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D M Shepherd

Publications and source records attributed to D M Shepherd.

At least 19 recordsLinked to original sources
PubMed2001

Anti-CD40 Treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 mice induces activation of antigen presenting cells yet fails to overcome TCDD-induced suppression of allograft immunity.

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.

Animals↗
PubMed2000

The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11.10 transgenic CD4(+) T cells in adoptively transferred mice.

Exposure to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the generation of T cell-dependent immunity, both humoral and cell-mediated. However, the mechanism of TCDD-induced immune suppression remains to be defined. We hypothesized that exposure to TCDD suppresses the activation of naive CD4(+) T cells and prevents their expansion and differentiation into effector T-helper cells capable of driving T cell-dependent immune responses. To test this hypothesis, we adoptively-transferred DO11.10 OVA-specific T-cell receptor (TCR) transgenic T cells into syngeneic recipients and used a TCR-specific monoclonal antibody to track the in vivo activation of naive CD4(+) T lymphocytes following exposure to OVA. The production of OVA-specific antibodies was suppressed in a dose-dependent manner in adoptively transferred mice that had been exposed to TCDD. Although TCDD exposure had little effect on the expansion or activation of the adoptively transferred, OVA-specific CD4(+) T cells, these cells disappeared from the spleen more rapidly in TCDD-treated mice and produced significantly decreased levels of the T cell-derived cytokines IL-2 and IL-10. There was also a trend towards reduced IFN-gamma and IL-4 production following in vitro re-stimulation. These data suggest that TCDD may interfere with the survival and/or differentiation of OVA-specific T-helper cells. These results demonstrate for the first time the potential of the DO11.10 adoptive transfer system to directly assess immunotoxic effects of xenobiotics on antigen-specific CD4(+) T cells in vivo.

Adoptive Transfer↗
PubMed1999

Disruption of CD154:CD40 blocks generation of allograft immunity without affecting APC activation.

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-gamma, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154-/- mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.

Animals↗
PubMed1996

Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and prototypic ligand for the aryl hydrocarbon receptor, is a potent immunotoxicant. To understand the underlying mechanisms of TCDD immunotoxicity, we have characterized the time course of changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in C57B1/6 mice treated with 0 or 15 microg TCDD/kg. Suppression of CTL activity by TCDD directly correlated with reduced numbers of splenic CTL effector cells identified by their CD8+CD44 high CD45RB low phenotype, while suppression of the alloantibody response correlated with a lack of expansion of the B220+ splenocyte population. Cytokine production was differentially modulated following TCDD treatment. Although type 1 cytokine production (IFN-gamma, IL-2, and TNF) was initially induced in TCDD-treated mice, production failed to increase normally after day 5. In contrast, the production of IL-1 beta, IL-4, and IL-6 was mostly unaffected by TCDD exposure. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of specific alloantibody isotypes. TCDD abrogated the production of IgG2a (promoted by IFN-gamma), but had much less effect on the level of IgG1 (promoted by IL-4). IgM Ab titers were also highly suppressed. CD8+ cells were the exclusive producers of IFN-gamma and IL-2 when spleen cells from P815-injected mice were cultured in vitro on days 4 to 7 after P815 injection. However, CD4+ cells were shown to play a crucial role in the generation of both CTL and alloantibody responses, since their depletion in vivo abolished both responses. Based on similar temporal effects produced by TCDD and anti-CD4 Ab on alloimmune responses, we postulate that TCDD interferes with the initial activation of CD4+ T cells, which leads to downstream inhibition of the activation and/or differentiation of CD8+ T cells and B cells. In addition, since delayed treatment with either anti-CD4 Ab or TCDD suppressed the alloantibody but not the CTL response, TCDD may also affect later CD4+ T helper-B cell interactions.

Animals↗
PubMed1994

Signaling events during helper T cell-dependent B cell activation. I. Analysis of the signal transduction pathways triggered by activated helper T cell in resting B cells.

gp39 is expressed on anti-CD3-activated Th, and binds CD40 on the B cell, driving B cell cycle entry. In this study, the signal-transduction pathway initiated in B cells as a consequence of interacting with activated Th is examined. Unlike anti-membrane Ig (anti-mlg) or anti-MHC class II, plasma membranes (PM) isolated from anti-CD3-activated Th, PMAct did not trigger an increase in the B cell intracellular concentrations of cAMP or calcium. In addition, PMAct did not stimulate protein kinase C activation as measured by myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation and protein kinase C translocation. The failure to detect these biochemical events may be caused by the asynchrony with which PMAct induce these normally transient biochemical changes. Alternatively, PMAct may not trigger these events. PMAct did induce the tyrosine phosphorylation of several B cell substrates. Neutralizing Abs directed against gp39 inhibited PMAct-induced protein tyrosine phosphorylation of B cell substrates. These results suggest that cognate interactions in B cells initiate a signal-transduction pathway that is different from the pathway initiated by cross-linking of mlg or MHC class II.

Animals↗
PubMed1993

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.

Animals↗
PubMed1993

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39.

The ligand for CD40 has been recently identified as a 39-kd protein, gp39, expressed on the surface of activated CD4+ T helper cells (Th). In vitro, soluble CD40 and anti-gp39 have been shown to block the ability of Th to activate B cells, suggesting that gp39-CD40 interactions are important to T cell-dependent B cell activation. Here it is shown that in vivo administration of anti-gp39 dramatically reduced both primary and secondary humoral immune responses to erythrocytes and soluble protein antigens without altering responses to the T-independent type II antigen, trinitrophenyl-Ficoll. Treatment of mice for 4 d with anti-gp39 inhibited the anti-sheep red blood cell (SRBC) response for at least 3 wk and inhibited the expression of all immunoglobulin isotypes in secondary responses to the protein antigen, keyhole limpet hemocyanin. To examine the direct effect of anti-gp39 on Th function, SRBC-immune Th cells from anti-gp39-treated mice were adoptively transferred and shown to be fully capable of providing help. These results suggest that anti-gp39 treatment does not cause Th deletion or anergy. Anti-gp39 may mediate its profound immunosuppressive effects on humoral immunity by blocking gp39-CD40 interactions. Moreover, these studies establish gp39-CD40 as an important receptor-ligand pair for the targeting of therapeutic antibodies to control thymus-dependent humoral responses.

Animals↗
PubMed1993

Immunotoxicology of cadmium and mercury on B-lymphocytes--I. Effects on lymphocyte function.

Heavy metals have been shown to exert immunotoxic effects on humoral immunity. To ascertain the mechanisms by which these immunotoxic effects are exerted, the effects of CdCl2 and HgCl2 on the biology of murine B-lymphocytes were studied. It was shown that CdCl2 and HgCl2 inhibited B-cell RNA and DNA synthesis. The IC50 (the concentration required to inhibit a specific B-cell function by 50%) for CdCl2 was 30 microM for RNA synthesis and DNA synthesis. The IC50 for HgCl2 was 50 and 120 nM for RNA and DNA synthesis, respectively. Cell cycle analysis revealed that B-cells were arrested throughout the cell cycle with CdCl2 and HgCl2. The inhibitory effects exerted by CdCl2 and HgCl2 were rapid, inhibiting RNA synthesis within 2 h of activation. Differentiation to Ig secretion was inhibited by CdCl2 and HgCl2 in culture and there appeared to be selective effects on specific Ig isotypes. IgG3 production was most sensitive to inhibition by CdCl2 and HgCl2 followed by IgG1 and IgG2b and then IgM and IgG2a. Changes in the expression of B-cell surface antigens induced by LPS were also influenced by CdCl2. LPS-induced increases in class II MHC expression was inhibited by CdCl2, as was the constitutive expression of class I MHC antigen. A summary of the IC50 for CdCl2 and HgCl2 are presented. In summary, both CdCl2 and HgCl2 exert early, inhibitory effects on B-cell activation. This is manifested by the inhibition of RNA, DNA and antibody synthesis. However, selective effects on the production of specific Ig isotypes by these metals may influence the ability of B-cells to mount effective immune responses to pathogens.

Animals↗
PubMed1992

Cognate interaction between T helper cells and B cells. VII. Role of contact and lymphokines in the expression of germ-line and mature gamma 1 transcripts.

Complete reconstitution of Th cell function for B cell growth and differentiation was provided by plasma membranes (PM) derived from activated Th (PMAct) in combination with IL-4 and IL-5 (IL-4/IL-5). IL-5 has been shown to be essential for the secretion of all Ig isotypes by resting, conventional B cells activated by PMAct and IL-4. It was shown that in the presence of PMAct/IL-4/IL-5, a high frequency of resting B cells differentiated to express IgG1. IL-4 alone transiently induced the expression of germ-line gamma 1 transcripts. PMAct alone were ineffective at inducing germ-line gamma 1 transcript expression by resting B cells suggesting that Th-B cell contact was an insufficient signal to cause a detectable increase in the steady-state levels of gamma 1 germ-line transcripts. PMAct alone or PMAct/IL-4 did not induce the appearance of transcripts for secreted mu or mature gamma 1. IL-5, in combination with PMAct/IL-4, provided the necessary signal(s) required for the expression of secreted mu and mature gamma 1 transcripts. Therefore, IL-5 appeared to be an important if not essential differentiation factor for conventional B cells that have been activated by cognate help. It appeared that IL-5 promoted the secretion of Ig by inducing the synthesis of mature Ig transcripts.

Animals↗
PubMed1992

A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells.

CD40 is a B-cell surface molecule that has been shown to induce B-cell growth upon ligation with monoclonal antibodies. This report shows that triggering via CD40 is essential for the activation of resting B cells by helper T cells (Th). A soluble fusion protein of CD40 and human immunoglobulin, CD40-Ig, inhibited the induction of B-cell cycle entry, proliferation, and differentiation by activated Th1 and Th2. The ligand for CD40 was identified as a 39-kDa membrane protein that was selectively expressed on activated Th. A monoclonal antibody specific for the 39-kDa protein inhibited CD40-Ig binding and also inhibited the activation of B cells by Th. These data indicate that the 39-kDa membrane protein expressed on activated Th is a binding protein for CD40 and functions to transduce the signal for Th-dependent B-cell activation.

Animals↗
PubMed1991

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.

Animals↗
PubMed1991

The lack of memory B cells in immune bone marrow.

Adoptive transfers of lymphoid populations between Ig-congenic strains of mice were performed to evaluate the presence of memory cells in the bone marrow of immune animals. Transfer of immune splenic B cells or immune peripheral blood mononuclear cells effectively transferred antigen-specific IgG1 antibody responses of donor origin. In contrast, BM from immune animals did not transfer a memory response. The lack of memory transfer with immune BM was not due to a suppressive activity of BM since (1) Purified antigen-specific B cells from BM did not transfer memory while purified antigen-specific B cells from immune spleen was a potent source of memory cells, and (2) cotransfer of immune BM with a source of memory B cells (immune PB) did not suppress the transfer of memory. Although immune BM did not have cells capable of transferring antigen-specific memory, copious amounts of antigen-specific antibody were spontaneously produced by immune BM in vitro. These studies suggest that the transfer of immunological memory observed in human BM transplant cases may be the consequence of PB contamination of BM preparations when collected.

Animals↗
PubMed1990

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

Animals↗
PubMed1982

Gastric mucosal histamine and histamine methytltransferase in patients with duodenal ulcer.

Histamine and histamine methyltransferase (HMT) have been measured in gastric mucosa from 110 patients with duodenal ulcer and 62 control subjects. Both antral and fundic mucosa had similar levels of HMT activity despite antral mucosa containing significantly less histamine. Patients with duodenal ulcer had significantly lower levels of fundic mucosal HMT activity and lower concentrations of fundic mucosal histamine than control subjects. An additional finding was that men who were cigarette smokers had significantly lower concentrations of fundic mucosal histamine (but not HMT) than non-smokers and, as there was an excess of cigarette smokers among patients with duodenal ulcer, this may be the explanation for the reduced concentrations of fundic mucosal histamine in these patients.

Adult↗