Search PubMed⌕ Search

Biomedical subjects

J F Gauchat

Publications and source records attributed to J F Gauchat.

At least 55 records · Page 3Linked to original sources
PubMed1994

CD23 interacts with a new functional extracytoplasmic domain involving N-linked oligosaccharides on CD21.

Human CD21 has been described as a receptor for the C3d,g and iC3b proteins of complement, for the Epstein-Barr virus, and also for IFN-alpha. We reported recently that CD23, a low affinity receptor for IgE (Fc epsilon R2), is a new functional ligand for CD21. To determine the site of interaction of CD23 on CD21, we analyzed the ability of purified recombinant CD23 incorporated into fluorescent liposomes to bind CD21 mutants bearing various deletions of extracytoplasmic short consensus repeats (SCRs). We found that the site of interaction of CD23 on CD21 is on SCRs 5 to 8, with contribution of SCRs 1 and 2. Tunicamycin treatment of CD21-transfected K562 cells strongly inhibited the binding of CD23-liposomes, suggesting that an N-linked sugar, present on SCRs 5 to 8, is involved in the CD23/CD21 interaction. By mutating together or individually, the three asparagines present on SCRs 5 to 8, asparagines (Asn) 370 and 295, but not Asn 492, were shown to be involved critically in the binding of CD23. Furthermore, we mapped the binding sites of a panel of anti-CD21 mAbs and found that at least six epitopes can be detected on CD21. The mAbs that inhibit the most CD23 binding to CD21 map in SCRs 5 to 8. This study indicates that SCRs 5 to 8 represent a novel functional domain on the CD21 molecule, and is the first demonstration of an activity of an extracytoplasmic region of the CD21 outside of SCRs 1 to 4.

Antibodies, Monoclonal↗
PubMed1994

Similar CD40 ligand expression on EL-4 thymoma cell lines with widely different helper activity for B lymphocytes.

A mutagenized subclone of the murine EL-4 thymoma (clone B5) is approximately 30 times more potent than parental EL-4 cells in stimulating proliferation and Ig secretion of murine or human B cells by direct cell contact in the presence of appropriate cytokines. In this study we found that CD40 ligand (CD40L) expression was constitutive and very similar on EL-4 B5 and parental EL-4 cells according to Northern blot and flow cytometry. Activation with phorbol 12-myristate 13-acetate (PMA) alone, PMA and ionomycin, interleukin-1 (IL-1) or human T-cell supernatant did not lead to significant CD40L up-regulation. A receptor-binding assay with soluble CD40 did not reveal different ligand affinities. However, murine and human soluble CD40-IgFc fusion proteins inhibited human B-cell stimulation by EL-4 B5 cells in the presence of human T-cell supernatant. Inhibition was 96% when soluble CD40 was added on day 0 of culture and progressively decreased when the CD40 was added subsequently. Ig secretion by cytoplasmic Ig-positive cells was no longer inhibited. These findings imply that, although CD40 ligand is necessary for B-cell activation by EL-4B5 cells, additional molecule(s) must be responsible for the increased helper activity of the EL-4 B5 clone.

Animals↗
PubMed1994

Stimulation of human IgE production by a subset of anti-CD21 monoclonal antibodies: requirement of a co-signal to modulate epsilon transcripts.

CD21, the receptor for Epstein-Barr virus (EBV) and the complement receptor-2 (CR2), was recently found to interact specifically with CD23, a low-affinity receptor for IgE, and to regulate IgE production. Therefore, the effect of different anti-CD21 monoclonal antibodies (mAb) on IgE synthesis by blood mononuclear cells was investigated. One anti-CD21 mAb, BU-33, was able to increase significantly (more than threefold) interleukin-4 (IL-4)-induced IgE synthesis, whereas HB-5, OKB-7 and B2 anti-CD21 mAb had no effect. BU-33 had no effect on IgG and IgA production and produced only a moderate increase in IgM production. Recombinant, 29,000 MW, soluble CD23 (sCD23) expressed in COS cells exhibited the same IgE-enhancing activity. BU-33 was the best inhibitor of CD23-liposome binding to the CD21-positive cell line RPMI-8226 when compared to the other anti-CD21 mAb tested. BU-33 identified a different epitope on CD21. The effect of BU-33 on IgE production by purified tonsillar B cells and highly purified germinal centre B cells, was dependent on the presence of T cells or anti-CD40 mAb stimulation. Molecular analysis revealed that BU-33 alone failed to induce germline epsilon mRNA but increased the IL-4-induced germline epsilon transcription levels. Moreover, BU-33 had a synergistic effect on anti-CD40 mAb or T-cell-induced productive epsilon transcript expression. These results therefore indicate that the CD23-CD21 interaction needs a co-signal for B-cell differentiation towards IgE production.

Antibodies, Monoclonal↗
PubMed1993

Induction of human IgE synthesis in B cells by mast cells and basophils.

Immunoglobulin E (IgE) is central to the induction of allergic diseases through its binding to the high-affinity receptor (Fc epsilon R1) on mast cells and basophils. Crosslinking by allergens of the bound IgE leads to the release of various inflammatory mediators. IgE production by B cells requires a physical interaction with T cells, involving a number of surface adhesion molecules, as well as the soluble factors interleukin-4 (IL-4) and IL-13 (ref. 5) produced by T cells, basophils and mast cells. Here we report that, in the presence of IL-4, mast and basophilic cell lines can provide the cell contact signals that are required for IgE synthesis. The human cell lines HMC-1 (mast) and KU812 (basophilic) both express the ligand for CD40 (CD40L) which is shown to be responsible for the IgE production. Moreover, freshly isolated purified human lung mast cells and blood basophils are also shown to express CD40L and to induce IgE production. This evidence suggests that mast cells and basophils may therefore play a key role in allergy not only by producing inflammatory mediators, but also by directly regulating IgE production independently of T cells.

Animals↗
PubMed1993

Inhibition of an in vivo antigen-specific IgE response by antibodies to CD23.

Immunoglobulin E (IgE) mediates many allergic responses. CD23 is a 45-kilodalton type II transmembrane glycoprotein expressed in many cell types. It is a low-affinity IgE receptor and interacts specifically with CD21, thereby modulating IgE production by B lymphocytes in vitro. In an in vivo model of an allergen-specific IgE response, administration of a rabbit polyclonal antibody to recombinant human truncated CD23 resulted in up to 90 percent inhibition of ovalbumin-specific IgE synthesis. Both Fabs and intact IgG inhibited IgE production in vitro and in vivo. Thus, CD23 participates in the regulation of IgE synthesis in vivo and so could be important in allergic disease.

Amino Acid Sequence↗
PubMed1993

IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes.

In the present study it is demonstrated that IL-10 and viral (v)-IL-10 inhibit IL-4-induced IgG4 and IgE synthesis in cultures of PBMC from healthy nonatopic donors. The inhibition occurred at the mRNA level. IL-10 strongly reduced IL-4-induced expression of both germline and productive epsilon transcripts in PBMC. The inhibitory effects were completely neutralized, or IgG4 and IgE synthesis was even enhanced, by anti-IL-10 mAb, demonstrating the specificity of the reaction. IL-4-induced IgG4 and IgE synthesis by PBMC was monocyte dependent. IL-4 failed to induce IgG4 or IgE synthesis by monocyte-depleted PBMC, but the production of these isotypes was completely restored by reconstitution with autologous monocytes. However, monocytes preincubated with IL-10 for 24 h failed to provide the accessory signals required for IL-4-induced IgG4 and IgE synthesis, indicating that the inhibitory effects of IL-10 are indirectly mediated via monocytes. This notion was further supported by the finding that IL-10 and v-IL-10 failed to inhibit IL-4-induced IgE synthesis in the absence of monocytes, i.e., when highly purified B cells were cultured in the presence of anti-CD40 mAb or cloned activated CD4+ T cells. Moreover, IL-10 failed to inhibit IL-4-induced germline epsilon mRNA synthesis in highly purified B cells. The inhibitory effects of IL-10 could not be restored by exogenous TNF-alpha or IL-6, indicating that the inhibitory effects were not mediated through inhibition of production of these cytokines. This is compatible with the observation that monocytes preincubated with IL-10 did not inhibit IgG4 or IgE synthesis in a monocyte- and T cell-independent culture system, in which purified B cells were cultured in the presence of anti-CD40 mAb and IL-4. Collectively, these data indicate that IL-10 mediates a potent, monocyte-dependent, inhibitory effect on IL-4-induced IgG4 and IgE production by human PBMC.

Antigen-Presenting Cells↗
PubMed1993

CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM.

Signalling for the B-cell immunoglobulin isotype switch requires T-cell-derived cytokines and T-B cell interaction, which operates primarily through the CD40 molecule on B cells with its ligand (CD40L) on activated T cells (reviewed in ref. 1). The CD40L is a type II membrane protein with homology to tumour necrosis factor-alpha and -beta, and has important functions in B-cell activation and differentiation. Human CD40L maps on Xq26.3-27.1 (ref. 3), the region where a primary immunodeficiency characterized by an immunoglobulin isotype switch defect (the hyper-IgM immunodeficiency syndrome, HIGM1) has been localized. The hypothesis that HIGM1 involves an abnormality of the CD40L has been tested. We report here the lack of CD40L expression in four unrelated male children with the hyper-IgM syndrome. CD40L transcripts in these patients showed either deletions or point mutations clustered within a limited region of the CD40L extracellular domain. These genetic alterations with abnormal CD40L expression provide a molecular basis for immunoglobulin isotype switch defects observed in this immunodeficiency.

Base Sequence↗
PubMed1993

Human CD40-ligand: molecular cloning, cellular distribution and regulation of expression by factors controlling IgE production.

Here we report the cloning of the cDNA for human CD40-Ligand (CD40-L) from a CD4-positive T cell clone. The deduced amino acid sequence predicts a type II membrane protein of 261 amino acids. Northern blot and FACS analysis of PBMNC revealed that the human CD40-L can be detected on T cells and is absent from B cells and monocytes. The human CD40-L is expressed on both CD4- and CD8-positive T cells, (CD45R0+) and (CD45RA+) subsets. We observed that IL-4, an inducer of IgE production, upregulated CD40-L mRNA level while IFN gamma, an inhibitor of IgE synthesis, reduced the expression of CD40-L mRNA. These data suggest a the correlation between human CD40-L expression and IgE production.

Amino Acid Sequence↗
PubMed1993

CD21 expressed on basophilic cells is involved in histamine release triggered by CD23 and anti-CD21 antibodies.

Recombinant full-length human CD23 incorporated into fluorescent liposomes was used to detect a ligand for CD23 on the basophilic leukemia cell line, KU 812. Based on our recent finding that CD23 interacts with CD21 on subsets of B and T cells, we investigated if the same ligand was involved on KU 812 cells. An anti-CD21 monoclonal antibody (mAb) BU-33, was able to totally block CD23-liposome binding to KU 812 cells. Moreover, KU 812 cells express CD21 mRNA and have a cell surface molecule that reacts with anti-CD21 mAb. The CD23/CD21 interaction was not merely physical but was also associated with an increase in histamine release by KU 812 cells. Both recombinant soluble CD23 and an anti-CD21 mAb-mediated effect on histamine release was not restricted to and anti-CD21 mAb-mediated effect on histamine release was not restricted to the leukemic cell line, but was also observed with normal human blood basophils. These data demonstrate that CD21 is expressed on basophilic cells and that CD21 controls histamine production upon ligand-induced stimulation (CD23 or anti-CD21 mAb).

Antibodies, Monoclonal↗
PubMed1993

A new pair of surface molecules involved in human IgE regulation.

The molecules controlling IgE production are the subject of intense study in the effort to find new ways to treat allergic diseases. One candidate, the CD23 molecule, a low affinity receptor for IgE, was recently identified to interact with another molecule, named CD21, in the regulation of IgE production.

B-Lymphocytes↗
PubMed1993

Receptors for IgE.

Following advances during the past 5 years in our understanding of the molecular structure of receptors for IgE, progress has been made in elucidating the structure and function of IgE receptors and the signalling events through these receptors. IgE is not the only ligand for some of these receptors, leading to their having unexpected and interesting biological activities.

Animals↗
PubMed1992

Regulation of germ-line epsilon transcription and induction of epsilon switching in cloned EBV-transformed and malignant human B cell lines by cytokines and CD4+ T cells.

IL-4 induces germ-line epsilon transcript synthesis in normal human B cells, but a second signal provided by CD4+ T cells is required for subsequent production of productive epsilon mRNA and IgE synthesis. In the present study we demonstrate that IL-4 specifically induces germ-line epsilon transcripts in most EBV lymphoblastoid (LCL) and EBV+ and EBV- Burkitt's lymphoma (BL) cells without inducing IgE synthesis. However, cocultivation of cloned sIgM+, sIgE- EBV-LCL or BL cells with activated CD4+ T cell clones in the presence of IL-4 resulted in IgE production in 13 to 24% of the wells. Analysis of rearranged DNA in IgE-producing cloned BL cells indicated that epsilon switching occurred through a recombination deletion event. IgE production is a stable feature of the cloned switched EBV-LCL and BL cells because these cells continued to produce relatively high levels of IgE in the absence of IL-4 and CD4+ T cells. Induction of IgE synthesis was blocked by IFN-gamma, IFN-alpha, and transforming growth factor beta (TGF-beta), but only TGF-beta inhibited IL-4-induced germ-line epsilon mRNA expression. These results suggest that the inhibitory effects of TGF-beta are mediated via inhibition of germ-line epsilon expression, whereas IFN-alpha and IFN-gamma block other steps in the regulatory processes resulting in induction of IgE synthesis by established human B cell line cells. Our results indicate that the same set of signals that induce normal B cells to switch to IgE synthesis also induce high frequency epsilon switching in cloned established EBV transformed and malignant B cell lines including classical cell lines such as JY and BL-2.

B-Lymphocytes↗
PubMed1992

Immunoregulatory functions of paf-acether. VIII. Inhibition of IL-4-induced human IgE synthesis in vitro.

In the present study we showed that paf-acether (paf), a naturally occurring phospholipid cytokine, down-regulated IL-4-induced IgE production by mononuclear cells from healthy nonatopic donors in a dose-dependent fashion from 10 microM to 10 pM. Kinetic studies indicated that addition of paf together with IL-4 strongly decreased IgE synthesis from day 7 up to day 14. By contrast, paf had little or no inhibitory effect on the levels of IgM, total IgG, and IgA production. The inhibition of IgE synthesis by paf occurred independently of known inhibitors of IgE synthesis, IFN-gamma, PGE2, and transforming growth factor-beta because the addition of anti-IFN-gamma (10 micrograms/ml) mAb, indomethacin (0.1 microM), or anti-transforming growth factor-beta (10 micrograms/ml) mAb, together with paf and IL-4, did not overcome the inhibition of IgE synthesis. Finally, paf not only inhibited IL-4-induced IgE production but also reduced both germ-line and productive IgE transcripts levels by 77 and 67%, respectively, suggesting that modulation of IgE production by paf occurred at the transcriptional level. Taken together, these results suggest a novel IgE regulatory mechanism by phospholipid cytokine.

Dinoprostone↗
PubMed1992

Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.

Antibodies, Monoclonal↗
PubMed1992

Modulation of IL-4 induced germline epsilon RNA synthesis in human B cells by tumor necrosis factor-alpha, anti-CD40 monoclonal antibodies or transforming growth factor-beta correlates with levels of IgE production.

To determine the role of germline epsilon transcription in IgE synthesis, the effects of cytokines on germline epsilon RNA synthesis in IL-4 dependent epsilon switching in B cells was investigated. Induction of germline epsilon transcription in highly purified B cells seems to be a specific property of IL-4, since none of the other cytokines tested [IL-1 alpha, beta, IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-10, G-CSF, GM-CSF, M-CSF, IFN-gamma, IFN-alpha, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta] were effective. TGF-beta, IFN-gamma, and IFN-alpha inhibit IL-4 dependent IgE synthesis, but only TGF-beta blocked germline epsilon RNA synthesis in purified B cells, indicating that this may be the mechanism by which TGF-beta inhibits IgE synthesis, and that IFN-gamma and IFN-alpha act on other stages of the regulatory process resulting in IgE production. IL-5, IL-6, and TNF-alpha enhance IL-4 dependent IgE synthesis, but only TNF-alpha enhanced IL-4 induced germline epsilon RNA synthesis. Finally, anti-CD40 mAbs and the non-IL-4 producing CD4+ T cell clone A3, which in the presence of IL-4 induce IgE synthesis by purified B cells, both strongly enhanced germline epsilon transcription. These data, together with the observation that epsilon switching in cultures initiated with single sIgM+, sIgE- B cells in all instances was preceded by germline epsilon RNA synthesis, indicate that there is a strong relationship between germline epsilon transcription and IgE synthesis.

Antibodies, Monoclonal↗
PubMed1991

Anti-CD40 monoclonal antibodies or CD4+ T cell clones and IL-4 induce IgG4 and IgE switching in purified human B cells via different signaling pathways.

IL-4 induces IgE and IgG4 synthesis, but in addition to IL-4, a second signal provided by CD4+ T cells is required. Here we demonstrate that the signal provided by CD4+ T cells can be replaced by anti-CD40 mAb. Highly purified surface (sIgM+) human B cells cultured with soluble anti-CD40 mAb in the presence of IL-4 produced IgM, total IgG, IgG4, and relatively high levels of IgE, indicating that production of these isotypes represented H chain switching and was not the result of a selective outgrowth of isotype committed B cells. No IgA was produced in these cultures. However, the T cell signal was different from the signal provided by anti-CD40 mAb, because in contrast to CD4+ T cells, anti-CD40 mAb failed to induce germ-line epsilon transcripts. However, anti-CD40 mAb strongly enhanced germ-line epsilon mRNA expression induced by IL-4. In addition, IFN-gamma, IFN-alpha, and anti-CD23 mAb, which block IL-4-induced IgE production by PBMC, or B cells cocultured with CD4+ T cell clones, failed to inhibit IgG4 and IgE synthesis induced by anti-CD40 mAb. Finally, anti-CD40 mAb and CD4+ T cell clones had strong synergistic effects on IgG4 and IgE synthesis. These results indicate that different B cell activation pathways can result in IgG4 and IgE switching in the presence of IL-4.

Antibodies, Monoclonal↗
PubMed1991

Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones.

In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.

Antibodies, Monoclonal↗