In vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation.

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Human thymus contains IFN-alpha-producing CD11c(-), myeloid CD11c(+), and mature interdigitating dendritic cells.

Three distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-alpha upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-alpha/beta-producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin(-), HLA-DR(int), IL-3R alpha(hi), CD45RA(hi), CD11c(-), CD13(-), and CD33(lo)) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR(int), CD11c(+), CD13(+), CD33(+)), and the other represented HLA-DR(hi) CD11c(+) mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-T alpha, lambda-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.

Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7.

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.

Functional Fas expression in human thymic epithelial cells.

Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. We report that Fas antigen is constitutively expressed in medullary epithelial cells of the human thymus. Expression is decreased in cultured thymic epithelial cells (TEC), similarly to HLA-DR antigen. TEC are resistant to anti-Fas-induced apoptosis after 4 days of primary culture, and this resistance is reversed by concomitant addition of cycloheximide. Cycloheximide also downregulated the expression of Fas-associated phosphatase-1, which has been found to inhibit Fas-induced apoptosis. This phosphatase could be involved in the resistance to Fas-induced apoptosis observed on day 4 of TEC culture. When TEC were subcultured after 10 to 13 days of primary culture, exposure to interleukin-1-beta, tumor necrosis factor-alpha, and interferon-gamma, alone or together, reinduced Fas mRNA and protein expression. In coculture with activated thymocytes, TEC also upregulated Fas protein expression. Cytokine-activated TEC became sensitive to apoptosis induced by an agonistic anti-Fas antibody. This apoptosis was inhibited by Z-VAD-fmk but not by Z-DEVD-fmk and DEVDase activity was slightly increased in Fas-stimulated TEC, suggesting that DEVDase activity is not sufficient to induce TEC apoptosis. Taken together, these data show that the Fas receptor is expressed in medullary epithelial cells of the human thymus and is able to induce apoptosis.

Fas/APO-1/CD95 in health and autoimmune disease: thymic and peripheral aspects.

Fas is a relative of the tumor necrosis factor receptor super-family. The receptors of this family play an important role in decisions of survival and cell death by apoptosis. It has become clear that Fas has multiple roles in the regulation of the immune response. In the peripheral immune system, Fas/Fas ligand interactions control cell-mediated cytotoxicity, activation-induced cell death and could confer immune privilege. In the thymus, it may regulate apoptosis during negative selection in a thymocyte subset. Autoimmune diseases are often associated with deregulated Fas expression through different mechanisms: genetic defects, Fas-mediated cytotoxicity or up-regulation of expression as a result of lymphocyte activation. Thus, in both the peripheral and central compartments, the Fas system controls the balance between lymphocyte life and death and failure to keep this balance may contribute to autoimmune disease.

Two signaling pathways can increase fas expression in human thymocytes.

Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. Fewer than 3% of human thymocytes strongly express Fas. We report that Fas antigen expression can be upregulated by two signaling pathways in vitro, one mediated by anti-CD3 and the other by interleukin-7 + interferon-gamma. The two signaling pathways differed in several respects. (1) Fas expression increased in all thymic subsets after cytokine activation, but only in the CD4 lineage after anti-CD3 activation. (2) Fas upregulation was inhibited by cyclosporin A (a calcineurin inhibitor) in anti-CD3-activated but not in cytokine-activated thymocytes. (3) Cycloheximide (a metabolic inhibitor) inhibited Fas upregulation in cytokine-activated thymocytes but not in anti-CD3-activated thymocytes. (4) Cytokine-activated thymocytes were more susceptible than anti-CD3-activated thymocytes to Fas-induced apoptosis, a difference mainly accounted for by CD4(+) cells. The nature of the stimulus might thus influence the susceptibility of human thymocytes to Fas-induced apoptosis.

Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes.

Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.

Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis.

In the granule exocytosis pathway of cell-mediated cytotoxicity, rapid apoptotic nuclear damage in target cells has been unequivocally linked to granzyme B activity. Direct cleavage and activation of caspase-3 and related proteases by granzyme B have been identified as a central event in apoptosis induction by cytotoxic granules. The Bcl-2 oncoprotein has been recently shown to act at the level or upstream of caspase-3 family activation to inhibit apoptosis induced by various stimuli including Fas ligation, an alternative cell-mediated lytic pathway. In this study, we have investigated whether activation of this caspase family by granzyme B, during human NK and lymphokine-activated killer cell granule-mediated apoptosis, could be influenced by Bcl-2 expression. Bcl-2-overexpressing clones were generated from parental K562 and U937 cell lines (K6 and U4 clones, respectively). Bcl-2 expression abrogated early 125I-DNA release and DNA fragmentation, these defects being compensated for by extended incubation times. Cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 family substrate, was detected in parental K562 cells exposed to lymphokine-activated killer effectors but not in K6 targets, indicating that caspase-3 and related proteases function was inhibited by Bcl-2. Functional inhibition of caspase-3 family with benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe) fluoromethylketone led to similar consequences on apoptotic nuclear events as for Bcl-2 expression. Thus, Bcl-2 antagonizes granzyme B-mediated apoptosis by a mechanism that interferes with caspase-3 activity. Finally, Bcl-2 expression or the Asp-Glu-Val-Asp peptide was much less efficient in preventing phosphatidylserine externalization, suggesting that despite impaired nuclear apoptosis, immediate recognition and elimination of Bcl-2-expressing cells by tissue phagocytes should remain partly unaffected.

Phenotypic and immunohistological analyses of the human adult thymus: evidence for an active thymus during adult life.

We analyzed cellular content of thymic samples from 26 human healthy donors, ranging from 1 week postnatal to 49 years old. Our results showed that there was an overall decrease in cellular density, beginning early during life, but with two peaks of cellular density, at 9 months and 10 years of age. Histological and immunohistological analyses showed that variations in cellular density were correlated with the morphological changes observed during thymic involution, namely the enlargement of interlobular trabeculae and the development of adipocytic tissue. However, the adult thymus still contained thymocytes, up to 49 years. Phenotypic analysis showed no significant variations according to the age of donors in the distribution of the main thymocyte subsets, both precursors and more mature cells. These results suggest that the human thymus remains active during adult life.

Thymocyte Fas expression is dysregulated in myasthenia gravis patients with anti-acetylcholine receptor antibody.

Myasthenia gravis (MG) is a human autoimmune disease mediated by anti-acetylcholine receptor (AChR) antibodies. The thymus is probably the site where the autoimmune response is triggered and maintained. Recent reports have linked various autoimmune disease with defective Fas expression. We thus analyzed Fas expression in thymocytes and peripheral blood lymphocytes (PBL) from MG patients. The proportion of a thymocyte subpopulation with strong Fas expression (Fas(hi)) was markedly enhanced in MG patients with anti-AChR antibodies (P < .0003, compared with controls). In this group of patients, the proportion of CD4+ Fas(hi) and CD4+ CD8+ Fas(hi) thymocytes were significantly increased (P < .002 for both subsets). Fas(hi) thymocytes were enriched in activated cells and showed intermediate CD3 expression. They were preferentially Vbeta5.1-expressing cells, previously shown to be enriched in potentially autoreactive cells. The proliferative response of thymocytes from MG patients to peptides from the AChR was abolished after depletion of Fas(hi) cells. Fas(hi) thymocytes were sensitive to an agonistic anti-Fas antibody. In peripheral blood, Fas(hi) lymphocytes proportion was not significantly modified in MG patients whatever their anti-AChR antibody titer, compared with controls. Altogether, these results indicate that Fas(hi) thymocytes, which accumulate in MG patients with anti-AChR antibodies, could be involved in the autoimmune response that targets the AChR.

Cyclosporin A affects functions concerning acetylcholine release of cholinergic Torpedo synaptosomes.

The effect of cyclosporin A was investigated on Torpedo synaptosomes. Cyclosporin A inhibits KCl-evoked acetylcholine release (up to 50% at 1 mu M) and was inactive on acetylcholine release induced by a Ca2+ ionophore, A23187. Interestingly, when the synaptosomes were pretreated with cyclosporin A, this immunosuppressor did abolish the modulation of A23187-induced acetylcholine release produced by two other drugs, cetiedil (alpha-cyclohexyl-3-thienyl acetic acid 2-(hexahydro-1H-azepin-1-yl) ethyl ester, citrate salt) and MR16728 (N-(N'-hexamethylene imino)-propyl-phenyl-cyclohexyl-methyl acetamide, chlorhydrate), which were previously shown to be inhibitory and stimulatory, respectively. Moreover, cyclosporin A and MR16728 are competitive inhibitors of [3H]cetiedil binding to purified synaptosomal presynaptic membranes (dissociation constant of 181.9 nM). These results suggest that presynaptic proteins involved in acetylcholine release (directly or indirectly through cyclophilin) are potential targets of cyclosporin A in Torpedo synaptosomes.

Spontaneous release of acetylcholine from Torpedo synaptosomes: effect of cetiedil and its analogue MR 16728.

The effects of cetiedil and its analogue MR 16728 were examined on spontaneous acetylcholine release measured with a chemiluminescent assay using choline oxidase in a synaptosomal suspension obtained from Torpedo marmorata electric organ. Evoked acetylcholine release is inhibited by cetiedil, whereas this drug enhances spontaneous extracellular Ca(2+)-independent acetylcholine release (up to 340%). This effect was examined as a function of cetiedil concentration and incubation time. On the other hand, the analogue MR 16728, which enhances A23187-evoked acetylcholine release, also enhances spontaneous Ca(2+)-independent acetylcholine release. Cetiedil and MR 16728 effects on spontaneous acetylcholine release were also examined in the presence of Ca2+. Addition of Ca2+ enhanced spontaneous acetylcholine release by 75%, and cetiedil and MR 16728 stimulation was maintained but with different levels of enhancement. Thus, these results show that the processes responsible for evoked and spontaneous acetylcholine release are sensitive but in different ways to drugs of the cetiedil family.

Ciguatoxin extracted from poisonous moray eels Gymnothorax javanicus triggers acetylcholine release from Torpedo cholinergic synaptosomes via reversed Na(+)-Ca2+ exchange.

Ciguatoxin (CTX) (0.1 pM to 10 nM) added to a suspension of Torpedo synaptosomes incubated in Ca(2+)-free medium caused no detectable acetylcholine (ACh) release. However, subsequent addition of Ca2+ caused a large ACh release that depended on time of exposure, dose of CTX and on [Ca2+]. Tetrodotoxin completely prevented CTX-induced Ca(2+)-dependent ACh release. Simultaneous blockade of Ca2+ channel subtypes by FTX, a toxin extracted from the venom of the spider Agelenopsis aperta, omega-conotoxin and Gd3+ did not prevent ACh release caused by CTX, upon addition of Ca2+. These results suggest that CTX activates the reversed operation of the Na+/Ca2+ exchange system allowing the entry of Ca2+ in exchange for Na+. It is concluded that Torpedo synaptosomes are endowed with Na+ channels sensitive to pico- to nanomolar concentrations of CTX.

Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes.

The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FTX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FTX and synthetic FTX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FTX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FTX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using omega-conotoxin GVIA. At a concentration of 20 microM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FTX + omega-conotoxin GVIA were applied together, the inhibitory effect on KCl-evoked acetylcholine release was not significantly increased in comparison with the one observed with FTX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KCl depolarization and followed continuously with a chemiluminescent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of MR16728, a cetiedil analogue, on acetylcholine release in Torpedo synaptosomes.

MR16728, a cetiedil analogue, enhanced acetylcholine (ACh) release (up to 145% of control) from Torpedo synaptosomes when the release was triggered by a Ca2+ ionophore, A23187 or ionomycin, in the presence of 4 mM Ca2+ in the release medium, but inhibited ACh release induced by KCl depolarization of the presynaptic membrane. MR16728 also inhibited Ca(2+)-ATPase activity measured in purified synaptosomal presynaptic membranes. We studied the stimulation by MR16728 as a function of its concentration; the half-maximal effect was reached at the concentration of 13.5 microM. Moreover the stimulation was more pronounced (up to 300%) when a low concentration of Ca2+ (in the 10-micromolar range) was added to the medium. The enhancement of ACh release was also observed in proteoliposomes having incorporated mediatophore, a presynaptic membrane protein, able to release ACh in a Ca(2+)-dependent manner. Thus, mediatophore is a potential presynaptic target for MR16728.