Biomedical subjects

J Grooten

Publications and source records attributed to J Grooten.

At least 37 records · Page 2Linked to original sources

PubMed1998

Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity.

Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)-inducing agent atractyloside of Percoll-purified mitochondria released a caspase-processing activity. Following detection of procaspase-11 processing, we further purified this caspase-processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase-11, caspase-1 is also readily processed by cathepsin B. Procaspase-2, -6, -7, -14 are weak substrates and procaspase-3 is a very poor substrate, while procaspase-12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin-permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z-VAD.fmk, z-DEVD.fmk and to a lesser extent by Ac-YVAD.cmk.

Amino Acid Sequence↗

PubMed1998

Cathepsin B-mediated activation of the proinflammatory caspase-11.

Members of the caspase (CASP) family of cysteine proteases can be subdivided in proapoptotic caspases and proinflammatory caspases. Whereas the apical activation pathways for the caspases that are involved in the execution of the apoptotic process are beginning to be understood, the pathways that lead to the activation of proinflammatory caspases are still largely unknown. Analysis of subcellular fractions for their ability to process and activate several caspases in vitro led to the identification of lysosomes as the source for a protease that could proteolytically activate the proinflammatory CASP-11. Although this lysosomal activity was sensitive to caspase inhibitors, affinity purification with the biotinylated broad spectrum caspase inhibitor z-VAD.fmk revealed the CASP-11 activating protease as cathepsin B. Activation of CASP-11 by cathepsin B as well as its sensitivity to several caspase inhibitors was further confirmed with purified proteases. Similar to the role of mitochondrial factors in the activation of proapoptotic caspases, our results suggest a potential role for lysosomes and cathepsin B as activators of specific proinflammatory caspases. In addition, the aspecific inhibition of cathepsin B by so-called specific caspase inhibitors implicates that results obtained with these inhibitors should be interpreted with care.

Animals↗

PubMed1998

Quiescence-inducing and antiapoptotic activities of IL-15 enhance secondary CD4+ T cell responsiveness to antigen.

IL-15 shows functional redundancy with IL-2 due to its usage of the beta and gamma c subunit of the IL-2R. Yet, the requirement of IL-15 for an IL-15R alpha chain for high affinity interaction and the separate cellular sources of IL-2 and IL-15 suggest divergent activities for both cytokines. We compared the growth-inducing and proapoptotic or antiapoptotic activities of IL-15 and IL-2 on mature CD4+ T lymphocytes in the presence or absence of TCR occupancy. We found that the nature of IL-15 activity was critically dependent on the activation status of the T cells. In the absence of TCR triggering, IL-15 did not exert the growth factor activity of IL-2, but induced a quiescent phenotype, characterized by maintenance of the cells in the G0/G1 phase of the cell cycle and down-regulation of CD25, CD71, and CD95 expression. In the presence of appropriate TCR engagement, the IL-15-induced quiescent T cells were resistant against TCR-induced cell death and proliferated strongly. IL-2-treated cells, on the contrary, were sensitized to cell death, resulting in a negative feedback on cellular expansion and weak proliferative responsiveness. Consecutive action of IL-15 during the distinct phases of an in vitro immune response markedly increased the cell output of a second antigenic stimulation, as compared with IL-2. These results imply that during immune reactivity in vivo, IL-15 may take over from the transiently available IL-2 the role of survival factor but not of growth factor, hence promoting the long term maintenance of resting, Ag-experienced CD4+ T cells.

Adjuvants, Immunologic↗

PubMed1998

Macrophages induce cellular immunity by activating Th1 cell responses and suppressing Th2 cell responses.

Differentiation of naive CD4+ T cells (Th0) into Th1 or Th2 cells determines whether antigen will raise a cellular or a humoral immune response. The maturation pathway chosen by the Th0 cell is often decisive for the outcome of disease and depends among others on the (co-)stimulatory attributes of the APC and the nature and abundance of cytokines provided by the APC and the microenvironment. In this study, we used macrophages, loaded ex vivo with antigen, for inciting Th0 activation and differentiation in vivo. The macrophages were derived from a clonal, immortalized population that both functionally and phenotypically expressed features characteristic of mature macrophages. Injection into syngeneic mice of IFN-gamma-treated, Ag-loaded macrophages induced a primary T cell response, indicated by the occurrence of a proliferative response in vitro after restimulation of splenocytes with Ag. Analysis of the accompanying cytokine secretion revealed high numbers of IFN-gamma-producing Th1 cells and only a few IL-4-secreting Th2 cells. This dominance of Th1 cells had functional implications, reflected in the high titer of Th1 cell-dependent IgG2 Abs and the absence of IgG1, characteristic of humoral immunity. Moreover, administration of Ag-loaded macrophages to mice with an ongoing Th1/Th2 response resulted in a complete suppression of IgG1 production, whereas IgG2 levels remained unaffected. These results demonstrate that macrophages exert APC activity in the organism, strongly skew primary responses to cellular immunity, and in addition suppress an already generated Th2-dependent humoral response, thus characterizing these cells as Th1-oriented APC.

Animals↗

PubMed1998

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

Amino Acid Chloromethyl Ketones↗

PubMed1998

The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region.

The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria.

Animals↗

PubMed1998

Induction of unresponsiveness to tumor necrosis factor (TNF) after autocrine TNF expression requires TNF membrane retention.

Tumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer.

Antigens, CD↗

PubMed1998

pH-dependent aggregation and secretion of soluble monomeric influenza hemagglutinin.

We previously reported the expression of soluble A/Victoria/3/75 (H3N2) hemagglutinin in insect cells and the molecular and immunological structure of an aggregated fraction, only observed in cell supernatant when expression was performed at low pH [23]. Here we report that besides this aggregated a monomeric and possibly a trimeric structure is detected in cell supernatant, irrespective of the pH of the medium. Evidence is presented that the aggregated fraction is generated out of monomeric HAOs molecules due to a low intracellular pH encountered during secretion.

Animals↗

PubMed1997

Differential role of calcium in tumour necrosis factor-mediated apoptosis and secretion of granulocyte-macrophage colony-stimulating factor in a T cell hybridoma.

The authors investigated the dependence on extracellular and intracellular free Ca2+ in the induction of apoptosis and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by tumour necrosis factor (TNF) in a rat/mouse T cell hybridoma PC60 R55/R75, using the Ca2+ chelators EGTA and BAPTA/AM, respectively. TNF-induced apoptosis still occurred in the absence of free Ca2+, while GM-CSF production required the continuous presence of Ca2+. The latter was also true for GM-CSF production driven by interleukin 1 (IL-1). The dependence on Ca2+ in the induction of GM-CSF, but not of apoptosis, was further confirmed by the inhibition of TNF- or IL-1-induced cytokine production by cyclosporin A or FK506, drugs that block the Ca2+/calmodulin-dependent protein Ser/Thr phosphatase calcineurin. This differential requirement for Ca2+ illustrates the partial functional redundancy between TNF and IL-1, showing the activation of cytokine gene expression through a Ca(2+)-dependent activation of calcineurin, and a Ca(2+)-independent activation of apoptosis, exerted solely by TNF.

Animals↗

PubMed1996

The oxidative metabolism of glutamine. A modulator of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fibrosarcoma cells.

Treatment of the mouse fibrosarcoma cell line L929 with tumor necrosis factor (TNF) induces necrotic cell death. A crucial step in the cytotoxic action mechanism of TNF involves perturbation of mitochondrial functions leading to the formation of reactive oxygen intermediates (ROI). L929 cells have energy requirements adapted to a high proliferation rate. Glutamine (Gln) is utilized as a major energy source and drives mitochondrial ATP formation, while glucose is mainly converted to lactate through glycolysis. We investigated the role of the bioenergetic pathways involved in substrate utilization on the cytotoxic action of TNF and established a link between Gln oxidation and TNF-induced mitochondrial distress. Omission of Gln from the medium desensitizes the cells to TNF cytotoxicity, while the lack of glucose in the medium does not alter the TNF response. Sudden depletion of Gln from the culture medium results in a sharp decline in mitochondrial respiration in the cells, which might explain the decreased TNF responsiveness. However, when L929 cells are adapted to long term growth under conditions without Gln, these so-called L929/Gln- cells have restored respiration, but they still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF responsiveness of L929 cells depends on bioenergetic reactions that are specifically involved in the oxidation of Gln. This is further confirmed by the desensitizing effect of specific inhibitors of these Gln-linked enzyme reactions on TNF cytotoxicity in the parental cells, but not in the L929/Gln- cells. Analysis of the induction of mitochondrial ROI formation by TNF in parental and L929/Gln- cells suggests that the effect of Gln on the sensitivity to TNF cytotoxicity involves a mechanism that renders the mitochondria more susceptible to TNF-induced mediators, resulting in enhanced ROI production and accelerated cytotoxicity.

Animals↗

PubMed1995

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

Animals↗

PubMed1995

Generation and biological characterization of membrane-bound, uncleavable murine tumor necrosis factor.

Tumor necrosis factor (TNF) is produced as a membrane-bound, 26-kDa proform from which the mature, 17-kDa TNF subunit is released by proteolytic cleavage. In order to compare the biological activity of membrane-bound versus soluble TNF, mutational analysis of potential cleavage sites in murine TNF was carried out. The biological activity was assessed after transfection in L929 cells. Deletion of the first nine codons of the mature part of the murine TNF gene still led to the production of secretable TNF, indicating alternative cleavage sites separate from the -1/+1 junction. However, an additional deletion of 3 amino acids, generating TNF delta 1-12, resulted in a membrane-bound form of TNF. Site-directed mutagenesis revealed Lys11 as the critical residue for alternative cleavage. Mutation of this residue to Glu in a TNF delta 1-9 mutant gave rise to uncleavable, membrane-bound TNF with biological activities similar to wild-type TNF. Induction of apoptosis, proliferation, or cytokine production by triggering of either 55-kDa or 75-kDa TNF receptors in appropriate cell lines occurred efficiently both with soluble and with membrane-bound TNF. The latter was, however, less active in the cytotoxic assays on U937 cells in which the 75-kDa TNF receptor is not signaling, but contributes to maximal TNF activity by ligand passing. This indicates that membrane-bound TNF cannot be passed from the 75-kDa to the 55-kDa TNF receptor.

Amino Acid Sequence↗

PubMed1995

Involvement of IFN-gamma in Bacillus Calmette-Guérin-induced but not in tumor-induced sensitization to TNF-induced lethality.

In healthy mice, murine (m) TNF is fairly lethal, whereas human (h) TNF (a selective murine TNF-R55 agonist) is rather harmless. However, we and others observed that mice suffering from a bacterial infection, such as Bacillus Calmette-Guérin (BCG), or bearing i.m. some types of tumor, develop a hypersensitivity to the IL-6-inducing and lethal properties of hTNF. This is a cardinal problem as it severely limits the potential use of hTNF-R55-specific agonists for systemic treatment of human cancer. Using mice carrying a targeted disruption in the gene encoding the IFN-gamma receptor (IFN-gamma Ro/o), we here report that endogenous IFN-gamma plays a crucial role in the development of TNF hypersensitivity during BCG infection. Indeed, both the lethality and the IL-6 induced by hTNF were drastically reduced in IFN-gamma Ro/o mice as compared with control mice. These results demonstrate that the enhancement of TNF effects is at least an equally important mechanism by which IFN-gamma contributes to BCG-induced hypersensitivity as the previously described augmentation of TNF production. Experiments in athymic nude mice, either depleted of NK cells or not, revealed that the latter cell population is an important source of the sensitizing IFN-gamma during BCG infection. In contrast, IFN-gamma Ro/o mice were as susceptible as control mice to the sensitizing effects of tumors. mTNF, which interacts with both mTNF-R55 and mTNF-R75 and causes lethality on its own, is as toxic in IFN-gamma Ro/o mice as in wt control mice; this means that TNF-induced IFN-gamma does not play a role in mTNF-induced lethality.

Animals↗

PubMed1995

Both TNF receptors are required for TNF-mediated induction of apoptosis in PC60 cells.

A rat/mouse T cell hybridoma (PC60) was transfected either with human (h) TNF-R p55 (TNF-R55), p75 (TNF-R75) or both cDNAs. hTNF-R55 expression was below 50 molecules/cell, whereas the number of hTNF-R75 reached about 4000 molecules/cell. Only cells co-expressing the two types of receptor showed TNF-dependent apoptosis, in contrast to cells expressing similar levels of only one receptor type, indicating that both TNF-R55 and TNF-R75 are required. Stable co-transfection of the bcl-2 proto-oncogene largely prevented this TNF-mediated induction of apoptosis. We found that a high level of hTNF-R75 expression was essential for obtaining TNF-dependent apoptosis in PC60 cells in addition to a low number of hTNF-R55. Both receptors are signal transducing because simultaneous triggering of hTNF-R55 and hTNF-R75 by agonistic mAbs or by TNF-R-specific TNF muteins induced similar levels of apoptosis as wild-type hTNF. Apoptotic killing of only those lymphocytes expressing a high, induced level of TNF-R75, in addition to TNF-R55, may play a physiologically important role.

Animals↗

PubMed1995

Cytotoxicity in L929 murine fibrosarcoma cells after triggering of transfected human p75 tumour necrosis factor (TNF) receptor is mediated by endogenous murine TNF.

We compared the biological function of the human tumor necrosis factor receptors p55 (hTNF-R55) and p75 (hTNF-R75) expressed in the murine (m) fibrosarcoma cell line L929. Receptor-specific triggering of hTNF-R55 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-R32WS86T, a hTNF-R55-specific mutant of hTNF, resulted in cytotoxicity. Specific clustering of hTNF-R75 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-D143F, a hTNF-R75-specific mutant of hTNF also induced cytotoxicity, albeit at low level. In both cases, the cytotoxic activity of receptor clustering could be synergized by addition of 20 mM LiCl. Remarkably, cytotoxicity induced after R75 triggering in transfected L929 cells could be completely abolished by addition of neutralizing anti-mTNF antibodies, in contrast to cell killing seen after specific R55 clustering. No soluble mTNF could be demonstrated using a sensitive biological assay, although L929 cells were expressing low levels of mTNF-specific mRNA as shown by PCR. These data clearly demonstrate that minute amounts of endogenously produced TNF can be a key mediator in R75-mediated cytotoxicity. Presumably, the latter efficiently traps the ligand and transfers it to TNF-R55, and/or by binding it, protects the endogenously made TNF from inactivation.

Animals↗

PubMed1994

Functional requirement of the two TNF receptors for induction of apoptosis in PC60 cells and the role of mitochondria in TNF-induced cytotoxicity.

The rat/mouse T-cell hybridoma PC60 was transfected either with hTNF-R55 cDNA, hTNF-R75 cDNA, or both. Receptor-specific stimulation was achieved using agonistic monoclonal antibodies or receptor-specific muteins of hTNF. Either hTNF-R55 or hTNF-R75 could mediate the activation of NF-kappa B and the induction of GM-CSF, IL-6, and IFN-gamma. But only in cells carrying both hTNF-R55 and hTNF-R75, was TNF able to induce apoptosis. This apoptosis could be inhibited almost completely by cotransfection with human bcl-2 cDNA. Functional cooperation was observed between liganded and unliganded receptors for the induction of apoptosis. In vitro protein kinase activity was detected only in TNF-R75 immunoprecipitates from cells in which the receptor was signaling. Direct evidence was obtained for reactive oxygen intermediates of mitochondrial origin responsible for TNF-induced cytotoxicity in L929 cells.

Animals↗

PubMed1993

Increased IL-6 production and IL-6-mediated Ig secretion in murine host-vs-graft disease.

BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.

Animals↗

PubMed1993

Cell membrane permeabilization and cellular collapse, followed by loss of dehydrogenase activity: early events in tumour necrosis factor-induced cytotoxicity.

Early events in the cytotoxic response to tumour necrosis factor (TNF) of the murine fibrosarcoma cell lines L929 and WEHI164cl13 were assessed on a cell by cell basis using the fluorescent exclusion dye propidium iodide (PI) and analysis by flow cytometry. The rationale of this approach is based on the exclusion of PI by cells with intact membranes. PI-positive cells appeared within a few hours of TNF treatment and further accumulated with time at a TNF dose-dependent rate. Thus, TNF rapidly caused a breakdown of the barrier function of the membrane in these TNF-sensitive fibrosarcoma cell lines. On a time basis, membrane permeabilization was immediately followed by a sudden shrinkage of the cell and was accompanied by cell death, but preceded the inactivation of the mitochondrial succinate dehydrogenase by several hours. The latter enzymatic activity was measured by the MTT chromogenic assay. Cell death was determined on the basis of the capability of individual cells to produce a progeny in a clonogenicity assay. Both membrane permeabilization and cellular collapse were fast events that were completed within a very short time and may represent the direct cause for cell death. Opposed to this, loss of mitochondrial succinate dehydrogenase activity evolved more slowly, was initiated at a later time and apparently represents a post-lethal event, not directly linked to the TNF signal transduction pathway. Finally, the enhancing effect of the protein synthesis inhibitor cycloheximide on the various features of TNF-induced cytotoxicity was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗