Resistance of activated human Th2 cells to NO-induced apoptosis is mediated by
-glutamyltranspeptidase
Ramon Roozendaal,
Edo Vellenga1,,
Marian A. de Jong,
Kristine F. Traanberg,
Dirkje S. Postma2,,
Jan G. R. de Monchy and
Henk F. Kauffman
Divisions of Allergology,
1 Hematology and
2 Pulmonology, Department of Internal Medicine, Faculty of Medicine, Groningen University, Groningen University Hospital, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Correspondence to:
H. F. Kauffman
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Abstract
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Activation-induced death of inflammatory cells (AICD) has an important function in immune maintenance. Type 1 Th cells are known to be more susceptible to AICD than Th2 cells. In the current study we examined whether NO-induced apoptosis also preferentially eliminates Th1 cells over Th2 cells. Naive human Th lymphocytes (CD4+CD45RO) were activated in vitro for 1 week in the presence of IL-12 plus anti-IL-4 or IL-4 plus anti-IL-12 to generate Th1- and Th2-polarized cultures respectively. Cultures were exposed to the NO donors Spermine-nonoate (Sper) and DPTA-nonoate to study NO-induced apoptosis. We found that NO preferentially induced apoptosis in Th1-polarized cells as demonstrated by Annexin staining in the presence of 10 µM Sper (70 ± 16 versus 23 ± 4.4% in Th2 cells P < 0.01) and by DioC6 staining (38 ± 10 versus 11 ± 5% in Th2 cells, P < 0.01). The mechanism of NO-induced apoptosis in Th1/Th2-polarized cells was distinct from AICD and Fas-induced apoptosis. Differential sensitivity between Th1- and Th2-polarized cultures originated at the level of intracellular glutathione (GSH) metabolism. GSH levels were higher in Th2 cells (1.6 ± 0.2-fold Th1, P < 0.01). High intracellular GSH in Th2-polarized cells did not account for reduced susceptibility to NO per se, since the inhibition of
-glutamyltrans-peptidase (
-GT), which is involved in GSH import, sensitized Th2 cells to NO-induced apoptosis without GSH depletion. Therefore, higher activity of
-GT in Th2 cells (2.1 ± 0.4-fold Th1, P < 0.001) specifically protects Th2 cells against NO-induced apoptosis. Preferential NO-induced elimination of human Th1 cells at sites of inflammation may thus select Th2 cells and contribute to immune deviation.
Keywords:
-glutamyltranspeptidase, apoptosis, differentiation, glutathione, NO, T lymphocyte, Th2 skewing
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Introduction
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Activation-induced inflammatory cell death (AICD) is an important mechanism to limit an inflammatory response (1,2). With regard to human T cell subsets it has been demonstrated that the Th1 lymphocytes are more prone to elimination by AICD than their Th2 CD4+ counterparts (35). It has been suggested that the preferential elimination of Th1 cells can result in a Th2 skewing of the immune response (6), which is indeed observed in some inflammatory diseases (7,8).
The inflammatory mediator NO has been shown to initiate, but also inhibit programmed cell death (apoptosis) in a variety of cell types, including lymphocytes (9,10). In murine and human T lymphocytes NO was reported to suppress the production of Th1-type cytokines (8,11), which might in part be due to the preferential loss of Th1 cells as a result of apoptosis. Since a connection between NO and AICD has also been reported we questioned whether the preferential NO-induced apoptosis of Th1-type cells could contribute to Th2 skewing. Naive human Th lymphocytes (CD4+CD45RO) were cultured for 1 week with phytohemagglutinin/IL-2 in the presence of IL-12 plus anti-IL-4 or IL-4 plus anti-IL-12 to generate Th1- and Th2-type cells respectively. The mechanism of NO-induced apoptosis was then studied in these polarized cells.
The present study shows that NO preferentially causes apoptosis of activated human Th1-polarized cells, although by a mechanism distinct from AICD and Fas-induced apoptosis. The Fas/caspase-8 pathway is not activated by NO in these human Th1- and Th2-polarized cells.
NO-activated apoptosis in Th1 rather than Th2 cells is regulated at the level of intracellular glutathione (GSH) metabolism. Th2 cells are specifically protected from NO-induced apoptosis by higher expression of
-glutamyltranspeptidase (
-GT). Intracellular GSH levels are elevated in Th2 cells relative to Th1 cells and are likely to confer a more general protection against the induction of apoptosis in Th2 cells.
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Methods
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Chemicals
DPTA-nonoate (DPTA) and Spermine-nonoate (Sper) were obtained from Kordia (Leiden, The Netherlands). BD-FMK (caspase inhibitor III) was purchased from Omnilabo (Breda, The Netherlands). MnTBAP was obtained from Biomedical Diagnostics (Brugge, Belgium) and Trolox from Fischer Scientific (Den Bosch, The Netherlands). Buthionine sulfoximide (BSO),
-glutamylcysteine (
-GC), acivicin, L- and L-N-monomethylarginine (L/D-NMMA) and all other chemicals used in assays were from Sigma-Aldrich (Zwijndrecht, The Netherlands), unless stated otherwise.
Th1/Th2 polarization
Lymphocytes were isolated from the peripheral blood of healthy volunteer platelet donors as previously described (12). Briefly, mononuclear cells were obtained by Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway) density-gradient centrifugation. Lymphocytes were isolated from these, by 2-aminoethylisothiouronium-treated sheep red blood cell rosetting, followed by an additional step of density-gradient centrifugation. The sheep red blood cells were lysed with 155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA. Naive Th lymphocytes were sorted after staining with anti-CD45ROFITC (UCHL1) and anti-CD4CyQ (B-F5) (ImmunoQuality Products, Groningen, The Netherlands), using a MoFlo flow cytometer (Cytomation, Fort Collins, CO) calibrated using Flow-Check fluorospheres (Beckman Coulter, Paris, France). Purity was always >95% by re-analysis. Cells were cultured in RPMI 1640 medium, using phytohemagglutinin (PHA) and IL-2 in the presence of irradiated allogenic PBMC and 10% FBS, and either IL-12 (2 ng/ml; ITK Diagnostics, Uithoorn, The Netherlands) plus anti-IL-4 (200 ng/ml; Becton Dickinson Benelux, Erembodegem-Aalst, Belgium) or IL-4 (200 U/ml; Becton Dickinson Benelux) plus anti-IL-12 (2 µg/ml; ITK Diagnostics, Uithoorn, The Netherlands) to generate Th1- or Th2-polarized lymphocytes respectively. After 1 week cells were analyzed for polarization, and re-plated in the presence of PHA/IL-2 and feeder cells for other experiments.
FACS analysis of intracellular cytokines
FACS analysis of intracellular cytokines was performed as previously described (13). Polarized cells were stimulated for 4 h with phorbol myristate acetate (PMA) (10 ng/ml) and ionomycin (1 µg/ml) in the presence of monensin (2 µM; Alexis, Laufelfingen, Switzerland). Cells were then fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin/ 0.1% azide, and stained using anti-CD4CyQ (B-F5) (ImmunoQuality Products), anti-IFN-
FITC (45-15) (ImmunoQuality Products) and anti-IL-4phycoerythrin (PE) (B-T4) [Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, The Netherlands]. Irrelevant specificity antibodies of the same isotype were used for gate setting. Analysis was performed using an Elite flow cytometer (Beckman Coulter) calibrated using Flow-Check fluorospheres (Beckman Coulter) in combination with Winlist software (Verity, Topsham, CO) for all FACS analyses described. Lymphocyte events were gated on the basis of forward and sideward scatter characteristics.
Determination of IL-4 and IFN-
protein
IL-4 and IFN-
protein were quantified in cell-free supernatants of polarized cells stimulated with PMA/ionomycin (1 ng/ml and 1 µM respectively) using a human IL-4 and human IFN-
ELISA kit (CLB Amsterdam), according to the manufacturer's instructions.
Apoptosis assays
Apoptosis was induced by incubating polarized cells with the NO-donor compounds Sper and DPTA for various time periods. In order to modulate NO-induced apoptosis Trolox, L/D-NMMA, MnTBAP and acivicin were added 30 min prior to the NO-generating compounds.
-GC and BSO were added 2 h prior to Sper and DPTA.
Plasma membrane asymmetry was assessed with Annexin staining as previously described (14). Briefly, cells were washed, resuspended in 140 mM NaCl/2.5 mM CaCl2 and stained with FITC-conjugated Annexin V (Nexins Research, Kattendijke, The Netherlands). Propidium iodide (PI) counter-staining was used to check for membrane integrity. Control samples (without the addition of NO donor compounds) were used for gate setting.
DioC6(3) (Molecular Probes, Leiden, The Netherlands) fluorescence was taken as a measure of mitochondrial membrane potential (
m) (15). Briefly, cells were washed, incubated with 0.1 µM DioC6(3) for 30 min at 33°C, washed again and resuspended in cold RPMI 1640 culture medium until FACS analysis. Viable lymphocytic events (based on forward/sideward scatter and PI) were used for gate setting.
DNA content of cells was determined by PI staining after ethanol (40%) fixation and RNase (0.2 µg/ml in PBS) treatment. Briefly, cells were washed, fixed for at least 3 h at 4°C, RNase treated for 30 min at 37°C, washed again and kept at 4°C after staining with PI in 3.8x102 M sodium citrate.
Fas ligand (FasL) assay and blocking
FasL expression was assayed using FACS analysis as previously described (16). Cells were washed, stained with biotin-conjugated anti-FasL (G247-4) or isotype control (Becton Dickinson Benelux), washed, and stained with streptavidinPE (Becton Dickinson Benelux). The metalloprotease inhibitor KB8301 (10 µM; Becton Dickinson Benelux) was included during culture, washing and staining. FasL blocking was performed using anti-FasL (NOK-1) (Becton Dickinson Benelux), as previously described (17).
Caspase-8 assay and blocking
Caspase-8 activity was determined using an AFC-labeled substrate based kits (Becton Dickinson Benelux), according to the manufacturer's instructions, with anti-Fas IgM (CH11, 1 µg/ml) included as a positive control. All measurements were performed using a FL600 spectrofluorometer (Bio-Tek Instruments) at 380 nm excitation and 520 nm emission wavelengths. The specific cell permeable caspase-8 inhibitor Ac-AAVALLPAVLLALLAP-IETD-CHO (10 µM; Omnilabo, Breda, The Netherlands) was used to block caspase-8 function in apoptosis assays.
Bcl-2-expression
Bcl-2 expression was assessed by FACS analysis. Cultured Th1 and Th2 subsets were fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.1% saponin in PBS. Cells were stained with FITC-conjugated anti-Bcl-2 (6C8) or isotype control (Ha4/8) (Becton Dickinson Benelux).
GSH assays
Monochlorobimane (MCB; Omnilabo)-dependent fluorescence was taken as a measure of reduced intracellular GSH. Th1 and Th2 cells were washed, stained with 100 µM MCB on ice for 1 h, washed, resuspended in RPMI, and kept on ice until analysis. Alternatively, we determined total cellular GSH using a modified Tietze procedure (18). Briefly, cultured Th1 and Th2 cells were separated from feeder cells by FACS sorting. Cells were washed and lysed in 5% trichloroacetic acid for protein precipitation. Lysates were centrifuged and total GSH was determined in the supernatants by kinetic measurement of OD405 in the presence of GSH reductase (1.5 U/ml), 5,5'-dithiobis(2-nitrobezoic acid) (0.6 mg/ml) and NADPH (0.4 mg/ml).
Multidrug-resistance protein (MRP)-1 assay
MRP function analysis was performed essentially as previously described, to asses the capability of cells to extrude GSH conjugates (19). Cells were loaded with the MRP-1 substrate carboxyfluorescine diacetate (0.1 µM) in the presence of the MRP-1 inhibitor MK571 (10 µM; a gift of Dr A. W. Ford-Hutchinson, Merck-Sharp, Kirkland, PQ, Canada) for 20 min at 37°C. Cells were washed to remove MK571 and incubated for 1 h at 37°C in the presence or absence of MK571. MRP-1 activity was calculated from the fluorescence ratio with/without MK571 and called efflux blocking factor.
-GT assay
Polarized cell cultures (5x105 cells/ml) were incubated for 24 h at 37°C and 5% CO2 in RPMI containing 0.5 µCi/ml [35S]GSH (New England Nuclear, Zaventem, Belgium) plus 100 µM carrier GSH in the presence or absence of 100 µM acivicin. Cells were harvested and washed twice. Total cell associated radioactivity was determined by scintillation counting.
Statistical methods
P < 0.05 was assumed to represent significant differences. Student's t-test for paired and unpaired observations were used to calculate P values. Normal distribution was assumed.
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Results
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Polarization of naive Th lymphocytes
In order to generate Th1- and Th2-type cells we expanded naive human Th lymphocytes (CD4+CD45RO, purity >95% by re-analysis) under polarizing conditions, as previously described (20). Highly divergent cytokine production profiles were obtained after 1 week of culture (Fig. 1
). Th1-polarized cells consisted of 39.3 ± 9.5% IFN-
+/IL-4 and 0.56 ± 0.24% IL-4+/IFN-
-, while Th2-polarized cells were 9.4 ± 3.4% IL-4+/IFN-
- and 0.72 ± 0.49% IFN-
+/IL-4 respectively (n = 22). Virtually all cells had a CD45RO+ phenotype (98.3 ± 0.8 and 95.0 ± 0.2% positive for Th1 and Th2 respectively, n = 4). Polarization was also clear at the cytokine secretion level, with large ranges (11.5186 and 1.322.6 ng/106 cells IFN-
; 11252 and 591269 pg/106 cells IL-4, for Th1- and Th2-polarized cells respectively, n = 22); IFN-
secretion by Th1 cells was 7.5 ± 4.3-fold higher than secretions by Th2 cells (P < 0.01) and IL-4 production by Th2 cells was 7.9 ± 4.7-fold higher compared to Th1 cells (P < 0.01). Thus, an average 58-fold difference in IFN-
/IL-4 ratios between Th1- and Th2-polarized cells was obtained.

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Fig. 1. Characterization of cytokine profiles of polarized cells. (Upper panel) Intracellular cytokine staining in Th1 (hatched bars)-and Th2 (open bars)-polarized cells. Data are presented as mean + SD, n = 22. (Lower panel) Secretion of IFN- and IL-4 by Th1- and Th2-polarized cells. Tracings represent individual polarizations with paired observations for Th1/Th2, n = 22. *P < 0.01.
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NO-induced apoptosis of activated T cells
Analysis of NO-induced apoptosis was performed on total polarized cell populations, which will be referred to as Th1 and Th2 cells, under activating conditions (PHA/IL-2). In the absence of NO, basal apoptosis was observed in both polarized cell populations under these conditions as assessed in both the Annexin V and the DioC6 assay. Basal apoptosis was generally higher in Th1 cells than in Th2 cells in the absence of NO (20.4 ± 11.4 and 8.2 ± 3.3% Annexin V+ cells, P < 0.01, and 4.5 ± 1.8 and 2.3 ± 1.0% DiOC6low cells, P < 0.01) respectively).
NO-induced apoptosis was observed in Th1 and Th2 subsets, as demonstrated by a dose-dependent increase in the number of Annexin+ cells in the presence of the NO-generating compounds Sper and DPTA) (Fig. 2
, upper and middle). Both NO-donor compounds induced the formation of Annexin single-positive cells, although secondary necrosis was also observed, as demonstrated by reduced plasma membrane integrity (PI positivity). At every concentration of Sper and DPTA (displayed for 10 and 25 µM respectively, Fig. 2
, bottom) the level of apoptosis induced was higher in Th1 than in Th2 cells (70.0 ± 15.7 versus 23.3 ± 4.4% at 10 µM Sper, P < 0.01, and 52.6 ± 9.0 versus 32.5 ± 10.9% at 25 µM DPTA, P < 0.01 respectively).

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Fig. 2. NO-induced apoptosis of Th1- and Th2-polarized cells, Annexin V/PI staining. NO generated from Sper (Upper panel) and (Middle panel) from DPTA; Annexin+ cells in Th1 (closed circles) and Th2 (closed squares), PI+ cells in Th1 (open circles)- and Th2 (open squares)-polarized cells 24 h after exposure to Sper and DPTA. One representative experiment out of four is shown. (Bottom panel) Annexin+ cells in Th1 (open bars) and Th2 (black bars), 24 h after exposure to 10 µM Sper and 25 µM DPTA, data are presented as mean + SD, n = 6. *P < 0.01.
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In accordance with Annexin V positivity, an NO-mediated decrease in DioC6 fluorescence could be detected at a lower concentration of Sper and DPTA, and in a larger proportion of the cells for Th1 than for Th2 cells (Fig. 3
) (37.8 ± 9.7% versus 11.4 ± 5.2% at 10 µM Sper, P < 0.01, 43.4 ± 15.8 versus 16.5 ± 6.6% at 50 µM DPTA, P < 0.01). Similar to Annexin V posititivity, observed levels of NO-induced decrease in DioC6 were also lower for DPTA than for Sper at the same concentration. Thus, both NO donor compounds caused a marked dose-dependent decrease in plasma membrane asymmetry (Annexin V) and increased mitochondrial depolarization (DioC6) as hallmarks of apoptosis. Sper was used to further investigate the mechanisms of NO-induced apoptosis in these cells.

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Fig. 3. NO-induced apoptosis of Th1- and Th2-polarized cells, DioC6 staining. DioC6low cells in Th1 (open bars) and Th2 (black bars) 24 h after exposure to 10 µM Sper and 50 µM DPTA, data are presented as mean + SD, n = 6. *P < 0.01.
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Role of Fas in NO-induced apoptosis
NO-induced apoptosis can be mediated by both Fas- dependent and -independent mechanisms (21). Since Th1 cells have been reported to be more sensitive towards Fas-mediated apoptosis than Th2 cells we first investigated the involvement of FasFasL interaction in NO-mediated apoptosis. Th1 cells had higher levels of Fas expression than Th2 subsets [230.7 ± 8.7 versus 113.5 ± 9.4 arbitrary units (AU) respectively, n = 4], while >95% of cells was positive for Fas in both cell types. NO-induced up-regulation of FasL could not be detected at any time point tested (t = 2, 4, 6 and 12 h at 10 µM Sper), even though the metalloprotease inhibitor KB8301 (10 µM) was included. In accordance with these observations, NO-induced apoptosis could not be blocked using an anti-FasL antibody (NOK-1, 10 µg/ml) (Fig. 4
, upper). Significant caspase-8 activity was detected in both Th1 and Th2 cells, being higher in Th1 cells (965 ± 238 AU/106 cells) than in Th2 cells (117 ± 15 AU/106 cells, P < 0.01) (Fig. 4
, bottom). However, caspase-8 activity was not enhanced by NO either in Th1 or in Th2 cells, while anti-Fas clearly enhanced caspase-8 activity (2144 ± 28 and 1673 ± 261 AU/106 cells in Th1 and Th2 respectively). Additionally, the specific caspase-8 inhibitor IETD-CHO (10 µM) did not inhibit the NO-mediated increase in Annexin+ cells, cells with decreased
m and cells with sub-G0 DNA content (results not shown). In contrast, the broad-spectrum caspase inhibitor (BD-FMK) inhibited NO-mediated DNA fragmentation (Fig. 5
). Thus, NO-induced apoptosis is caspase dependent, but does not involve caspase-8 activation.

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Fig. 4. No interaction of NO with Fas signaling. (Top panel) Annexin/PI staining of Th1 cells after exposure to 5 µM Sper for 24 h in the presence or absence of blocking anti-FasL antibody (NOK-1, added 30 min prior to Sper). Hatched bars, control; black bars, 5 µM Sper; open bars, anti-FasL; crossed bars, 5 µM Sper + anti-FasL. Data are presented as mean + SD, n = 4. (Bottom panel) Caspase-8 activity (AU) in Th1 (hatched bars) and Th2 (open bars)-polarized cells 8 h after exposure to 10 µM Sper or anti-Fas IgM (CH11, 1 µg/ml). Data are presented as mean + SD, n = 4.
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Fig. 5. Blocking of NO-induced DNA fragmentation by caspase inhibition in Th1-polarized cells. PI staining for DNA content of Th1 cells 24 h after exposure to 20 µM Sper in the presence or absence of BD-FMK (added 30 min prior to Sper). Hatched bars, control; black bars, BD-FMK; open bars, 20 µM Sper; crossed bars, 20 µM Sper + BD-FMK. Data are presented as mean + SD, n = 3. *P < 0.05.
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Mechanism of NO-induced apoptosis
The expression level of Bcl-2 has been described to correlate with apoptotic sensitivity at the mitochondrial level (22). Bcl-2 expression did not differ between the polarized Th1 and Th2 cells (60.2 ± 10.4 and 63.3 ± 15.4 AU respectively, n = 6) and was not altered by NO exposure. Blocking the formation of endogenous NO with L-NMMA and D-NMMA (16) as a control did not affect NO-induced apoptosis in either Th1 or Th2 cells (results not shown). Oxygen radical formation, while being central to AICD (23), likewise did not play a dominant role in NO-induced apoptosis, since MnTBAP (50 µM) and Trolox (100 µM) did not block NO-induced apoptosis in either Th1 or Th2 cells (results not shown). GSH levels have also been correlated with apoptotic sensitivity (24). Basal GSH levels were higher in Th2 relative to Th1 cells as assayed by FACS analysis with MCB (155 ± 16% of Th1, P = 0.002) (Fig. 6
, left). The same result was found using the Tietze assay for total cellular GSH (114 ± 10 of Th1, P = 0.01) (Fig. 6
, right). The absolute difference in GSH detected was smaller, probably due to the fact that the Tietze assay detects oxidized GSH apart from reduced GSH. Furthermore, when GSH levels of Th1 and Th2 cells were increased, by using
-GC (1 mM), the sensitivity towards NO-mediated mitochondrial depolarization in both Th1 and Th2 cells was reduced to below control values (Fig. 7A and B
). Alternatively, GSH depletion with BSO (50 µM) enhanced sensitivity towards NO-induced apoptosis in both Th1 and Th2 cells (41 96 versus 2285% DioC6low cells at 10 µM Sper respectively). NO alone did not cause GSH depletion in either Th1 or Th2 cells (Fig. 7C and D
). In summary, a significant elevation of the GSH levels in Th2 cells relative to Th1 cells was found to be correlated with decreased sensitivity towards NO-induced apoptosis.

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Fig. 6. Elevated intracellular GSH in Th2-polarized cells. (Left panel) MCB staining of Th1- and Th2-polarized cells, n = 7. (Right panel) Tietze assay for total cellular GSH isolated from Th1- and Th2-polarized cells, n = 6. Tracings represent individual polarizations with paired observations for Th1/Th2. *P < 0.01.
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The enzyme
-GT, which is involved in GSH import (33), has previously been reported to modulate NO-related effects (40). Furthermore,
-GT expression is known to be related to elevated GSH levels. Therefore, we assessed the function of
-GT in Th1 and Th2 cells, using the specific inhibitor acivicin. NO only induced significant depolarization in Th1 cells (26.9 ± 7.9 versus 10.1 ± 4.1% in control, P < 0.01) (Fig. 8A
). In the presence of acivicin Th2 cells were sensitized to NO-induced depolarization (23.6 ± 7.5 versus 11.6 ± 2.6% in control, P < 0.05), whereas NO-induced depolarization was further elevated in Th1 cells by acivicin (P < 0.05). NO also preferentially induced DNA fragmentation in Th1 cells (131 ± 9% of control, P < 0.05, not significant for Th2). Acivicin exposure alone elevated the number of cells with sub G0 DNA content in both Th1 and Th2 cells (Fig. 8B
) (132 ± 11 and 155 ± 16% of control respectively, P < 0.05 in both cases). Sensitization of Th2 cells to NO-induced apoptosis was also apparent at the level of DNA fragmentation, since the number of Th2 cells with sub-G0 DNA was increased when acivicin and NO were combined (P < 0.05 relative to acivicin and NO alone). This was not due to modulation of intracellular GSH levels, since significant GSH depletion could only be observed in Th1 cells (71 ± 15 of control and 51 ± 4% of control for Sper and acivicin + Sper respectively, P < 0.01 in both cases) (Fig. 8C
).
-GT activity was indeed higher in Th2- than in Th1-polarized cells since 2-fold more activity from [35S]GSH was incorporated in Th2 cells than in Th1 cells [54.0 ± 1.0 versus 33.6 ±3.4 disintigrations/s (d.p.s.) respectively, P < 0.001] (Fig. 9
). In the presence of acivicin incorporated activity was reduced in both Th1- and Th2-polarized cells (18.6 ± 1.5 versus 24.1± 1.0 d.p.s. respectively, P < 0.01). Differences between incorporation in Th1 and Th2 cultures were largely abolished in the presence of acivicin, indicating that the higher incorporation in Th2 cells relative to Th1 cells could be ascribed to
-GT activity.

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Fig. 9. -GT activity is elevated in Th2 relative to Th1 cells. Analysis of acivicin inhibitable uptake of [35S]GSH in Th1- and Th2-polarized cells in the presence (white bars) or absence (black bars) of 100 µM acivicin. Data are expressed in d.p.s. and presented as mean + SD, n = 6.
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MRP-1 activity, which has been associated with apoptotic resistance through export of GSH conjugates (25), and recently with Th1 activation (26), was assessed in Th1- and Th2-polarized cells. Th1 and Th2 cells both expressed significant and identical MRP-1 activity (efflux blocking factors of 3.2 ± 0.6 and 3.2 ± 0.8 respectively, n = 4).
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Discussion
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It is well known that Th1 lymphocytes are more sensitive to both Fas-induced apoptosis and to AICD than Th2 lymphocytes (35), which is thought to promote Th2 skewing in some inflammatory diseases. Results from the literature suggest that the augmented sensitivity of Th1 cells might not be restricted to AICD, but could also include sensitivity towards NO-induced apoptosis (6,27). The present study demonstrates that NO preferentially induces apoptosis in activated human Th1 cells, through a mechanism distinct from AICD. This is in accordance with previous observations demonstrating a selective and persistent suppression of IFN-
production from human T cells by NO (11). Furthermore, a differential sensitivity towards NO-induced apoptosis between Th1 and Th2 cells is shown to originate at the level of intracellular GSH metabolism. GSH levels are elevated in Th2 relative to Th1 cells. However, GSH levels per se do not appear to account for the differential sensitivity to NO-induced apoptosis between Th1 and Th2 cells. NO exposure does not result in decreased GSH levels in both Th1 and Th2 cells. Furthermore, Th2 cells become sensitized to NO-induced apoptosis in the absence of any changes in intracellular GSH when the specific
-GT inhibitor acivicin is used. These findings demonstrate that
-GT specifically protects Th2 cells against NO induced apoptosis, apart from its well-known function in GSH import.
We have used cells that were polarized for 1 week from naive human Th lymphocytes. Intracellular cytokine detection showed that polarization at this stage is not complete, but highly divergent cytokine profiles are obtained. Percentages of IL-4-producing cells in Th2-polarized cultures were always smaller than percentages of IFN-
-producing cells in Th1-polarized cultures. This is in accordance with the literature (28), since it has been reported that two cell divisions more are required to generate IL-4-producing cells than IFN-
-producing cells. Interestingly, the number of IL-4-producing cells was found to be 4-fold lower than the number of IFN-
-producing cells, which corresponds exactly to two cell divisions. Cells that do not detectably express IL-4 or IFN-
have proliferated, since they have acquired a memory phenotype (CD45RO+). Therefore, it may be expected that cells have at least progressed to Th1/Th2-like even though this is not yet apparent in the cytokine profile.
The role of NO in either activation or suppression of apoptosis has been the subject of intensive study in the past few years. In various cell types the duality in actions of NO appears to depend upon variables such as concentration (9) and redox state of NO (29). Furthermore, NO can both induce and inhibit apoptosis through different pathways (9,10). Even though NO donor concentrations used in the present study are relatively low (28,30) we only observed pro-apoptotic effects. In these polarized T cells the augmented apoptosis in response to NO is not mediated by Fas signaling even though Th1 cells display increased basal levels of caspase-8 activity in accordance with what has previously been described. Mitochondrial permeability transition does appear to be a critical event in this process, since differential sensitivity of Th1 and Th2 cells towards NO-mediated apoptosis could also be demonstrated at the level of mitochondrial depolarization.
AICD has recently been described to commence with the generation of reactive oxygen species in mitochondria, which could only be inhibited using the superoxide dismutase mimic MnTBAP (23). However, MnTBAP did not inhibit NO-induced apoptosis in either Th1 or Th2 cells in our study. Moreover, the GSH-elevating agent
-GC did inhibit NO-induced apoptosis both in Th1- and Th2-polarized cells, while several GSH precursors have previously been reported to be unable to inhibit AICD of T cells (23). Th2 cells had higher basal intracellular GSH levels than Th1 cells, in parallel with their relatively lower sensitivity towards NO-induced apoptosis. The elevated GSH in Th2 cells was predominantly of the reduced form, since the difference detected with the Tietze assay for total cellular GSH was less pronounced than with MCB staining, which only detects reduced GSH. Therefore, it is likely that Th1 cells have reduced oxidative stress coping in general, in accordance with the fact that they have an increased susceptibility to AICD relative to Th2 cells (23).
Elevated GSH might protect Th2 cells against apoptosis via several mechanisms. Apart from its function in detoxification, GSH has been described to interact with the important anti-apoptotic protein Bcl-2 (41). Up-regulation of Bcl-2 has been described to result in elevated levels and redistribution of GSH. However, in the current study we show that Bcl-2 levels do not differ between Th1 and Th2 cells. Secondly, GSH has recently been described to be directly involved in the regulation of mitochondrial membrane potential (42). Since we find differential apoptosis induction by NO between Th1 and Th2 cells at the mitochondrial level, the latter mechanism appears to be consistent with our findings. However, when Th1 and Th2 cells are exposed to NO, this does not result in GSH depletion in either cell type. Furthermore, Th2 cells become sensitized to NO in the presence of acivicin without the occurrence of GSH depletion occurring. Therefore, it is likely that elevated GSH per se is not responsible for the specifically reduced sensitivity towards NO-induced apoptosis in Th2 cells, although it does contribute to a general protective effect. Rather, elevated GSH levels in Th2 cells reflect enhanced import, as a consequence of high
-GT expression.
-GT has been described to modulate the effects of NO by degradation of nitrosoglutathione (GSNO) that is formed upon NO exposure (34,35). Since GSH depletion is not observed during the course of the experiments, sensitization of Th2 cells to NO-induced DNA fragmentation could be due to the reduced breakdown of GSNO, leading to its intracellular accumulation. The time course of NO-induced apoptosis in Th1/Th2-polarized cells supports this notion, since NO-induced apoptosis can first be observed at 6 h and is maximal at 24 h after exposition, which parallels the cellular half-life of GSNO (>4 h) (36), rather than that of Sper (30 min) (30). GSNO has been reported to interact directly with the permeability transition pore in the mitochondrial membrane, increasing its open frequency and thus inducing mitochondrial depolarization (37,38), which is differentially observed between Th1- and Th2-polarized cells. This hypothesis is consistent with recent findings of Ushmorov and co-workers, which describe resistance of subpopulations of Jurkat cells to NO-induced apoptosis to be associated with increased levels of GSH via a complex mechanism (43).
A role for NO in immune maintenance has originally been suggested by experiments in mice, where knock-out of the inducible NO synthase gene resulted in enhanced Th1 responses (8,39). The current study further supports the view that NO functions as a mediator in maintenance of the Th1/Th2 balance in the human system. Human Th1 cells are more sensitive to NO-induced apoptosis due to lower intracellular GSH levels and probably by less efficient detoxification of GSNO. Preferential NO-induced elimination of human Th1 cells at sites of inflammation can in conjunction with AICD preferentially select Th2 cells and subsequently modulate the inflammatory response.
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Acknowledgments
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We thank G. Mesander and H. Moes for technical assistance, B.-J. Wierenga for helpful discussions, S. de Jong for the gift of the NOK-1 antibody, and G. H. Koenderink for critical reading of the manuscript. This work was supported by research grants from the Netherlands Asthma Foundation and `Stichting Astma Bestrijding'.
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Abbreviations
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AICD activation-induced cell death |
AU arbitrary unit |
BSO buthionine sulfoximide |
d.p.s. disintegrations/s |
DPTA-nonoate dipropyltetraamino nonoate |
FasL Fas ligand |
-GC -glutamylcysteine |
-GT -glutamyltranspeptidase |
GSH glutathione |
GSNO nitrosoglutathione |
L/D-NMMA L/D-N-monomethylarginine |
MCB monochlorobimane |
MRP multidrug-resistance protein |
NO nitric oxide |
PE phycoerythrin |
PI propidium iodide |
PHA phythemagglutinin |
PMA phorbol myristate acetate. |
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Notes
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Transmitting editor: J. Borst
Received 24 August 2000,
accepted 5 January 2001.
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