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J. Biol. Chem., Vol. 275, Issue 25, 19282-19287, June 23, 2000

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Tumor Necrosis Factor- Production Is Differently Regulated in and Human T Lymphocytes^*

Virginie Lafont, Janny Liautard, Antoine Gross, Jean Pierre Liautard, and Jean Favero

From INSERM U431, Microbiologie et Pathologie Cellulaire Infectieuse, Université Montpellier 2, Place Eugène Bataillon, cc 100, 34095 Montpellier cedex 05, France

Received for publication, December 30, 1999, and in revised form, March 31, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tumor necrosis factor- (TNF-) plays a crucial role in the early defense against pathogens. This cytokine is produced by several cell types including T lymphocytes expressing the as well as the T cell receptor (TcR). In human, the circulating T cells, which mostly express V9V2 TcR, have been strongly suggested to play an important protective role against infectious agents. These activated cells early produce high amounts of TNF-, which induce a determinant beneficial effect against development of intracellular pathogens; however, sustained production of this cytokine can result in immunopathological diseases. The signals that regulate TNF- production in V9V2 T cells are totally unknown. In primary T cells, TNF- production was shown to necessitate engagement of the TcR and CD28, and to be independent of the p38 mitogen activated protein kinase pathway. We demonstrate herein that, in contrast to T cells, TNF- production in V9V2 T lymphocytes is independent of CD28 costimulation and highly dependent on TcR-induced p38 kinase and extracellular signal-regulated kinase 2 pathway activation for optimal cytokine release. Moreover, we bring elements supporting the idea that the "activation threshold" of T cells leading to cytokine production is lower than that of T cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TNF-¹ is crucial for the development of an early defense against many pathogens. This cytokine is produced by several cell types including monocytes/macrophages, lymphocytes, and mast cells. In monocytes, large quantities of TNF- are produced in response to bacterial endotoxin via activation of members of the mitogen-activated protein kinase (MAPK) family (1) including extracellular signal regulated kinase (ERK) (2, 3, 8), c-Jun N-terminal kinase (JNK) (4, 5, 9), and p38 MAPK (5-7). In T lymphocytes, TNF- production requires engagement of both the T cell receptor (TcR)·CD3 complex and CD28 antigen (10). However, little is known about the signaling pathways regulating expression of this cytokine in T cells. Recent results obtained with a T lymphoma cell line have shown that ERK, JNK, and p38 MAP kinase signaling pathways cooperate to mediate synthesis of TNF- (11). However, another report using human peripheral blood T cells has demonstrated that TNF- release was only modestly inhibited by the p38 inhibitor SB 203580 (10).

Among T cells, a minor population representing <10% of the circulating lymphocytes are T cells (see Ref. 12 for review); the majority of these cells express V9V2 TcR. The exact role of this subpopulation is not yet totally understood. However, the fact that their percentage dramatically increases in peripheral blood of patients infected by pathogens of viral, bacterial, or parasitic origin (13-23), suggest that these cells may be directly engaged in the response to infectious agents. A particular feature of these lymphocytes is that they are stimulated by nonpeptidic ligands in a major histocompatibility complex-unrestricted manner (24-29). Stimulation by such nonpeptidic molecules involves the T cell receptor (28). In vitro activation of human V9V2 T cells by nonpeptidic ligands rapidly induces a massive production of interferon- (30-33) and TNF- (30, 34). Early and high level TNF- production can have beneficial effects against development of intracellular pathogens; however, these TNF--induced beneficial effects are actually dependent on the strength and duration of its expression. An overactivation of the cells, leading to sustained high TNF- serum levels, could result in immunopathology (35). Indeed TNF- has been implicated in several diseases including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis (see, for review, Refs. 36-39). In this perspective, therapeutic potentialities for inflammatory diseases have been given to p38 kinase inhibitors, which blocked TNF- production in animal models (40, 41). Intracellular signals regulating TNF- production in V9V2 T cells are totally unknown.

In the present paper, we questioned whether TNF- production by V9V2 T cells requires, like in T cells, CD28-induced costimulatory signals, and whether the intermediary transducing molecules in T cells are different from those involved in T lymphocytes; particularly, we studied the possibility that p38 kinase which is poorly involved in the cytokine production by primary lymphocytes, could be considered as a major pharmacological target in V9V2 T lymphocytes. We show herein that, in contrast to what was demonstrated in T cells, TNF- production in V9V2 T cells is triggered through the T cell receptor complex independently of CD28 signaling; we also demonstrate that TcR·CD3-induced p38 and ERK-2 MAP kinase pathways are both essential for TNF- production. Moreover, we bring elements showing that signals triggered through the T cell receptor complex are more intense in V9V2 than in T cells supporting the idea already suggested by others (35) that the "activation threshold" of T cells leading to cytokine production is lower than that of T cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Reagents-- Recombinant IL-2 (rIL-2) was purchased from Chiron (Emeryville, CA). Isopentenyl pyrophosphate (IPP) was from Sigma, SB 203580 from Calbiochem Corp. (Nottingham, United Kingdom). PD 98059, anti-phospho-p38 MAP kinase antibody (Ab), anti-p38 MAP kinase Ab, anti-phospho-CREB-1 Ab, anti-CREB-1 Ab, anti-phospho-ATF-2 Ab, anti-ATF-2 Ab, and anti-phospho-p42/44 MAP kinase Ab were from New England Biolabs (Beverly, MA). Anti-ERK-2 Ab was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse Ab and anti-rabbit Ab were from Amersham Pharmacia Biotech (Paris, France). UCHT1 (anti-CD3 monoclonal antibody (mAb)), anti-TcR V2, anti-TcR V9, anti-TcR pan-, anti-CD28 mAb conjugated or not conjugated were purchased from Immunotech (Marseille, France). Unlabeled anti-CD28 was kindly provided by Dr. Olive (INSERM, Marseille, France).

Cell Culture-- Peripheral blood mononuclear cells were isolated from healthy donors. Human peripheral blood-derived T cells were generated as described previously (42) and maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and rIL-2 (20 ng/ml) at 37 °C in a 5% CO₂ humidified atmosphere. T lymphoblasts were allowed to return to quiescent state by washing three times and reculturing in RPMI 1640 with 10% serum in the absence of IL-2 for 48 h. T lymphocytes were purified from peripheral blood mononuclear cells by positive immunoselection, using anti-TcR V2 mAb and magnetic beads coated with anti-mouse IgG. After spontaneous detachment, T cells were specifically activated in presence of syngeneic monocytes, IPP (50 µM) and rIL-2 (20 ng/ml). Human peripheral blood-derived T lymphoblasts were generated as described above and maintained in RPMI 1640 supplemented with 5% fetal calf serum, 5% human AB serum, 2 mM glutamine, and rIL-2 (20 ng/ml) at 37 °C in 5% CO₂ humidified atmosphere for 4 or 5 weeks.

Preparation of Supernatants for Measurement of TNF- Production-- and T cells (2 × 10⁶ cells/ml) were cultured in 24-well tissue culture plates in RPMI 1640 supplemented with 10% FCS (for T cells) or 5% FCS + 5% human AB serum (for T cells) in a total volume of 0.5 ml/well. When mentioned cells were pretreated with inhibitors (SB 203580 or PD 98059) for 30 min at 37 °C at indicated concentrations, then stimulated with IPP, UCHT1 mAb, and/or anti-CD28 mAb at indicated concentrations. At different times, supernatants were harvested and assayed for TNF- using a human TNF- enzyme-linked immunosorbent assay kit (OptEIA set: human TNF-, PharMingen, San Diego, CA) according to the manufacturer's instructions.

Flow Cytometry-- 0.5 million cells were incubated with 10% human AB serum for 30 min. T cells were then stained with 1 µg of phycoerythrin (PE)-labeled anti-TcR V9 and fluorescein isothiocyanate-labeled anti-CD28; T cells were stained with 1 µg of PC5-conjugated anti-TcR pan- and PE-anti-CD28 in PBS supplemented with 10% FCS, 0.02% NaN₃, on ice in a total volume of 50 µl. After 30 min, the cells were washed once, fixed in 1% paraformaldehyde, and analyzed on a FACScalibur (Becton Dickinson) with Cell Quest software.

Cell Extract Preparation and Western Blot Analysis-- After stimulation, 5 × 10⁶ cells were lysed in 1 ml of lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM NaF, 10 mM iodoacetamide, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na₂VO₃, and 1 µg/ml each protease inhibitor (leupeptin, aprotinin, chymostatin). Proteins were concentrated by precipitation with 1.5 volumes of acetone. Proteins were separated by 10% SDS-PAGE (or 15% for visualization of ERK-2 shift) and then transferred to polyvinylidene difluoride membranes (Millipore), and detected with the indicated antibodies: anti-phospho- and pan-p38 MAP kinase Abs (1:1000), anti-ERK-2 Ab (1:5000), anti-phospho-p42/44 MAP kinase Ab (1:1000), anti-phospho- and pan-CREB-1 Abs (1:1000), anti-phospho- and pan-ATF-2 Abs (1:1000). Immunoreactive bands were visualized with the chemiluminescence Western blotting system (Amersham Pharmacia Biotech).

Measurement of Intracellular Calcium-- and T cells were suspended (5 × 10⁶ cells/ml) in RPMI 1640 without phenol red and loaded with 5 µl/ml of fura-2AM (1 mg/ml in Me₂SO) at 37 °C for 30 min. After washing, the cells were resuspended (2.5 × 10⁶ cells/ml) in RPMI 1640 without phenol red. Calcium measurement was done in a Hitachi F-2000 spectrofluorimeter set at 340 and 380 nm excitation and 560 nm emission wavelengths. The stimulating agent UCHT1 (1 µg/ml) was added directly in the cuvette. The maximum and the minimum fluorescence were measured after addition of 0.1% Triton X-100 and 1 mM EGTA, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TNF- Production by V9V2 T Cells-- Purified human peripheral blood-derived V9V2 T cells were stimulated through the TcR·CD3 complex either with UCHT1 (anti-CD3 mAb) or with IPP a specific V9V2-stimulating nonpeptidic phospholigand, and assayed for TNF- production. Fig. 1A shows that, at a concentration commonly used in previous studies, both stimulating agents induce cytokine synthesis; however, TNF- production is maximum after 3-6 h of CD3-stimulation, whereas with IPP, the maximum occurs after 16 h of activation. At the respective optimal incubation time, maximum production is reached at 1 µg/ml UCHT1 (Fig. 1B) and 20 µM IPP (Fig. 1C). It must be noticed that, even at low concentrations of anti-CD3 mAb (0.2 µg/ml) (Fig. 1B) or IPP (5 µM) (Fig. 1C), V9V2 T cell do not need any external costimulatory signal to produce TNF-.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   TNF- production by T cells. A, human peripheral blood-derived V9V2 T cells were stimulated or not by IPP (50 µM) or UCHT1 (2 µg/ml). After different times of stimulation as indicated, TNF- production was measured in the culture supernatants using an enzyme-linked immunosorbent assay kit. B, human peripheral blood-derived V9V2 T cells were stimulated for 6 h by different concentrations of UCHT1 ranging from 0.02 to 10 µg/ml; TNF- production was then measured in the culture supernatants. C, human peripheral blood-derived V9V2 T cells were stimulated by different concentrations of IPP ranging from 5 to 100 µM for 16 h, and then TNF- production was measured in the supernatants. Each experiment is representative of at least three.

CD28-independent TNF- Production in V9V2 T Cells-- In contrast to the preceding results, we confirmed that T cells need a CD28 costimulatory signal in addition to TcR·CD3 activation to produce TNF- (Fig. 2A), even when high doses of anti-CD3 mAb (10 µg/ml) are used (Fig. 2B). However, because B7 molecules, which normally interact with CD28, have been demonstrated to be expressed also on T cells (43), a possibility existed that TNF- produced in V9V2 T cells occurred upon a costimulatory signal via CD28 interacting with its counterreceptors present on neighboring T cells. To approach this particular point, we analyzed expression of CD28 on V9V2 T cells. Flow cytometry analysis showed that the expanded V9V2 T cell population we used in this study was 90% CD28-negative (Fig. 3), supporting the conclusion that TNF- production by V9V2 T cells is independent of CD28 stimulation (the same result was obtained with CD28-positive depleted cells; data not shown). We cannot, however, totally rule out the possibility that other self-costimulatory interactions in these cells may have contributed to TNF- production.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   TNF- production by T cells. A, human peripheral blood-derived T cells were stimulated either by UCHT1 (filled circles) (10 µg/ml) or by anti-CD28 (open circles) (10 µg/ml) or by UCHT1 (10 µg/ml) + anti-CD28 mAb (10 µg/ml) (triangles) for different times as indicated and then TNF- production was measured in the supernatants. B, human peripheral blood-derived T cells were stimulated either by different concentrations of UCHT1 (1-10 µg/ml) (filled circles) or by different concentrations of anti-CD28 mAb (1-10 µg/ml) (open circles) or by different concentrations of UCHT1 (1-10 µg/ml) + anti-CD28 (10 µg/ml) (triangles); after 16 h of stimulation, TNF- production was measured in the supernatants. Each experiment presented is representative of three.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Comparison of CD28 antigen expression by human peripheral blood-derived and T cells. Human peripheral blood-derived T cells were stained with both PC5-conjugated anti-pan-TcR mAb and PE-labeled anti-CD28 mAb; the cells were then analyzed by flow cytometry (right panel). Human peripheral blood-derived T cells were stained with both fluorescein isothiocyanate-conjugated anti-V9 TcR mAb and PE-anti-CD28 mAb, and analyzed by flow cytometry (left panel). Each analysis has been repeated at least three times.

TNF- Production by V9V2 T Cells Is Dependent on TcR·CD3-induced p38 Kinase Activation-- It was shown that the blockade of p38 MAP kinase by its specific inhibitor SB 203580 only slightly impaired TNF- synthesis in T lymphocytes (10), whereas it totally blocked it in monocytes. We therefore questioned whether p38 kinase might play a role in TNF- production in V9V2 T cells. We first demonstrated that p38 MAP kinase can be activated through TcR·CD3 ligation using anti-CD3 mAb. As can be seen in Fig. 4A, UCHT1 stimulation induces p38 phosphorylation/activation; after 5 min of stimulation, this activation reaches a plateau that lasts at least for 30 min. In parallel, we showed (Fig. 4B) that TNF- production induced upon anti-CD3 stimulation (left panel) is almost totally inhibited by 10 µM SB 203580 and totally blocked at 20 µM; at this latter concentration, TNF- release induced by IPP is also completely inhibited (right panel). In order to correlate inhibition of TNF- production with the blockade of p38 activity, we studied inhibition of phosphorylation of CREB-1 and ATF-2, two substrates of P38 kinase (44-46), by several concentrations of SB 203580. Fig. 4C shows that SB 203580-induced inhibition of TNF- production closely parallels that of p38 activity (IC₅₀ ~ 5 µM). A possibility existed that TNF- production in V9V2 T cells was regulated by the JNK pathway and that this pathway could have been inhibited by 20 µM SB 203580. We actually demonstrated that c-Jun, one of the JNK substrates that must be phosphorylated both at Ser-63 and Ser-73 for optimal transcription activity (47), was not phosphorylated on Ser-63 upon TcR·CD3 stimulation (data not shown). This result makes unlikely the involvement of JNK pathway in TcR·CD3-induced TNF- production by V9V2 T cells. Altogether, these data clearly indicate that p38 activation is a key event for TNF- production in V9V2 T cells.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Effect of SB 203580 inhibitor on TNF- production and activation of p38 MAP kinase in human peripheral blood-derived T cells. A, human peripheral blood-derived T cells were stimulated or not by UCHT1 for the indicated time. Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-p38 MAP kinase antibody (which specifically reveals the phosphorylated and active form of p38) and reprobed with an anti-p38 MAP kinase after Ab stripping. This experiment is representative of three. B, human peripheral blood-derived T cells were pre-incubated 30 min with different concentrations of SB 203580 inhibitor (1-100 µM) and then stimulated by UCHT1 (2 µg/ml) for 6 h. The production of TNF- were then measured in the supernatants (left panel). Human peripheral blood-derived T cells were pre-incubated 30 min with SB 203580 inhibitor (20 µM) and then stimulated by IPP (50 µM) for 16 h. TNF- production was then measured in the supernatants (right panel). This experiment is representative out of four. C, human peripheral blood-derived T cells were pre-incubated 30 min with different concentrations of SB 203580 inhibitor (0.1-20 µM) and then stimulated by UCHT1 (10 µg/ml) for 10 min. Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-CREB-1 Ab (a commercial Ab that cross-reacts with phospho-ATF-1) or anti-phospho-ATF-2 Ab, and reprobed with anti-pan-CREB-1 or anti-pan-ATF-2 Abs, respectively. These experiments were repeated twice.

TcR·CD3-induced Production of TNF- Is Dependent on MEK/ERK Activation Pathway in V9V2 T Cells-- In T cells, TNF- production is thoroughly dependent on the MEK/ERK pathway (10). We therefore studied whether activation of V9V2 T cells also led to activation of this pathway and whether this kinase cascade could be involved in TNF- production. As can be seen in Fig. 5A, stimulation of T cells through TcR·CD3 complex led to phosphorylation of ERK-2, as assessed by the appearance of a slowly migrating electrophoretic band. It had to be noticed that, when the cells are pretreated with PD 98059, the specific inhibitor of MEK1, the upstream kinase that phosphorylates and activates ERK-2, phosphorylation of ERK-2 is totally blocked, whereas it is not impaired by SB 203580, the inhibitor of p38 kinase (Fig. 5A).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of PD 98059 on TNF- production and activation of ERK-2 in human peripheral blood-derived T cells. A, human peripheral blood-derived T cells were stimulated or not for the indicated times by UCHT1 and were pretreated or not for 30 min in the presence or absence of SB 203580 (20 µM) or PD 98059 (20 µM) inhibitors. Total cellular proteins were separated on 15% SDS-PAGE and revealed by Western blot analysis using an anti-ERK-2 Ab. B, human peripheral blood-derived T cells were pre-incubated 30 min with different concentrations of PD 98059 inhibitor (1-100 µM) and then stimulated by UCHT1 (2 µg/ml) for 6 h. TNF- production was then measured in the supernatants (left panel). Human peripheral blood-derived T cells were pre-incubated 30 min with PD 98059 inhibitor (20 µM) and then stimulated by IPP (50 µM) for 16 h. TNF- production was then measured in the supernatants (right panel). Each experiment is representative of four.

To correlate activation of ERK-2 with TNF- production, we inhibited ERK-2 activation by pretreating the cells with PD 98059. As shown in Fig. 5B, 20 µM PD 98059 totally blocked production of TNF- induced either with UCHT1 (left panel) or IPP (right panel).

Comparison of Cell Signal Intensities Triggered via CD3 in and T Cells-- We demonstrated that in T cells CD3 stimulation alone is sufficient for TNF- production, whereas in T cells the cytokine release needs, in addition to CD3 stimulation, a costimulatory signal delivered via CD28. As already suggested by others, these data favor the hypothesis that the "activation threshold" of T cells might be lower than that of T cells. To approach this point, we compared activation of ERK-2 as well as calcium mobilization, two intracellular signals triggered via the T cell receptor complex in either or T cells. Fig. 6A shows ERK-1/ERK-2 activation (5 min) as a function of anti-CD3 concentration in both populations. To detect MAP kinase activation, we used a specific antibody that recognizes the active form of the kinase. As can be seen in the figure, for the same amount of detected ERK-2 protein, phosphorylation/activation of this kinase is detectable at 0.1 µg/ml UCHT1 stimulation and maximum at 0.2 µg/ml in T cells whereas, in T cells, activation is detectable only at 0.5 µg/ml and is maximum at 1 µg/ml. In parallel we tested calcium mobilization induced by optimal concentration of UCHT1. Fig. 6B shows that calcium response is 6 times higher in T cells than in T cells. These results are elements showing that at least some intracellular cell signals triggered via the T cell receptor complex are more intense in than in T cells. It is noteworthy that tyrosine phosphorylation electrophoretic profiles of total lysates from unstimulated and T cells are identical, ruling out the hypothesis that higher signal intensities triggered in T cells could be due to a preactivated state of some transducing molecules as is the case in transformed cells (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Comparison of activation threshold of and T cells upon TcR·CD3 stimulation. A, human peripheral blood-derived and T cells were stimulated or not for 5 min by different concentrations of UCHT1. Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-p42/44 MAP kinase Ab (which recognizes the phosphorylated and active forms of ERK-1 and ERK-2), and reprobed with anti-ERK-2 Ab, after Ab stripping. B, measurement of calcium response upon UCHT1 stimulation in human peripheral blood-derived and T cells. UCHT1 (1 µg/ml) was added in the cuvette at the time indicated by the arrow. Each experiment has been repeated twice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present paper we show that, in contrast to T cells, TNF- production in V9V2 T cells does not need CD28-triggered signals. This result first supports the idea that human V9V2 T cells behave differently than other T cells from other species; indeed, it was demonstrated that mouse T cells necessitated a CD28-mediated costimulation for their activation (48). One of the elements we brought to assess that TNF- production is a CD28-independent process in V9V2 T cells, was to show that the cells we used were CD28-negative cells. Previous analysis of CD28 expression on human T lymphocytes led to contradictory results; indeed, on the one hand, it was shown that long term culture of human peripheral blood T cells do not express CD28 (49), whereas, on the other hand, other results have demonstrated that 40-60% of freshly isolated T cells do express CD28 (50, 51). This apparent discrepancy was explained by the fact that CD28 was progressively lost during culture (50). We actually confirmed this conclusion since we noticed that 40-50% of the freshly isolated V9V2 T cells did express CD28, whereas the expanded long term cultured cells we used in our study have become CD28 negative cells. In peripheral blood T cells, it was demonstrated that ligation of CD28, which is a prerequisite for TNF- production, induced activation of the p38 MAP kinase; however, blockade of this kinase only slightly impairs TNF- production. From such a result, it can be concluded that in primary T cells, ligation of CD28 induces signals in addition to p38 activation that are involved in TNF- release. A possibility exists that these putative co-signals may act with p38 to regulate TNF- production in cooperation with the TcR-induced signals, and that specific blockade of p38 has only a limited effect on TNF- release. A recent paper has demonstrated that in T lymphoma cell lines, ERK, JNK, and p38 pathways cooperatively contribute to transcription and synthesis of TNF-, and it has been shown that specific blockade of p38 has no effect on TNF- promoter activity and a mild effect on TNF- biosynthesis; in contrast, the blockade of the three pathways abolished TNF- promoter induction (11). In V9V2 T cells, we established that TNF- production is dependent on ERK-2 and p38 pathways triggered upon TcR/CD3 engagement, whereas JNK pathway is not involved. In parallel, we showed that the activation threshold appears to be lower in V9V2 T cells than in T cells, as already suggested by others (35). From these results, it can be hypothesized that the high intensity signals triggered in T cells can lead to cytokine production without the need of costimulatory signaling pathways, and therefore the TcR/CD3-induced activation of p38 kinase pathway, which is a prerequisite for TNF- production in V9V2 T cells, could be more sensitive to inhibition than it is in T cells. However, one can question why ligation of the TcR/CD3 complex by the same anti-CD3 antibody (UCHT1) used at the same concentration triggers different signals in or T cells. Indeed, we showed that the calcium response is about 6 times higher in than T cells, and a strong activation of ERK-2 occurs at lower concentrations in than T cells. Actually, the stronger intracellular signaling we observed in V9V2 T cells can be related to the observation that human peripheral blood lymphocytes express about twice more TcR·CD3 complexes than lymphocytes (52). Indeed, among the earliest biochemical events detected after TcR stimulation is the phosphorylation of immunoreceptor tyrosine-based activation motifs on cytoplasmic tails of CD3 and TcR subunits, which promotes the recruitment of ZAP-70 for its phosphorylation and activation. Evidence has been brought that these events are crucial in TcR signaling, and the strength of TcR signal transduction depends on the number of the transducing molecules involved (53, 54). These data are in line with a recent report, which demonstrated using enterotoxin superantigens, i.e. low affinity TcR ligands, that there is a cooperative activation of the TcRs, which increases the number of signal transduction molecules and therefore the magnitude of the intracellular signals (55). This could be an element explaining how non-major histocompatibility complex-presented small nonpeptidic molecules like IPP could trigger strong activation of T cells leading to production of high amounts of TNF-.

In conclusion, our data indicate that early and high production of TNF- by V9V2 T cells is independent on the CD28-induced costimulatory signals but highly dependent on TcR·CD3-induced activation of both MEK/ERK and p38 MAP kinase pathways. Evidence of the involvement of these two pathways in the regulation of TNF- production brings arguments supporting the development of therapeutic strategies involving MAP kinase inhibitors aiming at modulating release of the cytokine in a beneficial way.

	ACKNOWLEDGEMENTS

We thank Dr. Olive (INSERM, Marseille, France) for sending us anti-CD28 mAb and Dr. J. Dornand and Dr. Rouot (INSERM U431, Montpellier, France) for helpful discussion of the results.

	FOOTNOTES

^* This work was supported by the Fondation Singer Polignac (France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-467-14-42-44; Fax: 33-467-14-33-38; E-mail: favero@crit.univ-montp2.fr.

Published, JBC Papers in Press, April 6, 2000, DOI 10.1074/jbc.M910487199

	ABBREVIATIONS

The abbreviations used are: TNF-, tumor necrosis factor ; IPP, isopentenyl pyrophosphate; TcR, T cell receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated ERK kinase; Ab, antibody; mAb, monoclonal antibody; PE, phycoerythrin; rIL-2, recombinant interleukin-2; CREB-1, cyclic AMP response element-binding protein; ATF, activating transcription factor; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum.

	REFERENCES

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V. Jubier-Maurin, R.-A. Boigegrain, A. Cloeckaert, A. Gross, M.-T. Alvarez-Martinez, A. Terraza, J. Liautard, S. Kohler, B. Rouot, J. Dornand, et al. Major Outer Membrane Protein Omp25 of Brucella suis Is Involved in Inhibition of Tumor Necrosis Factor Alpha Production during Infection of Human Macrophages Infect. Immun., August 1, 2001; 69(8): 4823 - 4830. [Abstract] [Full Text] [PDF]