Immunomodulatory and protective effects of N-acetylcysteine in mitogen-activated murine splenocytes in vitro

ELSEVIER

Toxicology

116 (1997) 219-226

Immunomodulatory and protective effects of N-acetylcysteine mitogen-activated murine splenocytes in vitro Felix Olima OmaraaT*, Barry Raymond

Blakleyb, Jacques

Bernier”,

in

Michel Fournier”

“Dipartement des Sciences Biologiques et TOXEN, Universite du Quebec ir Montt&l, C.P. 8888 Montriul. Qubbec H3C 3P8, Canada b University of’ Saskatchewan, 52 Campus Drive, Department of’ Veterinary Physiological Sciences. Suskatoon, Saskatchewan S7N 5B4. Canada Received

9 April

1996; accepted

25 July 1996

Abstract N-Acetylcysteine (NAC) is a pro-glutathione drug used to treat chronic lung disorders and because of its anti-AIDS virus activity in vitro, has been proposed for AIDS therapy. The effect of NAC on mitogen-activated-lymphocyte blastogenesis in C57B1/6 mouse splenocytes and ability of NAC to protect lymphocytes against mitogen-induced cytotoxicity was examined in vitro. NAC increased splenocyte proliferation in the presence of optimal and suboptimal concentrations of concanavalin A (Con A) and lipopolysaccharide (LPS). Stimulatory and costimulatory effects of NAC on mitogen-induced responses were also evident. The dose-response relationship describing the effects of NAC on lymphocyte proliferation with Con A-induced responses were enhanced in a dose-dependent manner, whereas the corresponding LPS-induced responses increased to a maximum level followed by decline in responses at higher concentrations of NAC. When splenocytes were incubated with inhibitory supraoptimal concentrations of Con A (10 pg/ml) or LPS (150 fig/ml), NAC partially enhanced the Con A-induced response but completely prevented the inhibitory effect of supraoptimal concentrations of LPS on splenocyte blastogenesis. Optimal and supraoptimal concentrations of Con A caused activation-induced cell death in the splenocytes whereas comparable concentrations of LPS did not produce a similar effect. Splenocyte cell death produced by the optimal mitogenic concentrations of Con A was completely blocked by the addition of NAC to cultures. Immunomodulation and protective effects of NAC were observed in mitogen-activated lymphocytes in vitro. Copyright 0 1997 Elsevier Science Ireland Ltd. Keywords:

N-acetylcysteine;

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F.O. Omara et al. / Toxicology 116 (1997) 219-226

1. Introduction A variety of thiol-containing compounds such as 2-mercaptoethanol (2-ME), L-cysteine, glutathione and many others augment various lymphocyte functions (Noelle and Lawrence, 1981; Ohomori and Yamamoto, 1982; Fidelus et al., 1986; Gmunder et al., 1990). Glutathione is required for normal activation and proliferation of lymphocytes (Hamilos et al., 1991). Studies by Messina and Lawrence (1992) demonstrated that in contrast to mouse lymphocytes, thiols did not enhance proliferation of human lymphocytes to mitogens. They suggested that differential thiol requirements exist between human and mouse lymphocytes (Messina and Lawrence, 1992). Earlier studies by Hamilos et al. (1991) showed that although 2-ME increased cysteine uptake in both human and murine lymphocytes, it inhibited proliferation in human cells. A recent study showed that glutathione supplementation (2- 10 mmol/l) in vitro enhances T-cell-mediated mitogenic responses in human lymphocytes which was due in part to increased interleukin-2 and decreased prostaglandin E, production (Wu et al., 1994). In addition, thiols did not influence antigen-driven lymphocyte proliferation (Gmunder et al., 1990) suggesting that their effects are signal-dependent. Many of the studies describing the effects of thiols on lymphocyte function have evaluated Tcells and provide little information concerning the effect thiols on B-cells. The impact of thiols on lymphocyte function varies with the thiol-containing compound. For example; thiol-bearing compounds such as aminothiol cysteamine and 2-ME inhibit mitogen proliferation of human T-cells mitogen proliferation whereas a similar effect was not observed with N-acetylcysteine (Messina and Lawrence, 1992; Jeintner et al., 1994). Antioxidant thiols like N-acetylcysteine and gluthatione protect T-cell lines against activation-induced cell death but dithiothreritol and L-cysteine do not possess these properties (Sandstroma et al., 1994). The thiol compound N-acetylcysteine, a Nacetyl derivative of cysteine and a precursor of glutathione, is a potent antioxidant and a free radical scavenger and is used clinically to treat chronic inflammatory lung disorders in humans

(Christman and Bernard, 1994; Riise et al., 1994). N-acetylcysteine was reported to enhance in vitro killing of Staphylococcus aureus by human alveolar macrophages and blood polymorphonuclear leukocytes (Oddera et al., 1994) and to reduce the incidence of bacterial infection in patients with chronic bronchitis (Meyer et al., 1994; Riise et al., 1994). N-acetylcysteine has also has been demonstrated to inhibit the replication of AIDS virus in vitro (Raju et al., 1994; Simon et al., 1994) and therefore has been proposed as a therapeutic agent for AIDS patients. In addition to its known antioxidant effect, NAC may influence immune function. In the present study, the effect of NAC on murine lymphocyte mitogenic response in vitro was evaluated as well as the ability of NAC to protect lymphocytes against mitogen-induced cytotoxicity and cell death.

2. Materials and methods 2.1. Chemicals and animals N-Acetylcysteine (NAC), concanavalin A (Con A), propidium iodide (PI) and E. coli lipopolysaccharide (LPS) serotype 011 l:B4 were purchased from Sigma (St. Louis, MO). Female C57B1/6 mice 6-8 weeks old were obtained from Charles Rivers (Bar Harbor, Maine). Upon arrival, the mice were given a 2-week acclimatization period prior to starting the experiments. Mice were maintained in a standard environment (temperature, 22 + 2°C; humidity, 55 f 5%; 12-h light/dark cycle) and given standard pelleted rodent diet and tap water ad libitum. 2.2. Splenocytes Mice were killed by CO, gas. Spleen cell suspensions were obtained by gently teasing the spleens in Hank’s balanced salt solution (HBSS). Splenocytes were cultured at 37”C, 5% CO, for 72 h in 96-well flat bottom Falcon polystyrene plates (Becton Dickinson) at 5 x 10’ viable splenocytes/ well in RPMI- 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 100 U/ml penicillin and 100 ,ug/ml

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streptomycin (Gibco). Cell viability was determined by the trypan blue dye exclusion test.

basis of the percentage of total cells stained with propidium iodide.

2.3. NAC and mitogen-induced blastogenesis

2.5. Statistical analysis

Splenocytes were incubated in the presence of 50- 1000 hg/ml NAC alone or with optimal concentrations of concanavalin A (Sigma, St. Louis, MO) or E. coli , lipopolysaccharide serotype 011 l:B4. The optimal concentrations of Con A and LPS used in this study were 2.5 pgg/ml and 30 pg/ml, respectively. To study the effect of NAC on suboptimal concentrations of Con A (0.31 pg/ml) and LPS (1.5 pg/ml), or a supraoptimal concentration of Con A (10 ,Ug/ml) or LPS (150 pug/ml), splenocytes were cultured in the presence of these mitogens with NAC at 1000 pg/ml for Con A or 400 pg/ml NAC for LPS. N-acetylcysteine was added to splenocyte cultures with at 0, 6, 24, 48 and 54 h after stimulation of the cells with Con A (2.5 pug/ml) during the 72 h culture period to evaluate the temporal effect of NAC on blastogenesis. During the last 18 h of the 72 h culture period, the splenocytes were pulsed with 0.5 pCi/well of [3H]thymidine (SA: 6.7 Ci/pmol; ICN, Biochem, Canada). The cells were harvested onto filters by an automatic cell harvester (Tetratex Cell Harvetster 550). Each filter disc was placed into a vial. Three ml of scintillation liquid was dispensed into each vial and [3H]thymidine incorporated by the cells was determined with a p-scintillation counter (Beckman LS1801).

Results were expressed as disintegration per min (DPM) or stimulation index (SI), which is the DPM in mitogen-stimulated cultures divided by the mean DPM in non mitogen-stimulated cultures in each experiment. Data are presented as the mean f standard deviation (SD.) from at least two experiments. The results were analysed by one-way analysis of variance (ANOVA) and significance between experimental and control values was determined by the Scheffe’s test (P < 0.05) using the statistical computer software StatView (Brainpower Inc, Calabasas, CA).

2.4. NAC and mitogen activation-induced splenocyte cell death Splenocytes were incubated simultaneously with NAC and Con A (2.5 or 10 pg/ml) or LPS (30 or 150 pg/ml) for 72 h. Cell death (cytotoxicity) was determined by membrane permeability to propidium iodide (PI). Fluorescence was measured by flow cytometry using the FACScan (Becton-Dickinson) after a light scatter acquisitation gate was optimized for identification of lymphoid cells. Splenocytes were labelled for 15 min with 5 ,ug/ml PI before data acquisitation by flow cytometry to distinguish live and dead cells. Data were expressed as relative percentage cell death on the

3. Results 3.1. Augmentation of lymphocyte prohferation by NAC Incubation of splenocytes with NAC caused a dose-dependent linear increase in proliferation (as measured by thymidine incorporation) indicating that NAC is a weak mitogen (Fig. 1). N-acetylcysteine produced increased mitogenic responses of spleen cells to the T-cell mitogen, Con A and the B-cell mitogen, LPS (Fig. 2). However, the kinetics of the NAC effect on spleen cell proliferation to the T-cell and the B-cell mitogens were different. N-acetylcysteine enhanced the Con A-induced splenocyte proliferation in a linear and a dose-dependent manner, whereas, the LPS response was increased at 50-400 @g/ml of NAC followed by a progressive decrease in the response to near control values at higher concentrations of NAC (Fig. 2A and B). 3.2. Effect of time of NAC addition in culture on blastogenesis The results of all the above experiments were obtained from splenocyte cultures incubated simultaneously with NAC and the mitogens for 72 h. Therefore, experiments were conducted to de-

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termine if the stimulatory effect of NAC on mitogenie responses was time dependent. Splenocytes were incubated for 72 h in the presence of Con A. N-acetylcysteine was added to the cultures at 0, 6 h, 24 h, 48 h or 54 h after cell activation with Con A. Lymphocyte blastogenesis which was measured by radioactive thymidine uptake was augmented by NAC in a time-independent fashion (Fig. 3). The relatively low stimulation indices in this later part of the experiment were due to high background counts (DPM) in the control cultures.

116 (1997) 219-226

8x

T

(A)

in Culture

NAC

3.3. NAC effect on blastogenesis induced by

(pglml)

suboptimal and supraoptimal concentrations of mitogens

(B)

The effect of NAC on splenocyte blastogenesis (expressed as stimulation indices) induced by suboptimal and supraoptimal concentrations of Con A or LPS are shown in Fig. 4. Splenocyte blastogenie responses induced by suboptimal and supraoptimal concentrations of Con A (0.31, 10 pg/ml) or LPS (1.5, 150 pg/ml) were lower than responses induced by optimal concentrations of 0

200

400

NAC

6fxl in

Km

1aJo

4

1200

culture(~.~glml)

Fig. 2. The effect of N-acetylcysteine (NAC) on T-lymphocyte (A) and B-lymphocyte (B) proliferation stimulated by optimal concentrations of Con A (2.5 fig/ml) and LPS (30 pg/ml), respectively. Data are represented as the mean + SD. of quadruphcate cultures expressed as stimulation index. *P < 0.05; **P-C 0.01 with control group.

01 0

200

400

NAC

6fxl

in

sol3

loo0

1200

culture (pglml)

Fig. 1. The effect of N-acetylcysteine (NAC) alone on murine splenocyte proliferation. Splenocytes were cultured without or with various concentrations of NAC for 72 h. Data are represented as the mean f S.D. of quadruplicate cultures expressed as DPM. *P i 0.05 with other groups.

these mitogens (Fig. 4A and B). N-acetylcysteine produced a costimulatory response with suboptima1 concentrations of LPS or Con A and resulted in greater splenocyte blastogenic responses. Although NAC increased the mitogenic responses with supraoptimal concentrations (10 pg/ml) of Con A, it did not restore the response to the control optimal values. In contrast the inhibitory effect of supraoptimal concentration of LPS (150 pug/ml) on spleen cell blastogenic response was completely reversed by the addition of NAC (Fig. 4A and B).

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F.O. Omara et al. / Toxicology 116 (1997) 219-226

3.4. NAC and mitogen activation-induced cell death

Incubation of splenocytes for 72 h with optimal (2.5 fig/ml) and supraoptimal (10 pg/ml) concentrations of Con A increased activation-induced cell deaths (Fig. 5A). The proportion of age-related control cell death in splenocyte cultures without Con A was 53.1 + 2.8% whereas, 78.3% and 84.9% cell death was present in splenocytes stimulated with optimal and supraoptimal concentrations of Con A, respectively. In splenocytes treated with optimal (30 pg/ml) or supraoptimal (150 pg/ml) concentrations of LPS, the percentage of cell death was not altered as compared to the control cultures (Fig. 5B). At concentrations of 400 and 1000 ,~g/ml, NAC did not affect spleen cell viability or aging-related controlled cell death (Fig. 5A and B). In addition, NAC (1000 pg/ml) protected the splenocytes against the activationinduced cell death observed at optimal concentrations of Con A (2.5 pug/ml) but not at supraoptimal concentration (10 pg/ml) of Con A (Fig. 5A).

0 0,31

2,5 Con

A in culture

1.5

x) LPS in Culture

lZO1

10 (pglml)

150 (pglml)

Fig. 4. The effect of N-acetylcysteine on splenocyte blastogenesis stimulated by suboptimal, optimal and supraoptimal concentrations of Con A (A) and LPS (B). Suboptimal concentrations of Con A (0.31 ,ug/ml) or LPS (1.5 pg/ml), optimal concentrations of Con A (2.5 fig/ml) or LPS (30 pg/ml) and supraoptimal concentrations of Con A (10 fig/ml) or LPS (150 pgg/ml) were used to stimulate splenocytes with or without NAC (400 or 1000 pg/ml). Data are expressed as the mean + S.D. of six cultures. Bars with different letters superscripts are significantly different (P < 0.01).

4. Discussion 0

ConAonly Time

6

NAC added stler

24

48

Con A activation

54 (hr)

Fig. 3. The effect of NAC added at various times of stimulation of splenocytes with Con A on T-cell blastogenesis. Splenocytes were stimulated with Con A (2.5 pg/ml) and NAC (1000 pg/ml) or NAC which was added to cultures at 0, 6, 24, 48 or 45 h after stimulation of the cells with Con A during the 72 h culture period. Data are represented as the mean x SD. of six cultures expressed as stimulation index. *P < 0.01 with other groups.

We have demonstrated that NAC, a thiol-containing compound is a weak mitogen which augments murine lymphocyte blastogenesis to Con A and LPS mitogens in vitro. Although several studies have investigated the effects of thiols on Tcells, data on B-cells are scarce. Our study indicated that the stimulatory effects of NAC on Con A and LPS-induced lymphocyte blastogenesis follow different kinetic profiles. The T- and B-cells showed different optimal concentration re-

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quirements of NAC to induce maximal mitogeninduced lymphocyte blastogenesis. This difference may be related to different thiol requirements for the respective cell types. Human and mouse lymphocytes have different requirements for DNA synthesis (Messina and Lawrence, 1992). The differences in the dose-response curves for NAC on T- and B-cell blastogenic responses to mitogens in the present study provide an indirect evidence that thiol requirements for blastogenesis or DNA synthesis may be different for T- and

p

85

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75

= ’ 2 E 1

65

55

:: 45

0

IO

23

ConA in culture

(pghl)

65

0

150

30 LPS in culture

helmI)

Fig. 5. The effect of NAC on mitogen activation-induced splenocyte cell death by Con A (A) and LPS (B). Splenocytes were incubated simultaneously with NAC (400 or 1000 pg/ml) and Con A (2.5 or 10 pg/ml) or LPS (30 or 150 fig/ml) for 72 h. Cell viability was determined by propidium iodide fluorescence using a flow cytometer (Beet). Data are expressed as the mean f S.D. of five cultures. Bars with different letters are significantly different (P < 0.01).

B-lymphocytes. The biologic significance of these differences in vivo is not known. It is not known whether the kinetic disparity associated with the stimulatory effect of NAC on B and T-cell blastogenesis in our study is related to differences in cysteine transport or release cysteine intracellularly in the B- and T-cells. The explanation which accounts for these differences on T- and B-cell proliferation need to be elucidated. N-acetylcysteine was able to augment Con A-induced blastogenie responses in a time-independent fashion. This suggests that NAC produces a more generalized effect on lymphocyte activation and does not specifically affect early or late event in the activation cycle. Stimulatory and costimulatory effects of NAC on lymphocyte blastogenesis and the ability of NAC to protect the splenocytes against the inhibitory effects of supraoptimal concentrations of Con A and LPS were demonstrated in the present study. N-acetylcysteine enhanced blastogenic responses stimulated by suboptimal mitogenic levels of B- and T-cell mitogens to a greater extent than responses stimulated by optimal levels of the same mitogens indicating its costimulatory effect. Although NAC enhanced the blastogenic response in the presence of inhibitory supraoptimal concentrations of Con A (10 pg/ml), it did not restore the response completely suggesting that oxidative stress which may be prevented by NAC is only one factor in the Con A-induced cytotoxicity of splenocytes or T-cells. In contrast, NAC completely blocked the inhibitory effect of supraoptima1 concentrations of LPS on splenocyte blastogenesis, suggesting that oxidative factors play a more significant role in LPS-induced splenocyte cytotoxicity. Previous studies have demonstrated that NAC protects pulmonary and cardiac lipids from LPS-induced oxidative stress (Nowak et al., 1993) or from LPS-induced pulmonary edema in mice (Faggioni et al., 1994) which is consistent with the observations in the present study. Glutathione protects cells from damage by reactive oxygen species and has been shown to participate in cell activation and proliferation (Hamilos et al., 1991; Liang et al., 1991). Glutathione may influence lymphocyte function by

F.O. Omara et al. / Toxicology 116 (1997) 219-226

increasing synthesis of interleukin-2 and decreasing prostaglandin E, production (Chen et al., 1994; Wu et al., 1994). In the present study, activation of splenocytes with an optimal concentration of the T-cell mitogen, Con A caused cell death in splenocytes which was blocked by the addition of NAC to the cultures. Therefore, NAC may also augment T-cell mitogenic blastogenesis by preventing cell death associated with activation of T-cells by mitogens. In contrast, activation of splenocytes at optimal and supraoptimal mitogenie levels of LPS caused no cell death. Thus, mechanisms associated with the inhibitory effects of supraoptimal mitogenic concentrations of LPS or Con A on lymphocyte blastogenesis appear to be different. N-acetylcysteine has anti-AIDS viral activity in vitro (Raju et al., 1994; Simon et al., 1994) and AIDS patients have decreased plasma cysteine and intracellular glutathione levels which correlate with a decrease in the number of CD4+ T-cells (Kinscherf et al., 1994). Some studies have shown that the decrease in the number of CD4+ T-cells in AIDS could by prevented by treatment with NAC with a resultant increase in CD4+ cells (Kinscherf et al., 1994). Our study demonstrated that NAC blocks cell death in splenocytes activated by Con A, a T-cell mitogen. The ability of NAC to increase the number of CD4+ T-cells reported in AIDS (Kinscherf et al., 1994) may in part be related to its protective effect against T-cell activation-induced cell death. The depletion of CD4+ T-cells in AIDS patients is believed to be associated with T-cell activation-induced cell death (Gougeon and Montagnier, 1993). In the present study it is not certain whether cell death in splenocytes induced by Con A occurred by apoptosis. Apoptosis in T-cells can be induced by T-cell activators such as antibodies to CD3/T-cell receptor complex, and calcium ionophores and phorbol esters (Kizaki et al., 1989; Smith et al., 1989).

Acknowledgements

This work was supported by TOXEN, Universite du Quebec a Montreal, Canadian Network of

225

Toxicology Research Centres (CNTC) and Health Canada.

References Chen, G., Wang, S.-H. and Converse, C.A. (1994) Glutathione increases interleukin-2 production in human lymphocytes. Int. J. Immunopharmacol. 16, 7555760. Christman, B.W. and Bernard, G.R. (1994) Antilipid mediator and antioxidant therapy in adult respiratory distress syndrome. N. Horizons 1, 623-630. Faggioni, R., Gatti, S., Demitri, M.T., Delgado, R., Echatenacher, B. and Gnocchi, P. (1994) The role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary oedema. J. Lab. Clin. Med. 123. 394-399. Fidelus, R.K., Ginouves, P., Lawrence, D. and Tsan, M.-F. (1986) Modulation of intracellular glutathione concentration alters lymphocye activation and proliferation. Cell. Immunol. 97, 1555163. Gmunder, H., Eck, H.-P., Benninghoff, B., Roth, S. and Droge, W. (1990) Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine. Cell. Immunol. 129. 32-46. Gougeon, M.L. and Montagnier, L. (1993) Apoptosis in AIDS. Sciences 260, 1269. Hamilos, D., Mascali, J.J. and Wender, H.J. (1991) The role of glutathione in lymphocyte activation. Int. J. Immunopharmacol. 13, 75-90. Jeintner, T.M., Kneale, C.L., Christopherson, R.I. and Hunt, N.H. (1994) Thiol-bearing compounds selectively inhibits protein kinase C-dependent oxidative events and proliferation in human T-cells. Biochim. Biophys. Acta 1223. 15. 22. Kinscherf, R., Fishbach, T., Mihm, S., Roth. S., HohenhausSievert, E., Weiss, C., Edler, L., Bartsch, P. and Drogue, W. (1994) Effect of glutathione depletion and oral Nacetylcysteine treatment on CD4+ and CD8+ cells. FASEB. J. 8. 448-451. Kizaki, H.. Tadakuma, T., Odaka. C.. Muramatsu. J. and Ishimura, Y. (1989) Activation of of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters. J. Immunol. 143, 17901794. Liang, S.-M., Liang, C.-M., Hargrove, M.E. and Ting, C.-C. (1991) Regulation by glutathione of the effect of lymphocytes on differentiation of primary activated lymphocytes. J. Immunol. 146, 190991913. Messina, J.P. and Lawrence, D.A. (1992) Effects of 2-mercaptoethanol and buthionine sulfoxime on cystine metabolism by and proliferation of mitogen-stimulated human and mouse lymphocytes. Int. J. Immunopharmacol. 14, 1221 1234.

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Meyer, A., Buhl, R. and Magnussuen, H. (1994) The effect of oral N-acetylcysteine on lung glutathione levels in idiopathic pulmonary fibrosis. Eur. Respir. 7, 431-436. Noelle, R. and Lawrence, D.A. (1981) Modulation of T-cell functions. II. Chemical basis for the involvement of cell surface thiol-reactive sites in control of T cell proliferation. Cell. Immunol. 60, 453-469. Nowak, D., Pietras, T., Antczak, A., Krol, M. and Piasecka, G. (1993) Ambroxol inhibits endotoxin-induced lipid peroxidation in mice. Pol. J. Pharmacol. 45, 317-322. Oddera, S., Silverstri, M., Sacco, O., Eftimiadi, C. and Rossi, G.A. (1994) N-acetylcysteine enhances in vitro the intracellular killing of Staphylococcus uureus by human alveolar macrophages and blood polymorphonuclear leukocytes and partially protects phagocytes from self-killing. J. Lab. Clin. Med. 124, 293-301. Ohomori, H. and Yamamoto, I. (1982) Mechanisms of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes. I. The uptake of cysteine. J. Exp. Med. 155, 1277-1290. Raju, P.A., Herzenberg, L.A., Herzenberg, L.A. and Roederer, M. (1994) Glutathione precursor and antioxidant activities of N-acetylcysteine and oxothiazolidine carboxy-

116 (1997) 219-226

late compared in in vitro studies of HIV replication. AIDS. Res. Hum. Retroviruses 10, 961-967. Riise, G.C., Larsson, S., Larsson, P., Jeansson, S. and Andersson, B.A. (1994) The intrabronchial microbial flora in chronic bronchitis patients: a target for N-acetylcysteine therapy?. Eur. Respir. J. 7, 94-101. Sandstroma, P.A., Mannie, M.D. and Buttke, T.M. (1994) Inhibition of activation-induced death in T-cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis. J. Leukoc. Biol. 55, 221-226. Simon, G., Moog, C. and Obert, G. (1994) Effects of glutathione precursors on human immunodeficiency virus replication. Chem.-Biol. Interact. 91, 217-224. Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J. and Owen, J.J.T. (1989) Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T-cells in thymic cultures. Nature (London) 337, 181- 184. Wu, D., Meydani, S.N., Sastre, J., Hayek, M. and Meydani, M. (1994) In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J. Nutr. 124, 655-663.