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D H Conrad

Publications and source records attributed to D H Conrad.

At least 19 recordsLinked to original sources
PubMed2001

Bryostatin-1 specifically inhibits in vitro IgE synthesis.

Bryostatin-1, a macrocyclic lactone, is an antineoplastic agent that potently activates protein kinase C. Bryostatin-1 (Bryo) had an immunomodulatory effect on murine B cells in that it specifically inhibited IgE production. IgE levels were inhibited in a B cell dose-response curve, whereas IgM and IgG1 were induced by Bryo treatment. Taken together, ELISPOT and surface Ig staining data suggested that Bryo inhibition occurred at the level of class switching. RT-PCR and real time PCR data showed that this inhibition was achieved at an early step in switch recombination, namely, the appearance of Iepsilon germline transcripts. Although Bryo caused a delay in the proliferative response of IL-4/CD40 ligand trimer-stimulated B cells, CFSE studies revealed that the Bryo-mediated inhibition of class switching to IgE occurred independently of the number of division cycles. Notably, Bryo showed the same specific IgE inhibition in human B cells. This study provides evidence for a unique mechanism regulating IgE production possibly downstream of PKC by specifically modulating Iepsilon germline transcription.

Animals↗
PubMed2001

Regulation of IgE production requires oligomerization of CD23.

Here we describe the production of a rabbit polyclonal Ab (RAS1) raised against the stalk of murine CD23. RAS1 inhibits release of CD23 from the surface of both M12 and B cells resulting in an increase of CD23 on the cell surface. Despite this increase, these cells are unable to bind IgE as determined by FACS. CD23 has previously been shown to bind IgE with both a high (4-10 x 10(7) M(-1)) and low (4-10 x 10(6) M(-1)) affinity. Closer examination by direct binding of (125)I-IgE revealed that RAS1 blocks high affinity binding while having no effect on low affinity binding. These data support the model proposing that oligomers of CD23 mediate high affinity IgE binding. These experiments suggest that RAS1 binding to cell surface CD23 results in a shift from oligomers to monomers, which, according to the model, only bind IgE with low affinity. These experiments also suggest that high affinity binding of IgE is required for IgE regulation by CD23 and is demonstrated by the fact that treatment of Ag/Alum-immunized mice treated with RAS1 results in a significant increase in IgE production similar to the levels seen in CD23-deficient mice. These mice also had significantly decreased levels of serum soluble CD23 and Ag-specific IgG1. RAS1 had no effect on IgE or Ag-specific IgG1 production in CD23-deficient mice.

Animals↗
PubMed2001

Suppression of IgE responses in CD23-transgenic animals is due to expression of CD23 on nonlymphoid cells.

Serum IgE is suppressed in CD23-transgenic (Tg) mice where B cells and some T cells express high levels of CD23, suggesting that CD23 on B and T cells may cause this suppression. However, when Tg B lymphocytes were compared with controls in B cell proliferation and IgE synthesis assays, the two were indistinguishable. Similarly, studies of lymphokine production suggested that T cell function in the Tg animals was normal. However, adoptive transfer studies indicated that suppression was seen when normal lymphocytes were used to reconstitute Tg mice, whereas reconstitution of controls with Tg lymphocytes resulted in normal IgE responses, suggesting that critical CD23-bearing cells are irradiation-resistant, nonlymphoid cells. Follicular dendritic cells (FDC) are irradiation resistant, express surface CD23, and deliver iccosomal Ag to B cells, prompting us to reason that Tg FDC may be a critical cell. High levels of transgene expression were observed in germinal centers rich in FDC and B cells, and IgE production was inhibited when Tg FDCs were cultured with normal B cells. In short, suppressed IgE production in CD23-Tg mice appears to be associated with a population of radioresistant nonlymphoid cells. FDCs that interface with B cells in the germinal center are a candidate for explaining this CD23-mediated IgE suppression.

Adoptive Transfer↗
PubMed2001

Metalloprotease inhibitor-mediated inhibition of mouse immunoglobulin production.

High levels of membrane CD23 have been shown to decrease immunoglobulin E (IgE). CD23 is a very labile molecule and is cleaved from the cell surface by an unknown metalloprotease. Two metalloprotease inhibitors, compound A (N-[4-hydoxyamino-2-(R)-isobutyl-3-(S)propargylthiomethylsuccinyl]-(S)-phenylalnine-N'-methyl-amide) and compound B (N-[3-(S)-hydroxy-4-hydroxyamino-2-(R)-(2-naphthylmethyl) succinyl]-(S)-tert-leucinamide), were chosen for their ability to inhibit human CD23 cleavage and selectively inhibit IgE production. The ability of these inhibitors to block cleavage of murine CD23 and immunoglobulin production in an in vitro system was examined. The inhibitors blocked sCD23 release from B cells. The inhibitors also decreased IgE production by B cells; however, 20-30 times more inhibitor was needed to give a similar amount of inhibition as compared with sCD23 release. The effects on immunoglobulin production did not require the presence of CD23 in that these inhibitors also blocked in vitro immunoglobulin production when B cells from CD23-/- mice were used. The inhibitors decreased production of all other immunoglobulin isotypes examined and reduced the number of IgE antibody-forming cells (AFC) while having no effect on cell proliferation or viability. The level of Iepsilon transcripts in cells treated with compounds A and B were not different as compared with control cells. These results suggest that while these inhibitors effectively inhibit IgE production in a CD23-specific manner in the human, these compounds, in the mouse, inhibit immunoglobulin production by an unknown mechanism that is unrelated to CD23.

Animals↗
PubMed2001

Enhanced transepithelial antigen transport in intestine of allergic mice is mediated by IgE/CD23 and regulated by interleukin-4.

BACKGROUND & AIMS: We previously described a system for enhanced transepithelial transport of antigen in which both the amount of specific antigen and its rate of transport were dramatically increased in intestine of sensitized rats compared with controls. This study investigated the essential components mediating antigen uptake in mice genetically deficient for interleukin (IL)-4 or CD23. METHODS: Mice were actively or passively sensitized to horseradish peroxidase (HRP). Jejunal segments from control or sensitized mice were mounted in Ussing chambers and challenged with HRP from the luminal side. Tissues were processed for electron microscopy, and photomicrographs were analyzed for antigen uptake (location and area of HRP-containing endosomes). Immunohistochemistry and reverse-transcription polymerase chain reaction were used to detect epithelial CD23 expression. RESULTS: Actively sensitized IL-4(+/+), but not IL-4(-/-) mice, displayed increased transepithelial antigen transport and CD23 expression on enterocytes. Passively sensitized IL-4(+/+) and IL-4(-/-) mice displayed elevated antigen transport after transfer of immune serum but not if the serum was depleted of immunoglobulin (Ig) E or IL-4. IL-4 added to cultured IEC-4 cells up-regulated expression of CD23 messenger RNA. The augmented antigen uptake was inhibited by anti-CD23 and was absent in sensitized CD23(-/-) mice. CONCLUSIONS: Our studies indicate that IL-4 regulates IgE/CD23-mediated enhanced transepithelial antigen transport in sensitized mouse intestine.

Animals↗
PubMed2000

Androstenetriol and androstenediol. Protection against lethal radiation and restoration of immunity after radiation injury.

Androstenetriol (AET) and Androstenediol (AED) upregulate host immunity, leading to increased resistance against infections. AET augments IL-2, IL-3, IFN gamma levels, and counteracts hydrocortisone immune suppression. AET and AED at a dose of 0.75 mg/- and 8.0 mg/25-g mouse, protected 60 and 70%, respectively, of C57/BL/6J mice irradiated with a lethal dose. These hormones also protected mice irradiated with 6 Gy and infected with a coxsackievirus B4 LD50. AET significantly increased spleen lymphocyte numbers at 7, 14, and 21 days after a 6-Gy exposure. Fluorescent activated cell-sorter analysis of irradiated mice, spleen, and bone marrow showed that AET significantly augmented the myeloid precursor markers, CD11b/Mac-1, and B220 (pan B), as well as the absolute numbers of CD4+/CD8+ cells over the 21 days of testing. Overall, the data are consistent with AET/AED inducing a more rapid recovery of all hematopoietic precursors from the small number of surviving stem cells.

Anabolic Agents↗
PubMed2000

Enhanced intestinal transepithelial antigen transport in allergic rats is mediated by IgE and CD23 (FcepsilonRII).

We previously reported that active sensitization of rats resulted in the appearance of a unique system for rapid and specific antigen uptake across intestinal epithelial cells. The current studies used rats sensitized to horseradish peroxidase (HRP) to define the essential components of this antigen transport system. Sensitization of rats to HRP stimulated increased HRP uptake into enterocytes (significantly larger area of HRP-containing endosomes) and more rapid transcellular transport compared with rats sensitized to an irrelevant protein or naive control rats. Whole serum but not IgE-depleted serum from sensitized rats was able to transfer the enhanced antigen transport phenomenon. Immunohistochemistry demonstrated that sensitization induced expression of CD23, the low-affinity IgE receptor (FcepsilonRII), on epithelial cells. The number of immunogold-labeled CD23 receptors on the enterocyte microvillous membrane was significantly increased in sensitized rats and was subsequently reduced after antigen challenge when CD23 and HRP were localized within the same endosomes. Finally, pretreatment of tissues with luminally added anti-CD23 antibody significantly inhibited both antigen transport and the hypersensitivity reaction. Our results provide evidence that IgE antibodies bound to low-affinity receptors on epithelial cells are responsible for the specific and rapid nature of this novel antigen transport system.

Animals↗
PubMed1999

Humoral response suppression observed with CD23 transgenics.

CD23, also known as the low affinity IgE receptor (FcepsilonRII), has been hypothesized to have a role in IgE regulation. A new CD23 transgenic mouse was generated using the MHC class I promoter and IgH enhancer to further test the hypothesis that CD23 plays a role in the down-regulation of IgE. Study of three founder lines by FACS showed overexpression to varying extents on both B and T lymphocytes. No alterations in lymphocyte populations was observed. All three founder lines exhibited strong suppression of IgE in response to DNP-keyhole limpet hemocyanin/alum and Nippostrongylus brasiliensis infection compared with that in parental or littermate controls. The founder line exhibiting the highest level of suppression also was less susceptible to Ag-induced systemic anaphylactic shock. Overall, the data support the concept that enhancing CD23 levels can be used to suppress IgE-mediated disease. The mechanism involves decreased IgE synthesis, because the serum half-life of IgE was not altered in transgenics, and enzyme-linked immunospot analysis demonstrated lower IgE-producing cells stimulated by injection of anti-IgD. Transgenics also exhibited significantly decreased IgG1 responses and exhibited lower levels of all Ig isotypes, although this was more variable in different founder lines.

Alum Compounds↗
PubMed1998

Production of a chimeric form of CD23 that is oligomeric and blocks IgE binding to the Fc epsilonRI.

The low affinity receptor for IgE (Fc epsilonRII/CD23) has previously been shown to interact with IgE with a dual affinity. Three chimeric constructs were created containing the lectin domain (amino acids 172-188) or the "neck" and lectin domain (amino acids 157-188) attached to subunits of oligomeric proteins. All chimeras were incapable of interacting with IgE with either a high or low affinity, indicating that the alpha-helical stalk of CD23 is important for orienting the lectin heads such that an interaction with IgE can occur. This concept received further support in that a chimeric CD23 composed of the human CD23 stalk and the mouse CD23 lectin head bound mouse IgE with a dual affinity, but could only bind rat IgE with a low affinity. Effort was next concentrated on a construct consisting of the entire extracellular (EC) region of CD23. A mutation to the first cleavage site of CD23 (C1M) resulted in a more stable molecule as determined by a decrease of soluble CD23 release. A soluble chimeric EC-C1M was prepared by attaching an isoleucine zipper to the amino terminus (lzEC-C1M). The interaction with IgE by lzEC-C1M was found to be superior to that seen with EC-CD23. The lzEC-C1M could inhibit binding of IgE to both CD23 and the high affinity receptor for IgE, Fc epsilonRI, providing further evidence for a strong interaction with IgE. Fc epsilonRI inhibition (approximately 70%) was seen at equimolar concentrations of lzEC-C1M, implying the effectiveness of this chimera and suggesting its potential therapeutic value.

Animals↗
PubMed1998

Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes.

Lymphocyte activation is often accompanied by changes in cell morphology, for example, in cell adhesion or motility. IL-4 is a cytokine exerting many effects on B lymphocytes. In this study, we show that stimulation with LPS in combination with IL-4, but not LPS or IL-4 alone, results in a pronounced dendritic morphology of B cells. Using a culture system in which Abs directed to B cell surface markers are immobilized on the tissue culture plastic, we find that cell spreading can be mediated by a variety of Abs, including anti-CD44, -CD23, -LFA-1, -VLA-4, -ICAM-1, and -Ig. B cells stimulated with anti-Ig Abs plus IL-4, or anti-CD40 Abs in the presence or absence of IL-4, are also induced to spread, while IL-2, IL-5, or IL-10 in combination with LPS or alone fail to induce this. Spreading correlates with induction of tight cell aggregation. It is sensitive to cytochalasin B, indicating a requirement for intact actin cytoskeleton. CD44 is selectively detected in the detergent-insoluble fraction of cell lysates prepared from LPS plus IL-4-stimulated B cell cultures after Ab cross-linking of CD44, suggesting a membrane protein-cytoskeleton interaction. Interestingly, electron microscopy studies reveal induction of microvilli-like structures on LPS plus IL-4-stimulated blasts, suggesting that IL-4 can influence cell morphology on an ultra-structural level. In summary, our data show that stimulation with LPS plus IL-4 or ligation of CD40 is capable of inducing dramatic morphologic changes in murine B cells, which correlates with in vitro induction of strong cell adhesion.

Animals↗
PubMed1998

STAT6, NF-kappaB and C/EBP in CD23 expression and IgE production.

STAT6, NF-kappaB (p50) and C/EBPbeta transcription factors (TF) were examined with respect to CD23 regulation. Electrophoretic mobility shift assay (EMSA), competition and supershift analysis demonstrated that STAT6 binds the CD23a promoter but with a lower affinity than the consensus site. STAT6-/- mice were analyzed for CD23 levels and showed reduced expression after CD40 ligand trimer (CD40LT) stimulation. However, normal CD23 expression and even some IgE production was induced in STAT6-/- mice with CD40LT/IL-4. EMSA analysis indicated that the CD23a STAT site was bound by a protein in nuclear extracts from CD40+/-IL-4-stimulated STAT6-/-B cells. Western blot analysis of these nuclear extracts demonstrated the presence of STAT3 and STAT5, suggesting that these STATs can induce CD23 in this situation. Further supporting evidence was obtained by showing that IL-2 and IL-4 both synergize with CD40 in an identical manner for CD23 induction on STAT6-/- B cells. EMSA analysis of the two putative NF-kappaB sites confirmed binding to both, although one site bound with a higher affinity than the second. Analysis of p50-/-mice indicated that this subunit was not necessary for CD23 induction or CD40/IL-4-induced IgE production. Finally, no role for C/EBP was observed in CD23 induction by EMSA or by CD23 induction analysis in C/EBPbeta-/- mice, whereas the absence of C/EBP, did have an effect on IgE production and lipopolysaccharide-induced B cell proliferation. Based on these data, a model is presented which suggests that CD23 superinduction results from STAT and NF-kappaB interaction.

Animals↗
PubMed1997

Modulation of antigen-induced B and T cell responses by antigen-specific IgE antibodies.

Ag-specific IgE Abs not only mediate immediate hypersensitivity through mast cell activation, but also enhance in vitro Ag presentation and in vivo specific Ab responses in mice. To delineate the role of IgE Ab in the modulation of Ag-specific responses, spleen cells from OVA-sensitized BALB/C mice were cultured together with OVA-specific IgE (or IgG isotypes). OVA-dependent proliferative responses and anti-OVA IgE production were enhanced in the presence of anti-OVA IgE. A significant decrease in IFN-gamma secretion in OVA-stimulated cultures was observed in the presence of anti-OVA IgE, but no changes in IL-4 production were detected. Anti-OVA IgG isotypes or anti-TNP IgE showed no significant effect on any of these Ag-dependent responses. Addition of anti-CD23 Ab abolished these effects of anti-OVA IgE. Further, OVA-sensitized spleen cells from CD23-deficient mice responded to in vitro stimulation with OVA, but demonstrated no modulation by anti-OVA IgE. These results demonstrate that Ag-specific IgE not only augments Ag presentation and T cell proliferation, but also alters the pattern of cytokine production and increases specific IgE synthesis. These modulatory effects of Ag-specific IgE appear to be mediated by binding to Fc epsilon RII/CD23.

Adjuvants, Immunologic↗
PubMed1997

B cell activation and Ig, especially IgE, production is inhibited by high CD23 levels in vivo and in vitro.

The capacity of CD23 to regulate IgE production was evaluated in both an in vitro and an in vivo system. The decreased IgE response seen in CD23 transgenic mice was confirmed and observed to occur at all antigen doses used. In addition, purified B cells from the Tg animals in general exhibited lower IgE production when stimulated with CD40L and IL-4. To examine this down-regulating activity of CD23 an in vitro model system was developed. CHO cells were transfected with CD23, ICAM-1, or both CD23 and ICAM-1. ICAM-1 was chosen to enhance B cell-B cell interaction. Purified resting B cells were placed into culture with the mitomycin C-treated transfected or control CHO cells and activated with IL-4, IL-5, and CD40L-CHO. A dose-dependent decrease in IgE production was observed with increasing cell numbers of the CHO transfectants that expressed CD23. The effect lasted up to Day 3 of culture. B cell proliferation was also inhibited in a dose-dependent fashion by increasing numbers of CD23-expressing cells suggesting a potential effect of CD23 on B cell apoptosis. In contrast, ICAM-1-transfected or CHO control cells had minimal effects on either Ig production or B cell proliferation. While IgE production was inhibited up to 95% by high numbers of CD23-transfected CHO cells, some inhibition of IgG and IgM production was also seen. Finally, the mechanism of CD23-mediated inhibition of IgE production was compared with the inhibition in IgE production seen when B cell were coactivated with multivalent anti-IgD in conjunction with CD40L plus optimal IL-4. To this end we used RT-PCR to compare the relative levels of epsilon-germline transcripts in control cultures and cultures coactivated by anti-IgD, CD40L, and IL-5 or activated in the presence of high levels of CD23-expressing cells. CD22 was used as an internal standard since levels change little with B cell activation. Coactivation strongly inhibited epsilon-germline transcript levels but the presence of CD23-expressing cells did not. Thus, coactivation potentially operates prior to isotype switching, while high CD23 coculture blocks either recombination or more likely B cell differentiation to high Ig producers stage. Our data support the hypothesis that IL-4 induces both IgE and a controlling agent for IgE, namely, CD23.

Animals↗
PubMed1997

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Animals↗
PubMed1997

A new multivalent B cell activation model--anti-IgD bound to Fc gamma RI: properties and comparison with CD40L-mediated activation.

CHO cells permanently transfected with mouse Fc gamma RI alpha chain were prepared and used as a model to polyclonally activate murine B cells. The transfected CHO cells were treated with mitomycin C and placed into culture with varying quantities of anti-IgD. Using this model, murine splenic B cells (from BALB/c or C57Bl/6) were activated by mouse IgG2a-anti-IgD (10.4.22 or AF3.33) in a manner that is analogous to the activation of B cells seen with highly polyvalent anti-IgD (H delta(a)/1) prepared by chemical cross-linking to dextran. Efficient B cell activation was seen with nanogram quantities of anti-IgD. In the presence of IL-4 and IL-5, IgG1 production levels were equivalent to or better than seen when stimulation was with H delta(a)/1-dextran; however, IgE induction was not seen in either situation. The Ig production capacity was compared to that seen when B cells were activated with CD40L, using either CD40L-transfected CHO or a soluble CD40L construct. In the presence of IL-4 and IL-5, once a critical threshold of B cells was present, IgE and to a lesser extent IgG1 production was inversely proportional to B cell number when CD40L was the activating agent. In contrast, with Fc gamma RI-anti-IgD, IgM and IgG1 production was directly proportional to B cell number, while IgE production was never seen. Finally, when B cells were co-activated with immobilized anti-IgD and CD40L simultaneously, the IgE production from B cells induced by CD40L was strongly inhibited, while IgG1 and IgM production were not affected. Since B cell co-activation via sIg and CD40L would be a common scenario in secondary follicles, this inhibition of IgE production may be one of the reasons why serum IgE levels are much below IgG in normal immune situations.

Animals↗