is distinct from that seen by co-cross-linking of the Fc gamma RII and sIg. In addition, these results suggest a means by which antigen-specific IgE can down-regulate additional B cell activation and IgE synthesis.

Animals↗
PubMed1995

Analysis of murine soluble Fc epsilon RII sites of cleavage and requirements for dual-affinity interaction with IgE.

The low-affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal sequence homologous to C-type animal lectins. Between this region and the membrane is a repetitive sequence predicted to form an alpha-helical coiled-coil and is termed the stalk region. The Fc epsilon RII is proteolytically cleaved when at the cell surface in this stalk region. Both the 38 Kd and 28 Kd major released fragments were isolated from culture media and N-terminal sequencing demonstrated that the cleavage sites were in the third and fourth repeat domains, respectively. The identified sites show no apparent similarity with the cleavage sites previously identified in human Fc epsilon RII. Recent studies have demonstrated that the intact Fc epsilon RII interacts with IgE with a dual-affinity, resulting from a multivalent interaction with the IgE Fc region; mutant Fc epsilon RII that have a disruption of the alpha-helical coiled-coil have a single low-affinity interaction consistent with a monomeric interaction with IgE. The soluble Fc epsilon RII were shown to interact with IgE with an affinity similar to these mutant Fc epsilon RII. Preparation of a chimeric Fc epsilon RII in which the transmembrane and cytoplasmic regions were replaced with sequences from Ly-49 revealed that these regions played no role in the multimeric association of the Fc epsilon RII necessary for dual-affinity interaction with IgE. In addition, a full-sized soluble Fc epsilon RII construct was expressed, and this molecule demonstrated increased capacity to interact with IgE.

Amino Acid Sequence↗
PubMed1995

Homotypic aggregation of murine B lymphocytes is independent of CD23.

CD23 is a low-affinity receptor for IgE (Fc epsilon RII). Functions attributed to CD23 not involving IgE suggest that it interacts with ligands other than IgE. CD21 has recently been described as a counter ligand for CD23. A number of lines of evidence have implicated CD23 as an adhesion molecule in human B cells. We have investigated the role of CD23 in homotypic B cell aggregation in the mouse, using lipopolysaccharide plus interleukin-4-induced aggregation as a model system. In this system high levels of aggregation are accompanied by a massive up-regulation of CD23 expression. However, in contrast to what has been observed in human B cells, we find no evidence of a role for CD23 in homotypic adhesion of murine B cells.

Animals↗
PubMed1995

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on interleukin-4-mediated mechanisms of immunity.

Because of similarities in the independent actions of the pleiotropic cytokine, interleukin-4 (IL-4), and the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on murine B-lymphocytes suggested in earlier studies, we have investigated whether the immunosuppression mediated by direct exposure to TCDD in vitro is due to an IL-4-like biological activity. In particular, the ability of TCDD to mimic hallmark responses of B-cells to IL-4, such as upregulation of major histocompatibility complex (MHC) antigens of the class II type, increases in cell surface expression of the low affinity form of the Fc receptor for IgE (CD23) and induction of immunoglobulin class switching, was tested. At concentrations that readily suppress B-cell proliferative and antibody-forming cell responses, TCDD failed to demonstrate any of the activities of IL-4 observed in parallel cultures. Further, in experiments in which TCDD was preincubated with B-cells before addition of IL-4, no evidence of increased IL-4 activity was observed. Rather, TCDD preincubation resulted in decreased secretion of IgG1 and IgE in B-cell cultures stimulated to undergo immunoglobulin class switching by incubation with bacterial lipopolysaccharide (LPS) and IL-4. Because TCDD produced comparable suppression of IgM secretion induced by LPS alone (i.e., no IL-4), it appears that TCDD inhibits the formation of fully differentiated B-cells capable of secreting antibody and has no effects on class switching events per se. Coupled with previous reports from this and other laboratories, these observations indicate that TCDD is able to suppress secretion of several classes of immunoglobulin.

Animals↗
PubMed1994

Antigen presentation is enhanced by targeting antigen to the Fc epsilon RII by antigen-anti-Fc epsilon RII conjugates.

Targeting Ag to the Fc epsilon RII by Ag-specific IgE has been shown to be an efficient means of enhancing Ag presentation by B cells to Ag-specific T cells. To take advantage of the Fc epsilon RII as a targeting molecule and to investigate whether IgE was required for mediation of the enhanced stimulation, Ag was covalently coupled to anti-Fc epsilon RII by using heterobifunctional crosslinking reagents. These Ag-Ab conjugates were used with T cell lines specific for the Ags, OVA (BB6.5) or rabbit gamma-globulin (CDC35 and D1.6), and splenic B cells to examine both B cell and T cell proliferation in vitro. Significant presentation of Ag-anti-Fc epsilon RII conjugates was apparent at doses of Ag 1,000- to 10,000-fold lower than seen with unconjugated Ag alone. Ag presentation with the use of anti-Fc epsilon RII-Ag conjugates was as good as or better than conjugates with Ab to the adhesion molecule Pgp-1 or control Ab in T cell proliferation and better than those conjugates in B cell proliferation assays (10- to 100-fold). Anti-Fc epsilon RII-Ag conjugates were clearly more effectively presented than Ag-anti-Fc gamma RII conjugates (> 100-fold). Mouse Fc epsilon RII is presently known to be expressed on B cells and follicular dendritic cells and these in vitro results suggest that the conjugates would be useful tools for investigating the role of IgE-mediated B cell Ag presentation in vivo. BALB/c mice immunized with OVA-anti-Fc epsilon RII conjugates made a quite significant OVA-specific IgG1 response and a detectable IgE response. No detectable Ab was produced in response to OVA alone and a minimal response was seen when an isotype-matched control conjugate was used. Thus, the results indicate that Fc epsilon RII targeting is operative both in vivo and in vitro.

Animals↗
PubMed1994

Molecular mechanisms of murine Fc epsilon RII/CD23 regulation.

The low affinity receptor for IgE (Fc epsilon RII or CD23), expressed primarily on mouse B cells, is known to be upregulated by interleukin-4 (IL-4) at both the mRNA and protein levels. Fc epsilon RII expression is superinduced when the IL-4 is combined with cell activation. In order to explore the molecular regulation of Fc epsilon RII expression, mouse B cell lines were screened to develop a cell line model. The B cell lymphoma A20.1, was found to behave in a manner similar to mouse B cells in that Fc epsilon RII levels are very low on cells cultured in media alone (< 10(3)/cell), increased by culture in the presence of IL-4, and superinduced by LPS and IL-4 (> 10(5)/cell). The steady state mRNA levels for Fc epsilon RII corresponded to the level of cell surface expression. Transcription assays indicated that the Fc epsilon RII level increases could be explained entirely by increased transcription rates. The A20.1 cell line was subsequently used to analyse the Fc epsilon RII promoter. Nested deletion analysis of the 1.3 kB 5' of the mouse Fc epsilon RII transcription start site, using CAT reporter plasmids transfected into A20.1 cells, identified major elements activating the Fc epsilon RII promoter within 250 bp of the transcription start site. Constructs containing greater than 250 bp of 5' sequence showed significantly reduced CAT activity suggesting negative regulatory regions. Coincident with the restricted tissue expression of murine Fc epsilon RII, the promoter was B cell specific in that little CAT expression was seen in fibroblast, mast cells or T cell lines. Expression was seen, however, in both mouse and human B cell lines. Finally, the promoter was analysed for response to IL-4. Stimulation with IL-4 plus LPS resulted in only a modest increase in CAT activity (approximately 2-fold), in contrast to transcription assays, where increases approximated that seen at the cell surface. Thus, the IL-4 response must also require sequences distal to the regions examined.

Animals↗
PubMed1993

The oligomeric nature of the murine Fc epsilon RII/CD23. Implications for function.

The low affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal region homologous to C-type animal lectins. Immediately adjacent to this lectin homology region is a sequence that is predicted to form an alpha-helical coiled-coil stalk leading to dimer or trimer formation. This provides an explanation for the known self-associative capacity for the Fc epsilon RII. In this study the self-association to a trimer or tetramer is shown with rFc epsilon RII by chemical cross-linking and affinity purification on IgE columns. The data indicate that only the oligomeric form of Fc epsilon RII has sufficient affinity/avidity to bind to an IgE adsorbent. In contrast, Fc epsilon RII that is purified using anti-Fc epsilon RII mAb adsorbents has largely lost its capacity to bind IgE, as well as its capacity to self-associate, indicating that IgE recognizes the oligomeric form of the Fc epsilon RII. This phenomenon was further examined by performing detailed binding analysis of the mouse IgE/Fc epsilon RII interaction. A biphasic binding curve with high (2-7 x 10(7) M-1) and low (2-7 x 10(6) M-1) affinity binding was seen. Fc epsilon RII mutants were prepared that lack one or more of the 21 amino acid homologous repeat domains in the stalk region of the molecule. These mutant Fc epsilon RII molecules bound IgE with only a single low affinity (5-10 x 10(6) M-1). In addition, cross-linking analysis of one of these mutants demonstrated that it does not exhibit the receptor self-association seen for the intact Fc epsilon RII. Two chimeric Fc epsilon RII molecules were prepared having the mouse Fc epsilon RII lectin homology (carboxyl-terminal) region and the stalk region of either the related human Fc epsilon RII or the corresponding domain of Ly-49. Chimeric molecules using the former (alpha-helical coiled-coil) stalk supported normal binding of IgE although the Ly-49/Fc epsilon RII chimera failed to bind IgE. Taken together, the results indicate that high (approximately 10(8) M-1) affinity IgE binding results from interaction of multiple lectin domains with (presumably) symmetrical sites on the IgE molecule. Specificity for IgE is determined by the lectin domain although the binding avidity is determined by oligomerization through the coiled coil stalk.

Animals↗
PubMed1993

Follicular dendritic cells and B cell costimulation.

Ag-bearing follicular dendritic cells (FDC) are found throughout secondary lymphoid tissues in close association with rapidly proliferating germinal center B lymphocytes. We reasoned that FDC might provide costimulatory signals that would enhance the ability of Ag to stimulate B cell proliferation in the germinal centers. To test this, FDC were cultured with B cells activated by a slg-dependent (goat anti-mouse mu conjugated to dextran (anti-mu-dex)) or -independent (LPS) pathway and their proliferation was measured by using [3H]thymidine incorporation. The addition of FDC markedly augmented B cell proliferation in a dose-dependent fashion. Depletion of FDC from cultures abrogated the increased proliferation. Addition of highly purified FDC obtained from cell sorting resulted in B cell costimulation, whereas addition of other sorted cells was without effect. The FDC accessory activity was apparent over the entire culture period and over a wide range of either polyclonal B cell activator. When B cells and activators were cultured in the absence of FDC, only about one fourth of the cells remained viable after 3 days. In contrast, virtually all cells in cultures containing FDC, B cells, and activator were viable. Cultures containing FDC and B cells from nude mice proliferated normally in the presence of anti-mu-dex plus rIL-4, implying that IL-4 provides adequate T cell help in this system. The costimulatory activity of the FDC could not replace either the anti-mu-dex or IL-4 in this system and was not MHC restricted. These data support the concept that FDC not only provide Ag but also facilitate B cell proliferation by means of other costimulatory interactions that contribute to make the microenvironment in the germinal center favorable for B cells to proliferate.

Animals↗
PubMed1993

Chromosomal location and isoform analysis of mouse Fc epsilon RII/CD23.

The gene for the mouse low affinity receptor for IgE (Fc epsilon RII, also known as CD23) was mapped on Chromosome (Chr) 8 proximal to Plat. This gene, symbolized Fcer2 (formerly Fce2) resides in a region of Chr 8 with linkage homology with human chromosomes 8 and 19. The mouse Fc epsilon RII was examined for the presence of alternate N-terminal forms such as seen in humans. An antisense RNA probe was prepared from the 5' end of the cDNA through the first 660 bp of the cDNA and was used to analyze message from Fc epsilon RII+ B cells and B cell hybridomas both before and after treatment with interleukin 4 (IL-4). Using RNase protection analysis, a major 640 bp band corresponding to the full length probe was seen, even after activation of the cells with LPS in the presence of IL-4, which is known to give high expression levels of the Fc epsilon RII. This result suggests that the mouse does not produce significant levels of an alternate IL-4 inducible Fc epsilon RII, as seen in man, and this may explain the more restricted cell lineage expression of the Fc epsilon RII in the mouse.

Animals↗
PubMed1992

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.

Animals↗
PubMed1992

Murine follicular dendritic cells and low affinity Fc receptors for IgE (Fc epsilon RII).

The present study was undertaken to determine whether mouse follicular dendritic cells (FDC) bear Fc epsilon RII (CD23) and whether IgE-immune complexes are retained by FDC. Mouse Fc epsilon RII was localized by both L and electron microscopy using the mAb B3B4. In lymph nodes of normal mice, Fc epsilon RII was low but detectable on FDC. By 14 days after Nippostrongylus brasiliensis infection, the level of Fc epsilon RII increased on B lymphocytes located in the cortex of draining mesenteric lymph nodes. However, the Fc epsilon RII level on FDC remained low. Although numerous IgE-producing plasma cells were seen at day 14, very little IgE was associated with FDC. By 26 days after infection, Fc epsilon RII was observed on FDC in increased levels and IgE binding was clearly associated with FDC. Unexpectedly, FDC of control mice immunized with albumin in CFA to elicit an IgG response showed intense labeling for Fc epsilon RII. In contrast, the B cells exhibited very little Fc epsilon RII. IgE immune complexes were observed in association with FDC in the CFA-immunized mice. When mice were given a hapten-specific monoclonal of the IgE isotype, hapten carrier complexes were trapped and retained on Fc epsilon RII-bearing FDC. In conclusion, FDC were clearly one of the major murine cell types bearing Fc epsilon RII. IgE immune complexes were found in association with FDC and Fc epsilon RII appeared to play a major role in trapping and retaining IgE immune complexes. FDC Fc epsilon RII was subject to regulatory control, but the Fc epsilon RII level on FDC was regulated very differently from the Fc epsilon RII level on B cells.

Animals↗
PubMed1992

Co-crosslinking Fc epsilon RII/CD23 and B cell surface immunoglobulin modulates B cell activation.

Previous studies have shown that a highly multivalent from of anti-IgD or anti-IgM, prepared by conjugating the respective antibodies to dextran, causes extensive B cell proliferation with ng/ml concentrations of the anti-immunoglobulin (Ig). A modification of this system has been exploited to investigate the effect of co-crosslinking the Fc epsilon RII and surface Ig by binding DNP to the dextran backbone (DNP-dextran) and employing a DNP-specific monoclonal IgE of either rat or mouse origin. Addition of anti-IgD-(H delta a/1)[DNP-dextran] or anti-IgM-[DNP-dextran] to purified, resting murine B cells resulted in B cell proliferation over a broad dose (0.03-30 micrograms/ml). Addition of DNP-specific rat or mouse IgE dramatically modulated the proliferative response. Proliferation in response to doses greater than 0.3 microgram/ml H delta a/1-[DNP-dextran] was consistently reduced in a dose-dependent manner in the presence of increasing amounts of IgE while proliferation to lower concentrations of H delta a/1-[DNP-dextran] was slightly enhanced or not influenced at all by the IgE anti-DNP. Interleukin-4 (IL-4) significantly increased the IgE effect, in line with its known enhancing effects on Fc epsilon RII levels. Experiments measuring Ig production rather than proliferation demonstrated that in the presence of IgE anti-DNP, B cells produced lower amounts of immunoglobulin (IgG1 or IgM) in response to an anti-Ig signal. Control experiments demonstrated that the IgE effect on proliferation was blocked by monoclonal anti-Fc epsilon RII, but not anti-Fc gamma RII, thus demonstrating the necessity for IgE/Fc epsilon RII interaction. In addition, the necessity for co-crosslinking was shown by the inability of IgE anti-DNP to affect the proliferative response to H delta a/1-dextran even in the presence of various doses of DNP-dextran. These results demonstrate that co-crosslinking of sIg and the Fc epsilon RII results in an altered B cell response to anti-Ig mediated activation. IL-4 does not ablate this inhibition, in contrast to the effect of co-crosslinking Fc gamma RII and surface Ig, suggesting a model whereby IgE can modulate its own production.

Animals↗
PubMed1991

The mast cell-committed progenitor. In vitro generation of committed progenitors from bone marrow.

Mast cell-committed progenitors are detected in the unique microenvironment of the mesenteric lymph node (MLN) of Nippostrongylus brasiliensis-infected mice but not in naive bone marrow. We have determined that MLN cells, after infection, produce high levels of IL-3, IL-4, and IgE, presumably in the form of immune complexes with antigens produced by the infecting helminth. After N. brasiliensis infection, peak production of these factors occurs several days before the peak appearance of mast cell-committed progenitors in the MLN. To determine if these factors play a role in mast cell commitment, we recreated these conditions, in vitro. Naive bone marrow cells were cultured with combinations of IL-3, IL-4, and IgE immune complexes, or on IgE-coated plates, and then assayed for acquisition of the ability to form mast cell colonies when supplemented with fibroblast-conditioned medium alone. IL-3 and IgE immune complexes, and, unexpectedly, IgE immune complexes alone were found to be capable of producing mast cell-committed progenitors, i.e., cells responsive to fibroblast-conditioned medium alone, from bone marrow, whereas IL-4 did not enhance production of mast cell-committed progenitors from bone marrow. Production of IFN-gamma peaked at the same time point as committed progenitor activity and may be responsible for down regulating the response.

Animals↗
PubMed1991

Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.

Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI.

Animals↗
PubMed1991

Fc epsilon receptors.

Advances in our understanding of the molecular structure of Fc receptors have been made at a rapid pace. Details of how Fc receptors are involved in cell triggering, e.g. allergic mediator release from mast cells, and IgE synthesis are also continuing to be elucidated, although much work is still required. Recent highlights of investigations of mast-cell and lymphocyte IgE Fc receptors will be outlined.

Antigens, Differentiation, B-Lymphocyte↗