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Inducible Nitric Oxide Synthase (iNOS) Expression in Human Monocytes Triggered by β-Endorphin through an Increase in cAMP
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
245, 717–721 (1998)
RC988127
Inducible Nitric Oxide Synthase (iNOS) Expression in Human Monocytes Triggered by b-Endorphin through an Increase in cAMP M. Soledad Aymerich, M. Teresa Bengoechea-Alonso, MarıB a J. Lo´pez-Zabalza, Esteban Santiago, and Natalia Lo´pez-Moratalla1 Department of Biochemistry, University of Navarra, 31080 Pamplona, Spain
Received December 30, 1997
Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of b-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. b-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides b-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by b-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected. q 1998 Academic Press
Macrophages play a variety of roles in the immune system. They may display their phagocytic capacity as direct effector cells against pathogens, or behave as immunoregulatory cells, processing and presenting antigens, or releasing, locally or systemally, different mediators (1). As part of their mechanism of action, macrophages express high levels of inducible nitric oxide synthase (iNOS) after stimulation with various agents (2-5). During the last few years, a number of findings have 1 To whom correspondence should be addressed. Department of Biochemistry, University of Navarra, Apdo. 177, Pamplona, Spain. Fax: (/34 48) 42 56 00; E-mail: [email protected].
provided evidence for a bidirectional relationship between neuroendocrine and immune systems (6,7). Available data suggest that the neuroendocrine-immune relationship influences the pathophysiology of disease processes. Some reports indicate that the nervous system may play a role in the regulation of the immune response through the action of neuropeptides (8,9). A growing body of evidence points out that bendorphin, a neuropeptide released by the pituitary in response to physical or mental stress (10), alters the function of the immune system (11). Opioid receptors are present in the cells of the immune system (12), and their activation results in an inhibition of the adenylate cyclase leading to a decrease in cAMP; the effect of opioid peptides on this receptor is reversed by naloxone. A specific non-opioid receptor for b-endorphin has been described in cultured human lymphocytes (13); and it has been found that neither opioid agonists or antagonists affect the binding of b-endorphin to this receptor. Recently, a naloxone insensitive receptor has been found on murine peritoneal macrophages (14). The study now reported was aimed at determining if b-endorphin, known to be released under stress conditions, could induce iNOS expression in monocytes. That was indeed the case. The results obtained strongly support the idea that the monocyte might be one of the links between neuroendocrine and immune systems. This expression of the inducible nitric oxide synthase was activated through the binding of b-endorphin to a receptor, sensitive to naloxone and leading to an increase in cAMP. MATERIALS AND METHODS Cell sources and cultures. Lymphomononuclear cells, obtained from peripheral blood of donors following the technique described by Bøyum (15), were incubated at 1 1 106 per ml in RPMI-1640 medium supplemented with 10% of fetal calf serum and 2 mM L-glutamine in a 5% CO2 humidified atmosphere. Isolation and purification of
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monocytes were carried out taking advantage of their adherence to culture plates. After a period of 24 hours at 377C nonadherent cells were removed with the medium, and adherent cells washed three times with saline solution. Then, adherent cells, consisting mainly of monocytes (approximately 90 per cent of the cells were CD14/), were carefully detached from the surface of culture plates by gentle friction. Immunocytochemistry. Cells were pelleted by centrifugation, fixed in formaldehyde-picric mixture, and processed for immunochemistry as described previously (5). A polyclonal rabbit antibody against a synthetic peptide (QNGSPQLLTGTAQNVPESLDKLHVTC) of mouse macrophage inducible NO synthase (kindly provided by V. Riveros-Moreno and S. Moncada) was used in the present study. The specificity of this antibody for the detection of human inducible NO synthase has been previously shown (16). The specificity of the immunoreaction was tested by preabsorption of the antiserum with the synthetic antigenic peptide. Determination of nitrite/nitrate. For the determination of nitrites and nitrates, spontaneous degradation products of nitric oxide, a commercial kit was used (Alexis Corporation, Switzerland). The method consists of a simple two-step process. The first step is the conversion of nitrate to nitrite utilizing nitrate reductase. The second step is the addition of Griess reagent which converts nitrite into a deep purple azo compound. Photometric measurement was then carried out at 540 nm. Assays were performed on samples containing 2 1 106 lymphomononuclear cells. An aliquot of the incubation medium was used as reaction blank. Measurement of cAMP. After blood sampling, lymphomononuclear cells were allowed to adhere to the flask surface for a period of 2-4 hours. Adherent cells were incubated at room temperature in culture medium containing 1mM 1-isobutyl-3-methyl xanthine (Sigma) for 20 minutes. Cholera toxin (5mg/mL) (Sigma), 1008 M bendorphin (Sigma), or 1008 M naloxone (Sigma) were added to the medium, when indicated, and cells incubated for 4 minutes. Untreated monocytes were used as controls. The medium was discarded and cells lysed with ice-cold 95% ethanol at 47C for 5 minutes. Lysates were centrifuged at 13,200 rpm at 47C for 5 minutes and supernatants dried under vacuum. Cyclic nucleotide content in the dried samples was quantified using a radioreceptor assay from Amersham Corporation.
RESULTS Induction of iNOS by b-endorphin. Lymphomononuclear cells freshly isolated from healthy donors were incubated for 24 hours in the absence or in the presence of 1008M b-endorphin. Then, monocytes were collected and processed for immunohistochemistry. In the absence of stimulating agents only a small percentage of monocytes (5-10%) was found to be iNOS positive (Fig. 1A and 1B). However, a large number of monocytes turned iNOS immunoreactive after being cultured in the presence of b-endorphin (Fig. 1C and 1D). The number of brown color positive cells increased clearly as compared with control monocytes; we have observed a wide variability of immunoreactive monocytes in five different experiments (30-95%). The specificity of the immunoreaction in monocytes was tested by preabsorption of the antiserum with the synthetic antigenic peptide used to prepare it. Different b-endorphin concentrations (1008M, 10010M and 10012M) were also tested;
the percentage of iNOS positive cells decreased when the concentration of the neuropeptide was lowered (data not shown). Finally, b-endorphin did not induce iNOS, if isolated monocytes were incubated under the same conditions as the entire population of lymphomononuclear cells. With the purpose of verifying if the expressed iNOS was active NO related products (nitrite/nitrate) were measured in the in the supernatant of the incubated cells as indicated in Materials and Methods. For this determination 0.5 mM L-arginine was present in the incubation medium as exogenous substrate. The amount of nitrite/nitrate, NO degradation products, released to the medium by PBMC incubated for 24 hours in the presence of 1008M b-endorphin was 5.4 nmoles per 105 cells (average of three different determinations). Nitrite/nitrate were not detected in the supernatants of cells from the same subjects if incubated for the same period in the absence of b-endorphin. b-endorphin increased cAMP levels in monocytes. Naloxone sensitive opioid receptors are known to inhibit adenylate cyclase activity and lower cAMP levels within the cells. However, we observed that the addition of 1008M b-endorphin to monocytes resulted in an increase in cAMP production (Fig. 2). Cholera toxin (5mg/mL) increased also cAMP levels. When adding both cholera toxin and b-endorphin, cAMP levels did not decrease with respect to those reached with cholera toxin alone. When, besides b-endorphin, 1008M naloxone was added to the medium, a decrease in the elevated levels of cAMP, caused by b-endorphin, was observed (Fig. 2). An inhibitor of protein kinase A prevented iNOS induction. The possible involvement of cAMP mediating iNOS expression was further investigated. To prevent the b-endorphin effect through cAMP we added H-89 (Calbiochem), an inhibitor of protein kinase A, to the incubation medium. When lymphomononuclear cells were cultured for a period of 24 hours with b-endorphin in the presence of 5mM H-89, immunostaining for iNOS strongly decreased (Fig. 1G and 1H). Lymphomononuclear cells belonging to the same donors, cultured for the same period of time and processed simultaneously, were used as negative control. Effect of naloxone on iNOS induction. Lymphomononuclear cells were also incubated for a period of 24 hours with b-endorphin in the presence of 1008M naloxone. When, besides b-endorphin, naloxone was added to the medium, iNOS expression was maintained, although a slight decrease in immunoreactivity (25-60%) was observed (Fig. 1E and 1F). DISCUSSION Macrophages play a central role in immune and stress responses, both processes being apparently me-
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FIG. 1. Inducible nitric oxide synthase in human monocytes detected by immunocytochemistry. Photographs are representative of an experiment in wich all cells corresponded to the same healthy donor and were processed simultaneously. Basal monocytes (A and B) did not express iNOS unless activated with 1008M b-endorphin (C and D). When besides b-endorphin, 1008M naloxone was present in the medium, iNOS continued to be expressed (E and F), although the niumber of stained cells slightly decreased. However, if lymphomononuclear cells were incubated in the presence of b-endorphin and 5mM H-89, an inhibitor of protein kinase A, immunostaining for iNOS did not appear (G and H). Photographs have been taken at two different magnifications (1 440 and 1 1.100) for each treatment.
FIG. 2. Effect of b-endorphin on cAMP levels in human monocytes. b-endorphin. C, control monocytes; CT, monocytes incubated in the presence of cholera toxin (5mg/mL); b-end, monocytes incubated in the presence of 1008M b-endorphin. b-end / NLX, monocytes incubated in the presence of 1008M b-endorphin and 1008M naloxone. Data represent mean { SD of duplicated of four experiments with different donors.
diated by a common pool of molecules: pro-opiomelanocortin-derived (POMC) peptides, such as b-endorphin, neurohormones, small bioactive molecules, cytokines, such as IL-1, IL-6 and TNFa, and NO (17). b-endorphin is a 31 amino acid endogenous opioid peptide synthesized by the pituitary and released into the circulation under stress situations (10). Recently there has been growing evidence that b-endorphin alters the function of a variety of cells of the immune system. This effect appears to be mediated through both naloxone sensitive and naloxone insensitive mechanisms. The first mechanism seems to be underlying monocyte chemotaxis (18), macrophage cytoxicity (19) and NO formation (20). A naloxone insensitive mechanism has been implicated in human lymphomononuclear cell production of IFNg (21) and macrophage phagocytosis (22). Data now presented describe for the first time the inducion of iNOS expression on human monocytes by b-endorphin (Fig 1C and 1D) and confirm that there is an interaction between neuropeptides produced by the central nervous system and immune responses. It has been suggested that this POMC peptide neither affects nor suppresses monocyte activity (23, 24). However,
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we found a striking induction of iNOS triggered by b-endorphin. This induction slightly decreased in the presence of naloxone (Fig. 1E and 1F). We observed an increase in cAMP when monocytes were incubated with b-endorphin (Fig. 2). The increase in cAMP was lowered if besides b-endorphin naloxone was also present in the medium. We believe that the cAMP elevation, caused by b-endorphin, contributed to iNOS induction. Several lines of evidence support the idea that cAMP plays a role in the induction of iNOS. Recent studies performed on a murine macrophage cell line demonstrate that cAMP increases nitrite levels in cells previously stimulated with LPS (25). It has been observed that in rat peritoneal macrophages cAMP analogues stimulate the LPS and cytokine induction of NO. It seems also that protein kinase A modulates the induction of iNOS (26). Our results strongly suggest that the induction of iNOS by b-endorphin could take place through a receptor mediating an increase in cAMP levels within the monocyte; thus when lymphomononuclear cells were incubated in the presence of H-89, a protein kinase A inhibitor, together with b-endorphin, the effect of this neuropeptide was completely reversed (Fig. 1G and 1H). The release of nitrite/nitrate into the incubation medium of cells treated with bendorphin shows that the enzyme detected by immunocytochemistry was active. The amount of nitrite/nitrate produced was of the same order as that described for activated human monocytes (5,29). However, the elevation of cAMP levels was necessary, but not enough to induce iNOS: when b-endorphin was added to isolated monocytes, an increase in cAMP took place, but not an induction of iNOS expression; something else, provided by the entire mononuclear popultion, factors or cells, seem to be required for this effect to take place. This observation is consistent with previous data showing that immunopotentiating peptides are capable of increasing cAMP (27) in isolated monocytes, but iNOS is induced only if the lymphomononuclear population or cytokines released by activated Th1 cells are present (28). It has been observed that iNOS is expressed on monocytes freshly obtained from patients with multiple sclerosis (MS) (29) and also that NO production is involved in the cell damage taking place in MS: studies performed on extracts of brains from patients who died of MS detected significant levels of iNOS (30). Furthermore, macrophages isolated from active demyelinating MS lesions showed a strong immunoreactivity for iNOS, suggesting (31) that the destruction of myelin and oligodendrocytes in MS could be the result of a cytokine-mediated increase in nitric oxide production by macrophages/microglia (32). It has been demonstrated that stress is an important factor in the relapses of MS, and even in the development of the disease (33). It may be suggested that b-endorphin induc-
tion of iNOS expression on monocytes is one of the possible links bringing together neuroendocrine and immune systems in MS and other autoimmune diseases. The results now reported lead us to conclude that bendorphin is capable of inducing iNOS on peripheral blood monocytes through an elevation of cAMP levels triggered by its binding to a naloxone sensitive. ACKNOWLEDGMENT This work was supported by CICYT, Spain, Grant SAF97-0233. M. S. Aymerich was recipient of a fellowship from the Government of Navarra, Spain.
REFERENCES 1. Celada, A., and Nathan, C. (1994) Immunol. Today 15, 100–102. 2. Kolb, J. P., Paul-Eugene, N., Damais, C., Yamaoka, K., Drapier, J. C., and Dugas, B. (1994) J. Biol. Chem. 269, 9811–9816. 3. Munoz-Ferna´ndez, M. A., Ferna´ndez, M. A., and Fresno, M. (1992) Immunol. Lett. 33, 35–40. 4. De Maria, R., Cifone, M. G., Trotta, R., Rippo, M. R., Festuccia, C., Santoni, A., and Testi, R. (1994) J. Exp. Med. 180, 1999– 2004. 5. Perez- Mediavilla, L. A., Lo´pez-Zabalza, M. J, Calonge, M., Montuenga, L., Lo´pez-Moratalla, N., and Santiago, E. (1995) FEBS Lett. 357, 121–124. 6. Blalock, E. E. (1989) Physiological reviews. 69, 1–32. 7. Brown, S. L., and Van Epps, D. E. (1986) Cell Immunol. 103, 19–26. 8. Chang, K. J. (1984) Trends Neurosci. 7, 234–237. 9. Pert, C. B., Ruff, M. R., Weber, R. T., and Herkenham, M. (1985) J. Immunol. 135, 820–826. 10. Guillemin, R., Vargo, J., Rossier, J., Minick, S., Ling N., Rivier C., Vale W., and Bloom F. (1977) Science 197, 1367–1369. 11. Lynch, D. R., and Snyder, S. H. (1986) Ann. Rev. Biochem. 55, 773–799. 12. Sanders, J. M. (1995) The pharmacology of opioid peptides. Harwood Academic Publishers. 13. Hazum, E., Kwen- Jen, C., and Cuatrecasas, P. (1979) Science 205, 1033–1035. 14. Woods, J. A., Shahabi, N. A., and Sharp, B. M. (1997) Life Sci. 60, 573–586. 15. Bøyum, A. (1983) Scand. J. Immunol. 17, 429–436. 16. Hamid, Q., Springall, D. R., Riveros-Moreno, V., Chanez, P., Howarth, P., Redington, A., Bousquet, J., Godard, P., Holgate, S., and Polak J. (1993) Lancet 342, 1510–1513. 17. Ottaviani, E., Franchini, A., and Franceschi, C. (1997) Int. Rev. Cyt. 170, 79–141. 18. Van Epps, D. E., and Saland, L. (1984) J. Immunol. 132, 3046– 3053. 19. Hagi, K., Uno, K., Kayo, I., and Muramatsu, S. (1994) J. Neuroimmunol. 50, 71–76. 20. Iuvone, T., Capasso, A., D’Aquisto, F., and Canuccio, R. (1995) Biochem. Biophys. Res. Comm. 212, 975–980. 21. Brown, J. L., and Van Epps, D. E. (1986) Cell. Immunol. 103, 19–26.
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22. Ichinose, M., Asai, M., and Sawada, M. (1995) J. Immunol. 42, 311–316. 23. Bhardwaj, R. S., Schwart, A., Becher, E., Mahnke, K., Araganes, Y., Schwart, T., and Luger, T. A. (1996) J. Immunol. 156, 2517– 2521. 24. Lieb, K., Fiebich, B. L., Busse-Grawitz, M., Hull, M., Berger, M., and Bauer, J. (1996) J. Neuroimmunol. 67, 77–81. 25. Mullet, D., Fertel, R. H., Kniss, D., and Cox, G. W., (1997) J. Immunol. 158, 897–904. 26. Pahan, K, Namboodini, A. M., Sheikh, F. G., Smith, B. T., and Singh, I. (1997) J. Biol. Chem. 272, 7786–7791. 27. Lo´pez-Zabalza, M. J., MartıB nez-LausıB n, S., Bengoechea-Alonso, M. T., Lo´pez-Moratalla, N., Gonza´lez, A., and Santiago, E. (1996) Arch. Biochem. Biophys. 15, 136–142.
28. Lo´pez-Moratalla, N., Calleja, A., Gonza´lez, A., Pe´rez-Mediavilla, L. A., Aymerich, M. S., Burrell, M. A., and Santiago, E. (1996) Biochem. Biophys. Res. Comm. 226, 723–729. 29. Lo´pez-Moratalla, N., Gonza´lez, A., Aymerich, M. S., Lo´pez-Zabalza, M. J., PıB o, R., de Castro, P., and Santiago, E. (1996) Nitric Oxide 1, 95–104. 30. Bagasra, O., Michaels, F. H., Zheng, Y. M., Bobroski, L. E., Spitsin, S. V., Fu, Z. F., Towadros, R., and Koprowski, H. (1995) Proc. Natl. Acad. Sci. 92, 12041–12045. 31. De Groot, C. J. A., Ruuls, S. R., Theeuwes, J. W. M., Dijkstra, C. D., and Van der Valk, P. (1997) J. Neuropathol. Exp. Neurol. 56, 10–20. 32. Sherman, M. P., Griscavage, J. M., and Ignarro, L. S. (1992) Med. Hyphotheses 39, 143–146. 33. Van Noort, J. M. (1996) J. Mol. Med. 74, 285–296.
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245, 717–721 (1998)
RC988127
Inducible Nitric Oxide Synthase (iNOS) Expression in Human Monocytes Triggered by b-Endorphin through an Increase in cAMP M. Soledad Aymerich, M. Teresa Bengoechea-Alonso, MarıB a J. Lo´pez-Zabalza, Esteban Santiago, and Natalia Lo´pez-Moratalla1 Department of Biochemistry, University of Navarra, 31080 Pamplona, Spain
Received December 30, 1997
Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of b-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. b-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides b-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by b-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected. q 1998 Academic Press
Macrophages play a variety of roles in the immune system. They may display their phagocytic capacity as direct effector cells against pathogens, or behave as immunoregulatory cells, processing and presenting antigens, or releasing, locally or systemally, different mediators (1). As part of their mechanism of action, macrophages express high levels of inducible nitric oxide synthase (iNOS) after stimulation with various agents (2-5). During the last few years, a number of findings have 1 To whom correspondence should be addressed. Department of Biochemistry, University of Navarra, Apdo. 177, Pamplona, Spain. Fax: (/34 48) 42 56 00; E-mail: [email protected].
provided evidence for a bidirectional relationship between neuroendocrine and immune systems (6,7). Available data suggest that the neuroendocrine-immune relationship influences the pathophysiology of disease processes. Some reports indicate that the nervous system may play a role in the regulation of the immune response through the action of neuropeptides (8,9). A growing body of evidence points out that bendorphin, a neuropeptide released by the pituitary in response to physical or mental stress (10), alters the function of the immune system (11). Opioid receptors are present in the cells of the immune system (12), and their activation results in an inhibition of the adenylate cyclase leading to a decrease in cAMP; the effect of opioid peptides on this receptor is reversed by naloxone. A specific non-opioid receptor for b-endorphin has been described in cultured human lymphocytes (13); and it has been found that neither opioid agonists or antagonists affect the binding of b-endorphin to this receptor. Recently, a naloxone insensitive receptor has been found on murine peritoneal macrophages (14). The study now reported was aimed at determining if b-endorphin, known to be released under stress conditions, could induce iNOS expression in monocytes. That was indeed the case. The results obtained strongly support the idea that the monocyte might be one of the links between neuroendocrine and immune systems. This expression of the inducible nitric oxide synthase was activated through the binding of b-endorphin to a receptor, sensitive to naloxone and leading to an increase in cAMP. MATERIALS AND METHODS Cell sources and cultures. Lymphomononuclear cells, obtained from peripheral blood of donors following the technique described by Bøyum (15), were incubated at 1 1 106 per ml in RPMI-1640 medium supplemented with 10% of fetal calf serum and 2 mM L-glutamine in a 5% CO2 humidified atmosphere. Isolation and purification of
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monocytes were carried out taking advantage of their adherence to culture plates. After a period of 24 hours at 377C nonadherent cells were removed with the medium, and adherent cells washed three times with saline solution. Then, adherent cells, consisting mainly of monocytes (approximately 90 per cent of the cells were CD14/), were carefully detached from the surface of culture plates by gentle friction. Immunocytochemistry. Cells were pelleted by centrifugation, fixed in formaldehyde-picric mixture, and processed for immunochemistry as described previously (5). A polyclonal rabbit antibody against a synthetic peptide (QNGSPQLLTGTAQNVPESLDKLHVTC) of mouse macrophage inducible NO synthase (kindly provided by V. Riveros-Moreno and S. Moncada) was used in the present study. The specificity of this antibody for the detection of human inducible NO synthase has been previously shown (16). The specificity of the immunoreaction was tested by preabsorption of the antiserum with the synthetic antigenic peptide. Determination of nitrite/nitrate. For the determination of nitrites and nitrates, spontaneous degradation products of nitric oxide, a commercial kit was used (Alexis Corporation, Switzerland). The method consists of a simple two-step process. The first step is the conversion of nitrate to nitrite utilizing nitrate reductase. The second step is the addition of Griess reagent which converts nitrite into a deep purple azo compound. Photometric measurement was then carried out at 540 nm. Assays were performed on samples containing 2 1 106 lymphomononuclear cells. An aliquot of the incubation medium was used as reaction blank. Measurement of cAMP. After blood sampling, lymphomononuclear cells were allowed to adhere to the flask surface for a period of 2-4 hours. Adherent cells were incubated at room temperature in culture medium containing 1mM 1-isobutyl-3-methyl xanthine (Sigma) for 20 minutes. Cholera toxin (5mg/mL) (Sigma), 1008 M bendorphin (Sigma), or 1008 M naloxone (Sigma) were added to the medium, when indicated, and cells incubated for 4 minutes. Untreated monocytes were used as controls. The medium was discarded and cells lysed with ice-cold 95% ethanol at 47C for 5 minutes. Lysates were centrifuged at 13,200 rpm at 47C for 5 minutes and supernatants dried under vacuum. Cyclic nucleotide content in the dried samples was quantified using a radioreceptor assay from Amersham Corporation.
RESULTS Induction of iNOS by b-endorphin. Lymphomononuclear cells freshly isolated from healthy donors were incubated for 24 hours in the absence or in the presence of 1008M b-endorphin. Then, monocytes were collected and processed for immunohistochemistry. In the absence of stimulating agents only a small percentage of monocytes (5-10%) was found to be iNOS positive (Fig. 1A and 1B). However, a large number of monocytes turned iNOS immunoreactive after being cultured in the presence of b-endorphin (Fig. 1C and 1D). The number of brown color positive cells increased clearly as compared with control monocytes; we have observed a wide variability of immunoreactive monocytes in five different experiments (30-95%). The specificity of the immunoreaction in monocytes was tested by preabsorption of the antiserum with the synthetic antigenic peptide used to prepare it. Different b-endorphin concentrations (1008M, 10010M and 10012M) were also tested;
the percentage of iNOS positive cells decreased when the concentration of the neuropeptide was lowered (data not shown). Finally, b-endorphin did not induce iNOS, if isolated monocytes were incubated under the same conditions as the entire population of lymphomononuclear cells. With the purpose of verifying if the expressed iNOS was active NO related products (nitrite/nitrate) were measured in the in the supernatant of the incubated cells as indicated in Materials and Methods. For this determination 0.5 mM L-arginine was present in the incubation medium as exogenous substrate. The amount of nitrite/nitrate, NO degradation products, released to the medium by PBMC incubated for 24 hours in the presence of 1008M b-endorphin was 5.4 nmoles per 105 cells (average of three different determinations). Nitrite/nitrate were not detected in the supernatants of cells from the same subjects if incubated for the same period in the absence of b-endorphin. b-endorphin increased cAMP levels in monocytes. Naloxone sensitive opioid receptors are known to inhibit adenylate cyclase activity and lower cAMP levels within the cells. However, we observed that the addition of 1008M b-endorphin to monocytes resulted in an increase in cAMP production (Fig. 2). Cholera toxin (5mg/mL) increased also cAMP levels. When adding both cholera toxin and b-endorphin, cAMP levels did not decrease with respect to those reached with cholera toxin alone. When, besides b-endorphin, 1008M naloxone was added to the medium, a decrease in the elevated levels of cAMP, caused by b-endorphin, was observed (Fig. 2). An inhibitor of protein kinase A prevented iNOS induction. The possible involvement of cAMP mediating iNOS expression was further investigated. To prevent the b-endorphin effect through cAMP we added H-89 (Calbiochem), an inhibitor of protein kinase A, to the incubation medium. When lymphomononuclear cells were cultured for a period of 24 hours with b-endorphin in the presence of 5mM H-89, immunostaining for iNOS strongly decreased (Fig. 1G and 1H). Lymphomononuclear cells belonging to the same donors, cultured for the same period of time and processed simultaneously, were used as negative control. Effect of naloxone on iNOS induction. Lymphomononuclear cells were also incubated for a period of 24 hours with b-endorphin in the presence of 1008M naloxone. When, besides b-endorphin, naloxone was added to the medium, iNOS expression was maintained, although a slight decrease in immunoreactivity (25-60%) was observed (Fig. 1E and 1F). DISCUSSION Macrophages play a central role in immune and stress responses, both processes being apparently me-
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FIG. 1. Inducible nitric oxide synthase in human monocytes detected by immunocytochemistry. Photographs are representative of an experiment in wich all cells corresponded to the same healthy donor and were processed simultaneously. Basal monocytes (A and B) did not express iNOS unless activated with 1008M b-endorphin (C and D). When besides b-endorphin, 1008M naloxone was present in the medium, iNOS continued to be expressed (E and F), although the niumber of stained cells slightly decreased. However, if lymphomononuclear cells were incubated in the presence of b-endorphin and 5mM H-89, an inhibitor of protein kinase A, immunostaining for iNOS did not appear (G and H). Photographs have been taken at two different magnifications (1 440 and 1 1.100) for each treatment.
FIG. 2. Effect of b-endorphin on cAMP levels in human monocytes. b-endorphin. C, control monocytes; CT, monocytes incubated in the presence of cholera toxin (5mg/mL); b-end, monocytes incubated in the presence of 1008M b-endorphin. b-end / NLX, monocytes incubated in the presence of 1008M b-endorphin and 1008M naloxone. Data represent mean { SD of duplicated of four experiments with different donors.
diated by a common pool of molecules: pro-opiomelanocortin-derived (POMC) peptides, such as b-endorphin, neurohormones, small bioactive molecules, cytokines, such as IL-1, IL-6 and TNFa, and NO (17). b-endorphin is a 31 amino acid endogenous opioid peptide synthesized by the pituitary and released into the circulation under stress situations (10). Recently there has been growing evidence that b-endorphin alters the function of a variety of cells of the immune system. This effect appears to be mediated through both naloxone sensitive and naloxone insensitive mechanisms. The first mechanism seems to be underlying monocyte chemotaxis (18), macrophage cytoxicity (19) and NO formation (20). A naloxone insensitive mechanism has been implicated in human lymphomononuclear cell production of IFNg (21) and macrophage phagocytosis (22). Data now presented describe for the first time the inducion of iNOS expression on human monocytes by b-endorphin (Fig 1C and 1D) and confirm that there is an interaction between neuropeptides produced by the central nervous system and immune responses. It has been suggested that this POMC peptide neither affects nor suppresses monocyte activity (23, 24). However,
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we found a striking induction of iNOS triggered by b-endorphin. This induction slightly decreased in the presence of naloxone (Fig. 1E and 1F). We observed an increase in cAMP when monocytes were incubated with b-endorphin (Fig. 2). The increase in cAMP was lowered if besides b-endorphin naloxone was also present in the medium. We believe that the cAMP elevation, caused by b-endorphin, contributed to iNOS induction. Several lines of evidence support the idea that cAMP plays a role in the induction of iNOS. Recent studies performed on a murine macrophage cell line demonstrate that cAMP increases nitrite levels in cells previously stimulated with LPS (25). It has been observed that in rat peritoneal macrophages cAMP analogues stimulate the LPS and cytokine induction of NO. It seems also that protein kinase A modulates the induction of iNOS (26). Our results strongly suggest that the induction of iNOS by b-endorphin could take place through a receptor mediating an increase in cAMP levels within the monocyte; thus when lymphomononuclear cells were incubated in the presence of H-89, a protein kinase A inhibitor, together with b-endorphin, the effect of this neuropeptide was completely reversed (Fig. 1G and 1H). The release of nitrite/nitrate into the incubation medium of cells treated with bendorphin shows that the enzyme detected by immunocytochemistry was active. The amount of nitrite/nitrate produced was of the same order as that described for activated human monocytes (5,29). However, the elevation of cAMP levels was necessary, but not enough to induce iNOS: when b-endorphin was added to isolated monocytes, an increase in cAMP took place, but not an induction of iNOS expression; something else, provided by the entire mononuclear popultion, factors or cells, seem to be required for this effect to take place. This observation is consistent with previous data showing that immunopotentiating peptides are capable of increasing cAMP (27) in isolated monocytes, but iNOS is induced only if the lymphomononuclear population or cytokines released by activated Th1 cells are present (28). It has been observed that iNOS is expressed on monocytes freshly obtained from patients with multiple sclerosis (MS) (29) and also that NO production is involved in the cell damage taking place in MS: studies performed on extracts of brains from patients who died of MS detected significant levels of iNOS (30). Furthermore, macrophages isolated from active demyelinating MS lesions showed a strong immunoreactivity for iNOS, suggesting (31) that the destruction of myelin and oligodendrocytes in MS could be the result of a cytokine-mediated increase in nitric oxide production by macrophages/microglia (32). It has been demonstrated that stress is an important factor in the relapses of MS, and even in the development of the disease (33). It may be suggested that b-endorphin induc-
tion of iNOS expression on monocytes is one of the possible links bringing together neuroendocrine and immune systems in MS and other autoimmune diseases. The results now reported lead us to conclude that bendorphin is capable of inducing iNOS on peripheral blood monocytes through an elevation of cAMP levels triggered by its binding to a naloxone sensitive. ACKNOWLEDGMENT This work was supported by CICYT, Spain, Grant SAF97-0233. M. S. Aymerich was recipient of a fellowship from the Government of Navarra, Spain.
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