Methionine-enkephalin andleucine-enkephalin increaseinterleukin-1b release in mixedglia cultures

Jan Kowalski, Bo _zzena Gabryel, Krzysztof Łabuzek, Zbigniew S. Herman

Department of Clinical Pharmacology, Medical University of Silesia, Medyk�oow, Poland

Summary Interleukin-1b (IL-1b) is synthesized in the brain in response to LPS. Excessive IL-1b expression isobserved in neurodegenerative diseases. The aim of this study was to evaluate the effects of methionine-en-kephalin (ME) and leucine-enkephalin (LE) on the baseline and LPS-activated release of IL-1b in rat mixed gliacultures. ME and LE increased LPS-induced IL-1b release, which was not blocked by naloxone. Both ME and LEincreased the baseline release of IL-1b, which was completely blocked by naloxone pretreatment. Mixed gliacultures deprived of microglia (by shaking and incubating with LL-leucine methyl ester) did not release IL-1b, whichindicates microglia as a source of the changes in IL-1b release. The results of the study suggest that neurons mayregulate glial activity through releasing enkephalins. ª 2002 Elsevier Science Ltd. All rights reserved.

INTRODUCTION

Interleukin 1-b (IL-1b) is found in two types of glialcells, namely microglia and astrocytes (Frei et al., 1988;Koenig et al., 1990), the former of which is the mainsource of this cytokine (Giulian and Corpuz, 1993).Microglia, like macrophages, originates from mononu-clear myeloid progenitors. In the CNS, they reside in aramified, quiescent state, but they can easily migrate toareas of inflammation in the CNS, where they releasenumerous cytokines and other molecules involved ininflammatory processes. Lipopolysaccharide (LPS) stim-ulates in vitro cultured microglial cells to release cyto-kines, such as TNF-a, IL-1b and IL-6 (Kong et al., 1997).LPS injections combined with interferon-c induce IL-1bmRNA expression in rat brain (Higgins and Olschowka,1991). IL-1b secreted by the CNS may increase the re-lease of norepinephrine, dopamine (Zalcman et al.,1994) and nerve growth factor (Leibrock et al., 1989).However, the knowledge of the regulatory mechanisms

of cerebral IL-1b release is relatively poor. Methionine-enkephalin (ME) and leucine-enkephalin (LE) are syn-thesized in the brain and peripheral tissues (Bhargavaet al., 1988). In many studies, opioid peptides havebeen found to affect the activity of the immune system.Both ME and LE increase (i) the activity of NK cells inhumans (Faith et al., 1984) and mice (Kowalski, 1997),(ii) the activity of lymphocytes in humans (Haegi et al.,1990) and mice (Kowalski, 1998) and (iii) the release ofIL-6 by mouse macrophages (Kowalski et al., 2000;Zhong et al., 1995). Despite these reports, the mecha-nisms of action of opioid peptides, such as ME and LE,on the activity of glial cells are still not fully under-stood (Das et al., 1995).The present study focused on the modulatory effects of

ME and LE on the release of IL1-b, a proinflammatorycytokine, in rat mixed glia cultures.

MATERIAL AND METHODS

Cell cultures

Primary mixed glia cultures were prepared from thecerebral cortices of newborn Wistar rats (Kong et al.,1997). Briefly, their brains were excised aseptically andseparated from the blood vessels and membranes.

Neuropeptides (2002) 36 (6), 401–406

ª 2002 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0143-4179(02)00109-9

Received 1 June 2002

Accepted 21 September 2002

Correspondence to: Dr. Jan Kowalski, Department of Clinical Pharmacology,

Medical University of Silesia, 40-752 Katowice, Medyk�oow 18, Poland. Tel./Fax:

+48-32-2523902; E-mail: farmklin@infomed.slam.katowice.pl

401

Cerebral cortical tissue was dissociated by trituration inice-cold medium containing Dulbecco’s/DMEM mediasupplemented with 10% FCS, 2mM glutamine, 100UI/ml penicillin, 100lg=ml streptomycin and 5lg=ml fun-gizone. The suspension was passed sequentially througha cell strainer (Becton–Dickinson) 70 and 40lm mesh.Then the cell concentration of the cell suspension wasadjusted to 1� 106 cells=ml and a volume of 0.1ml waspoured into each well of 96-well Becton–Dickinson tis-sue culture plates. The medium was replenished 1 and 4days after plating and changed every 3 days thereafter.After plating, the cells were cultured for 13–15 daysuntil confluence. The cultures were stained with anantibody against glial fibrillary acetic protein (GFAP)(SIGMA), a specific marker for astrocytes. The antibodylabelled 70–75% of the cells. About 20% cells in cul-tures reacted with Ricinus Communis Agglutinin-1, alectin that binds to surface glycoproteins on microglia(Vector, Burlingame, CA, USA). No neurons, as con-firmed by an immunocytochemical staining methodusing monoclonal antibodies against MAP-2 (Promega,USA), were detected.

Treatment of glial cell cultures

Prior to the experiment, the cells were incubated over-night with fresh medium. In order to determine the ef-fect of ME and LE on IL-1 release by mixed glialcultures, various concentrations of ME or LE (10�4 to10�14) either alone or together with LPS (100ng/ml)were added and the supernatants were collected 24hlater. In some experiments, the cells were incubatedwith LPS and opioids for 48h. To study a potentialopioid mechanism of ME and LE action, the cultureswere exposed to naloxone ð1� 10�5MÞ for 30min be-fore ME, LE/ or LPS was added, and the supernatantswere harvested after 24h.

Cytokine assay

Interleukin-1b levels were assayed using a rat IL-1bELISA Kit (R&D) according to manufacturer’s recom-mendation. The optical density of each well was mea-sured at 450nm using a UV microplate reader (DynexTechnologies). The detection limit of this assay wasdetermined to be 5pg/ml. The intra assay precision CVwas 8.7%.

Statistics

Results were analyzed with a one-way analysis of vari-ance test (ANOVA) followed by Bonferroni’s MultipleComparison test. All statistical procedures were per-formed using a Graph Pad Prism software (version 2.01).

The study was approved by the Ethical Committee ofthe Medical University of Silesia.

RESULTS

Effects of different LPS concentrations onIL-1b release in mixed glia cultures

To study the effect of LPS on IL-1b production by glialcells, primary mixed glia cultures were treated withvarious concentrations of LPS (10, 100 and 1000ng/ml).The culture supernatant were collected after 24h ofstimulation. LPS increased IL-1b levels in a concentration-dependent manner, indicating LPS stimulated glial cellsto produce IL-1b (Fig. 1).

Time course of LPS on IL-1b release

LPS at a concentration of 100ng/ml enhanced IL-1brelease after 8-, 24- and 72h of incubation (Fig. 2).

Effects of ME and LE on LPS-induced secretion of IL-1b

To determine whether opioid peptides are involved inCNS inflammatory process, we examined the effects ofME and LE on mixed glia cultures exposed to LPS. LE andME at concentrations of 10�6, 10�8, 10�10 and 10�12 in-creased IL-1b release. The maximum effect was observedfor LE and ME at a concentration of 10�10M. Naloxonepretreatment reversed partially the LE- and ME-inducedeffect (Fig. 3).In order to determine the duration of the enhanced IL-

1b release induced by enkephalins, we investigated theeffect of ME and LE at concentrations of 10�6, 10�8, 10�10

and 10�12 on IL-1b release in mixed glial cultures stimu-lated with 100ng/ml LPS for 48h. There were no differ-ences between the control cultures and those containingeither ME or LE (data not shown).

Effects of naloxone on LPS-induced release of IL-b

When given at a concentration of 1� 10�5, naloxonecompletely reversed IL-1b release induced by LPS given at100ng/ml (Fig. 4).

Effects of ME and LE on baseline IL-1b release

To determine how opioid peptides influence glial activity,we evaluated IL-1b release in mixed glia cultures exposedto LE or ME. LE at concentrations of 10�8, 10�10 and 10�12

augmented IL-1b release, and naloxone pretreatmentcompletely abolished the LE-induced effect. ME at con-centrations of 10�8 and 10�10M increased IL-1b release,and naloxone completely blocked this effect (Fig. 5).

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In order to determine whether astrocytes or microgliawere responsible for these results, petri dishes containing14-day mixed glia cultures were placed in an orbitalshaker for 5h (in these conditions microglial cells aredetached from the layer of astrocytes). The medium withdetached cells was replaced with medium containing l-leucine methyl ester, which destroys microglia but notastrocytes (Chao et al., 1995). After 24h, the medium wasremoved, the petri dishes were rinsed 5 times and filledwith medium supplemented with 1lg=ml LPS and incu-bated for another 24h. The medium from mixed gliacultures that were deprived of microglia did not contain

IL-1b, which suggests that it was microglia that releasedIL-1b after ME and LE stimulation (data not shown).

DISCUSSION

IL-1b, produced and released by glial cells, is involvedin the pathogenesis of many CNS diseases such asinflammatory conditions, multiple sclerosis, Parkinson’sdisease, AIDS and Alzheimer�s disease (Dickson et al.,1993; Gijbels et al., 1990; Hofman et al., 1989; Mogi et al.,1994; Tyor et al., 1992). Thus it is important to determine

Fig. 2 Time course of IL-1b release by LPS. Mixed glia cultures were incubated in medium alone or medium containing LPS (100 ng/ml).At indicated times, supernatants were assayed for IL-1b. Each bar represents the mean � SE of two experiments, each with four determinations.

Fig. 1 Concentration-dependent effects of LPS on the production of IL-1b in mixed glia cultures. The cultures were incubated with theindicated concentrations of LPS for 24 h. Each bar represents the mean � SE of two experiments, each with four determinations.

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what regulatory mechanisms are responsible for its pro-duction and release. There are many compounds thatstimulate glial cells to produce IL-1b. One of them is LPS,a bacterial endotoxin widely used in studies of experi-mental infections or inflammatory conditions. It has beenshown that the levels of endogenous opioid peptides in-creased in infection (Merill and Anderson, 1987), whichsuggests that enkephalins affect the course of infectionsand inflammatory conditions.

In this study, we evaluated the effects of ME and LEon the baseline and LPS-induced release of IL-1b in ratnewborn glial cell cultures. We found that ME and LEincreased IL-1b release by LPS-induced glial cells, whichdisagrees with a report by Das et al. (1995), in which MEhad no modulatory effect on LPS-stimulated microglia.However, Das et al. (1995) stimulated mixed brain cellcultures, but not mixed glia cultures, which were usedin our study. In other reports, the opioid peptides

Fig. 3 Concentration-dependent effects of LE or ME with LPS (100 ng/ml) on the release of IL-1b in mixed glia cultures after 24 h of exposure.The values are the means � SE of four determinations in each of two independent experiments. N – naloxone at concentration 10�5 M added30 min prior to LE or ME treatment. Significantly different from the vehicle treated group (LPS + medium) with ANOVA followed by Bonferroni�sMultiple Comparison test: �, P < 0:05. Naloxone pretreatment did not change significantly LPS + enkephalin-induced increase of IL-1b release.

Fig. 4 The effect of naloxone on the LPS-induced enhancement of IL-1b release. The cultures were incubated with 100 ng/ml LPSfor 24 h. Naloxone at concentration 10�5 M was added 30 min prior to LPS treatment. Each bar represents the mean � SE of twoexperiments, each with four determinations. Significantly different from the medium treated group with ANOVA followed by Bonferroni�sMultiple Comparison test:�, P < 0:001. Significantly different from LPS treated group; #, P < 0:001.

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b-endorphin and LE markedly increased LPS- or silica-induced IL-1 production and release by mouse bonemarrow macrophages (Apte et al., 1990), and ME in-creased IL-6 release by mouse peritoneal macrophagesactivated with LPS (Zhong et al., 1995).In this study, IL-1b release increased rapidly after

adding ME and LE to glial cell cultures not stimulatedwith LPS. Similar findings were reported by other authors.Das et al. (1995) found an ME-induced increase in mi-croglial IL-1b release in mixed brain cell cultures, andChao et al. (1995) found that dynorphin (1–13) increasedIL-6 and TNF-a release. As opioid receptors are located onthe surface of microglial and astrocytic cells (Dobreniset al., 1995; Low et al., 1992; Eriksson et al., 1990), it islikely that ME and LE directly activate these cells.We noted that naloxone completely abolished the ME

and LE-induced increase in IL-1b release, indicating thatclassic opioid receptors mediate this effect. In contrast,naloxone only slightly reduced the ME- and LE-inducedincrease in LPS-stimulated glial IL-1b release. It is notknown why naloxone blocks the effect of enkephalins oncells unstimulated with LPS, while it has no effect on LPS-stimulated cells. Other studies of the specificity of glialactivation by opioids also gave ambiguous results. Nal-oxone only partly reversed ME-induced microglial acti-vation (an increase in IL-1b release) (Das et al., 1995),and, on the other hand, naloxone and b-funaltrexaminecompletely abolished morphine- and LPS-induced mi-croglial activation (i.e., an increase in TNF-a release)(Chao et al., 1994). Furthermore, we observed, as did Daset al. (1995), that naloxone completely blocked LPS-

induced IL-1b release, which shows that endogenousenkephalins released by activated astrocytes in mixedglial cultures mediate this effect (Low et al., 1992, Negroet al., 1992) and suggests that enkephalins are involvedin glia–glia communication.We observed that enkephalins transiently enhance

IL-1b release, for their effect disappeared after 48h ofincubation. It seems that the duration of the enhanced IL-1b release depends, among other things, on the type ofcytokine, because Chao et al. (1995) found that, dynor-phin increased TNF-a release after 8h while IL-6 releaseincreased after 72, 96 and 120h.IL-1b is a cytokine that is excessively expressed in such

neurodegenerative diseases as multiple sclerosis, Alzhei-mer�s disease and Parkinson�s disease. The levels ofendogenous enkephalins are elevated in stress andinfection, which may unfavorably affect, via modulatingIL-1b release, the course of neurodegenerative diseases.In conclusion, it is likely that endogenous enkephalins

released by neurons and, to a lesser extent, by gliaregulate glial cell activity in the CNS.

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Fig. 5 The effects of LE or ME alone on the IL-1b production in mixed glia cell cultures after 24 h of exposure. The values are themeans � SE of four measurements in each two independent experiments. N – naloxone ð10�5 MÞ added 30 min prior to LE or ME treatment.Significantly different from the medium treated group with ANOVA followed by Bonferroni�s Multiple Comparison test; �, P < 0:05; #, comparedto the corresponding LE or ME concentration, P < 0:05.

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