[20] Redox processes regulate intestinal lamina propria T lymphocytes

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Redox

Processes Propria

[20]

Regulate Intestinal T Lymphocytes

Lamina

B y BERND SIDO, RAOUL BREITKREUTZ, CORNELIA SEEL, CHRISTIAN HERFARTH, a n d STEFAN MEUER

Introduction Due to the special architecture of the gut, the mucosal surface is more than 200 times larger than the surface of the skin. The largest cellular immune system can be found on this "inner" surface of the body. At this mucosal barrier, the effector T-cell compartment (lamina propria) is permanently exposed to exogenous antigens of nutritional and microbial origin. This antigen challenge will not lead to pathology only if a systemic immune response with proliferation and cytokine secretion is prevented. The physiological hyporeactivity of lamina propria T lymphocytes (LP-T) in the normal gut in vivo is indicated by their low proliferative potential, t Analogously, LP-T are defective in their ability to proliferate in vitro in response to antigen receptor stimulation, 2,3 although these cells are predominantly of the memory phenotype (CD45R0 +) and express a full range of cell surface receptors necessary for immune activation. 4'5 Evidence is accumulating that the special mucosal environment regulates the functional state of effector cells. We have shown that coculture of peripheral blood T lymphocytes with the intestinal mucosa supernatant induces a similar functional behavior as found in freshly recovered LP-T. 6 It was suggested that small, nonprotein, nonpeptide molecules with oxidative capacities downregulate antigen receptorinduced T lymphocyte proliferation. In contrast, the antioxidant 2-mercaptoethanol (2-ME) could reverse the suppressive effect of mucosa supernatant and could restore the CD3 reactivity of LP-T. This finding suggests that regulation of the intracellular redox state in LP-T may represent a versatile physiological control mechanism to adjust the mucosal lymphocyte reactivity to particular local requirements. Glutathione (GSH) is the most abundant intracellular low molecular weight thiol. Due to its strong antioxidative capacities, it controls several cellular immune 1 F. Autschbach, G. Schth'rnann, L. Qiao, H. Merz, R. Wallich, and S. C. Meuer, Virch. Arch. 426, 51 (1995). 2 M. Zeitz, T. Quinn, A. S. Graeff, and S. E James, Gastroenterology 94, 353 (1988). 3 L. Qiao, G. Schtirrnann, M. Betzler, and S. C. Meuer, Gastroenterology 101, 1529 (1991). 4 S. P. James, W. C. Kwan, and M. Sneller, J. Immunol. 144, 1551 (1990). 5 U. Pirzer, G. Schth'rnann, S. Post, M. Betzler, and S. C. Meuer, Eur. J. Immunol. 20, 2339 (1990). 6 L. Qiao, G. Schth'rnann, F. Autschbach, R. Wallich, and S. C. Meuer, Gastroenterology 105, 814 (1993).

METHODSIN ENZYMOLOGY,VOL.352

Copyright2002,ElsevierScience(USA). All fightsl~sel~ed. 0076-6879/02 $35.00

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REDOX REGULATION OF INTESTINAL T LYMPHOCYTES

233

functions, such as lymphocyte proliferation and cytotoxic activity, and modulates the activation of redox-regulated transcription factors such as NF-tcB and AP-1. A 10-30% decrease of the GSH content in lymphocytes disrupts the proximal signal cascade after TCR stimulation with complete inhibition of the increase of intracellular free calcium] Because lymphocytes are defective in cystine uptake, s,9 the availability of the reduced derivative cysteine becomes limiting for the synthesis of GSH. However, cysteine circulates at extremely low concentrations in plasma and is not contained in standard tissue culture medium. Therefore, dynamic changes in the local supply of cysteine by other cells that can take up cystine (and subsequently release cysteine) may evolve as a physiological mechanism involved in the regulation of intestinal lymphocyte reactivity. P r e p a r a t i o n of L a m i n a P r o p r i a Cells f r o m H u m a n G u t Large bowel specimens are obtained from patients undergoing resection for colon cancer. Normal mucosa (5 × 5 cm) is dissected from the submucosa near the resection margin. Lamina propria mononuclear cells are isolated according to a modification of the method of Bull and B ookman.t° The fresh tissue is washed extensively in HBSS, without Ca 2+ andMg 2+ (GIBCO, Paisley, Scotland), containing penicillin (100 U/ml; Sigma, Taufkirchen, Germany), streptomycin (100 #g/ml; Sigma, Taufkirchen, Germany), gentamycin (59 #g/ml; Sigma, Taufkirchen, Germany), and amphotericin B (2.5 #g/ml; GIBCO) at 4 °. The mucus can be removed by incubation in HBSS, without Ca 2+ and Mg 2+, containing i mM dithiothreitol (DTT) for 15 rain at 37 °. For the study ofredox regulation, however, the use of the strong antioxidant DTT should be avoided and, instead, the mucus should be scraped off gently with a scalpel. The tissue is cut into 2- to 4-ram pieces and incubated in a shaking water bath in HBSS, without Ca 2+ and Mg 2+, containing 0.7 mM EDTA (Sigma, Taufkirchen, Germany) and the aforementioned antibiotics at 37 ° for 45 rain. This incubation is repeated twice with fresh medium until the supernatant is free of epithelial cells. The tissue is then washed four times for 10 rain at 37 ° in HBSS, without Ca 2+ and Mg 2+, until the supernatant becomes clear. Subsequently, the mucosal tissue is enzymatically digested in RPMI 1640 (GIBCO) containing 2% fetal calf serum (FCS; Sigma) 45 U/ml collagenase (type IV; Sigma), 27 U/ml deoxyribonuclease I (Sigma), 2% glutamine, antibiotics, and amphotericin B in a shaking water bath at 37 ° for 10 hr. The digest is passed through a 70-#m nylon mesh (Becton Dickinson, Heidelberg, Germany)

7 F. J. Staal, M. T. Anderson, G. E. Staal, L. A. Herzenberg, C. Gitler, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 91, 3619 (1994). 8 T. Ishii, Y. Sugita, and S. Bannai, J. Cell. Physiol. 133, 330 (1987). 9 H. Gmtinder, H.-E Eck, and W. Dr6ge, Eur. J. Biochem. 201, 113 (1991). lo D. M. Bull and M. A. Bookman, J. Clin. Invest. 59, 966 (1977).

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and washed in RPMI 1640/2% FCS. After washing twice (4°), the cells are resuspended in 67.5% Percoll (Pharmacia Biotech, Uppsala, Sweden) and overlayed by 30% Percoll. After centrifugation at 3000 rpm and 4 °, lamina propria mononuclear cells are recovered from the interphase and washed twice. Viable cells are separated by Ficoll-Hypaque (Sigma) density gradient centrifugation at room temperature (2500 rpm, 20 min) and washed twice. Cells are resuspended in a 1 : 1 mixture of RPMI 1640/2% FCS and autologous serum and are allowed to adhere to a plastic tissue culture petri dish (Greiner, Frickenhausen, Germany) for 3 hr at 37 °. Adherent cells are harvested with a rubber policeman (Becton Dickinson) and used as lamina propria macrophages (LP-MO). Nonadherent cells are pelleted, mixed with 40 #1 of a 5% suspension of sheep red blood cells (SRBC; ICN Biomedicals, Eschwege, Germany) per 106 cells, spun at 700 rpm for 5 min, and incubated for 50 min at room temperature to allow E rosette formation. Subsequently, the pellet is resuspended gently and centrifuged on Ficoll-Hypaque for 20 min at 1000 rpm and at 1800 rpm. Supernatant and E rosette-negative cells are discarded, and the pellet is treated with lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.13 mM EDTA, pH 7.27) for 5 min to lyse SRBC. E rosette-positive cells are washed three times and finally resuspended in RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin, and 2% glutamine for use. This cell population is 90% positive for CD3 as shown by immunofluorescent staining and is used as lamina propria T cells (LP-T). Viability is consistently more than 95%. Venous blood is collected from the same patient, and peripheral blood mononuclear cells are obtained by Ficoll-Hypaque density gradient centrifugation. Adherent cells are isolated according to the adherence step described earlier and are used as peripheral blood monocytes (PB-MO). S t i m u l a t i o n of I n t e s t i n a l L a m i n a P r o p r i a T L y m p h o c y t e s /n Vitro LP-T (5 x 104/well) are cultured in 96-well round-bottomed microtiter plates (Costa, Bodenheim, Germany) at 37 ° and 7% CO2. PB-MO and LP-MO, respectively, are irradiated immediately before use (50 Gy) and added to LP-T at 30% of total cell number. LP-T cells are stimulated via CD3 according to standard procedures employing mitogenic monoclonal antibodies in the absence or presence of recombinant human IL-2 (10 U/ml; Biotest, Dreireich, Germany). For CD3 stimulation, we immobilize the mouse antibody OKT3 (IgGza) on beads (Irvine Scientific, Santa Ana, CA) or, alternatively, on 96-well fiat-bottomed microtiter plates (Costar) that are precoated with goat antimouse immunoglobulin. After 4 days of culture, wells are pulsed with 1 #Ci [3H]thymidine (Amersham, Karlsruhe, Germany) for 16 hr and then harvested on glass fiber filters using an automatic cell harvester (FilterMate, Packard, Meriden, CT). [3H]Thymidine

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REDOX REGULATION OF INTESTINAL T LYMPHOCYTES

235

incorporation is measured in a microplate scintillation counter (TopCount, Packard, Meriden, CT). E x p e r i m e n t a l P r o c e d u r e s to S t u d y t h e I n f l u e n c e of R e d o x Milieu o n P r o l i f e r a t i o n of I n t e s t i n a l L a m i n a P r o p r i a T L y m p h o c y t e s Hydrogen peroxide is a very simple, stable, and naturally occurring potent oxidant. Due to its lipophilicity, it can permeate cell membranes easily and react slowly with organic substances. It is physiologically produced intracellularly and is supposed to function as a second messanger in signal transduction in cells. Moreover, hydrogen peroxide is produced in vivo in large amounts in areas of inflammation by polymorphonuclear phagocytes due to the catalytic dismutation of superoxide anion radicals and can be converted to highly reactive hydroxyl radicals in the presence of transition metals. Micromolar concentrations (0-100 #M) of hydrogen peroxide (Merck, Darmstadt, Germany) suppress the proliferation of LP-T in a dose-dependent manner after stimulation with OKT3 plus IL-2 (10 U/ml). The suppressive effect of 10-25 # M is only marginal, whereas 50 # M hydrogen peroxide decreases proliferation by 61%. In the presence of 75 #M, the proliferation of LP-T drops down to nearly background values (Fig. 1). Even a partial depletion of the intracellular GSH pool in lymphocytes has dramatic effects on blast transformation and proliferation and suppresses the

OKT3 + IL-2 + 2-ME (50 ~tM) + DTT (0.5 raM) + GSH (3 mM) + H20 ~ (75 ~M)

+ BSO (50 ~M) + BCNU (50 ~xM)

10

20

30

40

50

60

70

80

[3H]Thymidine uptake (cpm x 10 -3) FIG. 1. Effect of antioxidant (2-ME, DTT, GSH) and prooxidant (H202, BSO, BCNU) culture conditions on the proliferative immune response of LP-T after stimulation via CD3. If added, the concentration of IL-2 was 10 U/ml. 2-ME, 2-mercaptoethanol (50 /,M); DTT, dithiothreitol (0.5 raM); H202, hydrogen peroxide (75/*M); BSO, buthionine-[S,R]sulfoximine (50/,M); BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea (50/*M).

236

CELLULAR RESPONSES

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activation of cytotoxic T cells and LAK cells.l 1-14 The water-soluble buthionine[S,R] sulfoximine (BSO; Sigma, Taufldrchen, Germany) is a specific inhibitor of the rate-limiting enzyme of GSH synthesis (y-glutamylcysteine synthetase) 15 and, therefore, can be used to study the specific consequences of GSH deficiency. BSO inhibits proliferation of LP-T by 48% at 10 # M when added at the beginning of culture and drops to background values at 50 # M (Fig. 1). 1,3-Bis(2-chloroethyl)- 1-nitrosourea (BCNU; Bristol Laboratories, Evansville, IN) is an irreversible enzyme inhibitor of glutathione reductase and leads to longlasting inhibition of the NADPH-dependent regeneration of GSH from glutathione disulfide (GSSG). 16 Inhibition of glutathione reductase in LP-T 120 rain after the addition of 50 # M BCNU is more than 90% of the control. As a consequence, the concentration of GSSG increases and the redox balance of glutathione (ratio GSH/GSSG) is shifted toward a prooxidant state. In an attempt to restore a nearly physiological balance of GSH/GSSG, the cell exports GSSG, leading to a decrease in the total intracellular glutathione content. A stock solution of BCNU in 100% ethanol (40 raM) is stable at - 2 0 ° for at least 2 weeks. 16 BCNU inhibits proliferation of LP-T at concentrations as low as 10 # M and blocks proliferation completely at 50 # M (Fig. 1). In contrast, an antioxidative environment increases the proliferation of LP-T. This can be demonstrated with the use of more than 0.1 m M D T T (Fig. 1). Concentrations above 1 mM DTT are toxic for the cells as proliferation decreases again. Similar stimulatory effects can be achieved with the addition of more than 1 mM GSH (Fig. 1). Note that GSH is acidic and the pH of the solution needs to be adjusted to 7.3 before use. The well-established potentiating effect of 2-ME on lymphocyte proliferation is due to an increased uptake of cysteine in lymphocytes. 17 2-ME thus compensates for the low inherent membrane transport activity of lymphocytes for cystine (transport system Xc-). 8'9 Although the membrane transport activity for cysteine is high in lymphocytes (transport system A S C ) , 8'9 the uptake of cysteine is insufficient because cysteine is not present in the culture medium. The oxidized derivative cystine is abundantly present in RPMI 1640 (204 # M = 408 cysteine equivalents). However, this cannot compensate for the cysteine deficiency because the activity of unstimulated and stimulated lymphocytes (both T and B cells) to take up cystine is more than 10 times lower than for cysteine, s,9 Therefore, the

11 j. E Messina and D. A. Lawrence, J. Immunol. 143, 1974 (1989). 12 M. Suthanthiran, M. E. Anderson, V. K. Shat-ma, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 87, 3343 (1990). 13 A. Yamauchi and E. T. Bloom, J. Immunol. 151, 5535 (1993). 14 H. Gmtinder and W. Drtge, Cell. Immunol. 138, 229 (1991). 15 O. W. Griffith and A. Meister, J. Biol. Chem. 254, 7558 (1979). 16 K. Becket and H. Schh-mer, Methods Enzymol. 251, 173 (1995). 17 T. Ishii, S. Bannai, and Y. Sugita, J. Biol. Chem. 256, 12387 (1983).

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REDOX REGULATION OF INTESTINAL T LYMPHOCYTES

237

availability of cysteine becomes limiting for glutathione synthesis 8,t8 and lymphocyte proliferation, t9,2° 2-ME forms a mixed disulfide with cysteine derived from cystine.t7 This mixed disulfide can be taken up easily by lymphocytes via the transport system shared by neutral amino acids such as leucine and phenylalanine, t7 The mixed disulfide 2-ME-cysteine is reduced intracellularly to liberate cysteine, whereas 2-ME recycles to the extracellular space as a carrier molecule. By promoting cysteine uptake, 2-ME preserves a critical intracellular GSH content, 8 which is a prerequisite for cell cycle progression from the Gt to S phase, tt At concentrations as low as 5 #M, 2-ME potentiates the proliferation of LP-T, which only slightly increases further at 50 # M 2-ME (Fig. 1). E x p e r i m e n t a l P r o c e d u r e s to A n a l y z e D i f f e r e n t i a l C a p a c i t y of P e r i p h e r a l B l o o d M o n o c y t e s v e r s u s R e s i d e n t I n t e s t i n a l M a c r o p h a g e s to P r o d u c e C y s t e i n e Because cysteine circulates at extremely low concentrations in human plasma (8-10 #M) 21 and because lymphocytes cannot produce cysteine by themselves, even moderate changes of extracellular cysteine concentrations influence lymphocyte immune functions. 11-14 LP-T, therefore, depend on an alternative cellular source of cysteine in vivo to establish a proliferative immune response like in inflammatory bowel disease. Lymphocytes deficient in cystine transport can be supplied with cysteine by other cells that can efficiently take up cystine; mouse peritoneal macrophages have a strong membrane transport activity for cystine 22 and, by release of substantial amounts of cysteine, these cells augment the GSH content and DNA synthesis in murine lymphocytes in vitro. 2° In inflammatory bowel disease, a sustained recruitment of PB-MO to severely inflamed areas has been well documented by immunohistochemical s t u d i e s . 23'24 W e , therefore, investigated the differential capacity of PB-MO versus LP-MO to release cysteine into the supernatant. PB-MO and LP-MO, respectively, are plated at 5 x 105 cells/well in a total volume of 1 ml/well in a 48-well culture plate (Costar, Bodenheim, Germany). Cells are either left untreated or stimulated with 1 #g/ml LPS from Escherichia coli (serotype 055:B5; Sigma). After 40 hr of culture (37 °, 7% CO2), the supernatant is harvested and centrifuged (4 ° ) to remove cells. 18 S. Bannai and N. Tateishi, J. Membr. Biol. 89, 1 (1986). 19 W. Dr6ge, R. Kinscheff, S. Mihm, D. Galter, S. Roth, H. Gmtinder, T. Fischbach, and M. Bockstette, Methods Enzymol. 251, 255 (1995). 2o H. Gmtinder, H. E Eck, B. Benninghoff, S. Roth, and W. Dr6ge, Cell. Immunol. 129, 32 (1990). 21 M. A. Mansoor, A. M. Svardal, and E M. Ueland, Anal. Biochem. 200, 218 (1992). 22 H. Watanabe and S. Bannai, J. Exp. Med. 165, 628 (1987). 23 j. Rugtveit, E Brandtzaeg, T. S. Halstensen, O. Fausa, and H. Scott, Gut 35, 669 (1994). 24 g. L. Btu'gio, S. Fais, M. Boirivant, A. Perrone, and F. Pallone, Gastroenterology 109, 1029 (1995).

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Method 1: Determination of Cysteine in the Supernatant as Acid-Soluble Thiol Principle. This spectrophotometric assay is simple to perform and very sensitive for the detection of reduced thiol compounds after protein sulfhydryls have been removed by acid. However, this assay is not specific for cysteine and does not discriminate between various acid-soluble thiols such as cysteine and GSH. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) reacts with thiols to give the yellow product 5'-thionitrobenzoic acid, which has an absorbance maximum at 412 nm.tS Reagents NaOH, 1 N EDTA, 80 mM Trichloroacetic acid (TCA), 30% Sodium phosphate buffer, 0.5 M, pH 7.0 DTNB (Serva, Heidelberg, Germany), 10 mM in buffer L-Cysteine (Serva, Heidelberg, Germany), 10 mM in RPMI 1640 (for preparation of standards)

Procedure. The cell-free supernatant (600/zl) is mixed with 150/zl of EDTA and 150 #1 of TCA to precipitate protein, followed by incubation on ice for 15 min and centrifugation. The deproteinized supernatant (267 #1) is mixed with 400 #1 of buffer and 100 #1 of NaOH. Finally, 33 #1 of DTNB is added, and absorption is recorded spectrophotometrically at 412 nm. Standard solutions of cysteine are subjected to the same analytical procedure as described. PB-MO constitutively produce micromolar amounts of cysteine that increase three to four times when the ceils are stimulated with LPS. IFN-y (200 U/ml), however, another potent activator of monocytes, does not enhance constitutive production of cysteine (Table I). We have demonstrated that receptor-ligand interactions TABLE I CYSTEINE (ACID-SOLUBLETHIOL) CONTENT IN SUPERNATANT OF PB-MO VERSUS LP-MO a Treatment Medium

q-H202 (50/*M) LPS (1/*g/ml) q-H202 (50/*M) IFN-v (200 U/ml) q-H202 (50/*M)

PB-MO (/*M)

LP-MO (/*M)

6.06 6.04 20.05 18.79 5.80 4.85

0.08 ND b 0.16 ND 0.06 ND

a After 40 hr of culture; PB-MO and LP-MO were isolated fl'om the same patient and cultured at 5 x 105/ml. b Not determined.

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REDOX REGULATION OF INTESTINAL T LYMPHOCYTES

239

100

80 e =I.

60

100 ~tM (tl/2 = 45 min)

- - < > - 25 ~tM (tl/2 = 36 min)

O

..= 40

i

20

I

. . . . .

I,

/

0

I /

0

50

100

150

200

250

' 450

'~

' 500

Incubation time (min) FIG. 2. Degradation of cysteine added to RPM11640 at 25 and 100/zM, respectively, under standard culture conditions (37 °, 7% CO2). Cysteine was determined as acid-soluble thiol (method 1). The dashed line indicates the half-life of cysteine.

between CD2 on LP-T and the ligand CD58 on PB-MO are involved in cysteine production comparable to LPS.25 This indicates that CD2-mediated costimulation of T cells is not restricted to the CD2 signal transduced in T cells, but also induces metabolic changes in professional antigen-presenting cells, which by the release of cysteine enhance the proliferative immune response of activated lymphocytes. It is of note that LP-MO, in clear contrast to PB-MO, are defective in cysteine production both constitutively and after activation with LPS (Table I). Cysteine oxidizes rapidly to cystine in culture. The half-life of cysteine depends on the concentration and is 36 and 45 rain at 25 and 100 # M cysteine, respectively (Fig. 2). It can therefore be concluded that cysteine (acid-soluble thiol) does not accumulate in the supernatant during the 40-hr culture of PB-MO and that the cysteine level represents the actual and long-lasting production of cysteine.

Method 2: Determination of Cysteine by HPLC Principle. This analytical procedure is based on the method described by Mansoor eta/. 2t It is very sensitive and, in contrast to method 1, allows measurement of the reduced, oxidized, and protein-bound forms of cysteine, 25 B. Sido, J. Braunstein, R. Breitkreutz, C. Heffarth, and S. C. Meuer, J. Exp. Med. 192, 907 (2000).

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cysteinylglycine, homocysteine, and glutathione. Thus, it is possible to determine the redox balance of various thiols as defined, e.g., by the ratio GSH/GSSG or cysteine/cystine. Here, we present the protocol for the specific detection of nonprotein-reduced thiols in the supernatant of PB-MO.

Reagents NaOH, 0.05 M Sulfosalicylic acid (SSA), 50% Perchloric acid, 70% N-Ethylmorpholine (Sigma), 1 M in water Dithioerythritol (DTE; BioMol, Hamburg, Germany), 50 # M in 5% SSA Monobromobimane (mBrB; Calbiochem, Bad Soden, Germany), 40 mM in acetonitrile L-Cysteine (Serva, Heidelberg, Germany), 10 mM in 5% SSA containing 50/zM DTE Glutathione (GSH; Serva), 10 mM in 5% SSA containing 50 # M DTE Solution B: 65% DMSO and 35% water (v/v) containing 51 mM NaC1 and 140 mM hydrobromic acid Elution solvent A: 0.25% glacial acetic acid (pH adjusted to 3.4 with 2 M NaOH) Elution solvent B: 80% acetonitrile (for chromatography; Merck, Darmstadt, Germany)

Procedure. The supernatant of PB-MO (930/zl) is deproteinized by the addition of 70 #1 of SSA (50%), followed by incubation on ice (10 rain) and centrifugation. To 60 #1 of supernatant are added 10 #1 of water, 30 #1 of NaOH, 130 #1 of solution B, 50 #1 of N-ethylmorpholine, and 10 #1 mBrB. The mixture is incubated for 30-40 min in the dark at room temperature. After centrifugation, the sample is stored at - 8 0 ° and analyzed within 48 hr. Samples (40 #1) are injected into a 4.6 x 150-mm column packed with 3.5-#m particles (Symmetry C18; Waters, Milford, MA), equipped with a 3.9 x 20-mm guard column packed with 5-#m particles (Symmetry C18; Waters). The temperature is 20 ° and the flow rate is 1.0 ml/min. The elution profile is as follows: 0-13 min, 7.5% solvent B; 13-23 min, 8.75% solvent B; 23-30 min, 23.75% solvent B; 30-40 min, 25% solvent B; 40-50 min, 100% solvent B. Fluorescent material is detected by a Shimadzu RF-551 fluorometer detector at an excitation wavelength of 400 nm and an emission wavelength of 480 nm. The software GOLD Nouveau (Beckman, Coulter, Fullerton, CA) is used for plotting and integration of peaks. L-Cysteine and GSH are used as standards. The retention time for cysteine and GSH is 12 and 23 min, respectively. Linearity of the assay for both thiols is demonstrated in Fig. 3. For the first time, acid-soluble thiol present in the supernatant of PB-MO has been identified as cysteine by HPLC. No reduced GSH can be detected in

[20]

REDOX REGULATION OF INTESTINAL T LYMPHOCYTES 2O

40-

,-- 15

30"

241

205

10-

0)

0

lb

0,

2b

1'0

0

Cysteine (btM)

15

GSH (gM)

FIG. 3. Standard curves for cysteine and reduced glutathione (GSH). The standards were dissolved in 5% sulfosalicylic acid containing 50/*M dithioerythritol and were analyzed by HPLC (method 2).

the supernatant of PB-MO (Fig. 4). Indeed, under the experimental conditions described here, both analytical procedures yield identical results (Fig. 5). For routine analysis, method 1 is preferred. However, the specificity of the assay has to be evaluated by HPLC depending on the experimental setting and the questions to be answered.

Medium (RPMI 1640/10 % FCS)

Standards R Cysteine(50 i.tM)

A

1'0

15

20

25

30

10

15

GSrt (20 paM)

20

25

30

SupernatantD ~, o f PB-MO, LPS-stimu~ted

Supernatant o f PB-MO, unstimulated

C

1'0

15 20 25 Elution time (min)

30

10

15 20 Elution time (min)

25

FIG. 4. Reversed-phase high-pefforl'nance liquid chromatography of a deproteinized and monobromobimane-derivatized sample of (A) RPMI 1640/10% FCS (blank); (B) a standard solution of 50/zM cysteine and 20/zM reduced glutathione in 5% sulfosalicylic acid containing 50/zM dithioerythritol; (C) supernatant of unstimulated PB-MO in RPMI 1640/10% FCS after 40 hr of culture; and (D) supernatant of PB-MO simulated by LPS (1/zg/ml) in RPMI 1640/10% FCS for 40 hr.

30

242

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35

30

25

20

15 10

0

Exp. 1

Exp. 2

Acid-soluble thiol, without LPS HPLC, without LPS Acid-soluble thiol, with LPS (1 ~tg/ml) HPLC, with LPS (1 I.tg/ml) FIG. 5. Comparative analysis of cysteine as acid-soluble thiol (method 1) or by HPLC (method 2) in the supernatant of PB-MO after 40 hr of culture. Cells were either left untreated or stimulated with LPS (1/zg/ml). Results of two independent experiments are presented.

E x p e r i m e n t a l P r o c e d u r e s to D e m o n s t r a t e D i f f e r e n t i a l C a p a c i t y of P e r i p h e r a l B l o o d M o n o c y t e s v e r s u s I n t e s t i n a l L a m i n a P r o p r i a M a c r o p h a g e s to R e s t o r e C D 3 R e a c t i v i t y of L a m i n a Propria T Lymphocytes PB-MO and LP-MO, respectively, are irradiated (50 Gy) immediately before use and are added to LP-T at 30% of total cell number. In accordance with their capacity to produce cysteine, PB-MO restore the CD3 reactivity of LP-T in the absence of IL-2, whereas LP-MO, in clear contrast, fail to do so (Fig. 6). PB-MO have to be added at more than 20% of total cell number to provide significant costimulation, 25 suggesting a metabolic or mediator-driven mechanism of costimulation. In conformance with the hypothesis of thiol (cysteine)-mediated redox regulation of LP-T by PB-MO, 2-ME (50 #M) is able to fully substitute for the

[20]

REDOX REGULATION OF INTESTINAL T LYMPHOCYTES

243

30

m

~:~

&

25

II

20

,~_--

15 ©

.'2. 10

=Z 5

without

with

8202

H202 (50 >M)

L _ _ l unstimulated ,/////a O K T 3 OKT3 + LP-MO

L\~ OKT3 + PB-MO , , OKT3 + 2-ME ~ O K T 3 + IL-2

FIG. 6. Effect of 2-mercaptoethanol (2-ME; 5 0 / , M ) versus P B - M O versus L P - M O on the C D 3 reactivity o f L P - T (5 x 104/well) u n d e r standard a n d prooxidant culture conditions, respectively. PBM O and L P - M O , respectively, were hl"adiated (50 G y ) and added to LP-T at 3 0 % o f total cell n u m b e r in a total volume of 200/*l/well. If added, the concentration of IL-2 was 10 U/ml. Cells were cultured in either the absence o1" the presence of h y d r o g e n peroxide.

costimulatory activity of PB-MO in the absence IL-2. Moreover, PB-MO, similar to the antioxidant 2-ME, allow proliferation of LP-T even under prooxidant conditions in the presence of 50 # M hydrogen peroxide (Fig. 6). LP-MO do not actively suppress the proliferation of LP-T, as LP-MO do not influence the restoration of CD3 reactivity of LP-T by 2-ME.

E x p e r i m e n t a l P r o c e d u r e s to D e m o n s t r a t e T h i o l - M e d i a t e d R e g u l a t i o n of DNA S y n t h e s i s of L a m i n a Propria T L y m p h o c y t e s The use of cystine-deficient culture medium provides a simple and convenient experimental system to investigate the consequences of cysteine and/or GSH defciency. To this end, experiments are set up in cystine-deficient RPMI 1640

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CELLULAR RESPONSES

[20]

(BioWhittaker, Verviers, Belgium) to which 10% FCS, 2% glutamine, and antibiotics are added. Cells are washed in cystine-free medium before use. For supplementation of cystine-free cultures with graded amounts of cystine, we use a 20 mM cystine stock solution (cystine 100 x for RPMI 1640; GIBCO, Paisley, Scotland). Contaminating cystine may result from the FCS. Therefore, it might be necessary to dialyze the FCS against cystine-free RPMI 1640 before use. Because such a procedure might change the biological activity of FCS in an unpredictable way, we tested batches of FCS from different companies to avoid dialysis. Best results were obtained in our experimental series using FCS from GIBCO (Paisley, Scotland). PB-MO produce cysteine by uptake and intracellular reduction of cystine. Therefore, the costimulatory potential of PB-MO strictly depends on the availability of extracellular cystine (Fig. 7). In the absence of cystine, PB-MO completely fail to mediate any reactivity of LP-T to CD3 stimulation. According to

40

35

~,

, , OKT3 ........... OKT3 + PB-MO OKT3 + 2-ME

30

X

E

25

20

.9 15

10

0

12.8

51.2

204

1500

C y s t i n e (~tM) FIG. 7. Dependence of CD3 reactivity of LP-T on the availability of extracellular cystine in the absence and presence of 2-ME (10/zM) and PB-MO, respectively. It'radiated PB-MO were added at 30% of total cell number.

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REDOX R E G U L A T I O N OF INTESTINAL T LYMPHOCYTES

245

the molecular action, similar results are obtained with 2-ME (10 #M), although some minor proliferation of LP-T can be observed consistently in cystine-deficient medium. At physiological cystine concentrations (60-70 # M in human plasma), both PB-MO and 2-ME allow a significant proliferative immune response of LP-T after CD3 stimulation. In the absence of 2-ME or PB-MO, excessive high and unphysiological cystine concentrations would be needed (1.5 mM) to partly compensate for the low membrane transport activity for cystine in lymphocytes (Fig. 7). Is it possible to substitute the thiol compound cysteine in cystine-deficient medium for the stimulatory function of PB-MO or 2-ME in cystine-containing medium? To address this question, LP-T (8 x 105/well) are cultured in 24-well plates (Costar, Bodenheim, Germany) in an initial volume of 1 ml/well. Cultures are primarily set up in cystine-free medium and are supplemented with cysteine in 15-#1 volumes every 6 hr to reach a final concentration of 30 # M each time. Given the short half-life of cysteine of about 36 min in culture (see Fig. 2), it follows that the cysteine concentration is below 15 # M for nearly 90% of the culture period. Other cultures receive equimolar amounts of cystine (15 # M = 30 # M cysteine equivalents). After 84 hr of culture, wells are pulsed individually with [3H]thymidine (5 #Ci/ml). Cells are harvested at 18 hr postmetabolic labeling, at which time cumulative cystine concentrations have reached the unphysiologic level of 255 # M (510 cysteine equivalents). However, the proliferation of LP-T in the presence of very low but physiological relevant cysteine concentrations is nearly twofold higher as compared to supplementation with equimolar amounts of cystine (Table 1I). The specific requirement for cysteine for the synthesis of

TABLE II INFLUENCE OF CYSTEINE VERSUS EQUIMOLAR AMOUNTS OF CYSTINE ON THE CD3 REACTIVITY OF LP-T a Treatment Cystine-deficient medium a Cysteine (30/zM, every 6 ha')/' + B S O (100 i z M ) c Cystine (15/zM, every 6 hr) b

[3H]Thymidine uptake (cpm x 10 -4) 1.02 42.51 0.98 22.43

4444-

0.11 ° 1.44 0.12 1.08

a Cultures were primarily set up in cystine-deficient RPMI 1640 in 24-well plates at 8 x 105/ml. FCS used in this experiment was fi'om Sigma (Taufldrchen, Germany) and allowed some minor proliferation when added at 10% to cystine-deficient RPMI 1640. b Cysteine was added every 6 ha" at a final concentration of 3 0 / z M each time during the entire culture period. Alternatively, equimolar amounts of cystine were added ( 1 5 / z M = 3 0 / z M cysteine equivalents). c BSO was added at the beginning of culture. Results are presented as means 4- SD of triplicate cultures.

246

CELLULAR RESPONSES

[20]

GSH is demonstrated by the finding that addition of the glutathione synthesis inhibitor BSO (100 #M) abolishes the cysteine-driven restoration of CD3 reactivity of LP-T. The increase in glutathione content in lymphocytes due to the increased availability of cysteine is well documented. 19,2oAs an alternative to HPLC, intracellular GSH and GSSG can be differentially determined on a large scale by the spectrophotometric method of Griffith26 using 96-well ELISA plates. Principle. DTNB reacts with GSH to form the yellow product 5'-thionitrobenzoic acid, which can be detected at 412 rim. GSSG formed is converted to GSH in a cycling NADPH-dependent enzymatic reaction catalyzed by glutathione reductase. For determination of GS SG, GSH is derivatized by 2-vinylpyridine prior to reaction with DTNB.

Reagents Sulfosalicylic acid, 2.5% Sodium phosphate buffer, 150 mM containing 0.6 mM EDTA, pH 7.5 NADPH (Serva, Heidelberg, Germany), 0.6 mM in phosphate buffer DTNB (Serva), 6 mM in phosphate buffer Triethanolamine (Merck, Darmstadt, Germany) 2-Vinylpyridine (Sigma, Taufkirchen, Germany) Glutathione reductase (Sigma), 14 U/ml in phosphate buffer GSH (Serva), 20 mM in 2.5% SSA (for preparation of standards) GSSG (Serva), 20 mM in 2.5% SSA (for preparation of standards) Solution A: 9 ml NADPH and 2.17 ml DTNB are diluted with water to 20 ml

Procedure. At least 2 x 106 cells are lysed in 400 #1 of SSA and incubated for 10 rain on ice. After centrifugation, 10 #1 of supernatant is mixed with 10 #1 of sodium phosphate buffer and 165 #1 of solution A in a 96-well ELISA plate. One minute later, absorbance is recorded at 412 nm using an ELISA reader (VICTOR 2, Wallac, Freiburg, Germany). The reaction is started by the addition of 40 #1 of glutathione reductase 5 rain later (total volume 225 #l/well). The total glutathione (tGSH) content is proportional to the increase in absorbance after 6 rain at 25 °. For determination of GSSG, 100 #1 of each sample is preincubated with 4 #1 of triethanolamine and 2 #1 of vinylpyridine for 20 rain at room temperature. Twenty microliters of this mixture is then used for the determination of glutathione according to the procedure described earlier. GSH and GSSG are used as standards. The amount of reduced GSH is calculated as follows: GSH = tGSH - 2 x GSSG.

26 0. W. Griffith, Anal. Biochem. 106, 207 (1980).

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Biological I m p l i c a t i o n s The availability of cysteine to lymhocytes is a critical parameter for the synthesis of glutathione, which, in turn, is essential for cell cycle progression.tt The capacity of antigen-presenting cells to produce cysteine by uptake and intracellular reduction of cystine modulates the redox state in the microenvironment of intestinal T lymphocytes. This thiol-mediated redox regulation represents a novel mechanism that allows a dynamic modulation of the responsiveness of intestinal LP-T after antigen receptor-induced activation. The incapability of resident LP-MO to produce cysteine contributes to the hyporesponsiveness and low proliferative potential of LP-T in the normal gut, despite their continuous exposure to luminal antigens. In inflammatory bowel disease, macrophage populations with a different phenotype dominate in severely inflamed areas, especially near vessels, and result from a sustained recruitment of monocytes from peripheral b l o o d . 23'24 As a consequence, the release of cysteine by recently recruited PB-MO in the microenvironment of LP-T might contribute to the increased lymphocyte reactivity in inflammatory bowel disease. In addition to bacterial wall products (LPS), receptor binding (CD2) to CD58 on PB-MO--as it occurs during cellular interaction with LP-T--enhances cysteine release by PB-MO considerably, 25 thereby initiating a bidirectional signal that provides sufficient constimulation to LP-T to increase intracellular glutathione synthesis and to allow antigen receptor-induced T-cell proliferation. Oxidative stress due to the infiltration of large numbers of granulocytes is regarded to be an important mediator of cytotoxic tissue damage in inflammatory bowel disease. 27 PB-MO continue to produce cysteine even in the presence of hydrogen peroxide and thus maintain a balanced redox state in their microenvironment, allowing mononuclear cells to escape oxidative damage and to carry out a proliferative immune response. Acknowledgments This work was supported by a grant fl'om the Medical Research Council of the University of Heidelberg and fl'om the Deutsche I%rschungsgemeinschaft (SFB 405).

27 g. Gross, H. Arndt, T. Andus, K. D. Palitzsch, and J. Sch61merich, Hepato-Gastroenterol. 41, 320 (1994).