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T Miethke

Publications and source records attributed to T Miethke.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rapid clearance of the bacterial superantigen staphylococcal enterotoxin B in vivo.

Following bolus injection of the superantigen Staphylococcus enterotoxin B (SEB) into mice, ligand-reactive T cells are triggered to release toxic amounts of lymphokines. Subsequently, within 6 to 10 h ligand-reactive Vbeta8+ T cells become anergic and clonally expand and thereafter are deleted via apoptosis. Since the binding affinities of SEB to major histocompatibility complex (MHC) class II molecules are as low as 1.7 microM, the concentration of SEB in the fluid phase dictates the presentation via MHC class II molecules. Here, we study the pharmacokinetics of SEB in vivo and correlate pharmacokinetics with immunogenicity. We describe here how after a bolus injection of SEB, the superantigen becomes systemically distributed, with peak levels within 5 to 30 min in blood and in lymph nodes. Clearance occurs within 10 to 24 h, with the kidneys being a major route. To induce T-cell activation in vivo, SEB must be present in concentrations above 10(-4) microg/ml. These concentrations exist for only 15 h. Manifestation of functional outcomes such as anergy, clonal expansion, and clonal deletion begins after 24 h. We conclude that the SEB model system can be used to separate the phase of T-cell receptor ligation from the phase of manifestation of functional outcomes.

Animals↗
PubMed1995

Negative-acting factor and superantigen are separable activities of the mouse mammary tumor virus long terminal repeat.

The open reading frame contained within the long terminal repeat (LTR) of mouse mammary tumor virus encodes Naf, a negative regulator of transcription, as well as a superantigen activity, Sag, which causes the deletion of specific classes of T cells. In the present study, the effect of Naf expression on different promoters and the coding requirements for Naf and Sag have been investigated. Sag activity was found to require only sequences in the LTR, whereas sequences located within the gag gene were additionally required for functional Naf activity. Surprisingly, both the classic promoter and a recently described promoter located in the LTR can give rise to both functional Naf and Sag. Further analysis of Naf revealed that the downregulatory effect was mediated by sequences located in the LTR and that heterologous promoters were also affected by Naf.

Animals↗
PubMed1995

Bacterial superantigens induce T cell unresponsiveness in B cell-deficient mice.

Bacterial superantigens such as staphylococcal enterotoxin B (SEB) cause in vivo a profoud and long-lasting state of unresponsiveness in ligand-reactive T cells. To test whether presentation of SEB by small resting B cells to ligand-reactive T cells is essential for the induction of T cell unresponsiveness, we analyzed the effect of SEB in B cell-deficient mice. We observed T cell deletion and T cell unresponsiveness in both B cell-deficient mice and control mice. We conclude that presentation of SEB by resting B cells is not a prerequisite for the induction of T cell unresponsiveness in vivo.

Animals↗
PubMed1995

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo.

Clonal deletion and/or inactivation establishes tolerance to self antigens. Endogenous and exogenous (bacterial) superantigens, like the staphylococcal enterotoxins, induce ligand-specific clonal anergy in vivo and thus are believed to mirror aspects of post-thymic tolerance mechanisms in mature peripheral T cells. Here we analyzed the level of anergy of ligand-responsive V beta 8+ T cells from staphylococcal enterotoxin B (SEB)-primed mice in vivo and in vitro. Upon in vitro restimulation with SEB, CD4+V beta 8+ and CD8+V beta 8+ T cells failed to produce IL-2. However, functional IL-2 receptors were triggered, since supplementation with IL-2 induced clonal growth in virtually all CD4+V beta 8+ and CD8+V beta 8+ T cells as determined by limiting dilution analyses. Thus in vitro unresponsiveness of lymphocytes from SEB-primed mice reflects the inability of SEB-reactive V beta 8+ T cells to produce IL-2. Surprisingly, anergy as defined in vitro was at variance with that in vivo. Following further challenge with SEB, systemic and acute lymphokine production (including IL-2 and tumor necrosis factor) occurred with almost identical peak values and kinetics to primary in vivo responses, and D-galactosamine-sensitized mice succumbed to lethal shock. Polymerase chain reaction analyses revealed that CD4+V beta 8+ expressed IL-2-specific mRNA in vivo upon restimulation with SEB. While lymphokine production and expression of the IL-2 receptor was similar to the response to in vivo primary stimulation, only CD8+V beta 8+ T cells expanded clonally upon reintroduction of SEB in vivo. Hence primed V beta 8+ T cells challenged with SEB display in vitro anergy yet in vivo responsiveness, at least in part. We conclude that the state of anergy is reversible, dependent upon the quality of activation signals provided in in vivo rather than in in vitro culture conditions.

Animals↗
PubMed1995

Superantigens: the paradox of T-cell activation versus inactivation.

Superantigens are interesting for several reasons. They exhibit unusual T-cell activation characteristics, they allow to follow in vivo ligand-reactive T cells, they cause T-cell-mediated shock symptoms and they may be causally involved in certain human diseases. This review focuses on the paradox of T-cell activation versus T-cell inactivation as caused by bacterial superantigens in vivo. T-cell activation leads to the acute release of toxic concentrations of lymphokines including tumor necrosis factor alpha/beta, gamma-interferon, interleukin (IL)-2, IL-4 and IL-10. T-cell inactivation mirrors induction of anergy, T-cell receptor downregulation, and that of CD2, CD4 and CD8 cell surface molecules, as well as of apoptosis. Two waves of apoptosis cripple the T-cell repertoire of superantigen-reactive T cells, an immediate one being induced within 24 h and a late one occurring at days 3-4. In between, clonal expansion of anergic ligand-reactive T cells takes place.

Down-Regulation↗
PubMed1994

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship.

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive V beta 8+ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001-10 micrograms) V beta 8+ T cells become anergic within 6-16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001-0.1 microgram) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1-10 micrograms) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1-10 mg) about 50% of the anergic V beta 8+ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotype-negative but genotypically V beta 8+ T cells are generated. The T cell phenotype-negative cells reacquire their V beta 8+ T cell phenotype upon culture in vitro. In vivo, a subset of V beta 8+ cells, defined by an intermediate stage of TcR down-regulation, i.e. V beta 8lowCD3+ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.

Animals↗
PubMed1994

Serum levels of end products of nitric oxide synthesis correlate positively with tumor necrosis factor alpha and negatively with body temperature in patients with postoperative abdominal sepsis.

Nitric oxide (NO) has been implicated as the principal mediator of the catecholamine resistant vasodilation in septic shock. In this pilot study, we wanted to know if the serum values of nitrite/nitrate (NO2/NO3), the stable endproducts of NO biosynthesis, are elevated in patients with septic shock. Furthermore, we investigated whether there is a correlation between NO2/NO3 serum levels and tumor necrosis factor alpha or interleukin 6. NO2/NO3 serum values were significantly elevated in septic patients compared to controls (72.1 +/- 6.1 vs. 35.7 +/- 9.2 microM, p < .001). There was a significant positive correlation between serum values of NO2/NO3 and tumor necrosis factor alpha (rs = 0.59, p < .001). In contrast, no correlation between NO2/NO3 and interleukin 6 was found. With the exception of body core temperature, which showed a negative correlation with NO2/NO3 levels, no clinical variable turned out to be significantly related to NO biosynthesis. These data indicate a potential role for NO in the clinical course of abdominal sepsis, but points out that more specific data has to be evaluated by prospective clinical studies in order to understand the complex pathophysiologic role of this novel mediator.

Abdomen↗
PubMed1994

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.

Animals↗
PubMed1994

A transient assay for gene expression studies in B lymphocytes and its use for superantigen assays.

Superantigen assays are used to determine the stimulation of classes of T cells in response to the presence of a superantigen. This requires that antigen-presenting cells, such as B-lymphocytes, are co-cultured with syngeneic T cells over a four-day period. This prerequisite rules out conventional transient transfection procedures, since the cells must remain metabolically active for around five days. Consequently, stably transfected B cells have previously been used to test recombinant DNA constructs for superantigen activity. Here we present a protocol for the transient transfection of B cells that results in metabolically viable cells. Mouse mammary tumor virus (MMTV) encodes an endogenous superantigen. Using this transient transfection procedure, we show that a number of MMTV-based constructs give rise to functional superantigen activity. The ability to transiently transfect lymphocytes and maintain their viability should greatly facilitate studies of genes encoding products, such as superantigens, that can only be indirectly assayed on a second cell type as a result of expression in the recipient cell.

Animals↗
PubMed1993

Bacterial superantigens induce rapid and T cell receptor V beta-selective down-regulation of L-selectin (gp90Mel-14) in vivo.

Upon challenge of mice with bacterial superantigens such as staphylococcal enterotoxin B, several facets of TCR V beta-selective acute T-cell alterations can be observed, which include acute T cell priming, and systemic lymphokine release followed by ligand-specific unresponsiveness. Prompted by experiments showing that stimulation of T cells by phorbol esters in vitro results in rapid shedding of the L-selectin homing receptor, we investigated the expression of adhesion molecules on superantigen-responsive T cells in vivo. Here we show that bacterial superantigens cause TCR V beta-specific loss of L-selectin. Down-regulation of L-selectin was selective, since the expression of other lymphocyte surface receptors was not changed. L-Selectin down-regulation represents a superantigen-induced immediate cell surface alteration and was not observed on T cells stimulated by TCR-specific antibodies. Loss of expression was almost complete within 30 min, and recovered 50 h after challenge. The results suggest that acute loss of L-selectin is a hallmark of T cell activation by bacterial superantigens that may result in profound changes of T lymphocyte recirculation pathways.

Animals↗
PubMed1993

Endogenous superantigen expression controlled by a novel promoter in the MMTV long terminal repeat.

Endogenous superantigens are encoded by the open reading frame contained within the mouse mammary tumour virus long terminal repeat (MMTV LTR). Superantigen expression results in T-cell proliferation and, during early ontogeny, T-cell deletion. Here we identify a novel promoter located upstream of the previously described MMTV promoter. Transcripts from this promoter initiate within the U3 region of the MMTV LTR and splice to the acceptor for endogenous superantigen coding region. The novel U3 promoter is active in B lymphocytes, which are cognate antigen-presenting cells for endogenous superantigen, and is able to direct expression of superantigen in the absence of the previously described MMTV promoter.

Animals↗
PubMed1993

Acquired resistance to superantigen-induced T cell shock. V beta selective T cell unresponsiveness unfolds directly from a transient state of hyperreactivity.

TCR V beta selective T cell activation and systemic release of T cell-derived lymphokines causing lethal shock in D-galactosamine (D-Gal)-sensitized mice depicts only one facet of in vivo challenge with the superantigen staphylococcal enterotoxin B (SEB). An immediate second major aspect represents the induction of peripheral unresponsiveness in SEB-reactive V beta 8+ T cells. SEB causes in vivo within 4 h resistance to an otherwise lethal challenge with SEB plus D-Gal, as well as to a challenge with the heterologous ligand toxic shock syndrome toxin 1 plus D-Gal. Contrary to the first challenge, no serum-borne IL-2 and TNF are discernible during the second challenge. On the other hand, kinetic analyses in vitro of LN cells draining the site of the first in vivo challenge indicate that SEB-reactive T cells develop via a transient state of hyperreactivity into a profound state of ligand-specific unresponsiveness. Yet unresponsive V beta 8+ T cells express IL-2R and are responsive to the growth-promoting effect of IL-2. Cyclosporin A does not impair sequential induction of hyperreactivity and unresponsiveness with concomitant IL-2R expression, but effectively blocks systemic IL-2 and TNF release during the initial hyperreactive phase. Taken together, the in vitro data imply that ligand-specific hyperreactivity followed immediately by ligand-specific unresponsiveness represents a hallmark of in vivo challenge with the superantigen SEB. The in vivo data suggest the existence of additional suppressive elements masking the ligand specificity of the state of unresponsiveness induced by SEB.

Animals↗
PubMed1993

Clonal deletion as direct consequence of an in vivo T cell response to bacterial superantigen.

To date clonal deletion of peripheral mature T cells is restricted to in vivo model systems characterized by prolonged exposure of mice to antigens and clonal T cell expansion preceding clonal deletion. Here we describe that upon challenge of mice with the superantigen staphylococcal enterotoxin B two immediate events become imposed on ligand-reactive V beta 8+ T cells in lymph node cells draining the local site of injection. First, and within hours V beta selective clonal deletion is initiated via an apoptotic process. Second, the remaining V beta 8+ T cells first develop a profound state of ligand-specific unresponsiveness and subsequently initiate clonal in vivo growth. It is suggested that the dichotomy of events observed reflects a direct consequence of T cell receptor occupancy in the context of inappropriate signalling.

Animals↗
PubMed1993

Pathogenesis of the toxic shock syndrome: T cell mediated lethal shock caused by the superantigen TSST-1.

The pathogenesis of the toxic shock syndrome (TSS) is only incompletely understood. We now present evidence that TSS toxin-1 (TSST-1), one of the superantigens produced by Staphylococcus aureus, induces lethal shock in D-galactosamine sensitized mice. In this model TSS is dependent on T cells, since cyclosporin A (CsA) completely blocked development of shock, and since T cell-deficient SCID mice did not show signs of disease upon injection with TSST-1. However, SCID mice repopulated with T cells succumbed to lethal shock. The disease is characterized by a burst of lymphokines like interleukin-2 (IL-2) and tumor necrosis factor (TNF) released into the sera of TSST-1-treated animals. Already 1-2 h after TSST-1 application TNF serum levels peaked and IL-2 levels peaked around 4 h after treatment. TNF appears as key mediator of TSS, because anti-TNF monoclonal antibodies protected TSST-1-challenged mice. Interestingly, the burst of TNF in serum was noted well in advance of detectable markers of T cell activation. Thus, about 5% of all peripheral T cells started to express the IL-2 receptors as late as 4 h after treatment. Comparing TSST-1- and endotoxin-induced shock we conclude that TNF effects shock in both diseases. However, the type of cells involved appears distinct in that T cells cause TSS triggered by the exotosin TSST-1 while macrophages mediate the shock induced by endotoxins.

Animals↗
PubMed1993

Superantigen mediated shock: a cytokine release syndrome.

Treatment of animals with superantigens results in profound immunological changes. A major fraction of all peripheral T cells becomes activated in vivo. Subsequently, successive waves of cytokines are produced with TNF playing a central pathophysiologic role. In addition, if the liver is damaged by an as yet poor defined mechanism the consequences of the cytokine syndrome are life threatening. However, TNF alone is not sufficient to cause death, instead synergizing interactions with cytokines like IL-1, IL-6, and IFN-gamma are probably involved. On the other hand, certain experimental conditions prevent these waves of cytokines and consequently lethal shock. Furthermore, a significant fraction of SA reactive T cells are deleted by programmed cell death 10 to 24 hours after treatment. Thereafter the surviving cells proliferate vigorously until day 2 or 3, followed by a second wave of apoptosis resulting in reduced SA reactive T cell numbers as compared to pretreatment levels. Of course, many aspects of the complicated events are only marginally understood and deserve further investigation.

Animals↗
PubMed1993

Induction of unresponsiveness to the superantigen staphylococcal enterotoxin B: cyclosporin A resistant split unresponsiveness unfolds in vivo without preceding clonal expansion.

The continuous presence of antigen and powerful immune responses (exhaustive cell proliferation) of ligand reactive T cells are currently thought to condition clonal deletion and/or induction of unresponsiveness to endogenous or exogenous superantigens (SAg). Here we report that in vivo induction of unresponsiveness to the SAg staphylococcal enterotoxin B (SEB) can be an immediate process. Within ours a large portion of ligand reactive V beta 8+ T cells becomes clonally deleted by apoptosis. In parallel, the remaining V beta 8+ T cells are unresponsive to SEB, yet at the same time express functional IL-2 receptors (IL-2R) and thus are highly responsive to the growth promoting effects of IL-2. In a subsequent step refractory IL-2R+V beta 8+ T cells undergo a wave of cell proliferation for 48 h, presumably driven by IL-2. Thereafter a large proportion of V beta 8+ T cells succumb to apoptosis, the remaining cells display the hallmarks of split unresponsiveness, i.e. they display a selective failure to produce IL-2 upon SEB stimulation in vitro combined with a preserved capability to express functional IL-2R. Early deletion and induction of unresponsiveness to SEB are cyclosporin A (CsA) resistant, while clonal expansion with subsequent cell deletion is blocked by CsA, yet the development of split unresponsiveness is not impaired by CsA. The results suggest that IL-2 driven growth of refractory T cells may mimic powerful immune responses of ligand reactive V beta 8+ T cells. Since unresponsiveness to SEB precedes in vivo expansion, the results as such question the concept of 'exhaustive cell proliferation' as a prerequisite for induction of unresponsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor.

Because mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine- (D-Gal) sensitized mice as a model system to evaluate potential toxic shock symptoms triggered by the superantigen staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 micrograms SEB, respectively. The lethal shock triggered by the superantigen SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T cell-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL-2). Serum-borne lymphokines were detected well in advance of signs of T cell activation, as assessed by IL-2 receptor expression of SEB-reactive V beta 8+ T cells. Passive immunization with anti-TNF-alpha/beta-neutralizing monoclonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)- and exotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T cells. Yet the pathogenesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock.

Animals↗