March 2009.indd SQU MED J, APRIL 2009, VOL. 9, ISS. 1, PP. 70-74, EPUB 16TH MAR 2009 SUBMITTED - 10TH JUNE 08 REVISION REQ. 18TH AUG 08 & 20TH JAN 09, REVISION RECD. 25TH OCT 08 & 28TH JAN 09 ACCEPTED - 2ND FEB 2009 Objectives: This study investigates the in vitro effect of the antioxidant drug, N-acetyl-L-cysteine (NAC), on cytokine production by peripheral blood mononuclear cells (PBMC). Methods: PBMC were isolated by Ficoll-Hypaque, and stimulated with anti-CD3 anti- bodies, phytohaemagglutinin (PHA), lipopolysaccharide (LPS) for 24 hours in the presence or absence of 5 mM NAC. The cytokines produced were measured by enzyme-linked immunosorbent assay (ELISA). Results: Treatment with NAC significantly up-regulates the secretion of IL-β, IL-5 (interleukin) and IFN-γ (interferon) and down regulates IL-0 production, after anti-CD3 or PHA (p<0.05), but not after LPS stimulation. NAC also significantly increased total IL-2 secretion after anti-CD3 (but not PHA or LPS) stimulation and IL-2p40 after anti-CD3, PHA, and LPS stimulation (p <0.05). Conclusion: These results indicate that NAC up-regulated the production of pro-inflammatory cytokines, and down regulated anti-inflammatory cytokine production by PBMC, in a process which may be associated with increased levels of glutathione (GSH). Further work is required to determine whether this increase or decrease in cytokine production is due to direct effect of NAC. Key words: Antioxidant; N-acetylcysteine; Cytokines; Peripheral blood mononuclear cells. Effect of N-acetyl-L-cysteine on Cytokine Production by Human Peripheral Blood Mononuclear Cells *Ahmed Al-Shukaili,1 Suad Al-Abri,2 Alia Al-Ansari,2 Michele A Monteil1 1Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of Oman; 2Department of Biology, College of Science, Sultan Qaboos University, Muscat, Sultanate of Oman *To whom correspondence should be addressed. Email: shukaily@squ.edu.om قبل اخلاليا من السايتوكني في إنتاج L - سيستني N - أسيتايل تأثير النواة في دم اإلنسان احمليطي وحيدة مونتيل ميشيل األنصاري، عالية العبري، سعاد الشكيلي، أحمد قبل اخلاليا من السايتوكني بإنتاج L - سيستني) ــيتايل (N - أس ــدة لألكس املضادة األدوية في تأثير تبحث اتبرية ــة الدراس هذه الهدف: امللخص: ،( CD3) نوع من األجسام فيكول هايبيك وحفزت مبضادات اخلاليا وحيدة النواة بواسطة على الطريقة: مت احلصول . احمليطي اإلنسان دم في �وحيدة النواة ــايتوكني الس قياس مت . ــتني L سيس - ــيتايل N - أس من 5 مل وبدون مع ــاعة 24 س ملدة يّ مِ ــحْ الشَّ ــكاريدِ السَّ ديدُ وعَ ، النَباتِيَّة وِيَّةٌ مَ الدَ ةٌ والراصَّ IL-1β, IL-5, IFN-γ)) من إنتاج L سيستني زاد - N - أسيتايل النتائج: إضافة ُرْتَبِط. امل لإلِنْزميِ يَّةُ َناعِ امل يَّةُ تِصاصِ االمْ ــةُ ُقايَسَ امل ــطة بواس املنتج ديدُ عَ بعد حتفيز ليس لكن إحصائيا ، النَباتِيَّة بشكل معتد وِيَّةٌ مَ الدَ ةٌ الراصَّ أو (CD3) األجسام مضادات إضافة بعد ، ( IL-10) إنتاج قلل من بينما ( CD3) األجسام مضادات إضافة بعد (IL-12) إفراز مجموع إحصائيا معتد L سيستني بشكل - كذلك يزيدN - أسيتايل . يّ مِ حْ كاريدِ الشَّ السَّ وِيَّةٌ النَباتِيَّة� مَ الدَ ةٌ والراصَّ ، ( CD3) مضادات حتفيز بعد (IL-12p40) و يّ ، مِ حْ كاريدِ الشَّ السَّ ديدُ عَ أو النَباتِيَّة وِيَّةٌ مَ الدَ ةٌ الراصَّ بتحفيز ليس ولكن ويقلل إنتاج ، االلتهابي قبل ما السايتوكني إنتاج يزيد L - سيستني N - أسيتايل إلى أن تشير اخلالصة: هذه النتائج . يّ مِ ــحْ الشَّ ــكاريدِ السَّ ديدُ وعَ املطلوب . اجللوتاثايون مستويات بزيادة مرتبطة تكون رمبا ــيلة بوس ــان احمليطي ، اإلنس دم في النواة وحيدة اخلاليا قبل من املضاد لاللتهاب ــايتوكني الس . L - سيستني N - أسيتايل ل املباشر التأثير بسبب كانت السايتوكني انتاج قلة أو زيادة كانت لتبيان فيما إذا من البحث مزيدا احمليطي. اإلنسان دم في النواة وحيدة سايتوكني، خاليا L - سيستني، N - أسيتايل األكسدة، الكلمات: مضاد مفتاح B R I E F C O M M U N I C A T I O N N-ACETYL-L-CYSTEINE (NAC) IS A well-known medication which is common-ly used as a mucolytic agent in a variety of respiratory illnesses, in the treatment of paracete- mol overdose, and in conditions characterised by de- creased antioxidants or oxidative stress such as HIV infection, cancer, heart disease and cigarette smoking. This drug works as a free radical scavenger, as a glu- tathione (GSH) precursor, or as structural analogue of GSH, enriching the GSH pool in the cellular envi- ronment. The beneficial effects of NAC are attributed either to its ability to reduce extracellular cysteine to cysteine, or as a source of sulfhydryl (SH) metabolites, which are important for GSH synthesis.1,2 E F F E C T O F N - A C E T Y L - L - C Y S T E I N E O N C Y T O K I N E P R O D U C T I O N B Y H U M A N P E R I P H E R A L B L O O D M O N O N U C L E A R C E L L S 71 There are limited studies investigating the effects of NAC on cytokine production by peripheral blood mononuclear cells (PBMC). A study by Viora et al., us- ing PBMC, demonstrated that NAC up-regulated the secretion of interleukin, IL-1β, IL-2, IL-12, and IL-15 after phytohaemagglutinin (PHA) stimulation.3 Sever- al reports have shown that NAC treatment induced a significant up-regulation of IL-12 production by isolat- ed monocytes.4,5 In addition, pre-treatment with NAC reduced lipopolysaccharide (LPS)-mediated secretion of TNF-α (tumour necrosis factor) and IL-6 by human alveolar macrophages6 and by rat epithelial cells.7 Based on the hypothesis that antioxidant drugs such as NAC can act as modulators of the immune re- sponse and can be used in antioxidant therapy in many human diseases with oxidative imbalance,1,2 we stud- ied the in vitro effect of NAC (as an active antioxidant agent) on the production of some of pro/anti-inflam- matory cytokines by peripheral blood mononuclear cells isolated from normal healthy individuals. Also, we examined whether NAC can influence several cell- activation pathways using different stimuli. M E T H O D S Peripheral blood (5ml) was collected in sodium heparin-containing tubes from 14 normal volunteers (10 males and 4 females, aged between 23-37 years) at the College of Medicine and Health Sciences, Sultan Qaboos University (SQU), Muscat, Oman. All the vol- unteers provided informed consent, and completed a questionnaire to assess their health status. The study was approved by the Research Committee of the Col- lege of science Medicine at SQU. PBMC were isolated by Ficoll density gradient centrifugation (Sigma Diagnostics, USA) following the manufacturer’s instructions. Buffy coats were har- vested and cells were washed twice (centrifuged at 300g for 5 minutes) in Hanks solution (GIBCO, Pais- ley, UK) and resuspended in an RPMI-1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (FCS), (Sigma Diagnostics, USA), L- glutamine and penicillin/streptomycin (GIBCO, USA, UK) (complete medium). The antioxidant compound NAC (Sigma Diagnos- tics, USA) was dissolved in RPMI1640, the pH was ad- justed to 7.4 by addition of sodium hydroxide (NaOH), and used at final concentration of 5 mM, after addition to cell suspension. The trypan blue dye exclusion test was performed in order to exclude any drug toxicity. All the materials for this assay were purchased from Sigma Diagnostics (USA). Total glutathione (reduced and oxidized GSH) was measured in PBMC lysates af- ter 24 hour incubation with 5 mM NAC. Cells were lysed in 2ml of 0.05% v/v Triton- X-100 in buffer (125 mM sodium phosphate, 6.3 mM sodium ethylenedi- amine tetra acetic acid (Na-EDTA), adjusted to pH 7.5). The samples were assayed by enzymatic recycling procedure as described previously.8 PBMC were resuspended at concentration of 1 x 106 cell/ml (for IL-1β, IL-1ra, IL-10, IL-12p40, and IFN-γ) or 5 x 106 cell/ml (for IL-5 and IL-12) in com- plete medium, and cultured in 24-well plates (1ml/ well). Cells were stimulated with mouse anti-human CD3 (Serotec, UK, MCA 463XZ; 10µg/ml), PHA, Sig- ma Diagnostics; 10µg/ml), or lipopolysaccharide (LPS, Escherichia coli serotype 055:B5, Sigma Diagnostics; 200ng/ml), in the presence or absence of 5 mM NAC. Cells were incubated for 24 hr at 37oC / 5% CO2. Su- pernatants were harvested and the cytokine content (except for IL-1β) was measured using commercially available sandwich enzyme-linked immunosorbent assays (ELISA) (R&D Systems, USA), performed ac- cording to the manufacturer’s instructions. Monocytes were isolated by an adherence method as previously described.9 The plastic-adherent cells were cultured for 24 hours in a complete medium con- taining 100 ng/ml LPS in the presence or absence of 5 mM NAC. A sandwich ELISA was developed in our laborato- ry using a method previously described.10 ELISA was performed to determine the production of IL-1β in the supernatant of the cell cultures (PBMC and isolated monocytes). The SPSS (Statistical Package for the Social Scienc- es) software programme was used for data analysis. As all the data were normally distributed, pair-wise group (NAC-treated cells and un-treated cells) comparison was carried out using Student’s t-test. P values of less than 0.05 were considered statistically significant. R E S U L T S Incubation of PBMC with 5mM NAC for 24 hours re- sulted in a two-fold increase in total GSH levels in all treated samples. The range of GSH concentration was 0.56 - 0.85 and 1.2 - 1.5nmol/106 cells before and after treatment with 5mM NAC respectively. As shown in Figure 1, NAC induced significant secretion of IL-1β after anti-CD3 and PHA stimula- A H M E D A L -S H U K A I L I , S UA D A L - A B R I , A L I A A L - A N S A R I A N D M I C H E L E A M O N T E I L 72 4000 3500 3000 2500 2000 1500 1000 500 0 IL -1 β p g /m l * * 60 50 40 30 20 10 0 IL -1 2 p g /m l * 40 35 30 25 20 15 10 5 0 IL -5 p g /m l * * 2500 2000 1500 1000 1500 1000 500 0 IL -1 2p 40 p g /m l * * * 1000 800 700 600 500 400 300 200 100 0 IL -1 2p 40 p g /m l Anti - CD 3 Anti CD3+NAC PHA PHA+NAC LPS LPS+NAC NAC PBMC only * * IF -1 γ p g /m l Anti - CD 3 Anti CD3+NAC PHA PHA+NAC LPS LPS+NAC NAC PBMC only 0 5 0 0 10 0 0 15 0 0 20 0 0 25 0 0 30 0 0 35 0 0 40 0 0 45 0 0 * * IL = interleukin; NAC = N-acetyl-L-cysteine ; PHA = phytohaemagglutinin; LPS = lipopolysaccharide ; PBMC = peripheral blood mononuclear cells Figure 1: N-acetyl-L-cysteine (NAC) modulates cytokine production. Anti-CD3/PHA/LPS-stimu- lated peripheral blood mononuclear cells (PBMC) were treated with 5 mM NAC for 24 hr. Results are expressed aspg/ml. Data presents the mean value ± S.D. of at least six experiments for six different blood volunteers. * = Significant p value <0.05. E F F E C T O F N - A C E T Y L - L - C Y S T E I N E O N C Y T O K I N E P R O D U C T I O N B Y H U M A N P E R I P H E R A L B L O O D M O N O N U C L E A R C E L L S 73 tion (p <0.05), and statistically insignificant secretion after LPS stimulation. Incubation of plastic-adhered monocytes with NAC in the presence of LPS resulted in increased IL-1β production. Although this increase is not statistically significant, it was a consistent in all five experiments (p value = 0.061). Low concentrations of IL-5 were detected after stimulation of PBMC with anti-CD3 (mean value of 15pg/ml), PHA, (12pg/ml) and LPS (5pg/ml) when compared to other cytokines. In the presence of NAC, significant IL-5 secretion was further increased after anti-CD3 or PHA (but not with LPS) stimulation, this achieved statistical significant (p <0.05) [Figure 1]. NAC had an inhibitory effect on IL-10 secretion by PBMC. In the presence of NAC, a significant decrease (p <0.05) in IL-10 production was observed after anti- CD3, and PHA stimulation, but not after LPS stimula- tion. IL-10 production, following LPS stimulation in the absence or presence of NAC, was twice the con- centration achieved after anti-CD3 or PHA stimula- tion [Figure 1]. The levels of IL-12 obtained after all three stimu- lants were low, similar to those of IL-5. However, stim- ulation with anti-CD3 or PHA induced better IL-12 secretion than with LPS. The addition of NAC caused a significant increase in IL-12 production after anti- CD3 (p <0.05); a slight increase was observed after PHA stimulation, but this was not significant. There was no significant change in Il-12 levels after LPS stimulation [Figure 1]. Unlike total IL-12, stimulation of PBMC with anti- CD3, PHA, or LPS induced substantial amounts of IL- 12p40. The presence of NAC with all three stimulants elicited higher levels (p <0.05) of IL-12p40. This was highest after anti-CD3 stimulation [Figure 1]. Stimulation of PBMC with anti-CD3 or PHA (but not LPS), induced high levels of IFN-γ, compared to un-stimulated cells. LPS (100 ng/ml) failed to induce a significant amount of IFN-γ. As shown in Figure 1, exposure of PBMC to NAC elicited a significant up- regulation (p <0.05) of IFN-γ after anti-CD3 or PHA stimulation and a slight increase after LPS stimula- tion. D I S C U S S I O N NAC significantly up-regulated IFN-γ, IL-1β, IL-5, IL- 12, and IL-12p40, and significantly down-regulated IL- 10 production by PBMC after cellular stimulation. A number of studies investigated the effect of both NAC on cytokine production by several cell types including alveolar epithelial cells,4 alveolar macrophages,11 and PBMC.3 Some of our results (increase in IL-1β and de- crease in IL-10 production) are in partial agreement with a study carried out Viora et al. which demon- strated a significant up-regulation of IL-1β, IL-2, IL-12 and IL-15 production and an insignificant increase in IL-10 and IFN-γ by PHA-stimulated PBMC in pres- ence of NAC.3 This discrepancy in the production of IL-10, may be related to the genetic variation among individuals, hence, immense differences in response to various stimulations and cytokine production was observed. T cells and monocytes are major sources of inflam- matory mediators and can communicate with each other as well as with other cells12 This communication is mediated by soluble factors and by direct cell-cell contact.12 As traditionally known, potent cytokine polypeptides have pleiotropic activities and func- tional redundancy, acting in a complex intermingled network where one cytokine can influence the pro- duction of, and response to, many other cytokines.13 For example, Th1 cells release IFN-γ, and to a lesser extent IL-2 and IL-12, promoting a proinflammatory response. Th2 cells secrete IL-4, and sometimes IL-5, IL-6, IL-10 and IL-13, and enhance anti-inflammatory responses.14 Therefore, we were not able to prove that the decrease in IL-10 or the increase of other inflam- matory cytokines in our study is due to the direct ef- fect of NAC. Moreover, our data showed that an increase in IL- 1β production after anti-CD3 activation, is most likely due to the effect of T lymphocytes activation in this system. The effects of NAC on cytokine production are still controversial. On the one hand, several studies (in- cluding this study), showed that, NAC up-regulated proinflammatory cytokines.3 On the other hand, some studies reported that NAC down regulated pro-in- flammatory cytokines such IL-6, TNF-α IL-1β, and IL- 8.7,15,16 In addition, Stanislaus et al. reported that NAC treatment blocked induction of TNF-α, IL-1β, and IFN-γ.17 From the above discussion, it appears that, the effect of NAC on cytokine production depends on cell types. C O N C L U S I O N NAC up-regulates the production of pro-inflamma- tory cytokines and down regulates anti-inflammatory A H M E D A L -S H U K A I L I , S UA D A L - A B R I , A L I A A L - A N S A R I A N D M I C H E L E A M O N T E I L 74 cytokines by PBMC, in a process associated with in- creased levels of GSH. Also this study indicated a side effect of NAC when used as an anti-inflammatory drug. Further studies are required to confirm whether this increase or decrease in cytokine production is due to a direct effect of NAC. A C K N OW L E D G M E N T S We gratefully thank the Medical Research and Ethics Committee (MREC), College of Medicine and Health Sciences, Sultan Qaboos University, for financially supporting this research. We appreciatively thank Dr. Abdullah Al-Maniri and Dr. Shyam S Ganguly for their help in statistical analysis. 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