{gamma}-Glutamyl transpeptidase is up-regulated on memory T lymphocytes

David R. Karp, Margaret L. Carlisle, Angela B. Mobley1, Timothy C. Nichols2, Nancy Oppenheimer-Marks, Ruth I. Brezinschek and V. Michael Holers2

The Simmons Arthritis Research Center and
1 Department of Microbiology, UT Southwestern Medical Center, Dallas, TX 75235, USA
2 The Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA

Correspondence to: D. R. Karp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 
The ectoenzyme {gamma}-glutamyl transpeptidase (GGT) hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH levels and modifies the activity of GSH-containing adducts. Previous data suggested that this enzyme was present on mitogen-activated T lymphocytes. However, the level of GGT protein expression on human mononuclear cell subsets has not been determined. A novel mAb to human GGT, 3A8, was developed. 3A8 was used to show that the expression of GGT is, in fact, highest on resting T cells that express markers of the memory phenotype, specifically CD45RO and decreased expression of CD45RB. The peripheral blood of patients with rheumatoid arthritis was found to have expanded numbers of T cells expressing levels of GGT up to 10-fold higher than controls. In addition, the CD4+ T cell subset with the capacity to migrate across a human endothelial cell monolayer expresses high GGT levels. GGT expression was up-regulated on peripheral blood T cells following activation in vitro by either superantigen, phorbol ester, or IL-15, a stimulatory cytokine synthesized in rheumatoid synovium. Resting peripheral blood T cells that express GGT have higher levels of intracellular thiols than those that do not. These observations suggest that GGT may play an important role in the regulation of lymphocytes that are at a particular developmental stage.

Keywords: cellular differentiation, cell surface molecules, glutathione, human, T lymphocytes, rheumatoid arthritis


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 
Several characteristics define naive and memory T lymphocytes. Naive T cells which have not been exposed to their specific antigen express low levels of adhesion and activation markers such as CD11a/CD18, CD44 and CD25, as well as high levels of CD62L (L-selectin) (1). They also express particular isoforms of the protein phosphatase, CD45. In humans, naive T cells express CD45RA. In contrast, antigen-experienced memory T cells that are CD4+ express high levels of adhesion molecules, low levels of L-selectin and have converted to the expression of the CD45RO isoform. After repeated exposure to antigen, memory CD4+ T cells also express reduced levels of CD45RB and CD27 (25). Functionally, memory cells respond more quickly to antigen, have enhanced ability to migrate across endothelial barriers and promote Ig synthesis by B cells (6,7).

The expression of several ectoenzymes has been shown to vary on populations of T cells at different stages of differentiation. For example the expression of CD26 (dipeptidyl dipeptidase IV) is up-regulated on T cells after activation (810) and has been shown to be involved in RANTES activation (11). Enzyme activity also enhances T cell stimulation through the TCR (12). The 5'-nucleotidase (CD73, VAP2) is found primarily on naive CD8+ cells and is co-stimulatory for the generation of alloreactive cytotoxic T lymphocytes or activation of T cells by CD3 (1315). CD38 is a multi-functional protein expressed on activated, but not resting T and B cells (16). As an enzyme, it catalyzes the formation and hydrolysis of cyclic adenosine diphosphate-ribose. It also activates T and B cell lines by directly promoting the phosphorylation of a number of protein tyrosine kinases (17,18). These enzymes have also been reported to be involved in adhesive interactions between T cells and the extracellular matrix (CD26) (19) or between T cells and other cells (CD38, CD73) (20,21). The regulated expression of these multifunctional proteins suggests that they may be important mediators of the differentiated activity of lymphocytes.

{gamma}-Glutamyl transpeptidase (GGT, EC 2.3.2.2) is an 87 kDa type II transmembrane ectoenzyme (reviewed in 22). It is a heterodimer composed of 65 and 22 kDa subunits. The majority of the protein is extracellular, with only the N-terminal methionine and three lysine residues located inside the plasma membrane (23). The major role of GGT at the cell surface is to cleave glutamyl groups from glutathione (GSH) or glutathione-S-conjugates. In doing so, it can transfer glutamyl groups to an amino acid, di-peptide, water or another molecule of GSH. The remaining cysteinyl-glycine can be cleaved by a membrane-bound dipeptidase. The glutamyl adducts, cysteine and glycine, can be transported into cells, where a series of enzymes re-synthesize GSH (24). This `glutamyl cycle' has been postulated to be necessary to maintain levels of intracellular GSH that are much higher than that in the extracellular space. Support for this hypothesis has been provided by recent experiments in which the single mouse gene for GGT was inactivated (25). Intracellular GSH levels in all tissues examined were ~50% of the levels seen in the wild-type mouse.

GGT enzyme activity has previously been demonstrated on peripheral blood lymphocytes (PBL) and some lymphocytic cell lines (26). This activity was increased in human PBL and rat splenocytes by concanavalin A (ConA) activation. More recent studies that looked at resting mononuclear cells have suggested that CD14+ cells have the highest levels of GGT enzyme activity, while CD4+ T cells have intermediate, and CD8+ T cells, NK cells and B cells have very low levels (27). In another study, a polyclonal antibody raised to a GGT–Protein A fusion protein has been used to analyze human peripheral blood mononuclear cells (PBMC) and bone marrow (28). Monocytes expressed higher levels of GGT than T or B lymphocytes. No consistent data showing the expression of GGT on different functional subsets of human mononuclear cells are available, however.

To examine the relationship between GGT expression and the differentiation state of lymphocytes, a mAb (3A8) to human GGT was developed. Using this reagent, a previously unknown association of GGT with a subset of cell surface proteins including CD81, CD53, and CD82 was demonstrated (29). The function of these proteins is unknown, although they form clusters on the cell surface and associate in distinct groups with other proteins including CD21, CD19, MHC class I and II, and VLA-4 (3033). In the present report, the expression of GGT on human mononuclear cells was examined.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 
Cells and antibodies
Peripheral blood was obtained from normal donors as well as patients with active rheumatoid arthritis (RA). All individuals gave informed consent and the UT Southwestern Institutional Review Board approved this study. Arthritis patients all had active synovitis at the time of blood collection, but were not further stratified. PBMC were separated from venous blood by density gradient centrifugation over Histopaque-1077 (Sigma, St Louis, MO). Purified T lymphocyte populations were obtained as described (7). T cells were prepared from PBMC by immunomagnetic depletion using mAb to CD14 (clone 63D3), CD16 (clone B73.1), CD19 (clone HD37) and HLA-DR (clone L243). 3A8 is an IgG2a mAb to human GGT (29). It was coupled to FITC using standard techniques. UPC-10 is an IgG2a control mAb of unknown specificity. Phycoerythrin (PE)-conjugated anti-human CD4 (clone 13B8.2) and CD8 (clone B9.11) were from CoulterImmunotech (Miami, FL). PE-conjugated anti-CD45RA (clone HI100), CD45RO (clone UCHL1), CD45RB (clone MT4) and CD27 (clone M-T271) were from PharMingen (San Diego, CA). PE-conjugated anti-CD25 (clone L78) and CD69 (clone 2A3) were from Becton Dickinson (San Jose, CA). Quantum Red (QR)-conjugated anti-human CD3 (clone UCHT-1) was from Sigma.

Flow cytometry
Flow cytometry of cell surface phenotypes was performed on a FACScan (Becton Dickinson). Forward scatter was measured with the amplifier in linear mode; side scatter and all fluorescence channels were measured with the amplifiers in logarithmic mode. Both acquisition and analysis were done using CellQuest software. The cytometer was not calibrated. Amplifier gains were set by eye so that the fluorescence histogram for cells stained with an isotype-matched control antibody for each fluorochrome was in the first decade of the relevant amplifier. Purified mononuclear cells were stained with FITC-3A8, QR–anti-CD3 and the panel of PE-conjugated antibodies described above. An acquisition gate was set on cells expressing CD3 and either CD4, CD8, CD27 or CD45 isoforms, and 10,000 events were collected.

Lymphocyte stimulation
Several methods of T cell stimulation were used. Purified T cells were stimulated with 10 ng/ml phorbol myristate acetate (PMA) and 0.5 µM ionomycin in RPMI 1640 containing 10% fetal bovine serum (RPMI-10) for 1–14 days as indicated. Alternatively, they were stimulated with 20 U/ml recombinant human IL-2, 25 ng/ml recombinant human IL-15 or both for up to 3 days. T cells in cultures of un-separated PBMC were stimulated by the addition of 125 ng/ml of staphylococcal enterotoxin A (SEA; Toxin Technologies, Sarasota, FL) in RPMI-10 for 1–14 days.

Endothelial transmigration
Human umbilical vein endothelial cells (HUVEC) were grown to confluence on hydrated collagen gels in 36 mm tissue culture wells as previously described (34). Resting or phorbol ester-activated CD4+ peripheral blood T cells isolated from normal donors by negative selection (7) were incubated (4 h at 37°C) with endothelial monolayers that had been formed on the surfaces of hydrated collagen gels. CD4+ T cells were recovered in three populations—cells non-adherent to the endothelial cells, cells adherent to the endothelial cells and removed by chelation, and cells that migrated through the endothelial cell monolayer into the collagen gels and released by collagenase digestion. The harvested T cells were then stained for the presence of GGT using the 3A8 mAb and the expression was compared to that on T cells in the starting population.

Intracellular thiol measurement
N-ethyl maleimide (NEM; Sigma) 10 mM in PBS and monobromobimane (MBB; Molecular Probes, Eugene, OR) 4 mM in 100% ethanol were prepared just prior to use. After surface staining with mAb, the T cell suspensions were allowed to equilibrate to room temperature. For each pair of identically stained samples, one tube received 10 µl of NEM (100 µM final concentration) and 10 µl of MBB (40 µM final concentration). The other tube received MBB alone. Data acquisition was performed 10 min after the cells were labeled with MBB. The NEM/MBB-stained sample and the MMB (only)-stained sample were analyzed at the same time after their respective staining. Data were collected on a FACStar Plus (Becton Dickinson) equipped with dual lasers and controlled by Lysys II software. The argon ion laser was used for light scatter, and for excitation of FITC, PE and QR. MBB/thiol fluorescence was excited by the UV laser tuned to 320 nm and emission measured through a 450 nm bandpasss filter. FL1, FL2 and FL3 were calibrated by eye as above. MBB/thiol fluorescence was measured on a linear scale, with the amplifier set so that Fluoresbrite carboxy BB 6 µm microspheres (Polysciences, Warrington, PA) gave a mean fluorescence intensity of 3500. For each sample, 10,000 events that satisfied both forward/orthogonal scatter and CD4+ resting lymphocytes were collected. Data analysis was performed using CellQuest (Becton Dickinson) software.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 
Expression of GGT on resting PBL
We determined the expression of GGT on subsets of human peripheral blood T cells by two- and three-color flow cytometry. Mononuclear cells were separated from whole blood and stained with antibodies to CD3, GGT and CD4 or CD8. The expression of GGT on resting CD4 and CD8 T cells is shown in Fig. 1Go. GGT expression is present at a very low level on the majority of both CD4+ and CD8+ T cells. Ten normal individuals aged 22–96 were studied. The percentage of T cells staining with 3A8 at a level greater than the isotype control was 38.30 ± 11.7% (mean ± SEM) for CD4+ cells with a range of 21.08–60.18% and 24.12 ± 6.9% for CD8+ cells with a range of 13.2–35.22%. The relative level of expression (median fluorescence intensity of 3A8/median fluorescence intensity of control) was 4.35 ± 1.09 for CD4+ cells and 3.03 ± 0.49 for CD8+ cells.



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Fig 1. Expression of GGT on peripheral blood T lymphocytes of a representative normal individual. PBMC were stained with 3A8–FITC (anti-GGT), anti-CD3–QR and PE-conjugated antibodies to CD4, CD8, CD45RA or CD45RO. Cells were gated for expression of CD3 and the appropriate T cell subset marker. Thick line, 3A8–FITC staining; thin line, UPC-10–FITC (isotype control) staining.

 
While preliminary studies had shown that GGT was up-regulated with T cell activation (see below), this did not account for the resting T cells with higher levels of GGT as they did not express markers of early (CD69) or late (CD25) activation (not shown). Staining by antibodies that identify CD45 isoforms was used to determine whether GGT was differentially expressed on naive or memory T cells. PBMC were stained with antibodies to CD3 and GGT along with anti-CD45RA or CD45RO (Fig. 1Go). In contrast, CD3+ CD45RA+ cells expressed the lowest levels of GGT, nearly identical with the isotype control. CD3+ CD45RO+ cells expressed uniformly higher levels of GGT. Moreover, GGT was expressed on the CD45RBdim population of T cells (Fig. 2AGo). T cells accumulate in this population following repeated antigenic stimulation (6). T cells also lose expression of CD27 after repeated stimulation (3,4). These CD27 cells are also GGT+ (data not shown). Taken together, these data demonstrate that expression of GGT is up-regulated on resting T cells that have a surface phenotype previously associated with the acquisition of immune memory.



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Fig. 2. Expression of GGT is highest on CD45RBdim T lymphocytes. PBMC from a normal donor (A) and from an individual with active rheumatoid synovitis (B) were stained with 3A8–FITC, anti-CD45RB–PE and anti-CD3–QR. A gate was set for CD3+ cells, and the levels of GGT and CD45RB expression were measured. The upper limits of the isotype control antibodies are indicated by dotted lines.

 
Expression of GGT on T lymphocytes from RA patients
The population of resting peripheral blood T cells exhibiting a memory phenotype has been shown to be expanded in a significant percentage of patients with RA (2). We analyzed the expression of GGT on this population of T cells in a panel of RA patients. For this study, peripheral blood T cells from 10 patients with active synovitis were analyzed for the expression of GGT (Fig. 3Go). Synovial fluid T cells isolated from two additional patients were also examined. In contrast to the 18–25% of T cells from normal control donors that express GGT, 50.18 ± 17.08% (range 23.27–82.44%) of the CD3+ cells from the peripheral blood of these patients had very high levels of GGT expression. The relative level of 3A8 staining (see above) was 9.36 ± 4.53 (range 1.40–15.97) times the level of control fluorescence (P < 0.01 compared to non-RA controls using Student's t-test). In the synovial fluid samples, nearly 100% of the T cells expressed GGT. The phenotype of the GGT expressing cells in RA patients was similar to that seen in the normal controls. That is, the cells that express high levels of GGT are CD45RO+ (not shown) and CD45RBdim (Fig. 2BGo). These observations extend the previous phenotypic analysis of T lymphocytes in patients with RA suggesting an accumulation of effector and/or memory cells. This expanded population also contains cells with more GGT.



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Fig. 3. GGT expression on T lymphocytes of three representative patients with RA. PBMC (A–C) or synovial fluid mononuclear cells (D) were stained with PE-conjugated anti-CD3 and 3A8–FITC. Cells gated for CD3 expression were analyzed for surface levels of GGT. Thick line, 3A8–FITC staining; thin line, isotype control staining.

 
Up-regulation of GGT on activated T lymphocytes
Peripheral blood of RA patients often contains a higher than normal number of activated T cells (35). Although the RA patient T cells that expressed a higher level of GGT did not express markers of recent activation, it was possible that activation, per se, could result in long-term alteration in GGT expression. For these studies, resting peripheral blood T cells were first activated by SEA, a polyclonal T cell mitogen, or by phorbol ester and ionomycin. At different times after activation, cells were analyzed for CD3, CD4, CD8 and GGT expression by three-color flow cytometry. The data in Fig. 4Go show that up-regulation of GGT surface expression on SEA-stimulated CD4+ T cells began 24–48 h after exposure to the superantigen and continued for 5–7 days. Stimulation of T cells with phorbol ester and ionomycin similarly increased GGT expression (data not shown). GGT expression remained elevated 2 weeks after stimulation with either SEA or phorbol ester and ionomycin. The kinetics of GGT up-regulation was similar for both CD4+ and CD8+ T cells.



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Fig. 4. Expression of GGT on activated T lymphocytes. PBMC were cultured for the indicated times with the bacterial superantigen, SEA, to cause polyclonal T cell activation. The cells were then stained with anti-CD3–PE and 3A8–FITC. CD3+ cells were gated for analysis. Thick line, 3A8–FITC staining; thin line, isotype control staining.

 
IL-15 up-regulation of GGT on T cells
IL-15 has been shown to activate T cells and increase their migration (3639). It acts through a receptor that shares common ß and {gamma} chains with the IL-2 receptor (CD25) but has a distinct {alpha} chain (40). This receptor similarity belies the similar functions of IL-2 and IL-15. However, IL-15 is not produced by activated T cells as is IL-2 but is produced by macrophages and endothelial cells (38,40). IL-15 has been identified at sites of inflammation including rheumatoid synovium, and it has been proposed that IL-15 production by synovial macrophages drives recruitment and activation of T cells in RA (41,42). Despite the fact that T cells at sites of inflammation express markers of activation and differentiation, IL-2 production is not expressed to a significant extent. We determined whether these stimulatory cytokines could alter GGT expression. Purified T cells were exposed to either 20 U/ml of IL-2, 25 ng/ml of IL-15 or both for periods of up to 3 days. The typical concentration of IL-15 in synovial fluid of patients with active RA is 20–100 ng/ml (41) although levels of 1200 ng/ml have been detected. The T cells were double-stained for GGT and CD4, CD45RA, CD45RO or CD69. In the absence of cytokines there was no change in the expression of GGT and spontaneous activation was insignificant. The percentage activated (CD69+) T cells increased from an initial value of 2.8 to 6.2% at 3 days in the absence of either IL-2 or IL-15 (Fig. 5Go, top row). The expression of GGT on the activated (CD69+) cells is shown in the bottom three rows of Fig. 5Go. These histograms are all scaled identically, so that the area of each one represents the relative degree of activation in the cultures as determined by up-regulation of CD69. IL-2 alone causes little T cell activation (only 6.7% of the T cells became CD69+ at 3 days) and only a small shift in expression of GGT was observed. Exposure of the T cells to IL-15, however, caused 16.1, 19.8 and 25.9% of the T cells to express CD69 after 1, 2 and 3 days respectively. This was accompanied by an upward shift in the expression of GGT on the CD69+ cells. In the activated (CD69+) T cell population, 10.3, 43.8 and 63.2% of the cells expressed GGT at a level higher than the unstimulated cells at 1, 2 and 3 days respectively. There was no increase in the expression of GGT on the cells that remained CD69 (data not shown). Treatment of the cells with IL-2 in addition to IL-15 had little effect over IL-15 alone. The percentage of T cells activated by the combination of cytokines that expressed up-regulated levels of GGT were 18.7, 34.9 and 62.2% at 1, 2 and 3 days respectively. The up-regulation of GGT caused by IL-15 alone or by IL-15 plus IL-2 was equivalent in all activated T cells regardless of whether they were CD4+ or CD4, or expressed CD45RA+ or CD45RO+ (data not shown).



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Fig. 5. Up-regulation of GGT expression on T lymphocytes by IL-15. Resting peripheral blood T cells were isolated and cultured in medium alone (top row), medium plus 20 U/ml of IL-2 (second row), medium plus 25 ng/ml IL-15 (third row) or both IL-2 and IL-15 (bottom row). Initially and at the indicated times the cells were stained with 3A8–FITC (or isotype control) and anti-CD69–PE. For the un-stimulated cells, the level of GGT expression on all T cells is shown. For the cells that were stimulated with IL-2 and/or IL-15, the GGT expression on only the CD69+ cells is displayed. The scales of the ordinate axes of the bottom three rows are identical. Therefore, the area of the histograms indicates the relative number of CD69+ (activated) cells for each condition and time. The dotted lines indicate the upper boundary of the isotype control antibody staining.

 
Transendothelial migration of resting lymphocytes
The data indicate that subsets of differentiated lymphocytes express GGT. Moreover, levels of GGT expression are increased by activation stimuli, including IL-15. In addition, RA peripheral blood T cells express more GGT than do normal T cells and all synovial T cells are GGT+. Recently, we observed that a subset of differentiated CD4+ T cells exhibits a capacity to extravasate through the vascular endothelium. The phenotype of the migrating cells is similar to that expressed by T cells at inflammatory sites (7,34). Using a previously developed assay, we determined whether GGT expression is an additional feature of T cells that exhibit an intrinsic capacity for transendothelial migration and perivascular accumulation. Resting CD4+ T cells were tested for GGT expression before and after their transendothelial migration through HUVEC monolayers grown on collagen gels. The results of a representative experiment are shown in Fig. 6Go. Initially, 21% of the T cells expressed GGT. Following a 4 h incubation with HUVEC, the T cells were recovered in distinct populations. Only 15% of the T cells that did not adhere to HUVEC were GGT+. In contrast, when T cells in the adherent and migrated subsets were analyzed, 71 and 75% or the cells respectively were GGT+. The proportion of GGT-expressing T cells in the latter subsets did not change regardless of whether the HUVEC were stimulated with tumor necrosis factor (TNF)-{alpha} (4 h at 37°C), the T cells were stimulated with PMA (1 h at 37°C) or both the HUVEC and the T cells were stimulated (data not shown).



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Fig. 6. GGT expression on migrating T cells. Resting CD4+ T lymphocytes were isolated from peripheral blood and cultured for 4 h on a monolayer of human endothelial cells grown on a collagen gel. The cells were then separated into non-adherent, adherent and migrated populations. The cells were stained with 3A8 or an isotype control antibody. Lymphocytes were differentiated from endothelial cells by forward and side scatter characteristics, and gated for analysis. Thick line, 3A8 staining; thin line, isotype control antibody staining.

 
Intracellular thiol concentrations in naive versus memory peripheral blood CD4+ T cells
MBB reacts non-enzymatically with intracellular thiols to form a fluorescent compound that can be measured by flow cytometry. Although MBB can react with low mol. wt thiols other than GSH (e.g. cysteine) or with protein thiols, the use of low MBB concentrations and a short staining time produces a fluorescent signal that is proportional to intracellular GSH (43). The analysis of identical samples treated with NEM controls for background (non-GSH) staining.

We examined the differences in intracellular thiol (largely GSH) levels in resting peripheral blood T cell populations from normal donors using MBB. The cells were also stained with 3A8 to compare GGT expression, and with antibodies to CD4 and either CD45RA or CD45RO to identify naive and memory populations. The results of a representative analysis comparing GGT expression with MBB staining are shown in Fig. 7Go. Two populations of CD4+ T cells are seen (Fig. 7Go, upper panel). The cells with a low level of GGT on their surface have the lowest staining with MBB. The second population has a higher expression of GGT and a broader, more intense staining with MBB. Gating of the cells for expression of either the RA or RO isoforms of CD45 distinguished between these two populations. CD4+ T cells gated for expression of CD45RA have both lower expression of GGT and less intense MBB staining (Fig. 7Go, lower left panel). Resting CD4+ T cells expressing CD45RO have increased GGT expression as seen in Fig. 1Go, as well as more intense staining with MBB (Fig. 7Go, lower right panel). Although the MBB staining of the memory population appears more heterogeneous than that of the naive population, no difference in cell size was detectable by light scatter characteristics that would account for this difference.



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Fig. 7. MBB staining of human T cell subsets. Resting T cells were isolated from peripheral blood, and stained with antibodies to GGT, CD45 isoforms and CD4. They were then stained with MBB to label intracellular GSH. The top panel shows the analysis of all CD4-expressing cells within the lymphocyte gate determined by light scatter characteristics. GGT expression is on the ordinate and MBB staining on the abscissa. The GGT expression and MBB staining on naive and memory T cells were determined by gating on CD45RA (lower left panel) and CD45RO (lower right panel) respectively in addition to CD4. The dashed lines indicate the upper limits of the isotype control for 3A8 (GGT) staining and MBB staining in the presence of NEM to abolish thiol reactivity.

 
We analyzed the MBB staining pattern on resting CD4+ T cells in 10 normal individuals. All 10 analyses were performed on the same day under identical conditions. The results from this analysis are summarized in Table 1Go. There is a variation among the individuals in the absolute level of intracellular thiols. In nine of the 10 PBMC samples, the level of MBB-thiol fluorescence was greater in the CD4+ CD45RO+ cells than in the CD4+ CD45RA+ cells. The mean level of intracellular thiol in the RO+ cells was 14% greater than the mean level of thiol in the RA+ cells. These data support the hypothesis that up-regulation of GGT on memory T cells provides these cells with a mechanism for more efficient recapture of released GSH.


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Table 1. Intracellular thiol levels in human peripheral blood T cells
 

    Dicussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 
GGT was first described on lymphocytes in 1976 using an enzymatic assay (26). Activity was detected on both T cell (sheep red blood cell binding) and B cell (nylon wool binding) mononuclear cells. At that time, it was not possible to characterize the cells further. Stimulation of un-separated human lymphocytes with Con A caused an increase in GGT enzyme activity. The authors also documented the presence of GGT activity on a large number of T and B lymphoblastic cell lines. Cytochemical analyses have been performed by other groups, documenting the variable level of GGT activity in normal and malignant cells of hematopoetic origin (28,44). The lack of appropriate contemporary serological reagents has prevented the further analysis of GGT expression on these lymphocytes.

Important questions regarding expression of GGT on distinct mononuclear cell subsets and the role of GGT up-regulation upon lymphocyte activation have not been previously addressed. GSH is the major non-protein thiol in cells and it is the most abundant natural anti-oxidant, with millimolar intracellular levels. Therefore it is the major determinant of intracellular redox potential. The mechanism of regulating GSH levels is not firmly established in all cell types. However, data from `knockout' mice (25) and GGT-deficient humans (22) suggest that GGT plays a major role. As the initial enzyme of the glutamyl cycle, GGT cleaves GSH into cysteinyl-glycine and glutamate or a glutamyl-amino acid adduct. This reaction is clearly necessary for recovery of extracellular GSH by hepatocytes under physiological conditions (45,46). The demonstration of higher intracellular thiol (GSH) levels in GGT-expressing T cells is consistent with this. Therefore, knowledge of the expression of GGT is relevant to our understanding of the regulation of GSH and redox potential by lymphocytes.

It is not known whether the up-regulation of GGT expression as assayed by 3A8 reactivity represents transcriptional or post-transcriptional events. The latter is unlikely to account for the increase seen here. Immunopreciptitation with 3A8 removes >80% of the GGT enzymatic activity from lymphocyte cell lysates (28), suggesting that there is not a significant population of GGT molecules that do not react with the antibody. Second, electron microscopic analysis of GGT activity in lymphoid cells failed to demonstrate any intracellular localization of the enzyme outside the synthetic pathway (ER–Golgi) (44). Thus, while it is likely that increased transcription is responsible for the up-regulation of GGT expression, the signals that mediate activate the GGT gene remain to be elucidated.

Numerous studies have shown that GSH levels and/or redox potential modulate the differentiation and activation of T lymphocytes. Early studies showed that T cells chemically depleted of GSH before stimulation with lectins develop a partial activation phenotype characterized by up-regulation of amino acid transport and synthesis of IL-2 and IL-2R, but fail to synthesize DNA and enter the cell cycle (4754). More recent experiments have addressed this issue at the molecular level, documenting cellular responses to oxidative stress. Treatment of T cells or fibroblasts with N-acetyl-cysteine (NAC), which increases intracellular levels of GSH (5557), blocks their activation of NF{kappa}B by TNF-{alpha} or phorbol ester. Conversely, NF{kappa}B is activated by hydrogen peroxide in intact cells. Protein kinase activation is enhanced in cells that are depleted of GSH and activated by TNF-{alpha}, whereas anti-CD3 induced intracellular calcium fluxes are abolished (58). Lastly, it has been reported that reactive oxygen intermediates are critical for the generation of co-stimulatory signals initiated by ligation of CD28 on T cells (59). Together, these data demonstrate that regulation of redox potential plays a critical role in modulating well-established signal transduction mechanisms in lymphocytes and as a result may alter specific functions of lymphocytes or lymphocyte subsets.

The results in this study show that GGT expression on resting T cells is highest in the population that expresses other markers of memory cells such as CD45RO and intermediate expression of CD45RB. These cells have higher levels of intracellular thiols as judged by their increased MBB fluorescence. Others have found greater GSH levels in fixed CD45RA+ cells using the related compound, MCB (60). Although MCB is highly specific for GSH in rodent cells, it fails to label human cells adequately because of its low affinity for human glutathione-S-transferases (43,61). Higher concentrations of MCB achieve more complete labeling of the human cellular GSH pool but also result in increased background fluorescence due to non-GSH binding (61). In addition, fixation of cells with paraformaldehyde causes permeabilization of the cell membrane that allows low mol. wt thiols, such as GSH, or their fluorescent adducts to leak out of the cells (43). We have not been able to measure differences between naive and memory T cells using MCB. While GSH is the major intracellular thiol by several orders of magnitude, it is possible that our results with MBB indicate the accumulation of other intracellular thiols.

Inflammatory tissues, such as rheumatoid synovium, are rich in reactive oxygen intermediates (6365). There is evidence that this oxidative stress influences the function of T cells in RA. T cells from the synovial fluid of RA patients have been shown to be hyporesponsive to CD3 stimulation (66), with markedly reduced Ca2+ mobilization. This correlated with a decrease in measured intracellular GSH levels. When the GSH levels were corrected with the addition of NAC, the functional responsiveness of the T cells was restored. The protein tyrosine phosphorylation patterns in these cells were also different from healthy controls or peripheral blood T cells of RA patients. In particular, there was diminished expression of the TCR {zeta} chain (67). These results suggest that the oxidative stress associated with rheumatoid inflammation may serve to inhibit the responsiveness of T cells, a feature of those T cells present in the synovium. The ability of T cells to survive in this environment would be enhanced by the ability to maintain intracellular redox potential. The higher expression of GGT on T cells in synovial fluid provides one mechanism to maintain the intracellular redox potential at a site of chronic inflammation. Experiments to test this hypothesis are underway.

Although high GGT expression is found on a minority of T cells in normal individuals, results from these studies show that this fraction of T cells is specifically recruited to migrate through the endothelium. Based on this, it is predicted that the T cells that reside on the tissue side of the endothelium have a markedly different surface enzymology than those in the intravascular space, with increased ability to metabolize GSH and GSH adducts. The increased expression of GGT would offer those T cells the ability to more efficiently re-capture GSH and withstand oxidative stress. The role of the endothelium in this process could be 2-fold. First, endothelial cells are responsible for the selection of the CD26bright, CD44bright, CD11abright memory T cell subset that has the highest capacity for transendothelial migration. These are also the cells with the highest levels of GGT. Second, endothelial cells produce IL-15 that both activates T cells and promotes their migration (38). As shown in this study, IL-15 alone can cause an increase in the expression of GGT on T cells. Therefore, the T cell–endothelial cell interaction can deliver T cells to tissue sites that are enriched for high levels of GGT expression. This would result in an increase in the GGT enzyme activity within the tissue, potentially altering both the redox potential, as well as metabolizing other GSH adducts.

Together, these data establish GGT as a regulated ectoenzyme on subsets of T lymphocytes. The full impact of this regulated expression is under investigation. GGT affects GSH transport and we have found increased thiols in GGT-expressing cells. This is one mechanism whereby memory T cells can maintain their intracellular redox potential in the face of oxidative stress, and may underlie functional differences between naive and memory T cells.


    Acknowledgments
 
This work was supported by a Biomedical Science Grant from the Arthritis Foundation (D. R. K.), a local chapter grant from the Rocky Mountain Chapter of the Arthritis Foundation (V. M. H.) and NIH R01-AI42772 (D. R. K.). The authors wish to thank Drs Michael Lieberman, Laurie Davis and Peter Lipsky for helpful discussions.


    Abbreviations
 
Con A concanavalin A
GGT {gamma}-glutamyl transpeptidase
GSH glutathione
HUVEC human umbilical vein endothelial cell
MBB monobromobimane
MCB monochlorobimane
NAC N-acetyl cysteine
NEM N-ethyl maleimide
PBL peripheral blood lymphocyte
PBMC peripheralblood mononuclear cell
PE phycoerythrin
PMA phorbol myristate acetate
QR Quantum Red
RA rheumatoid arthritis
SEA staphylococcal enterotoxin A
TNF tumor necrosis factor

    Notes
 
Transmitting editor: A. Weiss

Received 28 May 1999, accepted 21 July 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Dicussion
 References
 

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