antagonist in clonal cultures of mouse bone marrow cells

Biomed & Pharnmcother 0 Elsevier, Paris

(1995)

49, 27-31

Original

Naloxone

behaves

as opioid agonist/antagonist in clonal of mouse bone marrow cells

L Kriianac-Bengez ‘Ruder

BoSkoviC

Institute,

POB

1016,

41001

(Received

Zagreb, 26 January

I, M BoraniC’, Croatia; 1994;

2Paterson accepted

article

cultures

NG Testa

Institute

for

Cancer

Research,

Manchester,

lJK

15 June 1994)

Summary - The opioid peptide methionine (Met)-enkephalin and the opioid-receptor blocking agent naloxone were added to unseparated or to progenitor-enriched cell suspensions of mouse bone marrow before assay in clonal cultures. Bone marrow samples harvested at 18:OO hours produced more granulocyte-macrophage (GM) colonies than the 06:OO hour samples, and were more sensitive to the proliferation inhibition by both agents. Additive inhibitory effects of naloxone with the enkephalin were occasionally seen. Thus, in this experimental system, naloxone could behave as an opioid agonist. However, there were examples as a true opioid antagonist. Significant of naloxone diminishing (blocking) the suppressive effect of the enkephalin, naloxone/enkephalin interactions occurred in opioid-sensitive (18.00 h) samples of unseparated bone marrow. The interactions were virtually absent in progenitor cell-enriched populations, indicating a significant role of accessory cells in opioidergic regulation of hematopoietic progenitors. naxolone

I enkephalins

I hone

marrow

cells I cell proliferation

INTRODUCTION In a preceding article, we described inhibitory effects of opioid peptides methionine (Met)- and leucine (Leu)-enkephalin on mouse bone marrow progenitor cells, dependent on circadian variation in their proliferative activity [7]. Modulatory effects were also seen with a progenitor cell- enriched population of marrow cells, obtained by means of fluorescence-activated cell sorting (FACS). Continuing that work, we wished to see whether the effects of opioid peptides on hematopoietic cells were mediated by specific opioid mechanisms, and to that end used naloxone as a receptor-blocking agent. Naloxone is able to antagonize the effects of opioid peptides on macrophage effector functions [4], lymphocyte proliferation [6], chemotaxis of peripheral blood leukocytes [18], and O2 production by the polymorphonuclears [ 11, 161. Specific binding of naloxone to opioid receptors has been demonstrated on the polymorphonuclears [3] and lymphocytes [9].

We treated suspensions of mouse bone marrow cells (unseparated or progenitor cell-enriched) with naloxone and/or Met-enkephalin before assaying their growth in clonal cultures.

MATERIALS

AND METHODS

Mice Male (C57Bl x DBAR)Fl mice eight to ten weeks old were used as donors of bone marrow. They were maintained in a controlled environment with a regular 12hour dark/light cycle, and sacrificed at fixed times of the cycle performed

(06:OO h or IS:00 h). The experiments were in the

Bone marrow

summer

(July,

August).

cells

Bone marrow cells were obtained by flushing the femoral shafts with chilled Iscove’s medium. Cells from six femora were pooled for the clonal assays, and from 40 for FACS purification. Cell suspensions for the

28

L Krizanac-Berger

FACS procedure were collected at 06:OO h, and were depletedof the erythrocytes by lysis in ammoniumchloride (0.83%, IO minutes).Bone marrow cells were labeled with wheat germ agglutinin (WGA) and fluorescein

isothiocyanate

(FITC),

and were separated

initially on a metrizamidedensity cut (1.080 g/cm3). They were then subjectedto a double FACS separation, as described

by Lord and Spooncer

[8].

Clonal assays The cells were grown

in Iscove’s

medium

containing

20% pretestedfetal calf serum(Sigma) and 0.33% agar (Difco). The medium was supplemented with 2% rIL-3 (Biogen; specific activity 100 LJ/mL). One milliliter of the final mixture was pipetted into 35-mm Petri dishes (Falcon). Unseparatedbone marrow cells were seeded at a concentration of 5 x lo4 cells/ml, and the enriched

progenitor population at I O3cells/ml. Granulocyte-macrophage(GM) colonies(> 50 cells) where countedunder 20x magnification after 7 days of culture for unseparatedmarrow, or after 14 days for enriched populations. Enkephalin and naloxone Thesesubstanceswere kept in dry form at -20°C (enkephalin) or -4°C (naloxone). Appropriate amounts were dissolved in Iscove’s medium immediately before use, and serial dilutions were made. Aliquots of these were added to samples of the cell suspension (5% of the volume) to achieve the molar concentrations specified in the Results section. Control samples received the same volume of medium. The cells were then incubated for 20 minutes.If the enkephalinwasusedafter naloxone, the incubation was continued for 20 minutes.

The cell suspensions were then added(10% of the final volume) to the complete culture medium,mixed with agar, and plated.

RESULTS Effects of Met-enkephalin and naloxone bone marrow ceil suspensions

Each sample was cultured in triplicate. The results from repeated experiments were pooled, and the number of

samplesper group varied from 7 to 27. The resultswere expressedas colony counts per plate, and analyzed by nonparametricstatistical methods (the Kruskal-Wallis analysisof variance, and if significant, by the MannWhitney U test for two samples).The proportionswere was set

in

The cells were incubated with Met-enkephalin (lo-“, IO-“, lo-‘, 10mh M), naloxone (lo-‘“-IO-‘M), or naloxone preceding the enkephalin in a IO’-, IO’-, and 103-fold molar excess. The enkephalin concentrations were chosen so as to include the “physiological” range (lo-‘” and lo-” M) and values below and above it. The incidence of granulocyte-macrophage (GM) colonies in control samples harvested at 18.00 h was approximately 30% higher than that of the 06.00 h samples (table I). Incubation of the cells with Met-enkephalin resulted in reduction of the colony numbers, without a clear dose-response relationship. The 18.00 h samples appeared to be more sensitive to the proliferation inhibition, in that the inhibitory effect was seen with all four concentrations of the enkephalin, whereas only two higher ones (lo-” and 10e6M) caused significant reduction in the 06.00 h samples (table I). Naloxone also interfered with colony formation, reducing the colony count by lo-50% (average, 30%), also without a clear dose-response pattern. All twelve concentrations used were effective in the 18.00 h samples (table II), and 9 of 12 were effective in the 06.00 h samples (data not shown). Incubation of the cells with naloxone followed by Met-enkephalin also reduced the colony count; however, interactions of the two agents occurred, Table I. Effect of methionine-enkephalin on the number of granulocyte-macrophage (GM) colonies in clonal cultures of unseparated mouse bone marrow cells harvested at 06.00 h or 18.00 h. Harvest (06.00 h)

Enkephalin concentrarion i-log M)

Statistics

tested by the n2 test. The level of significance at p i 0.05.

rf nl

None

(controls)

29 (25-3

15 12 9 6

30 11 23 9

5 x 10’ cells per plate. of plates per group. * Significant difference groups.

Harvest (18.00 h)

I ): 38

(24-33): (10-30); (19-29); (7-12);

Medians, (p < 0.05)

9 8 18’ 7* interquartile from

42 (38-45);

38*

22 21 21 25

(14-27); (17-26); (16-27); (17-42);

4‘ 8’ 12’ 6’

ranges,

and no.

the respective

control

Naloxone

in bone

marrow

29

cultures

and lo-‘? M) was ineffective. In six groups above that range, there were two significant interactions of naloxone and Met-enkephalin (blocking effects) (data not shown).

particularly in the 18.00 h samples (table II). In five of twelve combinations, the joint effect of both agents on the colony count was significantly different from the effect of Met-enkephalin alone, as judged by Kruskall-Wallis analysis of variance and the Mann-Whitney U test for two samples. The incidence of the interactions (5 of 12, ie, 40%) was statistically significant (x’ = 6.153, p = 0.025). In four of five points, naloxone diminished (partially blocked) the suppressive effect of Met-enkephalin, and in one example (at the highest dilutions of both agents), the joint effect was additive. In bone marrow samples harvested at 06.00 h, Met-enkephalin at the lower concentrations ( 10mL5

Effect of Met-enkephalin and naloxone on a population eriched for progenitor cells That population produced, in average, 25 colonies per 10’ plated cells (table III). It should be noted that logistic reasons required the bone marrow samples for the FACS procedure to be harvested in the morning (06.00 h), that is, when the cells are, as shown earlier, less sensitive to opioid actions. Incubation with Met-enkephalin lo-”

Table 11. Effect of methionine-enkephalin, naloxone, or both agents on the number of granulocyte nacrophage (GM) colonies in cultures of unseparated hone marrow cells harvested at 18.00 h. Median colony counts per plate (5 x lo8 cells). interquartile ranges, and the no. of samples per group. Pooled data of three experiments (methionine-enkephalin with or without naloxone) and six experiments (naloxone alone). No

treatment (controls)

Enkephalin

and (-10~

42 (38-45);

Significant * control

Naloxone

Ml

(-la

Naloxone enkephalin

Mi

15

22 (14-27);

14*

14 13 12

29 (22-30); 26 (24-28); 30 (24-35);

21* 25‘ 24*

14 (1 I-18); 26 (24-28); 27 (25-30);

12

21 (17-26);

8*

11 10 9

26 (1740); 26 (23-35); 31 (23-38);

15’ 13‘ 13’

26 (13-36); 25 (21-32); 26 (21-38):

7* 18: 7’

9

21 (16-27);

12*

8 7 6

28 (25-32); 25 (23-29); 28 (21-34);

24’ 26’ 24’

21 (19-23); 27 (26-31); 27 (21-32):

14’ 14*.+ 15*,+

6

25 (17-42);

16*

5 4 3

26 (1640); 24 (18-34); 27 (22-34);

15’ 14* 13’

24 (15-35;; 21 (15-31); 28 (23-36);

8* 8’ 8*

38

13*.+ 14*+ 14”

differe+nce (p < 0.05 or better) from: group; enkephalin-treated group (Mann-Whitney).

Table III. Effect of methionine-enkephalin, naloxone, or both agents on the number of granulocyte-macrophage (GM) colonies in cultures of enriched progenitor cells harvested at 18.00 h. Median colony counts per IO3 plated cells, interquartile ranges, and the no. of samples per group. Pooled data of three experiments. No

treatment (controls)

Enkephalin

Naloxone and

(-1o.q M)

25 (20-28);

Significant control

(-10~

Naloxe enkephalin

M)

12

14 (10-16);

9’

11 10 9

15 ( 8-22): 20 (16-25): 20 (17-25);

13’ 15* 13’

14 ( 8-16); 13 ( 3-26); 13 (10-19);

7* 9’ 8’

9

II (10-14):

9’

8 7 6

9 ( 3-13): 12 ( 9-22); 17 (11-18);

13’ 13 12’

15 ( 5-21); I5 (12-19); 18 (15-21);

9* 8’ 9*.+

13

differtnce (p < 0.05) from: group; enkephalin-treated group

(Mann-Whitney).

30

L Kriaanac-Bergez

and 10e9 M reduced the colony count by 45 and 55%, respectively, of the control value. Naloxone (10-“-10-6 M) reduced the colony count by 2070%, without clear dose-effect relationship. There was only one example of significant interaction of naloxone with Met-enkephalin (a blocking), in the highest concentrations of the agents.

DISCUSSION Confirming our previous report [7], opioid peptide Met-enkephalin modulated GM colony formation in cultures of mouse bone marrow cells. As before [7], the 18.00 h samples produced more colonies, and these appeared more sensitive to inhibition. Higher plating efficiency of bone marrow samples harvested in the evening has also been described by Aardal [l]. A suppressive (but smaller) effect on GM colony formation was also seen with naloxone, an agent that binds to the opioid receptors and blocks opioid effects in other systems [13, 171. In spite of its own interference with bone marrow cell growth, naloxone was capable of blocking the (suppressive) effect of the enkephalin, that is, it did show features of an opioid antagonist; however, naloxone also behaved as an opioid agonist. The interactions occurred preferentially in the opioid-sensitive (18.00 h) population of bone marrow cells. Examples of naloxone exerting opioidlike effects on immune cell functions in vitro have been reported. Naloxone modulates NK cell activity [ 12, 141, superoxid anion release from the polymorphonuclears [IO], generation of’ cytotoxic T cells [2], and of hemolytic plaque-forming cells [5, 10, 151. Such opioidergic actions of naloxone may take place via opioid receptors, but apparently naloxone may bypass that route [see 11 for literature review]. There are also examples, however, of naloxone blocking the effects of opioid peptides on immune cells. Thus, naloxone abrogated stimulatory effects of Met-enkephalin on lymphocyte proliferation [6], antibody-dependent cytotoxicity of the macrophages [4], chemotaxis and 02- production by the polymorphonuclears [ll, 181. The susceptibility of Met-enkephalin effects to naloxone inhibition depends on Met-enkephalin concentration [4]. At 10-9-10m7 M, Met-enkephalin stimulated antibody-dependent cellular cytotoxicity (ADCC) and reduced the number of Fc-

et

al

gama receptors of rat peritoneal macrophages via a naloxone-sensitive mechanism (opioid receptors); at the concentration range of 1O-6-1Oms M, however, the effect was the opposite, and insensitive to naloxone. The effect of the lower concentrations was mediated by the activation of cGMP, and the effect of the higher ones by CAMP [41. Interactions of naloxone with the enkephalin were virtually absent in the progenitor-cell population obtained by the FACS technique. This indicates that joint effects occur at the level of the accessory cells regulating the progenitor cell, rather than at the progenitor cells themselves. In another experimental system [12], naloxone and the enkephalins modulated the NK-cell function indirectly by interfering with the release and/or regulatory actions of IFN-alpha. In conclusion, this work has shown that the opioid antagonist naloxone modulates the effects of Met-enkephalin on hematopoietic cell proliferation. The results lend further support to the concept [7] that opioidergic mechanisms in the accessory cells participate in regulation of hematopoiesis. ACKNOWLEDGMENTS NG Testa is supported by the CRC, Great Britain; L Kriianac-Bengez was supported by a Croatian Science Foundation grant. REFERENCES Aardal NP. Circannual variations of circadian periodicity in murine colony-forming cells. Exp Hemarol 1984; 12:617 Carr DJ, Klimpel GR. Enhancement of the generation of cytotoxic T cells by endogenous opioids. Neuroimmunol 1986;12:75-83 Falke NE, Fischer EG, Martin R. Stereospecific opiate binding in living human polymorphonuclear leucocytes. Cell Sioj In? Rep-1985;9:lb41-7 Foris G. Medgvesi GA, Hauck M. Bidirectional effect of Met-enkephalin on macrophage effector functions. Moi Cell Biochem 1986;69:127-37 Heijnen CJ, Bevers C, Kaavelaars A, Balieux R. Effect of alfa-endorphin on the antigen-induced primary antibody response of human blood B cells in vitro. J Immunol 1986;136:213-6 Hucklebridge HF, Hudspith BN, Muhamed J, Lydyard PM, Brostoff I. Methionine-enkephalin stimulates in vitro proliferation of human peripheral lymphocytes via opioid receptors. Brain Behav Immun 1989;3: 183-9 Kriianac-Bengez Lj, BoraniC M, Testa NG, Marotti T. Effect of enkephalins on bone marrow cells. Biomed Pharmacother 1992;46:367-73

Naloxone

in bone

8 Lord BI, Spooncer E. Isolation of haemopoietic spleen colony forming cells. L~mphokine Res 1986;5:59 9 Madden JJ, Donahoe RM, Zwemer Collins J, Shafer DA, Falek A. Binding of naloxone to human T lymphocytes. Biochem Pharmacol 1987;36:4103-9 10 Marotti T, Batinic D. Enkephalins regulate in vitro antibody response of human lymphocytes. Period Biol 1990;92: 197-202 I I Marotti T, Haberstok H. Naloxone is an inappropriate antagonist of Met-enkephalin-modulated superoxide anion release. Brain Behav lmmun 1992;6:233-33 12 Martin-Kleiner 1, Gabrilovac J. Naloxone modulates NK cell activity of human peripheral blood lymphocytes like an opioid agonist. Immunopharmacol Immunotoxicol 1993;15:179-97 13 Mather LE. Pharmacology of opioids. Part I. Basic aspects. Med J Aust 1986;144:424

marrow

cultures

31

14 Oleson DR, Johnson DR. Regulation of human natural killer cytotoxicity by enkephalins and selective opiate agonists. Brain Behav Zmmun 1988;2:171 15 Rowland RRR, Chukwuocha, R, Tukuda S. Modulation of the in vitro murine immune response by Met-enkephalin. Brain Behav Immun 1987;1:342-8 16 Sharp BM, Keane WF, Such HJ, Gekker G, Tsukayama D, Peterson PK. Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leucocytes and macrophages. Endocrinology 1985;J 17:793-5 17 Sibinga NES, Goldstein A. Opioid peptides and opioid receptors in cells of the immune system. Ann Rev Immunol 1988;6:219 18 Vann Epps DE, Saland L. Beta-endorphin and Met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis. J /mmunol 1984: 132:3046-52