International Immunopharmacology 2 (2002) 751 – 757 www.elsevier.com/locate/intimp
Inhibition of antigen specific lymphocyte proliferation and cytokine stimulation by peptidomimetic opioid compound P. Narayan a, R. Tandon b, W. Haq b, M.M. Dhar b, V.K. Singh a,* a
Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow-226 014, India b Central Drug Research Institute, Lucknow-226 001, India Received 30 October 2001; received in revised form 31 January 2002; accepted 4 February 2002
Abstract In sequel to our preliminary observations with peptidomimetic opioid compounds, we have further investigated immunomodulatory activity of one peptidomimetic compound (Tyr-NH-CH2-CH2-O-Phe-NH2) with peripheral blood mononuclear cells (PBMCs) of healthy volunteers/tuberculosis patients. This peptidomimetic compound was evaluated for its effect on purified protein derivative (PPD) stimulated lymphocyte proliferation in vitro, production of Th1 and Th2 cytokines by ELISA and ribonuclease protection assay. Our study shows the immunosuppressive potential of above synthetic peptidomimetic compound. This compound inhibited PPD stimulated human lymphocyte proliferation and this inhibition was reversed by opioid receptor antagonist, naloxone. Its immunosuppressive effect was further demonstrated by inhibition of interleukin-9 (IL-9), IL-10 but failed to influence IL-2, IL-15 and interferon-g (IFN-g) in PPD stimulated human PBMCs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytokines; Immunomodulator; Lymphocyte proliferation; Peptidomimetic compound
1. Introduction The effect of the neuroendocrine system on the immune system is well accepted [1,2]. This effect is mediated through both glucocorticoids and peptides . Significant among these are the enkephalins and endorphins [4– 6]. The pentapeptide met and leu-enkephalins were discovered as a result of investigation for the natural ligand for the morphine receptor in the brain . Morphine causes immunosuppression and has preference for the A-opioid receptor [8,9], while met*
Corresponding author. Current address: Radiation Medicine Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889, USA. Tel.: +1-301-2959972; fax: +1-301-295-0292. E-mail address: [email protected]
enkephalin causes immunostimulation [4,10,11] and has a propensity for y-opioid receptor . Elegant studies using monoclonal antibodies have confirmed the latter . The presence of these receptors has been demonstrated on cells of immune system, suggesting receptor selective compounds may be potent immunomodulators . Despite substantial information regarding the biosynthesis, structure, distribution and effects of endogenous opioid peptides and various synthetic peptidomimetic compounds, the knowledge about their definitive role in immune regulation is still limited. Although there is no unanimity, ligands of A- and nopioid receptors primarily down regulate the immune response [15,16], while y- and q-ligands stimulate the immune system [5,17 – 19]. The identification of endogenous peptide ligands for these opioid receptors has
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 0 11 - 5
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757
provided leads for designing receptor selective, biologically stable peptide molecules. The classical approach of replacement of amino acids has provided various opioid peptides with enhanced receptor selectivity and potency [20 – 22]. Structure –activity relationship studies with enkephalins had concluded that pharmacophores required for interaction with opioid receptors are: (a) a phenolic ring and terminal NH2 function of Tyr, (b) phenyl ring of Phe and (c) appropriate distance between the two aromatic residues Tyr and Phe [23 – 26]. On the basis of these requirements and earlier findings, it is likely that the tyramine part of a morphine molecule binds in the same manner at both opiate receptors. One of us  has suggested from a review of literature on enkephalins and immunomodulation that y- and Areceptor selective compounds should provide potent immunostimulants and immunosuppressants, respectively. Accordingly, the effect on lymphocyte proliferation of the cyclic y- and A-receptor selective compounds DPDPE and TOPA were examined and found to possess potent immunostimulatory and immunosuppressive activity . Working on similar line, we have designed peptidomimetics to enkephalins where phenylalanine and tyrosine moieties were placed on a bifunctional linker template to obtain the non-peptide peptidomimetics. The bifunctional linker used could impart certain restriction in flexibility for predominant orientation of the phenyl rings. In our preliminary study, we have demonstrated that one of the peptidomimetic compounds (Tyr-NH-CH2-CH2-O-Phe-NH2) has potential to inhibit mixed lymphocyte reaction through A-opioid receptor. It also inhibited tumor necrosis factor (TNFa) and nitric oxide (NO) production . In sequel to this observation, we further investigated the effects of this compound on antigen (PPD) stimulated lymphocyte proliferation response and stimulation of various Th1 and Th2 cytokines in vitro by ELISA and ribonuclease protection assay.
2. Material and methods 2.1. Cells and reagents Mouse macrophage cells (RAW 264.7) were grown in DMEM supplemented with 10% fetal bovine serum,
penicillin (100 U/ml) and streptomycin (100 Ag/ml). DMEM, RPMI-1640, LPS, naloxone, TriReagent were purchased from Sigma (St. Louis, MO, USA). Lymphoprep was obtained from Nycomed Pharma (Oslo, Norway). IL-10 and IFN-g ELISA kits were purchased from R&D systems (Minneapolis, MN, USA). PPD was gift from Span Diagnostics (Mumbai, India). Ribonuclease protection assay kit was procured from Pharmingen (San Diego, CA, USA). 2.2. Synthesis of peptidomimetic opioid compounds Compound (Tyr-NH-CH2-CH2-O-Phe-NH2) was synthesized by standard solution phase methodology. In the first step, amino group of ethanolamine was selectively blocked by acylation using di-tert-butyldicarbonate to get N-Boc protected amino alcohol. The N-Boc ethanolamine thus obtained was allowed to react with Z-Phe using DCC/DMAP to get the corresponding ester derivative. The Boc group was removed and resulting amine was coupled with Z-Tyr using DCC/HOBt procedure to yield the Z-Tyr-NHCH2-CH2-O-Phe-NH-Z. The protected compound thus obtained was purified by silica gel column chromatography and subjected to catalyze hydrogenation on 10% Pd/c which yielded the desired free compound as white hygroscopic solids in good yield and purity. Purity of the compound was assessed by analytical HPLC using RP-18 column and found to be more than 95% pure, which gave correct molecular ion on characterization of compounds by FAB-MS. 2.3. Lymphocyte proliferation assay Normal healthy volunteers showing delayed type hypersensitivity to PPD or tuberculosis patients were selected and PBMCs were separated from heparinized peripheral blood as described earlier . Cells were resuspended at a concentration of 0.5 106 cells/ml in complete RPMI-1640 and five well cultures were set up with 200 Al media containing 105 cells/well in 96 well round bottom plates. Opioid receptor antagonist naloxone was used at a concentration of 10 5 M. Peptidomimetic compound and PPD were added simultaneously to the culture in the beginning, or cells were preincubated with peptidomimetic compound (10 10 – 10 6 M) for 8 h at 37 jC, thereafter, washed thrice with phosphate buffer saline (PBS) and furthered
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757 Table 1 Effect of peptidomimetic compound on PPD stimulated lymphocyte proliferation and its reversal by naloxone Treatment
3 H-Thymidine incorporation (mean CPM F SD)
Control PPD Naloxone Peptidomimetic compound (10 6 M) Peptidomimetic compound (10 6 M) + PPD Peptidomimetic compound (10 6 M) + PPD + naloxone Peptidomimetic compound (10 8 M) Peptidomimetic compound (10 8 M) + PPD Peptidomimetic compound (10 8 M) + PPD + naloxone Peptidomimetic compound (10 10 M) Peptidomimetic compound (10 10 M) + PPD Peptidomimetic compound (10 10 M) + PPD + naloxone
4614 F 369 7932 F 951 4758 F 446 4783 F 523 5004 F 714** 7369 F 683# 4544 F 511 5469 F 300* 7043 F 372# 4511 F 757 5759 F 441* 6505 F 682
Naloxone and PPD were used at 10 5 M and 40 Ag/ml, respectively. Values represent mean CPM F SD. Represents statistical significance of inhibition of PPD stimulated proliferation by peptidomimetic compound as compared to PPD stimulated culture ( * p < 0.05; ** p < 0.01). PPD and peptidomimetic compound were added simultaneously to culture. Statistical significance of reversal of PPD stimulated peptidomimetic treated culture by naloxone (#p < 0.01).
cultured with PPD (40 Ag/ml). The culture was incubated at 37 jC for 5 days in 5% CO2 and 100% humidity. Thereafter, cells were pulsed with 0.5 ACi 3 H-thymidine/well (LCT 53, 6 Ci/mmol, BARC, Mumbai, India) and further cultured for 16 h. Cultures were harvested and 3H-thymidine uptake was measured in scintillation counter. The mean counts per minute (CPM) of five well cultures were calculated for each set of cultures. Results are presented as CPM F SD (standard deviation). 2.4. Stimulation and estimation of IL-10 and IFN-g In brief, PBMC suspension was cultured in RPMI1640 in the presence or absence of peptidomimetic compound (10 10 – 10 6 M) either alone or in combination with PPD (40 Ag/ml). Supernatant was har-
vested after 48 h and stored at 70 jC until tested. The quantity of IL-10 and IFN-g present in supernatants was estimated by ELISA. A standard curve was obtained by plotting known concentrations of IL-10 and IFN-g vs. absorbance. IL-10 and IFN-g concentrations in experimental samples were determined with standard curve drawn using SPSS 9.0 software. Results are presented as concentrations of cytokine in pg/ml. 2.5. Ribonuclease protection assay Total RNA was isolated from PBMCs using TriReagent. In vitro transcription was performed for preparation of [32P]-labeled anti-sense RNA probe, using multi probe RPA template set for human cytokines (hCK-1; Pharmingen) following manufacturer instructions. In brief, T7 RNA polymerase-directed synthesis of a high specific activity RNA probe was carried out using [a-32P] UTP, GACU nucleotide pool, DTT, 5 transcription buffer, and RPA template set followed by its purification using phenol and chloroform, and quantitation in liquid scintillation counter . Thereafter, probe set was hybridized in excess to target RNA in solution for 16 h at 56 jC, after which free probe and other single-stranded RNA was digested with RNAase. The remaining ‘‘RNAase-protected’’ hybridized products were purified as earlier and resolved on 5% sequencing denaturing urea-PAGE. A dilution of the
Table 2 Effect of peptidomimetic compound on PPD stimulated lymphocyte proliferation Treatments
Untreated cells Peptidomimetic compound (10 6 M) Peptidomimetic compound (10 8 M) Peptidomimetic compound (10 10 M)
3 H-Thymidine incorporation (mean CPM F SD)
PPD (40 mg/ml)
3260 F 2936 4155 F 819
6966 F 259 4785 F 390 ( p < 0.001) 5621 F 379 ( p < 0.01) 7290 F 468
2874 F 372 3560 F 399
The PBMCs of tuberculosis patients were cultured with or without peptidomimetic compound for 8 h at 37 jC. Thereafter, cells were washed thrice with PBS (pH 7.2) and then cultured again in the presence or absence of PPD (40 Ag/ml) for 5 days at 37 jC. The figure in brackets represent statistical significance of inhibition of PPD stimulated proliferation by peptidomimetic compound as compared to only PPD treated cells.
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757
probe set in loading buffer (4000 CPM/lane) was also loaded to serve as size markers. The gel was run at 50 W constant power until the leading edge of the front dye reached 30 cm. Detection of specific bands of protected mRNA of various cytokines were carried out by autoradiography and quantitated by spot densitometry using ALPHA Imager v. 5.5 software. 2.6. Statistical analysis Six donors have been used in each experiment. Data are expressed as mean F standard deviation of replicates. Significance differences between means were determined by Student’s t-test. The significance level was set at p < 0.05.
3. Results 3.1. Effect of peptidomimetic compounds on PPD stimulated lymphocyte proliferation PBMCs of PPD positive healthy volunteers/tuberculosis patients (0.5 106 cells/ml) were cultured for 5 days in the presence of peptidomimetic compound (10 10 –10 6 M) either alone or with PPD (40 Ag/ ml). Cell proliferation was determined by 3H-thymidine uptake as described above and data is presented in Tables 1 and 2. Peptidomimetic compound suppressed the PPD stimulated lymphocyte proliferation significantly in dose dependent manner and optimal inhibition was observed at 10 6 M concentration (Tables 1 and 2). Table 3 Effect of peptidomimetic compound on PPD stimulated IL-10 and IFN-g production S. No
1 2 3
Control 514.72 F 50 PPD 4868.9 F 42 PPD + peptidomimetic 4652 F 86 compound (10 6 M) PPD + peptidomimetic 3869.9 F 130 compound (10 8 M) (p < 0.05) PPD + peptidomimetic 3520.68 F 574 compound (10 10 M)
IFN-g (pg/ml) 2.4 F 5.4 322.06 F 1.4 355.8 F 9.8 316.1 F 4.2 290.28 F 4.2
The effect of peptidomimetic compound (10 6 – 10 10 M) on PPD (40 Ag/ml) stimulated IL-10 and IFN-g production from PBMCs (48 h) as estimated by ELISA. The figure in bracket represents statistical significance of inhibition of IL-10 by peptidomimetic compound.
Fig. 1. Autoradiograph showing mRNA expression (48 h) of cytokines by ribonuclease protection assay. Cells only (lane 3); cells stimulated with PPD (40 Ag/ml, lane 4); cells stimulated with PPD (40 Ag/ml) and peptidomimetic compound (10 8 M, lane 5). Lane 1 is RNA probe (4000 CPM) alone and lane 2 is human control RNA supplied with kit to serve as integrity controls for sample RNA.
Inhibitory effect of this compound on PPD stimulated proliferation was investigated under two different conditions. First, when this compound was added simultaneously with PPD (40 Ag/ml) at the beginning of culture (Table 1), and second, when cells were pretreated with peptidomimetic compound for 8 h and washed before addition of PPD (40 Ag/ml) (Table 2). Naloxone (10 5 M), opioid receptor antagonist, significantly reversed the inhibitory effect of this compound on PPD stimulated lymphocyte proliferation (Table 1). 3.2. Effect of peptidomimetic compound on PPD induced IL-10 and IFN-g production in PBMC culture Human PBMCs were cultured for 48 h in RPMI1640 media with PPD (40 Ag/ml) either in the presence or absence of peptidomimetic compound. Quantity of IL-10 and IFN-g produced was measured in supernatant by ELISA. This compound at 10 10 – 10 6 M concentrations inhibited PPD stimulated IL-10 production (Table 3). However, any concentration of this compound did not show any effect on PPD stimulated IFN-g production.
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757
Fig. 2. Histograms showing the densitometric analysis of various cytokine mRNA in PPD stimulated human PBMCs in the absence or presence of peptidomimetic compound after correcting for housekeeping gene L32 expression, as detected by ribonuclease protection assay. Results are expressed as a ratio integrated density mRNA of cytokines and L32.
3.3. Effect of peptidomimetic compound on PPD stimulated mRNA of Th1 and Th2 cytokines Total RNA was isolated from PBMCs stimulated with PPD (40 Ag/ml) either alone or in combination with peptidomimetic compound (10 8 M) for 48 h as described earlier. The mRNA of various cytokines in 5 Ag of total RNA samples of each treatment group as indicated in figure was detected by ribonuclease protection assay (Figs. 1 and 2). The mRNA of IL2, IL-9, IL-10, IL-15 and IFN-g were observed in PPD stimulated PBMCs. This compound moderately suppressed PPD stimulated mRNA of IL-10 and IL-9 but did not show any effect on PPD induced IL-2, IL15 and IFN-g mRNA (Figs. 1 and 2). This finding is in conformity with results of ELISA for IL-10 and IFN-g presented in Table 3.
4. Discussion Several investigators have shown the effect of opioids on mitogen and antigen activated T cell proliferation in vitro through opioid receptors [29,30]. Both T- and B-lymphocytes have been shown to possess opioid receptors in functional state. We have recently shown inhibition of two-way mixed lymphocyte reactions by receptor selective peptidomimetic opioid compounds in vitro . In sequel to
our above observations, we further evaluated the immunomodulatory effect of peptidomimetic compound (10 10 –10 6 M) on antigen (PPD) stimulated responses in vitro. We found that our peptidomimetic compound significantly suppressed PPD stimulated lymphocyte proliferation over a wide range of concentrations (10 10 –10 6 M). Naloxone, opioid receptor antagonist, significantly reversed the inhibition of PPD stimulated lymphocyte proliferation by this peptidomimetic compound suggesting that this compound acts through opioid receptors present on cells (Table 1). This compound also inhibited antigen specific lymphocyte proliferation when cells were exposed for only 8 h, and washed cells were cultured with antigen (Table 2). Recently, we have observed that naloxone also reverses inhibition of mixed lymphocyte reaction by this peptidomimetic compound . This observation also suggests that this compound inhibit cell proliferation through opioid receptors. Furthermore, we have demonstrated earlier using h-funaltrexamine (h-FNA) that this compound acts specifically through A-opioid receptor. In addition, this peptidomimetic compound significantly inhibited IL-10 production stimulated by PPD in human PBMCs culture (48 h). Various opioid receptor selective agonists have been shown to modulate the antigen induced Th2 cytokines [31 – 33]. Lower concentrations of our compound were found
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757
to be more inhibitory. Such observations have been made by other investigators in case of other opioid peptides for different parameters of immune response [34 – 36]. Results of ELISA supported the observations with ribonuclease protection assay where this compound was found to inhibit message of IL-10. The modulating effect of peptidomimetic compound on IL-10 and IL-9 may have effect on host – defense system in various pathophysiologic conditions. As observed with ELISA and ribonuclease protection assay, this compound did not show any effect on PPD stimulated Th1 cytokine (IFN-g) production in culture supernatant. Since this compound inhibits cell proliferation, we expected that it would inhibit IL-2 production also. Ribonuclease protection assay did not show inhibition of IL-2 message. Thus opioid receptor selective compound may modulate disease in favor of host in Th2 biased infectious diseases like tuberculosis. Our present study suggests modulation of T cell mediated immune responses by receptor selective opioid compounds. Such compound may find use in the development of therapeutic strategies in immune mediated disorders.
We are thankful to Dr. C.M. Gupta and Prof. S.S. Agarwal for their interest in this study. Financial assistance from Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. P.N. is a senior research fellow of CSIR. The laboratory infrastructure was provided by a JICA grant-in-aid to the SGPGI project.
References  Blalock JE. A molecular basis for bi-directional communication between the immune and neuroendocrine systems. Physiol Rev 1989;69:1 – 32.  Morley JE, Kay NE, Solomon GF, Plotnikoff NP. Neuropeptides: conductors of the immune orchestra. Life Sci 1987;41: 527 – 44.  Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982;65:133 – 55.  Wybran J, Appelboom T, Famaey JP, Govaerts A. Suggestive evidence for receptors for morphine and methionine-enkepha-
lin on normal human blood T lymphocytes. J Immunol 1979; 123:1068 – 70. Plotnikoff NP. The Ying-Yang hypothesis of opioid peptide immunomodulation. Pshychopharmacol Bull 1985;21:489. Sibinga NE, Goldstein A. Opioid peptides and opioid receptors in cells of the immune system. Annu Rev Immunol 1988; 6:219 – 49. Hughes J, Smith TW, Kosterlitz HW, Fothergill LA, Morgan BA. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 1975;258: 577 – 80. Shavit Y, Depaulis A, Martin FC, Terman GW, Pechnick RN, et al. Involvement of brain opiate receptors in the immunesuppressive effect of morphine. Proc Natl Acad Sci U S A 1986;83:7114 – 7. Lord JA, Waterfield AA, Hughes J, Kosterlitz HW. Endogenous opioid peptides: multiple agonists and receptors. Nature 1977;267:495 – 9. Plotnikoff NP, Murgo AJ, Miller GC, Corder CN, Faith RP. Enkephalins: immunomodulators. Fed Proc 1985;44:118 – 22. Mandler RN, Biddison WE, Mandler R, Serrate SA. h-Endorphin augments the cytolytic activity and interferon production of natural killer cells. J Immunol 1986;136:934 – 9. Carr DJ, Bost KL, Blalock JE. An antibody to a peptide specified by an RNA that is complementary to g-endorphin mRNA recognizes an opiate receptor. J Neuroimmunol 1986;12:329 – 37. Carr DJ, Blalock JE, Bost KL. Monoclonal antibody against a peptide specified by [Met]-enkephalin complementary RNA recognizes the y-class opioid receptor. Immunol Lett 1989; 20:181 – 6. Dhar MM. Enkephalins and immunomodulation. Ind J Chem 1991;30B:278 – 80. Holt V. Opioid peptide processing and receptor selectivity. Annu Rev Pharmacol Toxicol 1986;26:59 – 77. Gilman SC, Schwartz JM, Milner RJ, Bloom FE, Feldlman JD. h-endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci U S A 1982;79:4226 – 30. Singh VK, Bajpai K, Biswas S, Haq W, Khan MY, et al. Molecular biology of opioid receptors: recent advances. Neuroimmunomodulation 1997;4:285 – 97. Mazumder S, Nath I, Dhar MM. Immunomodulation of human T cell responses with receptor selective enkephalins. Immunol Lett 1993;35:33 – 8. Singh VK, Bajpai K, Biswas S, Mathur KB, Haq W, et al. Met-enkephalin and its analogs as immunomodulators. Anim Biol 1998;7:45 – 58. Bajpai K, Singh VK, Agarwal SS, Dhawan VC, Naqvi T, et al. Immunomodulatory activity of met-enkephalin and its two potent analogs. Int J Immunopharmacol 1995;17:207 – 12. Bajpai K, Singh VK, Dhawan VC, Haq W, Mathur KB, et al. Immunomodulation by two potent analogs of met-enkephalin. Immunopharmacology 1997;35:213 – 20. Singh VK, Bajpai K, Narayan P, Yadav VS, Dhawan VC, et al. y-Opioid receptor antagonist inhibits immunomodulation by met-enkephalin analogs. Neuroimmunomodulation 1999;6: 355 – 60.
P. Narayan et al. / International Immunopharmacology 2 (2002) 751–757  Gorin FA, Balasubramanium TM, Cicero TJ, Schwietzer J, Marshall GR. Novel analogues of enkephalin: identification of functional groups required for biological activity. J Med Chem 1980;23:1113 – 22.  Morley JS. Structure – activity relationship of enkephalin like peptides. Annu Rev Pharmacol Toxicol 1980;20:81 – 110.  Morley JS. Chemistry of opioid peptides. Br Med Bull 1983; 39:5 – 10.  Roques BP, Fournie-Zaluski MC, Gacel G, David M, Meunier JC, et al. Rational design and biological properties of highly specific A and y-opioid peptides. Adv Biochem Psychopharmacol 1982;33:321 – 31.  Narayan P, Singh VK, Agarwal SS, Tandon R, Haq W, et al. Immunomodulation by opioid peptidomimetic compound. Neuroimmunomodulation 2001;9:134 – 40.  Gilman M. Ribonuclease protection assay. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Siedman JG, Smith JA, Stuhl K, editors. Current Protocols in Molecular Biology, vol. 1. New York: John Wiley and Sons, Inc., 1993. p. 4.7.1. – 8.  Kowalski J. Immunomodulatory action of class A-, y- and nopioid receptor agonists in mice. Neuropeptides 1998;32: 301 – 6.  Heagy W, Teng E, Lopez P, Finberg RW. Enkephalin receptor
and receptor-mediated signal transduction in cultured human lymphocytes. Cell Immunol 1999;191:34 – 48. Pacifici R, di Carlo S, Bacosi A, Pichini S, Zuccaro P. Pharmacokinetics and cytokine production in heroin and morphinetreated mice. Int J Immunopharmacol 2000;22:603 – 14. Fimiani C, Magazine H, Welters ID, Bilfinger TV, Salsano F, et al. Antagonism of LPS and IFN-g induced iNOS statement in human atrial endothelia by morphine, anandamide, and estrogen. Acta Pharmacol Sin 2000;21:405 – 9. Roy S, Balasubramanian S, Sumandeep S, Charboneau R, Wang J, et al. Morphine directs T cells toward T(H2) differentiation. Surgery 2001;130:304 – 9. Singh S, Singh PP, Dhawan VC, Haq W, Mathur KB, et al. Lymphokines production by concanavalin A-stimulated mouse splenocytes: modulation by Met-enkephalin and a related peptide. Immunopharmacology 1994;27:245 – 51. Brown SL, Van Epps DE. Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin. J Immunol 1985;134:3384 – 90. Roscetti G, Ausiello CM, Palma C, Gulla P, Roda LG. Enkephalin activity on antigen-induced proliferation of human peripheral blood mononucleate cells. Int J Immunopharmacol 1988;10:819 – 23.