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The Journal of Immunology, 2000, 164: 1689-1694.
Copyright © 2000 by The American Association of Immunologists

TNF-{alpha} Is the Critical Mediator of the Cyclic AMP-Induced Apoptosis of CD8+4+ Double-Positive Thymocytes1

José A. Guevara Patiño2,*, Vladimir N. Ivanov2,3,*, Elizabeth Lacy{dagger}, Keith B. Elkon{ddagger}, Michael W. Marino§ and Janko Nikolic-Zugic4,*

* Immunology and {dagger} Molecular Biology Programs, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; {ddagger} The Hospital for Special Surgery, Weill Medical College of Cornell University, New York, NY 10021; and § Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Apoptosis is one of the key regulatory mechanisms in tissue modeling and development. In the thymus, 95–98% of all thymocytes die by apoptosis because they failed to express a TCR with an optimal affinity for the selecting intrathymic peptide-MHC complexes. We studied the possible role of two prominent nerve growth factor (NGF-TNF) family member systems, Fas ligand (FasL)-Fas receptor (FasR) and TNF-{alpha}-TNFR, in apoptosis of murine CD8+4+ double-positive (DP) thymocytes induced via TCR-CD3- and cAMP-mediated signaling. TCR-CD3{epsilon}-mediated apoptosis of DP thymocytes was found not to be dependent on either of the two systems. The FasL-FasR system was also found to be dispensable for the cAMP-mediated apoptosis. By contrast, cAMP agonists (dibutyryl-cAMP and forskolin) induced apoptosis via TNF-{alpha}, as evidenced by 1) the ability of anti-TNF-{alpha} mAbs to abrogate cAMP analogue-induced DP apoptosis in a dose-dependent manner; and 2) increased resistance of DP thymocytes from TNF-{alpha}-/- and TNFR I-/-II-/- animals to cAMP agonist-mediated apoptosis. cAMP agonists induced DP thymocyte death by a combination of two mechanisms: first, they induced selective up-regulation of TNF-{alpha} production, and, second, they sensitized DP thymocytes to TNF-{alpha}. The latter effect may be due to the down-regulation of TNFR-associated factor 2 protein. These results identify TNF-{alpha} as the critical mediator of cAMP-induced apoptosis in thymocytes and provide a molecular explanation for how the cAMP stimulators, including the sex steroids, may modulate T cell production output, as observed under physiological and pharmacological conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Apoptosis plays a critical role at several points in the life of a T cell. First, apoptosis weeds out useless and harmful thymocytes during T cell development. Second, apoptosis is in charge of maintaining mature T cell homeostasis in the course of an immune response by eliminating the majority of activated lymphocytes after the pathogen has been cleared. Finally, apoptosis is likely to be involved in maintaining peripheral T cell tolerance. Molecular events during the activation-induced cell death (AICD)4 of mature T lymphocytes occur in two phases. The first is initiated by primary TCR-mediated signaling pathways that activate preexisting transcription factors or induce the expression of new transcription factors. These factors regulate expression of many genes, including the genes encoding ligand-receptor pairs belonging to the nerve growth factor-TNF (NGF-TNF) and the corresponding receptor (NGFR-TNFR) families, many of which (e.g., Fas ligand (FasL):Fas receptor (FasR)-CD95: TNF-{alpha}:TNFR(I and II), and CD30 ligand:CD30) have been implicated in the regulation of T cell death (reviewed in Refs. 1, 2, 3, 4). The second phase of AICD begins with the interaction of these secondary ligands with their receptors, and culminates in the activation of preexisting proteins, including the caspase family of proteases (reviewed in Refs. 5, 6, 7) and endonucleases (8), that execute cell death.

Molecular details of thymocyte apoptosis are, by contrast, less well defined (3). Developing thymocytes (particularly CD8+4+ double-positive, or DP, cells) readily undergo apoptosis in response to not only TCR stimulation, but also when stimulated via cAMP, corticosteroid, and other pathways. These pathways may or may not share downstream signaling cascades (reviewed in Refs. 6, 9). Although thymocyte apoptosis remains one of the favorite models to study apoptosis, neither the involvement nor the identity of secondary apoptosis-effecting signaling cascades has been defined for any of the apoptotic pathways. Inhibition of thymocyte apoptosis by transcription and translation blockers argues in favor of the primary-secondary scenario. But definitive evidence for the obligatory involvement of the TNF-TNFR family members and their identification is missing.

Here, we studied TCR-CD3{epsilon}-mediated and cAMP-mediated apoptosis of DP thymocytes. The TCR-CD3{epsilon}-mediated apoptosis is an accepted model for negative selection by AICD, and cAMP-mediated apoptosis operates physiologically in the thymus in vivo, where it can be induced, among other stimuli, by sex steroids and ß-adrenergic stimulation. We focused on the TNF-TNFR family member systems: TNF-TNFR and FasL-FasR. Although both TNF-{alpha} and FasL, as well as their cognate receptors, are expressed in the thymus (10, 11, 12, 13, 14), thymic selection and apoptosis were not perturbed in mice with deficient expression-function of FasR-CD95, FasL, nor in any of the two TNFR knockouts or their combination (see Refs. 15, 16, 17, 18 and see below). Thus, if these molecules play a role in TCR-mediated apoptosis in vivo, this role is neither exclusive nor obligatory.

Thymocytes express high levels of cAMP (19, 20, 21), and a number of molecules (e.g., PGs PGE1 and PGE2; reviewed in Ref. 22) and receptors (e.g., the ß2 adrenergic, glucagon, and estrogen receptors; Refs. 22, 23, 24, 25, 26) that directly induce cAMP signaling are expressed in the thymus. The neurohumoral axis that operates via the cAMP pathway, and in particular the sex steroids, have long been known to negatively modulate thymic cellularity and T cell production output, as documented by transient thymic involution in physiological (e.g., pregnancy and menstrual cycle-estrus) and pharmacological or experimental (sex hormone therapy; castration, that leads to hypercellularity of the thymus) situations (reviewed in Ref. 2 7). cAMP signaling was also shown to block thymocyte maturation (28) and to regulate thymocyte adhesion (29). These changes affect immature cortical thymocytes and have fewer, if any, effects on mature T cells (27). In this study, we provide compelling evidence that TNF-{alpha} plays a fundamental role in apoptosis induced via the cAMP pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

C57BL/6.faslpr/+, C57BL/6.faslpr/lpr (lpr/+ and lpr/lpr, respectively, in the text), C57BL/6-Tnfr1-/- (17) and C57BL/6-Tnfr2-/- (18) (TNFRI- and TNFRII-, respectively, in the text) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). TNFR I-/-II-/- mice were obtained from the F2 offspring of the (TNFRI- x TNFRII-)F1 mice by breeding in the Memorial Sloan-Kettering Cancer Center Core Animal Facility. TNF-{alpha}- mice were described previously (30). All mice were used at 6–10 wk of age.

Thymocyte preparation, activation, and apoptosis detection

All experiments were performed in RPMI 1640 medium supplemented with pyruvate, 2-ME, L-glutamine, antibiotics, and 7.5% FBS (RP 7.5). CD4+CD8+ DP thymocytes were enriched from total thymocytes by "panning" as described previously (31). DP thymocytes were treated (unless otherwise indicated) with anti-CD3{epsilon} (145-2C11; PharMingen, San Diego, CA) mAb (10 µg/ml), forskolin (10 µM), dibutyryl (db)-cAMP (10 µM), or dexamethasone (1 µM) (Sigma, St. Louis, MO) with or without 50 µM benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk; Calbiochem, La Jolla, CA) in 24-well flat-bottom plates. Anti-mouse TNF-{alpha} Abs (neutralizing) were obtained from PharMingen (clone MP6-XT22) or Upstate Biotechnology (catalogue no. 05-168; Lake Placid, NY) were added as indicated. Following overnight incubation, apoptosis was assessed by quantifying the percentage of hypodiploid nuclei undergoing DNA fragmentation (32) or by monitoring the inversion of phosphatidylserine to the outer leaf of the plasma membrane by annexin V staining in the presence of propidium iodide (PI) (performed according to the manufacturer’s instructions, TACS annexin-V-FITC kit; Trevigen, Gaithersburg, MD). Flow cytometric analysis was performed on a FACScan flow cytometer using Lysis II or CellQuest 3.1 software (Becton Dickinson, Mountain View, CA), by analyzing 5 x 103 cells/sample, using wide scatter gates, to include late apoptotic cells. For the sake of simplicity, the results are reported as a percentage of total annexin+ cells, of which <23% (range, 9.3–22.7%) was also PI+ (i.e., late apoptotic or necrotic). Mean values ± SD of at least three samples per group are reported.

ELISA for the detection of secreted TNF-{alpha}

A TNF-{alpha} capture ELISA was used to detect secreted TNF-{alpha} in cell culture supernatants from DP thymocytes incubated in the presence or the absence of 10 µM forskolin and 10 µg ml-1 of plate-bound mAb 2C11 (18 h at 37°C), respectively. Wells of polyvinyl chloride plates (Immulon 4; Fisher Biotech, Pittsburgh, PA) coated with 10 µg ml-1 of mAb anti-TNF-{alpha} (PharMingen) in carbonate coating buffer (pH 9.6) and blocked with 2% FCS-PBS were incubated with 200 µl of cell culture supernatant. The plates were then washed and an optimal concentration of biotinylated anti-TNF-{alpha} polyclonal Ab (PharMingen) was added and incubated for 1 h at 37°C. Bound TNF-{alpha} was detected using avidin-conjugated HRP (Pierce, Rockford, IL), developed using the substrate o-phenylenediamine dihydrochloride. The developing reaction was stopped by adding 50 µl of 3 M H2SO4, and the OD at 490 nm was analyzed by a MCC/340 Multiskan microplate reader (Fisher Biotech). The assay was conducted in quadruplicate. Results are reported as mean values (±SD).

Northern blot analysis

Total RNA (~10 µg) extracted from DP B6 thymocytes after an 8-h incubation in the presence of complete medium, 10 µM forskolin, and plate-bound anti-CD3{epsilon} mAb at 10 µg ml-1, respectively, was electrophoretically fractionated in a 1% agarose formaldehyde gel and then transferred to a nylon membrane (Ambion, Austin, TX). A 260-bp [32P]dCTP probe was generated using PCR primers specific for the partial length cDNA templates of TNF-{alpha}. Probe was applied to the blot, with hybridization occurring at 65°C for 16 h. RNA loading was confirmed by the intensity of the 18S RNA bands. Autoradiography of the blot was performed at -70°C for 72 h on Kodak X-OMAT-AR (Eastman Kodak, Rochester, NY). Optic densitometric analysis of TNF-{alpha} mRNA was standardized according to the RNA 18S OD value using the GS-700 Imaging densitometer (Bio-Rad, Hercules, CA) and the accompanying bimolecular analysis software.

Western blot analysis

B6 DP thymocytes were harvested after an 18-h incubation with increasing concentrations of forskolin and db-cAMP. Total cellular extracts were subjected to electrophoresis under nonreducing conditions on a 12.5% polyacrylamide gel (Pharmacia Biotech, Piscataway, NJ) before being electrophoretically transferred to a nitrocellulose (0.2-mm pore size; Schleicher & Shüll, Dasel, Germany). Blots were blocked in PBS containing 5% (w/v) BSA and probed with a 1/1000 dilution of rat anti-TNFR-associated factor 2 (TRAF-2) or anti-p34 antisera (Santa Cruz Biotechnology, Santa Cruz, CA). After washing three times in PBS containing 0.05% (v/v) Tween 20, bound Ab was incubated with goat anti-rat IgG conjugated to HRP (1/4000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ), developed, and visualized using the enhanced chemiluminescence technology (enhanced chemiluminescence system; Amersham Pharmacia Biotech).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
CD3-TCR and cAMP stimulation induce DP thymocytes apoptosis in normal and lpr mice

In this study, we investigated the role of FasL-FasR and TNF-TNFR in the induction of apoptosis by TCR and cAMP pathways as models of Ag-mediated negative intrathymic selection and of neurohumorally induced thymocyte death, respectively. The ability of the TCR agonist anti-CD3{epsilon} mAb and the cAMP agonist forskolin to induce apoptosis in overnight-cultured DP thymocytes is shown in Fig. 1GoA. This time point was elected because it allows an accurate assessment of apoptosis and a relatively acceptable spontaneous background. At later time points, in both normal and knockout mice used in this study, spontaneous apoptosis increases to 40–60%, disallowing the observation of specific effects of stimulators. Both stimuli induced profound DP apoptosis. Moreover, either forskolin or db-cAMP (cAMP analogue) induced apoptosis in a dose-dependent manner (Fig. 1GoB), demonstrating the equipotency of these two cAMP-agonists. (Although all experiments in this study were performed with both compounds with indistinguishable results, for the sake of brevity we elected to show the db-cAMP experiments only at certain critical points.) The fact that cAMP agonists caused substantial apoptosis of DP thymocytes was consistent with the effect of cAMP stimulators on thymic cellularity (33), but stood in contrast to the protective role of these compounds against the AICD of peripheral T cells (34, 35). As expected, cAMP-mediated apoptosis operated in a caspase-dependent manner, as this, like many other types of DP thymocyte apoptosis (6), could be inhibited by the specific caspase inhibitor (36) zVAD-fmk (Fig. 2Go).



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  		FIGURE 1. DP thymocyte apoptosis in response to TCR and cAMP stimulation. A, DP thymocytes from normal mice were treated for 18 h with immobilized anti-CD3{epsilon} mAb (10 µg/ml) or 10 µM forskolin. Apoptosis was determined by the PI method (29 ), and the percentage of cells with hypodiploid DNA, diagnostic of apoptosis, shown as determined by marker M1. B, A dose-dependent apoptotic effect of forskolin and db-cAMP on DP thymocytes. DP thymocytes in RP 7.5 were divided into equal aliquots and incubated at 37°C in the presence or absence of forskolin and db-cAMP at 0, 0.35, 0.75, 1.25, 2.5, 5, or 10 mM. Cell loss after an18-h incubation was evidenced by staining cells with annexin V and PI and expressed as a percentage of apoptotic (total annexin V+ cells). In this and all other experiments shown, the percentage of the PI+ annexin+ cells varied between 9 and 23% of the total annexin+ cells, with no systematic variation between the groups, and was not shown as it did not impact upon the interpretation of the results. Dotted line indicates spontaneous apoptosis. Results are representative of seven independent experiments.

 


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  		FIGURE 2. cAMP-mediated death is dependent on caspase activation. DP thymocytes from B6 mice were treated for 18 h with 10 µM forskolin in the absence or the presence of 50 µM zVAD-fmk, a specific caspase inhibitor. Apoptotic cells were detected as in Fig. 1GoA. Comparable results were obtained in another experiment.

 
We next investigated the involvement of Fas-dependent signaling in the two types of apoptotic death by stimulating DP thymocytes from normal and Fas-defective lpr/lpr B6 mice. Overall, stimulation with dexamethasone, PMA, ionomycin, anti-CD3{epsilon}, forskolin, and their combination induced comparable apoptosis in both normal and lpr/lpr thymocytes (data not shown), corroborating previous results (15, 37) and excluding the obligatory role of Fas signaling in these types of apoptosis in vitro.

cAMP agonists induce DP thymocyte apoptosis via TNF-{alpha}

To investigate the role of TNF-TNFR in cAMP- or TCR-dependent apoptosis, we initially used a neutralizing anti-TNF-{alpha} mAb (MP6-XT22) that was introduced to the DP thymocytes simultaneously with primary stimuli. Fig. 3Go shows that this Ab abrogated, in a dose-dependent manner, either db-cAMP or forskolin-mediated apoptosis in DP thymocytes, whereas the control rat IgG1 had no effects (data not shown). By contrast, TNF-{alpha} neutralization had no effect on DP apoptosis induced by dexamethasone (data not shown, but see Fig. 4Go). Furthermore, TNF-{alpha} neutralization had variable and inconclusive effects on apoptosis induced by TCR agonists (data not shown), consistent with the findings that this pathway may be dispensable for the induction of TCR-mediated apoptosis of thymocytes (17, 18, 37). The above data were confirmed in six separate experiments and using two different anti-TNF-{alpha} Abs (MP6-XT22 in Fig. 3Go; catalogue no. 05-168, Upstate Biotechnology; data not shown). These results strongly suggested that TNF-{alpha} is the key mediator of cAMP-mediated apoptosis.



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  		FIGURE 3. Anti-TNF-{alpha} blocking reverses the apoptotic activity of cAMP agonists in a dose-dependent manner. Assays were performed and results displayed as in Fig. 1GoB, except that 5 µM forskolin or db-cAMP was added and cells then incubated in the absence or the presence of increasing concentrations of mAb anti-TNF-{alpha} (MP6-XT22). Dotted line indicates spontaneous apoptosis observed during 18-h incubation in RP 7.5. Results are expressed as in Fig. 1GoB. Results are representative of three experiments.

 


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  		FIGURE 4. cAMP-mediated signaling induces DP thymocyte apoptosis via TNF-{alpha}. DP thymocytes from B6, TNFR I-/-II-/-, and TNF-{alpha}-/- were incubated for 18 h at 37°C with RP 7.5, 10 µM forskolin, or 1 µM dexamethasone. Apoptotic cells were scored as in Fig. 1GoB. Results are representative of three independent experiments. WT, wild type.

 
To test the relevance of the above observations by a genetic approach, we took advantage of the TNF-{alpha}-/- and TNFR I-/-II -/- knockout mice that have normal thymocyte numbers and phenotype, and are otherwise indistinguishable from normal counterparts (17, 18, 30). Of note, in DP thymocytes of these animals, db-cAMP- (data not shown) and forskolin-mediated (Fig. 4Go) apoptosis was inhibited by >70%. In contrast, as expected, dexamethasone-mediated apoptosis was not affected (Fig. 4Go). Although sex steroids (such as testosterone and estrogens, produced in the gonads) invariably induce the activation of cAMP signaling, corticosteroids (produced in the adrenal gland cortex) are known to induce apoptosis by a different mechanism, possibly via their nuclear receptor (8). A similar level of inhibition (62–81%) was observed in a dose titration experiment over a range of cAMP agonist concentrations (2–50 µM). In contrast, DP AICD induced by TCR agonists in DP TNFRI-/-II-/- thymocytes was indistinguishable from that observed in the wild-type controls (V. N. Ivanov, unpublished observations), corroborating the redundant role of the TNF-TNFR pathway in negative selection. These results definitively demonstrate that TNF-{alpha} is the central player in the induction of cAMP-mediated DP thymocyte death.

Mechanism of action of cAMP agonists

Mechanistically, the most straightforward explanation for the effect of cAMP agonists would be that they stimulate DP thymocytes to produce TNF-{alpha} that then kills them by fratricide or suicide. To test this hypothesis, we investigated TNF-{alpha} mRNA and protein production in stimulated DP cells. Northern blot analysis was conducted to investigate whether specific cAMP agonists could induce mRNA TNF-{alpha} up-regulation. The results reveal a 5- and 9-fold increase of TNF-{alpha} mRNA following cAMP agonist and anti-CD3{epsilon} stimulation, respectively (Fig. 5Go, A and B). These results speak to the effect of TNF-{alpha} mRNA induction by cAMP agonists. The remaining question, however, was whether this was translated into protein production. Several reports have shown that TNF-{alpha} is processed from the membrane-bound precursors into soluble effector molecules by a metalloproteinase (38, 39, 40, 41, 42). A TNF-{alpha} capture ELISA was conducted to assess whether such processing of TNF-{alpha} into a soluble form occurred following stimulation of DP thymocytes. Stimulation with either cAMP agonist or immobilized anti-CD3{epsilon} mAb of DP thymocytes induced processing of the membrane TNF-{alpha} form and releasing of TNF-{alpha} (Fig. 5GoC). We therefore conclude that cAMP agonist apoptotic activity is likely to be due to an increase of TNF-{alpha} protein. However, given the relatively unremarkable effect of TNF-{alpha} on unstimulated thymocytes (24, 43) and given that {alpha}TCR-induced apoptosis is not TNF-dependent despite large induction of TNF-{alpha}, it was likely that cAMP agonists may also facilitate DP apoptosis by other means.



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  		FIGURE 5. Forskolin-induced apoptosis is mediated by TNF-{alpha}. A, Northern blot analysis of DP B6 thymocytes incubated for 8 h in the presence of RP 7.5, 10 µM forskolin, and anti-CD3{epsilon} mAb at 10 µg/ml concentration, respectively. RNA loading was controlled by assaying the abundance of 28S and 18S RNA. Densitometric analysis was conducted using 18S to standardize loading. Corresponding ODs are graphically represented in B. C, The 18-h accumulation of soluble TNF-{alpha} following incubation of DP in the presence of RP 7.5, forskolin, or plate-bound anti-CD3{epsilon} mAb, as measured by a TNF-{alpha} capture ELISA, performed as described in Materials and Methods. All results are representative of a minimum of two experiments.

 
Recent studies have shown that inhibition or targeted deletion of the TNFR-associated factor TRAF-2 enhanced TNF-{alpha}-induced apoptosis of murine lymphocytes or fibroblast, suggesting that TRAF-2 may be responsible for recruitment of antiapoptotic proteins (44, 45, 46). To determine the relationship between TRAF-2 and cAMP analogue-induced apoptosis, we studied TRAF-2 protein expression levels in DP thymocytes treated with cAMP agonists. Fig. 6Go shows that the expression of TRAF-2 in DP thymocytes from wild-type mice incubated with increasing amounts of forskolin and db-cAMP was impaired in a dose-dependent manner. These data suggest that cAMP agonists in DP thymocytes not only induce an increase of the TNF-{alpha} production, but also a decrease of TRAF-2 protein. The reduction of TRAF-2 protein was observed at cAMP agonist levels slightly higher than those inducing apoptosis in our assays. This may mean that the protein is functionally disabled by cAMP agonist treatment before its disappearance or that longer time periods are required for the decay to be evident (we measured TRAF-2 after 12 h in this experiment). Alternatively, it is possible that we are observing an unrelated epiphenomenon, and additional experiments will be required to address this issue. Regardless, there is an inversely proportional association between TRAF-2 and DP thymocyte cAMP-mediated apoptosis. These findings are in agreement with previous studies in which TRAF-2 has been determined to mediate antiapoptotic signaling (44, 45, 46).



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  		FIGURE 6. cAMP agonist-mediated down-regulation of TRAF-2 in DP thymocytes. Cellular extracts from DP thymocytes incubated with increasing concentrations of either forskolin or db-cAMP were analyzed by Western blot with an anti-TRAF-2 mAb. A decrease of TRAF-2 protein was observed concomitant with increasing concentrations of the cAMP analogue in a dose-dependent manner. Induction of a typical cAMP target protein, p34, is shown at the bottom.

 
It is well established that the mere presence of murine TNF-{alpha} has rather marginal effects on DP thymocyte apoptosis. How, then, do cAMP agonists exert apoptotic activity? The above data were consistent with the possibility that cAMP agonists both induce TNF-{alpha} secretion and sensitize DP thymocytes to its apoptotic effects, possibly by down-regulating TRAF-2. To address this possibility directly, recombinant murine TNF-{alpha} (rTNF-{alpha}) was added to DP thymocytes from TNF-{alpha}-/- mice in the presence or the absence of forskolin. DP cells from TNF-{alpha}-/- did not undergo apoptosis in the presence of forskolin or rTNF-{alpha} alone. Remarkably, DP thymocytes from TNF-{alpha}-/- underwent significant apoptosis in the presence of rTNF-{alpha} when incubated simultaneously with cAMP agonists (Fig. 7Go). These data indicate that cAMP agonists induce DP apoptosis by a two-pronged action: by inducing TNF-{alpha} secretion and by sensitizing DP thymocytes to the apoptotic effect of TNF-{alpha}.



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  		FIGURE 7. cAMP agonist sensitizes DP thymocytes to TNF-{alpha}. DP thymocytes from TNF-{alpha}-/- mice were incubated either individually or simultaneously with 10 ng/ml-1 rTNF-{alpha} and 10 µM forskolin. Dexamethasone was used as positive control. Neither rTNF-{alpha} nor forskolin alone induced apoptosis in DP thymocytes. However, incubation with rTNF-{alpha} and forskolin induced significant apoptosis as compared with control cells. Apoptosis was assessed as in Fig. 1GoB. Results are representative of two experiments.

 
The above results, like the previous reports (17, 18, 30), show that TCR-mediated apoptosis of DP thymocytes does not depend on the TNF-TNFR pathway. However, the increased levels of mRNA and soluble TNF-{alpha} detected following anti-CD3{epsilon} stimulation (Fig. 5Go) suggest that this pathway might still be one of the many redundant pathways that induces DP apoptosis in vivo. The other possibility is that the TNF-{alpha} activity on DP thymocytes is countered by a concomitant TNF-{alpha}-mediated activation of NF-{kappa}B. In several recent publications, an essential role of NF-{kappa}B in preventing TNF-{alpha}-induced death was established in RelA-/- cells or by inhibition of RelA in normal cells (47, 48, 49, 50). Consistent with these results, we observed that Bcl-2 protection against cAMP-induced apoptosis was always accompanied by RelA-p50 up-regulation (V. N. Ivanov, unpublished results). Of note, anti-CD3{epsilon} stimulation was accompanied by both TNF-{alpha} expression and NF-{kappa}B induction (data not shown), perhaps providing an explanation for protection against TNF-{alpha}-induced death.

The major conclusion from this study is that TNF-{alpha} acts as one of the main mediators of cAMP apoptosis in normal DP thymocytes. The fact that TNF-{alpha}-/- mice do not have enlarged thymi most likely reflects the action of compensatory mechanisms that frequently operate in knockout animals. In the normal mouse, where such mechanisms are not operative, TNF-{alpha} neutralization completely abrogates cAMP-mediated apoptosis (Fig. 3Go), whereas the gene disruption in TNF-{alpha}-/- DP cells inhibits this death by only 70%. Regardless of the existence and the importance of the compensatory mechanisms, the main conclusion of this work is that TNF-{alpha} plays a very prominent, and probably critical, role in mediating cAMP-induced apoptosis of DP cells. In the context of the extensive sympathic innervation of the thymus, the presence of neurohumoral receptors and high thymic cAMP cellular content (20, 21, 22, 23, 24, 25, 26), and the recent findings on the effects of cAMP on thymocyte development (28), our results elucidate perhaps the key molecular mechanism explaining the negative influence of the cAMP axis on thymocyte production-output, including the well-known phenomena of thymocyte depletion following stimulation with the sex steroids in pregnancy and menstrual cycle-estrus and thymocyte hypercellularity following experimental and pharmacological castration (Ref. 33 ; reviewed in Ref. 27). Physiological relevance of our findings is further confirmed by the demonstration that testosterone can mediate all of the effects ascribed to cAMP agonists in this study, and that it does so in a TNF-{alpha}-dependent manner (J. A. Guevara, M. W. Marino, V. N. Ivanov, and J. Nikolic-Zugic, manuscript in preparation).


   Acknowledgments
 
We thank Dragana Nikolic-Zugic for help with flow cytometry and Dr. L. J. Old for support.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants AI-32064 (to J.N.-Z.), P50 SCOR in SLE AR-42558 (to K.B.E.), and Memorial Sloan-Kettering Cancer Center Core Grant CA-08253 from the National Institutes of Health, the DeWitt Wallace Fund (to J.N.-Z.), and the Ludwig Institute (M.W.M.). Back

2 J.A.G.P. and V.N.I. contributed equally to this work. Back

3 Current address: Ruttenberg Cancer Center, Box 1130, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Back

4 Address correspondence and reprint requests to Dr. Janko Nikolic-Zugic, Box 98, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: Back

5 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; FasR, Fas receptor; db, dibutyryl; DP, double positive; TRAF-2, TNFR-associated factor 2; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PI, propidium iodide. Back

Received for publication September 10, 1999. Accepted for publication November 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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