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The Journal of Immunology, 2005, 175: 2158-2166.
Copyright © 2005 by The American Association of Immunologists

Redox Equilibrium in Mucosal T Cells Tunes the Intestinal TCR Signaling Threshold1

Brenda M. Rivera Reyes*, Silvio Danese{dagger}, Miquel Sans{dagger}, Claudio Fiocchi{dagger} and Alan D. Levine2,*,{dagger},{ddagger}

* Department of Pathology, {dagger} Department of Medicine, and {ddagger} Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mucosal immune tolerance in the healthy intestine is typified by lamina propria T cell (LPT) functional hyporesponsiveness after TCR engagement when compared with peripheral blood T cell (PBT). When LPT from an inflamed intestine are activated through TCR cross-linking, their responsiveness is stronger. LPT are thus capable of switching from a tolerant to a reactive state, toggling between high and low thresholds of activation. We demonstrate that in normal LPT global tyrosine phosphorylation upon TCR cross-linking or an increase in intracellular H2O2, an inhibitor of protein tyrosine phosphatases, is muted. Thus, we propose that LPT have a greater reducing capacity than PBT, shifting the balance between kinases and protein tyrosine phosphatases in favor of the latter. Surface {gamma}-glutamyl transpeptidase, an indirect indicator of redox potential, and glutathione are significantly elevated in LPT compared with PBT, suggesting that elevated glutathione detoxifies TCR-induced reactive oxygen species. When glutathione is depleted, TCR-induced LPT tyrosine phosphorylation rises to PBT levels. Conversely, increasing glutathione in PBT attenuates tyrosine phosphorylation. In LPT isolated from inflamed mucosa, TCR cross-linking induces greater phosphorylation, and {gamma}-glutamyl transpeptidase levels are reduced compared with those from autologous noninflamed tissue. We conclude that the high TCR signaling threshold of mucosal T cells is tuned by intracellular redox equilibrium, whose dysregulation may mediate intestinal inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
As distinct lymphocyte populations are distributed among various secondary and tertiary lymphoid organs, the local microenvironments within each tissue affect the functional status of resident T cells (1, 2). The tunable activation thresholds hypothesis proposes that a T cell regulates its sensitivity to activation by tuning its threshold for signaling (3, 4). The essence of this model is the existence of minimum requirements for activation that integrate environmental stimuli with the previous stimulatory experience of the cell. A prime example of tunability is the behavior of intestinal lamina propria T cell (LPT),3 which possesses a distinct proliferative hyporesponsiveness to activation through the TCR/CD3 complex as compared with peripheral blood T cells (PBT) (5). One component of the environment unique to the intestinal lamina propria is the physiologically high oxidative milieu in the mucosa (6, 7). Increased concentrations of reactive oxygen species (ROS) in the gut (8) may also be enhanced by the low local secretion of cysteine, a precursor of the reducing agent glutathione (GSH), by macrophages (9). We propose that the low responsiveness of LPT compared with that of PBT is due to different thresholds for TCR signaling.

Current models of TCR signaling advocate that T cell activation is tuned by the balance of protein tyrosine kinases and protein tyrosine phosphatases (PTP) (10). In particular, tyrosine phosphorylation is critical for the excitation of the T cell, and PTP mediate both de-excitation and nonresponsiveness (4). Because LPT are hyporesponsive to TCR engagement, it is likely that investigating the regulation of protein tyrosine phosphorylation in this signaling cascade may reveal mechanisms underlying the establishment of the LPT activation threshold. TCR cross-linking in mouse thymocytes, T cell hybridomas, and human PBT blasts generates both hydrogen peroxide and superoxide anion, which can be rapidly converted into H2O2 (11, 12). In addition, rapid intracellular formation of ROS is required for CD28-mediated activation of the NF-{kappa}B/CD28-responsive complex and IL-2 expression, which is mediated by 5-lipoxygenase (13). TCR stimulation of primary mouse and human T lymphoblasts induces three distinct events to generate ROS: rapid hydrogen peroxide production independent of Fas or NADPH oxidase; sustained hydrogen peroxide production dependent on both Fas and NADPH oxidase; and delayed superoxide production that is dependent on Fas ligand and Fas, yet independent of NADPH oxidase (14). Because H2O2 reversibly inactivates PTP (15, 16), its presence mimics the stimulatory effects of growth factors and environmental agonists in a variety of cells, including the tyrosine phosphorylation pattern unique to the TCR (17). The efficacy of H2O2 as a secondary messenger is likely to be regulated by the cell’s intracellular redox environment. PTP exist in two states: an active state with a reduced cysteine, presumably maintained by such antioxidants as GSH and thioredoxin, and an inactive state induced by oxidation of the active site cysteine (18). We hypothesize that LPT maintain a highly reducing cytosolic redox equilibrium, potentially in response to their reduced exposure to cysteine during maturation (9). This reduced LPT redox potential may therefore contribute to a weak signaling capacity, because the stability of ROS generated in LPT after TCR/CD3 engagement will be decreased, as the duration of action for these ROS will be transient.

Although the normal LPT exhibit subdued proliferation after TCR engagement, physiologic unresponsiveness to luminal Ags is abrogated in the lesions of patients with inflammatory bowel disease (IBD), a chronic T cell-mediated disease of the large or small bowel (19, 20, 21). These findings suggest that LPT retain the capacity to toggle between a hypo- and hyperresponsive state, possibly by modifying their threshold for activation, as proposed in the tunable activation threshold hypothesis (3, 4). Therefore, we investigated the threshold for physiological, hyporesponsive LPT activation through TCR cross-linking in the context of the cell’s intracellular redox equilibrium and its potential dysregulation in IBD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

PBMC were purified from venous blood of healthy donors by Ficoll-Hypaque density separation. T lymphoblasts were prepared by PHA (0.5%; Invitrogen Life Technologies) stimulation for 48 h in the presence of IL-2 (5 ng/ml; R&D Systems) in RPMI 1640, 10% heat-inactivated FCS, and 25 mM HEPES, and thereafter carried in IL-2 (5 ng/ml) for >8 days to obtain a population of >99% pure CD4+CD45RO+ peripheral blood lymphoblasts (22). Surgical specimens from patients undergoing bowel resection at the University Hospitals of Cleveland and the Cleveland Clinic Foundation were used as a source of LPT. Control LPT from patients admitted for malignant and nonmalignant conditions of the large bowel, including colon cancer and benign polyps, were isolated from histologically normal areas of the mucosa at least 10 cm from the lesion, as previously described (22). In IBD patients, LPT were isolated from macroscopically involved and noninvolved, dysplasia-free segments of the bowel. All diagnoses were confirmed by clinical, radiological, endoscopic, and histological criteria. Institutional review boards of the University Hospitals of Cleveland and the Cleveland Clinic Foundation approved this study. Briefly, the dissected mucosa was freed of mucus and epithelial cells by sequential washings with DTT and EDTA and digested overnight at 37°C with collagenase and DNase. Mononuclear cells were separated from the crude cell suspension by layering on a Ficoll-Hypaque density gradient. If necessary, further purification was achieved by subsequent Percoll density gradients. For generation of LPT lymphoblasts, lamina propria mononuclear cells were stimulated, as described above, for peripheral blood T lymphoblasts. For fresh LPT purification, macrophage-depleted lamina propria mononuclear cells were incubated for 30 min at 4°C with magnetically labeled CD19, CD14, and CD16 Abs directed against B lymphocytes, monocytes, and neutrophils, respectively (Miltenyi Biotec). CD3+ T cells (>95% purity (23)) were then collected by negative selection using the magnetic cells sorting systems (MACS; Miltenyi Biotec).

Reagents

The following reagents were used: anti-phosphotyrosine (PY20; BD Transduction Laboratories), rabbit anti-GAPDH (Trevigen), and HRP-conjugated secondary Abs (Santa Cruz Biotechnology). Reducing and oxidizing agents, 1,3-bis(2-chloroethyl)-1-nitrosurea (BCNU), N-acetylcysteine (NAC), hydrogen peroxide, glucose oxidase, 2-vinylpyridine, and N-ethylmaleimide, were purchased from Sigma-Aldrich. Monobromobimane (MBB) was obtained from Molecular Probes.

Cytokine analysis

LPT and PBT (1 x 106 cells/ml) were cultured in 24-well plates (Costar) in 2 ml of complete medium alone or with 1.5 µg of plate-bound murine monoclonal anti-CD3 (OKT3; Ortho Biotech). At 48 h, supernatants were collected and stored at –20°C until analysis. Levels of IFN-{gamma} were measured with a two-site sandwich ELISA using 1 µg/ml anti-human IFN-{gamma} mAb (M700A; Pierce) for coating and 0.5 µg/ml biotinylated Ab (M701B; Pierce) for detection. The wells were developed with a HRP-streptavidin conjugate (Zymed Laboratories) at 62.5 ng/ml, followed by ABTS peroxidase substrate (Kirkegaard & Perry Laboratories). Spectrophotometric absorbance was measured at 405 nm. This assay was sensitive to IFN-{gamma} levels of 31 pg/ml.

T cell stimulation

LPT and PBT, rested overnight in the absence of IL-2 (RPMI 1640, 10% FCS, 25 mM HEPES) at 37°C, were resuspended (5 x 106/100 µl) in RPMI 1640 and 25 mM HEPES and incubated at 37°C for 5 min. Sheep anti-mouse F(ab')2 (10 µg/ml; Sigma-Aldrich) was added and incubated with the cells for 2 min. Cells were then stimulated via the CD3 receptor with OKT3 Ab (10 µg/ml) at 37°C, immediately followed by the addition of 100 µl of 2x Laemmli sample buffer, and boiled for 10 min. Unstimulated cells received only the sheep anti-mouse F(ab')2.

Immunoblotting

Proteins were separated by SDS-PAGE on a 10% gel under reducing conditions and transferred to nitrocellulose membranes (Invitrogen Life Technologies) in a buffer consisting of 20 mM Tris-HCl, 150 mM glycine, and 20% methanol. Membranes were incubated at room temperature for 1 h in blocking buffer (5% nonfat milk, 0.1% Tween 20 in PBS). Primary and secondary Abs were diluted, as recommended by the manufacturer, in blocking buffer and incubated with the membranes for 1 h at room temperature with six washes in between. Detection of HRP-conjugated Abs was performed using Supersignal (Pierce). Phosphotyrosine analysis was performed, as follows. Membranes were blocked 1 h at room temperature in an alternate blocking buffer (3% BSA, 10 mM Tris, pH 7.6, 100 mM NaCl, and 0.1% Tween 20). HRP-conjugated PY20 Ab was diluted 1/2500 in blocking buffer and incubated with the membranes for 1 h at room temperature. Chemiluminescence for all membranes was detected using Hyperfilm ECL (Amersham Biosciences).

Flow cytometry

The {gamma}-glutamyl transpeptidase (GGT) expression on the T cell surface was assessed using the Elite ESP and Epics XL flow cytometers (BD Biosciences). Lymphocytes were washed and suspended in 0.1 ml of PBS containing 1 µg of murine anti-GGT mAb (a kind gift of D. Karp, University of Texas Southwestern Medical Center, Dallas, TX). After 30-min incubation on ice, the cells were washed and resuspended in 0.1 ml of PBS containing FITC-labeled goat anti-mouse IgG (BioSource International). Following an additional 30-min incubation at 4°C in the dark, lymphocytes were washed and suspended in 0.1 ml of PBS containing 10 µl of PE-labeled mouse anti-human CD3 mAb (DakoCytomation). After another 30-min incubation at 4°C in the dark, the cells were washed, fixed with 1% paraformaldehyde, and analyzed. Background immunofluorescence was determined by incubating the cells with FITC- or PE-conjugated mouse IgG (AMI4408; AMI4407; BioSource International). PE-labeled CD3+ T cells were identified and gated by single-parameter analysis. Data from 15,000 events were collected and analyzed for FITC-labeled GGT expression using WinList software (BD Biosciences).

GSH measurements

Intracellular thiol was measured using flow cytometry, as previously described, with the following modifications (24). T cells were stained with allophycocyanin-labeled anti-CD3, washed with PBS, and allowed to equilibrate to room temperature. One tube received MBB alone (10 µM), and the other tube received N-ethylmaleimide (40 µM) and MBB (10 µM). Allophycocyanin-labeled CD3+ T cells were identified and gated. Data from 15,000 events were recorded 10 min after the cells were labeled with MBB and analyzed using WinList software. Intracellular GSH levels, both GSH and GSH disulfide (GSSG), were also measured with a colorimetric assay that relies on enzymatic recycling using GSH reductase and 5,5'-dithiobis-2-nitrobenzoic acid as a substrate, as described by the manufacturer (Cayman Chemical).

Statistics

The percentage of T cells positive for GGT expression, the mean fluorescence intensity (MFI), and intracellular GSH levels were plotted as means and SE of the data. Based on the measurements, the unpaired t test was used to examine the difference between LPT and PBT (Fig. 4). Differences between autologous inflamed vs noninflamed segments of the same bowel were determined using the paired t test (Fig. 6). All tests were two tailed, and p values <0.05 were considered significant. All analyses were done using the GraphPad statistics software.



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  		FIGURE 4. Enhanced reductive capacity in LPT. A, LPT lymphoblasts from normal segments of the colon (n = 14) and PBT lymphoblasts from healthy volunteers (n = 8) were cultured in IL-2 for 10 days and rested overnight in the absence of IL-2. Cells were stained for cell surface GGT with a specific mouse mAb, followed by FITC-labeled goat anti-mouse. Cell fluorescence was measured by flow cytometry. The percentage of positive cells ({cjs2108}) and MFI ({blacksquare}) plus SEM are s hown. #, p < 0.0001; {dagger}, p = 0.005 (unpaired, two-tailed t test). B, Representative histograms of PBT (left) and LPT (right), stained for surface GGT expression, are shown. C, Total intracellular GSH was measured in PBT (n = 3) and LPT (n = 3) using MBB staining and quantitation by flow cytometry. #, p < 0.004 (unpaired, two-tailed t test). D, Both cytosolic GSH and GSSG from 30 x 106 cells were measured in PBT (n = 4) and LPT (n = 4) using a colorimetric kit. *, p < 0.03 (unpaired, two-tailed t test).

 


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  		FIGURE 6. Dysregulated intracellular redox equilibrium in LPT from inflamed mucosa lowers the threshold for T cell activation. A, LPT lymphoblasts from inflamed and noninflamed colonic mucosa (n = 4) from IBD patients were cultured in IL-2 for 10 days and rested overnight in the absence of IL-2. Inflamed and noninflamed freshly isolated LPT or LPT lymphoblasts were left unstimulated or stimulated by anti-CD3 cross-linking for 1, 5, or 15 min. B, Inflamed and noninflamed LPT lymphoblasts were left unstimulated or treated with 1 or 5 mM H2O2 for 1 or 5 min. Cell extracts from a representative donor were analyzed by phosphotyrosine immunoblotting. GAPDH loading control was performed. C, Inflamed and noninflamed LPT were stained for surface GGT, and percentage of GGT-positive cells ({cjs2108}) and MFI ({blacksquare}) were measured by flow cytometry. Statistical differences between inflamed and noninflamed LPT were significant for both percentage of positive (*, p = 0.01) and MFI (#, p = 0.03) (paired, two-tailed t test).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPT hyporesponsiveness: unique function, distinct signal transduction pathways

A reduced proliferative response of LPT compared with PBT after CD3 cross-linking is the hallmark functional phenotype of the mucosal T cell. Because PBT comprise both naive (CD45RA+) and memory (CD45RO+) T cells, whereas LPT are mostly memory (25), we reported that LPT hyporesponsiveness is not due to their memory phenotype alone (22). In addition, other biological responses of LPT, as defined by cytokine synthesis, are similarly diminished after TCR engagement (26). To extend this earlier report, freshly isolated PBT and LPT, purified by negative selection to >95% CD3+ (23), were cultured in 24-well plates in medium alone or with anti-CD3. Induction of IFN-{gamma} secretion after TCR cross-linking was detected in both T cell types (Fig. 1), yet the level of cytokine production was significantly reduced when LPT were compared with PBT (p < 0.01). We therefore hypothesized that the reduced biological responses associated with activation of LPT through TCR/CD3 cross-linking are linked to modification in the membrane-proximal signal transduction cascade, resulting in protein tyrosine phosphorylation. To investigate this hypothesis, freshly isolated T cells from the intestinal mucosa and blood were purified by negative selection (23) and rested for either 5 h or overnight in complete medium. The T cells were stimulated for 1 or 5 min by cross-linking the CD3 complex, as we described previously (10), and the cell extract was analyzed for global tyrosine phosphorylation by immunoblotting with an anti-phosphotyrosine Ab (10). As expected, PBT responded vigorously to CD3 cross-linking (lane 1 vs lanes 2 and 3, Fig. 1B, right panel), while the level of tyrosine phosphorylation in activated LPT was markedly lower (lane 1 vs lanes 2 and 3, Fig. 1B, left panel). This reduced tyrosine phosphorylation after TCR/CD3 engagement in LPT was present between 1 and 30 min, after which PBT and LPT phosphorylation status returns to basal levels. The muted membrane-proximal signaling in freshly isolated LPT was also observed in LPT lymphoblasts (Fig. 1C). PBT and LPT lymphoblasts (>99% CD4+CD45RO+ (22)) were maintained in culture with IL-2 for 10 days, rested overnight in the absence of cytokine, and subsequently activated by anti-CD3 cross-linking. Whole cell lysates were prepared after 5 min, and the tyrosine phosphorylation response was analyzed by immunoblotting. A reduced intensity of phosphotyrosine proteins was also observed in activated LPT lymphoblasts, far less than the level of protein phosphorylation detected with PBT lymphoblasts (lane 2 vs 4, Fig. 1C). The observation that diminished membrane-proximal phosphotyrosine induction after TCR engagement is preserved in IL-2-expanded LPT lymphoblasts after 1- to 2-wk cultures suggests that the regulatory mechanisms for this decreased response are imprinted on the resident LPT. This muted global tyrosine phosphorylation in CD3-cross-linked LPT reflects the functional hyporesponsiveness exhibited by this T cell subset.



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  		FIGURE 1. Muted cytokine production and tyrosine phosphorylation in LPT after TCR engagement. A, Freshly isolated LPT from normal colonic mucosa and PBT from healthy volunteers were left unstimulated ({blacksquare}) or stimulated ({cjs2108}) by plate-bound anti-CD3 cross-linking for 48 h. Cell-free conditioned medium was harvested and analyzed for IFN-{gamma} production by ELISA (n = 6; *, p < 0.01). B, LPT (left panel) and PBT (right panel) were freshly isolated from control donors by Ficoll-Hypaque sedimentation and negative selection. The cells were left unstimulated or stimulated by anti-CD3 cross-linking for 1 or 5 min. Cell extracts were analyzed by phosphotyrosine immunoblotting. Protein loading was controlled by anti-GAPDH immunoblotting (data not shown). C, LPT lymphoblasts from normal colonic mucosa and PBT lymphoblasts from healthy volunteers, maintained in IL-2 for 10 days and rested overnight without IL-2, were left unstimulated or stimulated by anti-CD3 cross-linking for 5 min. Cell extracts were analyzed by immunoblotting for phosphotyrosine. Immunoblot for GAPDH was used as a control for total protein loading. The m.w. markers are indicated. These findings are representative of a minimum of six donors.

 
Exogenous ROS induce attenuated signal transduction in LPT

Attenuated signal transduction emanating from the TCR in LPT may be mediated by a variety of molecular alterations, including reduced protein tyrosine kinase (PTK) activity, increased PTP activity, and enhanced activity of negative regulators such as Csk or Shp-1, among others. Although each of these mechanisms may contribute to the overall LPT hyporesponsive phenotype, we chose to focus on the potential effects of ROS because a dominant role for exogenous thiol-mediated redox regulation of intestinal T lymphocytes has been reported (9). In addition, it has been proposed that low m.w. nonprotein mediators with oxidative capacities produced in the gut milieu may be involved in decreased Ag-mediated stimulation of LPT (6). However, the mechanisms behind these observations have not been reported. Because exogenous application of hydrogen peroxide mimics the stimulatory effects of growth factors and environmental agonists in a variety of cells, including the tyrosine phosphorylation pattern unique to the TCR (17), we used short-term stimulation with low concentrations of hydrogen peroxide to mimic the reported effects of redox mediators on LPT (9), as a tool to investigate the ability of LPT and PBT to respond to environmental changes in oxidative capabilities (Fig. 2). Rested LPT and PBT lymphoblasts were stimulated via anti-CD3 cross-linking or with 5 mM H2O2, the threshold concentration for mimicking the TCR tyrosine phosphorylation pattern in Jurkat cells and PBT (17). Immunoblotting for phosphotyrosine revealed minimal activation of LPT for either stimulus (lanes 2 and 3, Fig. 2), especially when compared with the robust phosphorylation induced in PBT (lanes 5 and 6, Fig. 2). A similar evaluation of the effects of H2O2 on T cell biology is unfortunately not possible, due to the inability of T cells to remain functional after long-term exposure to ROS in culture. Alternately, T cells can be exposed to an exogenous oxidative stress generated by adding 100 mU glucose oxidase to the culture medium (27). In fibroblasts, within 10 min H2O2 concentrations in the cell reach a steady state level of 30 µM (28). As we showed with H2O2 treatment, the differences in membrane-proximal tyrosine phosphorylation were observed reproducibly in numerous samples (data not shown), demonstrating that LPT hyporesponsiveness is not only limited to inefficient engagement of the most apical step in TCR cross-linking, but must also be associated with a generalized defect in signal transduction pathways regulated by ROS.



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  		FIGURE 2. Attenuation of H2O2-induced tyrosine phosphorylation in LPT. LPT and PBT lymphoblasts were left unstimulated, stimulated by anti-CD3 cross-linking for 5 min, or treated with 5 mM H2O2 for 5 min. Cell extracts were analyzed by phosphotyrosine and anti-GAPDH immunoblotting. These results are representative of at least six donors.

 
Rapid reversal of PTP activity in H2O2-treated LPT

The weak tyrosine phosphorylation response of LPT exposed to hydrogen peroxide may reflect an inherent resistance of LPT to environmental oxidative stress, due in large part to the highly oxidative milieu of the gastrointestinal tract (9). It is possible that higher concentrations of peroxide are necessary to elicit a similar response as that seen with PBT. Alternatively, the intracellular redox environment of LPT may be highly reductive, and thus, any effects of ROS would be rapidly reversed. To distinguish between these possibilities, LPT and PBT were activated at two concentrations of H2O2, and the stability of the tyrosine phosphorylation was evaluated (Fig. 3). A concentration of 1 mM hydrogen peroxide, saturating for signal transduction in PBT, had minimal effects on LPT phosphotyrosine induction (lanes 3 and 4, Fig. 3). We have previously reported that 0.2 mM hydrogen peroxide had no effect on protein tyrosine phosphorylation, and that the lowest threshold concentration for altered kinase and phosphatase activity in T cells was 1 mM (29). A higher concentration of H2O2 (5 mM) stimulated modest protein phosphorylation in LPT at 1 min, which was still less than the strong response elicited by 1 mM H2O2 in PBT (lane 5, Fig. 3B vs lane 3, Fig. 3A). Notably, global protein tyrosine phosphorylation was markedly reversed by 5 min in LPT, but not in PBT (lane 6, Fig. 3, B vs A). We interpret these findings as indicating that higher H2O2 concentrations are required in LPT than PBT for oxidation of the crucial cysteine within the active site of PTP. It is important to recognize that inhibiting PTP activity may also prevent the initiation of TCR signal transduction via PTP-mediated activation of p56Lck. However, it was reported that exposure of cells to H2O2 activates Lck by inducing phosphorylation on Tyr394, a conserved residue within the activation loop of the catalytic domain (30). Lck that has been activated by H2O2 is simultaneously phosphorylated at both the C-terminal Tyr505 and Tyr394. Dephosphorylation of Tyr505 is not a prerequisite for either phosphorylation of Lck at Tyr394 or catalytic activation of the kinase (30). Therefore, because the reducing potential within the LPT cytosol appears to be extremely high, rapid reversal of PTP inactivation by hydrogen peroxide occurs, thus crafting a similarly rapid removal of phosphotyrosine residues.



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  		FIGURE 3. Rapid reversal of PTP activity in H2O2-treated LPT. PBT (A) and LPT (B) lymphoblasts were left unstimulated, stimulated by anti-CD3 cross-linking for 5 min, or treated with 1 or 5 mM H2O2 for 1 or 5 min. Cell extracts were analyzed by phosphotyrosine immunoblotting. GAPDH loading control was performed. These findings are representative of a minimum of six donors.

 
Increased activity of glutamyl cycle and total intracellular GSH levels in LPT

To directly test our hypothesis that the cytosol of LPT is highly reductive, we evaluated their intracellular redox equilibrium. The predominant, nonenzymatic regulator of a cell’s cytosolic redox status is the reducing agent GSH, a cell membrane-impermeable tripeptide. One mechanism by which GSH levels are elevated within a cell is via the glutamyl cycle, in which the membrane-bound enzyme GGT cleaves glutamyl groups from extracellular GSH (31). The resulting dipeptide and glutamyl are then transported into the cell and rapidly resynthesized into GSH, thus maintaining intracellular levels of GSH higher than extracellular levels. Measuring cell surface GGT levels by flow cytometry is an accepted, indirect assessment of the reducing environment within a cell (24) (Fig. 4). GGT levels were significantly elevated on LPT when compared with those on PBT (p = 0.0001 for percentage of positive cells; p = 0.005 for MFI; Fig. 4A). A representative histogram shows the increase in percentage of GGT-positive cells and the MFI for GGT within LPT lymphoblasts (Fig. 4B). Because both fresh LPT and LPT lymphoblasts exhibit reduced cytokine production and muted protein tyrosine phosphorylation after TCR engagement, we demonstrated that the change in redox potential is preserved after culturing in IL-2 (Table I). GGT levels in freshly isolated LPT were indistinguishable from those measured in LPT lymphoblasts, and both were significantly increased when compared with fresh PBT and PBT lymphoblasts. These findings suggest that the cytosolic environment of the LPT is highly reductive. To confirm these results, we determined the levels of intracellular thiols by fluorescently labeling GSH with MBB, which reacts nonenzymatically with thiols, and recorded the degree of fluorescence by single cell analysis using flow cytometry (24). Low concentrations of MBB combined with a short staining time produce a fluorescent GSH-MBB compound whose signal is proportional to intracellular GSH (24, 32). GSH levels in LPT lymphoblasts, when contrasted with PBT lymphoblasts, were significantly elevated (Fig. 4C; p < 0.004). Increased intracellular GSH was observed in both freshly isolated LPT and LPT lymphoblasts (MFI range: 104–114) when compared with similarly prepared PBT (MFI range: 52–75). Strikingly, fluorescent labeling of both freshly isolated T cells and T lymphoblasts for intracellular GSH was homogeneous, indicating that elevated GSH in mucosal T cells is a distinct phenotype of these cells. In addition, the total intracellular GSH levels in PBT vs LPT lymphoblasts were determined using a colorimetric assay. As a third assessment of the relative redox equilibrium between PBT and LPT, intracellular GSH concentrations were compared with GSSG levels in both cell subsets using a colorimetric kit (Fig. 4D). Measurements directly on the lysates will record total glutathione (GSH and GSSG), while those taken after treating the lysates with 2-vinylpyridine will quantify GSSG only, allowing for a calculation of GSH levels. Normalizing all lysates to 30 x 106 T cells, cytosolic GSH concentration was >2-fold higher in LPT vs PBT (Fig. 4D; p < 0.03). No GSSG was detected. Together, these results demonstrate a greater reducing potential in LPT and implicate this change in redox equilibrium as a possible explanation for the muted LPT tyrosine phosphorylation response to exogenous or endogenous H2O2.


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  		Table I. Increased GGT expression on both fresh LPT and LPT lymphoblasts

 
Modulation of the intracellular redox equilibrium tunes the threshold of TCR signaling

Because TCR cross-linking generates ROS, especially hydrogen peroxide, it was proposed that H2O2 inhibits PTP, and thus prolongs the signaling cascade (12). We extend this concept by proposing that elevated GSH in LPT should rapidly detoxify both exogenous H2O2 and ROS induced by TCR cross-linking, yielding a hyporesponsive T cell. When GSH concentrations in LPT lymphoblasts (average MFI: 107) were reduced to a level less than that of PBT (average MFI: 75) by treatment with 50 µM BCNU (MFI: 28), a GSH reductase inhibitor (33, 34), the duration and intensity of both TCR-induced (Fig. 5) and H2O2 (data not shown) global protein tyrosine phosphorylation increased to the level of PBT. LPT lymphoblasts, maintained in the absence and presence of BCNU, were stimulated by anti-CD3 cross-linking for 1, 5, and 15 min, and signal transduction was analyzed by immunoblotting for phosphotyrosine. As we previously proposed (10), disrupting the balance between kinases and phosphatases in unstimulated LPT with BCNU increased the basal level of phosphorylation (lane 5 vs 1, Fig. 5A). Furthermore, the threshold for T cell activation was similarly lowered in BCNU-treated LPT in that the intensity of signaling at 1 and 5 min approached that of PBT (lanes 6 and 7, Fig. 5A), and the phosphorylation pattern remained stable for 15 min (lane 8 vs 4, Fig. 5A). Reducing GSH levels in LPT decreases the clearance of TCR-induced ROS, thereby increasing PTP inhibition and reversing LPT hyporesponsiveness. Conversely, when PBT (MFI: 75) were treated with the antioxidant, NAC, to increase GSH levels (MFI: 98) to that of LPT (MFI: 107), global tyrosine phosphorylation in both unstimulated and anti-CD3-cross-linked PBT was markedly attenuated (Fig. 5B). This reduced induction of tyrosine phosphorylation was also observed in H2O2-stimulated, NAC-pretreated PBT (data not shown). We conclude that hyporesponsiveness in LPT is modulated via intracellular redox equilibrium, making it essential in tuning TCR signaling threshold and cell function.



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  		FIGURE 5. Modulation of the intracellular redox equilibrium tunes TCR signaling threshold. A, Rested LPT lymphoblasts were incubated in the absence (Control) or presence of 50 µM BCNU for 90 min, washed, and then left unstimulated or stimulated by anti-CD3 cross-linking for 1, 5, or 15 min. B, Rested PBT lymphoblasts were incubated in the absence (Control) or presence of 25 mM NAC for 6 h, washed, and then left unstimulated or stimulated by anti-CD3 cross-linking for 5 min. Cell extracts were analyzed by phosphotyrosine and anti-GAPDH immunoblotting. These results were observed with three donors.

 
Dysregulated intracellular redox equilibrium in LPT from inflamed mucosa lowers the threshold for T cell activation

The pathogenesis of IBD, distinguished by chronic, relapsing inflammation in the intestinal mucosa of unknown etiology, has been attributed to an immune dysfunction in the balance between mucosal T cells with immunoprotective activity and those with regulatory functions. The chronicity of IBD is partially perpetuated by the influx and expansion of T cells into the lamina propria. Accompanying the increased number of LPT in IBD mucosa is the concomitant increase of mucosal immune activation, classically typified as a strong T cell functional response (19) after TCR engagement. LPT are thus capable of switching from a quiescent to an active state during the inflammatory process, exchanging a high threshold for initiation of signaling to a lower one. Based on our findings that the threshold for T cell responsiveness is tuned by the cell’s redox equilibrium, we propose that LPT in involved IBD mucosa have decreased intracellular reductive capacity. The regulation of membrane-proximal signal transduction was contrasted between LPT from autologous inflamed (I) and noninflamed (NI) intestinal tissues from patients with ulcerative colitis or Crohn’s disease (Fig. 6). When stimulated through the TCR for 1, 5, and 15 min, as expected I-LPT showed a greater and more sustained induction of tyrosine phosphorylation as compared with NI-LPT from the sample patient when assessed by immunoblotting for global phosphotyrosine (lanes 2–4 vs lanes 6–8, Fig. 6A). Because normal LPT are also hyporesponsive to H2O2 activation and rapidly reverse the effects of phosphatase oxidation, properties not observed with PBT, we investigated whether an inflamed LPT would exhibit a PBT-like response to H2O2. I-LPT and autologous NI-LPT from an IBD patient were stimulated with 1 and 5 mM H2O2, and at 1 and 5 min of treatment total tyrosine phosphorylation was analyzed by immunoblot (Fig. 6B). As shown for normal LPT, NI-LPT exhibited no to minimal phosphoprotein with 1 mM H2O2, while autologous I-LPT responded strongly (lanes 2 and 3 vs lanes 6 and 7, Fig. 6B). Furthermore, as expected, NI-LPT rapidly reversed phosphatase oxidation within 5 min (similar to normal LPT), while tyrosine phosphorylation, and thus oxidized phosphatases, persists at high concentrations of H2O2 in I-LPT (as seen with PBT) (lanes 4 and 5 vs lanes 9 and 10, Fig. 6B). To address whether the differences in TCR signaling between LPT from I and NI tissue are due to their intracellular reductive capacities, the percentage of cells positive for GGT surface expression (p = 0.01) and the MFI for GGT (p = 0.03) were observed to be significantly higher in NI vs autologous I-LPT (Fig. 6C). These results implicate dysregulated intracellular redox equilibrium as a potential mediator of chronic intestinal inflammation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Immune tolerance to nonpathogenic, commensal microbiota at mucosal surfaces is essential for survival. Similarly, the ability to toggle between normal hyporesponsiveness and fully activated effector mechanisms is critical for host defense. In this study, we demonstrate that mucosal LPT attain these distinct responsive states by modulating their intracellular redox equilibrium. A highly reductive capability attenuates tyrosine phosphatase inhibition, yielding a hyporesponsive T cell upon TCR cross-linking, while a lower intracellular reducing potential, as seen in the circulating PBT or inflamed LPT, leads to a full spectrum of protein tyrosine phosphorylation and immune effector function.

Random rearrangement of TCR gene segments not only yields variable CDRs that recognize peptide in the context of MHC, but also enables the T cell to sample an enormous variety of potential peptide ligands with low, intermediate, and high affinity (35, 36, 37, 38). The ability of the TCR to recognize multiple peptides led to a proposal that T cells are selected to be moderately autoreactive, and their degree of autoreactivity is continuously controlled through tuning an activation threshold (39). The intestinal mucosa represents an ideal paradigm in which microenvironmental stimuli engage T cells in subthreshold interactions, mediating functions such as maintenance of immunological memory (3). However, the molecular mechanisms that establish this threshold and tune its level of excitation and de-excitation were previously unknown. Our results reveal that high intracellular GSH levels modulate this threshold by decreasing the intensity and duration of global protein tyrosine phosphorylation after TCR cross-linking, possibly by tipping the balance between PTK and PTP in favor of the latter.

Intracellular oxidative potential has recently been implicated in B lymphocyte signaling amplification by a PTK Syk/ITAM-positive feedback loop. The Syk-dependent activation of the BCR is counterbalanced by PTP, whose activity in turn is regulated by H2O2 and the cytosolic redox equilibrium (40). Similar to our findings with TCR cross-linking of NAC-treated PBT, stimulation of rat vascular smooth muscle cells by platelet-derived growth factor increases tyrosine phosphorylation, MAPK stimulation, DNA synthesis, and chemotaxis, which were inhibited by increasing the intracellular concentration of the chemical antioxidant NAC (41). Furthermore, consistent with its reversal of LPT hyporesponsiveness shown in this report, BCNU enhanced JNK, p38 MAPK, the I{kappa}B kinases, NF-{kappa}B activation, and IL-2 transcription, by lowering the intracellular GSH/GSSG ratio. Together, these findings indicate that oxidation of the cellular thiol pool amplifies TCR signal transduction during an immune response (34) and suggest that this enhancement may be due to either an inhibition of PTP or an activation of specific PTK, or both. In fact, oxidant-induced modification of cellular enzymes or structural proteins is an underdeveloped area of research and may explain why some of the biological and biochemical responses to oxidizing agents parallel those of phosphatase inhibitors.

Hyporesponsiveness in LPT was initially identified by their diminished proliferation upon TCR cross-linking (42) and was recently extended to include decreased Ca2+ signaling in normal LPT, which was much higher in blood-derived T cells and T cells from inflamed mucosa (43). Furthermore, an earlier report also showed qualitative differences in induction of tyrosine phosphorylation by CD3 in LPT vs PBT and that CD3 ligation failed to induce substantive substrate phosphorylation in LPT (26). Our results further define the mechanism for these events by revealing that membrane-proximal signal transduction emanating from the TCR is dramatically reduced in LPT. This low protein tyrosine phosphorylation may be attributed to increased PTP activity due to the highly reductive redox equilibrium in the LPT. Although elevated PTP activity was shown to stimulate components of the TCR signal transduction pathway (29), phosphatases also play a key role in initiating or perpetuating the TCR response. The effects of H2O2 are readily discernible in this model for two reasons. First, dephosphorylation of Tyr505 is not a prerequisite for either phosphorylation of Lck at Tyr394 or catalytic activation of the kinase (30). In addition, the Src family kinase inhibitor PP2 inhibited the H2O2-induced phosphorylation of ERK, p38, and JNK (44), demonstrating that Lck activation is a critical step in the signal transduction cascade resulting from H2O2 exposure. We therefore propose that H2O2 inhibition of the PTP that dephosphorylates tyrosine394 guarantees the initiation of an accurate mimic of the TCR/CD3 signaling cascade (30, 44). The ability of redox potential to regulate Ca2+ influx is directly related to the loss of linker for activation of T cells phosphorylation (45). In addition, Ca2+ signaling is indirectly regulated by redox, as a recent report demonstrates that oxidative stress activates PI3K-dependent signaling via the inactivation of the PTEN phosphatase, leading to an increase in cellular phosphatidylinositol (3, 4, 5) triphosphosphate, a trigger for Ca2+ release (46).

Functional LPT hyporesponsiveness and the decrease in Ca2+ signaling are partially reversed in IBD (43), consistent with the increase in global tyrosine phosphorylation in TCR-activated LPT from inflamed, but not noninflamed mucosal tissue. Increased production of ROS has been reported in IBD mucosa, often associated with depleted antioxidant defenses (47, 48). Decreased {gamma}-glutamylcysteine synthetase activity in IBD mucosa results in decreased availability of cysteine for GSH synthesis contributing to a GSH deficiency (49). Furthermore, treatment of dextran sulfate sodium- or 2, 4, 6-trinitrobenzene sulfonic acid-induced experimental models of colitis either with the antioxidant NAC or by replenishing GSH attenuates the acute inflammation, increases mucosal GSH and cysteine levels, decreases colonic damage, and reduces the incidence of colitis-associated colorectal adenocarcinoma (50, 51, 52).

Both attenuated global tyrosine phosphorylation after TCR cross-linking and elevated GSH, representative of the normal, hyporesponsive mucosal T cell, were observed in freshly isolated LPT and in LPT lymphoblasts that had been expanded in IL-2 for 10 days. The striking nature of the latter finding indicates that once peripherally circulating T cells are recruited into the intestinal mucosa, its microenvironment educates them to express the unique functional LPT phenotype characterized by a high threshold and hyporesponsiveness (6). Culturing PBT with intestinal mucosa supernatant yields a similar proliferative response as that found in freshly isolated LPT, which can be reversed by reducing agents such as 2-ME or DTT, suggesting that oxidative substances are contained in these mucosal supernatants (6). We have previously reported that T cell migration through the Peyer’s patches (53) or exposure to the interstitial extracellular matrix (54) induces distinct mucosal immune functions. The results in this report further indicate that the changes in LPT phenotype are permanent, suggesting that unique pathways are engaged that trigger the reducing potential in the normal LPT, maintain elevated GSH levels, and facilitate the toggling of redox equilibrium to accommodate LPT hyporesponsiveness and immune protection.


   Acknowledgments
 
We thank Gail West and Michael Sramkoski for technical assistance; Andrew Schade for invigorating discussions; the Departments of Surgery, Pathology, and the Cooperative Human Tissue Procurement Facility, University Hospitals of Cleveland; and the Colorectal Surgery Department of the Cleveland Clinic Foundation. Virginia Wong provided assistance with the IFN-{gamma} ELISA.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (DK-54213 and AI-53188 to A.D.L.; DK30399 and DK50984 to C.F.; GM66499 and GM07250 to B.M.R.R.) and the Crohn’s and Colitis Foundation of America (to S.D.). Back

2 Address correspondence and reprint requests to Dr. Alan D. Levine, Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4952. E-mail address: alan.levine{at}case.edu Back

3 Abbreviations used in this paper: LPT, lamina propria T cell; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosurea; GGT, {gamma}-glutamyl transpeptidase; GSH, glutathione; GSSG, GSH disulfide; I, inflamed; IBD, inflammatory bowel disease; MBB, monobromobimane; MFI, mean fluorescence intensity; NAC, N-acetylcysteine; NI, noninflamed; PBT, peripheral blood T cell; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species. Back

Received for publication January 18, 2005. Accepted for publication May 30, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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