Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin

Immunology Letters, 40 (1994) 43-47 0165

2478 / 94 / $ 7.00 © 1994 Elsevier Science B.V. All rights reserved

IMLET 02106

Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin .a

*

T. M a r o t t l ' , B. B u r e k a, S. R a b a t i c b, T. B a l o g a a n d I. H r s a k a aRudjer Boskovic Institute, Bijenicka 54, 41000 Zagreb, Croatia; and blnstitute of lmmunology, University of Zagreb, Roekefellerova 10, Zagreb, Croatia (Received 30 June 1993; accepted 21 January 1994)

1. Summary

2. Introduction

In the present study, we have examined the effect of opioid peptide methionine enkephalin (MENK) on production of factors with interleukin-1 (IL-1) and tumor necrosis factor (TNF) activity by mouse peritoneal macrophages and assessed whether modification in the production of those cytokines could be related to alteration of phagocytosis by MENK. None of the MENK concentrations examined altered IL-1 or TNF activity alone. However, peritoneal macrophages co-stimulated with 1 #g of lipopolysaccharide (LPS) and 10-1° M MENK potentiated IL-1 activity, compared to LPS alone, but abrogated TNF activity induced by LPS. While MENK alone slightly decreased phagocytosis of sheep red blood cells (SRBC) by mouse peritoneal macrophages, cells simultaneously incubated with 1 #g of LPS and 10 - l ° M MENK had increased phagocytosis compared to LPS alone. Moreover, phagocytosis of SRBC by cells incubated overnight with the supernatant of the respective cell culture was significantly augmented. These results provide additional evidence for the immunoregulatory role of neuropeptides and suggest that the modulatory action of MENK could be mediated, at least in part, through the up-regulation of cytokines, most probably IL-I and TNF.

The immune and neuroendocrine systems affect each other via molecules and receptors shared by both systems [2, 4, 23]. Thus, opioid peptides primarily of neural origin, like cytokines originating primarily from monocyte/macrophage cells of the immune system, have a wide spectrum of biological activities and both represent endogenous mediators of immunologic host defense reactions [3, 22, 29]. Among cytokines, interleukin-1 (IL-1) and tumour necrosis factor (TNF) are factors with multiple and overlapping effects, both vital to the maintenance of immune homeostasis and both produced by macrophages which bear receptors for IL-1, TNF and opioid peptides [5, 10, 29]. While considerable and well-documented information exists on the direct effects of opioid peptides on the immune system [12, 25], there are only very limited data concerning the indirect effects of opioid peptides on the cells of the immune system via modulation of cytokine production. In the present study, we aimed to examine the role of methionine enkephalin (MENK) in the regulation of IL- 1 and TNF activity in mouse peritoneal macrophages. The purpose of our work was to investigate whether modifications in the production of those cytokines induced by MENK could be related to induction of phagocytosis.

3. Material and Methods Key words: Tumour necrosis factor; Interleukin-1; Methionine-en-

3.1. IL-1 activity assay

kephalin; Phagocytosis

*Corresponding author: T. Marotti, Rudjer Boskovic Institute, Bijenicka 54, 41000 Zagreb, Croatia.

Abbreviations: MENK, methionine enkephalin; IL-1, interleukin1; TNF, tumour necrosis factor; and LPS, lipopolysaccharide.

SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) 0 0 0 2 2 - J

Peritoneal macrophages from male CBA/HZgr mice aged 10--12 weeks were harvested 5 days after injection of 0.12% glycogen (Kemika, Zagreb, Croatia). After the remaining red cells were lysed with NH4C1, 3 x 106 ml of macrophages was cultivated

for 24 h in 35 × 10 mm plastic petri dishes with 1 #g/ ml lipopolysaccharide (LPS) (Escherichia coli Serotype 0111 :B4, Sigma, USA) and/or different concentrations (10 -14 M, 10 -12 M, 10 - l ° M, 10 - s M and 10 -6 M) of M E N K (Sigma, USA) in 2 ml of RPMI1640/10% fetal calf serum (FCS). Control cultures in RPMI-1640/10% FCS only were included in each trial. After incubation, supernatants were collected and stored at - 2 0 ° C until use. The presence of IL-1 in culture supernatants was detected in a C3H thymocyte (2 × 106/well) proliferation assay in the presence of suboptimal concentrations of ConA (0.5 #g/ml) according to the method of Rosenwasser et al. [26]. Proliferation of C3H thymocytes was measured by a colorimetric assay [24]. For the last 4 h of the 72 h incubation period, 20/al of 5 mg/ml M T T (Sigma, USA) stock solution was added to 140/A of culture in the wells. After incubation 100 #1 of 0.04 N HC1 isopropanol (Kemika, Zagreb, Croatia) was added to each well and mixed thoroughly to dissolve the dark blue crystals. The plates were read using an ELISA Microreader at 570 nm. Results are given for 1:6 dilution of supernatants and are expressed as percent of control supernatants.

3.2. TNF activity assay Spleens of male CBA/HZgr mice aged 10-12 weeks were aseptically removed. Splenocytes were resuspended in RPMI-1640 medium. Red cells were removed by NH4C1 lysis. The remaining cells were washed, resuspended in RPMI-1640 supplemented with 5% FCS, and plated in 100 x 20 mm tissue culture dishes (Falcon, Becton Dickinson, USA). Following 90 rain incubation at 37°C in 5% CO2 the dishes were washed with RPMI-1640 to remove nonadherent cells. The remaining adherent cells were recovered by scraping with a rubber policeman, washed, adjusted to 4 x 106 cells per well with RPMI-1640 and 10% FCS and incubated for 4 h at 37°C in 5% CO2 in the presence of 1 /~g LPS and/or 10 -12 M, 10 -1° M and 10 -8 M M E N K in 24-well tissue culture plates (Falcon, Becton Dickinson, USA). Control cells were incubated in medium and 10% FCS only. Supernatants were removed from these cultures, centrifuged and used immediately to assay T N F activity. The T N F activity was assessed in a cytotoxicity test on mouse fibroblast L929 cells [27]. Briefly, 5 x 104 L929 cells per well were seeded into 96 well microtest plates (Falcon, Becton Dickinson, USA) in RPMI-1640 and 5% heat-inactivated FCS. After 24 h incubation at 37°C in 5% CO2, medium was re44

moved and test supernatants were added (100 #1) followed by 100 #1 (1/~g/well) of actinomycin D (Sigma, USA). Plates were incubated additionally for 20 h at 37°C in 5% CO2. Viable cells were stained with crystal violet for 15 min. After washing the stained cells were lysed with 0.3% sodium dodecyl sulfate (SDS) (Sigma, USA) and the quantity of released stain was measured at 570 nm using an ELISA microplate reader.

3.3. Phagocytosis assay Peritoneal macrophages after 24 h incubation with 1 #g of LPS and/or different concentrations of M E N K (10 -14 M, 10 -12 M, 10 -10 M, 10 -8 M, 10 - 6 M), after removing the supernatant, were scraped, washed in Medium 199 and their ability to ingest the opsonized 51Cr-labeled sheep red blood cells (SRBC) was measured according to the methods described previously [28]. In part of the experiments freshly isolated mouse peritoneal macrophages were incubated overnight with the respective supernatant tested for IL-1 activity and then the phagocytosis assay was performed. Briefly, 51Cr-labeled SRBC, opsonized with rabbit antiserum to SRBC, was mixed with effector cells (1:1 ratio) in a total volume of 300/~1 of Medium 199 containing 8 mM L-glutamine (Gibco), (100 U/ml penicillin/100 #g/ml streptomycin) (Gibco) in 10 mM Hepes buffer and supplemented with 10% FCS, and incubated at 37°C for 90 rain. At the end of the incubation, phagocytosis was stopped by placing the cells on ice, and hypotonic lysis of non-phagocytosed SRBC was performed in one-half of the samples. Lysed and nonlysed samples were centrifuged, the supernatants removed and the radioactivity in supernatants and pellets was measured separately in a gamma-counter. The extent of phagocytosis was determined by expressing the radioactivity in the pellet after lysis as a percentage of total radioactivity (in the pellet and in the supernatant).

3.4. Statistical analysis The paired 2-tailed Student's t test was performed for statistical analysis using Statgraphics software (Statistical Graphics Corporation, STSC). Differences were considered significant for P<0.05. Values are calculated as mean_+ SD of 4 or 6 consecutive experiments each performed in triplicate.

with LPS and 10-1° M M E N K abrogated TNF activity induced by LPS.

4. Results

4.1. The effect of M E N K on IL-1 activity Mouse peritoneal macrophages were cultured in the presence of 1 ~g of LPS and/or different concentrations of M E N K (10 - 1 4 M , 10 - 1 2 M , 10 - l ° M , 10 -8 M and 10 -6 M) (Fig. 1). IL-1 activity, assessed in the supernatants after 24 h of culture, was significantly (P<0.05) different in supernatants of macrophages incubated with LPS, but was not altered by any M E N K concentration in comparison to control RPMI- 1640/10% FCS-treated cultures. However, the simultaneous presence of LPS and 10 -1° M M E N K exhibited IL-1 activity above the level of LPS alone (P < 0.02). No other concentration of M E N K co-stimulated IL-1 activity with LPS.

4.2. The effect of M E N K on TNF activity An adherent population of mouse spleen cells was cultured in the presence of 1/zg LPS and/or 10-12 M, 10 -1° M and 10 -8 M M E N K (Fig. 2). TNF activity, assessed in the supernatants after 4 h of culture, was not altered by any concentration of M E N K per se. LPS, as expected, significantly (P<0.01) stimulated TNF activity as measured by the decreased surviving fraction. TNF activity also remained elevated in supernatants in which cells were simultaneously cultured with LPS and 10 -12 M and 10 -8 M M E N K (P<0.01). On the contrary, co-stimulation of cells r

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4.3. The effect of M E N K on phagocytosis of mouse peritoneal macrophages The effect of LPS and/or 10 -14 M, 10 -12 M, 10 -1° M, 10 -8 M and 10 - 6 M M E N K on phagocytosis of SRBC by mouse peritoneal macrophages is illustrated in Fig. 3. LPS by itself, significantly (P<0.05) increased phagocytosis. Phagocytic ability of mouse peritoneal macrophages incubated with any concentration of M E N K was slightly (but statistically insignificantly) lower compared to phagocytosis of control macrophages incubated with RPMI1640/10% FCS. Co-stimulation of mouse peritoneal macrophages with LPS and M E N K had no effect on phagocytosis except when simultaneously incubated with LPS and 10-10 M MENK. Namely, the amount of phagocytosis with 10 -1° M M E N K and LPS was statistically (P < 0.02) higher than with LPS only.

4.4. The effect of supernatants on phagocytosis of mouse peritoneal macrophages To examine the effects of supernatants tested for IL-1 activity (Fig. 1) on phagocytosis of mouse peritoneal macrophages, the latter were incubated overnight with corresponding supernatants, the cells were washed and their capability to ingest SRBC was tested. The results are illustrated in Fig. 4. Supernatants of LPS-treated cultures increased phagocytosis of mouse peritoneal macrophages (P<0.02) as did the supernatants of cultures incubated with 10-10 M MENK. Supernatants of cells incubated with other M E N K concentrations were ineffective. Nevertheless, supernatants of cells co-stimulated with LPS and 10-10 M M E N K augmented phagocytosis of mouse peritoneal macrophages significantly ( P < 0.01) above the level of supernatants of LPS-treated cultures only.

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Fig. 1. IL-I activity in supernatants of mouse peritoneal macrophages cultured for 24 h with 1/~g LPS and/or different concentrations of M E N K (10 -14 M, 10 12 M, 10 - ] ° M, 10 -8 M and 10 -6 M). LPS, []; M E N K , •; LPS + M E N K , I . Results of experiments repeated 4 times are shown as mean + SD of triplicate cultures. Data are expressed as percentage of IL-I activity in the supernatant of control macrophages cultured in the presence of RPMI-1640/10% FCS only (the dashed line indicates the control level). *P < 0.05; **P < 0.02.

Functional links between the neuroendocrine and the immune system are often related to the cytokine network, IL-1 and TNF playing the central role [11]. Cytokines, besides being recognized as essential mediators of the immune system, also effect the neuroendocrine system. Krueger et al. [18] have reported pronounced and direct somnogenic and anorexic effects of IL-1. IL-1 stimulates hypothalamo-pituitary-adrenal axis [15], is mitogenic for astrocytes and is gener45

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Fig. 2. TNF-activity in supernatants of adherent mouse splenocytes cultured for 4 h with 1 /~g LPS and/or l0 -12 M, 10 -1° M and 10 -8 M MENK. LPS, El; MENK, B; LPS + MENK, i . Results of experiments repeated 6 times are shown as mean_+ SD of triplicate cultures. Data are expressed as percentage of T N F activity in the supernatant of control splenocytes cultured i n t h e presence of RPMI-1640/10% FCS only (the dashed line indicates the control level). ***P<0.01.

ated by astrocytes [14]. TNF also induces astrocyte proliferation and is highly involved in neurological diseases [29]. T N F and IL-1 induce one another in vivo and in vitro [7]. Recently, Fagarasan et al. [9] demonstrated that IL-1 stimulates fl-endorphin release of mouse and rat pituitary cell line. TNF and IL-1 elicited time-dependent increase in the content of vasoactive intestinal:polypeptide (VIP) and decrease in the content of M E N K [8]. In contrast, much less data exist concerning the relationship between opioid peptides and cytokines. In the present study we report an increase in IL-1 activity by a simultaneous stimulation of peritoneal macrophages with LPS and MENK. So far, it has been demonstrated that neuropeptides can induce the release of IL-1, TNF and !L-6 from human blood monocytes [23]. Substance P has been shown to induce IL-1, IL-6 and TNF production by human and murine monocytes/macrophages [17], the stimulated production occurring preferentially in activated cells [19]. Youkilis et al. [30] demonstrated that M E N K increased IL-1 production by cultured human monocytes. On the contrary, Apte et al. [1] demonstrated that in contrast to fl-endorphin, Leu-enkephalin and fl-neoendorphin, M E N K did not alter the patterns of IL-1 production of mouse bone marrow macrophages. The discrepancy in the results obtained by our group and the group of Youkilis and Apte could possibly be explained by the complexity of the opioid receptor system, since various cell types bear different receptors or have different affinities on 46

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Fig. 3. Phagocytosis of SRBC by mouse peritoneal macrophages incubated for 24 h with 1 #g LPS and/or different concentrations of M E N K (10 -14 M, 10 -12 M, l0 -I0 M, 10 -8 M and 10 -6 M). LPS, W; MENK, !~; LPS + MENK, II. Results of experiments repeated 4 times are shown as mean + SD of triplicate cultures. Data are expressed as percentage of phagocytosis in control peritoneal macrophages cultured in the presence of RPMI-1640/10% FCS only (the dashed line indicates the control level). *P<0.05; ** P < 0.02.

different cell types. In our study, different concentrations of M E N K alone did not stimulate IL-1 activity but potentiated IL-1 activity in macrophages concomitantly stimulated with LPS. This is not surprising since other neuropeptides also enhance the production of cytokines only when stimulated with LPS [1,17,19]. It is of interest that an enhancing effect of the opioid peptide was observed by physiological M E N K concen-

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Fig. 4. Phagocytosis of SRBC by mouse peritoneal macrophages incubated overnight with respective supernatants tested for IL-I activity. LPS, ~1; MENK, []; LPS + MENK, I . Results of experiments repeated 4 times are shown as mean + SD of triplicate cultures. Data are expressed as percentage of phagocytosis in control peritoneal macrophages cultured in the presence of RPMI1640/10% FCS only (the dashed line indicates the control level). **P<0.02; ***P
trations (10 -1° M). The enhancing effect was observed at the suboptimal concentration o f the inducer (LPS), suggesting that M E N K m a y serve as amplifying signal for IL-1. I n c u b a t i o n o f mouse peritoneal m a c r o p h a g e s with LPS enhanced, while incubation with M E N K decreased, phagocytosis o f SRBC. Those results are in accordance with the suppressed phagocytosis o f mouse peritoneal m a c r o p h a g e s incubated with M E N K as demonstrated by Foris et al. [13] and Casellas et al. [6]. Unexpectedly, peritoneal m a c r o phages incubated simultaneously with 10 -1° M M E N K and LPS, exhibited statistically significantly higher phagocytosis than macrophages incubated only with LPS. These results implicated an indirect rather than direct effect o f M E N K , possibly mediated by induction o f a second mediator, probably IL-1. The possibility that the increased phagocytosis resulted from M E N K - s t i m u l a t e d IL-1 activity was therefore tested by incubating peritoneal macrophages with supernatants tested for IL-1 activity. Indeed, incubation o f m a c r o p h a g e s with only the supernatant in which increased IL-1 activity was demonstrated (macrophages incubated with 10 - t ° M M E N K and LPS) increased phagocytosis above the level o f LPS alone. This result suggests that at least some o f the effects o f M E N K u p o n immune functions could be due to induction o f mediators (cyto: kines) rather than to direct influences o f M E N K . In contrast to M E N K - and LPS-induced increase in IL-1 activity, T N F activity o f mouse adherent spleen cells simultaneously incubated with 10 -1° M M E N K and LPS, was decreased up to the level o f control cells incubated with m e d i u m only. The possible mechanism o f down-regulation o f T N F activity might be the M E N K - i n d u c e d increase o f IL-1 activity. Namely, IL-1 is k n o w n to inhibit the expression o f T N F receptors and down-regulate cellular responsiveness [16] as well as the release o f T N F [20]. In vivo a single IL-1 injection completely protected mice against a lethal dose o f T N F [21]. To be absolutely sure that the active molecules for the effects observed were indeed IL-1 and T N F , specific i m m u n o a s s a y s and m o n o c l o n a l antibodies to these cytokines should be induced. Nevertheless, we believe that the bioassays used support the conclusion that M E N K most p r o b a b l y has the capability to modulate p r o d u c t i o n o f these two cytokines by peritoneal macrophages. The data from our study contribute to a growing b o d y o f evidence suggesting a reciprocity o f regulatory functions between the nervous and immune systems predominantly by sharing molecules like cytor

kines. In addition, our observations add IL-1 and T N F regulation to the list o f m a c r o p h a g e functions which are shown to be m o d u l a t e d by opioids.

5. References [1] Apte, R.N., Durum, S.K. and Oppenheim, J.J. (1990) Immunol. Lett. 24, 141. [2] Blalock, J.E. and Smith, E.M. (1985) Fed. Proc. 44, 108. [3] Brandwein, S.R. (1986) J. Biol. Chem. 261, 8624. [4] Carr, D.J.J., De Costa, B.R., Kim, C.H., Jacobson, A.E., Guarcello, V., Rice, K.C. and Blalock, K.E. (1989) J. Endocrinol. 122, 161. [5] Carr, D.J.J., De Costa, B.R., Kim, C.H., Jacobson, A.E., Bost, K.L., Rice, K.C. and Blalock, J.E. (1990) Neuroendocrinology 51,552. [6] Casellas, A.M., Guardiola, H. and Renaud, L.F. (1991) Neuropeptides 18, 35. 17] Dinarello, C.A., Cannon, J.G., Wolff, S.M., Barheim, H.A., Beutler, B., Cerami, A., Figari, I.S., Palladino, M.A. and O'Connor, J.V. (1986) J. Exp. Med. 163, 1433. [8] Eskay, R.L. and Eiden, L.E. (1992) Endocrinology 130, 2252. [9] Fagarasan, O.M., Arora, P.K. and Axelrod, J. (1991) Prog. Neuro-Psychopharrnacol. Biol. Psyehiat. 15, 551. [10] Farrar, W.L., Kilian, P.L., Ruff, M.R., Hill, J.M. and Pert, C.B. (1987) J. Immunol. 139, 459. [11] Fibbe, E.W., Schaafsmo, M.R., Falkenburg, J.M.F. and Willemze, R. (1989) Blut 59, 147. [12] Fischer, E.G. (1988) Immunopharmacol. Immunotoxicol. 10, 265. [13] Foris, G., Medgyesi, G.A., Gyimesi, E. and Hauck, M. (1984) Mol. Immunol. 21,747. [14] Giulian, D. and Lachman, L.B. (1985) Science 228, 497. [15] Harbuz, M.S., Stephanou, A., Sarlis, N. and Lightman, S.L. (1991) J. Endocrinol. 133, 349. [16] Holtman, H. and Walach, D. (1987)J. Immunol. 139, 1161. [17] Kimball, E.S. (1990) Ann. NY Acad. Sci. 294, 293. [18] Krueger, J.M., Walter, J., Dinarello, C.A., Wolff, S.M. and Chedid, L. (1984) Am. J. Physiol. 246, R994. [19] Laurenzi, M.A., Persson, M.A.A., Dalsgaard, G.J. and Haegerstrand, A. (1990) Scand. J. Immunol. 31, 529. [20] Le Contel, C., Parant, F. and Parant, M. (1992) Immunobiol. 186, 199. [21] Libert, C., Van Bladel, S., Brouckaert, P., Shaw, A. and Fiers, W. (1991) J. Immunol. 146, 2625. [22] Loh, H.H. and Smith, P. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 123. [23] Lotz, M., Vaughan, J.H. and Carson, D.A. (1988) Science241, 1218. [24] Mosmann, T. (1983) J. Immunol. Methods 65, 55. [25] Olson, G.Y., Olson, R.D. and Kastin, A.J. (1990) Peptides 11, 1277. [26] Rosenwasser, L.J. and Dinarello, C.A. (1981) Cell Immunol. 63, 134. [27] Ruff, M.R. and Gifford, G.E. in: (E. Pick ed.) Lymphokines. Academic Press, New York, 1981, p. 235. [28] Sabioncello, A., Rabatic, S., Kadrnka-Lovrencic, M. and Dekaris, D. (1981) Biomedicine 35, 227. [29] Vassali, P. (1992)Annu. Rev. Immunol. 10, 411. [30] Youkillis, E., Chapman, J., Woods, E. and Plotnikoff, N. (1985) Int. J. Immunopharmacol. 7, 79. 47