gy 178 (2006) 62–75www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

Amphetamine triggers an increase in met-enkephalin simultaneouslyin brain areas and immune cells

María A. Assis a,1, César Collino b,c, María de L. Figuerola d,Claudia Sotomayor b, Liliana M. Cancela a,⁎

a Departamento de Farmacología, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,Ciudad Universitaria, X5000HUA, Córdoba, Argentina

b Departamento de Bioquímica Clínica, CIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,Ciudad Universitaria, X5000HUA, Córdoba, Argentina

c Centro de Química Aplicada, CEQUIMAP, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,Ciudad Universitaria, X5000HUA, Córdoba, Argentina

d Centro de Investigaciones Endocrinólogicas (CEDIE), CONICET, Hospital de Niños Ricardo Gutiérrez,Gallo 1360, 1425, Buenos Aires, Argentina

Received 21 October 2005; received in revised form 16 May 2006; accepted 17 May 2006

Abstract

We analyzed effects of amphetamine on proenkephalin-derived peptides in brain areas and immune cells in rats. Acute, as well as arepeated amphetamine treatment, decreased the concanavalin-A-induced lymphocyte proliferation, concomitantly with an increase of freemet-enkephalin in nucleus accumbens, prefrontal cortex, spleen, thymus and splenic macrophages. Proenkephalin protein increased inprefrontal cortex, thymus (32 kDa isoform), nucleus accumbens and spleen (44 kDa isoform), while proenkephalin mRNA levels decreasedin brain stem. The influence of met-ENK in key brain areas for sensitization and in immune organs is consistent with the idea that changes onmet-ENK could underlie amphetamine's effects on brain and IS.© 2006 Elsevier B.V. All rights reserved.

Keywords: Psychostimulants; Proenkephaline; Sensitization; T-cell proliferation; Rat

1. Introduction

Drug addiction is a devastating illness with deleteriousconsequences not only on behavior, but also on immune

Abbreviations: IS, immune system; DA, dopamine; NAc, nucleusaccumbens; PfC, prefrontal cortex; ENK, enkephalin; AMPH, amphet-amine; Con A, concanavalin-A; VEH, vehicle; ZT, zeitgeber; RIA,radioimmunoassay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;FITC, fluorescein isothiocyanate; MoAb, monoclonal antibody; PE, R-phycoerythrin; CREB, cAMP-response element binding protein; AP-1,activator protein-1.⁎ Corresponding author. Tel.: +54 351 4344974x161; fax: +54 351

4334420.E-mail address: lcancela@fcq.unc.edu.ar (L.M. Cancela).

1 Fellowship from CONICET.

0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2006.05.009

system (IS) functions, rendering the individuals morevulnerable to infectious diseases. It is currently known thatdrugs of abuse clearly perturb immune functions as do stress,mood and emotion (Woiciechowsky et al., 1999; Pruett,2001; Galinowski et al., 1992; Baldwing et al., 1998; Padgettand Glaser, 2003). Furthermore, the interchangeabilitybetween psychostimulant drugs and stress observed atbehavioral level, showing long-lasting changes in dopami-nergic neurons and in the pituitary functions (Diaz-Otañez etal., 1997; Knych and Eisenberg, 1979; Saal et al., 2003), wasalso found in the IS (Basso et al., 1999).

Sensitization is a well known adaptive process which hasbeen associated to the long-lasting behavioral consequencesfollowing exposure to psychostimulants (Robinson andBerridge, 2000; Vezina et al., 2002). Several of the changes

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that underlie this process occur in the mesocorticolimbicdopamine (DA) systems, of which the ventral tegmentalarea, nucleus accumbens (NAc) and prefrontal cortex (PfC)are the main components. However, enhanced dopaminer-gic transmission is only one component of the motivecircuit, which mediates behavioral sensitization to psychos-timulants. Other neurotransmitter systems, such as enke-phalins (ENKs) and glutamate, are also among the mostimportant neurotransmitters in the mesocorticolimbic sys-tems associated with the development and/or expression ofsensitization (Kalivas and Stewart, 1991; Pierce andKalivas, 1997a). Notwithstanding, no evidence has so farbeen provided regarding possible psychostimulant-inducedlong-lasting changes in the ENK levels in these brain areas.

It has been shown that the administration of centralnervous system (CNS) acting drugs into the lateralventricle of the brain, induced a similar effect on the ISas that observed following the systemic infusion of thedrug (Pellegrino and Bayer, 1998a, 2000) suggesting that acentral component could be involved. Related to this, itwas found that brain DA and opioid systems have beeninvolved in the impairment of IS functions followingpsychostimulants and/or stress (Basso et al., 1999; Saraviaet al., 1998; Kubera et al., 2002; Jankovic and Radulovic,1992). However, it is also possible that autocrine andparacrine actions of opioid peptides and DA produced byimmune cells may be responsible for the effects of drugabuse on IS (Gordon and Barnes, 2003; Pellegrino andBayer, 1998b).

ProENK mRNA and proENK derived peptides weredetected in immune cells of bone marrow, thymus, spleenand alveolar macrophages (Vindrola et al., 1990; Saravia etal., 1993; Linner et al., 1995, 1991; Padrós et al., 1989;Kuis et al., 1991; Roth et al., 1989; Rittner et al., 2001).However, the myeloid lineage cells are the only immunecells that contain the prohormone converting enzymes PC1and/or PC2, which are involved in the post-translationalprocessing of proENK to produce the opioid peptides (Rothet al., 1989; Vindrola and Lindberg, 1992; Mathis andLindberg, 1992). Because of this, only macrophages andneutrophils are able to synthesize opioid peptides derivedfrom proENK (i.e. met-ENK) (Saravia et al., 1998; Kuis etal., 1991). In agreement with this, it was shown that T-lymphocytes release cryptic met-ENK-containing peptidesbut they do not have opioid activity (Roth et al., 1989;Padrós et al., 1995). On the other hand, several physiolog-ical studies have demonstrated the immunosuppressiveeffect of opioid peptides (Jankovic and Radulovic, 1992).Interestingly, a stressful stimulus inducing immunosuppres-sion modified proENK-derived peptide levels and theirrelease from cells of peripheral immune tissues (Saravia etal., 1998) as it did in CNS (Borsook et al., 1994; Lightmanand Young, 1987). In addition to these neuropeptides, thecatecholamines, including DA, are also present in lympho-cytes, macrophages and neutrophils, which also express thetransporters, receptors and synthesis enzymes for these

neurotransmitters (Bergquist et al., 1994; Amenta et al.,2001; McKenna et al., 2002; Cosentino et al., 2002).

It has been shown that long-lasting behavioral sensitiza-tion is induced following either a single dose or a chronicamphetamine (AMPH) treatment (Vanderschuren and Kali-vas, 2000). In this work, we showed that a single dose ofAMPH is also able to induce effects on IS, which wereobservable after 4 days, as in the case of a repeated regimewith the drug. At the same time of observation, it could beseen that both acute and repeated AMPH treatments hadinduced a simultaneous increase of met-ENK in IS, as well asin CNS. We also found that AMPH was a sufficient stimulusfor splenic T-lymphocytes to produce almost as many crypticproENK-derived peptides as concanavalin-A (Con A).Interestingly, it was shown that splenic macrophages werethe cells that synthesized elevated amounts of met-ENKfollowing the drug. All these data suggest that the activationof enkephalinergic systems in CNS and IS may be involvedin the AMPH's effects on both systems. Furthermore,changes in the proENK-derived peptide levels fromperipheral immune cells might be an index reflecting anopioid central effect of AMPH.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (250–330 g) from the Facultad deCiencias Veterinarias of the Universidad Nacional de LaPlata (Buenos Aires, Argentina) were maintained at 20–24 °C under a 12 h light–dark cycle (lights on at 07:00 a.m.)with free access to food and water. Rats were collectivelyhoused in cages in the experimental room for at least 7 daysbefore starting the experiments. All procedures wereconducted in accordance with the NIH Guide for the Careand Use of Laboratory Animals as approved by Animal Careand Use Committee of the Facultad de Ciencias Químicas,Universidad Nacional de Córdoba.

2.2. Drugs

For all experiments D-AMPH sulfate (Sigma Co, St.Louis, MO) was dissolved in an isotonic saline solution(0.9% NaCl), which was also used for vehicle (VEH) controlinjections. All injections were administered intraperitoneallyin a volume of 1 ml/kg and the treatments were made at 11 a.m. (ZT 4) to avoid the influence of the circadian rhythm onthe immune response (Haus and Smolensky, 1999) and onthe behavioral sensitization to psychostimulants (Abarca etal., 2002).

2.3. Experimental procedures

2.3.1. Repeated AMPH treatmentRats were randomly assigned to one of three treatments:

VEH group, AMPH 5×1 (1 mg/kg/day i.p.) and AMPH 5×2

Table 1Amplification parameters of proENK, GAPDH and c-fos mRNA

ProENK: exons I–III GAPDH and c-fos

94 °C–5 min 94 °C–5 min60 °C–2 min 60 °C–2 min72 °C–3 min 72 °C–3 min1 cycle 1 cycle94 °C–1 min 94 °C–1 min60 °C–2 min 60 °C–1 min72 °C–3 min 72 °C–1.5 min30 cycles 30 cycles94 °C–1 min 94 °C–1 min60 °C–2 min 60 °C–2 min72 °C–10 min 72 °C–10 min1 cycle 1 cycle

64 M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

(2 mg/kg/day i.p.). The AMPH or VEH treatment wasadministered during days 1–5, and on day 9 animals werekilled by decapitation (4 days following the last druginjection) (Fig. 2A). Spleens were removed and mononu-clear cells were isolated as mentioned below. Since weobtained the same proliferative response in untreated controlanimals as in the VEH group in previous experiments (datanot shown), this indicated that there was no influence ofinjection on this parameter, therefore we decided to use VEHas the control group.

2.3.2. Acute AMPH treatmentRats were randomly assigned to one of five treatments:

VEH group, AMPH 1×0.5 (0.5 mg/kg i.p.), AMPH 1×1(1 mg/kg i.p.), AMPH 1×2.5 (2.5 mg/kg i.p.) and AMPH1×5 (5 mg/kg i.p.). The AMPH or VEH treatment wasadministered during day 1, and on day 5 animals were killedby decapitation (4 days following drug injection) (Fig. 2B).Spleens were removed and mononuclear cells were isolated asmentioned below.

As similar dose–response effects were obtained followingacute and repeated AMPH treatment, a single dose of AMPH(5mg/kg i.p.) was chosen to asses the possible effect on spleencellularity and to observe the long-lasting effects of proENKmRNA, proteins and derived peptides on IS and CNS.

Once rats were decapitated, the brain, spleen, thymus andbone marrow were removed. The following brain areas ofboth hemispheres were dissected: cerebellum, caudateputamen, PfC, hippocampus, NAc, septum and brainstem.

2.3.3. Isolation of mononuclear cellsSpleen cell suspensions were obtained by gently grinding

tissue into RPMI 1640 culture medium (Sigma-Aldrich,Steinheim, Germany) under sterile conditions. Mononuclearcells were separated by Ficoll–Hypaque density gradient(1.083 g/ml) centrifugation. The mononuclear cells concen-trated at the surface were collected and washed twice inRPMI 1640 medium. After cell counting, splenocytes weresuspended at a final concentration of 2×106 cells/ml inRPMI 1640 medium supplemented with 10% of inactivatedfetal bovine serum, 2 mM glutamine, 10 mM sodiumbicarbonate, 100 UI/ml penicillin and 100 μg/ml streptomy-cin (denominated complete RPMI 1640 medium).

To evaluate the proENK-derived peptides released fromcultured spleen cells, part of the splenic mononuclear cells

Fig. 1. The specificity of met-enkephalin-Arg-Phe (ME-Arg-Phe)-directedantiserum (dilution 1 :1000) was determined incubating it (previous to theWestern blot) with different concentrations of the synthetic peptide used toproduce the conjugate injected to the rabbits. 100 μg of proteins from ratthymus acidic extracts were electrophoresed and analyzed by Western blot.

were cultured in flasks (TPP, Switzerland) for 48 h withcomplete RPMI 1640 medium with or without Con A, asdescribed below. Then, the supernatants were separated fromthe non-adherent cells by centrifugation, and used forradioimmunoassay (RIA) (Saravia et al., 1998). The adherentcells (macrophages) from culture without Con A wereremoved with 2 M acetic acid after two washes with salinesolution, boiled for 15 min, homogenized by sonication,centrifuged at 50,000×g for 1 h, and used for RIA.

2.3.4. Mitogenic assayThe mitogen Con A (Sigma-Aldrich, Steinheim, Germany)

was used to evaluate the splenic T-cell response. Splenocyteswere added in quadruplicate to each well of a 96-well flat-bottom tissue culture plates (TPP, Switzerland) in the presenceof 5 μg/ml of Con A. The plates were incubated at 37 °C in ahumidified atmosphere with 5% CO2 for 48 h. 18 h beforeharvesting, cells were pulsed with 1 μCi [3H]-thymidine(PerkinElmer Life Sciences, Wellesley, MA). Finally, quad-ruplicates were collected onto glass fiber filter paper (Watt-man, UK) using a Scatron micro cell harvester (SIEM,Córdoba, Arg.). A 1205 Betaplate liquid scintillation counter(PerkinElmer Life Sciences, Wellesley, MA) was used todetect the incorporation of radioactive thymidine in a Packard,Tri-Carb Liquid Scintillation Analyzer. The radioactivity wasexpressed as cpm and the percentage of responses wascalculated relative to VEH-treated animals.

2.3.5. Flow cytometry studiesIn order to evaluate if the differences in the lymphopro-

liferative response could be produced by a direct AMPH effecton lymphocyte subpopulations, the spleens were weighed andtheir cellularity were assessed by flow cytometry. Cellsuspensions were obtained by gently grinding tissue intoRPMI 1640 culture medium without phenol red (Sigma-Aldrich, Steinheim, Germany), counted and suspended at afinal concentration of 2×106 cells/ml. To analyse the totalcount of T- and B- and NK-cells in these tissues, three-colourimmunofluorescence staining was performed and the intensityof stained cells was analysed by flow cytometry (CytoronAbsolute flow cytometer; Ortho Diagnostic System, Raritan,

Fig. 2. Schematic diagrams of repeated (A) and acute (B) AMPH treatments. C: Dose-dependent effects of repeated and acute AMPH treatment on theimmunoproliferative response Percentage of proliferative responses related to the control groups (VEH). Data show the mean±S.D. of five rats per group.⁎p<0.05 and ⁎⁎p<0.01. These data are representative of at least two independent experiments.

65M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

NJ, USA). 100 μL of cells suspension were incubated withfluorescein isothiocyanate-conjugated mouse anti-rat CD161amonoclonal antibody (MoAb), R-phycoerythrin-conjugatedmouse anti-rat CD3 MoAb and Cy-Chrome™-conjugatedmouse anti-rat CD45RAMoAb (BD Bioscience, NJ, USA) inorder to determine NK-, T- and B-cells, respectively. Theincubation was for 30 min in the dark, followed by 15 minwith lysis buffer and three washes in PBS (pH 7.2). After that,the cells were fixed with 2% formaldehyde, washed tree timesin PBS and finally resuspended in ISOTON prior to analyse inthe cytometer.

Lymphocytes were gated based on their characteristic light-scatter and corroborated by a morphologic analysis by May–Grünwald–Giemsa. Fluorescence intensity was depicted on athree-decade logarithmic scale and in single-parameteranalysis as histograms. Absolute numbers of lymphocyteswere calculated according to lymphocyte subset percentages

and the absolute value of leukocytes (analyzed in a Coulter T-540 hematology analyzer).

2.3.6. Total and free met-ENK radioimmunoassayFrozen immune tissues and brain areas were suspended in

1 M acetic acid containing 50 mM HCl, boiled for 15 min,homogenized with a Polytron, and centrifuged at 50,000×gfor 1 h. An aliquot of the supernatant was lyophilized andreconstituted in 50 mM Tris–HCl buffer, pH 8.4, and 2 mMCaCl2. Free and total immunoreactive met-ENK, thosebeing, previous to and after sequential enzymatic digestionwith trypsin and carboxypeptidase B, were determined byRIA (Vindrola et al., 1990; Padrós et al., 1989).

Culture medium was diluted in Tris buffer forenzymatic digestion and peptides assayed as describedbefore (Saravia et al., 1998; Padrós et al., 1989). Specificmet-ENK antiserum was obtained and characterized as

Table 2Effect of the acute AMPH treatment (5 mg/kg i.p.) on spleen weight(mg spleen/g body weight) and cellularity (cell number×106/mg spleen)

Spleens VEH treatment AMPH treatment

Weight 2.08±0.19 2.03±0.13Leucocytes 566.5±28.1 549.9±27.9Lymphocytes 447.3±24.9 437.2±21.3T-cells 141.5±23.4 135.4±9.6B-cells 247.7±8.0 258.0±16.0NK cells 1.5±0.5 1.7±0.2

The results are the means±S.D. of four rats.

66 M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

previously described (Vindrola et al., 1990; Padrós et al.,1989), and used to identify the carboxy-terminal portion ofmet-ENK.

2.3.7. ImmunoblottingAliquots of acidic tissue extracts were dried, suspended

in 2× Laemmli buffer and boiled for 5 min. 50 μg ofproteins were applied to 15% (proENK-derived proteins) or8.8% (PC1 and Fos) acrylamide gel using the mini-proteangel system (BioRad). Electrophoresis and immunoblottingwere carried out as described previously (Saravia et al.,1998). The second antibody was conjugated with alkalinephosphatase (BioRad Laboratories, Hercules, CA) anddeveloped with the respective color reagent (BioRad kit).Antibodies against met-ENK-Arg-Phe (final dilution1 : 1000) (Fig. 1), N-terminal PC1 (DAM3-B6) (finaldilution 1 :1000), Fos (final dilution 1 :500) and actin(1 :500) (Santa Cruz, Inc. CA) were used in these studies.Control experiments were run simultaneously, omitting thefirst antibody. PC1 enzyme was mostly found as a long andstable pool in neuroendocrine cells (Rittner et al., 2001),while Fos mainly appeared as a consequence of tissue or cellactivation. The measurement of these two proteins with aknown pattern of expression in rat brain served as areference of the specific response of proENK proteins. Sincean increase in proENK and met-ENK after AMPH treatmentwas only obtained in PfC, we also measured PC1 and Fos inthis structure.

2.3.8. RNA isolationTotal cellular RNA was extracted from the immune

tissues and brain areas with TRIZOL isolation solvent asdescribed by the manufacturer (Invitrogen, Inc.). RNA wasprecipitated and suspended in 50 μl diethylpyrocarbonate-treated-dH2O and the concentration was estimated after opticdensity lecture at 260 nm using a GeneQuantPro spectro-photometer (Amersham-Pharmacia, Buckinghamshire, UK).The integrity of RNA was determined by applying 1 μg in1.5% agarose gel containing ethidium bromide followed byUV visualization in a transiluminator (Stratagene, La Jolla,CA). mRNAwas purified by mini kit Oligotex, following thespecification described by the manufacturer (Qiagen Inc.,Hilden, Germany).

2.3.9. RT–PCR analysis5 μg of total RNAwere incubated for 5 min at 90 °C, and

reverse transcription was carried out in a final volume of20 μl, adding 500 ng of random hexamers, 100 units ofMoloney murine leukemia virus reverse transcriptase(Promega, Madison, WI), 0.5 mM dNTPs and 20 units ofRNasin (Promega, Madison, WI). The samples wereincubated for 60 min at room temperature, followed by45 min at 37 °C. Finally, tubes were heated to 95 °C for5 min. Samples containing 0.5 μg of RT were amplified inthe presence of 200 μM of dNTPs (Roche Diagnostics,Mannheim, Germany), 1 μM of sense and antisense primers

(Life Technology, USA), 1.5 mM MgCl2 and 1.25 units TaqDNA polymerase (Roche Diagnostics, Mannheim, Ger-many) and were placed in a thermal cycler (Robocycler 40,Stratagene, La Jolla, CA). The parameters of the amplifica-tion are specified in Table 1. A proENK-specific primers pairwas designed and assessed for specificity by a computer-assisted search of up-dated versions of Oligo-4 program. ThePCR primer sequences for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from aprevious publication (Saravia et al., 1998). All primers usedwere synthesized by Life Technologies.

ProENK exon I–exon III: 5′–CCAGCTACCTGCGC-CATCTGAACAA–3′ and 5′–AGAAGCTTTTCC-TGTTGGTGGCTATCTTT–3′ (545 bp).c- fos : 5′–TGCATGAATTCCCCAGCCGACTC-CTTCTCCA– 3 ′ a n d 5 ′ –TGCATAAGCTT-CAGCTCCCTCCTCCGATTCC–3′ (388 bp).GAPDH: 5′–TGCATGAATTCTGATTCTACCCACGG-CAAGT–3 ′ and 5 ′–TGCATAAGCTTGTCAT-GAGCCCTTCCACGAT–3′ (380 bp).

The PCR products were analyzed on ethidium bromide3% agarose gels followed by UV visualization in atransiluminator (Stratagene).

2.4. Statistical analysis

Cell proliferation was measured by [3H]-thymidineincorporation assay and results were plotted as thepercentage of changes between AMPH- and VEH-treatedrats. Data represent means±S.D., and correspond toquadruplicate values of five different rats. Student's t-test(the level of significance was set at p<0.05) was used toevaluate the statistical significance.

3. Results

3.1. Dose-dependent effects of repeated and acute AMPHtreatments on the immunoproliferative response

Exposure of rats to repeated AMPH treatment resulted ina dose-dependent decrease in the in vitro splenic proliferativeresponse to Con A, relative to VEH-treated controls. This

67M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

effect was seen by a significant decrease in 3H-thymidineincorporation, by 45% (p<0.05) and 65% (p<0.01), in ConA-stimulated splenocytes, for rats treated with 1 or 2 mg/kg/day of AMPH, respectively (Fig. 2C).

Similar results were obtained after exposure to acute AMPHtreatment. 3H-thymidine incorporation was significantly re-duced in a dose-dependentmanner, by 37% (p<0.05), 50% and65%(p<0.01) in rats treatedwith 1, 2.5 or 5mg/kg, respectively(Fig. 2C). No significant differences were detected in ratstreated with 0.5 mg/kg relative to the control group.

As similar results were obtained after the repeated andacute VEH treatments in the proliferative response to ConA (p=NS), the data were pooled (5.6±0.5×104 cpm) andrepresented the maximum percentage of proliferation.

3.2. Effect of acute AMPH on the spleen weight andcellularity

No significant differences on spleen weights wereobserved after the acute AMPH (5 mg/kg) treatment, relativeto their controls (Table 2). In the same line, the lymphocytesubpopulations of spleens in rats treated with AMPH did notshow any difference, in absolute values as well as inpercentages, relative to their VEH-treated controls (Table 2).

3.3. Effect of AMPH on the proENK-derived peptide levelsin rat mesocorticolimbic areas

3.3.1. Radioimmunoassay proceduresThe acute AMPH treatment significantly increased the

free met-ENK levels in NAc (80%, p<0.05) and PfC (45%,p<0.05), compared with VEH-treated controls. This effectwas not seen in the other brain regions tested (Table 3,column 1 vs. 4).

No significant changes in total met-ENK levels weredetected in NAc and PfC after AMPH treatment (Table 3).However, free to total met-ENK ratios increased significant-ly in both regions (p<0.05) (Table 3, column 3 vs. 6).

To confirm if the changes after the acute AMPH treatmentin the met-ENK content are also observed after a repeatedAMPH treatment (AMPH 5×2), we studied the same brain

Table 3Effect of the acute (5 mg/kg i.p.) and repeated (2 mg/kg/day i.p.) AMPH treatmen

Brain areas IR-met-ENK levels (pg/mg protein)

Ac VEH Ac A

Free Total Ratio Free

Brainstem 3014±305 n.m. 3370±Caudate Putamen 1680±185 n.m. 1834±Cerebelum 1615±185 n.m. 1309±Hippocampus 1862±427 n.m. 1361±Nucleus accumbens 997±75 7663±96 0.13±0.02 1795±Prefrontal cortex 593±93 3436±295 0.17±0.03 861±Septum 3231±649 n.m. 2684±

The results are the means±S.D. of five rats. ap<0.01 and bp<0.05 compared to tAc=acute; Rep=repeated; IR= immunoreactive; n.m.=not measured; n.d.=not de

structures and immune tissues that showed a variation after theacute treatment. This AMPH repeated treatment was chosenbecause it produced a similar decrease of proliferativeresponse to that observed after acute AMPH (5 mg/kg i.p.).

The repeated AMPH treatment significantly increased thefree met-ENK levels in NAc (72%, p<0.01) and PfC (39%,p<0.01), compared with VEH-treated controls. (Table 3,column 7 vs. 8).

3.3.2. Western blotting studiesIn order to characterize high molecular weight proENK

molecules in NAc and PfC and their probable contentchanges induced by the acute AMPH treatment, weperformed immunoblotting analysis, using an antibodyagainst the C-terminal portion of proENK, which distin-guished three proteins of 32, 44 and 64 kDa in rat brain (Fig.3A). The 32 kDa molecule corresponded to the authenticproENK and the 64 kDa protein corresponds to therespective dimer (Lindberg and Shaw, 1992). The 44 kDamolecule corresponds to a novel isoform previouslydescribed in tumoral tissues and cells (Vindrola et al.,1998; Brar and Lowry, 1999). AMPH-treated rats showed asignificant increase in the 32 kDa proENK content in the PfC(p<0.05), while the other two proteins levels remainedpractically unchanged. On the other hand, the AMPHtreatment did not modify 32 kDa protein levels in NAc,where only a significant decrease (p<0.05) of the 44 kDamolecule was detected (Fig. 3).

PC1 enzyme appeared as the 68 kDa molecule and itshigh levels, as well as actin levels, were not affected inPfC and NAc of AMPH-treated rats, respectively.However, blotting results for Fos proteins in PfC ofAMPH-treated rats showed a significant increase in the35–37 kDa band, corresponding to highly stable isoformsof ΔFosB (Fig. 4).

3.4. Effect of acute AMPH on the proENK mRNA levels inrat mesocorticolimbic areas

The proENK mRNA content did not change in PfC andNAc, but showed a significant decrease in brainstem (p<0.05)

ts on brain IR-met-ENK levels (pg/mg protein)

MPH Rep VEH Rep AMPH

Total Ratio Free Free

640 n.m. n.m. n.m.160 n.m. n.m. n.m.249 n.m. n.m. n.m.334 n.m. n.m. n.m.226b 6952±1093 0.26±0.06a 1133±72 1952±177a

72b 3005±152 0.29±0.04a 724±67c 1008±55a

396 n.m. n.m. n.m.

heir controls (VEH-treated rats); cp<0.05 compared to the acute treatment;tectable levels.

Fig. 3. Regulation of proENK by AMPH in PfC and NAc. (A): Immunoblotting of proENK peptides in control rats (VEH) and in rats that received an acuteAMPH treatment (5 mg/kg i.p.) 4 days before. (B) Relative units of absorbance of 32, 44 and 64 kDa proENK isoforms. Data show the mean±S.D. of three ratsper group. ⁎p<0.05.

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(Fig. 5) after the acute AMPH treatment. The c-fos mRNAlevels were not changed in any of brain regions tested (Fig. 5).

In conclusion, the acute AMPH treatment increased freemet-ENK levels in PfC and NAc, as well as proENK content inPfC, and also decreased proENK mRNA levels in thebrainstem.

3.5. Effect of AMPH on proENK-derived peptide levels inrat immune organs

3.5.1. Radioimmunoassay proceduresThe acute AMPH treatment enhanced free met-ENK

levels in rat spleen (35%, p<0.05) and thymus (31%,p<0.05), relative to VEH-treated controls. This effect wasnot seen in the bone marrow (Table 4, column 1 vs. 4).However, total met-ENK (met-ENK plus cryptic met-ENK-containing peptides) did not change significantly in anytested organs. Free to total met-ENK ratios increasedsignificantly in the spleen and thymus, but not in the bonemarrow of the animals treated with AMPH (Table 4, column3 vs. 6). These results suggest an increased proENK post-translational processing towards low molecular weightpeptides in spleen and thymus.

Free met-ENK concentration was not detected in themedium of cultured splenocytes from all-treated animals(Table 4, column 1 vs. 4). However, total met-ENK levelsincreased significantly (65%, p<0.01) (Table 4, column 5 vs.2) in the medium of AMPH-treated rats splenocytes, culturedfor 48 h. This effect was observed in the absence of any kind ofmitogen, that is, with complete RPMI 1640 medium alone.Although the addition of T-lymphocyte mitogen Con A to theculture medium increased significantly the total met-ENKlevels in VEH-treated rats (66%, p<0.05, Table 4, column 2),it produced aminor non-significant effect (14%) on the already

elevated levels following AMPH. Free met-ENK concentra-tion was elevated in the macrophages (38%, p<0.01) fromacute AMPH-treated animals (Table 4, column 1 vs. 4).

The repeated AMPH-treated animals showed a similarpattern in met-ENK content. The repeated treatmentsignificantly increased the free met-ENK levels in spleen(35%, p<0.01), thymus (29%, p<0.01) and macrophages(45%, p<0.01) (Table 4, column 7 vs. 9), and also raisedthe total met-ENK levels in the medium of AMPH-treatedrats splenocytes (Table 4, column 8 vs. 10), cultured for48 h, compared with VEH-treated controls.

3.5.2. Western blotting studiesImmunoblotting studies of proENK proteins showed that

the spleen contained high levels of a 34–32 kDa doublet,together with 64 kDa (see before), 44 kDa (see before),27 kDa and 25 kDa proteins. Spleens of AMPH-treated ratshad a significant increase in the 44 kDa protein content,while the levels of the other proteins remained unchanged(Fig. 6). The thymus contained the same proteins asspleens, except for the 44 kDa molecule. Thymus ofAMPH-treated rats showed a significant increase in 34–32 kDa doublet content with no changes in the otherproteins (Fig. 6). Although both immune organs of AMPH-treated rats showed increased levels of proENK, differentisomers of this molecule were responsible for this change.Actin levels were not modified after AMPH treatment inany tissue assayed.

3.6. Effect of acute AMPH on the proENK mRNA levels inrat immune organs

RT-PCR for proENK, using exon I–exon III primers,amplified the expected fragment of 545 bp (Fig. 7A). The

Fig. 4. Regulation of PC1 and Fos-related proteins by AMPH in PfC. (A) Immunoblotting of PC1; (B) Fos-related proteins in control rats (VEH) and in rats thatreceived an acute AMPH treatment (5 mg/kg i.p.) 4 days before. (C) Relative units of absorbance of PC1 and 35–37 kDa isoform of ΔFosB. Data show themean±S.D. of four rats per group. ⁎p<0.05. MW: molecular weight marker.

69M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

proENK mRNA was detected in the thymus but not in thespleen. In this last organ, proENK was not even observedafter RT–PCR of purified mRNA (data not shown). Theseresults verified previous publications, indicating thatproENK mRNA expression in splenocytes may dependon cell activation (Linner et al., 1995). ProENK mRNAcontent did not change in immune organs 4 days after onedose of AMPH (Fig. 7), while this drug only induced asignificant decrease of c-fos mRNA levels in the thymus(p<0.05) (Fig. 7).

4. Discussion

This work has demonstrated that a single dose, as well asa repeated treatment with AMPH, can induce a decrease inthe splenocyte proliferation, as well as a simultaneousincrease in met-ENK levels in immune organs and in brainareas. In addition, the non-activated cultured splenocytesfrom AMPH-treated rats released nearly the same amount ofcryptic met-ENK-containing products as Con A-stimulated

control splenocytes. Within the cultured splenocytes, themacrophages were the cells where the increase in met-ENKwas observed. Since met-ENK has been consistently relatedto a decrease in immune function, our current results stronglysuggest that an increased in vivo release of met-ENK byperipheral monocytes/macrophages may be the primer forthe inhibitory proliferative response of T-lymphocyte. Theinfluence of the met-ENK system in key brain areas forsensitization, and in immune organs, is consistent with theidea that similar biological changes could underlie theAMPH's effects on the CNS and IS.

It has been shown that a single, as well as a repeatedtreatment with AMPH, induces a phenomenon of behavioralsensitization, which has been proposed as a key process inthe addiction to psychostimulants and other abuse drugs(Vanderschuren et al., 1999b; Vanderschuren and Kalivas,2000; Robinson and Berridge, 2000). Several alterations inthe mesocorticolimbic dopaminergic nuclei have beenidentified in the sensitization induced by psychostimulants.Within these nuclei, the ventral tegmental area, NAc, and

Fig. 5. Levels of proENK, c-fos and GAPDH mRNA in brain areas of control rats (VEH) and rats that received an acute AMPH treatment (5 mg/kg i.p.) 4 daysbefore. A: The amplified products of proENK cDNA (545 bp), c-fos cDNA (388 bp) and GAPDH cDNA (380 bp), as generated from mRNA, are shown in brainstem. (B) Relative units of absorbance of proENK and c-fos mRNA. W: water; MW: molecular weight marker.

70 M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

PfC have been shown to undergo various physiological,neurochemical and molecular changes following adminis-tration of psychostimulants (Pierce and Kalivas, 1997b;Nestler, 2001). Interactions between DA, opioids andexcitatory amino acids have been shown in behavioralsensitization to psychostimulants (Kalivas and Stewart,1991; Pierce and Kalivas, 1997b; Wolf, 1998; Cador et al.,1999). In the current study, we have provided the firstevidence for a notable increase in free met-ENK levels in keybrain areas for sensitization, such as NAc and PfC, 4 days

Table 4Effect of the acute (5 mg/kg i.p.) and repeated (2 mg/kg/day i.p.) AMPH treatmen

Immune source IR-met-ENK levels (pg/mg protein)

Ac VEH Ac AMPH

Free Total Ratio Free T

Spleen 2731±139 6269±481 0.44±0.06 3698±285b 5Thymus 1184±7 4271±123 0.28±0.01 1551±223b 4Bone marrow 638±13 4110±349 0.16±0.05 534±10 3Spleen-culture

macrophages⁎1518±146 n.m. 2089±176a n

Spleen-cultureSupernatant⁎

n.d. 689±44 n.d. 1

Spleen-culture Supernatantwith Con A⁎

n.d. 1148±6d 0.13±0.02 n.d. 1

The results are the means±S.D. of five rats. ap<0.01 and bp<0.05 compared todp<0.05 compared to the non-stimulated culture; Ac=acute; Rep=repeated; IR=⁎ pg/2×106 cultured splenocytes.

after an acute or a repeated treatment with AMPH. As thetotal met-ENK levels were not changed, an increase inproENK post-translational processing towards low molecu-lar weight peptides can be suggested.

This increase in met-ENK, following a single dose ofAMPH, was accompanied by an increase in the 32 kDa bandcorresponding to the proENK in PfC but not in NAc. Thisdifference might be attributed to a differential turnover rateof the protein in each brain region throughout the timefollowing AMPH. Considering that the 44 kDa band

ts on immune IR-met-ENK levels (pg/mg protein)

Rep VEH Rep AMPH

otal Ratio Free Total Free Total

379±160 0.69±0.09a 2936±97 n.m. 3825±269a n.m.480±383 0.35±0.01a 1318±59c n.m. 1700±121a n.m.125±464 0.17±0.02 n.m. n.m. n.m. n.m..m. 1513±228 n.m. 2467±189a, c n.m.

136±87a n.d. 720±106 n.d. 1225±99a

306±26 0.26±0.06a n.m. n.m. n.m. n.m.

control rats (VEH-treated rats); cp<0.05 compared to the acute treatment;immunoreactive; n.m.=not measured; n.d.=not detectable levels.

Fig. 6. Regulation of proENK by AMPH in thymus and spleen. (A) Immunoblotting of proENK isoforms in control rats (VEH) and in rats that received an acuteAMPH treatment (5 mg/kg i.p.) 4 days before. (B) Relative units of absorbance of 32, 4nd 64 kDa proENK isoforms. Data show the mean±S.D. of three rats pergroup. ⁎p<0.05.

71M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

corresponds to a novel isoform whose function is not totallyunderstood, further studies are necessary to interpret thedecrease of this band in NAc after AMPH treatment.

The increase in proENK in PfC 4 days after AMPHtreatment is in line with that observed in proENK RNAm instriatum following 3 h of acute or repeated AMPH treatment(Wang and McGinty, 1995, 1996; Mao and Wang, 2003).However, a decrease in proENK mRNA levels was observedin the central nucleus of the amygdala, 3 h after the lastinjection following acute and chronic treatment with AMPH(Turchan et al., 2002). It has been suggested that the increasein proENK RNAm by AMPH could be through increasedcytosolic Ca2+ levels that activate the phosphorylation of thetranscription factor CREB (Konradi et al., 1994), whichmight result in an enhanced proENK gene transcription(Rajadhyaksha et al., 1999). However, since the proENKgene promoter also has an AP-1 site that can be activated byFos–Jun proteins (Monnier and Loeffler, 1998), thesetranscription factors might also be involved in the proENKgene regulation following AMPH. A recent study hasprovided evidence, that following AMPH, the DNA-bindingactivity of AP-1 was increased in the human astrocyte-likeU373 MG cells, concomitantly to the psychostimulant'seffects on the proENK gene regulation (Malaplate-Armandet al., 2005). In this work, the brain proENK mRNA levelswere not modified 4 days after an acute AMPH treatment,with the exception of the brainstem where a decrease wasobserved. The lack of effect of AMPH on proENK mRNA inmost of the CNS areas could be related to the long intervalbetween AMPH injection and the assay (i.e. 4 days in ourstudy vs. 3 h in the previous study mentioned above). Other

authors also failed to show changes in brain proENK mRNAfollowing morphine (Tjon et al., 1997) and cocaine (Daunaisand McGinty, 1994; Álvarez Fischer et al., 2001). Our dataalso indicate that c-fos mRNA levels were unchanged in anyregion tested after AMPH treatment. These results supportprevious data indicating that proENK mRNA may behavelike early gene mRNAs (Weisinger, 1995).

The measurement of other inducible proteins, such asFos-related proteins, showed a similar pattern of change tothe 32 kDa proENK band following AMPH. The highlystable isoform of ΔFosB was the only one that remainedelevated in PfC of AMPH-treated rats, in agreement withprevious data showing that after chronic exposure to AMPHor cocaine, a sustained increase in these protein levels wasobserved in specific brain areas for several weeks (Chen etal., 1997; Ehrlich et al., 2002; McClung et al., 2004). Ourresults suggest that although the other isoforms (c-Fos, Fra-1and -2, FosB) are soon activated by an AMPH treatment,only ΔFosB content may remain elevated after 4 days inPfC. It is important to address that this is the first evidence ofan increase of this protein after a single dose of AMPH. Inaddition, since a related constitutive protein such as PC1 didnot show any change in PfC after AMPH administration, it islikely that this drug exerts a specific effect on some proteinsrather than having a general influence on protein synthesis.

Interestingly, in this study we also showed that AMPHmodified the met-ENK in IS in a similar way to that observedin CNS. Our findings indicate an increase in free met-ENKlevels in immune organs such as the spleen and the thymusobservable 4 days after acute or repeated AMPH. Inagreement with this, an increased free met-ENK and free

Fig. 7. Levels of proENK, c-fos and GAPDH mRNA in immune organs of control rats (VEH) and rats that received an acute AMPH treatment (5 mg/kg i.p.)4 days before. (A) The amplified products of proENK cDNA (545 bp), c-fos cDNA (388 bp) and GAPDH cDNA (380 bp), as generated from mRNA, are shownin thymus. (B) Relative units of absorbance of proENK and c-fos mRNA. W: water; MW: molecular weight marker.

72 M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

syn-ENK level was observed in cultured splenocytes ofacutely and chronically stressed rats, suggesting that a stressstimulus also enhanced proENK-derived peptide releasefrom thymus, spleen and bone marrow macrophages(Saravia et al., 1998). Moreover, macrophages of chronicallystressed rats have been suggested to have a tonic increasedrelease of proENK products (Saravia et al., 1998). Thus,similar mechanisms may be triggered by stress and drugs onIS, as was previously described for behavioral andneurochemical effects (Kalivas and Stewart, 1991; Antelmanet al., 1980; Pacchioni et al., 2002, in press).

In the current study, the medium of cultured ratsplenocytes did not have detectable levels of free met-ENK, although it did contain proENK-derived peptides.These peptides increased following AMPH to a similarextent to that induced by the T-cell mitogen in control rats.Moreover, the addition of Con A in the splenocytes ofAMPH-treated rats only induced a low additional increase inproENK-derived peptides, indicating that the other spleencells (like macrophages and B-cells) had a minimalinfluence. This finding suggests that one dose of AMPHby itself was sufficient stimulus for splenic T-lymphocytes torelease proENK-derived peptides, as in the case of mitogenicactivation. Spleen and alveolar macrophages contained PC1(Vindrola et al., 1994), which is involved in proENKprocessing in endocrine cells (Rittner et al., 2001; Vindrolaand Lindberg, 1992). Moreover, a previous report indicatedthat peripheral blood monocytes, but not T-cells, produced

free met-ENK (Kuis et al., 1991; Roth et al., 1989). Sinceonly macrophages have the machinery (PC1) to produce lowmolecular weight biologically active opioid peptides, in thepresent study cryptic proENK-derived peptides may havebeen released by lymphocytes. Related to this, we demon-strated that the macrophages are specifically the cells thatshow an increase in met-ENK following acute or repeatedAMPH in the spleen culture.

Spleens of acute AMPH-treated rats had an increase in the44 kDa protein content, while the thymus showed anincrease in the 34–32 kDa doublet corresponding to proENKcontent. It could be that different turnover rates of proENKthroughout the immune organs are responsible for thesechanges.

ProENK mRNA has been described in uncultured ratbone marrow and cultured mouse bone marrow-derivedmacrophages (Saravia et al., 1993), in thymic CD4+ cellsafter 15-day activation with Con A (Linner et al., 1991), andin splenic monocyte–macrophage and B-cells (Saravia et al.,1993; Behar et al., 1991). A single dose of AMPH did notmodify the proENK or c-fos mRNA levels in IS which couldbe also attributed to the fact that the assays were performed4 days after the drug. The only exception was that AMPHinduced a decrease in c-fos mRNA levels in the thymuswhich could be related, at least in part, to the decreased T-proliferative response after the drug. Further studies shouldconfirm if the repeated AMPH treatment exerts any influenceon pro-ENK mRNA and protein content in IS and CNS.

73M.A. Assis et al. / Journal of Neuroimmunology 178 (2006) 62–75

This study demonstrates that a single dose, as well as arepeated treatment with AMPH, induces a dose-dependentdecreasing effect in the Con A-proliferative response 4 daysfollowing the AMPH injection. Since the spleen weight andcellularity were not modified following acute AMPH, it islikely that the drug is exerting a specific influence onlymphocyte responsivenessmore than an effect on the absolutenumber of cells. This is the first result showing a influence ofAMPH on IS observable 4 days later, which is consistent withprevious reports which indicated a decreased splenocyteproliferation immediately after a challenge with this drug(Kubera et al., 2002; Freire-Garabal et al., 1991). Previousfindings from our laboratory also showed that chronic AMPHtreatment induced a persistent sensitization to the stress-induced immunosuppressive effects (Basso et al., 1999).

Met-ENK synthesized by endocrine and/or nervous tissuehas been involved in stress-induced immunosuppression(Saravia et al., 1998; Roda et al., 1996; Roy and Loh, 1996).In rat spleen, only macrophages produced free met-ENK,which had led to suggest that these cells may be the source ofmet-ENK, and could participate in the modulation of theimmune response (Saravia et al., 1998). ProENK is notprocessed to met-ENK in bone marrow–mononuclear cells,while in thymus– and spleen–mononuclear cells met-ENKrepresented 10% and 26% of total met-ENK-containingpeptides, respectively (Saravia et al., 1998). In the currentstudy, it is possible that an enhanced met-ENK release fromperipheral monocyte/macrophages is actually the primer forthe inhibitory proliferative response of T-lymphocytesfollowing AMPH. In agreement with our present findings,antisense oligonucleotides against proENK reduced theopioid expression and enhanced the proliferation of ConA-stimulated splenocytes (Fulford et al., 2000). Furtherstudies are necessary to clarify the role played by central vs.peripheral met-ENK in the AMPH's effects on IS.

Besides met-ENK, other systems affected by AMPH couldalso be exerting an influence on IS. DA is the neurotransmitterprimarily involved in the long-term expression of behavioralsensitization to AMPH (Vanderschuren et al., 1999a) and thereis evidence of an immunomodulatory role for it (Basu andDasgupta, 2000). It is highly probable that the dose-dependentdecreasing effect on proliferative response showed in thisstudy may be also linked to this neurotransmitter. In vivostudies fromour lab have provided evidence of an involvementof DA in the chronic AMPH-induced enhancement of thestress-induced immunosupression (Basso et al., 1999).However, in the most of in vivo studies so far is has beenimpossible to separate the effects of the drug on DA of CNSfrom any that might target immune cells directly. Whendiscussing the possible contribution of DA, we should alsotake into account the peripheral role of this catecholamine inthe neuroimmune network. Thus, DA may act as a transmitterbetween splenic sympathetic nerve terminals and immuno-cytes (Bencsics et al., 1997), and it has been hypothesized thatendogenous DA detected in lymphocytes could underlieseveral functions (Cosentino et al., 2002, 2003).

Summing up, we conclude that similar biologicalmechanisms could be triggered by AMPH to underlie itseffects on brain and immune organs. We propose thatchanges in proENK-derived peptides in peripheral T-lymphocytes may be reflecting an opioid central effect ofAMPH. Furthermore, proENK-derived peptides could beconsidered as one of the main targets for changes in the ISfunctions induced by a psychostimulant drug. Since drugs ofabuse not only modify the behavior of the individuals, butalso compromise their immune functions, increasing thevulnerability to numerous infectious diseases (Baldwing etal., 1998; Friedman and Eisenstein, 2004), it is highlyrelevant to find similar biological changes in immune organsand brain and follow them up at peripheral level.

Acknowledgments

This work was supported by grants from Ministerio deSalud de la Nación, Agencia Córdoba Ciencia, CONICETand SeCyT (Argentina), and IBRO Latin-American Com-mittee. Dr Cancela, Dr Sotomayor and Dr Figuerola areSenior Researchers of CONICET (Argentina). The authorswish to express their sincere gratitude to Mrs. Estela Salde,Mrs. Paula Icely and Mrs. Elsa R. Pereyra for their excellenttechnical assistance, to Dr. German Roth for his collabora-tion with the micro cell harvester and to Dr. JacquelineMcGinty for kind suggestions to the manuscript.

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