Published online before print April 7, 2005
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* Department of Rheumatology, Division of Immunity and Infection, University of Birmingham, United Kingdom;
Department of Molecular Biology, University of Duisburg-Essen, Germany; and
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee
1 Correspondence: Department of Rheumatology, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. E-mail: L.D.Church{at}Bham.ac.uk
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(TNF-) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF- alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca2+]i) signal in Jurkat T cells after just 30 min exposure to TNF-, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca2+]i was also observed in human CD4+ T lymphocytes. The change in Ca2+ signal was related to a decrease in the plasma membrane Ca2+ influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca2+ influx. The role of TNF--induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF- led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca2+ response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca2+ responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF--induced inhibition of the Ca2+ influx in an ASM-deficient murine T cell line (OT-II+/+ASM–/–). This potent ability of TNF- to rapidly modulate the TCR Ca2+ signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.
Key Words: T lymphocyte • T cell receptor • signal transduction • cytokines • lipid mediators
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) is a pleiotrophic cytokine and a key mediator of inflammation. Since the early 1980s, TNF- has been studied, mainly for its proinflammatory and costimulatory effects on T cell function [1
2
3
4
]. However, as an understanding of immune regulation and chronic inflammatory disease has increased, it has become apparent that the effects of TNF- are more complex. Strong evidence has emerged demonstrating that TNF- mediates potent, negative immunomodulatory effects on T cell responses in addition to its proinflammatory activities. This is most evident in diseases associated with persistent inflammation such as rheumatoid arthritis (RA) [5
], Crohn’s disease, and systemic lupus erythematosis. In addition, chronic exposure of T cells to TNF- in vitro and in animal models has been shown to alter proximal and distal T cell receptor (TCR) signal transduction, impairing proliferation and cytokine production [6
7
8
]. Although the signaling pathways associated with the proinflammatory and costimulatory effect of TNF- are well-characterized, the mechanisms by which TNF- suppresses T cell function are yet to be defined fully [9
].
The biological effects of TNF- are transduced through two receptors: TNF receptor 1 (TNFR1; CD120a) and TNFR2 (CD120b) [10
]. These TNFRs are members of a much larger TNFR superfamily, which includes Fas, CD40, and TNF-related apoptosis-inducing ligand receptors. TNFR1 and TNFR2 share only a 28% homology, mostly in the extracellular domains; their intracellular sequences are largely unrelated, which accounts for the different signaling pathways regulated by each [11
]. TNFR1, unlike TNFR2, contains a so called "death domain" (DD), characteristic of most TNFR superfamily members. This region is responsible for the association of other DD-containing proteins involved in signaling for death by apoptosis [12
]. TNFR1 also contains an intracellular sequence that binds to the adaptor protein, factor associated with neutral sphingomyelinase (NSMase) activation, a key element in the activation of NSMase [13
]. TNFR1 is also reported to induce the activation of the acidic SMase (ASM), through DD-containing proteins [14
]. The generation of ceramide and ceramide-containing molecules, following activation of the SMase enzymes by members of the TNFR superfamily, has received much attention recently. In particular, the activation of Fas (CD95), a member of the TNFR family, has been shown to inhibit calcium (Ca2+) entry through the store-operated Ca2+ (SOC) channel known as ICa2+ release-activated Ca2+ current (CRAC) in T lymphocytes [15
]. Inhibition of Ca2+ entry was shown to be dependent on the hydrolysis of sphingomyelin (SM) in the plasma membrane by ASM, which generates secondary sphingolipids such as ceramide and sphingosine [16
, 17
] involved in several signaling cascades. This observation suggests that TNF- through TNFR1 might also be capable of inhibiting Ca2+ signaling in T lymphocytes following the activation of ASM.
Activation of T cells requires engagement of the TCR to induce an influx of Ca2+ ions. This influx is needed for proliferation to occur, and it also regulates differentiation into an effector T cell [18
]. The elevation in cytoplasmic intracellular Ca2+ ([Ca2+]i) is derived from two sources: intracellular and extracellular [19
]. The initial release of Ca2+ from intracellular stores follows a cascade of membrane proximal signaling, which results in the generation of the second messenger inositol 1,4,5 trisphosphate [20
21
22
], triggering the release of Ca2+ from internal stores by binding to its receptor, a Ca2+ release channel in the endoplasmic reticulum (ER) [23
, 24
]. In accordance with the capacitance entry hypothesis proposed by Putney [25
], the depletion of the intracellular stores rapidly triggers a sustained influx of Ca2+ through SOC channels in the plasma membrane, although the detailed mechanisms where this occurs are poorly understood. The magnitude and pattern of the Ca2+ signal have a crucial role in controlling activation of a large number of genes [26
] and thus, the subsequent functional responses made by the lymphocyte [19
]. For example, a sustained cytosolic rise in Ca2+, >500 nM, in T lymphocytes is required to induce interleukin (IL)-2 synthesis [27
]. Thus, modulation of the magnitude of the Ca2+ responses by TNF- can have a profound effect on lymphocyte gene activation.
To understand the mechanisms whereby TNF- may modulate T lymphocyte activation, we investigated the specific role of TNFR1 in regulating TCR signal transduction. We have examined the effects of TNF- on T lymphocyte [Ca2+]i signaling, as a broad measure of early upstream TCR signaling events. We have shown for the first time that the Ca2+ influx across the plasma membrane in T lymphocytes is impaired directly following exposure to TNF- and that this effect is independent of earlier upstream TCR signaling events. Furthermore, we show that the inhibition of Ca2+ entry is related to the generation of the sphingolipid second messenger ceramide, following ASM activation, as the inhibition of Ca2+ influx by TNF- was completely abrogated in an ASM-deficient (ASM–/–) murine cell line. Taken together, these results indicate an integral role for TNFR1 activation of ASM in the regulation of T lymphocyte activation by inhibition of Ca2+ influx through SOC channels.
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Peripheral blood (PB) CD4+ T lymphocytes were obtained from healthy donors. PB mononuclear cell preparations were isolated by Ficoll-Paque (Pharmacia Ltd.) density gradient centrifugation. Adherent cells were removed by incubation in serum-coated, plastic Petri dishes for 1 h at 37°C. Nonadherent cells were then incubated with saturating concentrations of monoclonal antibodies (mAb) to CD19 (RFB9, kindly provided by Royal Free Hospital, London, UK), CD14 (MON1040, Bradshire Biologicals Ltd., UK), CD16 (0813, Coulter, Luton, UK), CD8 (RFT8
1, kindly provided by Royal Free Hospital), and glycophorin (PharMingen, San Diego, CA). A two-stage, negative selection with sheep anti-mouse immunoglobulin G-coated Dynabeads M-450 (Dynal UK Ltd., New Ferry) was performed. This procedure yielded CD4+ T lymphocytes with a purity of >90% when analyzed by flow cytometry (Epics XL, Coulter). PB T lymphocytes were stimulated with 6.7 µg/ml phytohemagglutinin (PHA) plus 10 units/ml IL-2 and were kept in culture in complete medium for 4 days prior to investigation.
Murine OT-II/ASM+/+ and OT-II/ASM–/– T cell lines were generated from mice expressing the OT-II T cell receptor transgene {ovalbumin (OVA) peptide 326–339-specific, H-2Ab-restricted; ref. [28 ]}, crossed with mice lacking ASM [29 ]. These cell lines were initially generated by stimulating splenocytes [2.5x106 cells/ml in Minimum Essential Medium Eagle (SMEM) plus 10% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, 100 µM nonessential amino acids, 5 mM HEPES, 5.5x10–5 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin (cSMEM)] with 10 µM OVA peptide 326–339. After 3 days, the cells were washed and passaged at 2 x 105 cells/ml in cSMEM containing 1.5 ng/ml IL-2. Subsequently, the T cell lines (1x106) were cultured with 2 x 107 irradiated C57BL/6 splenocytes and 10 µM OVA peptide 326–339 for 48 h. T cell blasts were counted and cultured at 2.5 x 105 cells/ml. Medium was replaced every 2–3 days.
Murine splenocytes were obtained from ASM-positive or -negative mice, purified via Ficoll-density centrifugation, and incubated for 3 days with 10 µg/ml IL-2 and 10 µg/ml PHA to obtain lymphoblasts.
Analysis of cell-surface molecules by flow cytometry
PB CD4+ T lymphocytes isolated as above were resuspended at 1 x 106 cells/ml. Cells were then washed and incubated with blocking buffer [phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA)] followed by incubation at 4°C with fluorescein isothiocyanate (FITC)-conjugated mAb against CD4 (Dako Ltd., UK) at a saturating concentration. Cells were washed in PBS, and fluorescence was analyzed by flow cytometry, collecting 10,000 events per sample. Analysis of TNFR expression on Jurkat T lymphoma cells was performed in the same manner using FITC-conjugated mAb specific to TNFR1 and TNFR2 (Serotec, UK) at saturating concentrations.
Exposure of T lymphocytes to TNF-, SMase, and ceramide
Fresh medium was added to Jurkat T cells at 24 h prior to each investigation. The cells (1x106/ml) were cultured with TNF- (0–100 ng/ml; R&D Systems, UK) for varying times (30 min–3 h) at 37°C and under 5% CO2 in a humidified incubator. Human PB CD4+ T lymphocytes were resuspended at 1 x 106 cells/ml and exposed to 100 ng/ml TNF- for 3 h. Murine OT-II/ASM+/+ and OT-II/ASM–/– T cell lines were also resuspended at 1 x 106 cells/ml and exposed to 100 ng/ml human TNF- (hTNF-) for 3 h. As hTNF- is only recognized by murine TNFR1, these studies are therefore only specific for the TNFR1 signaling pathways.
Following this preincubation, the T lymphocytes were washed and loaded with the Ca2+ indicator, Indo-1 AM, and Ca2+ responses were measured by fluorimetry. Investigations of the effect of exogenous SMase and ceramide on Ca2+ signals in Jurkat T cells were performed on cells previously loaded with Indo-1 AM. Jurkat T cells were exposed to SMase (1–1000 mU, Sigma-aldrich, UK) for 30 min at room temperature prior to Ca2+ measurement. Exposure to exogenous ceramide was performed by adding the semisynthetic C2-ceramide (160–330 nM N-acetyl-D-sphingosine, Sigma-aldrich, UK) directly into the cuvette in the fluorimeter for 60 s prior to addition of the agonist to induce the Ca2+ response.
Ca2+ signaling
Following incubation with and without TNF-, T lymphocytes were washed twice and resuspended at 2 x 106/ml in RPMI-1640 medium. Indo-1 AM ester (Molecular Probes, Eugene, OR), dissolved in anhydrous dimethyl sulfoxide, was added to a final concentration of 1 µM Indo-1 AM ester in the medium. The cells were then incubated in the dark for 40 min at 37°C to allow dye entry. After three washes in 1.4 mM, Ca2+-containing Hanks’ balanced salt solution (HBSS), supplemented with 25 mM HEPES, 7.5% NaHCO3, and 10 mM glucose at a pH of 7.4, the cells were resuspended at 1 x 106/1.5 ml in Ca2+-containing HBSS and were kept protected from light for a further 15 min prior to use.
A total of 1.5 ml resuspended cells was placed in disposable acrylic cuvettes and prewarmed in a water bath at 37°C for 5 min prior to being placed in an LS50B spectrofluorimeter (Perkin Elmer, Emeryville, CA), equipped with a fast-filter accessory in the emission path and a magnetic cuvette stirrer. The FL data manager software (FL Winlab, Version 2.1, Perkin Elmer) was used to configure and set up the dual-emission experimental method required for Indo-1-loaded T cells (excitation 
at 310 nm and emission at 405 nm for the Ca2+-bound dye and at 495 nm for the Ca2+-unbound dye) [30
]. Slit-widths were set at 10 nm, and all experiments were carried out at 37°C.
A calibration file was generated by the addition of 5.6 µM ionomycin (Calbiochem, La Jolla, CA), followed by 3 mM EGTA (pH 8.7) to Indo-1-loaded T cells in 1.4 mM Ca2+-containing HBSS. The Rmax and Rmin values and the fluorescent intensity of the unbound probe in saturating and limiting concentrations of Ca2+ were entered into a calibration file for calculation of [Ca2+]i in future experiments.
Investigations in Ca2+-containing medium
Before stimulation of Indo-1-loaded cells in Ca2+-containing HBSS, the fluorescence signal was observed for 50–100 s to acquire a Ca2+ baseline. Dose-response curves established the submaximal dose of PHA protein (6.7 µg/ml, Difco, Detroit, MI), which was used throughout these investigations. Stimulation of T cells with anti-CD3 cross-linking was performed by adding a submaximal dose of a mouse anti-human CD3 mAb (OKT3; 0.5 µg/ml) and cross-linking 30–40 s later by the addition of a rabbit anti-mouse (RM) polyclonal antibody (2.5 µg/ml, Dako Ltd). The Ca2+ response was observed for a period of 10 min, and the baseline peak and plateau rise in [Ca2+]i were recorded. The peak rise (peak [Ca2+]i–baseline [Ca2+]i) and the plateau rise (plateau [Ca2+]i–baseline [Ca2+]i) in signal were calculated and compared for TNF--treated and untreated T cells.
Investigations in Ca2+-free medium
To assess the relative contributions of the endoplasmic Ca2+ store and the SOCs in the plasma membrane to the overall Ca2+ response, investigations were performed under Ca2+-free conditions. Jurkat T cells were transferred into Ca2+-free HBSS and added to a cuvette as described earlier. Once in the fluorimeter, any remaining Ca2+ was removed by the addition of EGTA (0.4 mM) immediately prior to stimulation, thus minimizing the time that the cells spent in Ca2+-free medium. The activity of the SOC channels was examined by addition of a saturating concentration of the Ca-adenosinetriphosphatase (ATPase) inhibitor, thapsigargin (TG; 500 nM, Sigma-aldrich, UK), which induces a leakage of Ca2+ out of the ER, shown to activate the same SOC as TCR stimulation by antigen [31
]. TG was used in this investigation to bypass TCR signals upstream of the endoplasmic Ca2+ store and thus, to specifically assay the effect of TNF- exposure and exogenous ceramide on SOCs. Following the addition of TG to the cell suspension, the addition of Ca2+ to the medium causes an influx of Ca2+ through the open SOCs, enabling the rate and magnitude of Ca2+influx to be assessed, independent of upstream signaling events.
ASM activity
Cells were stimulated as indicated; lysed in 250 mM sodium acetate (pH 5.0), 1.3 mM EDTA, and 1% Nonidet P-40 (NP-40); sonicated three times for 10 s each; diluted with 250 mM sodium acetate (pH 5.0), 1.3 mM EDTA to 0.1% NP-40, and 30 µl/sample [14C] SM (55 mCi/mmol); and resuspended in 0.1% NP-40 and 250 mM sodium acetate (pH 5.0), and 1.3 mM EDTA was added. Samples were incubated for 30 min at 37°C and extracted in 5 vol chloroform:methanol (2:1), and radioactive choline chloride was measured in the upper phase by liquid scintillation counting.
Measurement of sphingolipids
Cells (1x106) were incubated overnight with 5 µCi [3H] choline chloride (60 Ci/mmol). The indicated cells were washed three times, resuspended in HEPES/saline [132 mM NaCl, 20 mM HEPES (pH 7.4), 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4), and exposed to 100 ng/ml TNF- for the indicated time. Stimulation was terminated by addition of 120 ml 0.22 N HCl and 2.7 ml CHCl3:CH3OH (2:1, v/v). The samples were then supplemented with 0.9 ml CHCl3 and 0.9 ml 1 M KCl. Lipids were extracted, and the organic phase was dried under vacuum. This was resuspended in 20 ml CHCl3:CH3OH (1:1, v/v) and separated on Silica G60 thin-layer chromatography (TLC) plates, which were developed with CHCl3:CH3OH:H2O:acetic acid (50:30:8:4, v/v/v/v) and dried, and SM was identified in iodine vapor by comigration with an authentic standard. The spots were scraped from the plates and quantified by liquid scintillation counting.
To measure ceramide, lipids were extracted with CHCl3:CH3OH:HCl (100:100:1, v/v/v), an aliquot of the organic phase was dried, and 1,2-diacylglycerol (DAG) was destroyed by mild alkaline hydrolysis in 0.1 M methanolic KOH. Samples were re-extracted with CHCl3:CH3OH:HCl (100:100:1, v/v/v), and ceramide levels were ascertained by the DAG kinase assay as described [32 , 33 ].
To measure sphingosine, cells were extracted in CHCl3:CH3OH (2:1, v/v), and the organic phase was collected, dried, and resuspended in 40 µl 4 mg/ml BSA, 2% ß-octylglucopyranoside, and 1.25 mM cardiolipin. Samples were vortexed and 60 µl of a buffer consisting of 200 mM Tris-HCl (pH 7.4), 20% glycerol, 1 mM ß-mercaptoethanol, 1 mM EDTA, 20 mM ZnCl2, 1 mM Na3VO4, 15 mM NaF, 10 µg/ml each aprotinin and leupeptin, and 0.5 mM deoxypyridoxine was added. Samples were bath-sonicated for 10 min, and 80 µl of a Swiss 3T3 extract containing sphingosine kinase (SKase) plus 2 µCi [32P] adenosine 5'-triphosphate (3000 Ci/mmol) and 10 mM MgCl2 was added. The lysates were prepared in 200 mM Tris-HCl (pH 7.4), 20% glycerol, 1 mM ß-mercaptoethanol, 1 mM EDTA, 20 mM ZnCl2, 1 mM Na3VO4, 15 mM NaF, 10 µg/ml each aprotinin and leupeptin, and 0.5 mM deoxypyridoxine, and cells were disrupted by five-times freeze-thawing, and the lysates were centrifuged for 60 min at 100,000 g and stored at 1 mg/ml at –80°C.
Samples were then incubated at 37°C for 60 min. The kinase reaction was terminated by addition of 20 µl 1 M HCl and 800 µl CHCl3:CH3OH:concentrated HCl (100:200:1, v/v/v). Samples were vortexed, 240 µl 2 M KCl was added, phases were separated and spotted onto silica G60 TLC plates, and sphingosine-1-phosphate (S1P) was isolated by separation with a running buffer consisting of CHCl3:CH3OH:acetic acid:H2O (90:90:15:5, v/v/v/v).
S1P was assayed by the methodology described by Edsall and Spiegel [34 ]. Briefly, cells were lysed in 12.5 mM HCl and 1 M NaCl and extracted by addition of CHCl3:CH3OH:3 M NaOH (1:1:0.1, v/v/v), and the phases were separated. The aqueous phase was brought to 30 mM Tris-HCl (pH 7.4), 11.25 mM MgCl2, and 0.3 M glycine (pH 9.0), 50 units alkaline phosphatase (Roche Diagnostics, Penzberg, Germany) were added, and the samples were incubated for 30 min at 37°C. The phosphatase reaction was stopped by addition of 50 µl concentrated HCl, and the lipids were extracted twice with 0.3 vol CHCl3. The organic fractions were pooled and dried, and sphingosine in the samples was phosphorylated as described above.
Statistical analysis
For statistical analysis, the means and SEM were calculated. In most cases, differences between groups were tested for statistical significance using paired t-test. P values less than 0.05 were considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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exposure on anti-CD3 and PHA-induced Ca2+ signals in Jurkat T cells
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Figure 1. TNF- exposure inhibits anti-CD3 and PHA-stimulated Ca2+ signals in Jurkat T cells. (A) Surface expression of TNFR1 and TNFR2 on Jurkat T cells was examined by flow cytometry with FITC-conjugated mAb specific for TNFR1 (thick, black line) and TNFR2 (thin, black line); negative control (shaded area). Jurkat T cells were cultured with 100 ng/ml TNF- for 3 h prior to the measurement of Ca2+ responses to (B) stimulation with PHA (6.7 µg/ml) and (C) stimulation by cross-linking anti-CD3 mAb (0.05 µg/ml) with a RM antibody (2.5 µg/ml). (D) Exposure of Jurkat T cells to 100 ng/ml TNF- (solid bars) for 3 h significantly impaired the magnitude of the peak rise in [Ca2+]i signal to both types of stimuli compared with untreated controls (open bars; P 0.0001). Shown are the mean ± SEM derived from nine (PHA) and seven (anti-CD3) independent experiments. We examined the effect of a 3-h TNF- exposure on CD4+ PB T lymphocytes isolated from healthy individuals to investigate whether the inhibition of [Ca2+]i responses could be induced in a nontransformed cell and was not an artifact of Jurkat T cells. [Ca2+]i responses to (E) PHA stimulation and (F) stimulation by cross-linking anti-CD3 mAb in normal CD4+ T lymphocytes were also impaired following exposure to 100 ng/ml TNF- (—) for 3 h compared with controls (—), indicating that the inhibition is not specific only for Jurkat T cells. Data shown are representative of three individual experiments.
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for 3 h before activation. The baseline, peak, and plateau Ca2+ signals for control and TNF--treated Jurkat T lymphocytes were obtained and analyzed. After exposure to TNF-, the [Ca2+]i signal evoked by PHA stimulation was decreased by up to 40% (Fig. 1B)
. TNF- exposure did not alter basal levels, which were comparable with control cells. Peak [Ca2+]i signals were significantly reduced by TNF-, as were the plateau [Ca2+]i responses. The TNF- effect was not confined to the response to mitogen, as the rise in [Ca2+]i induced by cross-linking anti-CD3 mAb was also impaired following TNF- exposure (Fig. 1C)
. Peak [Ca2+]i levels were reduced, and the baseline [Ca2+]i was unchanged, a consistent and significant finding over a number of experiments. The inhibition of the peak [Ca2+]i signal was reflected in the measurement of the peak rise in [Ca2+]i (peak rise=peak [Ca2+]i–baseline [Ca2+]i). The peak rise in [Ca2+]i following mitogen (P0.0001, n=9) or anti-CD3 stimulation (P0.0001, n=7) was impaired significantly compared with untreated controls (Fig. 1D)
.
The inhibition of Ca2+ responses by TNF- was not a result of an increase in cell death, as the proportion of viable cells, determined by propidium iodide incorporation, was comparable between TNF--treated and untreated Jurkat T cells (data not shown).
TNF- exposure inhibits PHA-induced [Ca2+]i signals in PB CD4+ T lymphocytes
To confirm that this phenomenon was not a vagary of the transformed Jurkat T cell line, we investigated the effects of TNF- on fresh human PB CD4 + T lymphocytes isolated from healthy donors. Before exposure to TNF-, the purified PB CD4+ T lymphocytes were first activated with PHA and IL-2 to up-regulate TNFR1 and TNFR2 expression. Resting T lymphocytes express relatively few or no TNFRs, but after activation, it is up-regulated rapidly, and expression is maintained for several days before declining back to basal levels (L. D. Church, unpublished data). The cells were therefore cultured for 4 days following PHA activation, after which time, they had regained responsiveness to TCR stimulation and retained TNFR expression. Exposure of these PB CD4+ T lymphocytes to TNF- for 3 h was performed under conditions identical to those used for the Jurkat T cells. Like the Jurkat T cells, the magnitude of the [Ca2+]i signal evoked following stimulation with PHA (Fig. 1E)
or by cross-linking anti-CD3 mAb (Fig. 1F)
was decreased regularly after exposure to TNF-. The PHA-induced Ca2+ response was impaired by nearly 30%, and the anti-CD3-evoked Ca2+ response was reduced by more than 50%, confirming that suppression of [Ca2+]i signaling by TNF- is not confined to the Jurkat T cell line. This observation is also intriguing, as the suppressive effect of TNF- on TCR signaling is clearly occurring in CD4+ PB T lymphocytes in which there is surface expression of both TNFRs.
TNFR1-dependent inhibition of TCR-induced Ca2+ signal is dose- and time-dependent
Having demonstrated that exposure of Jurkat and PB CD4+ T cells to TNF- for 3 h could inhibit TCR-induced Ca2+ responses, we next investigated the dose range of TNF-, which could impair Ca2+ signaling. Jurkat T cells were exposed to four different concentrations of TNF- (0.1, 1, 10, and 100 ng/ml) for 3 h, and their Ca2+ responses to PHA stimulation were measured as described before. The peak rise in [Ca2+]i following PHA stimulation was significantly inhibited (P0.05) in a dose-dependent manner, and 100 ng/ml TNF- induced the greatest inhibition (Fig. 2A
).
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Figure 2. Inhibition of PHA-induced Ca2+ signal by TNF- exposure in Jurkat T cells is dose- and time-dependent. (A) Jurkat T cells were cultured for 3 h with increasing concentrations of TNF- (0.1–100 ng/ml). TNF- exposure impaired PHA-induced [Ca2+]i responses in a dose-dependent manner, and the maximal inhibition in peak-rise observed was at 100 ng/ml (all concentrations of TNF- were impaired significantly, P0.05). (B) Jurkat T cells were exposed to 100 ng/ml TNF- for different durations prior to measuring PHA-triggered [Ca2+]i responses. Inhibition of [Ca2+]i responses by TNF- increased with time, and the maximal inhibition was observed following a 3-h exposure (P0.01). Inhibition of [Ca2+]i responses was evident following only 30 min incubation and was impaired significantly by 90 min exposure to TNF- (P0.05, data not shown). However, the inhibition of [Ca2+]i responses by TNF- was transient, as the inhibition had almost dissipated following 6 h exposure. The suppression of [Ca2+]i responses in TNF--treated Jurkat T cells was not evident at 12 h, indicating that the effect of TNF- is transient and reversible. Shown are the mean ± SEM derived from four independent experiments.
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exposure was also examined (Fig. 2B)
. Jurkat T cells were exposed to 100 ng/ml TNF- for different times, and then their Ca2+ responses to PHA stimulation were analyzed. Following exposure to TNF- for just 30 min, a reduction in the PHA-induced Ca2+ responses was evident. The suppression of [Ca2+]i signals increased with the duration of exposure, and maximal inhibition was seen at 3 h, at which time, the peak rise was decreased significantly by nearly 50%. However, this inhibition was transient, and after 6 or more hours of continuous exposure to TNF-, the magnitude of Ca2+ responses was again comparable with untreated controls.
TNF- exposure impairs SOC influx across plasma membrane
To investigate the mechanism by which TNF- exposure inhibits [Ca2]i signaling in human T lymphocytes, we examined the effect of TNF- on Ca2+ influx and efflux across the plasma membrane (Fig. 3A
). Jurkat T cells were exposed to 100 ng/ml TNF- for 3 h as before and stimulated with PHA. Once a plateau response had been achieved, EGTA was added to the cell suspension to chelate external Ca2+, thereby allowing the activity of Ca2+ efflux channels (PMCA) to be assessed directly. Addition of EGTA resulted in a sharp drop in the [Ca2+]i signal, as [Ca2+]i was pumped out of the cell cytoplasm rapidly. The rate of efflux was identical in T lymphocytes exposed to TNF- and the untreated controls, confirming that Ca2+ efflux channel activity is not altered following TNF- exposure and that changes in this activity do not contribute to the decreased Ca2+ signal.
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Figure 3. Ca2+ influx but not efflux is dysregulated in Jurkat T cells following 3 h TNF- exposure. (A) To assess whether TNF- exposure increased the efflux of Ca2+ from Jurkat T cells, the activity of plasma membrane Ca2+ ATPase (PMCA) efflux pumps was assessed by measuring the rate of chelation following the addition of EGTA. The rate of efflux from TNF--treated T cells was unaltered compared with untreated T cells, indicating that Ca2+ efflux pumps are not affected by TNF- exposure. (B) The sarcoplasmic and ER Ca2+-activated ATPase (SERCA) pump inhibitor TG was used to induce a release of Ca2+ from intracellular stores in the absence of TCR stimulation. Application of TG under Ca2+-free conditions enabled the measurement of plasma membrane Ca2+ channel activity, independent of TCR signals. The re-addition of Ca2+ to the cell suspension revealed that the plasma membrane Ca2+ channels were significantly impaired in TNF--treated Jurkat T cells and that Ca2+ influx is greatly reduced (P0.007). Data shown are representative of three independent experiments.
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, the magnitude of Ca2+ influx was diminished compared with untreated control cells (Fig. 3A)
. In accordance with the "capacitance model" proposed by Putney [25
], the influx of Ca2+ across the plasma membrane is dependent on the proportion of Ca2+ released from the internal stores. Therefore, the reduction in the magnitude of Ca2+ influx may be the result of a direct inhibition of the membrane SOC channels or a reduction in the magnitude of internal store release.
To distinguish between these two effects, TG was used to activate SOC entry independently (Fig. 3B)
. TG blocks the endoplasmic Ca2+ pump (SERCA) specifically, leading to maximal activation of SOC entry [36
]. Application of TG enabled us to assay the activity of SOC channels specifically, independent of upstream TCR signals, as these are bypassed with TG. The investigations were performed in Ca2+-free HBSS, allowing the independent measurement of first, the TG-induced Ca2+ release from ER stores and then, the Ca2+ influx across the plasma membrane by the later addition of Ca2+ to the cell suspension. The magnitude of the Ca2+ release from the ER stores was comparable between TNF--treated and untreated cells, suggesting that TNF- exposure does not alter the proportion of stored Ca2+ or the ability of TG to empty stores. However, subsequent addition of Ca2+ to the cell suspension revealed that TNF- exposure significantly impaired SOC influx, independent of early TCR signaling events (P0.007, n=3). As the influx of Ca2+ across the plasma membrane is essential for sustaining the [Ca2+]i levels above the required threshold for transcription factor activation, inhibition of this by TNFR1 signaling has profound implications for T cell activation and function.
Exogenous SMase and ceramide mimic the effect of TNF- on Ca2+ signals in Jurkat T lymphocytes
Breittmayer et al. [37
] showed that exogenous addition of SMase and sphingosine could modulate CD3 and TG and ionomycin Ca2+ signals in Jurkat T cells. To determine whether the mechanism by which TNFR1 signaling modulated PHA and CD3-mediated Ca2+ signals may be through the activation of SMase, we examined the effect of exogenous addition of SMase and ceramide on PHA-induced Ca2+ signaling in T lymphocytes. Jurkat T cells were exposed to SMase for 30 min prior to stimulation with PHA (Fig. 4A
). The magnitude of Ca2+ signals generated by stimulating Jurkat T cells with PHA was inhibited in a dose-dependent manner. Maximal inhibition was observed at the highest SMase concentration of 1000 mU, but nearly 50% inhibition was produced at 100 mU.
 View larger version (18K): [in a new window] |
Figure 4. Exogenous addition of SMase or ceramide mimics TNF- impairment of Ca2+ signaling in Jurkat T lymphocytes. (A) Incubation of Jurkat T cells for 30 min with exogenous SMase, prior to stimulation with PHA, impaired the magnitude of Ca2+ responses in a dose-dependent manner. (B) The effect of ceramide directly on Jurkat T cells was examined by exposing cells to increasing concentrations of semisynthetic ceramide (C2-ceramide) for 60 s prior to stimulation with PHA. The magnitudes of Ca2+ responses were impaired in a dose-dependent manner, shown as the mean ± SEM (n=7). (C) The effect of exogenous ceramide on Ca2+ influx was examined by measuring the response to TG under Ca2+-free conditions. C2-ceramide (25 µM, final concentration) was added directly to the cuvette 60 s prior to stimulation with TG (arrow under the trace). Ceramide did not impair TG-induced Ca2+ release from intracellular stores but significantly inhibited Ca2+ influx through SOC channels, similar to that observed for TNF--treated Jurkat T cells.
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(Fig. 1)
, we next examined whether the exogenous preaddition of ceramide could also inhibit PHA responses in Jurkat T cells. Direct addition of ceramide to Jurkat T cell inhibited PHA-induced Ca2+ signals in a concentration-dependent manner (Fig. 4B)
. In addition, the characteristics of inhibition of the Ca2+ responses by ceramide were the same as observed for TNF- involving Ca2+ influx. Jurkat T cells that had been exposed to ceramide for 1 min were stimulated with TG under Ca2+-free conditions (Fig. 4C) . The magnitude of the intracellular store releases was unaltered in comparison with untreated controls. However, the re-addition of Ca2+ to the cell suspension revealed an impaired influx of Ca2+ across the plasma membrane. This observation supports previous findings that ceramide can inhibit SOC channels and provides strong evidence that the TNF--induced inhibition of CD3 and PHA Ca2+ signals occurs following the activation of SMase and the generation of ceramide.
TNFR1 signaling increases ceramide and S1P content in Jurkat T lymphocytes
To confirm the activation of this pathway following TNFR1 ligation, the activity of ASM and NSMase was measured over time following exposure to 100 ng/ml TNF-. Within 5 min, there was a significant increase in ASM activity that was maintained for over 3 h (Fig. 5A
). ASM activity had returned to background levels by 5 h. NSMase activity was unchanged over this time course; however, a small increase in activity was detected following 7 h TNF- exposure (data not shown). ASM is a lysosomal hydralase that catalyzes the degradation of SM to phosphorylcholine and ceramide. In addition, ceramide can be subsequently metabolized to sphingosine and S1P, via ceramidase and SKase activity, respectively. To provide further evidence for the activation of this pathway, the cellular content of SM and its metabolites was also quantified in response to 100 ng/ml TNF- in Jurkat T cells over time (Fig. 5B 5C
5D
5E)
. Within a few minutes after exposure to TNF-, the cellular content of SM was reduced significantly (Fig. 5B
, P0.01). This reduction was maintained for over 3 h and only began to return to basal levels by 5 h. The ceramide content was accordingly increased rapidly, which correlated strongly with the decrease in SM content (Fig. 5C)
. The increase in ceramide content was statistically highly significant and remained elevated for over 3 h. Similar to the restoration of SM levels, the cellular content of ceramide had returned to control levels at 5 h. It is interesting that quantification of sphingosine revealed a slight decrease in the cellular content by 30 min (Fig. 5D)
consistent with a previous report by Pettus et al. [38
]. The decrease in sphingosine was also maintained for over 3 h and returned to basal levels by 5 h. The cellular content of S1P was elevated threefold by 30 min (Fig. 5E)
, a finding that is consistent with effects of TNF- on cellular S1P previously reported [38
]. This level declined over time and had nearly returned to baseline levels by 5 h.
 View larger version (29K): [in a new window] |
Figure 5. TNFR1 signaling induces the activation of ASM and accumulation of ceramide and S1P in Jurkat T lymphocytes. Activation of the SMase signaling pathway over time by TNFR1 was investigated following TNF exposure by examining (A) ASM activation (**, P0.001; *, P0.005), (B) SM concentration (850 pmol/106 cells of SM at time-point 0 corresponds to 510x103 cpm/106 cells; *, P0.01), and the cellular content of subsequent metabolites (C) ceramide (215 pmol/106 cells of ceramide at time-point 0 corresponds to 530x103 cpm/106 cells; **, P0.001; *, P0.005), (D) sphingosine (1.6 pmol/106 cells of sphingosine at time-point 0 corresponds to 11,988 cpm/106 cells), and (E) S1P (50 fmol/106 cells of S1P at time-point 0 corresponds to 375 cpm/106 cells). Data shown are the mean ± SD derived from three independent experiments.
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TNFR1-induced inhibition of Ca2+ influx is completely abrogated in ASM–/– T lymphocytes
As previously discussed, the hydrolysis of SM following TNFR1 engagement may occur by NSMase or ASM activation. Both isoforms may be activated rapidly through independent TNFR1 signaling pathways. As NSMase activity was only detected after 7 h exposure to TNF-, it is unlikely that it plays a role in the inhibition of Ca2+ influx. The activation of ASM, however, closely correlated with the decrease in the magnitude of [Ca2+]i. We therefore examined whether ASM activation by TNFR1 underlies the inhibition of TCR-induced Ca2+ influx by measuring Ca2+ responses following TNF- exposure in murine T cell lines derived from wild-type (OT-II/ASM+/+) and ASM knockout mice (OT-II/ASM–/–). OT-II/ASM+/+ and OT-II/ASM–/– murine T lymphocytes were exposed to 100 ng/ml hTNF- for 3 h. hTNF- was chosen over murine TNF- for this study, as hTNF- binds only to murine TNFR1 and not TNFR2 [39
]. This feature makes the use of these cells more relevant to our study of the mechanism of TNFR1-induced inhibition of TCR Ca2+ signaling.
First, to ensure that the ASM pathway is activated in the OT-II/ASM+/+ T cells and not activated in the OT-II/ASM–/– T cells following exposure to TNF-, we examined ASM activity in both these cell types. In the OT-II/ASM+/+ T cells, exposure to 100 ng/ml hTNF- induced a time-dependent increase in ASM activity (Fig. 6A
). The OT-II/ASM–/– T cells did not reveal any detectable activity following exposure to hTNF- (data not shown). These observations were also true for splenocytes isolated from ASM-deficient mice. Examination of the cellular content of SM and ceramide following hTNF- exposure showed a time-dependent decrease in SM expression in the OT-II/ASM+/+ T cells, which returned to baseline by 5 h (Fig. 6B)
. However, the expression of SM in the OT-II/ASM–/– T cells was unchanged throughout the duration of exposure to hTNF- (Fig. 6B) . The content of ceramide accumulated over time following hTNF- exposure in the OT-II/ASM+/+ T cells, again returning to baseline by 5 h (Fig. 6C)
. Conversely, the OT-II/ASM–/– T cells did not display any such accumulation of ceramide throughout the exposure to hTNF-. This observation was also seen with the splenocytes from the ASM-deficient mice, demonstrating that TNFR1 induces ASM activation, similar to that observed in the Jurkat T cells and that this pathway is not activated by hTNF- in the OT-II/ASM–/– T cells.
 View larger version (25K): [in a new window] |
Figure 6. TNFR1-induced accumulation of ceramide is abrogated in ASM–/– murine CD4+ T lymphocytes. To confirm that ASM is required for the generation of ceramide and inhibition of Ca2+ influx following TNFR1 ligation, we examined the (A) activation of ASM (*, P0.005), (B) cellular SM content (*, P0.001), and (C) cellular ceramide content (*, P0.01) in wild-type (ASM+/+) and ASM–/– murine T lymphocytes. Data shown are the mean ± SD derived from three independent experiments.
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-treated (395.7±140.8 nM) and untreated OT-II/ASM+/+ T lymphocytes (557.1±87.45 nM) revealed a marked suppression in magnitude by 29% (Fig. 7A
) similar to that observed in human T lymphocytes (Fig. 4B)
. The magnitude of TG-induced [Ca2+]i release was comparable between hTNF--treated (65.38±7.86 nM) and untreated OT-II/ASM+/+ T lymphocytes (64.98±21.36 nM). However, Ca2+ influx in the OT-II/ASM–/– T lymphocytes (673.8±50.2 nM) was not perturbed by treatment with TNF- compared with hTNF--treated cells (664.4±20.22 nM; Fig. 7B
). Again, the TG-induced [Ca2+]i release was comparable between hTNF--treated (64.92±3.3 nM) and untreated OT-II/ASM–/– T lymphocytes (57.92±4.4 nM). It is interesting that the addition of C2-ceramide to the OT-II/ASM–/– T lymphocytes did induce a suppression of Ca2+ influx (data not shown), showing that sphingolipid signaling downstream of SMase was not affected in these ASM–/– T cells. Taken together, these observations provide strong evidence that TNFR1 induces the suppression of TCR-provoked Ca2+ influx through the activation of ASM and subsequent generation of ceramide.
 View larger version (14K): [in a new window] |
Figure 7. Inhibition of Ca2+ influx by TNF- is completely abrogated in ASM–/–T lymphocytes. To determine whether TNFR1-induced ASM activation underlies the inhibition of Ca2+ influx, we examined the effect of TNF- on Ca2+ signaling in wild-type (ASM+/+) and ASM–/– murine T lymphocytes. ASM+/+ and ASM–/– T lymphocytes were exposed to 100 ng/ml hTNF- for 3 h prior to measurement of Ca2+ influx. (A) The magnitude of Ca2+ influx following TNF- exposure was impaired in ASM+/+ T lymphocytes compared with untreated controls; however, Ca2+ influx was unperturbed in (B) ASM–/– T lymphocytes, implying a specific role for ASM in the suppression of TCR-induced Ca2+ signaling. Data shown are representative of two independent experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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can regulate T lymphocyte activation by specifically modulating [Ca2+]i signals generated following TCR stimulation. The data gathered in Jurkat T cells were confirmed in fresh CD4+ T lymphocytes. The inhibition of TCR-induced Ca2+ signals by TNF- was dose- and time-dependent and occurred rapidly after stimulation and with concentrations of TNF-, which can be found in biological fluids during inflammation in vivo [40
]. This modulation is dependent on the TNFR1 signaling pathway and targets SOC influx channels directly. Under conditions using Ca2+-free medium, stimulation of TNF--treated T cells with TG to mobilize Ca2+ from intracellular stores revealed impairment of Ca2+ entry. The magnitude of [Ca2+]i released by TG was unaltered by TNF- exposure, whereas the magnitude of the Ca2+ influx across the plasma membrane when Ca2+ was added back to the cell suspension was reduced. As TG bypasses TCR signaling events upstream of an endoplasmic Ca2+ store, the inhibition of [Ca2+]i signals can be attributed directly to the inhibition of Ca2+ influx through SOC channels in the plasma membrane. TNF- alone did not induce a Ca2+ flux nor did it have any effect on resting [Ca2+]i levels (data not shown).
In most cell types, the elevation in [Ca2+]i level is an essential component of several physiological and functional responses, but the relative contribution of internal and external sources of Ca2+ to the rise in [Ca2+]i varies between cell types. In T lymphocytes, the Ca2+ entry across SOC channels in the plasma membrane contributes to the larger fraction of the TCR-induced rise in [Ca2+]i [41
]. Ca2+ entry is not only important for the elevation of the [Ca2+]i level but also for maintaining the [Ca2+]i level above a required threshold for the activation of specific transcription factors [27
] and thus, the activation of a large number of genes [26
]. The specificity of transcription factor activation is tightly regulated by the amplitude, frequency, and duration of [Ca2+]i signals [18
, 19
, 42
]. For example, a high transient peak rise in [Ca2+]i is associated with the preferential activation of the nuclear factor 
B (NF-B) and c-jun N-terminal kinase, and a sustained plateau in the [Ca2+]i response is required for nuclear translocation of NF of activated T cells and extracellular signal-regulated kinase activation [43
, 44
]. This differential regulation of transcription factors underlines the central importance of [Ca2+]i signals in determining the functional outcome of lymphocyte activation.
Signaling through CD95 (Fas), a member of the TNFR family, has been shown to block ICRAC channels in T lymphocytes by the activation of SMase and the generation of sphingolipids, in particular, ceramide [15
]. The inhibition of SOC entry following TNF- exposure, which we observed, was reminiscent of that induced by CD95 ligation. TNF- has not been shown previously to impair Ca2+ signaling through this pathway in T lymphocytes, although TNFR1 shares much homology with Fas. It is interesting that the addition of SMase or sphingosine exogenously to Jurkat T cells has been shown to inhibit Ca2+ signals generated by CD3 cross-linking, TG, and ionomycin [37
], implying that activation of this pathway by TNF- could mediate these signaling aberrations. In the latter study, the authors suggested that sphingosine was acting by enhancing Ca2+ extrusion, as the Ca2+ signal induced by ionomycin stimulation was also impaired. However, our data support a mechanism involving the activation of ASM by TNF- through TNFR1 in T lymphocytes and the impairment of Ca2+ influx. Measurement of SMase activity revealed a time-dependent activation in ASM but not NSMase. This increase in ASM activity was accompanied by a decrease in SM and an increase in its hydrolysis product ceramide in Jurkat T cells exposed to TNF-. The increase in ceramide was time-dependent and correlated strongly with the impaired Ca2+ responses. Furthermore, the exogenous addition of ceramide and SMase to T cells also impaired the magnitude of Ca2+ responses in a similar manner to that observed for TNF-. It is striking that the exogenous addition ceramide specifically inhibited Ca2+ entry, mimicking that observed following TNF- exposure. We did not observe any effect of TNF- on Ca2+ extrusion, and it is interesting that quantification of the cellular content of sphingosine revealed a marginal but time-dependent decrease in sphingosine following TNF- exposure. The decrease in sphingosine, however, was paralleled by an increase in S1P. It is unlikely that the increase in S1P following TNF- contributes to the observed suppression of Ca2+ influx, as no inhibitory effect of S1P on Ca2+ influx was observed in patch-clamp experiments [45
]. Our data are more consistent with the increase in ceramide following TNF- exposure, impairing the magnitude of Ca2+ responses by inhibiting Ca2+ influx specifically.
Thus, our data from human (Jurkat and CD4+ PB) T cells are consistent with a model of TNF- acting through TNFR1 to activate SMase, which in turn generates ceramide with the latter, or a subsequent sphingolipid product (i.e., sphingosine) acting directly on Ca2+ influx and thus modulates TCR-induced signaling.
To examine directly the putative role of SMase in the above model of TNFR1-induced suppression of TCR signaling, we examined the effect of hTNF- on a murine CD4+ T cell line deficient in ASM (OT-II/ASM–/–). The use of hTNF- in these studies enabled us to analyze the TNFR1 signaling pathway specifically, as murine TNFR2 is unable to bind to hTNF-. TNF- exposure induced a time-dependent increase in ASM activity in the wild-type OT-II/ASM+/+ T lymphocytes; however, no activation of NSMase was observed. The increase in ASM activity followed the same time course as observed in human T cells. Examination of SM and ceramide in the wild-type OT-II/ASM+/+ and ASM-deficient OT-II/ASM–/– T lymphocytes revealed a time-dependent decrease in SM levels in parallel with an increase in the cellular content of ceramide in the wild-type T lymphocytes. The cellular content of SM and ceramide did not change in the ASM-deficient T lymphocytes, indicating that activation of the ASM signaling pathway TNFR1 ligation is completely abrogated in these cells. Furthermore, exposure to TNF- suppressed the Ca2+ influx in the wild-type OT-II/ASM+/+ T lymphocytes, but the Ca2+ influx in OT-II/ASM–/– T lymphocytes was unperturbed. These data support the postulated model for TNF--modulating TCR signaling through TNFR1-induced SMase activation and generation of ceramide. The wild-type murine OT-II/ASM+/+ cells showed a pattern of depressed Ca2+ influx comparable with that observed in the human T lymphocytes. By contrast, the ASM–/– T lymphocytes (OT-II/ASM–/–) displayed no such depression following TNF- exposure. However, the OT-II/ASM–/– cells did show a depression in Ca2+ influx when exposed to exogenous ceramide (data not shown). This demonstrates that in the absence of ASM, the SOC channels in these cells may still be impaired by the SMase product ceramide. This confirms that it is the absence of ASM, rather than any other downstream defect, that was responsible for their lack of response to TNF-.
This is the first observation that activation of ASM through TNF- may regulate T cell responses by inhibiting Ca2+ influx in T lymphocytes specifically. The mechanism by which hydrolysis of SM and the generation of ceramide interferes with the lymphocyte Ca2+ plasma membrane influx remains obscure. Work on voltage-gated Ca2+ channels suggests that these channels are localized to caveolar microdomains [46
], and this relationship between ion channels and lipid domains may be quite widespread with, for example, different isoforms of voltage-gated potassium channels located within distinct lipid raft populations [47
]. Given the important role that SM and ceramide play in the structure and formation of rafts and membrane platforms, respectively [48
], their generation following TNF- activation of SMase may well modulate or disrupt the activity or distribution of the ion channels.
We have previously identified depressed Ca2+ signals in T lymphocytes from patients with RA [49
50
51
], a disease associated with elevated TNF- and chronic inflammation. Perturbed Ca2+ signaling is one of many TCR signaling aberrations characteristic of such CD4+ T lymphocytes isolated from patients with RA and contributes to their dysfunctional state in this disease. More recently, we have identified that a contributing factor to the depressed Ca2+ signaling is a marked reduction in Ca2+ influx in these cells [52
]. By investigating the role of TNFR1 in modulating TCR-induced Ca2+ signals, we have identified a potential mechanism to explain these signaling aberrations. Although this study revealed that the effect of acute TNF- exposure on Ca2+ signals is relatively short-lived, it is plausible to suggest that under chronic conditions, the activation of SMase may also provide a mechanism to explain the suppression of T lymphocyte responses following persistent TNF- exposure.
In conclusion, this study has identified ASM activation through TNFR1 and the inhibition of Ca2+influx as a novel mechanism, whereby TNF- may regulate T lymphocyte activation. This may therefore be an important component of not only acute inflammatory responses but also T lymphocyte dysfunction in chronic inflammatory diseases, such as RA and Crohn’s disease, and warrants further investigation.
Received October 3, 2003; accepted February 28, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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: induction of TNF receptors on human T cells and TNF--mediated enhancement of T cell responses J. Immunol. 138,1786-1790[Abstract] and IL-1 ß on mitogen-induced T cell activation J. Immunol. 140,2639-2644[Abstract] J. Immunol. 140,531-536[Abstract]
and expression of the TCR/CD3 complex at the cell surface J. Immunol. 166,5495-5507 1 J. Biol. Chem. 266,12135-12139 1 association with CD3 structure in T cells J. Exp. Med. 175,285-288- and ß-chain genes under the control of heterologous regulatory elements Immunol. Cell Biol. 76,34-40[CrossRef][Medline] FASEB J. 17,1411-1421 but not tumor necrosis factor ß in rheumatoid arthritis synovial fluid and serum Arthritis Rheum. 31,1041-1045[Medline]B transcriptional activity J. Biol. Chem. 274,33995-33998 exposure impairs store operated calcium influx in CD4+ T lymphocytes FEBs. Letters 579,1539-1544[CrossRef][Medline]This article has been cited by other articles:
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