lmmunophamtacotagv. 0

1992 Elsevier

IMPHAR

23 (1992)

51-61

Science Publishers

B.V. All rights reserved.

0162-31@9~92:SO5.00

57

00574

Jo&Luis

PCrez-Castrilkn, Jo&-Lois Pkez-Arellano, Jest-Daniel Garcia-Palomo, Antonio Jim&z-Epea Deportment ofMeditine, (Received

Abstract:

Intravenous

drug

abusers (IDA)

and Sisinio De Castro U~~iwi-sily of Suhmrca.

16 July 1391; accepted 24 October

normally

show functional

Spain 1991)

defects in monocyte

activity,

in particular

tactic response. The aim of the present work was to study the action of severat opioids on monocyte monocytes

from healthy individuals

were incubated with heroin and morphute et three different

their chemo-

chemotaxis.

To do so,

concentrations (JO- 5M, NJ-’M

and lo-’ M), with the finding of a signilicant depression in monocyte chemotaxis in all cases. This &e&on could be due to a receptor effect or, conversely. to a non-specific effect, Accordingly, in the second phase of the study, monocytes from controls were incubated with a selective agonist ofu receptors (DAGG) and a selective agonist of ii receptors (DPDPE). Ia both cases a decrease in chemotactic function was observed similar to that folIowing incubation with morphine or heroin. Preincubation of the monocytes with naloxone prevented the depression induced by bath specific agonists.Thesefindingssuggestthat opioids play an importnnt role in the depression of monocyte chemotaxis observed in IDA. The. results also suggest the presence ofp and 8 opiate receptors Key wards:

Intravenous

in the cells afthe drug abusers;

---

phagocytic Monoeyte

mononuctear chemotaxia;

In parenteral drug addicts a defect in monocyte chemotaxis has been reported (Poli et al., 1985). At our laboratory we have demonstrated a direct relationship between drug addictive behaviour artd chemotactic depression (Olmos et al., 1986); heroin and/or its contaminants may be implicated in the disturbance. The number and composition of substance added to brown heroin are highly variable and diicult to determine. Accordingly we attempted to establish the possible relationship between the opiate and the motility defect. Lop& et al. (1980) have reported the existence Cor~~po~&~ce: D. JosCLuis

Heroin;

Morphine;

Opioids

-

Introduction

P&z-Castrillbn, Servicio de Jtttema II, Cute&a de Patologia General. Hospital Clinic0Universitariq Salamauca, Spain. Medicina

system.

of preceptors in monocytes, although this has been questioned by others (Mehrishi and Mills, I985). Additionally, some opiate agonists Go_ endorphin, dinorphin, met-enkephalin o-ala-leuenkephalin) behave as chemoattractants (Van Epps and Saland, 1984; Rtiet al., 1985). The receptor-mediated nature of the effeci was suggested by the inhibition due to naloxone addition. These findings point to the possible existence of K and 6 opiate receptors in monocytes. However, morphine lacks chemotactic activity (Ruff et al., 1985). Moreover, in rats and rabbits addicted to morphine a depression in the activity of the phagocyte monocyte system has been observed; this is reflected by a decrease in the phagocyte index, a reduction in the production of the superoxide aoion, and a lower survival when drug abusers are challenged by infection. This dis-

turbance cannot be reversed with naloxone (Tubaro et al., 1983). The present study was designed to check whether morphine and heroin are able to depress monocyte chemotaxis in vitro. Morphine, the active metabolite of heroin (Yew, McQuinn and Gorodetzky, 1977),is ableto bind to two types of

receptors: 1 and 6 [Snyder, 1984).We therefore used a selectivep agonist,DAGO (@iron et al., 1983)and another d agonist;DPDPE (D-PEN’*~enkephalin)(Rodriguezet al., 1986),in the presence and absence of naloxone in an attempt to gain further insightinto the possible existenceof opiate receptors in monocytes and their relationship with monocyte cbemotaxis.

Materials and Methods All studies were carried out in control cells donated by healthy (normal monocyte chemotactic activity) hospital staff (two groups of nine subjects). The mononuclear fraction was obtained from heparinized venous blood (10 U/ml) using the method of Bbyum(Bbyum, 1969). The blood was diluted down to 50% in physiologicalsaline and 30 ml of this suspensionwas added to 15 ml of Ficoll-Hypaque (Lymphoprep Nygawd, Oslo, Norway) in sterile siliconizedpolystyrene tubes (30 x 15mm, Falcon Oxnard, CA). After 35 min centrifugation at 400 xg the mononuclear cells were obtained in the interphase. These were washed three times in RPM1 medium (RPMI1640,25 m Hepes, Grand Island BiologicalCo., NY) and resuspended in the same medium at a concentration of 1.5 x lo6 monocytes/ml. Before carrying out the chemotaxis experiments, the monocytes were incubated for 5 tin with the followingsubstances: heroin (10e5 M, 10m6M and 10w7M), morphine (loss M, 10-6 M and lo-‘M), DAGO (1O-5 M) and DPDPE (10m5M) at 37 “C in sterile siliconized polystyrene tubes. In the Iatter three cases, this was carried out in the presence and absence of naloxone (10m5M). After incubation had fin-

ished, the cells were washed three times in RPMI medium and were adjusted to a concentration of 1.5 x lo6 monocytes/ml.Prior to this we carried

out a comparative study in 6 controls of the chemotaxis of purified monocytes followingthe method of Recalde et al. (1984) (purity> SO%, according to non-specific esterase staining) as compared with the chemotaxis of total mononuclear cells. There were no differences among the numbersof migratedcells per highpower field. These results are similar to those reported by other authors (Snyderman, 1972). Morphologicalchemotaxis was performed in Boyden chambers (Blind well chambers of chemotaxis, Neuropore Unipore System, BioRad Laboratories, Richmond,VP’ followingthe technique reported by Snyderman (1972). The chambersweredividedinto two compartmentsby a polycarbonate membrane with 5 pm pores (Unipore membrties, Bio-Rad Laboratories, Richmond, VA), 2004 of the attractant, serum activatedwith 1y0 endotoxin (EAS), were placed in the!ower part, and 200~1of the cell suspension in the upper part. EAS was used since,in previous works on monocytechemotaxis,we had observed that there were no differencesamong the results obtained with several different chemoattractants (Jimenez, 1984).After 90 min in a humid atmosphere at 37°C with 5% CO,, the filters were removed and stained according to the method of Wilkinson (1982) Reading was made under a light microscope at a magnificationof x 100 by two independent observers, counting 20 fields randomly on the lowerpart ofthe filter.The mean values are expressed as the number of cells migrated per high power field. All assays were carried out in triplicate*At the sametime a chemotactic assay was performed against a control medium (200,ucl of RPM% (RPMI-1640 Grand

Island BiologicalCo., NY)) to evaluate random locomotion. The results obtained were processed on a Cii Honeywell Bull model 64 DPS computer(BMDP Program, Universityof California,CA),The nonparametric Mann-WhitneyU and Wilcoxon tests were employed in the statistical analysisof data.

59 Results Incubation of control monocytes with heroin led to a decrease in chemotaxisat allthree concentrations employed: 10e5 M (P-C 0.0001). 10m6M (P c 0.031) and lo-’ M (P-C 0.031). A similar decrease was observed with the morphiie: loss M (P c: O.OOl),1O-6M (P < 0.017)and IO-’ M (P < 0.005).The results are shown in Table I. Incubationofcontrol monocyteswiththe selective p agonist DAGO (lo- 5 M) produced a chemotactic deficit (P c 0.014) that was abolished when naloxonc was present in tineincubaTABLE

I

Effects of heroin and morphine on monocyte chemotaxis EASQ’

Conlrol”

-.

Control* Control Control Control Control Control Control

+ + + + + +

Heroin 10e5 M Heroin 10 _ b M Heroin IO” M Morphine 10-SM Morphine 10 -b M Morphine 10 -’ M

Mean

SD

Mean

SD

7.85 8.71 8,81 6.76 7.33 7.06 8.00

6.62 6.88 7,Zl 5.73 6.34 7.51 6.66

63.8 37.1 47.U 46.9 30.8 38.9 41.6

14.41 8.7 15.5 14.2 9.38 19.5 13.42

Results expressed as number of cells migrated/hpf in the presence of RPMI* or Endatoxin activated serum**.In all

experiments R = 9.

tion medium.A similareffect was seen when the selective delta agonist DPDPE (to-‘WI) was employed.The results are shown in Table II. It should be noted that, in the assays periormed, two groups of controls were wsedsince this technique carries a considerable inter-assay variability(Axtell, 1988).

The heroin addicts in the geographicalregion of our hospital institution consume an average of rlOQmg/day of brown heroin, at a purity of 10%. Each addict thus administershi/herself 40 mg of pure heroin,~hirh, accordingto the pharmacokinetic studies of inturrisi et al. (1984), would alkrd a serum concentrationof 10m6M/l. In the present work we have shown that this in vitro concentration is able to cause a depression in chemotaxis.Heroin may be said to play an essential role in the motor defectdescribedin the heroin addicts. Heroin is unable to bind to opiate receptors (Inturrisiet al., 1983).It behavesas a prodrug,its metabolites morphine and 6-acetyl morphine being those providing the drug action (Yew, McQuinn and Ciorodetzky, 1977).Accordingly, the degree of depression should be similar for both agonists, as in fact has been observed.

TABLE II Effects of DAGO and DPDPE OIImonocytechemotaxis EASH

CQntrol~

Control’ Control + DAGO IO-‘M Control + DAGO 10v5M t Naloxone 10a5M Control + DPDPE IO- ’ M Control t DPDPE IOmsM t Naloxone lO-s M Control t Morphine 10m5M Control + Morphine IO-’ M t Nalaxone W’M

Meall

SD

Mean

32.7 33.4 35.7

1.58 6.40 6.17

101.10 63.42 98.88

13. I

7.12

58.30-

6.34

83.95

26.2

5.3

46.3

6.5

32.5

4.2

92.9

8.5

1.3

SD

6.14 4.96 7.02 8.08

IO.10

Resultsexpressed as number ofcells migraked/hpf in the presence of RPMI*or Endotoxinactivatedserum**.IIIall experiments n = 9.

We employeda selectivep agonist,DAGOand another 6agonist, DPDPE, to observe their effect on monocyte chemotaxis because morphine is ab!c to bind to both receptors,althoughits action is 50 to 100times weaker on the 6 receptor (Snyder, 1984).The incubation of control cells with DAGO caused a depressionin the chemotaxis, there being no significantdifferenceswith respect to that produced by heroin and morphine at equal concentrations. Previous addition of naloxone ity induced

prevented by

the disturbance

in cell motil-

DAGO. Incubationof control cells with DPDPE led to an alteration in cell Iocomotionthat wasabolishedby previousadditionof naloxone to the incubation medium. With these findings,and taking into account that we used specificand selectivep and b agonists (Quiron et al., 1983; Rodriguezet al., 1986)it may be suggested that monocyteshave p and d receptors, .j down-regulationmechanismcould be postulated to attempt to explain the depression in chemotaxisinduced by the delta agonist. a-alaleu-enkephalinand met-enkephalin(both delta agonists} are potent chemotactic agents at a lo-‘M concentration (Van Epps and Saland, 1984;Ruffet al., 1985).The ligand-receptorbindingcausesan internalizationof the complex,leading to a transient loss of chemotactic capacity (Gianciolo and Snyderman, 1981).A desensitizationoccurs, accompaniedby a drop in the number of receptors. However,this modelis not valid in the case of the p agonist since morphinedoes not produce down-regulationphenomena (Din&dine et al., 1983) and lacks chemotactic activity(Ruffet al., 1985).The In-opiatereceptors is bound to the adenylate cyclase system; the bindingof the agonistcauseschangesin the intracellular levels of cyclic AMP (Rodbell, 1980) This nucleotideacts as modulatorin the chemotactic response at the level of cell polarity (Stephensand Snyderman, 1982);a rise in intracellularCAMPimprovesthis phase. On the other hand, si-bstances that increase the CAMP decrease monocytechemotaxis(GaIlinet al., 1978). We postulated that heroin and morphine could depress chemotaxis by emulating the concen-

tration of CAMP.In supportof this hypothesiswe have previously demonstrated a significantincrease of CAMPlevels in IDA’s monocyte vs. control’smonocytes(Castrillbnet al., 1989).Currently we are studyingthe precise mechanismof action of the different opiate agonists on the adenylate cyciase system and their relationship with the alterations observed in chemotaxis. In summary,the findingsof the present work suggestthe presenceof opiate receptors (p and S) in monocytesand the depressoreffecton chemotaxis of morphine and heroin in vitro through a receptor phenomenon.We propose that the effect of exogenous opioids (morphine and heroin) takes place through two differentmechanisms;a down-regulationin the case of the 6 receptors and a rise in intra-cellularCAMPin the case of the p receptors. References Axttll RA. Evaluation of the patient with a possible phagocytic disorder. tlematol/Oncol Clin North Am 1988; 2: l-12. Bliyum A. Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Invest 1968; 21 (suppl97): 77. Dingledine R,Valentino RJ, Bostock E, King MR,ChengKJ. Down-regulation of S but not p opioid receptors in the hipacampal slice associated with lass of physiological response. Life Sci 19ll3; 33 {Sup@ I): 333-336. Gallin JI, Sandier A, Clymau RI, Manganiello VC, Vaughan M. Agents that increase cyclic AMP inhibit accumulation ofcyclic GMP and depress human monocyte locomotion. J lmmunol 1978; 120: 492-496. Gianeiolo GJ, Snyderman R. Monacyte responsiveness to chemotactic stimuli is a property of a subpopulation of cells that respond to multiple chemoattractants. J Clin Invest1981: 67: 60-68. lntorrisi CE, Schultz M, Shin S, Umana JG, Angel L. Simon El. Evidence from opiate binding studies that heroin acts through its metabolites. Life Sci 1983; 33 (Suppl I): 773-176. MB, Foley KM, Schultz M, Shin S, Woude RW. The pharrnacokinetics of heroin in patients with chronic paid. N Engl I Med 1984; 310: 1213-1217. Jimenez Lbpez A, Olmos Martinez JM, Garcia-Palamo JD, Sanchez Hemandez IJ, De DiosMartin 8, Pedraz Garcia MI, Monocyte chemotaxis: A critical evaluation of two lnturrisi CE,Max

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