Influence of Beta-Endorphin on the Production of Reactive Oxygen and Nitrogen Intermediates by Rabbit Alveolar Macrophages

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(98)00031-7 All rights reserved

Gen. Pharmac. Vol. 31, No. 3, pp. 393–397, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

Influence of Beta-Endorphin on the Production of Reactive Oxygen and Nitrogen Intermediates by Rabbit Alveolar Macrophages H. Billert,1 D. Fiszer,2 L. Drobnik1 and M. Kurpisz2* 1

Institute of Anesthesiology and Intensive Therapy, University School of Medicine, Poznan´, Poland and 2Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyn´ska 32 60-479 Poznan´, Poland [Tel: (48 61) 8233-011; Fax: (48 61) 8233-235] ABSTRACT. 1. Alveolar rabbit macrophages were studied for superoxide and nitric oxide production at basal levels and upon stimulation with phorbol myristate acetate (PMA), zymosan, cytokines (two types of interferon), and lipopolysaccharide in the presence (or absence) of b-endorphin or hydroxylamine or both. 2. b-Endorphin diminished (statistically significant at concentration of 1028 M) superoxide production by PMA-stimulated macrophages but augmented reactive oxygen generation (10212 M b-endorphin) by zymosan-activated cells. 3. In the presence of hydroxylamine, b-endorphin had a visible (albeit not statistically significant) suppressive effect on nitrite production by PMA-activated cells. 4. Cytokine-stimulated macrophages enhanced nitric oxide production in the presence of hydroxylamine and b-endorphin in culture supernatants. 5. b-Endorphin exerted different modulatory effects on the production of reactive oxygen and nitrite intermediates by rabbit alveolar macrophages (suppression or enhancement) that was strictly dependent on the method of cell activation. gen pharmac 31;3:393–397, 1998.  1998 Elsevier Science Inc. KEY WORDS. Superoxide, nitrogen intermediates, b-endorphin, alveolar macrophages INTRODUCTION The complex multifactorial network connecting immune, endocrine and nervous systems has been intensively studied in recent years (Reichlin, 1993). Among the others, the endogenous opioids are recognized as important molecules linking these systems (Daniele et al., 1992; Olson et al., 1992). Immunocompetent cells can both synthesize the endogenous opioids and interact with them in a paracrine/autocrine manner. Cells of monocyte-macrophage origin are capable of synthesizing b-endorphin in lungs, because proopiomelanocortin mRNA is expressed in these cells, and they possess specific opioid receptors on their surfaces (Mechanick et al., 1992; Stein et al., 1990). Pulmonary macrophages play an important role in organ defense and prevent the invasion of pathogens through the secretion of reactive oxygen and nitrogen intermediates (Barnes and Liew, 1995; Sibille and Reynolds, 1990). Superoxide may influence the expression of genes involved in inflammatory and immune reactions (Segal and Abo, 1993). Oxidative stress may produce changes in the structure of both DNA and proteins. It may also induce glutathione and depletion and raise the level of intracellular free iron and lipid peroxidation, which may result in both cell and connective tissue damage, leading to noncardiogenic pulmonary edema (Cochrane, 1991). Reactive nitrogen intermediates are known to be cytotoxic, but they may also amplify asthmatic inflammatory reactions (Barnes and Liew, 1995). A number of studies have dealt with the effect of b-endorphin on the production of superoxide by both polymorphonuclear and mononuclear phagocytes, but results are still contradictory (Hagi et al., 1994; Ichinose et al., 1995; Peterson et al., 1987; Seifert et al., 1989; Slaoui-Hasnaouri et *To whom correspondence should be addressed. Received 10 November 1997.

al., 1992). The problem of the possible influence of b-endorphin on nitric oxide synthesis remains unresolved. The present study aims to provide information on the effect of b-endorphin on superoxide and nitric oxide production by rabbit alveolar macrophages. MATERIALS AND METHODS

Cell preparation The cells were obtained from Shinshilla rabbits by bronchoalveolar lavage with phosphate buffered saline (pH 7.2; total volume5400 ml). Cells were then washed spinning 10 min at 1,000 rpm and the remaining erythrocytes were lyzed by addition of 0.84% NH4Cl solution for 5 min in 378C. Then macrophages were repeatedly washed (in the aforedescribed conditions), counted and resuspended in Dulbecco minimal essential medium supplemented with 2 mM glutamine, 5% fetal calf serum and antibiotics. Cell preparations consisted of more than 95% viable macrophages, as judged by the Trypan blue dye exclusion assay.

Cell cultures Macrophages were placed into 96-well microtiter plates, at a concentration of 23105 cells per well and allowed to adhere for 1 hr. Then, naloxone (Sigma) was added to some wells at concentration 1026 M, and, after 30 min, b-endorphin (Sigma) at different concentrations (1028, 10210, 10212 M) was inserted. Then, the cells were incubated for 24 hr in a humidified atmosphere with 5% CO2 at 378C. To estimate the nitrite concentration, cells were stimulated by using phorbol myristate acetate (PMA, Sigma; final concentration, 0.5 mg/ml) or recombinant interferon g (IFNrg, Sigma, 1,000

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U/ml) together with lipopolysaccharide (LPS, Sigma, 1 mg/ml) in the presence of 5 mM hydroxylamine (HA, Sigma). All cultures were set up in triplicate and repeated from two to four times.

Analytical procedures Superoxide production was estimated by measuring the ferricytochrome c reduction with or without respiratory burst stimulus (PMA at 0.5 mg/ml or opsonized zymosan at 1 mg/ml; Sigma). Control wells contained superoxide dismutase (Sigma; 40 mg/ml), according to the micromethod described by Pick and Mizell (1981). Nitric oxide production was estimated through the nitrite concentrations in cell culture supernatants. Test samples were combined with equal volumes of Griess reagent for 10 min at room temperature, according to the method of Ding et al. (1988). Absorbance was read at 550 nm on a multiscan microreader.

Statistics Statistical analysis was performed by using the Mann Whitney test. RESULTS After 24 hr of incubation of rabbit alveolar macrophages with different concentrations of b-endorphin, we observed marked alterations in respiratory burst activity as measured by superoxide production. These changes were expressed by a significant decrease in superoxide production by PMA-activated macrophages owing to the addition of b-endorphin at 1028 M (Fig. 1A) and significantly elevated superoxide production by zymosan-stimulated macrophages in the presence of 10212 M b-endorphin (Fig. 1C). Although PMA-stimulated macrophages showed diminished superoxide production owing to b-endorphin (Fig. 6; statistically significant for b-endorphin at 1028 M) compared with the control cultures (without b-endorphin), in general, PMA-activated macrophages produced higher values of superoxide than did zymosan-stimulated macrophages. In contrast with the PMA-stimulated system, b-endorphin added to cultures with zymosan-stimulated cells induced higher superoxide production (statistically significant at 10212) M than in control cultures (no opioid). Naloxone itself did not affect the superoxide levels produced by rabbit alveolar macrophages (Fig. 2). We were not able to obtain any increase in the nitrite concentrations (culture supernatants) after 24–48 hr of incubation of rabbit alveolar macrophages stimulated with rabbit interferon (1,000 U/ ml), rat rIFNg (1,000 U/ml), LPS (1,000 ng/ml) or rIFNg1LPS together, or by using PMA at 500 ng/ml as a cell stimulator (data not shown). After addition of HA at a concentration of 5 mM, again there was no visible increase in nitrite concentrations in supernatants from the cytokine-stimulated cell cultures (Fig. 3). PMA consistently showed the best stimulatory effect (Fig. 3) For NO production, b-endorphin did not exert a statistically significant modulatory effect; however, the addition of the opioid to the cell cultures (Fig. 4) markedly diminished nitrite concentrations in PMA-stimulated cells (in dose-dependent fashion) but did not have a similar effect on NO production by rIFNg1LPS-stimulated macrophages. In fact, in the last combination, an increase in NO production in the presence of b-endorphin at a concentration of 10212 M was even observed. Again, naloxone (Fig. 5) did not significantly influence NO production.

FIGURE 1. Influence of b-endorphin on superoxide production by rabbit alveolar macrophages in 24-hr culture. Control, no opioid; N, naloxone; bE, b-endorphin. Concentrations of the stimulators: PMA, 0.5 mg/ml; opsonized zymosan, l mg/ml. Results expressed as means6SD. Statistical significance of differences: from respective control value * P,0.01.

DISCUSSION In this study, we tried to elucidate the possible influence of b-endorphin on the respiratory burst (superoxide) and nitric oxide production by rabbit alveolar macrophages. We found that b-endorphin, at a concentration of 1028, diminished superoxide production by PMA-stimulated cells (Fig. 6). However, superoxide production (in the presence of b-endorphin) by zymosan-stimulated alveolar mac-

FIGURE 2. Influence of naloxone on superoxide production by rabbit alveolar macrophages in 24-hr culture. Control, no naloxone; N, naloxone. Results expressed as means6SD.

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FIGURE 5. Influence of naloxone on NO production by rabbit alveolar macrophages in 24-hr culture. N, naloxone. Results as means6SD.

FIGURE 3. Influence of b-endorphin on NO production by rabbit alveolar macrophages in 24-hr culture. Control, no opioid; N, naloxone; bE, b endorphin. Concentrations of the stimulators: PMA, 0.5 mg/ml; rIFNg, 1,000 U/ml; LPS, l mg/ml. Results expressed as means6SD. rophages showed a slight but significant increase (Fig. 1A and Fig. 6; b-endorphin concentration of 10212 M). The problem of the possible modulation (by the endogenous opioids) of superoxide production by alveolar phagocytes has been already broadly discussed in the literature, but the results of previous

FIGURE 4. Influence of b-endorphin on NO production by rabbit alveolar macrophages in 24-hr culture. Concentrations of stimulators: PMA, 0.5 mg/ml; rIFNg, 1,000 U/ml; LPS, l mg/ml. Results expressed as means6SD.

studies are conflicting. Our findings partly confirmed the data obtained by Peterson et al. (1987), who evaluated the effect of a 48hr exposure to b-endorphin on the oxygen metabolism of cultured human peripheral blood cells. They observed a marked decrease in superoxide production in the respiratory enzymatic systems challenged by PMA and zymosan with a concomitant significant increase in the basal level of superoxide production. In our study, b-endorphin diminished superoxide formation by PMA-stimulated cells but not by zymosan-stimulated cells (Fig. 6). Suppression of superoxide production by b-endorphin upon PMA stimulation of human polymorphonuclear leukocytes also was reported earlier by Slaoui-Hasnaoui et al. (1992). However, this modulatory effect of b-endorphin on the respiratory burst of phagocytic cells has not been confirmed by others (Seifert et al., 1989). In a model in which rabbit peritoneal macrophages were stimulated with N-formyl-methionyl-leucyl-phenylalanine, it was clear that b-endorphin did not augment O22 production (Koshida and Kotake, 1994). Other reports suggest a biphasic effect of opioids, which could be stimulatory only at concentrations not exceeding 10 nM. The possible explanation of the biphasic phenomenon could be the presence of multiple receptors mediating opposite effects (Sibille and Reynolds, 1990). It is important to note that the highest concentration of b-endorphin used in our study (1028 M) is pharmacologically but not physiologically relevant; therefore it may mimic the condition of the acute stress. Superoxide anion production by phagocytic cells results primarily from the activation of the NADPH oxidase system, which can be stimulated by a variety of factors, such as the agonists interacting with surface receptors—namely, opsonized particles and phorbol esters (Rossi et al., 1986; Segal and Abo, 1993). During activation of

FIGURE 6. Influence of b-endorphin on superoxide production by rabbit alveolar macrophages in 24-hr culture. NS, no stimulation. Concentrations of stimulators PMA, 0.5 mg/ml; rIFNg, 1,000 U/ml; LPS, l mg/ml. Results expressed as means6SD. *Statistical significance of differences from respective control value P,0.01.

396 the oxidase system, NADPH and heme-binding sites become attached to an activating cytosolic protein complex. GTPase activity may regulate the duration of the process (Segal and Abo, 1993). The proximal activation pathway includes activation of phospholipases, which selectively hydrolyze phospholipids, thus leading to the formation of second messengers and protein kinases, with subsequent phosphorylation of the NADPH oxidase system proteins (Rossi et al., 1986; Thelen and Wirthmueller, 1994). PMA stimulation of respiratory burst in phagocytic cells is a result of a number of biochemical events connected with protein kinase C activation. In fact, protein kinase C constitutes a family of related enzymes expressing contradictory effects on the respiratory burst (Philips et al., 1992). Protein kinase C-b has been proved to take part in PMAinduced activation of the arachidonic acid cascade and in superoxide formation in macrophages, whereas the d-izoenzyme seems to be connected to zymosan-induced phosphoinositide hydrolysis and prostaglandin E2 formation (Duyster et al., 1993) (which in turn may influence superoxide formation). Possible influence of b-endorphin on superoxide formation may therefore affect all levels of phagocyte stimulation, and its modulatory effect on stimulated phagocytes may depend on the type of stimulation (as was clearly observed in our study as well—see Fig. 6). b-Endorphin, acting primarily through m and d opioid receptors, exerts its metabolic effect through G proteins and second messengers (Selley and Bidlack, 1992). Whether b-endorphin may act at the level of receptors, possibly modifying receptor–ligand interactions, remains unclear. In human lymphocytes, the opioids affect the phosphorylation of the T-cell receptor CD3 complex in a biphasic dose-dependent manner (Chiapelli et al., 1992a). In promonocyte-like 937 cells, activation of protein kinase C has been shown to rapidly down-regulate naloxone-resistant receptors for b-endorphin (Shahabi and Sharp, 1993), indicating an inverse relation between these two systems. b-Endorphin may also affect further stages of cell activation, interfering with second-messenger formation. In rat glioma cells, b-endorphin has been shown to reverse endothelin-induced Ca21 mobilization and phosphoinositide turnover (Barg et al., 1994). In activated human mononuclear cells, it blunts the formation of phosphatidylinositol turnover by about 20% (the effect is dose dependent and partly blocked by naltrexone) (Chiapelli et al., 1992b). Diminition of superoxide production by PMA-stimulated cells through b-endorphin might therefore be connected with its possible effect on protein kinase C through changes in Ca21 concentration of phosphatidylinositol turnover. It is also possible that the stimulation of cells with phorbol ester may mask the interaction of b-endorphin with other components of the membrane signaling system (Chiapelli et al., 1992a). The influence on cell metabolism through changes in cAMP concentrations also should be taken into consideration. In human lymphocytes, b-endorphin has been shown to both decrease and increase intracellular cAMP concentrations (Chiapelli et al., 1992a). In our studies, zymosan-stimulated alveolar macrophages, exposed to b-endorphin, increased superoxide formation (significantly at 10212 M of b-endorphin; Fig. 6). Enhanced phagocytosis of mouse peritoneal macrophages can be due to the action of b-endorphin, and this effect is largely dependent on intracellular Ca21 (Ichinose et al., 1995). In our study, naloxone only partly blocked the b-endorphin effect on superoxide formation, so we may presume that its effect is only partly mediated by classical, naloxone-sensitive receptors. In vitro superoxide formation may also be modified by a number of factors; that is, the separation procedure and the assay conditions. Slaoui-Hasnaoui et al. (1992) reported that, after exposure of

H. Billert et al. b-endorphin (and other endogenous opioids) to a reactive oxygen intermediate cascade, the opioid peptides enhanced the PMA-stimulated respiratory burst in human polymorphonuclear leukocytes. So, lack of any modulatory effect of b-endorphin on PMA-stimulated superoxide production by phagocytic cells may come from the opposite effects of both native and altered (by oxygen species) forms of the opioid agent (inhibition or stimulation may be simultaneous). The possible down-regulation of the respiratory burst by b-endorphin may be a protecting mechanism with respect to tissue injury in a condition of stress (elevated levels of the opioid). We also tried to elucidate the possible effect of b-endorphin on nitric oxide production by rabbit alveolar macrophages. We did not observe any increase in nitrite concentrations after 24 and 48 hr of culture, owing to opioid action both at the basal level or upon stimulation with LPS plus IFNg with or without PMA. Thus b-endorphin may not exert visible effect on nitrate production. These observations are in agreement with the report of Hey et al. (1995), who could neither detect NO synthase activity nor induce it with LPS plus IFN in rabbit alveolar macrophages. After the addition of HA (at a concentration of 50 mM), we could not find any stimulatory effect of cytokines on NO production. A decrease in nitrite production (in PMA-stimulated cells in the presence of b-endorphin; Fig. 4) was clearly visible (and dose dependent). HA has been previously demonstrated to strongly potentiate NO production in PMA-stimulated human neutrophils, owing to catalase and probably other heme protein inhibition, thus providing a useful tool for the determination of nitrite production by these cells (Koshida and Kotake, 1994). These findings, however, need further clarification. Our results suggest that NO production by rabbit alveolar macrophages might not be generated by a classical NO synthase system or NO may be scavenged by member proteins or both. We may assume that b-endorphin does not play a very significant role in the regulation of NO production by rabbit alveolar macrophages; however, both the nitrites and superoxide seemed to be suppressed by the addition of b-endorphin to PMA-activated cells. On the other hand, Hagi et al. (1994) reported an enhancement of tumoricidal activity by murine macrophages through the activation of b-endorphin (Duyster et al., 1993), and, regarding this observation, we can speculate that the tumoricidal activity of macrophages may correspond with NO production. A slight increase in NO production by cytokine-activated cells (rIFNg1LPS) due to b-endorphin was observed in our study but was not statistically significant (Fig. 4). Studies on b-endorphin modulation have revealed a very complex picture with complicated multifactorial relation. In both models, superoxide and nitrites were suppressed (with different intensity) by the addition of b-endorphin to PMA-activated cells but elevated when the cells were activated in a different way. It is clear therefore that b-endorphin may act differently according to different pathophysiological situations and may depend on the prevailing stimulus for cell activation. Thus final effector reactions may be driven in quite opposite directions (suppression or enhancement), depending on the domination of certain stimulating agent(s) at particular phases of cell activity. References Barg J., Belcheva M. M., Zimlichman R., Levy R., Saya D., McHale R. J., Johnson F. E., Coscia C. J. and Vogel Z. (1994) Opioids inhibit endothelin mediated DNA synthesis, phosphoinositide turnover and Ca21 mobilisation in rat C6 glioma cells. J. Neurosci. 14, 58–74. Barnes P. J. and Liew F. Y. (1995) Nitric oxide and asthmatic inflammation. Immunol. Today 16, 128–130. Chiapelli F., Nguyen L., Bullington R. and Fahey J. L. (1992a) Beta-endorphin blunts phosphatidylinositol formation during in vitro activation of

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