effects of fentanyl on cellular immune functions in man

E�ects of fentanyl on cellular immune functions in man

Roland Jacobsa,*, Matthias Karstb, Dirk Scheinichenb, Carmine Bevilacquac,Udo Schneiderc, JoÈ rn Heineb, Manfred Schedlowskid, Reinhold E. Schmidta

aDivision of Clinical Immunology, Hannover Medical School, D-30625, Hannover, GermanybDivision of Anaesthesiology, Hannover Medical School, D-30625, Hannover, Germany

cDivision of Clinical Psychiatry, Hannover Medical School, D-30625, Hannover, GermanydDivision of Medical Psychology, Medical Faculty, University of Essen, D-45122, Essen, Germany

Received 6 January 1999; accepted 29 March 1999

Abstract

In order to analyze the e�ects of the opioid agonist fentanyl on cells of the innate immune system,seven healthy individuals were treated intravenously with the opioid fentanyl and ®ve subjects received aplacebo. Respiratory burst of polymorphonuclear cells (PMNC) and phenotypes of peripheral bloodlymphocytes (PBL) were analyzed from blood samples drawn before, 15 and 30 min after fentanyl orplacebo application. In addition, in vitro e�ects of fentanyl on natural killer (NK) activity was assessed.Fentanyl administration a�ected neither superoxide production of PMNC nor circulating numbers of B-and T-lymphocytes. In contrast, NK cell (CD16+/CD56+) numbers signi®cantly increased in responseto fentanyl. However, no direct in¯uence of fentanyl on NK cell function in vitro could be detected.These results suggest a transient e�ect of fentanyl on NK cell circulation which seemed to be centrallymediated rather than a direct e�ect of this opioid on NK cells. # 1999 International Society forImmunopharmacology. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Opioid; Fentanyl; Lymphocyte subpopulations; Granulocytes; NK cell tra�cking

1. Introduction

NK cells play an important role in the ®rst line of immunologic defence against viral orbacterial infections and tumour cells [1±3]. NK cells are immediately active on site and do not

International Journal of Immunopharmacology 21 (1999) 445±454

0192-0561/99/$20.00 # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights

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PII: S0192-0561(99 )00025-9

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* Corresponding author.E-mail address: [email protected] (R. Jacobs)

require further maturation or sensitization for lysis of potential target cells and, in contrast toT cells, they are not MHC-restricted [4±7]. On the other hand, NK cell activity is triggered byMHC molecules present on target cells [8±10]. Although there is a regulation via MHCproteins, NK cells are attributed to the innate arm of the immune system.NK cells have been shown to respond quickly to physical and psychological stress [11,12].

These stress e�ects on NK cells seem to be mainly mediated via adrenergic mechanisms [13±16]. In addition to catecholamines, endogenous and exogenous opioids have been demonstratedto a�ect innate immune functions, in particular NK cell number and function [17±19]. This isalso of clinical interest since opioids are often applied as anaesthetics for surgical operations.These opioids act via speci®c receptors in the brain, which are also expressed on leukocytes.The e�ects of opioids on white blood cells however, are as yet not well understood. In additionto stress e�ects in¯uencing immune functions, surgical operations are always accompanied bydisruption of the skin enabling pathogens to penetrate the body. Perioperative infections,which are in most cases caused by bacteria, are still a serious clinical problem [20,21]. In thissituation an e�cient immune response is essential. Thus, the aim of this study was to elucidatethe in vivo e�ect of the clinically applied opioid fentanyl on cells of the non-adaptive immunesystem.

2. Experimental procedures

2.1. Subjects

Twelve healthy male volunteers participated in this study. All subjects, aged 27.3 2 4.92years, were free of any current or chronic serious ailments and did not ingest any other drugs.Five subjects were treated with saline as placebo and seven subjects received 0.2 mg/kg BWfentanyl (Janssen-Cilag, Neuss, FRG), a dose commonly applied for induction of balancedanaesthesia. The substances were applied intravenously as one single bolus injection and thesubjects were not aware of whether they received fentanyl or saline. The protocol wasapproved by the Ethical Committee for investigations involving human subjects of theHannover Medical School. For in vitro assays blood samples from seven additional healthycontrols were used.All experiments were conducted at the same time starting at 08:00 h. First, a peripheral port

system (14G Introcan1

) was inserted into a cubital vein. Immediately before (baseline), 15 and30 min after the injection of fentanyl heparinized blood samples were drawn. EDTA blood forwhite blood cell counts and serum for determination of fentanyl concentrations were obtainedat the same time.In addition, four fentanyl-treated subjects who displayed the most pronounced changes in

NK cell numbers in the ®rst experiment were retested in the same setting. However, thesesubjects received the opioid receptor antagonist naloxone (0.4 mg) immediately before fentanylinjection and 0.4 mg were administered again 15 min later.

R. Jacobs et al. / International Journal of Immunopharmacology 21 (1999) 445±454446

2.2. Cardiovascular monitoring

Blood pressure and heart rate were continuously monitored during the whole experimentusing a Sirecust intensive care monitoring equipment (Siemens, MA).

2.3. Fentanyl serum level determination

Serum levels of fentanyl were determined by high pressure liquid chromatography (HPLC)by a commercial laboratory (Gemeinschaftslabor Dr Schiwara, Bremen, Germany).

2.4. Separation of mononuclear cells

Peripheral blood mononuclear cells (PBMC) were separated from 20 ml blood by Ficoll-Hypaque density gradient centrifugation [22]. Collected cells from the interphase were washedthree times in RPMI 1640 and ®nally resuspended in RPMI 1640 supplemented with 10% fetalcalf serum (FCS).

2.5. Separation and activation of polymorphonuclear cells

Heparinized blood was layered on the same volume of Ficoll. After sedimentation for 45 minat room temperature, a leukocyte enriched supernatant was obtained. WBC were activated aspreviously described in detail [23]. Brie¯y, separated cells were adjusted to 5� 105 cells/ml withPBS; 30 ml of this suspension and 15 ml Dihydrorhodamine-123 (DHR) were added to 1 ml ofwarm PBS. After 20 min incubation at 378C with PMA, bacteria (E. coli ) or a mixture of TNFand FMLP, the reaction was terminated by transferring the samples onto ice. Viabilitydiscrimination was performed by addition of propidium iodide (PI) just before FACS analysis.In activated PMNC dihydrorhodamine is oxidated to rhodamine in the presence of H2O2. The¯uorescence intensity of rhodamine as assessed by FACS is proportional to the amount ofproduced H2O2 [24].

2.6. Phenotypic analyses

Freshly isolated PBMC were stained as previously described in detail [25]. Brie¯y, 1 to 3�105 cells/well were incubated with murine monoclonal antibodies at an optimal dilution for30 min. Isotype matched mab were used as negative controls. Nonspeci®c binding waseliminated by mixing the samples with a 1:5 solution of a commercial i.v. IgG (Intraglobin

1

,Biotest, Frankfurt, FRG). After three washes samples were resuspended and analyzed using aFACScan (Becton Dickinson, Heidelberg, FRG).

2.7. NK activity assay

Standard 4 h 51Chromium release cytotoxicity assays were performed using fresh PBMC intriplicates at four e�ector to target (E/T) ratios (60:1, 30:1, 15:1, and 7.5:1) in V-bottommicrotiter plates with 5� 103 51Cr-labelled K562 target cells per well as previously described in

R. Jacobs et al. / International Journal of Immunopharmacology 21 (1999) 445±454 447

detail [25]. Medium for cytotoxicity assays was RPMI 1640 supplemented with 5% FCS and1% penicillin-streptomycin. E�ector cells were preincubated with medium or fentanyl at twoconcentrations (20 and 40 mM, respectively) for 45 min. Targets were then added and theassays were incubated for another four hours in the presence of the substances. Speci®ccytotoxicity was measured following a 4 h incubation at 378C by determining released 51Cr.Background values were determined by incubating target cells without e�ector cells. Maximalvalues were obtained by lysing target cells with Triton X-100 (Sigma, St Louis, MO). Speci®clysis was calculated by:

experimental releaseÿ spontaneous release

maximum releaseÿ spontaneous release� 100

2.8. Statistical analyses

Data were analyzed with ANOVA with repeated measure as previously described in detail[16]. When not otherwise stated, signi®cant interaction e�ects (group� time) are given.

2.9. Materials

All monoclonal antibodies (mab) used for this study are commercially available, and werelabelled with either ¯uorescein-isothiocyanate (FITC) or phycoerythrin (PE) with the followingspeci®cities: W6/32 (FITC; sera-lab, Sussex, GB) binds to HLA-A, B, C present on allnucleated cells. CD2 (FITC; Dako, Hamburg, FRG) is an antigen detectable on T and NKcells. CD3 (PE; Immunotech, Hamburg, FRG) is a protein associated with the T cell receptorand reacts with all mature T cells and a subset of non-MHC-restricted T cells. CD4 (FITC;Dako, Hamburg, FRG) is expressed by T cells with helper/inducer function. CD8 (PE; Dako,Hamburg, FRG) reacts with T suppressor/cytotoxic cells. CD14 (FITC; Immunotech,

Fig. 1. E�ects of fentanyl on cardiovascular parameters. Heart rate (*, right y-axis), as well as systolic (Q) and

diastolic (R) blood pressure did not change signi®cantly from 10 min before to 45 min after injection of fentanyl.The arrows indicate the time points when blood samples for immunological analyses were drawn. Mean values andSEM (n=7) are depicted.


Hamburg, FRG) is the LPS receptor mainly on monocytes and granulocytes. CD16 (FITC;Dako, Hamburg, FRG) is a synonym for FcgRIII expressed on NK cells and granulocytes.CD19 (PE; Immunotech, Hamburg, FRG) is an antigen present on all mature B cells. CD56(PE; Becton & Dickinson, Heidelberg, FRG) is expressed on all NK cells and non-MHC-restricted T cells in peripheral blood.

3. Results

3.1. Cardiovascular variables

Cardiovascular data were monitored from 10 min before to 105 min after a single fentanylinjection using an intensive care monitoring equipment. Fentanyl did not a�ect heart rate orblood pressure signi®cantly (Fig. 1).

3.2. Fentanyl serum levels

Fentanyl serum levels were analyzed 15 and 30 min after drug administration. After 15 min1.91 ng/ml (21.17) fentanyl was detected, subsequently decreasing to 0.67 ng/ml (20.23) 30 minafter injection. These fentanyl plasma concentrations are comparable to patients' plasma levelswhen fentanyl is used as analgesic during anaesthesia [26].

3.3. PMNC function

PMNC from fentanyl treated subjects were activated with three di�erent stimuli (PMA, E.coli, TNF/FMLP) and analyzed by FACS. Percentages of ¯uorescent PMNC did not di�ersigni®cantly at all three time points for each stimulus indicating that respiratory burstmachinery was not a�ected by fentanyl administration. Moreover, the ¯uorescence intensity

Fig. 2. Changes of NK cell frequency in response to fentanyl. NK cell numbers (CD56+/CD16+) 15 min before,and 30 min after fentanyl injection q (0.2 mg/kg BW, n=7) or saline Q (n=5).


(mean channels) indicating the activity of the single cells was not a�ected by fentanyl (data notshown).

3.4. Phenotypic analyses

Injection of fentanyl induced a moderate increase in leukocyte count, mainly due to changesin lymphocyte numbers. There was a slight increase in PMNC counts (<10%) 30 min afterinjection. However, lymphocyte numbers increased signi®cantly by 30% (1739 vs 2260/F=5, P< 0.05) 15 min after drug administration. When lymphocyte changes were analyzed in moredetail, ¯ow cytometry analyses showed that absolute numbers of peripheral T (CD3+, CD4+)and B cells (CD19+) were not a�ected by fentanyl (data not shown). However, NK cellnumbers (CD16+/CD56+) increased by more than 250% (177 cells/ml vs 456 cells/ml) 15 minafter fentanyl injection (Fig. 2) and slightly decreased 30 min after injection (216% of thebaseline level; =177 cells/ml vs 382 cells/ml) (F=3.5, P<0.05).Since NK cell numbers more than doubled after fentanyl injection, further analyses were

undertaken to determine whether this enhanced NK cell circulation is mediated via opioidreceptors. Four subjects showing the most pronounced fentanyl induced increase in NK cellnumbers were retested with the same experimental paradigm. However, this time the opioidantagonist naloxone was administered prior to fentanyl injection. This pretreatment withnaloxone revealed di�erential e�ects. In two subjects naloxone completely abrogated thefentanyl-induced increase in NK cell numbers. In two other individuals increase in NK cellnumbers was not blocked by naloxone (Fig. 3).

3.5. In vitro e�ects on NK-activity

Circulation of NK cells was clearly in¯uenced by fentanyl in vivo. Whether this is a directreceptor mediated e�ect on NK cells or mediated by indirect mechanisms is not distinguishablein vivo. This prompted us to examine whether fentanyl can in¯uence NK cell function directly.

Fig. 3. NK cell numbers after naloxone blockade. NK cell numbers (CD16+/CD56+) in naloxone pretreated

subjects 15 min before and 30 min after injection of fentanyl are shown. Fentanyl induced increases in NK cellnumbers were completely abrogated in two subjects (open symbols, solid lines). However, in two other individualsnaloxone failed to antagonize NK cell changes after fentanyl injection (solid symbols and lines).


Therefore, fentanyl e�ects on cytotoxic capability of NK cells were tested in vitro. NK-assayswere performed with PBMC from seven healthy donors against standard target cells (K562 cellline). E�ector cells were preincubated with medium or fentanyl at two concentrations (20 and40 mM, respectively) and the assays were performed in the presence of the substances. In allcases tested, fentanyl did not in¯uence the cytotoxic activity of NK cells (data not shown).

4. Discussion

Perioperative infections are still a serious problem of surgical intervention. Duringanaesthesia opioids are commonly used as analgesic substances. These drugs act via speci®c d,k, and m receptors on cells of the nervous system. On the other hand it has been demonstratedthat cells of the human hematopoietic system also express d, k, and m opioid receptors [27±29].It is already known that these receptors can transmit signals into di�erent types of cellsinducing Ca2+ mobilization and/or cAMP metabolism [30,31]. However, the particular role ofthese molecules on white blood cells is still unknown. Therefore, the aim of this study was toinvestigate whether a potent opioid agonist (fentanyl) is a�ecting the circulation and/orfunction of peripheral leukocytes. Results of this study demonstrate that fentanyl did not a�ectgranulocyte function or circulation of T- and B-lymphocyte subsets. However, fentanylsigni®cantly increased NK cell numbers. This enhanced NK cell circulation seems to becentrally mediated since fentanyl did not a�ect NK activity in vitro. In addition, pretreatmentwith the opioid antagonist naloxone prevented the fentanyl-induced NK cell increase in twosubjects but was ine�ective in two other subjects.In these experiments we did not observe any in¯uence of fentanyl on PMNC. Granulocyte

counts did not change due to fentanyl nor was there a regulatory in¯uence of the drug upongranulocyte activity as shown by analyzing the respiratory burst ex vivo. This result is in linewith previous observations where fentanyl did not a�ect granulocyte functions likephagocytosis, killing of bacteria, or generation of superoxide anions [32,33].Fentanyl administration did not change the circulation of T- or B-lymphocytes. NK cell

tra�cking, however, was clearly a�ected by this opioid. NK cell numbers more than doubled(258%) shortly after fentanyl injection and slightly decreased thereafter. Surprisingly, this e�ectwas also seen after administration of opioid receptor antagonist naloxone in two subjects,whereas NK cell increases in two other individuals were totally abolished by naloxone. Themechanisms of this opioid-induced increase in peripheral NK cell numbers are still unknown.In previous studies we observed a similar pronounced increase of NK cell numbers after acutestress [34] or catecholamine infusion [15,16] which are mediated via b-adrenoceptors [14].Under these conditions NK cells are quickly recruited from lymphoid organs such as the spleenand in particular released from endothelia of the blood vessels, the so-called marginal pool.The detachment of NK lymphocytes is dependent on b2 adrenoceptors expressed on these cells[35]. Similar conditions may be responsible for the quick mobilization of NK cells observed inthis study. However, cardiovascular data did not signi®cantly change after fentanyl application.In addition, fentanyl does not a�ect peripheral catecholamine concentrations [36,37].Therefore, it is unlikely that fentanyl-induced increases in circulating catecholamines areresponsible for this quick NK cell mobilization.


NK activity is a function proportional to the e�ector:target (E/T) ratio. Increased NK cellcounts are therefore also accompanied by enhanced speci®c lysis of target cells. To elucidatewhether there is additionally a more direct e�ect of fentanyl on NK activity we performed 51Crcytotoxicity assays. The standard assay was varied by performing the test in the presence offentanyl with preincubated e�ector cells. Under these conditions we did not ®nd any in¯uenceof fentanyl on NK activity.The underlying mechanisms of opioid-induced changes of NK activity in vivo are still

unclear. A clear suppression of splenic ex vivo NK activity in rats that can be blocked bynaltrexone has been described [38,39]. In humans, the suppression of NK activity could beprolonged with increasing doses of fentanyl or morphine [40,41]. In vitro suppression of NKactivity by morphine was only achievable with very high doses of the opioid and was notnaltrexone-reversible suggesting that immunomodulary e�ects are not directly mediated by theopioid receptors on lymphocytes. In addition, s.c. doses of N-methylnaltrexone that do notgain access to the central nervous system (CNS) do not antagonize the morphine-mediatede�ects on lymphocytes whereas higher doses of the antagonist enter the CNS and block theimmunomodulatory e�ects of morphine [42]. Further evidence for a centrally mediated route ofopioid induced immunomodulation was demonstrated by suppression of NK activity bymicroinjection of opioids into the brain of rats. The suppressive e�ects could be blocked byprior administration of the opiate antagonist naltrexone [43]. However, the e�ects of opioidson lymphocyte functions seem to be concentration dependent [44]. In addition, it is believedthat the e�ects of opioid peptides on the immune response are highly dependent on theactivation status of lymphocytes, inhibiting functions of activated lymphocytes whilststimulating resting lymphocytes [45]. This dependency of the activation status of thelymphocytes may explain other observations where endogenous opioids did not induce anye�ects on lymphocyte proliferation in 50% of the blood donors [44].In summary, the opioid fentanyl induced immediate pronounced increases in circulating NK

cell numbers which could be antagonized with the opioid blocker naloxone. However, naloxonewas only e�ective in 50% of the subjects. In contrast, fentanyl did not a�ect NK activity invitro. Further experiments will focus on the lymphocyte-endothelium interactions to unravelthe mechanisms responsible for opioid-induced NK cell recruitment.

Acknowledgements

The authors would like to thank Dr Michael Exton for carefully reviewing the manuscript.This study was supported in part by Grant I/72031 from the Volkswagen-Stiftung.

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effects of fentanyl on cellular immune functions in man

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