lmmunopharmacology Immunopharmacology 29 (1995) 175-183

Morphine inhibits the development of allogeneic immune responses in mouse lymph node

Rita Maity *, Ranadev Mukherjee, Phil Skolnick Laborarory of Neuroscience. National Institute of Diabetes. Digestive and Kidney Diseases, National Institutes yf He&h, Bethesda,

MD 20892. USA

(Received 7 February 1994; accepted 28 September 1994)

Abstract

Morphine and related opiates are often administered to relieve post-operative and chronic pain following transplantation surgery. Opiates have been shown to suppress a variety of immune parameters in both animal models and man. In the present study, we investigated whether morphine affects allogeneic immune responses by injecting C57BL/6 mice in the footpad with allogeneic spleen cells and examining changes in the draining popliteal lymph node (PLN). Morphine (administered as subcutaneous implants) had profound inhibitory effects on the development of alloreactivity manifested as a suppression of: (1) lymph node hyperplasia, (2) mixed lymphocyte reactivity (MLR) in PLN cells and (3) the num- ber of CD4 t and Thy 1.2 lyrnphoid subsets. These inhibitory effects of morphine were abolished or dramatically reduced by co-administration of the opiate antagonist, naltrexone, indicating that suppression of allo-sensitization was opiate receptor mediated. In toto, these findings demonstrate that morphine administration interferes with the development of allogeneic immune response in mouse lymph node through an opiate receptor mediated mechanism.

Kqvwovds: Morphine; Naltrexone; Opiate receptor; Popliteal lymph node; Alloantigen; Tissue rejection

1. Introduction

Rejection of transplants between genetically dis- similar (allogeneic) subjects has been attributed in large part to alloantigens encoded by the major

* Corresponding author. Present address: Developmental Endo- crinology Branch, Bldg. 10, Rm lOD-15. National Institute of Child Health and Development, National Institutes of Health, Bethesda. MD 20892, USA. Tel: (301) 496-4686. Fax: (301) 402-0574. Abbreviations: HPA, hypothalamic-pituitary-adrenal axis; mAb, monoclonal antibodies; MLR, Mixed lymphocyte reaction; NK, natural killer cell; NIDA, National lnstitutc of Drug Abuse; PLN, popliteal lymph node; DP, double positive (CD4 + /CDS + ) cells.

Elsevier Science B.V. SSDI 0162-3109(94)0005X-1

histocompatibility complex (MHC) (Krensky et al., 1990; Clayberger et al., 1987; Lafferty et al., 1983; Lechler et al., 1983). Introduction of a graft initiates specific T lymphocyte responses against the foreign histocompatibility antigens of the donor (Mason and Morris, 1986; Swain, 1983; Steinmuller, 1985). Helper/inducer T lymphocytes (CD4 + ) recognize foreign MHC class II antigens and are activated to proliferate, differentiate, and secrete lymphokines (Dinarello and Mier, 1987). These events induce fur- ther expression of MHC class II antigens on grafted tissues, stimulate B lymphocytes to produce anti- bodies against determinants on the transplant (Auchincloss et al., 1988; Cobbold et al., 1986a) and

facilitate the development of effector functions in T cells such as cytotoxicity (Tyler et al., 1984; Mason et al., 1984; Whelahan and McKenzie, 1987; Cob- bold and Waldmann, 1986b). Cytotoxic T lympho- cytes (CD8 + ), usually specific for MHC Class I antigens, can directly damage the graft (Woodcock et al., 1986; Rosenberg et al., 1987; Madsen et al., 1987).

Opiates suppress a variety of immune functions in both experimental animals and man (Brown et al., 1974; Wybran et al., 1979; Bryant et al., 1988b; Weber and Pert, 1989) including the number and proliferative function of different subsets of T lym- phocytes as well as B lymphocytes (McDonough et al., 1980; Arora et al., 1990; Sei et al., 1991), mi- togen stimulated T and B cell responses (Bryant et al., 1987; Bryant et al., 1988a), NK activity (Shavit, 1984) antigen specific antibody production (Lefkowitz and Chiang, 1975; Weber et al., 1987), Con-A stimulated calcium mobilization in both T and B cells (Sei et al., 1991), and atrophy of im- munogenic organs like thymus and spleen (Bryant et al., 1987; Arora et al., 1990; Sei et al., 1991). The objective of the present study was to examine the effects of morphine on allo-sensitization after in vivo priming with alloantigens. We have utilized an in vivo assay in the mouse that allows the quantitative assessment of several early events in the activation of lymphocytes in response to alloantigen stimula- tion (Fanslow et al., 1990; Kroczek et al., 1987a,b; Pereira et al., 1990). We now report that adminis- tration of morphine as a subcutaneous implant pro- duces a robust suppression of alloantigen-induced reactivity in lymph node. These effects were mani- fested as: (1) a significant reduction in alloantigen induced increase in lymph node size and cellularity (lymphocyte population); (2) a decrease in CD4 + and Thy 1.2 lymphoid subsets and (3) a decrease in the functional status of T lymphocytes measured as a reduction in MLR.

2. Materials and Methods

2.1. Animals

BALB/c(H-~~) and C57BL/6J(H-2b) male mice (6-8 weeks of age) were obtained from the Jackson

laboratory, Bar Harbor, ME. Animals were housed in 32 x 25 x 15 cm transparent plastic cages with woodchip bedding material and maintained in a tem- perature and humidity controlled facility on a 0600- 1800 lights on schedule. Chow and water were freely available. All animals were treated according to NIH/AALAC guidelines.

2.2. Experimental design

The response to allogeneic cells in vivo was quan- tified by the popliteal lymph node (PLN) enlarge- ment assay. a measure of allograft transplant immu- nity (Fanslow et al., 1990; Kroczek et al., 1987a,b; Pereira et al., 1990). In brief, spleens were used as a source of injectable allogeneic cells (H-2d). They were minced and filtered through a nylon mesh, treated with NH,Cl solution (ACK lysing buffer, NIH Media Unit, Bethesda, MD) to lyse red blood cells, and washed twice in Hanks balanced salt SO-

lution (HB SS). The pellets were then resuspended in HBSS solution to give 10’ viable cells in 50 ~1. The irradiated (2000 rads) allogeneic cell suspension was then injected intra-cutaneously into the rear foot- pads of recipient (H-2b) mice (lo7 cells/footpad). Seven days after antigen administration, the mice were killed by cervical dislocation and both PLNs were removed and weighed. The PLNs were then mechanically dissociated and eluted through a nylon mesh to make a single cell suspension. They were washed twice in HBBS + 57; fetal bovine serum (FBS) and viable cell counts obtained by trypan blue exclusion method. Suspensions were adjusted to 2 x lo6 cells/ml. They were ultimately resus- pended in sterile RPM1 1640 medium (GIBCO) supplemented with 5% FBS, 10 mM Hepes buffer, 100 IU/ml penicillin and 100 pg/ml strepto- mycin (GIBCO), 1 Y0 sodium pyruvate and non es- sential amino acids, (M.A. Bioproducts, Walkers- ville, MD) and 5 x 10 - ’ M 2 mercaptoethanol (GIBCO).

2.3. Drug administration

Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were implanted subcutaneously with pellets containing either morphine (75 mg) or micro- crystalline cellulose (placebo), naltrexone (10 mg) or

a combination of pellets. The pellets were replaced after 72 h. Prior to pellet implantation, the ani- mals were anesthetized with pentobarbital solution (0.01 mligm body weight; 5 mglml) injected IP. Morphine, naltrexone and placebo pellets were ob- tained from NIDA (Rockville, MD). Seven days after pellet implantation, the morphine treated mice exhibited withdrawal signs consisting of hyperactiv- ity, vocalization, jumping and wet dog shakes. These behaviors were not present in placebo pelleted groups but much less severe than animals subjected to acute withdrawal by naltrexone.

2.4. Flow cytometry

A week after pellet implantation, single cell sus- pensions from PLN were prepared and stained with monoclonal antibodies (mAB) specific for cell sur- face markers (Thy 1.2, CD4 + , CDS + ) as described previously (Sei et al., 1991). In some experiments, the PLNs were pooled to obtain appropriate cell suspensions for analysis and culture. Cells were analysed on a FACScan (Becton Dickinson, Moun- tain View, CA) equipped with argon and krypton lasers.

2.5. Mixed (ywphocyte reaction (MLR)

MLR assay was performed in 96 well flat bottom microtiter plates (COSTAR, Carbundy, MA) using 2 x 105cells/well responder PLN cells. Cells were cultured in the presence or absence of irradiated (3000 rads) allogeneic male BALB/C stimulator spleen cells (5 x 105/well) which had been freed of erythrocytes by treatment with NH,Cl (ACK lysing buffer, NIH Media Unit, Bethesda, MD). The cul- ture plates were incubated at 37°C in 95% air, 59<, Co, for 96 h. Cultures were pulsed with 1 pCi/well of [ 3H] thymidine ([ 3H]Tdr. DuPont-New England Nuclear, Boston, MA) for the final 18 h. The cells were harvested with a PHD cell harvester (Cam- bridge Technology, Cambridge, MA) and [‘H]Tdr uptake measured in a liquid scintillation counter (Beckman LS 5801). MLR ratio (stimulation index) was defined as the quotient of [ 3H] incorporated in the presence or absence (basal uptake) of allogeneic stimulator cells.

2.6. Monoclonal antibodies

Anti Thy 1.2, phycoerythrin-conjugated (PE) anti CD4 (L3T4) mAb and fluorescein isothiocyanate (FITC) conjugated anti CD8 (Ly2) mAb were ob- tained from Becton Dickinson (Mountain View, CA).

3. Results

3.1. Efllicts of morphine administration on the PLN response to allogeneic cells

Injection of allogeneic cells produced a statisti- cally significant increase in both the weight (43 7; ; ~~0.05) and number ofviable cells (38%; ~~0.05) in PLN (Fig. 1). The effects of morphine implanta- tion were examined at the peak allogeneic response, 7 days after injection (data not shown). Morphine produced a robust inhibition of the allogeneic re- sponse with respect to both PLN size (60”/, reduc- tion; p< 0.05) and cell number (55% reduction; p < 0.05). The inhibitory effects of morphine on PLN size were completely reversed by simultaneous im- plantation of the opiate antagonist naltrexone (p < 0.05) while its inhibitory effects on cell number were significantly, albeit incompletely (to 859, of placebo values; p < 0.05 compared to morphine) re- versed (Fig. 1). Morphine by itself also decreases lymph node weight (82%; p< 0.05) and viable cell count (73 y< ; p < 0.05) which is partially antago- nized by naltrexone (209;, p< 0.05). Naltrexone by itself has no effect on any of the parameters ex- amined.

3.2. Efects of morphine on the suface phenotype of T cell subsets

Seven days following injection of alloantigen and pellet implantation, PLN cells were harvested and stained with PE/FITC conjugated mAB specific for cell surface markers. PLN cells were pooled from the appropriate experimental animals and labelled with antibodies for FACScan analysis. A represent- ative FACscan analysis is presented in Fig. 2, and the results of five experiments summarized in Table 1.

R. A-laity et al. j Immun(~p~ar~r~uc~l~gy 29 (1995) 175-183

I 1 A

** ,I I

MORPHINE MOR +I

**

I ‘JALTRX

B

1 PL 1 ACEBO

**

I MORPHINE

**

;I R+NAL ,TRX

Fig. 1. Effects of morphine on alloantigen induced increases in PLN size (A) and cell number (B). Mice were subcutaneously implanted with either morphine alone, morphine and naltrcxone (Mor + Naltrx), or placebo pellets. They were then injected with allogeneic cell suspensions of 10’ cells/50 ~1 in both hind footpads (black bars). A set of control groups with the same subcutaneous implants of either morphine, morphine and naltrexone or placebo pellets were taken in the absence of allogeneic injections or presence of syngeneic injections of the same concentrations as before (white bars). Seven days later, PLNs were removed and processed as described in Methods. Val- ues represent X k SEM of 8-9 animals. These experiments are representative, and were repeated > 5 times. Two way ANOVA with Students-Newman-Keuls post hoc comparison reveals difference in LN size and cell count in primed animals to be significant (*p < 0.05) compared to unprimed animals, whereas these parameters were significantly reduced (**p<O.O5) due to morphine. Naltrexone appears to block the effects of morphine on the primed animals (**p<O.Oj). Morphine itself has an inhibitory effect on the development of an immune response in the lymph node which is partially abrogated by naltrexone.

Allogeneic priming produced significant increases in both the percentage and number of THY 1.2 and CD4 + positive cells (Table 1) compared to unprimed mice. The percentage of Thy 1.2 and CD4 + cells increased by 55yj0 and 27% (p< 0.05) respectively compared to unprimed animals. In con- trast, the decrease in percentage and number of CD8 + T cells and double positive (CD4 + /CD8 + ) cells was not altered significantly by priming. Mor- phine implantation produced a sharp decrease in both the percentage reduction (487;) and number (73x), p< 0.05 respectively, of Thy 1.2 cells in primed animals. Morphine implantation produced a similar, dramatic reduction in CD4 + cells from primed animals. Thus, the percentage of CD4 + cells was reduced by 40% (~~0.05) while the number of cells was diminished by 68 y0 (p < 0.05). Morphine increases the CD8 + “/, in primed animals almost compensating for the decreased% due to priming).

Morphine also appears to increase double positive (CD4 + /CD8 +) T cell population in the lymph nodes of primed animals. But these values did not achieve statistical significance. Co-administration of naltrexone produced a partial blockade of the effects of morphine in primed animals (Table 1; Fig. 2). This opiate antagonist partially restored both the number and”/, of LN cell population to values that were comparable to those obtained in primed mice respectively (Table 1). The effect of morphine alone in unprimed LN cell population was by itself strik- ing - a significant decrease (69:~) in CD4 + y/, but 50:~ increase in immature DP population. There was slight change in Thy 1.2 and CDS + population but it was not significant. This effect was partially antagonized by naltrexone. Naltrexone by itself did not have significant effects on LN cell populations in either primed or unprimed animals.

R. Maity et al. / Immunophurnzacolog~ 29 (199.5) 175-183 179

2

I

I 3 4

-i4

a a 10’ lap

FL1 10*

Anti-CDB-FTC-

Fig. 2. Effects of morphine on allogen induced increase of single positive CD4’ lymphocytes in LN: FACscan analysis. Distribution of lymphocyte subpopulations was determined by analysis of CD4 + (ordinate) vs. CD8 + (abscissa) expression. A: alloprimed, B: alloprimed + morphine, C: alloprimed + morphine + naltrexone D: placebo (unprimed). This is a representative FACScan. The data are presented in Table 1. Animals were treated with allogenic cells and then surgically implanted with either morphine or placebo pellets. Cells were either stained with antibodies or assayed for MLR to determine the differential effect of surgical stress or vehicle on both groups. Surgical stress had no significant effect on these measures (data not shown).

3.3. Effects of morphine administration on functional activity of PLN cells from primed animals: mixed lym- phocyte reaction (MLR)

Allogeneic priming of PLN produced a 2.5fold increase in [3H]TdR incorporation into cells har- vested from PLN. The stimulation index was in- creased from 6.5 k 1.2 to 22.6 k 1.9 (p< 0.05). This

effect was abolished in primed animals that received morphine pellets (Table 2). [ 3H]TdR incorporation in primed animals receiving naltrexone and mor- phine pellets was not significantly different from primed animals receiving placebo implants but was significantly different from primed animals receiving morphine pellets (Table 2), indicating naltrexone partially blocks the effect of morphine in primed

180 R. M&y er al. / Inzmunopharmacoiog~ 29 (1 Y9CiJ 175-183

Table 1

The effect of morphine administration on the surface antigenic phenotypes of T cell subsets in PLN

Groups T lymphocyte phenotypes in LN

THY 1.2 CD4 + ;CD8 - CD8 + /CD4 - CD4 + CD8 +

0. _i io No.~ ‘;” No.~ “&” No.” “/b” No.~

Unprimed (20) 55* 1 11 49+ 1 10 25&2 5 2 2 0.03 0.4 Primed (30) 85k9 26* 62 f 2 19* 1522 4 2 f 0.05 0.6 Morphine (8) 4128 3* 15k 1 1* 22+4 2 4 * 0.001 0.3

M + N (10) 45k6 j** 2553 3** 24+6 2 3io.002 0.3

Unprimed (20) 55k 1 11 49* 1 10 25*2 5 220.03 0.4

Primed (30) 8529 26* 6222 19* 15*2 4 2 2 0.05 0.6

Primed (30) 85*9 26 62k2 19 1522 4 2+0.05 0.6

Primed + M (15) 44*8 7* 37*3 6* 26&3 4* 5 * 0.07 0.8

Primed + M + N (20) 6056 12** 52_+4 1 o** 2423 5 2_+0.002 0.4

Values represent mean + SEM of 5-6 animals/group. This representative experiment was replicated 5 times with similar results. Values for primed, morphine and morphine plus naltrexone treated mice were compared with those of unprimed mice; in the second series unprimed mice were compared to primed mice; and in the third block primed mice were compared to priming plus morphine or prim- ing plus morphine plus naltrexone (*p<O.O5). The reversal of the effect of morphine by naltrexone has been denoted by **pi 0.05) in the initial and last sets. Data were analysed by two way ANOVA with Student-Newman-Keuls posthoc comparisons. The number of cells (b) is calculated thus: percentage (a)/ 100 x total cellularity as expressed in the figures in parenthesis below each group. a The percentage this population contributes to the total LN population. ’ Number of cells (x 10’). M, morphine implants; N, naltrexone implants.

animals. No consistent drug or priming effects were observed on basal (unstimulated) [ 3H]TdR incor-

Table 2

The effect of morphine on MLR in primed PLN cells

Groups [ ‘H]TdR incorporation

@pm) Net incorp. (cpm)

Unstimulated Stimulated (Stim - Unstim)

Cnprimed 9682128 6195k1250 5227t693 Primed 7072237 15554+5141* 148472 1860* Morphine 922k99 1936?97* 1014*149* Primed + M 970 + 113 4X50&314** 3880+724** M+N 843 ?I8 33125439 2529+414 Primed + 999*450 13986+4258** 12987*755** M+N

Values represent mean + SEM of 5-6 experiments. * Primed or morphine vs. unprimed; p<O.O5. ** Primed + morphine vs. primed or primed + M + N: ~~0.05 as analysed by two-way ANOVA with Student-Newman-Keuls pos- thoc comparison. M, morphine; N, naltrexone implants,

poration. Morphine by itself produced a decrease in SI in basal unprimed animals (68%) (~~0.05) which was partially restored by coadministration of naltrexone (307;). Naltrexone by itself did not have any effect on this measure in primed or unprimed animals.

4. Discussion

Tissue transplantation between individuals with genetically diverse backgrounds initiates a sequence of specific immune responses (collectively referred to as rejection) resulting in destruction of the graft (Krensky et al., 1990; Clayberger et al., 1987; Lafferty et al., 1983; Lechler et al., 1983; Mason and Morris, 1986). Morphine is a potent analgesic that is widely used in clinical and surgical cases. This and related opiates (e.g. heroin) have well documented immunosuppressive properties (McDonough et al., 1980; Arora et al., 1990; Sei et al., 1991). The present experiments were designed to determine whether the

R. Mait) et al. : Immu~~opharmacology 29 (1995) 375-183 181

immunosuppressive properties of morphine could be used to modulate allogeneic immune responses in vivo. We employed a well characterized assay sys- tem for the assessment of lymphocyte proliferation in the draining popliteal lymph node (PLN) after injection of allogeneic cells in the footpads of mice.

The principal molecular targets of transplant re- jection are non-self allelic forms of class I and class II MHC molecules (Swain, 1983; Loveland et al., 1981; Sprent and Schaefer, 1985; Ascher et al., 1984). Since the reaction to foreign class I and II molecules can be analyzed using the MLR, we em- ployed this measure in the current studies. More- over, analysis of lymphocyte subpopulations was performed since foreign class I molecules generally stimulate alloreactive CD8 A cytotoxic T lymphoc- tyes (CTLs) whereas foreign class II molecules stim- ulate alloreactive CD4* helper T lymphocytes (Parnes, 1989), and graft rejection is generally con- sidered to be mediated by antibodies and CTLs (Tyler et al., 1984; Mason et al., 1984; LeFrancois and Bevan, 1984). Moreover, CD4+ helper T cells are often required to initiate the rejection phenom- ena (Mizuochi et al., 1985; McCarthy and Singer, 1986).

Using a regimen of morphine that was previously described to suppress a variety of immune functions (Arora et al., 1990; Sei et al., 1991) we observed a dramatic inhibition of alloantigen induced increases in PLN size and absolute cell number (Fig. 1) This was accompanied by significant reductions in alloantigen-stimulated increases in the percentage and number of lymphocytes bearing CD4 + and Thy 1.2 markers (Fig. 2; Table l), and an abolition of the increased MLR activity induced by allogeneic chal- lenge (Table 2). Morphine administered by itself in unprimed animals causes a suppression of immune parameters. and while similar effects have been documented (Falek et al., 1991; McDonough et al., 1980; Arora et al., 1990; Sei et al., 1991) in immuno- genic organs like spleen and thymus, to our knowl- edge, the effects in lymph node have not been pre- viously reported. While the molecular mechanisms responsible for these morphine-induced changes in alloreactivity are unknown, the ability of the opiate antagonist naltrexone to blunt or abolish the effects of morphine clearly indicates an opiate receptor- mediated action. These effects of morphine may be

directly mediated through opiate receptors present on lymphocytes (Madden et al., 1991; McDonough et al., 1990; Sibinga and Goldstein, 1988; Wybran et al., 1979; Mehrishi and Mills, 1983; Carr et al., 1989), or indirectly via opiate receptors that are widely distributed throughout the brain and immune tissues (Sibinga and Goldstein, 1988; Wybran et al., 1979; Mehrishi and Mills, 1983; Cat-r et al., 1989; Shavit et al., 1986). However, in vitro studies gen- erally indicate that opiates either stimulate or have no effect on immune function, in contrast to the effects observed in this and other in vivo studies (Wybran et al., 1979; Gilman et al., 1982; Johnson et al., 1982; Miller et al., 1984). Many of the immu- nosuppressive actions of this regimen of morphine have been attributed to a receptor mediated activa- tion of the HPA axis (George and Way, 1955) resulting in the release of immuno-suppressive glucocorticoids (Bryant, 1988~). The reduction in alloantigen induced increases in PLN size and cell number suggest changes in lymphocyte trafficking. Direct injection of allogeneic spleen cells into the footpads of mice led to the generation of antigen specific helper cells in the draining popliteal LN within 7 days (30) and morphine tends to inhibit this effect. Since these cells are believed to be important mediators of early graft rejection, morphine would be expected to delay the elimination of allogeneic cells, prolonging allograft survival in the host. It was observed that morphine given concurrently with allogen injection was a potent inhibitor of MLR ac- tivity, indicating it is an inhibitor of DNA synthesis, likely arresting cell growth at the Gl- > S interphase (Abbas et al., 1991). In toto, the present findings are consistent with the hypothesis that this widely used analgesic has the potential for use in allograft rejec- tion.

References

Abbas A, Lichtman A and Pober J. Molecular basis of T cell antigen recognition and activation. In: Wonsienicz M Ed. Cel- lular and Molecular Immunology. Philadelphia: Saunders, 1991; 166.

Arora PK, Fride E, Petit0 J, Waggie K, Skolnick P. Morphine induced immune alterations in vivo. Cell Immunol 1990; 126: 343.

Ascher NL, Hoffman R, Simmons R. Cellular basis of allograft rejection. Immunol Rev 1984: 77: 217.

.4uchincloss H Jr, Ghobrial RRM, Russell P, Winn HJ. Anti

182 R. Mait) et al. / Immunopharmacolog~ 29 11995J 175-183

L3T4 in viva prevents alloantibody formation after skin graft- ing without prolonging graft survival. Transplantation 1988; 45: 1118.

Brown SW, Stimmel B, Taub RN, Kochwa S, Rosenberg RE. Immunological dysfunction in heroin addicts. Arch Intern Med 1974; 134: 1001.

Bryant HU, Bernton EW, Holaday JW. Immunosuppressive ef- fects of chronic morphine treatment in mice. Life Sci 1987; 41: 1731.

Bryant HU, Yoburn BC, Inturrisi CE, Bernton EW, Holaday JW. Morphine induced immunosuppression is not related to serum morphine concentrations. Eur J Pharmacol 1988a; 149: 165.

Bryant HU, Bernton EW, Holaday JW. Morphine pellet induced immunosuppression in mice: temporal relationships. J Phar- macol Exp Ther 1988b; 245: 913.

Bryant HU, Bernton EW, Holaday JW. Morphine induced im- munouppression: Involvement of glucocorticoids and prolac- tin. Proceedings of the 50”’ Annual Scientific Meeting, the Committee on Problems of Drug Dependance, NIDA Res Monogr. 1988~.

Carr DJ, DeCosta B, Kim CH et al. Opioid receptors on cells of the immune system:evidence for delta and kappa classes. J Endocrinol 1989; 122: 161.

Clayberger C, Parham P, Rothbard J, Ludwig DS, Schoolnik GK, Krensky AM. HLA-A2 peptides can regulate cytolysis by human allogeneic T lymphocytes. Nature 1987; 330: 763.

Cobbold SP, Jayasuria A, Nash A, Prosper0 TD, Waldmann H. Therapy with monoclonal antibodies by elimination of T cell subsets in viva. Nature 1986a; 312: 548.

Cobbold S, Waldmann H. Skin allograft rejection by L3T4 + and Lyt-2 + T cell subsets. Transplantation 1986b; 41: 634.

Dinarello CA, Mier J. Lymphokines. N Engl J Med 1987; 317: 940.

Falek A, Donahoe RM, Madden JJ, Shafer DA. Opiates as immunosuppressive and genotoxic agents. Adv Exp Med Biol 1991; 288: 189-201.

Fanslow WC, Sims JE, Sassenfeld H, Morrissey PJ, Gillis S, Dower SK. Regulation of alloreactivity in vivo by a soluble form of the interleukin-1 receptor. Science 1990; 248: 739.

George R, Way E.L. Studies on the mechanism of pituitary- adrenal activation by morphine. Br J Pharcol 1955; 10: 260.

Gilman SC, Schwartz JM, Milner RJ, Bloom FE, Feldman JD. Beta endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci USA 1982; 79: 4226.

Johnson HM. Smith EM, Torres BA, Blalock JE. Neuroendo- crine hormone regulation of in vitro antibody production. Proc Nat1 Acad Sci USA 1982; 79: 4171.

Krensky AM, Weiss A, Crabtree G. Davis MM, and Parham P.T. Lymphocyte-antigen interactions in transplant rejection. The New Engl J of Med 1990; 322: 510.

Kroczek RA, Black CDV, Barbet J, Eddison LJ, Shevach EM. Induction of IL-2 receptor expression in vivo: response to al- logeneic cells. Transplantation 1987; 44: 547.

Kroczek RA, Black CDV, Barbet J, Shevach EM. Mechanism of action of cyclosporine A in vivo. I. Cy A fails to inhibit T lymphocyte activation in response to alloantigens. J Immunol 1987; 139: 3597.

Lafferty KJ, Prawse SJ, Simeonovic RJ, Warren HS. Immuno- biology of transplantation. Ann Rev Immunol 1983; 1: 143.

Lechler RI, Lombardi G, Batchelor JR, Reinsmoen N, Bach FH. The molecular basis of alloreactivity. Immunology Today 1990; 11: 83.

Lelkowitz SS, and Chiang CY. Effects of certain abused drugs on haemolysin forming cells. Life Sci 1975; 17: 1763.

LeFrancois L, Bevan MJ. A reexamination of the role of Ly-2 + T cells in murine skin graft rejection. J Exp Med 1984; 159: 57.

Loveland BE, Hogarth PM, Ceredig R, Mckenzie IFC. Cells mediating graft rejection in the mouse. I.Lyt-1 cells mediate skin graft rejection. J Exp Med 1981; 153: 1044.

Madden JJ, Falek A, Donahoe R, Ketelson D, Chappel CL. Opiate binding sites on cells of the immune system. NIDA Res Monogr 1991; 105: 103-108.

Madsen J, Peugh W, Wood K, Morris P. The effect of anti L3T4 monoclonal antibody treatmant on first set rejection of murine cardiac allografts. Transplantation 1987; 44: 849.

Mason DW, Dallman M, Arthur R, Morris P. Mechanisms of allograft rejection: The roles of cytotoxic T cells and delayed type hypersensitivity. Immunol Rev. 1984; 77: 177.

Mason DW, Morris P. Effector mechanisms in allograft rejection. Annu Rev Immunol 1986; 4: 119.

McCarthy SA. Singer A. Recognition of MHC Class I allo-determinants regulates the generation of MHC Class II specific CTL. J Immunol 1986; 137: 3087.

Mcdonough RJ, Madden JJ, Falek A et al. Alteration of T and null lymphocyte frequencies in the peripheral blood of human opiate addicts:in vivo evidence for opiate receptor sites on T lymphocytes. J Immunol 1980; 125: 1068.

Mehrishi JN, Mills IH. Opiate receptors on lymphocytes and platelets in man. Clin Immunol Immunopathol 1983; 27: 240.

Miller JC, Murgo AJ, Plotnikoff NP. Enkephalins enhancement of active T cell rosettes from normal volunteers. Clin Immunol Immunopathol 1984; 31: 132.

Mizuochi T, Golding H, Rosenberg AS, Glimcher LH, Malek TR, Singer A. Both L3T4 + and Ly-2 + helper T cells initiate cytotoxic T lymphocyte responses against allogeneic major histocompatibility antigens but not against trinitrophenyl modi- fied self. J Exp Med 1985; 162: 427.

Parnes JR. Molecular biology and function of CD4 and CDS. Adv Immunol 1989; 44: 265.

Pereira GMB. Miller J, Shevach E. Mechanism of action of Cyclosporine A in viva II. J Immunol 1990; 144: 2109.

Rosenberg A. Mizuochi T, Sharrow S, Singer A. Phenotype, specificity, and function of T cell subsets and T cell intreactions involved in skin allograft rejection. J Exp Med 1987; 165: 1296.

Sei Y, Yoshimoto K, McIntyre T, Skolnick P, Arora PK. Mor- phine induced thymic hypoplasia is glucocorticoid dependent. J Immunol 1991; 146: 194.

Sei Y, McIntyreT, Fride E, Yoshimoto K, Skolnick P, Arora PK. Inhibition of calcium mobilization is an early event in opiate- induced immunosuppression. FASEB J 1991; 5: 2194.

Shavit Y, Lewis JW, Terman GW, Gale RP, Liebeskind JC. Opioid peptides mediate the suppressive effect of stress on natural killer cell activity. Science 1984; 223: 188.

R. Maity et al. / It~~munopharnzacoiog~ 29 (1995) 175-183 183

Shavit Y, Depaulis A, Martin FC, et al. Involvement of brain Weber RJ, Ikejiri B, Rice KC, Pert A. Hagan AA. Opiate receptor opiate receptors in the immunesuppressive effect of morphine. mediated regulation of the immune response in vivo. Nat1 Inst Proc Nat1 acad Sci USA 1986; 83: 7114. Drug Abuse Res Monogr Ser 1987; 76: 341.

Sibinga NES, Goldstein A. Opioid peptides and opioid receptors Weber RJ, Pert A. The periaqueductal gray matter mediates opi- in cells of the immune system. Ann Rev Immunol 1988; 6: 219. ate induced immunosuppression. Science 1989; 245: 188.

Sprent J, Schaefer M. Properties of purified T cell subsets. I. In Whelahan J. McKenzie I. The role of T4 + and lyt-2 + cells in vitro responses to Class I and class II H-2 alloantigens. J Exp skin graft rejection in the mouse. Transplantation 1987; 44: Med 1985; 162: 2068. 273.

Steinmuller D. Which T cells mediate allograft rejection. Trans- plantation 1985; 40: 229.

Swain SL. T cell subsets and the recognition of MHC class. Immunol Rev 1983; 74: 129.

Tyler JD, Galli SJ, Snider M, Dvorak A, Steinmullcr D. Cloned ly-2 + cytolytic T lymphocytes destroy allogeneic tissue in vivo. J Exp Med 1984; 159: 234.

Woodcock J, Wofsy D, Eriksson E, Scott J, Seaman W. Rejec- tion of skin grafts and generation of cytotoxic T cells by mice depleted of L3T4 + cells. Transplantation 1986; 42:636.

Wybran J, Appelboom T, Famaey JP, Govaerts A. Suggestive evidence for receptors for morphine, and mcthionine-cnkepha- lin on normal human blood T lymphocytes. J Immunol 1979; 123: 1068.