Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine metabolism via the trans-sulphuration pathway in rats.

BACKGROUND AND PURPOSE: Elevated plasma total homocysteine (tHcy) level has been established as an independent risk factor for cardiovascular diseases. Danshensu, an active ingredient of Salvia miltiorrhiza, shows wide cardiovascular benefit. However, in terms of its own methylation, danshensu could elevate tHcy level, which would act against its cardiovascular benefit, thus posing a 'therapeutic paradox'. As this paradox has not been fully assessed, we have evaluated the effects of danshensu on tHcy levels to uncover the underlying mechanisms. EXPERIMENT APPROACH: We evaluated the influence of danshensu on homocysteine (Hcy) metabolism in rats with normal tHcy levels and in rat models of elevated tHcy (single intravenous methionine loading model and a hyperhomocysteinemic model after 3 weeks methionine dosing, with and without 3 weeks of danshensu treatment). We also quantified some metabolic intermediates (S-adenosyl methionine, S-adenosyl-l-homocysteine, cysteine and glutathione) relevant to Hcy metabolism in rat liver and kidney. KEY RESULTS: Acute treatment with a single dose of danshensu in rats with normal tHcy did not change plasma tHcy. In contrast, danshensu significantly lowered tHcy in rats with elevated tHcy. The relatively higher cysteine and glutathione levels after treatment with danshensu indicated that its tHcy-lowering effect was via increased activity of the trans-sulphuration pathway. CONCLUSIONS AND IMPLICATIONS: Our results suggested that danshensu may act both acutely to increase trans-sulphuration and after chronic exposure to up-regulate the activity of the trans-sulphuration enzymes. The tHcy-lowering effect of danshensu is another cardiovascular benefit provided by S. miltiorrhiza and suggests a potential tHcy-lowering therapy.

T-cell anergy and peripheral T-cell tolerance.

The discovery that T-cell recognition of antigen can have distinct outcomes has advanced understanding of peripheral T-cell tolerance, and opened up new possibilities in immunotherapy. Anergy is one such outcome, and results from partial T-cell activation. This can arise either due to subtle alteration of the antigen, leading to a lower-affinity cognate interaction, or due to a lack of adequate co-stimulation. The signalling defects in anergic T cells are partially defined, and suggest that T-cell receptor (TCR) proximal, as well as downstream defects negatively regulate the anergic T cell's ability to be activated. Most importantly, the use of TCR-transgenic mice has provided compelling evidence that anergy is an in vivo phenomenon, and not merely an in vitro artefact. These findings raise the question as to whether anergic T cells have any biological function. Studies in rodents and in man suggest that anergic T cells acquire regulatory properties; the regulatory effects of anergic T cells require cell to cell contact, and appear to be mediated by inhibition of antigen-presenting cell immunogenicity. Close similarities exist between anergic T cells, and the recently defined CD4+ CD25+ population of spontaneously arising regulatory cells that serve to inhibit autoimmunity in mice. Taken together, these findings suggest that a spectrum of regulatory T cells exists. At one end of the spectrum are cells, such as anergic and CD4+ CD25+ T cells, which regulate via cell-to-cell contact. At the other end of the spectrum are cells which secrete antiinflammatory cytokines such as interleukin 10 and transforming growth factor-beta. The challenge is to devise strategies that reliably induce T-cell anergy in vivo, as a means of inhibiting immunity to allo- and autoantigens.

CD152 ligation by CD80 on T cells is required for the induction of unresponsiveness by costimulation-deficient antigen presentation.

Two apparently contradictory observations have been made concerning peripheral T cell tolerance; costimulation-deficient Ag presentation leads to unresponsiveness, and CTLA4 (CD152) ligation is required for unresponsiveness to be induced. This issue was addressed using a CD80- CD86low B cell line to present Ag to DO.11.10 naive CD4+ T cells. Proliferation was substantially enhanced by anti-CD80 or anti-CD152, but was inhibited by anti-CD86. Furthermore, anti-CD80 partially, and anti-CD152 totally protected cloned DO.11.10 T cells from the induction of unresponsiveness following culture with peptide and Chinese hamster ovary H2-Ad+ CD80- CD86- cells. Fab of anti-CD80 caused similar enhancement, and coimmobilized anti-CD80 failed to costimulate the anti-CD3 response of purified T cells, indicating that direct signaling by anti-CD80 was not responsible for these effects. The possibility that anti-CD80 liberated CD28 molecules that were sequestered by the T cell-expressed CD80, enabling them to coaggregate with TCR:CD3 complexes was excluded by finding that anti-CD80 and anti-CD152 individually caused maximal enhancement, rather than having additive effects. These data suggest that T cell-expressed CD80 has a regulatory function and plays a key role in the induction of unresponsiveness due to costimulation-deficient Ag presentation by the ligation of CD152 on neighboring, or even the same, T cell.

Anergic T cells inhibit the antigen-presenting function of dendritic cells.

The phenomena of infectious tolerance and linked-suppression are well established, but the mechanisms involved are incompletely defined. Anergic T cells can inhibit responsive T cells in vitro and prolong skin allograft survival in vivo. In this study the mechanisms underlying these events were explored. Allospecific mouse T cell clones rendered unresponsive in vitro inhibited proliferation by responsive T cells specific for the same alloantigens. The inhibition required the presence of APC, in that the response to coimmobilized anti-CD3 and anti-CD28 Abs was not inhibited. Coculture of anergic T cells with bone marrow-derived dendritic cells (DC) led to profound inhibition of the ability of the DC to stimulate T cells with the same or a different specificity. After coculture with anergic T cells expression of MHC class II, CD80 and CD86 by DC were down-regulated. These effects did not appear to be due to a soluble factor in that inhibition was not seen in Transwell experiments, and was not reversed by addition of neutralizing anti-IL-4, anti-IL-10, and anti-TGF-beta Abs. Taken together, these data suggest that anergic T cells function as suppressor cells by inhibiting Ag presentation by DC via a cell contact-dependent mechanism.

Critical role of costimulation in the activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro.

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.

Anergic T cells act as suppressor cells in vitro and in vivo.

The potential suppressive effects of allospecific anergic T cells were investigated both in vitro and in vivo. Allospecific T cells were rendered unresponsive in vitro using immobilized anti-CD3 mAb. These anergic T cells profoundly inhibited proliferation of responsive T cells in an antigen-specific manner. The observed inhibition did not appear to be due to the release of inhibitory cytokines in that secretion of IL-2, IFN-gamma, IL-4, IL-10 and TGF-beta was greatly reduced following the induction of anergy, and neutralizing mAb specific for IL-4, IL-10 and TGF-beta failed to reverse the inhibition. Furthermore, the suppression mediated by anergic T cells required cell to cell contact. In vivo, adoptive transfer of anergic T cells into recipients of allogeneic skin grafts led to prolonged skin graft survival. Consistent with the lack of inhibitory cytokine production by the anergic cells, prolongation of skin allograft rejection was not influenced by the simultaneous administration of a neutralizing anti-IL-4 antibody. These results indicate that anergic T cells can function as antigen-specific suppressor cells both in vitro and in vivo.

T:T antigen presentation by activated murine CD8+ T cells induces anergy and apoptosis.

Using an IL-2-secreting, noncytolytic, H-Y-specific, CD8+ T cell clone, the functional consequences of Ag presentation by T cells to T cells were investigated. Incubation of the T cells with H-Y-soluble peptide led to nonresponsiveness to Ag rechallenge. This was due to the simultaneous induction of apoptosis, involving approximately 40% of the T cells, and of anergy in the surviving cells. These effects were strictly dependent upon bidirectional T:T presentation, in that exposure of C6 cells to peptide-pulsed T cells from the same clone induced proliferation but not apoptosis or anergy. The inhibitory effects of T:T presentation were not due to a lack of costimulation, since the T cells expressed levels of CD80 and CD86 higher than those detected on cultured dendritic cells and equipped them to function as efficient APCs for primary CD8+ T cell responses. Following incubation with soluble peptide, CD80 expression increased, and high levels of CTLA-4 (CD152) expression were induced. Although addition of anti-CTLA-4 Ab augmented proliferation in response to soluble peptide, no protection from apoptosis or anergy was observed. Neither Fas nor TNF-alpha was expressed/produced by the C6 cells, and coligation of MHC class I molecules and TCR failed to reproduce the effects of T:T presentation. Taken together, these data suggest that T:T Ag presentation induces anergy and apoptosis in murine CD8+ T cells and may reflect the regulatory consequences of T:T interactions in the course of clonal expansion in vivo.

Immobilized anti-CD3 mAb induces anergy in murine naive and memory CD4+ T cells in vitro.

The induction of non-responsiveness in resting murine CD4+ T cells was investigated using immobilized anti-CD3 mAb. Incubation of freshly isolated CD4+ T cells with immobilized anti-CD3 mAb led to apoptosis in 40-60% cells. The surviving cells were profoundly non-responsive to subsequent mitogenic stimulation. The non-responsive state was characterized by a lack of IL-2 production and hyper-responsiveness to added IL-2, but was not explained by further activation-induced cell death. The induction of non-responsiveness was not due to modulation of the TCR-CD3 complex, and required partial activation of the T cells in that it was accompanied by an increase in cell size and was inhibited by addition of cyclosporin A. Finally, analysis of anti-CD3-mediated responses in naive and memory CD4+ T cells, separated on the basis of CD44 expression, showed that both naive and memory T cells have similar sensitivity to immobilized anti-CD3 mAb-induced activation, apoptosis and anergy. These results demonstrate that TCR-CD3 engagement on freshly isolated resting CD4+ naive and memory T cells, In the absence of co-stimulation, as achieved by plastic-immobilized anti-CD3 mAb, induces both anergy and cell death.

Development of functional rat-derived T cells in SCID mice engrafted with the fetal thymus of LEC rats which are defective in CD4+ T cells.

We reported that LEC rats are genetically deficient in the development of thymic CD4+8- cells and that this defect is caused by bone marrow (BM)-derived stem cells. To determine which BM-derived cells are responsible for the arrest of T-cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC-TG and LEA-TG mice, respectively). We than examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat-derived CD4+ T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC-TG mice. Furthermore, the peripheral LN cells in LEC-TG mice appeared to be functional. These cells produced IL-2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL-2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC-TG as well as LEA-TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC-TG mice closely resembled that of LEA rat thymus and that in LEA-TG mice, suggesting that normal T-cell differentiation occurred in LEC-TG mice. The results indicated that BM-derived progenitor T cells of LEC rats could differentiate to functional CD4+ T cells.

Dissociation between the mitogenic effect and antitumor activity of seed extract from Aeginetia indica L.

The extract from seeds of Aeginetia indica L. (AIL), a parasitic plant, induces potent antitumor immunity against Meth A fibrosarcomas in BALB/c mice. AIL also possesses a thymocyte co-stimulatory effect in vitro with suboptimal dose of Con A, a B cell mitogenic effect, and stimulates AIL-primed CD4+ T cells to produce Th1-type cytokines. In this study, we investigated the relationship between mitogenicity and antitumor activity with AIL. When AIL was analyzed by SDS-PAGE, there was strong and diffuse staining in the region between 14 kDa and the bottom of polyacrylamide gel and it was unaffected when AIL was digested with proteinase K (PK) before SDS-PAGE. Some bands with different molecular mass were also found in silver-stained gel and they disappeared completely by incubating AIL with PK before SDS-PAGE. The in vitro thymocyte co-stimulatory and B cell mitogenic effects were not influenced by digesting AIL with PK but were completely suppressed by the oxidation of AIL with sodium periodate before culture. In contrast, the in vivo antitumor activity was completely abolished by PK, but it was not affected by periodate oxidation. We generated mAbs specific for AIL and investigated the influence on the antitumor activity of AIL in vivo. Around 60-80% of tumor-bearing mice failed to recover from a challenge tumor when they were treated with supernatants isolated from mAb-induced precipitation reactions. Immunoblotting (Western blotting) revealed that all the mAbs reacted exclusively with a 50-60 kDa protein and that this reactivity was not influenced after oxidizing the blots with sodium periodate. We demonstrated that AIL contains polysaccharides and proteins. The polysaccharides induced B cell mitogenic and thymocyte co-stimulatory effects in vitro, while the proteins, especially a 50-60 kDa protein containing non-carbohydrate epitopes recognized by the mAbs, mediated antitumor activity in vivo.

Seed extract of Aeginetia indica L induces cytokine production and lymphocyte proliferation in vitro.

We previously reported that the extract of seeds from Aeginetia Indica L (AIL), a parasitic plant, induces potent antitumor immunity in tumor-bearing mice and that CD4+ T cells appear to be the main contributors in the induction of antitumor resistance. The present study was set up to investigate the in vitro effects of AIL on various lymphoid cells. Spleen cells from mice pretreated with AIL every 2 days for 1 week produced interleukin 2 (IL-2), interferon gamma (IFN gamma), tumor necrosis factor (TNF) and interleukin 6 (IL-6) when these cells were stimulated in vitro by AIL. Further, we found that CD4+ T cells were main producers of IL-2 and TNF upon the stimulation with ALL in vitro, while both CD4+ and CD8+ T cells secreted IFN. On the other hand, ALL was mitogenic in vitro to T enriched splenic lymphocytes as well as B enriched splenic lymphocytes. Moreover, AIL also proliferated thymocytes and this activity was potently synergistic with a suboptimal dose of concanavalin A (Con A). Lipopolysaccharide (LPS) contamination in AIL preparation was negligible since proliferative activity of AIL to B enriched splenic lymphocytes was not influenced in the presence of an endotoxin antagonist, polymyxin B sulfate (PMB). Further, B cell mitogenic activity of AIL seems to be mediated by different mechanism(s) from that of LPS since ALL could proliferate B enriched lymphocytes of C3H/HeJ mice which do not respond to the stimulation with LPS. A well known biological response modifier (BRM), Krestin (PSK), had no ability in inducing either T or B lymphocyte activation in vitro as shown by AIL.(ABSTRACT TRUNCATED AT 250 WORDS)

An extract of seeds from Aeginetia indica L., a parasitic plant, induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice.

The antitumor activity of an extract of seeds from Aeginetia indica L., a parasitic plant, was investigated. BALB/c mice, inoculated i.p. 1 x 10(5) syngeneic Meth A tumor cells, were administered 2.5 mg/kg A. indica extract i.p. every 2 days from day 0. The untreated mice died of an ascitic form of tumor growth within 21 days, whereas all the treated mice completely recovered from tumor challenge without any side-effects. The extract did not exert direct cytotoxic activity against Meth A in vitro. Mice that survived after the first challenge as a result of A. indica treatment overcame the rechallenge with homologous Meth A without additional administration of the extract. On the other hand, those mice could not survive after rechallenge with Meth 1 tumor cells, which were also established in BALB/c mice but were different in antigenicity from Meth A, suggesting the development of antigen-specific concomitant immunity in the A. indica-cured mice. In the induction phase of antitumor resistance in this system, CD4+ T cells appeared to be the main contributors, since in vivo administration of anti-CD4 mAb completely abolished such resistance. In contrast, anti-CD8 mAb administration did not influence the effect of A. indica. The importance of CD4+ T cells in antitumor immunity was again clarified by Winn assay; that is, spleen and lymph node cells depleted of CD4+ T cells in vitro prior to assay abolished antitumor activity on co-grafted Meth A tumor cells in vivo.