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R S Geha

Publications and source records attributed to R S Geha.

At least 73 records · Page 4Linked to original sources
PubMed1995

CD40-mediated lymphotoxin alpha expression in human B cells is tyrosine kinase dependent.

The cytokine lymphotoxin (LT)alpha is known to play a role in B cell activation. As the engagement of the B cell antigen CD40 is known to lead to B cell proliferation and differentiation, we studied LT alpha expression in human B cells after CD40 ligation. We demonstrate that anti-CD40 monoclonal antibody (mAb) induces strong LT alpha mRNA and surface-expression in human tonsil B cells. Induction of LT alpha mRNA and surface expression by CD40 ligation is inhibited by the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein in a dose-dependent manner. The protein kinase C (PKC)-specific inhibitors sphingosine and bis-indolylmaleimide caused negligible inhibition of anti-CD40-induced LT alpha mRNA and surface expression. No inhibition is observed with the protein kinase (PKA) inhibitors H89 and HA1004. Cross-linking of the transmembrane phosphatase CD45 to CD40 by using goat-anti-mouse F(ab')2 fragments strongly inhibits CD40-mediated LT alpha expression in human B cells, confirming the role of PTK activation in CD40-mediated induction of LT alpha expression. Inhibitors of the serine/threonine protein phosphatases PP1 and PP2A, okadaic acid and calyculin induce LT alpha mRNA expression. In contrast, cyclosporin A, an inhibitor of the serine/threonine phosphatase calcineurin has no effect on anti-CD40-induced LT alpha expression. These results suggest that induction of LT alpha expression in B cells following engagement of CD40 involves activation of protein tyrosine kinases.

Antibodies, Monoclonal↗
PubMed1995

CD40 ligand expression is developmentally regulated in human thymocytes.

Thymic selection involves the interaction between developing thymocytes and the thymic microenvironment. CD40 is expressed on thymic epithelial cells and on bone marrow-derived antigen-presenting cells in the thymus. We have investigated the capacity of human thymocytes to express CD40 ligand. CD40 ligand was not detectable on resting immature double-positive (CD4+/CD8+) or resting mature single-positive (CD4+/CD8- or CD4-/CD8+) thymocytes. Immature thymocytes failed to express CD40 ligand on their surface after stimulation with PMA and ionomycin, whereas stimulated CD4+/CD8- mature thymocytes expressed CD40 ligand. Northern blot analysis revealed that CD40 ligand mRNA was detectable in activated mature single-positive thymocytes. CD40 ligand expression by mature thymocytes was associated with the capacity to drive immunoglobulin isotype switching in purified B cells. These results demonstrate that the ability to express CD40 ligand is acquired late in thymocyte development and that mature thymocytes are capable of providing B cells with the activation signals necessary for immunoglobulin isotype switching.

Antigens, CD↗
PubMed1995

Role of protein tyrosine kinases and phosphatases in isotype switching: crosslinking CD45 to CD40 inhibits IgE isotype switching in human B cells.

Protein tyrosine kinases and protein tyrosine phosphatases play an important role in the transduction of signals via antigen receptors in T and B cells, and in CD40-dependent B-cell activation. To examine the role of tyrosine kinases and phosphatases in B-cell isotype switching, we examined the effects of the engagement of the transmembrane phosphatase CD45 on the synthesis of IgE induced by IL-4 and anti-CD40 monoclonal antibody (mAb). Crosslinking CD45 to CD40 using biotinylated mAbs and avidin strongly inhibited CD40-mediated IgE synthesis in IL-4-treated human B cells. CD40/CD45 crosslinking did not affect epsilon germline transcription in B cells stimulated with IL-4, but strongly inhibited induction of S mu/S epsilon switch recombination as detected by a nested primer polymerase chain reaction assay. The B-cell src-type tyrosine kinase lyn, which is activated following CD40 engagement, is a potential target for the effects of CD45 observed in our experiments, because CD45/CD40 crosslinking resulted in the inhibition of CD40-mediated lyn phosphorylation and activation. These results suggest an important role for protein tyrosine kinases and phosphatases in CD40-mediated induction of isotype switching to IgE.

Antigens, CD↗
PubMed1995

CD40-CD40 ligand (CD40L) interactions and X-linked hyperIgM syndrome (HIGMX-1).

Interactions between the B cell surface antigen CD40 and its ligand (CD40L) expressed on activated T cells play a critical role in isotype switching. This is illustrated by failure of isotype switching in patients with X-linked hyperIgM syndrome in whom the CD40L gene is mutated and by failure of isotype switching of CD40-deficient mice in response to T-cell-dependent antigens. We review these findings and discuss the signaling mechanisms of CD40 and the developmental control and transcriptional regulation of CD40L expression.

Animals↗
PubMed1995

Interleukin-4 receptor expression by human B cells: functional analysis with a human interleukin-4 toxin, DAB389IL-4.

BACKGROUND: Studies of human IgE-secreting B cells have proven difficult because of the small size of this population. We have used an interleukin-4 (IL-4) fusion toxin to detect functionally IL-4 receptor (IL-4R) expression on B cells involved in IgE synthesis. METHODS: In diphtheria toxin IL-4 (DAB389IL-4) the receptor-binding domain of diphtheria toxin has been replaced with human IL-4. DAB389IL-4 cytotoxicity depends on IL-4R binding and internalization. RESULTS: Addition of DAB389IL-4 inhibited IgE synthesis induced by IL-4+ anti-CD40 monoclonal antibody or hydrocortisone. IgE inhibition resulted from DAB389IL-4 B-cell cytotoxicity because DAB389IL-4 inhibited IL-4-independent B-cell proliferation. Thus induction of human IgE synthesis involves IL-4R+ cells. In contrast, terminally differentiated, IgE-producing B cells no longer express functional IL-4R because DAB389IL-4 only modestly inhibited ongoing IgE synthesis by B cells from patients with hyper-IgE states and only minimally affected IL-4-induced IgE synthesis in normal B cells when the toxin was added at day 7. Pokeweed mitogen-induced IgM synthesis was sensitive to early but not to late addition of DAB389IL-4. Thus the loss of functional IL-4R immunoglobulin-secreting B cells is independent of isotype switching. CONCLUSIONS: IgE-secreting B cells no longer express functional IL-4R. Therapies for allergic disease that target the IL-4R would not affect IgE-secreting B cells but may block the recruitment of B cells into the IgE-secreting pool. For optimal benefits this approach may be combined with therapies that target IL-4R-, IgE-secreting B cells.

B-Lymphocytes↗
PubMed1995

Cross-linking of Fc gamma receptors activates HIV-1 long terminal repeat-driven transcription in human monocytes.

Elevation of the levels of circulating immune complexes frequently accompanies HIV-1 infection and is a prognostic indicator of clinical progression from asymptomatic infection to AIDS. Here we report that cross-linking of Fc gamma RI or Fc gamma RII by adherent human IgG or by specific anti-Fc gamma R mAb activates HIV-1 gene expression in the human monocytic cell line BF24 and increased HIV RNA expression in monocytes from HIV infected patients as assayed by reverse transcription-PCR. In THP-1 cells, Fc gamma R cross-linking induced NF-kappa B, which is known to bind to the regulatory region of the long terminal repeat (LTR) of HIV-1 and to activate HIV-1 transcription. Anti-TNF-alpha antibody but not anti-IL-1 beta antibody strongly inhibited both the induction of HIV-1-LTR-driven transcription and the induction of NF-kappa B by Fc gamma R cross-linking. These results indicate that Fc gamma R can mediate a TNF-alpha-dependent induction of HIV-1 gene transcription and suggest that immune complexes may contribute to the pathophysiology of HIV-1 infection by augmenting viral replication in monocytes.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Defective expression of early activation genes in cartilage-hair hypoplasia (CHH) with severe combined immunodeficiency (SCID).

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-gamma) synthesis and IL-2 receptor alpha-chain (IL-2R alpha) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2R alpha, IL-2 and IFN-gamma were markedly decreased in patient lymphocytes stimulated with PMA+ionomycin as compared to control lymphocytes. The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c-fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes.

Child↗
PubMed1995

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.

Amino Acid Sequence↗
PubMed1995

Bacterial superantigens induce the proliferation of resting gamma/delta receptor bearing T cells.

We observe that highly purified (> or = 97% pure) gamma/delta T cells isolated from normal peripheral blood proliferate to bacterial toxin supperantigens SEA, SEB, SED, and TSST-1. MHC class II molecules were necessary and sufficient for the recognition of superantigens by gamma/delta T cells because MHC Class II deficient B cell line failed to support the proliferation of gamma/delta T cells to toxins and murine L cells transfected with HLA-DR but not untransfected cells were capable of presenting toxins to gamma/delta T cells. As in the case with alpha/beta T cells, bacterial superantigens synergized with PMA in causing the proliferation of purified gamma/delta T cells rigorously depleted of accessory cells. Together, our findings suggest that gamma/delta T cells recognize and respond to bacterial superantigens in a manner similar to alpha/beta T cells.

Animals↗
PubMed1994

CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.

To study the role of B-cell antigen CD40 in immune responses, mouse embryonic stem (ES) cells in which both copies of the gene encoding CD40 had been disrupted by homologous recombination were injected in RAG-2 (recombination-activating gene-2)-deficient blastocysts to generate chimeras in which all mature lymphocytes are derived from the CD40-deficient ES cells. T- and B-cell number and phenotype were normal in the CD40-/- chimeras. However, B cells failed to proliferate and undergo isotype switching in vitro in response to soluble CD40 ligand (sCD40L) with interleukin 4 (IL-4) but responded normally to lipopolysaccharide (LPS) with IL-4. CD40-/- chimeras completely failed to mount an antigen-specific antibody response or to develop germinal centers following immunization with the T cell-dependent (TD) antigen keyhole limpet hemocyanin. In contrast, CD40-/- mutant mice responded normally to the T cell-independent (TI) antigens, 2,4,6-trinitrophenyl (TNP)-LPS and TNP-Ficoll. The most noticeable alteration in the serum immunoglobulin levels of young (6-8 weeks old) CD40-/- animals was absence of IgE and severe decrease of IgG1 and IgG2a. These results confirm the essential role of CD40- CD40L interactions in the antibody response to TD antigens and in isotype switching.

Animals↗
PubMed1994

Immunosuppressant deoxyspergualin inhibits antigen processing in monocytes.

Deoxyspergualin (DSG) is a novel immunosuppressive agent recently shown to bind to the constitutive heat shock protein 70, which is involved in binding and intracellular transport of antigenic peptides. In this study, we show that DSG inhibits the proliferation of PBMCs to the Ags tetanus toxoid and diphtheria toxoid, but not to the mitogens PHA and PMA/ionomycin, nor to the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. DSG's effect was specific for monocytes as preincubation of T cells with DSG did not inhibit their proliferation to monocytes pulsed with tetanus toxoid Ag for 16 h, whereas the presence of DSG during Ag pulsing of the monocytes inhibited their ability to stimulate T cell proliferation. DSG did not down-regulate the expression of MHC class II molecules by monocytes, and the inhibitory effect of DSG on T cell proliferation was not reversed by the addition of IL-2, nor by the addition of the costimulatory signals IL-1, IL-6, and anti-CD28. Studies with two human T cell clones, HA1.7 and PF5, specific, respectively, to peptides spanning amino acids 307-319 and 256-270 of influenza hemagglutinin, showed that DSG inhibited the proliferation of the clones to the native hemagglutinin molecule but minimally affected their proliferation to the peptides. These data suggest that DSG interferes with Ag processing and/or presentation.

Adult↗
PubMed1994

Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn.

The high affinity immunoglobulin G (IgG) receptor Fc gamma RI (CD64) is expressed constitutively on monocytes and macrophages, and is inducible on neutrophils. Fc gamma RI has recently been shown to be associated with the signal transducing gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma). Induction of cytoplasmic protein tyrosine phosphorylation by Fc gamma RI cross-linking is known to be important in mediating Fc gamma RI-coupled effector functions. Recently, syk has been implicated in this role. We now report that the src-type kinases hck and lyn are physically and functionally associated with Fc gamma RI. Hck and lyn coimmunoprecipitated with Fc gamma RI from detergent lysates of normal human monocytes and of the monocytic line THP-1. Hck and lyn showed rapidly increased phosphorylation and increased exogenous substrate kinase activity after cross-linking of Fc gamma RI. These results demonstrate both physical and functional association of the Fc gamma RI/Fc epsilon RI gamma receptor complex with hck and lyn, and suggest a potential signal transducing role for these kinases in monocyte/macrophage activation.

Amino Acid Sequence↗
PubMed1994

Engagement of the common leukocyte antigen CD45 induces homotypic adhesion of activated human T cells.

The transmembrane tyrosine phosphatase CD45 plays an important role in TCR/CD3-mediated signaling. We demonstrate in this study that ligand binding to the CD45 molecule induces homotypic cell adhesion of activated, but not resting, T lymphocytes. mAbs to CD45 (4B2 and 10G10) and to CD45RO (UCHL1), but not to CD45RA (IOL2), caused sustained adhesion of alloreactive T cell lines. In contrast, none of the anti-CD45 mAbs induced aggregation of resting peripheral T cells. CD45-mediated adhesion of activated T cells involved both CD11a/18-dependent as well as CD11a/18-independent mechanisms. mAb 4B2 induced a strictly CD11a/18-dependent adhesion that was completely inhibited by both the protein kinase C (PKC) inhibitor sphingosine and the protein tyrosine kinase (PTK) inhibitors genestein and herbimycin A. In contrast, mAb 10G10, which recognized an epitope on CD45 distinct from the one recognized by mAb 4B2, induced CD11a/18-independent adhesion that was inhibited by sphingosine, but not by genestein or herbimycin A. Biochemical studies revealed direct evidence for activation of protein kinase C and protein tyrosine kinase after engagement of CD45 on activated T cells by mAb 4B2. These results indicate that in addition to its role in TCR/CD3-mediated activation, engagement of CD45 transduces signals that result in enhanced adhesiveness of activated T cells.

Antigens, CD↗
PubMed1994

Disodium cromoglycate inhibits S mu-->S epsilon deletional switch recombination and IgE synthesis in human B cells.

IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4-treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti-CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper-IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG inhibits switching to IgE in B cells and suggest a novel potential mechanism for the prevention of allergic disease by DSCG.

B-Lymphocytes↗
PubMed1994

Signal transduction via CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C gamma 2.

CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti-CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3-kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

Engagement of MHC class II molecules by staphylococcal superantigens activates src-type protein tyrosine kinases.

Staphylococcal exotoxins (SE) are superantigens that bind to monomorphic determinants on major histocompatibility complex (MHC) class II molecules and stimulate human peripheral blood T lymphocytes in a V beta-specific manner. SE also deliver activation signals via MHC class II molecules that initiate cell adhesion and cytokine gene transcription. These events are preceded by tyrosine phosphorylation and are antagonized by inhibitors of tyrosine kinases, indicating an essential role for these kinases in signaling via class II molecules. We report that stimulation of human peripheral blood monocytes with SE induced rapid and selective activation of the src-related protein tyrosine kinases (PTK) fgr and hck. SE also induced the activation of fgr and lyn in B cells. PTK activation by SE required MHC class II expression, and was greatly potentiated in the presence of T cells bearing toxin-specific V beta chains. These results indicate that in addition to their antigen and superantigen-presenting function, MHC class II molecules act as signal-transducing receptors that are coupled to src-type PTK.

Bacterial Toxins↗